BioMed Research International: Microbiology The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Combating Pathogenic Microorganisms Using Plant-Derived Antimicrobials: A Minireview of the Mechanistic Basis Sun, 14 Sep 2014 12:15:47 +0000 The emergence of antibiotic resistance in pathogenic bacteria has led to renewed interest in exploring the potential of plant-derived antimicrobials (PDAs) as an alternative therapeutic strategy to combat microbial infections. Historically, plant extracts have been used as a safe, effective, and natural remedy for ailments and diseases in traditional medicine. Extensive research in the last two decades has identified a plethora of PDAs with a wide spectrum of activity against a variety of fungal and bacterial pathogens causing infections in humans and animals. Active components of many plant extracts have been characterized and are commercially available; however, research delineating the mechanistic basis of their antimicrobial action is scanty. This review highlights the potential of various plant-derived compounds to control pathogenic bacteria, especially the diverse effects exerted by plant compounds on various virulence factors that are critical for pathogenicity inside the host. In addition, the potential effect of PDAs on gut microbiota is discussed. Abhinav Upadhyay, Indu Upadhyaya, Anup Kollanoor-Johny, and Kumar Venkitanarayanan Copyright © 2014 Abhinav Upadhyay et al. All rights reserved. Molecular Identification and Quantification of Tetracycline and Erythromycin Resistance Genes in Spanish and Italian Retail Cheeses Thu, 11 Sep 2014 09:17:27 +0000 Large antibiotic resistance gene pools in the microbiota of foods may ultimately pose a risk for human health. This study reports the identification and quantification of tetracycline- and erythromycin-resistant populations, resistance genes, and gene diversity in traditional Spanish and Italian cheeses, via culturing, conventional PCR, real-time quantitative PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). The numbers of resistant bacteria varied widely among the antibiotics and the different cheese varieties; in some cheeses, all the bacterial populations seemed to be resistant. Up to eight antibiotic resistance genes were sought by gene-specific PCR, six with respect to tetracycline, that is, tet(K), tet(L), tet(M), tet(O), tet(S), and tet(W), and two with respect to erythromycin, that is, erm(B) and erm(F). The most common resistance genes in the analysed cheeses were tet(S), tet(W), tet(M), and erm(B). The copy numbers of these genes, as quantified by qPCR, ranged widely between cheeses (from 4.94 to ). DGGE analysis revealed distinct banding profiles and two polymorphic nucleotide positions for tet(W)-carrying cheeses, though the similarity of the sequences suggests this tet(W) to have a monophyletic origin. Traditional cheeses would therefore appear to act as reservoirs for large numbers of many types of antibiotic resistance determinants. Ana Belén Flórez, Ángel Alegría, Franca Rossi, Susana Delgado, Giovanna E. Felis, Sandra Torriani, and Baltasar Mayo Copyright © 2014 Ana Belén Flórez et al. All rights reserved. Bifidobacteria-Host Interactions—An Update on Colonisation Factors Thu, 11 Sep 2014 06:39:36 +0000 Bifidobacteria are one of the predominant bacterial groups of the human intestinal microbiota and have important functional properties making them interesting for the food and dairy industries. Numerous in vitro and preclinical studies have shown beneficial effects of particular bifidobacterial strains or strain combinations on various health parameters of their hosts. This indicates the potential of bifidobacteria in alternative or supplementary therapeutic approaches in a number of diseased states. Based on these observations, bifidobacteria have attracted considerable interest by the food, dairy, and pharmaceutical industries and they are widely used as so-called probiotics. As a consequence of the rapidly increasing number of available bifidobacterial genome sequences and their analysis, there has been substantial progress in the identification of bifidobacterial structures involved in colonisation of and interaction with the host. With the present review, we aim to provide an update on the current knowledge on the mechanisms by which bifidobacteria colonise their hosts and exert health promoting effects. Verena Grimm, Christina Westermann, and Christian U. Riedel Copyright © 2014 Verena Grimm et al. All rights reserved. Effect of High Hydrostatic Pressure Processing on Microbiological Shelf-Life and Quality of Fruits Pretreated with Ascorbic Acid or SnCl2 Thu, 11 Sep 2014 06:30:38 +0000 In the current study, the processing conditions required for the inactivation of Paenibacillus polymyxa and relevant spoilage microorganisms by high hydrostatic pressure (HHP) treatment on apricot, peach, and pear pieces in sucrose (22°Brix) solution were assessed. Accordingly, the shelf-life was determined by evaluating both the microbiological quality and the sensory characteristics (taste, odor, color, and texture) during refrigerated storage after HHP treatment. The microbiological shelf-life of apricots, peaches, and pears was prolonged in the HHP-treated products in comparison with the untreated ones. In all HHP-treated packages for apricots, peaches, and pears, all populations were below the detection limit of the method (1 log CFU/g) and no growth of microorganisms was observed until the end of storage. Overall, no differences of the , , or value among the untreated and the HHP-treated fruit products were observed up to the time at which the unpressurized product was characterized as spoiled. HHP treatment had no remarkable effect on the firmness of the apricots, peaches, and pears. With regard to the sensory assessment, the panelists marked better scores to HHP-treated products compared to their respective controls, according to taste and total evaluation during storage of fruit products. Anthoula A. Argyri, Chrysoula C. Tassou, Fotios Samaras, Constantinos Mallidis, and Nikos Chorianopoulos Copyright © 2014 Anthoula A. Argyri et al. All rights reserved. Epidemiology of West Nile Disease in Europe and in the Mediterranean Basin from 2009 to 2013 Thu, 11 Sep 2014 06:30:22 +0000 West Nile virus (WNV) transmission has been confirmed in the last four years in Europe and in the Mediterranean Basin. An increasing concern towards West Nile disease (WND) has been observed due to the high number of human and animal cases reported in these areas confirming the importance of this zoonosis. A new epidemiological scenario is currently emerging: although new introductions of the virus from abroad are always possible, confirming the epidemiological role played by migratory birds, the infection endemisation in some European territories today is a reality supported by the constant reoccurrence of the same strains across years in the same geographical areas. Despite the WND reoccurrence in the Old World, the overwintering mechanisms are not well known, and the role of local resident birds or mosquitoes in this context is poorly understood. A recent new epidemiological scenario is the spread of lineage 2 strain across European and Mediterranean countries in regions where lineage 1 strain is still circulating creating favourable conditions for genetic reassortments and emergence of new strains. This paper summarizes the main epidemiological findings on WNV occurrence in Europe and in the Mediterranean Basin from 2009 to 2013, considering potential future spread patterns. Daria Di Sabatino, Rossana Bruno, Francesca Sauro, Maria Luisa Danzetta, Francesca Cito, Simona Iannetti, Valeria Narcisi, Fabrizio De Massis, and Paolo Calistri Copyright © 2014 Daria Di Sabatino et al. All rights reserved. Attachment of Asaia bogorensis Originating in Fruit-Flavored Water to Packaging Materials Thu, 11 Sep 2014 00:00:00 +0000 The objective of this study was to investigate the adhesion of isolated spoilage bacteria to packaging materials used in the food industry. Microorganisms were isolated from commercial fruit-flavored mineral water in plastic bottles with flocks as a visual defect. The Gram-negative rods were identified using the molecular method through the amplification of a partial region of the 16S rRNA gene. Based on the sequence identity (99.6%) between the spoilage organism and a reference strain deposited in GenBank, the spoilage isolate was identified as Asaia bgorensis. Experiments on bacterial adhesion were conducted using plates made of glass and polystyrene (packaging materials commonly used in the beverage industry). Cell adhesion ability was determined using luminometry, plate count, and the microscopic method. The strain of A. bogorensis was characterized by strong adhesion properties which were dependent on the surface type, with the highest cell adhesion detected on polystyrene. Dorota Kregiel, Anna Otlewska, and Hubert Antolak Copyright © 2014 Dorota Kregiel et al. All rights reserved. Cytotoxicity of Probiotics from Philippine Commercial Dairy Products on Cancer Cells and the Effect on Expression of cfos and cjun Early Apoptotic-Promoting Genes and Interleukin-1β and Tumor Necrosis Factor-α Proinflammatory Cytokine Genes Sun, 07 Sep 2014 06:19:56 +0000 This study determined cytotoxicity of probiotic Lactobacillus spp. from Philippine dairy products on cancer cells and normal fibroblasts and their effects on expression of early apoptotic-promoting cfos, cjun and proinflammatory cytokine IL-1β, TNF-α genes. Cultures were from Yakult, Bear Brand Probiotic Drink, Nido3+ Powdered Milk. Filter-sterilized supernatants from cultures of Lactobacillus spp. were evaluated for cytotoxicity to colon cancer cells (HT-29 and HCT116), leukemia cells (THP-1), and normal human dermal fibroblasts (HDFn) using PrestoBlue. Bleomycin was the positive control. Absolute quantification of transcript levels was conducted using qRT-PCR. Cytotoxicity index profiles on HDFn, THP-1 of all probiotic supernatants and negative controls suggest nontoxicity to the cells when compared to bleomycin, whereas all probiotic supernatants were found to be cytotoxic to HT-29 and HCT-116 colon cancer cell lines. Expression of cfos, cjun transcripts was significantly upregulated in HT-29 and HCT116 cells treated with probiotic supernatants compared to untreated baseline levels (). Expression of IL-1β and TNF-α by lipopolysaccharide-treated macrophages was significantly downregulated in cells with probiotic supernatants compared to those exposed to MRS medium (). Results provide strong support for the role of Lactobacillus spp. studied in anticancer therapy and in prevention of inflammation that may act as precursor to carcinogenesis. Peter T. Shyu, Glenn G. Oyong, and Esperanza C. Cabrera Copyright © 2014 Peter T. Shyu et al. All rights reserved. Successive Nonstatistical and Statistical Approaches for the Improved Antibiotic Activity of Rare Actinomycete Nonomuraea sp. JAJ18 Wed, 03 Sep 2014 12:59:02 +0000 The selection and optimization of nutritional constituents as well as their levels for the improved production of antibiotic by Nonomuraea sp. JAJ18 were carried out using combination of both nonstatistical one-factor-at-a-time (OFAT) method and statistical response surface methodology (RSM). Using OFAT method, starch and (NH4)2SO4 were identified as suitable carbon and nitrogen sources, respectively. Subsequently, starch, NaCl, and MgSO47H2O were recognized as the most significant media components with confidence level of above 95% using the Plackett-Burman design. The levels of the three media components were further optimized using RSM employed with Box-Behnken design. Accordingly, a second-order polynomial regression model was fitted into the experimental data. By analyzing the response surface plots as well as using numerical optimization method, the optimal levels for starch, NaCl, and MgSO47H2O were determined as 15.6 g/L, 0.8 g/L, and 1.98 g/L, respectively. With the optimized medium, 15.5% increase was observed in antibiotic activity of JAJ18. Results further support the use of RSM for media optimization. To the best of our knowledge, this is the first report of statistical media optimization for antibiotic production in rare actinomycete Nonomuraea species, which will be useful for the development of Nonomuraea cultivation process for efficient antibiotic production on a large scale. Polpass Arul Jose and Solomon Robinson David Jebakumar Copyright © 2014 Polpass Arul Jose and Solomon Robinson David Jebakumar. All rights reserved. Origanum dictamnus Oil Vapour Suppresses the Development of Grey Mould in Eggplant Fruit In Vitro Mon, 01 Sep 2014 13:17:10 +0000 Grey mould rot (Botrytis cinerea) development in vitro or in eggplant (Solanum melongena L.) fruit was evaluated after treatment with dittany (Origanum dictamnus L.) oil (DIT) and storage at 12°C and 95% relative humidity during or following exposure to the volatiles. DIT volatiles used in different concentration (0-50-100-250 μL/L) and times of exposure (up to 120 h) examined the effects on pathogen development as well as fruit quality parameters. In vitro, fungal colony growth was inhibited with the application of DIT oil (during or after exposure) and/or time of application. Continuous exposure to oils reduced conidial germination and production with fungistatic effects observed in 250 μL/L. In vivo, fungal lesion growth and conidial production reduced in DIT-treated fruits. Interesting, in fruits preexposed to volatiles before fungal inoculation, DIT application induced fruit resistance against the pathogen, by reduced lesion growth and conidial production. Conidial viability reduced in >100 μL/L DIT oil. Fruits exposed to essential oil did not affect fruit quality related attributes in general, while skin lightness (L value) increased in 50 and 100 μL/L DIT oil. The results of the current study indicated that dittany volatiles may be considered as an alternative food preservative, eliminating disease spread in the storage/transit atmospheres. Andriana Stavropoulou, Kostas Loulakakis, Naresh Magan, and Nikos Tzortzakis Copyright © 2014 Andriana Stavropoulou et al. All rights reserved. Differences in Extended-Spectrum Beta-Lactamase Producing Escherichia coli Virulence Factor Genes in the Baltic Sea Region Thu, 28 Aug 2014 12:19:42 +0000 The aim of this study was to compare the prevalence of different virulence factor (VF) genes in extended-spectrum beta-lactamase (ESBL) producing Escherichia coli strains isolated from the Baltic Sea region. A total of 432 strains of phenotypically ESBL positive E. coli were collected from 20 institutions located in Estonia, Latvia, Lithuania, and the region of St. Petersburg in Russia from January to May 2012 and analyzed for phylogenetic group and prevalence of 23 VF genes. The strains were collected from clinical material (urine, blood, wound, and respiratory tract). Bacterial isolates were compared according to phylogenetic group, clinical material, and geographical origin. Most of the VF genes were concentrated within phylogenetic group B2 and/or D. When comparing strains isolated from different countries, it was found that strains originating from Estonia and Latvia belonged mainly to group B2 and strains from Lithuania and Russia mainly to groups B2 and D. The P-fimbrial adhesin gene papEF was more prevalent in Russian strains, colicin gene cvaC in Lithuanian strains, and capsular gene kpsMTII in Latvian strains; serum resistant gene traT was less prevalent in Estonian strains. The regional differences of VF genes remained statistically significant after taking into account the phylogenetic distribution in the countries. Jana Lillo, Kristiine Pai, Arta Balode, Mariia Makarova, Kristi Huik, Siiri Kõljalg, Marina Ivanova, Lidia Kaftyreva, Jolanta Miciuleviciene, Paul Naaber, Kristel Parv, Anastasia Pavelkovich, Tiiu Rööp, Karolin Toompere, Ludmila Suzhaeva, and Epp Sepp Copyright © 2014 Jana Lillo et al. All rights reserved. The Probiotic Bifidobacterium breve B632 Inhibited the Growth of Enterobacteriaceae within Colicky Infant Microbiota Cultures Thu, 28 Aug 2014 08:39:14 +0000 Infant colic is a common gastrointestinal disorder of newborns, mostly related to imbalances in the composition of gut microbiota and particularly to the presence of gas-producing coliforms and to lower levels of Bifidobacteria and Lactobacilli. Probiotics could help to contain this disturbance, with formulations consisting of Lactobacillus strains being the most utilized. In this work, the probiotic strain Bifidobacterium breve B632 that was specifically selected for its ability to inhibit gas-producing coliforms, was challenged against the Enterobacteriaceae within continuous cultures of microbiota from a 2-month-old colicky infant. As confirmed by RAPD-PCR fingerprinting, B. breve B632 persisted in probiotic-supplemented microbiota cultures, accounting for the 64% of Bifidobacteria at the steady state. The probiotic succeeded in inhibiting coliforms, since FISH and qPCR revealed that the amount of Enterobacteriaceae after 18 h of cultivation was 0.42 and 0.44 magnitude orders lower in probiotic-supplemented microbiota cultures than in the control ones. These results support the possibility to move to another level of study, that is, the administration of B. breve B632 to a cohort of colicky newborns, in order to observe the behavior of this strain in vivo and to validate its effect in colic treatment. Marta Simone, Caterina Gozzoli, Andrea Quartieri, Giuseppe Mazzola, Diana Di Gioia, Alberto Amaretti, Stefano Raimondi, and Maddalena Rossi Copyright © 2014 Marta Simone et al. All rights reserved. Possible Use of Bacteriophages Active against Bacillus anthracis and Other B. cereus Group Members in the Face of a Bioterrorism Threat Thu, 28 Aug 2014 08:38:43 +0000 Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat. Ewa Jończyk-Matysiak, Marlena Kłak, Beata Weber-Dąbrowska, Jan Borysowski, and Andrzej Górski Copyright © 2014 Ewa Jończyk-Matysiak et al. All rights reserved. Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota Thu, 28 Aug 2014 00:00:00 +0000 Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest. Chiara Devirgiliis, Paola Zinno, Mariarita Stirpe, Simona Barile, and Giuditta Perozzi Copyright © 2014 Chiara Devirgiliis et al. All rights reserved. Soil Fungal Resources in Annual Cropping Systems and Their Potential for Management Thu, 28 Aug 2014 00:00:00 +0000 Soil fungi are a critical component of agroecosystems and provide ecological services that impact the production of food and bioproducts. Effective management of fungal resources is essential to optimize the productivity and sustainability of agricultural ecosystems. In this review, we (i) highlight the functional groups of fungi that play key roles in agricultural ecosystems, (ii) examine the influence of agronomic practices on these fungi, and (iii) propose ways to improve the management and contribution of soil fungi to annual cropping systems. Many of these key soil fungal organisms (i.e., arbuscular mycorrhizal fungi and fungal root endophytes) interact directly with plants and are determinants of the efficiency of agroecosystems. In turn, plants largely control rhizosphere fungi through the production of carbon and energy rich compounds and of bioactive phytochemicals, making them a powerful tool for the management of soil fungal diversity in agriculture. The use of crop rotations and selection of optimal plant genotypes can be used to improve soil biodiversity and promote beneficial soil fungi. In addition, other agronomic practices (e.g., no-till, microbial inoculants, and biochemical amendments) can be used to enhance the effect of beneficial fungi and increase the health and productivity of cultivated soils. Walid Ellouze, Ahmad Esmaeili Taheri, Luke D. Bainard, Chao Yang, Navid Bazghaleh, Adriana Navarro-Borrell, Keith Hanson, and Chantal Hamel Copyright © 2014 Walid Ellouze et al. All rights reserved. Dyeing Industry Effluent System as Lipid Production Medium of Neochloris sp. for Biodiesel Feedstock Preparation Wed, 27 Aug 2014 05:23:57 +0000 Microalgae lipid feedstock preparation cost was an important factor in increasing biodiesel fuel hikes. This study was conducted with the concept of implementing an effluent wastewater as lipid production medium for microalgae cultivation. In our study textile dyeing industry effluent was taken as a lipid production medium for Neochloris sp. cultivation. The changes in physicochemical analysis of effluent before and after Neochloris sp. treatment were recorded using standard procedures and AAS analysis. There was especially a reduction in heavy metal like lead (Pb) concentration from 0.002 ppm to 0.001 ppm after Neochloris sp. treatment. Neochloris sp. cultivated in Bold Basal Medium (BBM) (specific algal medium) produced 41.93% total lipid and 36.69% lipid was produced in effluent based cultivation. Surprisingly Neochloris sp. cultivated in effluent was found with enhanced neutral lipid content, and it was confirmed by Nile red fluorescence assay. Further the particular enrichment in oleic acid content of the cells was confirmed with thin layer chromatography (TLC) with oleic acid pure (98%) control. The overall results suggested that textile dyeing industry effluent could serve as the best lipid productive medium for Neochloris sp. biodiesel feedstock preparation. This study was found to have a significant impact on reducing the biodiesel feedstock preparation cost with simultaneous lipid induction by heavy metal stress to microalgae. Vidyadharani Gopalakrishnan and Dhandapani Ramamurthy Copyright © 2014 Vidyadharani Gopalakrishnan and Dhandapani Ramamurthy. All rights reserved. Phenotypic Characterization of Mycoplasma synoviae Induced Changes in the Metabolic and Sensitivity Profile of In Vitro Infected Chicken Chondrocytes Tue, 26 Aug 2014 13:06:54 +0000 In infectious synovitis caused by Mycoplasma synoviae chicken chondrocytes (CCH) may come into direct contact with these bacteria that are also capable of invading CCH in vitro. In this study, phenotype microarrays were used to evaluate the influence of Mycoplasma synoviae on the global metabolic activity of CCH. Therefore, CCH were cultured in the presence of 504 individual compounds, spotted in wells of 11 phenotype microarrays for eukaryotic cells, and exposed to Mycoplasma synoviae membranes or viable Mycoplasma synoviae. Metabolic activity and sensitivity of normal cells versus infected cells were evaluated. Metabolic profiles of CCH treated with viable Mycoplasma synoviae or its membranes were significantly different from those of CCH alone. CCH treated with Mycoplasma synoviae membranes were able to use 48 carbon/nitrogen sources not used by CCH alone. Treatment also influenced ion uptake in CCH and intensified the sensitivity to 13 hormones, 5 immune mediators, and 29 cytotoxic chemicals. CCH were even more sensitive to hormones/immune mediators when exposed to viable Mycoplasma synoviae. Our results indicate that exposure to Mycoplasma synoviae or its membranes induces a wide range of metabolic and sensitivity modifications in CCH that can contribute to pathological processes in the development of infectious synovitis. Daliborka Dušanić, Dušan Benčina, Mojca Narat, and Irena Oven Copyright © 2014 Daliborka Dušanić et al. All rights reserved. Nonthermal Pasteurization of Fermented Green Table Olives by means of High Hydrostatic Pressure Processing Mon, 18 Aug 2014 11:18:10 +0000 Green fermented olives cv. Halkidiki were subjected to different treatments of high hydrostatic pressure (HHP) processing (400, 450, and 500 MPa for 15 or 30 min). Total viable counts, lactic acid bacteria and yeasts/moulds, and the physicochemical characteristics of the product (pH, colour, and firmness) were monitored right after the treatment and after 7 days of storage at 20°C to allow for recovery of injured cells. The treatments at 400 MPa for 15 and 30 min, 450 MPa for 15 and 30 min, and 500 MPa for 15 min were found insufficient as a recovery of the microbiota was observed. The treatment at 500 MPa for 30 min was effective in reducing the olive microbiota below the detection limit of the enumeration method after the treatment and after 1 week of storage and was chosen as being more appropriate for storing olives for an extended time period (5 months). After 5 months of storage at 20°C, no microbiota was detected in treated samples, while significant changes for both HHP treated and untreated olives were observed for colour parameters only (minor degradation). In conclusion, HHP treatment may introduce a reliable nonthermal pasteurization method to extend the microbiological shelf-life of fermented table olives. Anthoula A. Argyri, Efstathios Z. Panagou, George-John E. Nychas, and Chrysoula C. Tassou Copyright © 2014 Anthoula A. Argyri et al. All rights reserved. Implementation of a Computerized Decision Support System to Improve the Appropriateness of Antibiotic Therapy Using Local Microbiologic Data Sun, 17 Aug 2014 09:29:17 +0000 A prospective quasi-experimental study was undertaken in 218 patients with suspicion of nosocomial infection hospitalized in a polyvalent ICU where a new electronic device (GERB) has been designed for antibiotic prescriptions. Two GERB-based applications were developed to provide local resistance maps (LRMs) and preliminary microbiological reports with therapeutic recommendation (PMRTRs). Both applications used the data in the Laboratory Information System of the Microbiology Department to report on the optimal empiric therapeutic option, based on the most likely susceptibility profile of the microorganisms potentially responsible for infection in patients and taking into account the local epidemiology of the hospital department/unit. LRMs were used for antibiotic prescription in 20.2% of the patients and PMRTRs in 78.2%, and active antibiotics against the finally identified bacteria were prescribed in 80.0% of the former group and 82.4% of the latter. When neither LMRs nor PMRTRs were considered for empiric treatment prescription, only around 40% of the antibiotics prescribed were active. Hence, the percentage appropriateness of the empiric antibiotic treatments was significantly higher when LRM or PMRTR guidelines were followed rather than other criteria. LRMs and PMRTRs applications are dynamic, highly accessible, and readily interpreted instruments that contribute to the appropriateness of empiric antibiotic treatments. Manuel Rodriguez-Maresca, Antonio Sorlozano, Magnolia Grau, Rocio Rodriguez-Castaño, Andres Ruiz-Valverde, and Jose Gutierrez-Fernandez Copyright © 2014 Manuel Rodriguez-Maresca et al. All rights reserved. Antibacterial Activity of Pseudonocardia sp. JB05, a Rare Salty Soil Actinomycete against Staphylococcus aureus Thu, 14 Aug 2014 06:41:52 +0000 Staphylococcus aureus is a Gram-positive bacterium that causes many harmful and life-threatening diseases. Some strains of this bacterium are resistant to available antibiotics. This study was designed to evaluate the ability of indigenous actinomycetes to produce antibacterial compounds against S. aureus and characterize the structure of the resultant antibacterial compounds. Therefore, a slightly modified agar well diffusion method was used to determine the antibacterial activity of actinomycete isolates against the test microorganisms. The bacterial extracts with antibacterial activity were fractionated by silica gel and G-25 sephadex column chromatography. Also, the active fractions were analyzed by thin layer chromatography. Finally, the partial structure of the resultant antibacterial compound was characterized by Fourier transform infrared spectroscopy. One of the isolates, which had a broad spectrum and high antibacterial activity, was designated as Pseudonocardia sp. JB05, based on the results of biochemical and 16S rDNA gene sequence analysis. Minimum inhibitory concentration for this bacterium was 40 AU mL−1 against S. aureus. The antibacterial activity of this bacterium was stable after autoclaving, 10% SDS, boiling, and proteinase K. Thin layer chromatography, using anthrone reagent, showed the presence of carbohydrates in the purified antibacterial compound. Finally, FT-IR spectrum of the active compound illustrated hydroxyl groups, hydrocarbon skeleton, and double bond of polygenic compounds in its structure. To the best of our knowledge, this is the first report describing the efficient antibacterial activity by a local strain of Pseudonocardia. The results presented in this work, although at the initial stage in bioactive product characterization, will possibly contribute toward the Pseudonocardia scale-up for the production and identification of the antibacterial compounds. Nesa Jafari, Reza Behroozi, Davoud Farajzadeh, Mohammad Farsi, and Kambiz Akbari-Noghabi Copyright © 2014 Nesa Jafari et al. All rights reserved. Helicobacter pylori in Vegetables and Salads: Genotyping and Antimicrobial Resistance Properties Tue, 12 Aug 2014 12:06:40 +0000 From a clinical and epidemiological perspective, it is important to know which genotypes and antibiotic resistance patterns are present in H. pylori strains isolated from salads and vegetables. Therefore, the present investigation was carried out to find this purpose. Three hundred eighty washed and unwashed vegetable samples and fifty commercial and traditional salad samples were collected from Isfahan, Iran. Samples were cultured and those found positive for H. pylori were analyzed using PCR. Antimicrobial susceptibility testing was performed using disk diffusion method. Seven out of 50 (14%) salad and 52 out of 380 (13.68%) vegetable samples harbored H. pylori. In addition, leek, lettuce, and cabbage were the most commonly contaminated samples (30%). The most prevalent virulence genes were oipA (86.44%) and cagA (57.625). VacA s1a (37.28%) and iceA1 (47.45%) were the most prevalent genotypes. Forty different genotypic combinations were recognized. S1a/cagA+/iceA1/oipA+ (33.89%), s1a/cagA+/iceA2/oipA (30.50%), and m1a/cagA+/iceA1/oipA+ (28.81%) were the most prevalent combined genotypes. Bacterial strains had the highest levels of resistance against metronidazole (77.96%), amoxicillin (67.79%), and ampicillin (61.01%). High similarity in the genotyping pattern of H. pylori among vegetable and salad samples and human specimens suggests that vegetable and salads may be the sources of the bacteria. Emad Yahaghi, Faham Khamesipour, Fatemeh Mashayekhi, Farhad Safarpoor Dehkordi, Mohammad Hossein Sakhaei, Mojtaba Masoudimanesh, and Maryam Khayyat Khameneie Copyright © 2014 Emad Yahaghi et al. All rights reserved. Biodegradation Ability and Catabolic Genes of Petroleum-Degrading Sphingomonas koreensis Strain ASU-06 Isolated from Egyptian Oily Soil Sun, 10 Aug 2014 08:25:20 +0000 Polycyclic aromatic hydrocarbons (PAHs) are serious pollutants and health hazards. In this study, 15 PAHs-degrading bacteria were isolated from Egyptian oily soil. Among them, one Gram-negative strain (ASU-06) was selected and biodegradation ability and initial catabolic genes of petroleum compounds were investigated. Comparison of 16S rRNA gene sequence of strain ASU-06 to published sequences in GenBank database as well as phylogenetic analysis identified ASU-06 as Sphingomonas koreensis. Strain ASU-06 degraded 100, 99, 98, and 92.7% of 100 mg/L naphthalene, phenanthrene, anthracene, and pyrene within 15 days, respectively. When these PAHs present in a mixed form, the enhancement phenomenon appeared, particularly in the degradation of pyrene, whereas the degradation rate was 98.6% within the period. This is the first report showing the degradation of different PAHs by this species. PCR experiments with specific primers for catabolic genes alkB, alkB1, nahAc, C12O, and C23O suggested that ASU-06 might possess genes for aliphatic and PAHs degradation, while PAH-RHDαGP gene was not detected. Production of biosurfactants and increasing cell-surface hydrophobicity were investigated. GC/MS analysis of intermediate metabolites of studied PAHs concluded that this strain utilized these compounds via two main pathways, and phthalate was the major constant product that appeared in each day of the degradation period. Abd El-Latif Hesham, Asmaa M. M. Mawad, Yasser M. Mostafa, and Ahmed Shoreit Copyright © 2014 Abd El-Latif Hesham et al. All rights reserved. The Association of Toll-Like Receptor 4 Polymorphism with Hepatitis C Virus Infection in Saudi Arabian Patients Sun, 10 Aug 2014 08:19:43 +0000 Hepatitis C virus (HCV) is a single stranded RNA virus. It affects millions of people worldwide and is considered as a leading cause of liver diseases including cirrhosis and hepatocellular carcinoma. A recent study reported that TLR4 gene polymorphisms are good prognostic predictors and are associated with protection from liver fibrosis among Caucasians. This study aims to investigate the implication of genetic polymorphisms of TLR4 gene on the HCV infection in Saudi Arabian patients. Two SNPs in the TLR4 gene, rs4986790 (A/G) and rs4986791 (C/T), were genotyped in 450 HCV patients and 600 uninfected controls. The association analysis confirmed that both SNPs showed a significant difference in their distribution between HCV-infected patients and uninfected control subjects (; , 95% –0.581) and (; , 95% –0.443), respectively. More importantly, haplotype analysis revealed that four haplotypes, AC, GT, GC, and AT (rs4986790, rs4986791), were significantly associated with HCV infection when compared with control subjects. One haplotype AC was more prominently found when chronic HCV-infected patients were compared with cirrhosis/HCC patients (frequency = 94.7% and ). Both TLR4 SNPs under investigation were found to be significantly implicated with HCV-infection among Saudi Arabian population. Ahmed A. Al-Qahtani, Mashael R. Al-Anazi, Fahad Al-Zoghaibi, Ayman A. Abdo, Faisal M. Sanai, Mohammed Q. Khan, Ali Albenmousa, Hamad I. Al-Ashgar, and Mohammed N. Al-Ahdal Copyright © 2014 Ahmed A. Al-Qahtani et al. All rights reserved. Participation of Integrin α5β1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells Thu, 07 Aug 2014 09:28:27 +0000 Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin , in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin ; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin . To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin shRNA. Altogether, our data support the involvement of integrin in the fibronectin-mediated EAEC binding to intestinal cells. Mariana Izquierdo, Alejandra Alvestegui, James P. Nataro, Fernando Ruiz-Perez, and Mauricio J. Farfan Copyright © 2014 Mariana Izquierdo et al. All rights reserved. Eucalyptus Essential Oil as a Natural Food Preservative: In Vivo and In Vitro Antiyeast Potential Thu, 07 Aug 2014 07:21:23 +0000 In this study, the application of eucalyptus essential oil/vapour as beverages preservative is reported. The chemical composition of eucalyptus oil was determined by gas chromatography-mass spectrometry (GC-MS) and solid phase microextraction GC-MS (SPME/GC-MS) analyses. GC-MS revealed that the major constituents were 1,8-cineole (80.5%), limonene (6.5%), -pinene (5%), and -terpinene (2.9%) while SPME/GC-MS showed a relative reduction of 1,8-cineole (63.9%) and an increase of limonene (13.8%), -pinene (8.87%), and -terpinene (3.98%). Antimicrobial potential of essential oil was initially determined in vitro against 8 different food spoilage yeasts by disc diffusion, disc volatilization, and microdilution method. The activity of eucalyptus vapours was significantly higher than the eucalyptus oil. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) varied from 0.56 to 4.50 mg/mL and from 1.13 to 9 mg/mL, respectively. Subsequently, the combined efficacy of essential oil and thermal treatment were used to evaluate the preservation of a mixed fruit juice in a time-dependent manner. These results suggest eucalyptus oil as a potent inhibitor of food spoilage yeasts not only in vitro but also in a real food system. Currently, this is the first report that uses eucalyptus essential oil for fruit juice preservation against food spoiling yeast. Amit Kumar Tyagi, Danka Bukvicki, Davide Gottardi, Giulia Tabanelli, Chiara Montanari, Anushree Malik, and Maria Elisabetta Guerzoni Copyright © 2014 Amit Kumar Tyagi et al. All rights reserved. Generation of Dipeptidyl Peptidase-IV-Inhibiting Peptides from β-Lactoglobulin Secreted by Lactococcus lactis Sun, 03 Aug 2014 08:28:25 +0000 Previous studies showed that hydrolysates of β-lactoglobulin (BLG) prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus. Suguru Shigemori, Kazushi Oshiro, Pengfei Wang, Yoshinari Yamamoto, Yeqin Wang, Takashi Sato, Yutaka Uyeno, and Takeshi Shimosato Copyright © 2014 Suguru Shigemori et al. All rights reserved. N-Acyl Homoserine Lactone-Mediated Quorum Sensing with Special Reference to Use of Quorum Quenching Bacteria in Membrane Biofouling Control Thu, 24 Jul 2014 09:59:53 +0000 Membrane biofouling remains a severe problem to be addressed in wastewater treatment systems affecting reactor performance and economy. The finding that many wastewater bacteria rely on N-acyl homoserine lactone-mediated quorum sensing to synchronize their activities essential for biofilm formations; the quenching bacterial quorum sensing suggests a promising approach for control of membrane biofouling. A variety of quorum quenching compounds of both synthetic and natural origin have been identified and found effective in inhibition of membrane biofouling with much less environmental impact than traditional antimicrobials. Work over the past few years has demonstrated that enzymatic quorum quenching mechanisms are widely conserved in several prokaryotic organisms and can be utilized as a potent tool for inhibition of membrane biofouling. Such naturally occurring bacterial quorum quenching mechanisms also play important roles in microbe-microbe interactions and have been used to develop sustainable nonantibiotic antifouling strategies. Advances in membrane fabrication and bacteria entrapment techniques have allowed the implication of such quorum quenching bacteria for better design of membrane bioreactor with improved antibiofouling efficacies. In view of this, the present paper is designed to review and discuss the recent developments in control of membrane biofouling with special emphasis on quorum quenching bacteria that are applied in membrane bioreactors. Harshad Lade, Diby Paul, and Ji Hyang Kweon Copyright © 2014 Harshad Lade et al. All rights reserved. Biotechnological Applications Derived from Microorganisms of the Atacama Desert Wed, 23 Jul 2014 11:07:20 +0000 The Atacama Desert in Chile is well known for being the driest and oldest desert on Earth. For these same reasons, it is also considered a good analog model of the planet Mars. Only a few decades ago, it was thought that this was a sterile place, but in the past years fascinating adaptations have been reported in the members of the three domains of life: low water availability, high UV radiation, high salinity, and other environmental stresses. However, the biotechnological applications derived from the basic understanding and characterization of these species, with the notable exception of copper bioleaching, are still in its infancy, thus offering an immense potential for future development. Armando Azua-Bustos and Carlos González-Silva Copyright © 2014 Armando Azua-Bustos and Carlos González-Silva. All rights reserved. Alpha-Melanocyte Stimulating Hormone: An Emerging Anti-Inflammatory Antimicrobial Peptide Wed, 23 Jul 2014 00:00:00 +0000 The alpha-melanocyte stimulating hormone (-MSH) is a neuropeptide belonging to the melanocortin family. It is well known for its anti-inflammatory and antipyretic effects and shares several characteristics with antimicrobial peptides (AMPs). There have been some recent reports about the direct antimicrobial activity of -MSH against various microbes belonging to both fungal and bacterial pathogens. Similar to -MSH’s anti-inflammatory properties, its C-terminal residues also exhibit antimicrobial activity parallel to that of the entire peptide. This review is focused on the current findings regarding the direct antimicrobial potential and immunomodulatory mechanism of -MSH and its C-terminal fragments, with particular emphasis on the prospects of -MSH based peptides as a strong anti-infective agent. Madhuri Singh and Kasturi Mukhopadhyay Copyright © 2014 Madhuri Singh and Kasturi Mukhopadhyay. All rights reserved. Recombinant Lysostaphin Protects Mice from Methicillin-Resistant Staphylococcus aureus Pneumonia Wed, 16 Jul 2014 12:08:55 +0000 The advent of methicillin-resistant Staphylococcus aureus (MRSA) and the frequent and excessive abuse of ventilators have made MRSA pneumonia an inordinate threat to human health. Appropriate antibacterial therapies are crucial, including the use of lysostaphin as an alternative to antibiotics. To explore the potential use of lysostaphin as a therapeutic agent for MRSA pneumonia, mice were intranasally infected with MRSA and then treated with recombinant lysostaphin (rLys; 45 mg/kg in the high-dose group and 1 mg/kg in the low-dose group) (0.33 mg/mL, 15 mg/mL), vancomycin (120 mg/kg) (40 mg/mL), or phosphate-buffered saline (PBS, negative control) 4 h after infection. Therapeutic efficacy was assessed by mouse survival, lung histopathology, bacterial density in the lungs, bodyweight, lung weight, temperature, white blood cells counts, lymphocytes counts, granulocytes counts, and monocytes counts. The mice treated with rLys showed lower mortality, less lung parenchymal damage, and lower bacterial density at metastatic tissue sites than mice treated with PBS or vancomycin. The overall mortality was 100%, 60%, 40%, and 60% for the control, vancomycin, high-dose rLys, and low-dose rLys groups, respectively. These findings indicate that, as a therapeutic agent for MRSA pneumonia, lysostaphin exerts profound protective effects in mice against the morbidity and mortality associated with S. aureus pneumonia. Chen Chen, Huahao Fan, Yong Huang, Fan Peng, Hang Fan, Shoujun Yuan, and Yigang Tong Copyright © 2014 Chen Chen et al. All rights reserved. Diversity and Enzymatic Profiling of Halotolerant Micromycetes from Sebkha El Melah, a Saharan Salt Flat in Southern Tunisia Wed, 16 Jul 2014 12:04:04 +0000 Twenty-one moderately halotolerant fungi have been isolated from sample ashes collected from Sebkha El Melah, a Saharan salt flat located in southern Tunisia. Based on morphology and sequence inference from the internal transcribed spacer regions, 28S rRNA gene and other specific genes such as β-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase, the isolates were found to be distributed over 15 taxa belonging to 6 genera of Ascomycetes: Cladosporium (), Alternaria (), Aspergillus (), Penicillium (), Ulocladium (), and Engyodontium (). Their tolerance to different concentrations of salt in solid and liquid media was examined. Excepting Cladosporium cladosporioides JA18, all isolates were considered as alkali-halotolerant since they were able to grow in media containing 10% of salt with an initial pH 10. All isolates were resistant to oxidative stresses and low temperature whereas 5 strains belonging to Alternaria, Ulocladium, and Aspergillus genera were able to grow at 45°C. The screening of fungal strains for sets of enzyme production, namely, cellulase (CMCase), amylase, protease, lipase, and laccase, in presence of 10% NaCl, showed a variety of extracellular hydrolytic and oxidative profiles. Protease was the most abundant enzyme produced whereas laccase producers were members of the genus Cladosporium. Atef Jaouani, Mohamed Neifar, Valeria Prigione, Amani Ayari, Imed Sbissi, Sonia Ben Amor, Seifeddine Ben Tekaya, Giovanna Cristina Varese, Ameur Cherif, and Maher Gtari Copyright © 2014 Atef Jaouani et al. All rights reserved. A Novel Promising Strain of Trichoderma evansii (WF-3) for Extracellular α-Galactosidase Production by Utilizing Different Carbon Sources under Optimized Culture Conditions Sun, 13 Jul 2014 08:51:16 +0000 A potential fungal strain of Trichoderma sp. (WF-3) was isolated and selected for the production of α-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Basal media selected for the growth of fungal isolate containing different carbon sources like guar gum (GG), soya bean meal (SM), and wheat straw (WS) and combinations of these carbon substrates with basic sugars like galactose and sucrose were used to monitor their effects on α-galactosidase production. The results of this study indicated that galactose and sucrose enhanced the enzyme activity in guar gum (GG) and wheat straw (WS). Maximum α-galactosidase production (213.63 UmL−1) was obtained when the basic medium containing GG is supplemented with galactose (5 mg/mL). However, the presence of galactose and sucrose alone in the growth media shows no effect. Soya meal alone was able to support T. evansii to produce maximum enzyme activity (170.36 UmL−1). The incubation time, temperature, and pH for the maximum enzyme synthesis were found to be 120 h (5 days), 28°C, and 4.5–5.5, respectively. All the carbon sources tested exhibited maximum enzyme production at 10 mg/mL concentration. Among the metal ions tested, Hg was found to be the strongest inhibitor of the enzyme. Among the chelators, EDTA acted as stronger inhibitor than succinic acid. Aishwarya Chauhan, Nikhat Jamal Siddiqi, and Bechan Sharma Copyright © 2014 Aishwarya Chauhan et al. All rights reserved. Geodermatophilus poikilotrophi sp. nov.: A Multitolerant Actinomycete Isolated from Dolomitic Marble Wed, 09 Jul 2014 14:05:46 +0000 A novel Gram-reaction-positive, aerobic actinobacterium, tolerant to mitomycin C, heavy metals, metalloids, hydrogen peroxide, desiccation, and ionizing- and UV-radiation, designated G18T, was isolated from dolomitic marble collected from outcrops in Samara (Namibia). The growth range was 15–35°C, at pH 5.5–9.5 and in presence of 1% NaCl, forming greenish-black coloured colonies on GYM Streptomyces agar. Chemotaxonomic and molecular characteristics of the isolate matched those described for other representatives of the genus Geodermatophilus. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diaminoacid. The main phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and small amount of diphosphatidylglycerol. MK-9(H4) was the dominant menaquinone and galactose was detected as diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids iso-C16:0 and iso-C15:0 and the unsaturated C17:1ω8c and C16:1ω7c. The 16S rRNA gene showed 97.4–99.1% sequence identity with the other representatives of genus Geodermatophilus. Based on phenotypic results and 16S rRNA gene sequence analysis, strain G18T is proposed to represent a novel species, Geodermatophilus poikilotrophi. Type strain is G18T (= DSM 44209T = CCUG 63018T). The INSDC accession number is HF970583. The novel R software package lethal was used to compute the lethal doses with confidence intervals resulting from tolerance experiments. Maria del Carmen Montero-Calasanz, Benjamin Hofner, Markus Göker, Manfred Rohde, Cathrin Spröer, Karima Hezbri, Maher Gtari, Peter Schumann, and Hans-Peter Klenk Copyright © 2014 Maria del Carmen Montero-Calasanz et al. All rights reserved. Fungi Treated with Small Chemicals Exhibit Increased Antimicrobial Activity against Facultative Bacterial and Yeast Pathogens Wed, 09 Jul 2014 11:04:36 +0000 For decades, fungi have been the main source for the discovery of novel antimicrobial drugs. Recent sequencing efforts revealed a still high number of so far unknown “cryptic” secondary metabolites. The production of these metabolites is presumably epigenetically silenced under standard laboratory conditions. In this study, we investigated the effect of six small mass chemicals, of which some are known to act as epigenetic modulators, on the production of antimicrobial compounds in 54 spore forming fungi. The antimicrobial effect of fungal samples was tested against clinically facultative pathogens and multiresistant clinical isolates. In total, 30 samples of treated fungi belonging to six different genera reduced significantly growth of different test organisms compared to the untreated fungal sample (growth log reduction 0.3–4.3). For instance, the pellet of Penicillium restrictum grown in the presence of butyrate revealed significant higher antimicrobial activity against Staphylococcus (S.) aureus and multiresistant S. aureus strains and displayed no cytotoxicity against human cells, thus making it an ideal candidate for antimicrobial compound discovery. Our study shows that every presumable fungus, even well described fungi, has the potential to produce novel antimicrobial compounds and that our approach is capable of rapidly filling the pipeline for yet undiscovered antimicrobial substances. Christoph Zutz, Dragana Bandian, Bernhard Neumayer, Franz Speringer, Markus Gorfer, Martin Wagner, Joseph Strauss, and Kathrin Rychli Copyright © 2014 Christoph Zutz et al. All rights reserved. The Bacterial Contamination of Allogeneic Bone and Emergence of Multidrug-Resistant Bacteria in Tissue Bank Tue, 08 Jul 2014 12:49:31 +0000 Present study was carried out for the microbiological evaluation of allogeneic bone processed from femoral heads. A total 60 bacterial isolates comprising five different species including Streptococcus spp., Staphylococcus spp., Klebsiella spp., Bacillus spp., and Pseudomonas spp. were characterized based on their cultural and biochemical characteristics. Average bioburden was ranged from to  cfu/gm. The majority (81.7%) of the microbial contaminants were detected as Gram positive with the predominant organism being skin commensal coagulase negative Staphylococci (43.3%). Antimicrobial resistance was evaluated by the activities of 14 broad and narrow spectrum antibiotic discs. Comparing the overall pattern, marked resistance was noted against Penicillin and Amoxicillin 100% (60/60). The most effective single antibiotics were Gentamicin, Tobramycin, and Ofloxacin which were bactericidal against 100% (60/60) isolates. Multidrug resistance (MDR) was confirmed in 70% (42/60) of the samples. Among them, the most prevalent antibiotypes were Penicillin, Amoxicillin, Oxacillin, Polymyxin, and Cefpodoxime (80% of total MDR). The study results revealed higher contamination rate on bone allografts and recommend the implementation of good tissue banking practices during tissue procurement, processing, and storage in order to minimize the chances of contamination. Fahmida Binte Atique and Md. Masudur Rahman Khalil Copyright © 2014 Fahmida Binte Atique and Md. Masudur Rahman Khalil. All rights reserved. Specific Growth Rate Determines the Sensitivity of Escherichia coli to Lactic Acid Stress: Implications for Predictive Microbiology Tue, 08 Jul 2014 00:00:00 +0000 This study tested the hypothesis that sensitivity of Escherichia coli to lactic acid at concentrations relevant for fermented sausages (pH 4.6, 150 mM lactic acid, , temperature = 20 or 27°C) increases with increasing growth rate. For E. coli strain 683 cultured in TSB in chemostat or batch, subsequent inactivation rates when exposed to lactic acid stress increased with increasing growth rate at harvest. A linear relationship between growth rate at harvest and inactivation rate was found to describe both batch and chemostat cultures. The maximum difference in T90, the estimated times for a one-log reduction, was 10 hours between bacteria harvested during the first 3 hours of batch culture, that is, at different growth rates. A 10-hour difference in T90 would correspond to measuring inactivation at 33°C or 45°C instead of 37°C based on relationships between temperature and inactivation. At similar harvest growth rates, inactivation rates were lower for bacteria cultured at 37°C than at 15–20°C. As demonstrated for E. coli 683, culture conditions leading to variable growth rates may contribute to variable lactic acid inactivation rates. Findings emphasize the use and reporting of standardised culture conditions and can have implications for the interpretation of data when developing inactivation models. Roland Lindqvist and Gunilla Barmark Copyright © 2014 Roland Lindqvist and Gunilla Barmark. All rights reserved. Production of Conjugated Linoleic and Conjugated α-Linolenic Acid in a Reconstituted Skim Milk-Based Medium by Bifidobacterial Strains Isolated from Human Breast Milk Sun, 06 Jul 2014 12:09:59 +0000 Eight bifidobacterial strains isolated from human breast milk have been tested for their abilities to convert linoleic acid (LA) and α-linolenic acid (LNA) to conjugated linoleic acid (CLA) and conjugated α-linolenic acid (CLNA), respectively. These bioactive lipids display important properties that may contribute to the maintenance and improvement human health. Three selected Bifidobacterium breve strains produced CLA from LA and CLNA from LNA in MRS (160–170 and 210–230 μg mL−1, resp.) and, also, in reconstituted skim milk (75–95 and 210–244 μg mL−1, resp.). These bifidobacterial strains were also able to simultaneously produce both CLA (90–105 μg mL−1) and CLNA (290–320 μg mL−1) in reconstituted skim milk. Globally, our findings suggest that these bifidobacterial strains are potential candidates for the design of new fermented dairy products naturally containing very high concentrations of these bioactive lipids. To our knowledge, this is the first study describing CLNA production and coproduction of CLA and CLNA by Bifidobacterium breve strains isolated from human milk in reconstituted skim milk. María Antonia Villar-Tajadura, Luis Miguel Rodríguez-Alcalá, Virginia Martín, Aránzazu Gómez de Segura, Juan Miguel Rodríguez, Teresa Requena, and Javier Fontecha Copyright © 2014 María Antonia Villar-Tajadura et al. All rights reserved. Emerging Microbial Concerns in Food Safety and New Control Measures Sun, 06 Jul 2014 06:18:06 +0000 Moreno Bondi, Patrizia Messi, Prakash M. Halami, Chrissanthy Papadopoulou, and Simona de Niederhausern Copyright © 2014 Moreno Bondi et al. All rights reserved. Clonality and Resistome Analysis of KPC-Producing Klebsiella pneumoniae Strain Isolated in Korea Using Whole Genome Sequencing Thu, 03 Jul 2014 13:40:18 +0000 We analyzed the whole genome sequence and resistome of the outbreak Klebsiella pneumoniae strain MP14 and compared it with those of K. pneumoniae carbapenemase- (KPC-) producing isolates that showed high similarity in the NCBI genome database. A KPC-2-producing multidrug-resistant (MDR) K. pneumoniae clinical isolate was obtained from a patient admitted to a Korean hospital in 2011. The strain MP14 was resistant to all tested β-lactams including monobactam, amikacin, levofloxacin, and cotrimoxazole, but susceptible to tigecycline and colistin. Resistome analysis showed the presence of -lactamase genes including , , , and . MP14 also possessed aac(6′-)Ib, aadA2, and aph(3′-)Ia as aminoglycoside resistance-encoding genes, mph(A) for macrolides, oqxA and oqxB for quinolone, catA1 for phenicol, sul1 for sulfonamide, and dfrA12 for trimethoprim. Both SNP tree and cgMLST analysis showed the close relatedness with the KPC producers (KPNIH strains) isolated from an outbreak in the USA and colistin-resistant strains isolated in Italy. The plasmid-scaffold genes in plasmids pKpQil, pKpQil-IT, pKPN3, or pKPN-IT were identified in MP14, KPNIH, and Italian strains. The KPC-2-producing MDR K. pneumoniae ST258 stain isolated in Korea was highly clonally related with MDR K. pneumoniae strains from the USA and Italy. Global spread of KPC-producing K. pneumoniae is a worrying phenomenon. Yangsoon Lee, Bong-Soo Kim, Jongsik Chun, Ji Hyun Yong, Yeong Seon Lee, Jung Sik Yoo, Dongeun Yong, Seong Geun Hong, Roshan D’Souza, Kenneth S. Thomson, Kyungwon Lee, and Yunsop Chong Copyright © 2014 Yangsoon Lee et al. All rights reserved. Prophages in Enterococcal Isolates from Renal Transplant Recipients: Renal Failure Etiologies Promote Selection of Strains Thu, 03 Jul 2014 09:11:19 +0000 Infections caused by commensal bacteria may be fatal for the patients under immunosuppressive therapy. This results also from difficulty in identification of high risk strains. Enterococcal infections are increasingly frequent but despite many studies on virulence traits, the difference between commensal and pathogenic strains remains unclear. Prophages are newly described as important elements in competition between strains during colonization, as well as pathogenicity of the strains. Here we evaluate a difference in presence of pp4, pp1, and pp7 prophages and ASA (aggregation substance) gene expression in enterococcal isolates from renal transplant recipients (RTx) with different etiology of the end-stage renal failure. Prophages sequence was screened by PCR in strains of Enterococcus faecalis isolated from urine and feces of 19 RTx hospitalized at Medical University of Gdansk and 18 healthy volunteers. FLOW-FISH method with use of linear locked nucleic acid (LNA) probe was used to assess the ASA gene expression. Additionally, ability of biofilm formation was screened by crystal violet staining method. Presence of prophages was more frequent in fecal isolates from immunocompromised patients than in isolates from healthy volunteers. Additionally, both composition of prophages and ASA gene expression were related to the etiology of renal disease. Agnieszka Daca, Tomasz Jarzembowski, Jacek M. Witkowski, Ewa Bryl, Bolesław Rutkowski, and Alicja Dębska-Ślizień Copyright © 2014 Agnieszka Daca et al. All rights reserved. Probiotic Potential of Lactobacillus Strains with Antimicrobial Activity against Some Human Pathogenic Strains Thu, 03 Jul 2014 08:23:53 +0000 The objective of this study was to isolate, identify, and characterize some lactic acid bacterial strains from human milk, infant feces, and fermented grapes and dates, as potential probiotics with antimicrobial activity against some human pathogenic strains. One hundred and forty bacterial strains were isolated and, after initial identification and a preliminary screening for acid and bile tolerance, nine of the best isolates were selected and further identified using 16 S rRNA gene sequences. The nine selected isolates were then characterized in vitro for their probiotic characteristics and their antimicrobial activities against some human pathogens. Results showed that all nine isolates belonged to the genus Lactobacillus. They were able to tolerate pH 3 for 3 h, 0.3% bile salts for 4 h, and 1.9 mg/mL pancreatic enzymes for 3 h. They exhibited good ability to attach to intestinal epithelial cells and were not resistant to the tested antibiotics. They also showed good antimicrobial activities against the tested pathogenic strains of humans, and most of them exhibited stronger antimicrobial activity than the reference strain L. casei Shirota. Thus, the nine Lactobacillus strains could be considered as potential antimicrobial probiotic strains against human pathogens and should be further studied for their human health benefits. Parisa Shokryazdan, Chin Chin Sieo, Ramasamy Kalavathy, Juan Boo Liang, Noorjahan Banu Alitheen, Mohammad Faseleh Jahromi, and Yin Wan Ho Copyright © 2014 Parisa Shokryazdan et al. All rights reserved. Predictive Symptoms and Signs of Severe Dengue Disease for Patients with Dengue Fever: A Meta-Analysis Tue, 01 Jul 2014 11:44:09 +0000 The aim of the meta-analysis was to provide more solid evidence for the reliability of the new classification. A systematic literature search was performed using PubMed, Armed Forces Pest Management Board Literature Retrieval System, and Google Scholar up to August 2012. A pooled odds ratio (OR) was calculated using either a random-effect or a fixed-effect model. A total of 16 papers were identified. Among the 11 factors studied, five symptoms demonstrated an increased risk for SDD, including bleeding [OR: 13.617; 95% confidence interval (CI): 3.281, 56.508], vomiting/nausea (OR: 1.692; 95% CI: 1.256, 2.280), abdominal pain (OR: 2.278; 95% CI: 1.631, 3.182), skin rashes (OR: 2.031; 95% CI: 1.269, 3.250), and hepatomegaly (OR: 4.751; 95% CI: 1.769, 12.570). Among the four bleeding-related symptoms including hematemesis, melena, gum bleeding, and epistaxis, only hematemesis (OR: 6.174; 95% CI: 2.66, 14.334; ) and melena (OR: 10.351; 95% CI: 3.065, 34.956; ) were significantly associated with SDD. No significant associations with SDD were found for gender, lethargy, retroorbital pain, diarrhea, or tourniquet test, whereas headache appeared protective (OR: 0.555; 95% CI: 0.455, 0.676). The meta-analysis suggests that bleeding (hematemesis/melena), vomiting/nausea, abdominal pain, skin rashes, and hepatomegaly may predict the development of SDD in patients with DF, while headache may predict otherwise. H. Zhang, Y. P. Zhou, H. J. Peng, X. H. Zhang, F. Y. Zhou, Z. H. Liu, and X. G. Chen Copyright © 2014 H. Zhang et al. All rights reserved. Blood Stream Infections Tue, 01 Jul 2014 08:48:56 +0000 Renu Bharadwaj, Abhijit Bal, Ketoki Kapila, Vidya Mave, and Amita Gupta Copyright © 2014 Renu Bharadwaj et al. All rights reserved. Amelioration of Colitis in Mouse Model by Exploring Antioxidative Potentials of an Indigenous Probiotic Strain of Lactobacillus fermentum Lf1 Tue, 01 Jul 2014 07:38:49 +0000 Based on the preliminary screening of eight indigenous putative probiotic Lactobacilli, Lactobacillus fermentum Lf1 was selected for assessing its antioxidative efficacy in DSS colitis mouse model based on its ability to enhance the expression of “Nrf2” by 6.43-fold and malondialdehyde (MDA) inhibition by 78.1  ±  0.24% in HT-29 cells under H2O2 stress. The Disease Activity Index and histological scores of Lf1-treated mice were lower than the control group. However, expression of “Nrf2” was not observed in Lf1-treated mice. A significant increase in the expression of antioxidative enzymes such as SOD2 and TrxR-1 was recorded in both of the groups. The expression of SOD2 was significantly downregulated in colitis-induced mice by −100.00-fold relative to control group, and the downregulation was considerably reduced to −37.04-fold in colitis Lf1 treatment group. Almost, a similar trend was recorded in case of “thioredoxin” expression, though “CAT” was refractile to expression. The Lf1-treated group had decreased malondialdehyde level as compared to colitis control (37.92  ±  6.31 versus 91.13  ±  5.76 μM/g). These results point towards Lf1-induced activation of the antioxidant enzyme system in the mouse model and its prospects to be explored as a new strategy for IBD management. Ritu Chauhan, Aparna Sudhakaran Vasanthakumari, Harsh Panwar, Rashmi H. Mallapa, Raj Kumar Duary, Virender Kumar Batish, and Sunita Grover Copyright © 2014 Ritu Chauhan et al. All rights reserved. Reversal of Ampicillin Resistance in MRSA via Inhibition of Penicillin-Binding Protein 2a by Acalypha wilkesiana Mon, 30 Jun 2014 12:28:32 +0000 The inhibitory activity of a semipure fraction from the plant, Acalypha wilkesiana assigned as 9EA-FC-B, alone and in combination with ampicillin, was studied against methicillin-resistant Staphylococcus aureus (MRSA). In addition, effects of the combination treatment on PBP2a expression were investigated. Microdilution assay was used to determine the minimal inhibitory concentrations (MIC). Synergistic effects of 9EA-FC-B with ampicillin were determined using the fractional inhibitory concentration (FIC) index and kinetic growth curve assay. Western blot experiments were carried out to study the PBP2a expression in treated MRSA cultures. The results showed a synergistic effect between ampicillin and 9EA-FC-B treatment with the lowest FIC index of 0.19 (synergism 0.5). The presence of 9EA-FC-B reduced the MIC of ampicillin from 50 to 1.56 μg mL−1. When ampicillin and 9EA-FC-B were combined at subinhibitory level, the kinetic growth curves were suppressed. The antibacterial effect of 9EA-FC-B and ampicillin was shown to be synergistic. The synergism is due the ability of 9EA-FC-B to suppress the activity of PBP2a, thus restoring the susceptibility of MRSA to ampicillin. Corilagin was postulated to be the constituent responsible for the synergistic activity showed by 9EA-FC-B. Carolina Santiago, Ee Leen Pang, Kuan-Hon Lim, Hwei-San Loh, and Kang Nee Ting Copyright © 2014 Carolina Santiago et al. All rights reserved. Fresh-Cut Pineapple as a New Carrier of Probiotic Lactic Acid Bacteria Sun, 29 Jun 2014 08:30:31 +0000 Due to the increasing interest for healthy foods, the feasibility of using fresh-cut fruits to vehicle probiotic microorganisms is arising scientific interest. With this aim, the survival of probiotic lactic acid bacteria, belonging to Lactobacillus plantarum and Lactobacillus fermentum species, was monitored on artificially inoculated pineapple pieces throughout storage. The main nutritional, physicochemical, and sensorial parameters of minimally processed pineapples were monitored. Finally, probiotic Lactobacillus were further investigated for their antagonistic effect against Listeria monocytogenes and Escherichia coli O157:H7 on pineapple plugs. Our results show that at eight days of storage, the concentration of L. plantarum and L. fermentum on pineapples pieces ranged between 7.3 and 6.3 log cfu g−1, respectively, without affecting the final quality of the fresh-cut pineapple. The antagonistic assays indicated that L. plantarum was able to inhibit the growth of both pathogens, while L. fermentum was effective only against L. monocytogenes. This study suggests that both L. plantarum and L. fermentum could be successfully applied during processing of fresh-cut pineapples, contributing at the same time to inducing a protective effect against relevant foodborne pathogens. Pasquale Russo, Maria Lucia Valeria de Chiara, Anna Vernile, Maria Luisa Amodio, Mattia Pia Arena, Vittorio Capozzi, Salvatore Massa, and Giuseppe Spano Copyright © 2014 Pasquale Russo et al. All rights reserved. Differential Response of Oyster Shell Powder on Enzyme Profile and Nutritional Value of Oyster Mushroom Pleurotus florida PF05 Thu, 26 Jun 2014 13:27:56 +0000 Oyster mushroom Pleurotus florida was cultivated on different combinations of wheat straw (WS) as basal substrate and oyster shell powder (OSP) supplement. The OSP supplementation considerably responded to different cultivation phases. The mycelium grew fast and showed rapid growth rate (8.91 mmd−1) in WS + OSP (97 + 3) combination while WS + OSP (92 + 8) showed maximum laccase (3.133 U/g) and Mn peroxidase (MnP) activities (0.091 U/g). The climax level of laccase (5.433 U/g) and MnP (0.097 U/g) was recorded during fruit body initiation in WS + OSP (97 + 3) and WS + OSP (98 + 2) combinations, respectively. The WS + OSP (97 + 3) combination represented the best condition for mushroom cultivation and produced the highest biological efficiency (147%). In addition, protein and lipid contents in fruit bodies were slightly improved in response to OSP. The carbohydrate was significantly increased by raising concentration of OSP. The highest values of protein, carbohydrate, and lipid noted were 31.3 μg/g, 0.0639 (g/g), and 0.373 (g/g) correspondingly. Conclusively it was evident that lower concentrations of OSP acted positively and relatively to higher concentrations and improved nutritional content which may suitably be used to enhance both yield and nutritional values of mushroom. Ram Naraian, Om Prakash Narayan, and Jatin Srivastava Copyright © 2014 Ram Naraian et al. All rights reserved. In Vitro Antimicrobial and Antiprotozoal Activities, Phytochemical Screening and Heavy Metals Toxicity of Different Parts of Ballota nigra Thu, 26 Jun 2014 11:07:19 +0000 The study was done to assess the phytochemicals (flavonoids, terpenoids, saponins, tannin, alkaloids, and phenol) in different parts (root, stem, and leaves) of Ballota nigra and correlated it to inhibition of microbes (bacteria and fungi), protozoan (Leishmania), and heavy metals toxicity evaluation. In root and stem flavonoids, terpenes and phenols were present in ethanol, chloroform, and ethyl acetate soluble fraction; these were found to be the most active inhibiting fractions against all the tested strains of bacteria, fungi, and leishmania. While in leaves flavonoids, terpenes, and phenols were present in ethanol, chloroform, and n-butanol fractions which were the most active fractions against both types of microbes and protozoan (leishmania) in in vitro study. Ethanol and chloroform fractions show maximum inhibition against Escherichia coli (17 mm). The phytochemical and biological screenings were correlated with the presence of heavy metals in selected plant Ballota nigra. Cr was found above permissible value (above 1.5 mg/kg) in all parts of the plant. Ni was above WHO limit in B. nigra root and leaves (3.35 ± 1.20 mg/kg and 5.09 ± 0.47 mg/kg, respectively). Fe was above permissible value in all parts of B. nigra (above 20 mg/kg). Cd was above permissible value in all parts of the plant (above 0.3 mg/kg). Pb was above WHO limit (above 2 mg/kg) in all parts of Ballota nigra. Najeeb Ullah, Ijaz Ahmad, and Sultan Ayaz Copyright © 2014 Najeeb Ullah et al. All rights reserved. High-Level Antimicrobial Efficacy of Representative Mediterranean Natural Plant Extracts against Oral Microorganisms Thu, 26 Jun 2014 06:59:34 +0000 Nature is an unexplored reservoir of novel phytopharmaceuticals. Since biofilm-related oral diseases often correlate with antibiotic resistance, plant-derived antimicrobial agents could enhance existing treatment options. Therefore, the rationale of the present report was to examine the antimicrobial impact of Mediterranean natural extracts on oral microorganisms. Five different extracts from Olea europaea, mastic gum, and Inula viscosa were tested against ten bacteria and one Candida albicans strain. The extraction protocols were conducted according to established experimental procedures. Two antimicrobial assays—the minimum inhibitory concentration (MIC) assay and the minimum bactericidal concentration (MBC) assay—were applied. The screened extracts were found to be active against each of the tested microorganisms. O. europaea presented MIC and MBC ranges of 0.07–10.00 mg mL−1 and 0.60–10.00 mg mL−1, respectively. The mean MBC values for mastic gum and I. viscosa were 0.07–10.00 mg mL−1 and 0.15–10.00 mg mL−1, respectively. Extracts were less effective against C. albicans and exerted bactericidal effects at a concentration range of 0.07–5.00 mg mL−1 on strict anaerobic bacteria (Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Parvimonas micra). Ethyl acetate I. viscosa extract and total mastic extract showed considerable antimicrobial activity against oral microorganisms and could therefore be considered as alternative natural anti-infectious agents. Lamprini Karygianni, Manuel Cecere, Alexios Leandros Skaltsounis, Aikaterini Argyropoulou, Elmar Hellwig, Nektarios Aligiannis, Annette Wittmer, and Ali Al-Ahmad Copyright © 2014 Lamprini Karygianni et al. All rights reserved. Preliminary Evaluation of Probiotic Properties of Lactobacillus Strains Isolated from Sardinian Dairy Products Thu, 26 Jun 2014 05:28:19 +0000 Twenty-three Lactobacillus strains of dairy origin were evaluated for some functional properties relevant to their use as probiotics. A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyze conjugated bile salts was applied, and six strains were selected for further characterization including survival under gastrointestinal environmental conditions, adhesion to gut epithelial tissue, enzymatic activity, and some safety properties. All selected strains maintained elevated cell numbers under conditions simulating passage through the human gastrointestinal tract, well comparable to the values obtained for the probiotic strain Lactobacillus rhamnosus GG, and were able to adhere to Caco-2 cells to various extents (from 3 to 20%). All strains exhibited high aminopeptidase, and absent or very low proteolytic and strong β-galactosidase activities; none was found to be haemolytic or to produce biogenic amines and all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin, and amoxicillin/clavulanic acid. Our results indicate that the Lactobacillus strains analyzed could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial activity, adhesion to Caco-2 cell-line, and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products. Maria Barbara Pisano, Silvia Viale, Stefania Conti, Maria Elisabetta Fadda, Maura Deplano, Maria Paola Melis, Monica Deiana, and Sofia Cosentino Copyright © 2014 Maria Barbara Pisano et al. All rights reserved. Use of Antimicrobial Films and Edible Coatings Incorporating Chemical and Biological Preservatives to Control Growth of Listeria monocytogenes on Cold Smoked Salmon Wed, 25 Jun 2014 08:10:23 +0000 The relatively high incidence of Listeria monocytogenes in cold smoked salmon (CSS) is of concern as it is a refrigerated processed food of extended durability (REPFED). The objectives of this study were to compare and optimize the antimicrobial effectiveness of films and coatings incorporating nisin (Nis) and sodium lactate (SL), sodium diacetate (SD), potassium sorbate (PS), and/or sodium benzoate (SB) in binary or ternary combinations on CSS. Surface treatments incorporating Nis (25000 IU/mL) in combination with PS (0.3%) and SB (0.1%) had the highest inhibitory activity, reducing the population of L. monocytogenes by a maximum of 3.3 log CFU/cm2 (films) and 2.9 log CFU/cm2 (coatings) relative to control samples after 10 days of storage at 21°C. During refrigerated storage, coatings were more effective in inhibiting growth of L. monocytogenes than their film counterparts. Cellulose-based coatings incorporating Nis, PS, and SB reduced the population of L. monocytogenes, and anaerobic and aerobic spoilage flora by a maximum of 4.2, 4.8, and 4.9 log CFU/cm2, respectively, after 4 weeks of refrigerated storage. This study highlights the effectiveness of cellulose-based edible coatings incorporating generally regarded as safe (GRAS) natural and chemical antimicrobials to inhibit the development of L. monocytogenes and spoilage microflora thus enhancing the safety and quality of CSS. Hudaa Neetoo and Fawzi Mahomoodally Copyright © 2014 Hudaa Neetoo and Fawzi Mahomoodally. All rights reserved. Sinefungin, a Natural Nucleoside Analogue of S-Adenosylmethionine, Inhibits Streptococcus pneumoniae Biofilm Growth Mon, 23 Jun 2014 10:56:57 +0000 Pneumococcal colonization and disease is often associated with biofilm formation, in which the bacteria exhibit elevated resistance both to antibiotics and to host defense systems, often resulting in infections that are persistent and difficult to treat. We evaluated the effect of sinefungin, a nucleoside analogue of S-adenosylmethionine, on pneumococcal in vitro biofilm formation and in vivo colonization. Sinefungin is bacteriostatic to pneumococci and significantly decreased biofilm growth and inhibited proliferation and structure of actively growing biofilms but did not alter growth or the matrix structure of established biofilms. Sinefungin significantly reduced pneumococcal colonization in rat middle ear. The quorum sensing molecule (autoinducer-2) production was significantly reduced by 92% in sinefungin treated samples. The luxS, pfs, and speE genes were downregulated in biofilms grown in the presence of sinefungin. This study shows that sinefungin inhibits pneumococcal biofilm growth in vitro and colonization in vivo, decreases AI-2 production, and downregulates luxS, pfs, and speE gene expressions. Therefore, the S-adenosylmethionine (SAM) inhibitors could be used as lead compounds for the development of novel antibiofilm agents against pneumococci. Mukesh Kumar Yadav, Seok-Won Park, Sung-Won Chae, and Jae-Jun Song Copyright © 2014 Mukesh Kumar Yadav et al. All rights reserved. Potential Probiotic Escherichia coli 16 Harboring the Vitreoscilla Hemoglobin Gene Improves Gastrointestinal Tract Colonization and Ameliorates Carbon Tetrachloride Induced Hepatotoxicity in Rats Thu, 19 Jun 2014 07:05:36 +0000 The present study describes the beneficial effects of potential probiotic E. coli 16 (pUC8:16gfp) expressing Vitreoscilla hemoglobin (vgb) gene, associated with bacterial respiration under microaerobic condition, on gastrointestinal (GI) colonization and its antioxidant activity on carbon tetrachloride (CCl4) induced toxicity in Charles Foster rats. In vitro, catalase activity in E. coli 16 (pUC8:16gfp) was 1.8 times higher compared to E. coli 16 (pUC-gfp) control. In vivo, E. coli 16 (pUC8:16gfp) not only was recovered in the fecal matter after 70 days of oral administration but also retained antibacterial activities, whereas E. coli 16 (pUC-gfp) was not detected. Oral administration of 200 and 500 μL/kg body weight of CCl4 to rats at weekly interval resulted in elevated serum glutamyl pyruvate transaminase (SGPT) and serum glutamyl oxalacetate transaminase (SGOT) levels compared to controls. Rats prefed with E. coli 16 (pUC8:16gfp) demonstrated near to normal levels for SGPT and SGOT, whereas the liver homogenate catalase activity was significantly increased compared to CCl4 treated rats. Thus, pUC8:16gfp plasmid encoding vgb improved the growth and GI tract colonization of E. coli 16. In addition, it also enhanced catalase activity in rats harboring E. coli 16 (pUC8:16gfp), thereby preventing the absorption of CCl4 to GI tract. Prasant Kumar, Ayush V. Ranawade, and Naresh G. Kumar Copyright © 2014 Prasant Kumar et al. All rights reserved. Carbapenemases in Gram-Negative Bacteria: Laboratory Detection and Clinical Significance Sun, 15 Jun 2014 11:05:30 +0000 Branka Bedenić, Vanda Plečko, Sanda Sardelić, Selma Uzunović, and Karmen Godič Torkar Copyright © 2014 Branka Bedenić et al. All rights reserved. Inhibitory and Toxic Effects of Volatiles Emitted by Strains of Pseudomonas and Serratia on Growth and Survival of Selected Microorganisms, Caenorhabditis elegans, and Drosophila melanogaster Wed, 11 Jun 2014 12:02:08 +0000 In previous research, volatile organic compounds (VOCs) emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans). VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds—ketones (2-nonanone, 2-heptanone, 2-undecanone) and dimethyl disulfide—were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions. Alexandra A. Popova, Olga A. Koksharova, Valentina A. Lipasova, Julia V. Zaitseva, Olga A. Katkova-Zhukotskaya, Svetlana Iu. Eremina, Alexander S. Mironov, Leonid S. Chernin, and Inessa A. Khmel Copyright © 2014 Alexandra A. Popova et al. All rights reserved. Association between Giardia duodenalis and Coinfection with Other Diarrhea-Causing Pathogens in India Mon, 09 Jun 2014 15:53:56 +0000 Giardia duodenalis, is often seen as an opportunistic pathogen and one of the major food and waterborne parasites. Some insights of Giardia infestation in a diarrhoea-prone population were investigated in the present study. Our primary goal was to understand the interaction of this parasite with other pathogens during infection and to determine some important factors regulating the diarrhoeal disease spectrum of a population. Giardia showed a steady rate of occurrence throughout the entire study period with a nonsignificant association with rainfall . Interestingly coinfecting pathogens like Vibrio cholerae and rotavirus played a significant role in the occurrence of this parasite. Moreover, the age distribution of the diarrhoeal cases was very much dependent on the coinfection rate of Giardia infection. As per our findings, Giardia infection rate seems to play a vital role in regulation of the whole diarrhoeal disease spectrum in this endemic region. Avik K. Mukherjee, Punam Chowdhury, Krishnan Rajendran, Tomoyoshi Nozaki, and Sandipan Ganguly Copyright © 2014 Avik K. Mukherjee et al. All rights reserved. Statistical Optimization of Fibrinolytic Enzyme Production Using Agroresidues by Bacillus cereus IND1 and Its Thrombolytic Activity In Vitro Mon, 09 Jun 2014 12:50:30 +0000 A potent fibrinolytic enzyme-producing Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent. Ponnuswamy Vijayaraghavan and Samuel Gnana Prakash Vincent Copyright © 2014 Ponnuswamy Vijayaraghavan and Samuel Gnana Prakash Vincent. All rights reserved. Effect of Experimentally Induced Hepatic and Renal Failure on the Pharmacokinetics of Topiramate in Rats Mon, 09 Jun 2014 11:46:12 +0000 We aimed to investigate the effect of induced hepatic and renal failure on the pharmacokinetics of topiramate (TPM) in rats. Twenty-four Sprague-Dawley rats were used in this study. Renal or hepatic failure was induced by a single i.p. dose of 7.5 mg/kg cisplatin () or 0.5 mL/kg carbon tetrachloride (CCl4) (), respectively. Three days after cisplatin dose or 24 h after CCl4 dose, the rats were administered a single oral dose of 20 mg/kg TPM. The plasma samples were quantified by LC-MS/MS method. Compared to control, plasma concentration-time profile in CCl4-treated and, to a lesser extent, in cisplatin-treated rats decreased more slowly particularly in the elimination phase. TPM oral clearance (CL/F) in CCl4-treated group was significantly lower than that in control (), whereas , T1/2, and Vd/F were significantly higher in CCl4-treated rats compared to the control (). The CL/F was not significantly different between cisplatin-treated rats and control (). However, in cisplatin-treated rats, the T1/2 and Vd/F were significantly higher than that in the control group (). Both conditions failed to cause a significant effect on or . The present findings suggest that induced hepatic or renal failure could modify the pharmacokinetic profile of TPM in the rat. Kamal M. Matar and Yasin I. Tayem Copyright © 2014 Kamal M. Matar and Yasin I. Tayem. All rights reserved. Phenotypic and Molecular Characterization of Plasmid Mediated AmpC β-Lactamases among Escherichia coli, Klebsiella spp., and Proteus mirabilis Isolated from Urinary Tract Infections in Egyptian Hospitals Mon, 09 Jun 2014 11:44:52 +0000 The incidence of resistance by Enterobacteriaceae to β-lactam/β-lactamase inhibitors combination is increasing in Egypt. Three phenotypic techniques, comprising AmpC disk diffusion and inhibition dependent methods using phenylboronic acid (PBA) and cloxacillin, were compared to PCR based method for detection of plasmid mediated AmpC β-lactamase in common urinary tract isolates. A total of 143 isolates, including E. coli, Klebsiella pneumonia, and Proteus mirabilis, were collected from urinary tract infections cases in Egyptian hospitals. Plasmid encoded AmpC genes were detected by PCR in 88.46% of cefoxitin resistant isolates. The most prevalent AmpC gene family was CIT including CMY-2, CMY-4, and two CMY-2 variants. The second prevalent gene was DHA-1 which was detected in E. coli and Klebsiella pneumonia. The genes EBC, FOX, and MOX were also detected but in small percentage. Some isolates were identified as having more than one pAmpC gene. The overall sensitivity and specificity of phenotypic tests for detection of AmpC β-lactamase showed that AmpC disk diffusion and inhibition dependent method by cloxacillin were the most sensitive and the most specific disk tests. PCR remains the gold standard for detection of AmpC β-lactamases. This study represents the first report of CMY-2 variants of CMY-42 and CMY-102 β-lactamase-producing E. coli, Klebsiella pneumonia, and Proteus mirabilis isolates in Egypt. Mai M. Helmy and Reham Wasfi Copyright © 2014 Mai M. Helmy and Reham Wasfi. All rights reserved. Molecular Analysis of VanA Outbreak of Enterococcus faecium in Two Warsaw Hospitals: The Importance of Mobile Genetic Elements Mon, 09 Jun 2014 11:42:38 +0000 Vancomycin-resistant Enterococcus faecium represents a growing threat in hospital-acquired infections. Two outbreaks of this pathogen from neighboring Warsaw hospitals have been analyzed in this study. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA, multilocus VNTR analysis (MLVA), and multilocus sequence typing (MLST) revealed a clonal variability of isolates which belonged to three main lineages (17, 18, and 78) of nosocomial E. faecium. All isolates were multidrug resistant and carried several resistance, virulence, and plasmid-specific genes. Almost all isolates shared the same variant of Tn1546 transposon, characterized by the presence of insertion sequence ISEf1 and a point mutation in the vanA gene. In the majority of cases, this transposon was located on 50 kb or 100 kb pRUM-related plasmids, which lacked, however, the axe-txe toxin-antitoxin genes. 100 kb plasmid was easily transferred by conjugation and was found in various clonal backgrounds in both institutions, while 50 kb plasmid was not transferable and occurred solely in MT159/ST78 strains that disseminated clonally in one institution. Although molecular data indicated the spread of VRE between two institutions or a potential common source of this alert pathogen, epidemiological investigations did not reveal the possible route by which outbreak strains disseminated. Ewa Wardal, Katarzyna Markowska, Dorota Żabicka, Marta Wróblewska, Małgorzata Giemza, Ewa Mik, Hanna Połowniak-Pracka, Agnieszka Woźniak, Waleria Hryniewicz, and Ewa Sadowy Copyright © 2014 Ewa Wardal et al. All rights reserved. In Vitro Antifungal Evaluation of Seven Different Disinfectants on Acrylic Resins Thu, 05 Jun 2014 10:53:19 +0000 Objective. The aim of this study was to evaluate alternative methods for the disinfection of denture-based materials. Material and Methods. Two different denture-based materials were included in the study. Before microbial test, the surface roughness of the acrylic resins was evaluated. Then, the specimens were divided into 8 experimental groups , according to microorganism considered and disinfection methods used. The specimens were contaminated in vitro by standardized suspensions of Candida albicans ATCC#90028 and Candida albicans oral isolate. The following test agents were tested: sodium hypochlorite (NaOCl 1%), microwave (MW) energy, ultraviolet (UV) light, mouthwash containing propolis (MCP), Corega Tabs, 50% and 100% white vinegar. After the disinfection procedure, the number of remaining microbial cells was evaluated in CFU/mL. Kruskal-Wallis, ANOVA, and Dunn’s test were used for multiple comparisons. Mann Whitney U test was used to compare the surface roughness. Results. Statistically significant difference was found between autopolymerised and heat-cured acrylic resins. The autopolymerised acrylic resin surfaces were rougher than surfaces of heat-cured acrylic resin. The most effective disinfection method was 100% white vinegar for tested microorganisms and both acrylic resins. Conclusion. This study showed that white vinegar 100% was the most effective method for tested microorganisms. This agent is cost-effective and easy to access and thus may be appropriate for household use. A. Z. Yildirim-Bicer, I. Peker, G. Akca, and I. Celik Copyright © 2014 A. Z. Yildirim-Bicer et al. All rights reserved. Experimental Protection of Diabetic Mice against Lethal P. aeruginosa Infection by Bacteriophage Thu, 05 Jun 2014 08:26:44 +0000 The emergence of antibiotic-resistant bacterial strains has become a global crisis and is vulnerable for the exploration of alternative antibacterial therapies. The present study emphasizes the use of bacteriophage for the treatment of multidrug resistant P. aeruginosa. P. aeruginosa was used to induce septicemia in streptozotocin (STZ) induced diabetic and nondiabetic mice by intraperitoneal (i.p.) injection of 3 × 108 CFU, resulting in a fatal bacteremia within 48 hrs. A single i.p. injection of 3 × 109 PFU phage GNCP showed efficient protection in both diabetic (90%) and nondiabetic (100%) bacteremic mice. It was further noted that the protection rate was reduced in diabetic mice when phage GNCP was administered after 4 h and 6 h of lethal bacterial challenge. In contrast, nondiabetic bacteremic mice were rescued even when treatment was delayed up to 20 h after lethal bacterial challenge. Evaluation of results confirmed that a single intraperitoneal injection of the phage dose (3 × 109 PFU/mL) was more effective than the multiple doses of imipenem. These results uphold the efficacy of phage therapy against pernicious P. aeruginosa infections, especially in cases of immunocompromised host. Nagaveni Shivshetty, Rajeshwari Hosamani, Liyakat Ahmed, Ajay Kumar Oli, Syed Sannauallah, Shivshetty Sharanbassappa, S. A. Patil, and Chandrakanth R. Kelmani Copyright © 2014 Nagaveni Shivshetty et al. All rights reserved. Association of Levels of Antibodies from Patients with Inflammatory Bowel Disease with Extracellular Proteins of Food and Probiotic Bacteria Wed, 04 Jun 2014 11:10:55 +0000 Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, ), Crohn's disease patients (CD, ), and ulcerative colitis patients (UC, ). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (; ). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. Arancha Hevia, Patricia López, Ana Suárez, Claudine Jacquot, María C. Urdaci, Abelardo Margolles, and Borja Sánchez Copyright © 2014 Arancha Hevia et al. All rights reserved. Safe-Site Effects on Rhizosphere Bacterial Communities in a High-Altitude Alpine Environment Wed, 04 Jun 2014 07:37:33 +0000 The rhizosphere effect on bacterial communities associated with three floristic communities (RW, FI, and M sites) which differed for the developmental stages was studied in a high-altitude alpine ecosystem. RW site was an early developmental stage, FI was an intermediate stage, M was a later more matured stage. The N and C contents in the soils confirmed a different developmental stage with a kind of gradient from the unvegetated bare soil (BS) site through RW, FI up to M site. The floristic communities were composed of 21 pioneer plants belonging to 14 species. Automated ribosomal intergenic spacer analysis showed different bacterial genetic structures per each floristic consortium which differed also from the BS site. When plants of the same species occurred within the same site, almost all their bacterial communities clustered together exhibiting a plant species effect. Unifrac significance value () on 16S rRNA gene diversity revealed significant differences () between BS site and the vegetated sites with a weak similarity to the RW site. The intermediate plant colonization stage FI did not differ significantly from the RW and the M vegetated sites. These results pointed out the effect of different floristic communities rhizospheres on their soil bacterial communities. Sonia Ciccazzo, Alfonso Esposito, Eleonora Rolli, Stefan Zerbe, Daniele Daffonchio, and Lorenzo Brusetti Copyright © 2014 Sonia Ciccazzo et al. All rights reserved. MiR-15b Targets Cyclin D1 to Regulate Proliferation and Apoptosis in Glioma Cells Wed, 04 Jun 2014 00:00:00 +0000 Aim. To investigate the role and mechanism of miR-15b in the proliferation and apoptosis of glioma. Methods. The miR-15b mimics were transfected into human glioma cells to upregulate the miR-15b expression. Cyclin D1 was determined by both western blotting analysis and luciferase reporter assay. Methylthiazol tetrazolium (MTT) and flow cytometry were employed to detect the cell proliferation, cell cycle, and apoptosis. Results. Overexpression of miR-15b inhibits proliferation by arrested cell cycle progression and induces apoptosis, possibly by directly targeting Cyclin D1. Both luciferase assay and bioinformatics search revealed a putative target site of miR-15b binding to the 3′-UTR of Cyclin D1. Moreover, expression of miR-15b in glioma tissues was found to be inversely correlated with Cyclin D1 expression. Enforced Cyclin D1 could abrogate the miR-15b-mediated cell cycle arrest and apoptosis. Conclusions. Our findings identified that miR-15b may function as a glioma suppressor by targeting the Cyclin D1, which may provide a novel therapeutic strategy for treatment of glioma. Guan Sun, Lei Shi, Shushan Yan, Zhengqiang Wan, Nan Jiang, Linshan Fu, Min Li, and Jun Guo Copyright © 2014 Guan Sun et al. All rights reserved. Complex Links between Natural Tuberculosis and Porcine Circovirus Type 2 Infection in Wild Boar Tue, 03 Jun 2014 09:26:02 +0000 Individuals in natural populations are exposed to a diversity of pathogens which results in coinfections. The aim of this study was to investigate the relation between natural infection with tuberculosis (TB) due to infection by bacteria of the Mycobacterium tuberculosis complex and porcine circovirus type 2 (PCV2) in free-ranging Eurasian wild boar (Sus scrofa). Apparent prevalence for TB lesions and PCV2 infection was extremely high in all age classes, including piglets (51% for TB; 85.7% for PCV2). Modeling results revealed that the relative risk of young (less than 2 years old) wild boar to test positive to PCV2 PCR was negatively associated with TB lesion presence. Also, an interaction between TB, PCV2, and body condition was evidenced: in wild boar with TB lesions probability of being PCV2 PCR positive increased with body condition, whereas this relation was negative for wild boar without TB lesions. This study provides insight into the coinfections occurring in free-ranging host populations that are naturally exposed to several pathogens at an early age. Using TB and PCV2 as a case study, we showed that coinfection is a frequent event among natural populations that takes place early in life with complex effects on the infections and the hosts. Iratxe Díez-Delgado, Mariana Boadella, MariPaz Martín-Hernando, José Angel Barasona, Beatriz Beltrán-Beck, David González-Barrio, Marina Sibila, Joaquín Vicente, Joseba M. Garrido, Joaquim Segalés, and Christian Gortazar Copyright © 2014 Iratxe Díez-Delgado et al. All rights reserved. Pretreatment of Cottage Cheese to Enhance Biogas Production Tue, 03 Jun 2014 08:08:12 +0000 This study evaluated the possibility of pretreating selected solid fraction of an anaerobic digester treating food waste to lower the hydraulic retention time and increase the methane production. The study investigated the effect of different pretreatments (thermal, chemical, thermochemical and enzymatic) for enhanced methane production from cottage cheese. The most effective pretreatments were thermal and enzymatic. Highest solubilisation of COD was observed in thermal pretreatment, followed by thermochemical. In single enzyme systems, lipase at low concentration gave significantly higher methane yield than for the experiments without enzyme additions. The highest lipase dosages decreased methane yield from cottage cheese. However, in case of protease enzyme an increase in concentration of the enzyme showed higher methane yield. In the case of mixed enzyme systems, pretreatment at 1 : 2 ratio of lipase : protease showed higher methane production in comparison with 1 : 1 and 2 : 1 ratios. Methane production potentials for different pretreatments were as follows: thermal 357 mL/g VS, chemical 293 mL/g VS, and thermochemical 441 mL/g VS. The average methane yield from single enzyme systems was 335 mL/g VS for lipase and 328 mL/g VS for protease. Methane potentials for mixed enzyme ratios were 330, 360, and 339 mL/g VS for 1 : 1, 1 : 2, and 2 : 1 lipase : protease, respectively. Vidhya Prabhudessai, Bhakti Salgaonkar, Judith Braganca, and Srikanth Mutnuri Copyright © 2014 Vidhya Prabhudessai et al. All rights reserved. Comparative Examination of the Olive Mill Wastewater Biodegradation Process by Various Wood-Rot Macrofungi Mon, 02 Jun 2014 09:17:41 +0000 Olive mill wastewater (OMW) constitutes a major cause of environmental pollution in olive-oil producing regions. Sixty wood-rot macrofungi assigned in 43 species were evaluated for their efficacy to colonize solidified OMW media at initially established optimal growth temperatures. Subsequently eight strains of the following species were qualified: Abortiporus biennis, Ganoderma carnosum, Hapalopilus croceus, Hericium erinaceus, Irpex lacteus, Phanerochaete chrysosporium, Pleurotus djamor, and P. pulmonarius. Fungal growth in OMW (25%v/v in water) resulted in marked reduction of total phenolic content, which was significantly correlated with the effluent’s decolorization. A. biennis was the best performing strain (it decreased phenolics by 92% and color by 64%) followed by P. djamor and I. lacteus. Increase of plant seeds germination was less pronounced evidencing that phenolics are only partly responsible for OMW’s phytotoxicity. Laccase production was highly correlated with all three biodegradation parameters for H. croceus, Ph. chrysosporium, and Pleurotus spp., and so were manganese-independent and manganese dependent peroxidases for A. biennis and I. lacteus. Monitoring of enzymes with respect to biomass production indicated that Pleurotus spp., H. croceus, and Ph. chrysosporium shared common patterns for all three activities. Moreover, generation of enzymes at the early biodegradation stages enhanced the efficiency of OMW treatment. Georgios Koutrotsios and Georgios I. Zervakis Copyright © 2014 Georgios Koutrotsios and Georgios I. Zervakis. All rights reserved. Biofilm Formation by Mycobacterium bovis: Influence of Surface Kind and Temperatures of Sanitizer Treatments on Biofilm Control Sun, 01 Jun 2014 12:53:34 +0000 Mycobacterium bovis causes classic bovine tuberculosis, a zoonosis which is still a concern in Africa. Biofilm forming ability of two Mycobacterium bovis strains was assessed on coupons of cement, ceramic, or stainless steel in three different microbiological media at 37°C with agitation for 2, 3, or 4 weeks to determine the medium that promotes biofilm. Biofilm mass accumulated on coupons was treated with 2 sanitizers (sanitizer A (5.5 mg L−1 active iodine) and sanitizer B (170.6 g1 alkyl dimethylbenzyl ammonium chloride, 78 g−1 didecyldimethyl ammonium chloride, 107.25 g L−1 glutaraldehyde, 146.25 g L−1 isopropanol, and 20 g L−1 pine oil) at 28 and 45°C and in hot water at 85°C for 5 min. Residual biofilms on treated coupons were quantified using crystal violet binding assay. The two strains had a similar ability to form biofilms on the three surfaces. More biofilms were developed in media containing 5% liver extract. Biofilm mass increased as incubation time increased till the 3rd week. More biofilms were formed on cement than on ceramic and stainless steel surfaces. Treatment with hot water at 85°C reduced biofilm mass, however, sanitizing treatments at 45°C removed more biofilms than at 28°C. However, neither treatment completely eliminated the biofilms. The choice of processing surface and temperatures used for sanitizing treatments had an impact on biofilm formation and its removal from solid surfaces. Victoria O. Adetunji, Aderemi O. Kehinde, Olayemi K. Bolatito, and Jinru Chen Copyright © 2014 Victoria O. Adetunji et al. All rights reserved. Identification, Typing, Antifungal Resistance Profile, and Biofilm Formation of Candida albicans Isolates from Lebanese Hospital Patients Sun, 01 Jun 2014 08:37:36 +0000 As leading opportunistic fungal pathogens identification and subtyping of Candida species are crucial in recognizing outbreaks of infection, recognizing particularly virulent strains, and detecting the emergence of drug resistant strains. In this study our objective was to compare identification of Candida albicans by the hospitals through the use of conventional versus identification based on the ITS (Internal Transcribed Spacer) and to assess biofilm forming capabilities, drug resistance patterns and correlate these with MLST typing. ITS typing revealed a 21.2% hospital misidentification rate. Multidrug resistance to three drugs out of four tested was detected within 25% of the isolates raising concerns about the followed treatment regimens. Drug resistant strains as well as biofilm formers were phylogenetically related, with some isolates with significant biofilm forming capabilities being correlated to those that were multidrug resistant. Such isolates were grouped closely together in a neighbor-joining tree generated by MLST typing indicating phylogenetic relatedness, microevolution, or recurrent infection. In conclusion, this pilot study gives much needed insight concerning C. albicans isolates circulating in Lebanese hospitals and is the first study of its kind correlating biofilm formation, antifungal resistance, and evolutionary relatedness. Ibrahim Bitar, Roy A. Khalaf, Houda Harastani, and Sima Tokajian Copyright © 2014 Ibrahim Bitar et al. All rights reserved. Mass Spectrometry and Multiplex Antigen Assays to Assess Microbial Quality and Toxin Production of Staphylococcus aureus Strains Isolated from Clinical and Food Samples Thu, 29 May 2014 11:51:57 +0000 The aim of our study was to investigate the microbial quality of meat products and on some clinical samples in Abidjan focused on Staphylococcus genus and the toxin production profile of Staphylococcus aureus (S. aureus) isolated. Bacteria were collected from 240 samples of three meat products sold in Abidjan and 180 samples issued from clinical infections. The strains were identified by both microbiological and MALDI-TOF-MS methods. The susceptibility to antibiotics was determined by the disc diffusion method. The production of Panton-Valentine Leukocidin, LukE/D, and epidermolysins was screened using radial gel immunodiffusion. The production of staphylococcal enterotoxins and TSST-1 was screened by a Bio-Plex Assay. We observed that 96/240 of meat samples and 32/180 of clinical samples were contaminated by Staphylococcus. Eleven species were isolated from meats and 4 from clinical samples. Forty-two S. aureus strains were isolated from ours samples. Variability of resistance was observed for most of the tested antibiotics but none of the strains displays a resistance to imipenem and quinolones. We observed that 89% of clinical S. aureus were resistant to methicillin against 58% for those issued from meat products. All S. aureus isolates issued from meat products produce epidermolysins whereas none of the clinical strains produced these toxins. The enterotoxins were variably produced by both clinical and meat product samples. Paul Attien, Haziz Sina, Wardi Moussaoui, Gaëlle Zimmermann-Meisse, Thomas Dadié, Daniel Keller, Philippe Riegel, Vincent Edoh, Simeon O. Kotchoni, Marcellin Djè, Gilles Prévost, and Lamine Baba-Moussa Copyright © 2014 Paul Attien et al. All rights reserved. Development of a Potential Probiotic Fresh Cheese Using Two Lactobacillus salivarius Strains Isolated from Human Milk Thu, 29 May 2014 09:41:20 +0000 Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to the consumer. The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2) isolated from human milk during production and storage of fresh cheese for 28 days at 4°C. The effect of such strains on the volatile compounds profile, texture, and other sensorial properties, including an overall consumer acceptance, was also investigated. Both L. salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable counts was only observed after 21 days. Globally, the addition of the L. salivarius strains did not change significantly neither the chemical composition of the cheese nor texture parameters after the storage period, although cheeses manufactured with L. salivarius CECT5713 presented significantly higher values of hardness. A total of 59 volatile compounds were identified in the headspace of experimental cheeses, and some L. salivarius-associated differences could be identified. All cheeses presented good results of acceptance after the sensory evaluation. Consequently, our results indicated that fresh cheese can be a good vehicle for the two L. salivarius strains analyzed in this study. Nivia Cárdenas, Javier Calzada, Ángela Peirotén, Esther Jiménez, Rosa Escudero, Juan M. Rodríguez, Margarita Medina, and Leónides Fernández Copyright © 2014 Nivia Cárdenas et al. All rights reserved. Immune Modulating Capability of Two Exopolysaccharide-Producing Bifidobacterium Strains in a Wistar Rat Model Thu, 29 May 2014 06:30:22 +0000 Fermented dairy products are the usual carriers for the delivery of probiotics to humans, Bifidobacterium and Lactobacillus being the most frequently used bacteria. In this work, the strains Bifidobacterium animalis subsp. lactis IPLA R1 and Bifidobacterium longum IPLA E44 were tested for their capability to modulate immune response and the insulin-dependent glucose homeostasis using male Wistar rats fed with a standard diet. Three intervention groups were fed daily for 24 days with 10% skimmed milk, or with 109 cfu of the corresponding strain suspended in the same vehicle. A significant increase of the suppressor-regulatory TGF-β cytokine occurred with both strains in comparison with a control (no intervention) group of rats; the highest levels were reached in rats fed IPLA R1. This strain presented an immune protective profile, as it was able to reduce the production of the proinflammatory IL-6. Moreover, phosphorylated Akt kinase decreased in gastroctemius muscle of rats fed the strain IPLA R1, without affecting the glucose, insulin, and HOMA index in blood, or levels of Glut-4 located in the membrane of muscle and adipose tissue cells. Therefore, the strain B. animalis subsp. lactis IPLA R1 is a probiotic candidate to be tested in mild grade inflammation animal models. Nuria Salazar, Patricia López, Pablo Garrido, Javier Moran, Estefanía Cabello, Miguel Gueimonde, Ana Suárez, Celestino González, Clara G. de los Reyes-Gavilán, and Patricia Ruas-Madiedo Copyright © 2014 Nuria Salazar et al. All rights reserved. Phenotypic and Genotypic Characterization of Atypical Listeria monocytogenes and Listeria innocua Isolated from Swine Slaughterhouses and Meat Markets Wed, 28 May 2014 12:22:59 +0000 In the last decade, atypical Listeria monocytogenes and L. innocua strains have been detected in food and the environment. Because of mutations in the major virulence genes, these strains have different virulence intensities in eukaryotic cells. In this study, we performed phenotypic and genotypic characterization of atypical L. monocytogenes and L. innocua isolates obtained from swine slaughterhouses and meat markets. Forty strains were studied, including isolates of L. monocytogenes and L. innocua with low-hemolytic activity. The isolates were characterized using conventional phenotypic Listeria identification tests and by the detection and analysis of L. monocytogenes-specific genes. Analysis of 16S rRNA was used for the molecular identification of the Listeria species. The L. monocytogenes isolates were positive for all of the virulence genes studied. The atypical L. innocua strains were positive for hly, plcA, and inlC. Mutations in the InlC, InlB, InlA, PI-PLC, PC-PLC, and PrfA proteins were detected in the atypical isolates. Further in vitro and transcriptomic studies are being developed to confirm the role of these mutations in Listeria virulence. Luisa Zanolli Moreno, Renata Paixão, Debora Dirani Sena de Gobbi, Daniele Cristine Raimundo, Thais Sebastiana Porfida Ferreira, Andrea Micke Moreno, Ernesto Hofer, Cristhiane Moura Falavina dos Reis, Glavur Rogério Matté, and Maria Helena Matté Copyright © 2014 Luisa Zanolli Moreno et al. All rights reserved. Contrasted Reactivity to Oxygen Tensions in Frankia sp. Strain CcI3 throughout Nitrogen Fixation and Assimilation Wed, 28 May 2014 10:47:49 +0000 Reconciling the irreconcilable is a primary struggle in aerobic nitrogen-fixing bacteria. Although nitrogenase is oxygen and reactive oxygen species-labile, oxygen tension is required to sustain respiration. In the nitrogen-fixing Frankia, various strategies have been developed through evolution to control the respiration and nitrogen-fixation balance. Here, we assessed the effect of different oxygen tensions on Frankia sp. strain CcI3 growth, vesicle production, and gene expression under different oxygen tensions. Both biomass and vesicle production were correlated with elevated oxygen levels under both nitrogen-replete and nitrogen-deficient conditions. The mRNA levels for the nitrogenase structural genes (nifHDK) were high under hypoxic and hyperoxic conditions compared to oxic conditions. The mRNA level for the hopanoid biosynthesis genes (sqhC and hpnC) was also elevated under hyperoxic conditions suggesting an increase in the vesicle envelope. Under nitrogen-deficient conditions, the hup2 mRNA levels increased with hyperoxic environment, while hup1 mRNA levels remained relatively constant. Taken together, these results indicate that Frankia protects nitrogenase by the use of multiple mechanisms including the vesicle-hopanoid barrier and increased respiratory protection. Faten Ghodhbane-Gtari, Karima Hezbri, Amir Ktari, Imed Sbissi, Nicholas Beauchemin, Maher Gtari, and Louis S. Tisa Copyright © 2014 Faten Ghodhbane-Gtari et al. All rights reserved. Synergistic Antimicrobial Action of Chlorhexidine and Ozone in Endodontic Treatment Wed, 28 May 2014 00:00:00 +0000 Objectives. The aim of this study was to determine whether irrigation with sodium hypochlorite, chlorhexidine, and ozone gas, alone or in combination, were effective against Enterococcus faecalis and Candida albicans; these are microorganisms frequently isolated from teeth with periapical lesions resistant to endodontic treatment. Material and Methods. 220 single root teeth, recently extracted, were inoculated with Candida albicans and Enterococcus faecalis. The formulations tested were sodium hypochlorite at 1, 3, and 5% chlorhexidine at 0.2% and 2% and ozone gas applied for different periods of time. The combination of sodium hypochlorite at 5% and chlorhexidine at 2%, with gaseous ozone, were also assessed. For the most active treatments the mechanism of action was assessed through flow cytometry. Results. Sodium hypochlorite, chlorhexidine, and gaseous ozone alone were ineffective in completely eliminating the microorganisms. The association of chlorhexidine at 2% followed by ozone gas for 24 seconds promoted the complete elimination of Candida albicans and Enterococcus faecalis. Flow cytometry shows that ozone and chlorhexidine act differently, which could explain its synergic activity. Conclusions. This new disinfection protocol, combining irrigation with chlorhexidine at 2% and ozone gas for 24 seconds, may be advantageous when treating infected root canals. Rita Noites, Cidália Pina-Vaz, Rita Rocha, Manuel Fontes Carvalho, Acácio Gonçalves, and Irene Pina-vaz Copyright © 2014 Rita Noites et al. All rights reserved. Screening for Genes Coding for Putative Antitumor Compounds, Antimicrobial and Enzymatic Activities from Haloalkalitolerant and Haloalkaliphilic Bacteria Strains of Algerian Sahara Soils Tue, 27 May 2014 10:23:57 +0000 Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications. Okba Selama, Gregory C. A. Amos, Zahia Djenane, Chiara Borsetto, Rabah Forar Laidi, David Porter, Farida Nateche, Elizabeth M. H. Wellington, and Hocine Hacène Copyright © 2014 Okba Selama et al. All rights reserved. Statistical Approach for Production of PUFA from Kocuria sp. BRI 35 Isolated from Marine Water Sample Sun, 25 May 2014 11:02:48 +0000 In this study, Plackett-Burman design was used to identify the most influential parameters affecting PUFA production by Kocuria sp. BRI 35 isolated from Antarctic water sample. Amongst 10 variables evaluated, magnesium chloride, protease peptone, glucose, and temperature were significant. Response surface methodology consisting of a central composite design was developed to study the interactions between the variables and to determine optimal values of significant variables. A quadratic model (R = 0.9652, F = 14.64, P < 0.0001) was built. The contour plots indicated that the isolate produced maximum PUFA at lower concentrations of magnesium sulfate (0.9 g/L) and higher concentrations of protease peptone (5 g/L) and glucose (10 g/L) at 15°C. MgSO4 and glucose exhibited quadratic as well as interactive effect on PUFA production whereas protease peptone and temperature showed interactive effects only. After optimization, PUFA production per unit biomass increased from 0.94 mg/g to 11.12 mg/g. This represented an increase from 3% to 58.62% of the total fatty acids. Among PUFAs, the yield of ω-6 fatty acids increased from 9.66 mg/L to 107.71 mg/L with significant increase in linoleic acid (20.36 mg/L) whereas ω-3 fatty acids increased up to 12.37 mg/L with DHA being the major ω-3 fatty acid produced. Swanandi Pote and Rama Bhadekar Copyright © 2014 Swanandi Pote and Rama Bhadekar. All rights reserved. Evaluation of Microbial Enzymes in Normal and Abnormal Cervicovaginal Fluids of Cervical Dysplasia: A Case Control Study Thu, 22 May 2014 11:46:52 +0000 The aim of the present study was to evaluate the role of microbial enzymes in normal and abnormal cervicovaginal fluids of cervical dysplasia. The cervicovaginal infections were evaluated through the estimation of microbial enzymes in patients with and without abnormal cervical cytology like bacterial and fungal infections. The patients were categorized based on infection caused by organism and stages of dysplasia. The pH, Whiff test, and Pap smear tests were conducted for normal and abnormal cervical swabs based on standard protocols. Microbial enzymes include mucinase, sialidases, and proteases of the cervical swabs and are estimated according to standard methods. The results of abnormal cervical cytological smears showed increased pH and the presence of amines with different levels of Pap smear test. Increased levels of microbial enzymes were observed in patients with abnormal cytology than normal cytology. Three microbial enzymes mucinase, sialidase, and protease were significantly () more elevated in patients with bacterial infections (, , ) than without dysplasia (, , ). The results reinforce that the microbial infection seems to be more prone to cervical dysplasia and may act as risk-factor for the development of cervical cancer along with HPV infection. Subramanyam Dasari, Wudayagiri Rajendra, and Lokanatha Valluru Copyright © 2014 Subramanyam Dasari et al. All rights reserved. Three-Dimensional In Vitro Models of Granuloma to Study Bacteria-Host Interactions, Drug-Susceptibility, and Resuscitation of Dormant Mycobacteria Wed, 21 May 2014 07:15:24 +0000 Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium avium subsp. paratuberculosis can survive within host macrophages in a dormant state, encased within an organized aggregate of immune host cells called granuloma. Granulomas consist of uninfected macrophages, foamy macrophages, epithelioid cells, and T lymphocytes accumulated around infected macrophages. Within granulomas, activated macrophages can fuse to form multinucleated giant cells, also called giant Langhans cells. A rim of T lymphocytes surrounds the core, and a tight coat of fibroblast closes the structure. Several in vivo models have been used to study granuloma’s structure and function, but recently developed in vitro models of granuloma show potential for closer observation of the early stages of host’s responses to live mycobacteria. This paper reviews culture conditions that resulted in three-dimensional granulomas, formed by the adhesion of cell populations in peripheral blood mononuclear cells infected with mycobacteria. The similarities of these models to granulomas encountered in clinical specimens include cellular composition, granulomas’ cytokine production, and cell surface antigens. A reliable in vitro dormancy model may serve as a useful platform to test whether drug candidates can kill dormant mycobacteria. Novel drugs that target dormancy-specific pathways may shorten the current long, difficult treatments necessary to cure mycobacterial diseases. Liam E. Fitzgerald, Naiara Abendaño, Ramon A. Juste, and Marta Alonso-Hearn Copyright © 2014 Liam E. Fitzgerald et al. All rights reserved. Antibacterial Activity of pH-Dependent Biosynthesized Silver Nanoparticles against Clinical Pathogen Wed, 21 May 2014 00:00:00 +0000 Simple, nontoxic, environmental friendly method is employed for the production of silver nanoparticles. In this study the synthesized nanoparticles UV absorption band occurred at 400 nm because of the surface Plasmon resonance of silver nanoparticles. The pH of the medium plays important role in the synthesis of control shaped and sized nanoparticles. The colour intensity of the aqueous solution varied with pH. In this study, at pH 9, the colour of the aqueous solution was dark brown, whereas in pH 5 the colour was yellowish brown; the colour difference in the aqueous solution occurred due to the higher production of silver nanoparticles. The antibacterial activity of biosynthesized silver nanoparticles was carried out against E. coli. The silver nanoparticles synthesized at pH 9 showed maximum antibacterial activity at 50 μL. Kethirabalan Chitra and Gurusamy Annadurai Copyright © 2014 Kethirabalan Chitra and Gurusamy Annadurai. All rights reserved. In Vitro Synergistic Effect of Curcumin in Combination with Third Generation Cephalosporins against Bacteria Associated with Infectious Diarrhea Sun, 18 May 2014 12:47:08 +0000 Diarrhea is one of the leading causes of morbidity and mortality in humans in developed and developing countries. Furthermore, increased resistance to antibiotics has resulted in serious challenges in the treatment of this infectious disease worldwide. Therefore, there exists a need to develop alternative natural or combination drug therapies. The aim of the present study was to investigate the synergistic effect of curcumin-1 in combination with three antibiotics against five diarrhea causing bacteria. The antibacterial activity of curcumin-1 and antibiotics was assessed by the broth microdilution method, checkerboard dilution test, and time-kill assay. Antimicrobial activity of curcumin-1 was observed against all tested strains. The MICs of curcumin-1 against test bacteria ranged from 125 to 1000 μg/mL. In the checkerboard test, curcumin-1 markedly reduced the MICs of the antibiotics cefaclor, cefodizime, and cefotaxime. Significant synergistic effect was recorded by curcumin-1 in combination with cefotaxime. The toxicity of curcumin-1 with and without antibiotics was tested against foreskin (FS) normal fibroblast and no significant cytotoxicity was observed. From our result it is evident that curcumin-1 enhances the antibiotic potentials against diarrhea causing bacteria in in vitro condition. This study suggested that curcumin-1 in combination with antibiotics could lead to the development of new combination of antibiotics against diarrhea causing bacteria. Nishanth Kumar Sasidharan, Sreerag Ravikumar Sreekala, Jubi Jacob, and Bala Nambisan Copyright © 2014 Nishanth Kumar Sasidharan et al. All rights reserved. Effect of Er,Cr:YSGG Laser Irradiation with Radial Firing Tips on Candida albicans in Experimentally Infected Root Canals Thu, 15 May 2014 13:35:04 +0000 Aim. To compare the disinfection effect of Er,Cr:YSGG laser using radial firing tips with NaOCI in root canals infected with C. albicans and to evaluate the irradiation effect on the dentinal surfaces. Material and Methods. In total seventy-six mandibular premolar teeth were used. In order to standardize the incubation and sterilization procedure, eight teeth were used. Sixty-eight of the root canals were incubated with C. albicans suspension for 72 hours. The specimens were divided into 5 experimental groups. Two groups were constituted as Group 1 was irradiated with 1.5 W laser () and group 2, which was irradiated with 2 W laser (). Two more groups were formed as Group 3 (2 W laser () and Group 4 NaOCI (5%) (). Group 5 () did not receive any treatment. Mann-Whitney U and Kruskal-Wallis H tests were used to compare the different laser output powers. Wilcoxon Signed Ranks Test was used in order to compare the Candida cfu/ml levels according to treatment protocols (). Results. Both 1.5 W and 2 W laser resulted in a major reduction of C. albicans without a significant difference. The comparison of the dentin surfaces irradiated with Er,Cr:YSGG laser at two power settings resulted in similar morphological changes. However, NaOCI was found to be more effective in reduction of C. albicans than 2 W laser application. Conclusion. According to the results of the present study, the Er,Cr:YSGG laser with radial firing tips presented less antifungal effects on C. albicans in root canals of infected teeth than NaOCl solution. Leman Ozkan, Serap Cetiner, and Tamer Sanlidag Copyright © 2014 Leman Ozkan et al. All rights reserved. A Fast, Reliable, and Sensitive Method for Detection and Quantification of Listeria monocytogenes and Escherichia coli O157:H7 in Ready-to-Eat Fresh-Cut Products by MPN-qPCR Thu, 15 May 2014 06:38:04 +0000 In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g−1 could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 101 CFU g−1 after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype. Pasquale Russo, Giuseppe Botticella, Vittorio Capozzi, Salvatore Massa, Giuseppe Spano, and Luciano Beneduce Copyright © 2014 Pasquale Russo et al. All rights reserved. Antimicrobial Activity of Coronarin D and Its Synergistic Potential with Antibiotics Thu, 15 May 2014 00:00:00 +0000 Coronarin D is a labdane-type diterpene from the rhizomes of Hedychium coronarium. In the view of our ongoing effort to explore its novel biological activity, antimicrobial activity study of coronarin D was performed. The results showed that coronarin D was active against tested Gram-positive bacteria, inactive for tested Gram-negative bacteria, and weakly active against tested fungi. The antibacterial effect of the combination of coronarin D with nine classical antibiotics against four Gram-positive bacteria was also evaluated. The fractional inhibitory concentration indices (FICI) of coronarin D-antibiotics combinations, calculated from the checkerboard assay, were used as synergism indicator. Out of 36 combinations, 47% showed total synergism, 33% had partial synergistic interaction, 17% showed no effect, and 3% showed antagonism. By combination with coronarin D at concentration of 0.25 minimal inhibitory concentration (MIC), the activities of antibiotics were boosted to 4- to 128-fold. These finding suggested an attractive approach to combat the infectious diseases by using coronarin D-antibiotic drug combination. Nanthawan Reuk-ngam, Nitirat Chimnoi, Nisachon Khunnawutmanotham, and Supanna Techasakul Copyright © 2014 Nanthawan Reuk-ngam et al. All rights reserved. Antifungal and Antiproliferative Protein from Cicer arietinum: A Bioactive Compound against Emerging Pathogens Wed, 14 May 2014 08:36:46 +0000 The emergence of epidemic fungal pathogenic resistance to current antifungal drugs has increased the interest in developing alternative antibiotics from natural sources. Cicer arietinum is well known for its medicinal properties. The aim of this work was to isolate antimicrobial proteins from Cicer arietinum. An antifungal protein, C-25, was isolated from Cicer arietinum and purified by gel filtration. C-25 protein was tested using agar diffusion method against human pathogenic fungi of ATCC strains and against clinical isolates of Candida krusei, Candida tropicalis, and Candida parapsilosis, and MIC values determined were varied from 1.56 to 12.5 μg/mL. The SEM study demonstrated that C-25 induces the bleb-like surface changes, irregular cell surface, and cell wall disruption of the fungi at different time intervals. Cytotoxic activity was studied on oral cancer cells and normal cells. It also inhibits the growth of fungal strains which are resistant to fluconazole. It reduced the cell proliferation of human oral carcinoma cells at the concentration of 37.5 μg/mL (IC50) and no toxic effect was found on normal human peripheral blood mononuclear cells even at higher concentration of 600 μg/mL. It can be concluded that C-25 can be considered as an effective antimycotic as well as antiproliferative agent against human oral cancer cells. Suresh Kumar, Vaishali Kapoor, Kamaldeep Gill, Kusum Singh, Immaculata Xess, Satya N. Das, and Sharmistha Dey Copyright © 2014 Suresh Kumar et al. All rights reserved. Epidemiology of Carbapenemase-Producing Enterobacteriaceae and Acinetobacter baumannii in Mediterranean Countries Tue, 13 May 2014 13:04:43 +0000 The emergence and global spread of carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii are of great concern to health services worldwide. These β-lactamases hydrolyse almost all β-lactams, are plasmid-encoded, and are easily transferable among bacterial species. They are mostly of the KPC, VIM, IMP, NDM, and OXA-48 types. Their current extensive spread worldwide in Enterobacteriaceae is an important source of concern. Infections caused by these bacteria have limited treatment options and have been associated with high mortality rates. Carbapenemase producers are mainly identified among Klebsiella pneumoniae, Escherichia coli, and A. baumannii and still mostly in hospital settings and rarely in the community. The Mediterranean region is of interest due to a great diversity and population mixing. The prevalence of carbapenemases is particularly high, with this area constituting one of the most important reservoirs. The types of carbapenemase vary among countries, partially depending on the population exchange relationship between the regions and the possible reservoirs of each carbapenemase. This review described the epidemiology of carbapenemases produced by enterobacteria and A. baumannii in this part of the world highlighting the worrisome situation and the need to screen and detect these enzymes to prevent and control their dissemination. Nassima Djahmi, Catherine Dunyach-Remy, Alix Pantel, Mazouz Dekhil, Albert Sotto, and Jean-Philippe Lavigne Copyright © 2014 Nassima Djahmi et al. All rights reserved. Prevalence and Correlation with Clinical Diseases of Helicobacter pylori cagA and vacA Genotype among Gastric Patients from Northeast China Tue, 13 May 2014 08:59:39 +0000 Helicobacter pylori vacA and cagA genes have significant genetic heterogenicity, resulting in different clinical outcomes. Northeast part of China has reported high prevalence of H. pylori infections and gastric cancer. Hence, we investigated the H. pylori cagA and vacA genotypes with clinical outcomes in Northeast China. Gastric tissue samples (), chronic gastritis (GIs), gastric ulcer (GU), and gastric cancer (GC) were analysed for 16S rRNA ureA, cagA, and cagA genotypes by PCR. A total of 141 (84%) cases were found positive for H. pylori by 16S rRNA and ureA. GC showed high H. pylori infection (93%) compared with GIs (72%) and GU (84%). The vacAs1am1 was highly found in GC (40%) and GU (36%), vacAs1am2 in GIs (33%), vacAs1bm1 (14%) and vacAs1bm2 (8%) in GU cases, and s2m1 in normal cases (33%), while vacAs1cm1 showed low frequency in GIs (2%) and GU (3%) and GC showed negative result. The East-Asian cagA strain was highly observed in GC (43%), as compared to GIs (41%) and GU (20%). The East-Asian cagA/vacAs1am1 was significantly higher in GC (23%) than in GU (22%) and GIs (145) patients. The East-Asian type cagA with vacAs1a and vacAm1 is the most predominant genotype in H. pylori strains of Northeast China. Faisal Aziz, Xin Chen, Xuesong Yang, and Qiu Yan Copyright © 2014 Faisal Aziz et al. All rights reserved. Safety Characterization and Antimicrobial Properties of Kefir-Isolated Lactobacillus kefiri Tue, 13 May 2014 08:00:47 +0000 Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused - or -hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use. Paula Carasi, Mariángeles Díaz, Silvia M. Racedo, Graciela De Antoni, María C. Urdaci, and María de los Angeles Serradell Copyright © 2014 Paula Carasi et al. All rights reserved. Emetic Bacillus cereus Are More Volatile Than Thought: Recent Foodborne Outbreaks and Prevalence Studies in Bavaria (2007–2013) Thu, 08 May 2014 00:00:00 +0000 Several Bacillus cereus strains possess the genetic fittings to produce two different types of toxins, the heat-stable cereulide or different heat-labile proteins with enterotoxigenic potential. Unlike the diarrheal toxins, cereulide is (pre-)formed in food and can cause foodborne intoxications shortly after ingestion of contaminated food. Based on the widely self-limiting character of cereulide intoxications and rarely performed differential diagnostic in routine laboratories, the real incidence is largely unknown. Therefore, during a 7-year period about 4.300 food samples linked to foodborne illness with a preliminary report of vomiting as well as food analysed in the context of monitoring programs were investigated to determine the prevalence of emetic B. cereus in food environments. In addition, a lux-based real-time monitoring system was employed to assess the significance of the detection of emetic strains in different food matrices and to determine the actual risk of cereulide toxin production in different types of food. This comprehensive study showed that emetic strains are much more volatile than previously thought. Our survey highlights the importance and need of novel strategies to move from the currently taxonomic-driven diagnostic to more risk orientated diagnostics to improve food and consumer safety. Ute Messelhäusser, Elrike Frenzel, Claudia Blöchinger, Renate Zucker, Peter Kämpf, and Monika Ehling-Schulz Copyright © 2014 Ute Messelhäusser et al. All rights reserved. In Vitro Antibacterial, Antioxidant, and Cytotoxic Activities of Parthenium hysterophorus and Characterization of Extracts by LC-MS Analysis Wed, 07 May 2014 11:45:30 +0000 Present work reports the biological activities of P. hysterophorus leaf, stem, flower, and root. Dried samples were sequentially extracted with many solvents. Hexane (HX), benzene (BZ), and chloroform (CH) extracts of leaf showed considerable antibacterial activity against Streptococcus mutans (MTCC 497), Proteus vulgaris (MTCC 7299), and Salmonella typhi (MTCC 3917). Flower extracts exhibited presence of higher amount of flavonoids (13.9–59.6 μgQE/mg) followed by leaf, stem, and root. Stem (HX, BZ, and CH), leaf ethanol (ET), and root (HX, BZ, and CH) fractions showed noticeable antioxidant capacity in phosphomolybdate assay. Most of the extracts demonstrated beta carotene bleaching inhibition capability. BZ, ethyl acetate (EA), and ET fractions of leaves, stem aqueous (AQ), and flower EA extracts showed membrane protective activities (40–55%). Middle fractions of the plant parts displayed moderate antihemolytic potential. Most of the flower extracts exhibited cytotoxic activity (80–100%) against lung and colon cancer cell lines. Root (HX and ET) and leaf ET extracts showed considerable inhibition (90–99%) of colon and ovary cancer cell lines. The LC-MS scan demonstrated presence of different compounds showing 3–20 min retention time. The study revealed considerable antibacterial, antioxidant, lipo-protective, antihemolytic, and anticancer potential in all parts of P. hysterophorus. Shashank Kumar, Sanjay Pandey, and Abhay K. Pandey Copyright © 2014 Shashank Kumar et al. All rights reserved. Synthesis and Antimicrobial Activities of 5-Arylidene-thiazolidine-2,4-dione Derivatives Wed, 07 May 2014 07:48:51 +0000 Antibiotic resistance is considered one of the world's major public health concerns. The main cause of bacterial resistance is the improper and repeated use of antibiotics. To alleviate this problem, new chemical substances against microorganisms are being synthesized and tested. Thiazolidines are compounds having many pharmacological activities including antimicrobial activities. For this purpose some thiazolidine derivatives substituted at position 5 in the thiazolidine nucleus were synthesized and tested against several microorganisms. Using a disc diffusion method, antimicrobial activity was verified against Gram-positive, Gram-negative, and alcohol acid resistant bacteria and yeast. The minimum inhibition concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined. All derivatives showed antimicrobial activity mainly against Gram-positive bacteria, with MIC values ranging from 2 to 16 µg/mL. Ivanildo Mangueira da Silva, João da Silva Filho, Priscila Brandão Gomes da Silva Santiago, Micalyne Soares do Egito, Carlos André de Souza, Frederico Leite Gouveia, Rafael Matos Ximenes, Kêsia Xisto da Fonseca Ribeiro de Sena, Antonio Rodolfo de Faria, Dalci José Brondani, and Julianna Ferreira Cavalcanti de Albuquerque Copyright © 2014 Ivanildo Mangueira da Silva et al. All rights reserved. Bioactive Compounds Derived from the Yeast Metabolism of Aromatic Amino Acids during Alcoholic Fermentation Mon, 05 May 2014 14:08:43 +0000 Metabolites resulting from nitrogen metabolism in yeast are currently found in some fermented beverages such as wine and beer. Their study has recently attracted the attention of researchers. Some metabolites derived from aromatic amino acids are bioactive compounds that can behave as hormones or even mimic their role in humans and may also act as regulators in yeast. Although the metabolic pathways for their formation are well known, the physiological significance is still far from being understood. The understanding of this relevance will be a key element in managing the production of these compounds under controlled conditions, to offer fermented food with specific enrichment in these compounds or even to use the yeast as nutritional complements. Albert Mas, Jose Manuel Guillamon, Maria Jesus Torija, Gemma Beltran, Ana B. Cerezo, Ana M. Troncoso, and M. Carmen Garcia-Parrilla Copyright © 2014 Albert Mas et al. All rights reserved. Antagonistic Interactions of “Ya-Sa-Marn-Phlae” Ethanol Extract in Combination with Topical Antiseptics against Clinical Isolates of Staphylococcus aureus Mon, 05 May 2014 08:26:25 +0000 This investigation was aimed at assessing a possible interaction of a traditional Thai herbal recipe, “Ya-Sa-Marn-Phlae (YSMP),” used for wound treatments with topical antiseptics, povidone-iodine (PI) solution and hydrogen peroxide (H2O2), and effects of THR-SK010 alone and the combinations on Staphylococcus aureus. Antibacterial activities of ethanol extracts from the herbal recipe were determined against both methicillin resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). YSMP exhibited remarkable antistaphylococcal activity with MIC values of 3.9–7.8 μg/mL. This recipe possessed bacteriostatic activity and did not reduce the tolerance of both MRSA and MSSA isolates to the high ionic strength. Interaction between THR-SK010 and the antiseptics was carried out by checkerboard testing and time-kill assay. Both indifferent and slightly antagonistic effects were observed with THR-SK010/PI and THR-SK010/H2O2 combinations against the tested isolates. In addition to commercially available antiseptics, THR-SK010 offered additional therapeutic options for the decolonization of MRSA and MSSA. Topical application of plant extracts with antioxidant activity, such as THR-SK010, should not be used immediately with PI or H2O2 and further investigation on this interaction is needed. Sasitorn Chusri, Sirirat Tongrod, Julalak Chokpaisarn, Surasak Limsuwan, and Supayang Piyawan Voravuthikunchai Copyright © 2014 Sasitorn Chusri et al. All rights reserved. Recent Developments in Mycobacteriology: A Clinical and Diagnostic Perspective Wed, 30 Apr 2014 09:46:50 +0000 Tomasz Jagielski, Jakko van Ingen, Nalin Rastogi, and Jarosław Dziadek Copyright © 2014 Tomasz Jagielski et al. All rights reserved. The Kampo Medicine Rokumigan Possesses Antibiofilm, Anti-Inflammatory, and Wound Healing Properties Wed, 30 Apr 2014 09:09:36 +0000 Periodontal diseases, which are inflammatory diseases of bacterial origin affecting the tooth-supporting tissues, are characterized by inflammation and destruction of gingival connective tissue and alveolar bone, and may lead to tooth loss. The aim of the study was to investigate Rokumigan, a Kampo Japanese traditional medicine made of six different plants, for its capacity to prevent biofilm formation by Fusobacterium nucleatum, to inhibit interleukin-6 (IL-6) and interleukin-8 (IL-8) secretion by mucosal cells, and to promote wound healing in a fibroblast model. Using a microplate colorimetric assay, Rokumigan prevented biofilm formation by F. nucleatum, while it had no effect on bacterial growth. Rokumigan inhibited IL-6 secretion in both epithelial cells and fibroblasts stimulated with lipopolysaccharide. However, it caused no significant inhibition of IL-8 secretion by both cell types. Rokumigan significantly increased proliferation and migration of gingival fibroblasts in a wound healing assay. In conclusion, the Kampo formulation Rokumigan, through suppression of biofilm formation by F. nucleatum, inhibition of IL-6 secretion by gingival epithelial cells and fibroblasts, and promotion of wound healing in a fibroblast model, may have potential application for periodontal diseases. James Liao, Jabrane Azelmat, Lei Zhao, Masami Yoshioka, Daisuke Hinode, and Daniel Grenier Copyright © 2014 James Liao et al. All rights reserved. Emergent Bacteria in Cystic Fibrosis: In Vitro Biofilm Formation and Resilience under Variable Oxygen Conditions Tue, 29 Apr 2014 13:17:52 +0000 Concurrent to conventional bacterial pathogens, unusual microbes are emerging from cystic fibrosis (CF) airways. Nonetheless, little is known about the contribution of these newly microbes to the resilience of CF-associated biofilms, particularly under variable-oxygen concentrations that are known to occur in vivo in the mucus of CF patients. Two CF-emergent bacterial species, Inquilinus limosus and Dolosigranulum pigrum, and the major pathogen Pseudomonas aeruginosa were studied in terms of biofilm development and antibiotic susceptibilities under in vitro atmospheres with different oxygen availabilities. All species were able to develop in vitro biofilms under different oxygen-available environments, with D. pigrum accumulating high amounts of biomass and respiratory activities. When established, biofilms were of difficult eradication, with antibiotics losing their effectiveness in comparison with the corresponding planktonic populations. Surprisingly, biofilms of each emergent organism displayed multidrug resistance under aerobic environments, enduring even in low-oxygen atmospheres. This study suggests a potential prospect on the impact of nonconventional organisms I. limosus and D. pigrum on CF lung infections, demonstrating capacity to adapt to biofilm mode of life under restricted-oxygen atmospheres resembling CF airways, which may ultimately endanger the efficacy of currently used antibiotic regimens. Susana P. Lopes, Nuno F. Azevedo, and Maria O. Pereira Copyright © 2014 Susana P. Lopes et al. All rights reserved. The Structural Modeling of the Interaction between Levofloxacin and the Mycobacterium tuberculosis Gyrase Catalytic Site Sheds Light on the Mechanisms of Fluoroquinolones Resistant Tuberculosis in Colombian Clinical Isolates Mon, 28 Apr 2014 14:07:17 +0000 We compared the prevalence of levofloxacin (LVX) resistance with that of ofloxacin (OFX) and moxifloxacin (MFX) among multidrug resistant (MDR) MTB clinical isolates collected in Medellin, Colombia, between 2004 and 2009 and aimed at unraveling the underlying molecular mechanisms that explain the correlation between QRDR-A mutations and LVX resistance phenotype. We tested 104 MDR isolates for their susceptibility to OFX, MFX, and LVX. Resistance to OFX was encountered in 10 (9.6%) of the isolates among which 8 (7.7%) were also resistant to LVX and 6 (5.7%) to MFX. Four isolates resistant to the 3 FQ were harboring the Asp94Gly substitution, whilst 2 other isolates resistant to OFX and LVX presented the Ala90Val mutation. No mutations were found in the QRDR-B region. The molecular modeling of the interaction between LVX and the DNA-DNA gyrase complex indicates that the loss of an acetyl group in the Asp94Gly mutation removes the acid base interaction with LVX necessary for the quinolone activity. The Ala90Val mutation that substitutes a methyl for an isopropyl group induces a steric modification that blocks the LVX access to the gyrase catalytic site. N. Alvarez, E. Zapata, G. I. Mejía, T. Realpe, P. Araque, C. Peláez, F. Rouzaud, and J. Robledo Copyright © 2014 N. Alvarez et al. All rights reserved. Predicting Mycobacterium tuberculosis Complex Clades Using Knowledge-Based Bayesian Networks Wed, 23 Apr 2014 00:00:00 +0000 We develop a novel approach for incorporating expert rules into Bayesian networks for classification of Mycobacterium tuberculosis complex (MTBC) clades. The proposed knowledge-based Bayesian network (KBBN) treats sets of expert rules as prior distributions on the classes. Unlike prior knowledge-based support vector machine approaches which require rules expressed as polyhedral sets, KBBN directly incorporates the rules without any modification. KBBN uses data to refine rule-based classifiers when the rule set is incomplete or ambiguous. We develop a predictive KBBN model for 69 MTBC clades found in the SITVIT international collection. We validate the approach using two testbeds that model knowledge of the MTBC obtained from two different experts and large DNA fingerprint databases to predict MTBC genetic clades and sublineages. These models represent strains of MTBC using high-throughput biomarkers called spacer oligonucleotide types (spoligotypes), since these are routinely gathered from MTBC isolates of tuberculosis (TB) patients. Results show that incorporating rules into problems can drastically increase classification accuracy if data alone are insufficient. The SITVIT KBBN is publicly available for use on the World Wide Web. Minoo Aminian, David Couvin, Amina Shabbeer, Kane Hadley, Scott Vandenberg, Nalin Rastogi, and Kristin P. Bennett Copyright © 2014 Minoo Aminian et al. All rights reserved. Absence of Cospeciation between the Uncultured Frankia Microsymbionts and the Disjunct Actinorhizal Coriaria Species Tue, 22 Apr 2014 12:25:40 +0000 Coriaria is an actinorhizal plant that forms root nodules in symbiosis with nitrogen-fixing actinobacteria of the genus Frankia. This symbiotic association has drawn interest because of the disjunct geographical distribution of Coriaria in four separate areas of the world and in the context of evolutionary relationships between host plants and their uncultured microsymbionts. The evolution of Frankia-Coriaria symbioses was examined from a phylogenetic viewpoint using multiple genetic markers in both bacteria and host-plant partners. Total DNA extracted from root nodules collected from five species: C. myrtifolia, C. arborea, C. nepalensis, C. japonica, and C. microphylla, growing in the Mediterranean area (Morocco and France), New Zealand, Pakistan, Japan, and Mexico, respectively, was used to amplify glnA gene (glutamine synthetase), dnaA gene (chromosome replication initiator), and the nif DK IGS (intergenic spacer between nifD and nifK genes) in Frankia and the matK gene (chloroplast-encoded maturase K) and the intergenic transcribed spacers (18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA) in Coriaria species. Phylogenetic reconstruction indicated that the radiations of Frankia strains and Coriaria species are not congruent. The lack of cospeciation between the two symbiotic partners may be explained by host shift at high taxonomic rank together with wind dispersal and/or survival in nonhost rhizosphere. Imen Nouioui, Faten Ghodhbane-Gtari, Maria P. Fernandez, Abdellatif Boudabous, Philippe Normand, and Maher Gtari Copyright © 2014 Imen Nouioui et al. All rights reserved. Two-Dimensional PCA Highlights the Differentiated Antitumor and Antimicrobial Activity of Methanolic and Aqueous Extracts of Laurus nobilis L. from Different Origins Wed, 16 Apr 2014 16:06:46 +0000 Natural matrices are important sources of new antitumor and antimicrobial compounds. Species such as Laurus nobilis L. (laurel) might be used for this purpose, considering its medicinal properties. Herein, in vitro activity against human tumor cell lines, bacteria, and fungi was evaluated in enriched phenolic extracts. Specifically, methanol and aqueous extracts of wild and cultivated samples of L. nobilis were compared considering different phenolic groups. Principal component analysis (PCA) was applied to understand how each extract acts differentially against specific bacteria, fungi, and selected human tumor cell lines. In general, the extract type induced the highest differences in bioactivity of laurel samples. However, from the PCA biplot, it became clear that wild laurel samples were higher inhibitors of tumor cell lines (HeLa, MCF7, NCI-H460, and HCT15). HepG2 had the same response to laurel from wild and cultivated origin. It was also observed that methanolic extracts tended to have higher antimicrobial activity, except against A. niger, A. fumigatus, and P. verrucosum. The differences in bioactivity might be related to the higher phenolic contents in methanolic extracts. These results allow selecting the extract type and/or origin with highest antibacterial, antifungal, and antitumor activity. Maria Inês Dias, João C. M. Barreira, Ricardo C. Calhelha, Maria-João R. P. Queiroz, M. Beatriz P. P. Oliveira, Marina Soković, and Isabel C. F. R. Ferreira Copyright © 2014 Maria Inês Dias et al. All rights reserved. Molecular Screening of Virulence Genes in Extraintestinal Pathogenic Escherichia coli Isolated from Human Blood Culture in Brazil Tue, 15 Apr 2014 09:55:01 +0000 Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20 E. coli isolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes, iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC. Vanessa L. Koga, Geizecler Tomazetto, Paula S. Cyoia, Meiriele S. Neves, Marilda C. Vidotto, Gerson Nakazato, and Renata K. T. Kobayashi Copyright © 2014 Vanessa L. Koga et al. All rights reserved. Detection of Food Spoilage and Pathogenic Bacteria Based on Ligation Detection Reaction Coupled to Flow-Through Hybridization on Membranes Thu, 10 Apr 2014 11:02:14 +0000 Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific for Aeromonas spp., Pseudomonas spp., Shewanella spp., and Morganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality. K. Böhme, P. Cremonesi, M. Severgnini, Tomás G. Villa, I. C. Fernández-No, J. Barros-Velázquez, B. Castiglioni, and P. Calo-Mata Copyright © 2014 K. Böhme et al. All rights reserved. Evaluation of Three Formulations of Culture Media for Isolation of Brucella spp. regarding Their Ability to Inhibit the Growth of Contaminating Organisms Thu, 10 Apr 2014 08:16:26 +0000 Three culture media (Brucella agar, Farrell medium, and CITA) were compared for their effectiveness in inhibiting contamination and for isolating Brucella spp. One hundred lymph nodes from pigs (n = 50) and wild boars (n = 50) with lymphadenitis were collected in slaughterhouses in the State of São Paulo and were assessed on these three selective media for Brucella spp. All of the samples were negative for Brucella spp. on the three culture media. On the agar medium, fungal (70 plates) and Gram-positive bacterial (59 plates) contaminants were observed; in the CITA medium, the absence of fungal and Gram-positive bacteria on 15 plates was observed; no bacterial or fungal growth was observed on the Farrell media. The results demonstrated that the CITA and Farrell media inhibited the growth of contaminants better than the Brucella agar. Acácia F. Vicente, João M. A. P. Antunes, Gustavo H. B. Lara, Mateus S. R. Mioni, Susan D. Allendorf, Marina G. Peres, Camila M. Appolinário, Fernando J. P. Listoni, Marcio G. Ribeiro, and Jane Megid Copyright © 2014 Acácia F. Vicente et al. All rights reserved. Molecular Analysis of Ciprofloxacin Resistance Mechanisms in Malaysian ESBL-Producing Klebsiella pneumoniae Isolates and Development of Mismatch Amplification Mutation Assays (MAMA) for Rapid Detection of gyrA and parC Mutations Thu, 10 Apr 2014 08:08:42 +0000 Ninety-three Malaysian extended-spectrum -lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6′)-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs  μg/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone or in both gyrA and parC regions . aac(6′)-Ib-cr was the most common PMQR gene detected in this study , followed by qnrB and qnrS ( and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μg/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6′)-Ib-cr or a single gyrA 83 mutation . Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital. Farah Al-Marzooq, Mohd Yasim Mohd Yusof, and Sun Tee Tay Copyright © 2014 Farah Al-Marzooq et al. All rights reserved. Improving the Diagnosis of Bloodstream Infections: PCR Coupled with Mass Spectrometry Wed, 09 Apr 2014 09:11:26 +0000 The reference method for the diagnosis of bloodstream infections is blood culture followed by biochemical identification and antibiotic susceptibility testing of the isolated pathogen. This process requires 48 to 72 hours. The rapid administration of the most appropriate antimicrobial treatment is crucial for the survival of septic patients; therefore, a rapid method that enables diagnosis directly from analysis of a blood sample without culture is needed. A recently developed platform that couples broad-range PCR amplification of pathogen DNA with electrospray ionization mass spectrometry (PCR/ESI-MS) has the ability to identify virtually any microorganism from direct clinical specimens. To date, two clinical evaluations of the PCR/ESI-MS technology for the diagnosis of bloodstream infections from whole blood have been published. Here we discuss them and describe recent improvements that result in an enhanced sensitivity. Other commercially available assays for the molecular diagnosis of bloodstream infections from whole blood are also reviewed. The use of highly sensitive molecular diagnostic methods in combination with conventional procedures could substantially improve the management of septic patients. Elena Jordana-Lluch, Montserrat Giménez, M. Dolores Quesada, Vicente Ausina, and Elisa Martró Copyright © 2014 Elena Jordana-Lluch et al. All rights reserved. Antibacterial Potential of Northeastern Portugal Wild Plant Extracts and Respective Phenolic Compounds Tue, 08 Apr 2014 08:20:24 +0000 The present work aims to assess the antibacterial potential of phenolic extracts, recovered from plants obtained on the North East of Portugal, and of their phenolic compounds (ellagic, caffeic, and gallic acids, quercetin, kaempferol, and rutin), against bacteria commonly found on skin infections. The disk diffusion and the susceptibility assays were used to identify the most active extracts and phenolic compounds. The effect of selected phenolic compounds on animal cells was assessed by determination of cellular metabolic activity. Gallic acid had a higher activity, against gram-positive (S. epidermidis and S. aureus) and gram-negative bacteria (K. pneumoniae) at lower concentrations, than the other compounds. The caffeic acid, also, showed good antibacterial activity against the 3 bacteria used. The gallic acid was effective against the 3 bacteria without causing harm to the animal cells. Gallic and caffeic acid showed a promising applicability as antibacterial agents for the treatment of infected wounds. Eva Pinho, Isabel C. F. R. Ferreira, Lillian Barros, Ana Maria Carvalho, Graça Soares, and Mariana Henriques Copyright © 2014 Eva Pinho et al. All rights reserved. Genomic and Proteomic Characterization of Bacteriocin-Producing Leuconostoc mesenteroides Strains Isolated from Raw Camel Milk in Two Southwest Algerian Arid Zones Mon, 07 Apr 2014 16:33:57 +0000 Information on the microbiology of camel milk is very limited. In this work, the genetic characterization and proteomic identification of 13 putative producing bacteriocin Leuconostoc strains exhibiting antilisterial activity and isolated from camel milk were performed. DNA sequencing of the 13 selected strains revealed high homology among the 16S rRNA genes for all strains. In addition, 99% homology with Leuconostoc mesenteroides was observed when these sequences were analysed by the BLAST tool against other sequences from reference strains deposited in the Genbank. Furthermore, the isolates were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDITOF MS) which allowed for the identification of 2 mass peaks 6242 m/z and 5118 m/z that resulted to be specific to the species L. mesenteroides. Remarkably, the phyloproteomic tree provided more intraspecific information of L. mesenteroides than phylogenetic analysis. Accordingly, phyloproteomic analysis grouped L. mesenteroides strains into different subbranches, while all L. mesenteroides isolates were grouped in the same branch according to phylogenetic analysis. This study represents, to our knowledge, the first report on the use of MALDI-TOF MS on the identification of LAB isolated from camel milk. Zineb Benmechernene, Inmaculada Fernández-No, Marcos Quintela-Baluja, Karola Böhme, Mebrouk Kihal, Pilar Calo-Mata, and Jorge Barros-Velázquez Copyright © 2014 Zineb Benmechernene et al. All rights reserved. Bacteriological Profile and Drug Resistance Patterns of Blood Culture Isolates in a Tertiary Care Nephrourology Teaching Institute Mon, 07 Apr 2014 00:00:00 +0000 Blood stream infections can lead to life threatening sepsis and require rapid antimicrobial treatment. The organisms implicated in these infections vary with the geographical alteration. Infections caused by MDR organisms are more likely to increase the risk of death in these patients. The present study was aimed to study the profile of organisms causing bacteremia and understand antibiotic resistance patterns in our hospital. 1440 blood samples collected over a year from clinically suspected cases of bacteremia were studied. The isolates were identified by standard biochemical tests and antimicrobial resistance patterns were determined by CLSI guidelines. Positive blood cultures were obtained in 9.2% of cases of which Gram-positive bacteria accounted for 58.3% of cases with staph aureus predominance; gram negative bacteria accounted for 40.2% with enterobactereciea predominence; and 1.5% were fungal isolates. The most sensitive drugs for Gram-positive isolates were vancomycin, teicoplanin, daptomycin, linezolid, and tigecycline and for Gram-negative were carbapenems, colistin, aminoglycosides, and tigecycline. The prevalence of MRSA and vancomycin resistance was 70.6% and 21.6%, respectively. ESBL prevalence was 39.6%. Overall low positive rates of blood culture were observed. Kalpesh Gohel, Amit Jojera, Shailesh Soni, Sishir Gang, Ravindra Sabnis, and Mahesh Desai Copyright © 2014 Kalpesh Gohel et al. All rights reserved. Sequence and Apoptotic Activity of VacA Cytotoxin Cloned from a Helicobacter pylori Thai Clinical Isolate Wed, 02 Apr 2014 12:46:12 +0000 The vacuolating cytotoxin VacA produced by Helicobacter pylori induces the formation of large cytoplasmic vacuoles in host gastric epithelial cells as well as a release of cytochrome C from mitochondria resulting in cell apoptosis. Considerable sequence diversity in VacA relating to different degrees of disease severity is observed with clinical samples from a multitude of geographic places. In this study we describe expression in Escherichia coli, purification to homogeneity and in vitro assay of its apoptotic activity of a VacA toxin from a H. pylori isolate of a Thai patient with gastrointestinal lymphoma. Sequencing revealed that the deduced amino acid sequence of the cloned Thai isolate VacA is similar to H. pylori s1/m2 type strains. The percent sequence similarity to the model strain 60190 was lower due to the presence of extra amino acids in the mid (m) region. The purified VacA toxin exhibited significant apoptotic activity on both T84 and MDCK epithelial cell lines, as revealed by DAPI staining, whereby the observed activity was significantly higher on MDCK cells. These findings could relate to a modulation of VacA activity on host cells in the Thai isolate-VacA toxin that may differ from those of the model strain. Muhammad Junaid, Sarbast Al-Gubare, Muhammad Yousef, Mathukorn Na Ubol, Somphob Leetachewa, Chatchai Muanprasat, Chanan Angsuthanasombat, Wanpen Chaicumpa, Niaz Ali, and Gerd Katzenmeier Copyright © 2014 Muhammad Junaid et al. All rights reserved. Staphylococcus aureus and Staphylococcal Food-Borne Disease: An Ongoing Challenge in Public Health Tue, 01 Apr 2014 16:24:57 +0000 Staphylococcal food-borne disease (SFD) is one of the most common food-borne diseases worldwide resulting from the contamination of food by preformed S. aureus enterotoxins. It is one of the most common causes of reported food-borne diseases in the United States. Although several Staphylococcal enterotoxins (SEs) have been identified, SEA, a highly heat-stable SE, is the most common cause of SFD worldwide. Outbreak investigations have found that improper food handling practices in the retail industry account for the majority of SFD outbreaks. However, several studies have documented prevalence of S. aureus in many food products including raw retail meat indicating that consumers are at potential risk of S. aureus colonization and subsequent infection. Presence of pathogens in food products imposes potential hazard for consumers and causes grave economic loss and loss in human productivity via food-borne disease. Symptoms of SFD include nausea, vomiting, and abdominal cramps with or without diarrhea. Preventive measures include safe food handling and processing practice, maintaining cold chain, adequate cleaning and disinfection of equipment, prevention of cross-contamination in home and kitchen, and prevention of contamination from farm to fork. This paper provides a brief overview of SFD, contributing factors, risk that it imposes to the consumers, current research gaps, and preventive measures. Jhalka Kadariya, Tara C. Smith, and Dipendra Thapaliya Copyright © 2014 Jhalka Kadariya et al. All rights reserved. Biomarkers for Sepsis Sun, 30 Mar 2014 12:25:56 +0000 Bloodstream infections are a major concern because of high levels of antibiotic consumption and of the increasing prevalence of antimicrobial resistance. Bacteraemia is identified in a small percentage of patients with signs and symptoms of sepsis. Biomarkers are widely used in clinical practice and they are useful for monitoring the infectious process. Procalcitonin (PCT) and C-reactive protein (CRP) have been most widely used, but even these have limited abilities to distinguish sepsis from other inflammatory conditions or to predict outcome. PCT has been used to guide empirical antibacterial therapy in patients with respiratory infections and help to determine if antibacterial therapy can be stopped. New biomarkers such as those in this review will discuss the major types of biomarkers of bloodstream infections/sepsis, including soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), soluble urokinase-type plasminogen receptor (suPAR), proadrenomedullin (ProADM), and presepsin. Cesar Henriquez-Camacho and Juan Losa Copyright © 2014 Cesar Henriquez-Camacho and Juan Losa. All rights reserved. Worldwide Dissemination of the NDM-Type Carbapenemases in Gram-Negative Bacteria Wed, 26 Mar 2014 13:36:23 +0000 The emergence of one of the most recently described carbapenemases, namely, the New Delhi metallo-lactamase (NDM-1), constitutes a critical and growingly important medical issue. This resistance trait compromises the efficacy of almost all lactams (except aztreonam), including the last resort carbapenems. Therapeutical options may remain limited mostly to colistin, tigecycline, and fosfomycin. The main known reservoir of NDM producers is the Indian subcontinent whereas a secondary reservoir seems to have established the Balkans regions and the Middle East. Although the spread of -like genes (several variants) is derived mostly by conjugative plasmids in Enterobacteriaceae, this carbapenemase has also been identified in P. aeruginosa and Acinetobacter spp. Acinetobacter sp. may play a pivotal role for spreading genes for its natural reservoir to Enterobacteriaceae. Rapid diagnostic techniques (Carba NP test) and screening of carriers are the cornerstone to try to contain this outbreak which threatens the efficacy of the modern medicine. Laurent Dortet, Laurent Poirel, and Patrice Nordmann Copyright © 2014 Laurent Dortet et al. All rights reserved. Performance of Quantification of Modified Hodge Test: An Evaluation with Klebsiella pneumoniae Carbapenemase-Producing Enterobacteriaceae Isolates Wed, 26 Mar 2014 08:58:43 +0000 Modified Hodge Test (MHT) has been suggested as screening tests for carbapenemases, but concerns regarding its difficult interpretation and common false-positive results obtained in the presence of other β-lactamases have been noted. This study aimed to quantify the enhanced growth formed by the indicator strain and thus evaluate the performance of a quantitative interpretation of MHT for KPC screening. MHT was performed in 50 KPC-producing isolates and 334 non-carbapenemase-producing isolates, using ertapenem (ETP) and meropenem (MEM) as substrates. The size of enhanced growth of indicator strain was measured for each isolate tested and for the positive control used, and a ratio was calculated. Our results revealed 17 different ETP and MEM ratios, with distinct sensitivity (SN) and specificity (SP). Higher SN combined to higher SP was achieved when ETP and MEM ratios were 0.45, with a SN value of 96% for both substrates and SP values of 99.4% and 100% for ETP and MEM, respectively. The quantification with both substrates increased SP of the test for KPC detection. Considering that MHT is the unique phenotypic test that is referred to by CLSI, a more accurate approach for its interpretation could be applied to make it a more useful tool. Vanessa Bley Ribeiro, Adriano Rostirolla Linhares, Alexandre P. Zavascki, and Afonso Luis Barth Copyright © 2014 Vanessa Bley Ribeiro et al. All rights reserved. Potential Synergy Activity of the Novel Ceragenin, CSA-13, against Carbapenem-Resistant Acinetobacter baumannii Strains Isolated from Bacteremia Patients Mon, 24 Mar 2014 16:36:24 +0000 Carbapenem-resistant Acinetobacter baumannii is an important cause of nosocomial infections, particularly in patients in the intensive care units. As chronic infections are difficult to treat, attempts have been made to discover new antimicrobials. Ceragenins, designed to mimic the activities of antimicrobial peptides, are a new class of antimicrobial agents. In this study, the in vitro activities of CSA-13 either alone or in combination with colistin (sulphate), tobramycin, and ciprofloxacin were investigated using 60 carbapenem-resistant A. baumannii strains isolated from bacteremia patients blood specimens. MICs and MBCs were determined by microbroth dilution technique. Combinations were assessed by using checkerboard technique. The MIC50 values (mg/L) of CSA-13, colistin, tobramycin, and ciprofloxacin were 2, 1, 1.25, and 80, respectively. The MIC90 (mg/L) of CSA-13 and colistin were 8 and 4. The MBCs were equal to or twice greater than those of the MICs. Synergistic interactions were mostly seen with CSA-13-colistin (55%), whereas the least synergistic interactions were observed in the CSA-13-tobramycin (35%) combination. No antagonism was observed. CSA-13 appears to be a good candidate for further investigations in the treatment of A. baumannii infections. However, future studies should be performed to correlate the safety, efficacy, and pharmacokinetic parameters of this molecule. Cagla Bozkurt-Guzel, Paul B. Savage, Alper Akcali, and Berna Ozbek-Celik Copyright © 2014 Cagla Bozkurt-Guzel et al. All rights reserved. A Novel Electronic Nose as Adaptable Device to Judge Microbiological Quality and Safety in Foodstuff Mon, 24 Mar 2014 07:37:40 +0000 This paper presents different applications, in various foodstuffs, by a novel electronic nose (EN) based on a mixed metal oxide sensors array composed of thin films as well as nanowires. The electronic nose used for this work has been done, starting from the commercial model EOS835 produced by SACMI Scarl. The SENSOR Lab (CNR-INO, Brescia) has produced both typologies of sensors, classical MOX and the new technologies with nanowire. The aim of this work was to test and to illustrate the broad spectrum of potential uses of the EN technique in food quality control and microbial contamination diagnosis. The EN technique was coupled with classical microbiological and chemical techniques, like gas chromatography with mass spectroscopy (GC-MS) with SPME technique. Three different scenarios are presented: (a) detection of indigenous mould in green coffee beans, (b) selection of microbiological spoilage of Lactic Acid Bacteria (LAB), and (c) monitoring of potable water. In each case, the novel EN was able to identify the spoiled product by means of the alterations in the pattern of volatile organic compounds (VOCs), reconstructed by principal component analysis (PCA) of the sensor responses. The achieved results strongly encourage the use of EN in industrial laboratories. Finally, recent trends and future directions are illustrated. V. Sberveglieri, E. Nunez Carmona, Elisabetta Comini, Andrea Ponzoni, Dario Zappa, Onofrio Pirrotta, and A. Pulvirenti Copyright © 2014 V. Sberveglieri et al. All rights reserved. Aetiology of Bacteraemia as a Risk Factor for Septic Shock at the Onset of Febrile Neutropaenia in Adult Cancer Patients Thu, 20 Mar 2014 00:00:00 +0000 Septic shock (SS) at the onset of febrile neutropaenia (FN) is an emergency situation that is associated with high morbidity and mortality. The impact of the specific aetiology of bloodstream infections (BSIs) in the development of SS at the time of FN is not well established. The aim of this study was to evaluate the association between the aetiology of BSIs and SS at the time of FN in hospitalised adult cancer patients. This prospective cohort study was performed at a single tertiary hospital from October 2009 to August 2011. All adult cancer patients admitted consecutively to the haematology ward with FN were evaluated. A stepwise logistic regression was conducted to verify the association between the microbiological characteristics of BSIs and SS at the onset of FN. In total, 307 cases of FN in adult cancer patients were evaluated. There were 115 cases with documented BSI. A multivariate analysis showed that polymicrobial bacteraemia () was associated with SS. The specific blood isolates independently associated with SS were viridans streptococci () and Escherichia coli (). Neutropaenic cancer patients with polymicrobial bacteraemia or BSI by viridans streptococci or Escherichia coli are at increased risk for SS at the time of FN. Regis Goulart Rosa and Luciano Zubaran Goldani Copyright © 2014 Regis Goulart Rosa and Luciano Zubaran Goldani. All rights reserved. Biomarkers for Sepsis: A Review with Special Attention to India Wed, 19 Mar 2014 08:01:55 +0000 Sepsis is a serious infection and still a common cause of morbidity and mortality in resource-limited settings such as India. Even when microbiologic diagnostics are available, bacteremia is only identified in a proportion of patients who present with sepsis and bloodstream infections. Biomarkers have been used in a variety of disease processes and can help aid in diagnosing bacterial infections. There have been numerous biomarkers investigated to aid with diagnosis and prognostication in sepsis with the majority suffering from lack of sensitivity or specificity. Procalcitonin has been heralded as the biomarker that holds the most promise for bloodstream infections. Data are emerging in India, and in this review, we focus on the current data of biomarkers in sepsis with particular attention to how biomarkers could be used to augment diagnosis and treatment in India. George E. Nelson, Vidya Mave, and Amita Gupta Copyright © 2014 George E. Nelson et al. All rights reserved. Experimental Inoculation of BFDV-Positive Budgerigars (Melopsittacus undulatus) with Two Mycobacterium avium subsp. avium Isolates Thu, 13 Mar 2014 16:07:17 +0000 Beak and feather disease virus- (BFDV-) positive (naturally infected) but clinically healthy budgerigars (Melopsittacus undulatus) were inoculated with two isolates of Mycobacterium avium subsp. avium isolated from naturally infected golden pheasant (Chrysolophus pictus) and peafowl (Pavo cristatus). During a period of more than two months after inoculation, samples of cloacal and crop swabs, faeces, and blood were obtained for BFDV and Mycobacterium avium testing with PCR. Birds were euthanized nine weeks after inoculation. All infected budgerigars developed signs typical of mycobacteriosis, but more advanced clinical and pathological changes were visible in the group infected with the pheasant isolate. Only a few cloacal and crop swab samples were positive for Mycobacterium avium subsp. avium despite advanced pathological changes in the internal organs. In the groups infected with mycobacterium isolates the frequency of BFDV-positive samples was higher than in the control group. In the infected groups the frequency of BFDV was substantially higher in the cloacal swabs of birds inoculated with the pheasant isolate than in the peafowl-isolate-infected group. Aleksandra Ledwoń, Rafał Sapierzyński, Ewa Augustynowicz-Kopeć, Piotr Szeleszczuk, and Marcin Kozak Copyright © 2014 Aleksandra Ledwoń et al. All rights reserved. A Systematic Follow-Up of Mycobacterium tuberculosis Drug-Resistance and Associated Genotypic Lineages in the French Departments of the Americas over a Seventeen-Year Period Thu, 13 Mar 2014 13:52:08 +0000 The population of the French Departments of the Americas (FDA) is highly influenced by the intense migratory flows with mainland France and surrounding countries of the Caribbean and Latin America, some of which have high incidence rates of tuberculosis (Haiti: 230/100,000; Guyana: 111/100,000; and Suriname: 145/100,000) and drug resistance. Since the development of drug resistance to conventional antituberculous drugs has a major impact on the treatment success of tuberculosis, we therefore decided to review carefully Mycobacterium tuberculosis drug resistance and associated genotypic lineages in the FDA over a seventeen-year period (January 1995–December 2011). A total of 1239 cases were studied, including 153 drug-resistant and 26 multidrug-resistant- (MDR-) TB cases, representing 12.3% and 2.1% of the TB cases in our study setting. A significantly higher proportion of M. tuberculosis isolates among relapse cases showed drug resistance to isoniazid (22.5%, ), rifampicin (20.0%, ), or both (MDR-TB, 17.5%; ). Determination of spoligotyping based phylogenetic clades showed that among the five major lineages observed—T family (30.1%); Latin-American and Mediterranean (LAM, 23.7%); Haarlem (H, 22.2%); East-African Indian (EAI, 7.2%); and X family (6.5%)—two lineages, X and LAM, were overrepresented in drug-resistant and MDR-TB cases, respectively. Finally, 19 predominant spoligotypes were identified for the 1239 isolates of M. tuberculosis in our study among which 4 were significantly associated with drug resistance corresponding to SIT20/LAM1, SIT64/LAM6, SIT45/H1, and SIT46/undefined lineage. Julie Millet, Elisabeth Streit, Mylène Berchel, Anne-Gaël Bomer, Franziska Schuster, Delaina Paasch, Jessica Vanhomwegen, Gilbert Cadelis, and Nalin Rastogi Copyright © 2014 Julie Millet et al. All rights reserved. A First Assessment of Mycobacterium tuberculosis Genetic Diversity and Drug-Resistance Patterns in Twelve Caribbean Territories Mon, 10 Mar 2014 14:19:26 +0000 With the exception of some French-speaking islands, data on tuberculosis (TB) in the Caribbean are scarce. In this study, we report a first assessment of genetic diversity of a convenience sample of Mycobacterium tuberculosis strains received from twelve Caribbean territories by spoligotyping and describe their drug-resistance patterns. Of the 480 isolates, 40 (8.3%) isolates showed resistance to at least one anti-TB drug. The proportion of drug-resistant strains was significantly higher in The Bahamas (21.4%; ), and Guyana (27.5%; ), while it was significantly lower in Jamaica (2.4%; ) than in other countries of the present study. Regarding genetic diversity, 104 distinct spoligotype patterns were observed: 49 corresponded to clustered strains (2 to 93 strains per cluster), while 55 remained unclustered among which 16 patterns were not reported previously. Combining the study results with regional data retrieved from the international SITVIT2 database underlined a connection between frequency of certain M. tuberculosis phylogenetic lineages and the language spoken, suggesting historical (colonial) and ongoing links (trade, tourism, and migratory flows) with European countries with which they shared a common past. Julie Millet, Shirematee Baboolal, Elisabeth Streit, Patrick E. Akpaka, and Nalin Rastogi Copyright © 2014 Julie Millet et al. All rights reserved. Strain Diversity of Mycobacterium tuberculosis Isolates from Pulmonary Tuberculosis Patients in Afar Pastoral Region of Ethiopia Thu, 06 Mar 2014 13:24:29 +0000 Data on genotypic diversity of Mycobacterium tuberculosis complex (MTBC) is important to understand its epidemiology, human adaptation, clinical phenotypes, and drug resistance. This study aimed to characterize MTBC clinical isolates circulating in a predominantly pastoralist area in Ethiopia, a country where tuberculosis is the second leading cause of mortality. Culture of sputum samples collected from a total of 325 pulmonary TB suspects was done to isolate MTBC. Spoligotyping was used to characterize 105 isolates from culture positive slopes and the result was compared with an international database. Forty-four spoligotype patterns were observed to correspond to 35 shared-types (SITs) containing 96 isolates and 9 orphan patterns; 27 SITs containing 83 isolates matched a preexisting shared-type in the database, whereas 8 SITs ( isolates) were newly created. A total of 19 SITs containing 80 isolates were clustered within this study (overall clustering of 76.19%). Three dominant lineages (T, CAS, and Manu) accounted for 76.19% of the isolates. SIT149/T3-ETH was one of the two most dominant sublineages. Unlike previous reports, we show that Manu lineage strains not only constitute a dominant lineage, but are also associated with HIV infection in Afar region of Ethiopia. The high level of clustering suggests the presence of recent transmission that should be further studied using additional genotyping markers. Mulugeta Belay, Gobena Ameni, Gunnar Bjune, David Couvin, Nalin Rastogi, and Fekadu Abebe Copyright © 2014 Mulugeta Belay et al. All rights reserved. Effects of Growth Phase and Temperature on Activity within a Listeria monocytogenes Population: Evidence for RsbV-Independent Activation of at Refrigeration Temperatures Wed, 05 Mar 2014 12:22:23 +0000 The alternative sigma factor of Listeria monocytogenes is responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation of within populations of L. monocytogenes EGD-e wild-type, ΔsigB, and ΔrsbV throughout growth at both 4°C and 37°C, using a reporter fusion that couples expression of EGFP to the strongly -dependent promoter of lmo2230. A similar activation pattern within the population was observed in wt-egfp at both temperatures, with the highest induction of occurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction of activity was heterogeneous, with only a proportion of the cells in the wt-egfp population being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation of was observed during growth at 4°C. This result suggests that an alternative route to activation exists in the absence of RsbV, a finding that is not explained by the current model for regulation. Marta Utratna, Eoin Cosgrave, Claas Baustian, Rhodri H. Ceredig, and Conor P. O’Byrne Copyright © 2014 Marta Utratna et al. All rights reserved. Vancomycin-Resistant Enterococcus faecium Bacteremia in a Tertiary Care Hospital: Epidemiology, Antimicrobial Susceptibility, and Outcome Wed, 05 Mar 2014 08:38:55 +0000 Vancomycin-resistant Enterococcus faecium (VREF) has emerged as a relevant multidrug-resistant pathogen and potentially lethal etiology of health care associated infections worldwide. The objective of this retrospective cohort study was to assess factors associated with mortality in patients with VREF bacteremia in a major tertiary referral hospital in Southern Brazil. All documented cases of bacteremia identified between May 2010 and July 2012 were evaluated. Cox regression was performed to determine whether the characteristics related to the host or antimicrobial treatment were associated with the all-cause 30-day mortality. In total, 35 patients with documented VREF bacteremia were identified during the study period. The median APACHE-II score of the study population was 26 (interquartile range: 10). The overall 30-day mortality was 65.7%. All VREF isolates were sensitive to linezolid, daptomycin, and quinupristin-dalfopristin. Linezolid was the only antimicrobial agent with in vitro activity against VREF that was administered to the cohort. After multivariate analysis, linezolid treatment (HR, 0.08; 95% CI, 0.02–0.27) and presence of acute kidney injury at the onset of bacteremia (HR, 4.01; 95% CI, 1.62–9.94) were independently associated with mortality. Presentation with acute kidney injury and lack of treatment with an effective antibiotic poses risk for mortality in patients with VREF bacteremia. Regis G. Rosa, Alexandre V. Schwarzbold, Rodrigo P. dos Santos, Eduardo E. Turra, Denise P. Machado, and Luciano Z. Goldani Copyright © 2014 Regis G. Rosa et al. All rights reserved. Efficacy of Three Light Technologies for Reducing Microbial Populations in Liquid Suspensions Tue, 04 Mar 2014 08:25:39 +0000 The aim of the current study was to evaluate the effectiveness of three nonthermal light technologies (NUV-Vis, continuous UV, and HILP) on their ability to inactivate Escherichia coli K12 and Listeria innocua.  E. coli K12 was selected as a representative microorganism for the enterohaemorrhagic foodborne pathogen E. coli O157:H7 and L. innocua as a surrogate microorganism for the common foodborne pathogen Listeria monocytogenes, respectively. The liquid matrix used for the disinfection experiments was a liquid matrix (MRD solution). The results of the present study show that the HILP treatment inactivated both E. coli and L. innocua more rapidly and effectively than either continuous UV-C or NUV-vis treatment. With HILP at 2.5 cm from the lamp, E. coli and L. innocua populations were reduced by 3.07 and 3.77 log10 CFU/mL, respectively, after a 5 sec treatment time, and were shown to be below the limit of detection (<0.22 log10 CFU/mL) following 30 sec exposure to HILP (106.2 J/cm2). These studies demonstrate the bactericidal efficacy of alternative nonthermal light technologies and their potential as decontamination strategies in the food industry. Angeliki Birmpa, Apostolos Vantarakis, Spyros Paparrodopoulos, Paul Whyte, and James Lyng Copyright © 2014 Angeliki Birmpa et al. All rights reserved. Detection of Carbapenemase-Producing Enterobacteriaceae in the Baltic Countries and St. Petersburg Area Tue, 04 Mar 2014 00:00:00 +0000 The spread of carbapenemase-producing Enterobacteriaceae is a global problem; however, no exact data on the epidemiology of carbapenemase in the Baltic countries and St. Petersburg area is available. We aimed to evaluate the epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Baltic States and St. Petersburg, Russia, and to compare the different methods for carbapenemase detection. From January to May 2012, all K. pneumoniae   and E. coli   clinical isolates from 20 institutions in Estonia, Latvia, Lithuania, and St. Petersburg, Russia were screened for carbapenem susceptibility. The IMP, VIM, GIM, NDM, KPC, and OXA-48 genes were detected using real-time PCR and the ability to hydrolyze ertapenem was determined using MALDI-TOF MS. Seventy-seven strains were found to be carbapenem nonsusceptible. From these, 15 K. pneumoniae strains hydrolyzed ertapenem and carried the gene. All of these strains carried integron 1 and most carried integron 3 as well as genes of the CTX-M-1 group. No carbapenemase-producing E. coli or K. pneumoniae strains were found in Estonia, Latvia, or Lithuania; however, NDM-positive K. pneumoniae was present in the hospital in St. Petersburg, Russia. A MALDI-TOF MS-based assay is a suitable and cost-effective method for the initial confirmation of carbapenemase production. Anastasia Pavelkovich, Arta Balode, Petra Edquist, Svetlana Egorova, Marina Ivanova, Lidia Kaftyreva, Irina Konovalenko, Siiri Kõljalg, Jana Lillo, Lidia Lipskaya, Jolanta Miciuleviciene, Kristiine Pai, Kristel Parv, Katri Pärna, Tiiu Rööp, Epp Sepp, Jelena Štšepetova, and Paul Naaber Copyright © 2014 Anastasia Pavelkovich et al. All rights reserved. Suitability of IS6110-RFLP and MIRU-VNTR for Differentiating Spoligotyped Drug-Resistant Mycobacterium tuberculosis Clinical Isolates from Sichuan in China Mon, 03 Mar 2014 13:36:46 +0000 Genotypes of Mycobacterium tuberculosis complex (MTBC) vary with the geographic origin of the patients and can affect tuberculosis (TB) transmission. This study was aimed to further differentiate spoligotype-defined clusters of drug-resistant MTBC clinical isolates split in Beijing () versus non-Beijing isolates () from Sichuan region, the second high-burden province in China, by IS6110-restriction fragment length polymorphism (RFLP) and 24-locus MIRU-VNTRs. Among 274 spoligotyped isolates, the clustering ratio of Beijing family was 5.3% by 24-locus MIRU-VNTRs versus 2.1% by IS6110-RFLP, while none of the non-Beijing isolates were clustered by 24-locus MIRU-VNTRs versus 9.5% by IS6110-RFLP. Hence, neither the 24-locus MIRU-VNTR was sufficient enough to fully discriminate the Beijing family, nor the IS6110-RFLP for the non-Beijing isolates. A region adjusted scheme combining 12 highly discriminatory VNTR loci with IS6110-RFLP was a better alternative for typing Beijing strains in Sichuan than 24-locus MIRU-VNTRs alone. IS6110-RFLP was for the first time introduced to systematically genotype MTBC in Sichuan and we conclude that the region-adjusted scheme of 12 highly discriminative VNTRs might be a suitable alternative to 24-locus MIRU-VNTR scheme for non-Beijing strains, while the clusters of the Beijing isolates should be further subtyped using IS6110-RFLP for optimal discrimination. Chao Zheng, Yuding Zhao, Guoqiang Zhu, Song Li, Honghu Sun, Qin Feng, Mei Luo, Fanzi Wu, Xuefeng Li, Véronique Hill, Nalin Rastogi, and Qun Sun Copyright © 2014 Chao Zheng et al. All rights reserved. Molybdenum Reduction to Molybdenum Blue in Serratia sp. Strain DRY5 Is Catalyzed by a Novel Molybdenum-Reducing Enzyme Mon, 03 Mar 2014 12:20:05 +0000 The first purification of the Mo-reducing enzyme from Serratia sp. strain DRY5 that is responsible for molybdenum reduction to molybdenum blue in the bacterium is reported. The monomeric enzyme has an apparent molecular weight of 105 kDalton. The isoelectric point of this enzyme was 7.55. The enzyme has an optimum pH of 6.0 and maximum activity between 25 and 35°C. The Mo-reducing enzyme was extremely sensitive to temperatures above 50°C (between 54 and 70°C). A plot of initial rates against substrate concentrations at 15 mM 12-MP registered a for NADH at 12.0 nmole Mo blue/min/mg protein. The apparent for NADH was 0.79 mM. At 5 mM NADH, the apparent and apparent values for 12-MP of 12.05 nmole/min/mg protein and 3.87 mM, respectively, were obtained. The catalytic efficiency (/) of the Mo-reducing enzyme was 5.47 . The purification of this enzyme could probably help to solve the phenomenon of molybdenum reduction to molybdenum blue first reported in 1896 and would be useful for the understanding of the underlying mechanism in molybdenum bioremediation involving bioreduction. M. Y. Shukor, M. I. E. Halmi, M. F. A. Rahman, N. A. Shamaan, and M. A. Syed Copyright © 2014 M. Y. Shukor et al. All rights reserved. Evidence of Leishmania infantum Infection in Rabbits (Oryctolagus cuniculus) in a Natural Area in Madrid, Spain Mon, 03 Mar 2014 11:37:40 +0000 Leishmaniasis is one of the most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area. Nerea García, Inmaculada Moreno, Julio Alvarez, María Luisa de la Cruz, Alejandro Navarro, Marta Pérez-Sancho, Teresa García-Seco, Antonio Rodríguez-Bertos, María Luisa Conty, Alfredo Toraño, Antonio Prieto, Lucas Domínguez, and Mercedes Domínguez Copyright © 2014 Nerea García et al. All rights reserved. Molecular Epidemiology and Genotyping of Mycobacterium tuberculosis Isolated in Baghdad Wed, 26 Feb 2014 12:13:52 +0000 Tuberculosis (TB) remains a major health problem in Iraq but the strains responsible for the epidemic have been poorly characterized. Our aim was to characterize the TB strains circulating in Bagdad (Iraq). A total of 270 Mycobacterium tuberculosis complex (MTBC) strains isolated between 2010 and 2011 from TB patients attending the Center of Chest and Respiratory diseases in Baghdad were analyzed by Spoligotyping. The analysis indicated that 94.1% of the isolates belong to known genotype clades: CAS 39.6%, ill-defined T clade 29.6%, Manu 7.4%, Haarlem 7%, Ural 4.1%, LAM 3.3%, X 0.7%, LAM7-TUR 0.7%, EAI 0.7%, S 0.7%, and unknown 5.9%. Comparison with the international multimarker database SITVIT2 showed that SIT 309 (CAS1-Delhi) and SIT1144 (T1) were the most common types. In addition, 44 strains were included in SITVIT2 database under 16 new Spoligotype International Types (SITs); of these, 6 SITs (SIT3346, SIT3497, SIT3708, SIT3790, SIT3791, and SIT3800) (n = 32 strains) were created within the present study and 10 were created after a match with an orphan in the database. By using 24-loci MIRU-VNTR-typing on a subset of 110 samples we found a high recent transmission index (RTI) of 33.6%. In conclusion, we present the first unifying framework for both epidemiology and evolutionary analysis of M. tuberculosis in Iraq. Ruqaya Mustafa Ali, Alberto Trovato, David Couvin, Amina N. Al-Thwani, Emanuele Borroni, Fahim H. Dhaer, Nalin Rastogi, and Daniela M. Cirillo Copyright © 2014 Ruqaya Mustafa Ali et al. All rights reserved. Carbapenemase Genes among Multidrug Resistant Gram Negative Clinical Isolates from a Tertiary Hospital in Mwanza, Tanzania Mon, 24 Feb 2014 07:53:30 +0000 The burden of antimicrobial resistance (AMR) is rapidly growing across antibiotic classes, with increased detection of isolates resistant to carbapenems. Data on the prevalence of carbapenem resistance in developing countries is limited; therefore, in this study, we determined the prevalence of carbapenemase genes among multidrug resistant gram negative bacteria (MDR-GNB) isolated from clinical specimens in a tertiary hospital in Mwanza, Tanzania. A total of 227 MDR-GNB isolates were analyzed for carbapenem resistance genes. For each isolate, five different PCR assays were performed, allowing for the detection of the major carbapenemase genes, including those encoding the VIM-, IMP-, and NDM-type metallo-beta-lactamases, the class A KPC-type carbapenemases, and the class D OXA-48 enzyme. Of 227 isolates, 80 (35%) were positive for one or more carbapenemase gene. IMP-types were the most predominant gene followed by VIM, in 49 (21.59%) and 28 (12%) isolates, respectively. Carbapenemase genes were most detected in K. pneumoniae 24 (11%), followed by P. aeruginosa 23 (10%), and E. coli with 19 isolates (8%). We have demonstrated for the first time a high prevalence of MDR-GNB clinical isolates having carbapenem resistance genes in Tanzania. We recommend routine testing for carbapenem resistance among the MDR-GNB particularly in systemic infections. Martha F. Mushi, Stephen E. Mshana, Can Imirzalioglu, and Freddie Bwanga Copyright © 2014 Martha F. Mushi et al. All rights reserved. Antimicrobial Resistance Pattern and Their Beta-Lactamase Encoding Genes among Pseudomonas aeruginosa Strains Isolated from Cancer Patients Sun, 23 Feb 2014 09:09:01 +0000 This study was designed to investigate the prevalence of metallo-β-lactamases (MBL) and extended-spectrum β-lactamases (ESBL) in P. aeruginosa isolates collected from two different hospitals in Cairo, Egypt. Antibiotic susceptibility testing and phenotypic screening for ESBLs and MBLs were performed on 122 P. aeruginosa isolates collected in the period from January 2011 to March 2012. MICs were determined. ESBLs and MBLs genes were sought by PCR. The resistant rate to imipenem was 39.34%. The resistance rates for P. aeruginosa to cefuroxime, cefoperazone, ceftazidime, aztreonam, and piperacillin/tazobactam were 87.7%, 80.3%, 60.6%, 45.1%, and 25.4%, respectively. Out of 122 P. aeruginosa, 27% and 7.4% were MBL and ESBL, respectively. The prevalence of blaVIM-2, blaOXA-10-, blaVEB-1, blaNDM-, and blaIMP-1-like genes were found in 58.3%, 41.7%, 10.4%, 4.2%, and 2.1%, respectively. GIM-, SPM-, SIM-, and OXA-2-like genes were not detected in this study. OXA-10-like gene was concomitant with VIM-2 and/or VEB. Twelve isolates harbored both OXA-10 and VIM-2; two isolates carried both OXA-10 and VEB. Only one strain contained OXA-10, VIM-2, and VEB. In conclusion, blaVIM-2- and blaOXA-10-like genes were the most prevalent genes in P. aeruginosa in Egypt. To our knowledge, this is the first report of blaVIM-2, blaIMP-1, blaNDM, and blaOXA-10 in P. aeruginosa in Egypt. Mai M. Zafer, Mohamed H. Al-Agamy, Hadir A. El-Mahallawy, Magdy A. Amin, and Mohammed Seif El-Din Ashour Copyright © 2014 Mai M. Zafer et al. All rights reserved. Molecular Approaches for the Classification of Microbial Pathogens of Public Health Significance Tue, 18 Feb 2014 12:29:35 +0000 Hiroshi Asakura, Holger Brueggemann, Sou-ichi Makino, and Yoshiko Sugita-Konishi Copyright © 2014 Hiroshi Asakura et al. All rights reserved. Physical, Chemical, and Biological Methods for the Removal of Arsenic Compounds Mon, 17 Feb 2014 09:51:46 +0000 Arsenic is a toxic metalloid which is widely distributed in nature. It is normally present as arsenate under oxic conditions while arsenite is predominant under reducing condition. The major discharges of arsenic in the environment are mainly due to natural sources such as aquifers and anthropogenic sources. It is known that arsenite salts are more toxic than arsenate as it binds with vicinal thiols in pyruvate dehydrogenase while arsenate inhibits the oxidative phosphorylation process. The common mechanisms for arsenic detoxification are uptaken by phosphate transporters, aquaglyceroporins, and active extrusion system and reduced by arsenate reductases via dissimilatory reduction mechanism. Some species of autotrophic and heterotrophic microorganisms use arsenic oxyanions for their regeneration of energy. Certain species of microorganisms are able to use arsenate as their nutrient in respiratory process. Detoxification operons are a common form of arsenic resistance in microorganisms. Hence, the use of bioremediation could be an effective and economic way to reduce this pollutant from the environment. K. T. Lim, M. Y. Shukor, and H. Wasoh Copyright © 2014 K. T. Lim et al. All rights reserved. Comparison of Ligation-Mediated PCR Methods in Differentiation of Mycobacterium tuberculosis Strains Sun, 16 Feb 2014 14:35:15 +0000 Fast and inexpensive identification of epidemiological links between limited number of Mycobacterium tuberculosis strains is required to initially evaluate hospital outbreaks, laboratory crosscontaminations, and family or small community transmissions. The ligation-mediated PCR methods (LM-PCR) appear sufficiently discriminative and reproducible to be considered as a good candidate for such initial, epidemiological analysis. Here, we compared the discriminative power of the recently developed in our laboratory fast ligation amplification polymorphism (FLAP) method with fast ligation-mediated PCR (FLiP). Verification of the results was based on analyzing a set of reference strains and RFLP-IS6110 typing. The HGDI value was very similar for both LM-PCR methods and RFLP-IS6110 typing. However, only 52% of strains were correspondingly grouped by both FLiP and FLAP methods. Differentiation by FLAP method demonstrated a limited similarity to IS6110-RFLP (37,7%). As much as 78,7% of strains were grouped identically when differentiated by FLiP and IS6110-RFLP methods. The analysis differentiated 31, 35, and 36 groups when using FLAP, FLiP, and RFLP-IS6110 methods, respectively. Anna Zaczek, Anna Brzostek, Arkadiusz Wojtasik, Anna Sajduda, and Jaroslaw Dziadek Copyright © 2014 Anna Zaczek et al. All rights reserved. Mycobacterium avium Subsp. avium Infection in Four Veal Calves: Differentiation from Intestinal Tuberculosis Thu, 13 Feb 2014 11:15:23 +0000 Mycobacterium avium subsp. avium (Maa) is an intracellular pathogen belonging to the Mycobacterium avium-intracellulare complex (MAC). Reservoirs of MAC are the natural environment, wildlife and domestic animals. In adult bovine, MAC infections are typically caused by Mycobacterium avium subsp. paratuberculosis (Map). Maa infections in bovine are rarely reported but may cause clinical disease and pathological lesions similar to those observed in paratuberculosis or those induced by members of the Mycobacterium tuberculosis complex (MTBC). Therefore, differentiation of MAC from MTBC infection should be attempted, especially if unusual mycobacterial lesions are encountered. Four veal calves from a fattening farm dying with clinical signs of otitis media, fever, and weight loss were submitted for necropsy. Samples from affected organs were taken for histologic investigation, bacteriologic culture, and bacterial specification using PCR. Macroscopic thickening of the intestinal mucosa was induced by granulomatous enteritis and colitis. Intracytoplasmic acid-fast bacteria were detected by Ziehl-Neelsen stains and PCR revealed positive results for Mycobacterium avium subsp. avium. Clinical and pathological changes of Maa infection in veal calves had features of Mycobacterium avium subsp. paratuberculosis and the MTBC. Therefore, Mycobacterium tuberculosis complex infection should be considered in cases of granulomatous enteritis in calves. Christine Goepfert, Nadine Regenscheit, Vanessa Schumacher, Simone Roos, Christophe Rossier, Corinne Baehler, Sarah Schmitt, and Horst Posthaus Copyright © 2014 Christine Goepfert et al. All rights reserved. Effect of Plasma Processing and Organosilane Modifications of Polyethylene on Aeromonas hydrophila Biofilm Formation Thu, 30 Jan 2014 13:11:42 +0000 The aim of our research was to study how the modifications of polyethylene—a material commonly used in medicine and water industry—influence bacterial cell attachment and biofilm formation. The native surface was activated and modified using two-step process consisting in the activation of native surface with a H2O vapor plasma followed by its treatment with various organosilanes, namely, [3(tertbutylamine-2hydroxy) propyloxypropyl] diethoxymethylsilane, 1H,1H,2H,2H-perfluorooctylmethyldimethoxysilane, dimethoxydimethylsilane, and isobutylmethyldimethoxysilane. The effect of polyethylene modification after chemical treatment was analyzed using surface tension measurement. The adhesive properties of Aeromonas hydrophila LOCK0968 were studied in water with a low concentration of organic compounds, using luminometric and microscopic methods, and the viability of the adhered bacterial cells was evaluated using the colony forming units method. After two-week incubation the chemically modified materials exhibited better antiadhesive and antibacterial characteristics in comparison to the native surface. Among the examined modifying agents, dimethoxydimethylsilane showed the best desired properties. Dorota Kregiel and Kamila Niedzielska Copyright © 2014 Dorota Kregiel and Kamila Niedzielska. All rights reserved. Molecular Approach for Tracing Dissemination Routes of Shiga Toxin-Producing Escherichia coli O157 in Bovine Offal at Slaughter Thu, 30 Jan 2014 00:00:00 +0000 Bovine offal is currently recognized as one of the sources of human Shiga toxin-producing Escherichia coli (STEC) infection in Japan. Here, the prevalence and genetic characterization of STEC O157 in bovine feces, offal, and carcasses at slaughtering were examined between July and October in 2006. STEC O157 was detected in 31 of 301 cattle feces (10.3%) delivered from 120 farms. Simultaneously, 60 bovine-originated offal (tongue, liver, and omasum) and carcasses were randomly selected and the detection of O157 STEC was examined as well. STEC O157 was isolated from 4 tongues (6.7%), 1 liver (1.7%), 3 omasa (5.0%), and 2 carcasses (3.3%), respectively. All the O157 isolates were positive for eae and hlyA genes, and 37 of 41 isolates (90.2%) exhibited stx2c genotype. PFGE analysis revealed the identical macrogenotypes of 4-tongue- and 1-liver-originated isolates and among 2 fecal isolates from animals slaughtered consecutively. Considering their continuous detection according to the slaughtering order, we concluded that these distributions of O157 in bovine offal and feces might be due to cross-contamination at (pre)slaughter. Our data thus reposes implication of better sanitary control in diapedesis from both upper and lower sites to prevent spread of this pathogen to bovine offal at slaughtering. Hiroshi Asakura, Kazuya Masuda, Shigeki Yamamoto, and Shizunobu Igimi Copyright © 2014 Hiroshi Asakura et al. All rights reserved. Assessment of the BD MGIT TBc Identification Test for the Detection of Mycobacterium tuberculosis Complex in a Network of Mycobacteriology Laboratories Thu, 23 Jan 2014 12:42:07 +0000 We evaluate the performance of the TBcID assay in a panel of 100 acid-fast bacilli cultures. Sixty-four isolates were TBcID positive for Mycobacterium tuberculosis complex (MTBC), whereas 36 gave negative results. These included 28 nontuberculous mycobacteria, one nonmycobacterial isolate, one M. tuberculosis, and six M. bovis BCG strains. This corresponds to a sensitivity of 90.14%, specificity of 100%, and positive and negative predictive values of 100% and 80.55%, respectively. The test is rapid, easy to perform and interpret, and does not require sample preparation or instrumentation. However, a negative result does not exclude the presence of a strain belonging to MTBC, especially when mutations in mpb64 gene are present or some M. bovis BCG strains are isolated. The TBcID showed potential to assist in the identification of MTBC when the implementation and usage of molecular methods are often not possible, principally in resource-limited countries. Diana Machado, Jorge Ramos, Isabel Couto, Nureisha Cadir, Inácio Narciso, Elizabeth Coelho, Sofia Viegas, and Miguel Viveiros Copyright © 2014 Diana Machado et al. All rights reserved. Assessment of Mycobacterium bovis Deleted in p27-p55 Virulence Operon as Candidate Vaccine against Tuberculosis in Animal Models Tue, 21 Jan 2014 08:17:50 +0000 A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFN, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response. María V. Bianco, Simon Clark, Federico C. Blanco, Sergio Garbaccio, Elizabeth García, Angel A. Cataldi, Ann Williams, and Fabiana Bigi Copyright © 2014 María V. Bianco et al. All rights reserved. Antimicrobial Activity of Artemisinin and Precursor Derived from In Vitro Plantlets of Artemisia annua L. Sun, 19 Jan 2014 12:20:51 +0000 Artemisia annua L., a medicinal herb, produces secondary metabolites with antimicrobial property. In Malaysia due to the tropical hot climate, A. annua could not be planted for production of artemisinin, the main bioactive compound. In this study, the leaves of three in vitro A. annua L. clones were, extracted and two bioactive compounds, artemisinin and a precursor, were isolated by thin layer chromatography. These compounds were found to be effective in inhibiting the growth of Gram-positive and Gram-negative bacteria but not Candida albicans. Their antimicrobial activity was similar to that of antibactericidal antibiotic streptomycin. They were found to inhibit the growth of the tested microbes at the minimum inhibition concentration of 0.09 mg/mL, and toxicity test using brine shrimp showed that even the low concentration of 0.09 mg/mL was very lethal towards the brine shrimps with 100% mortality rate. This study hence indicated that in vitro cultured plantlets of A. annua can be used as the alternative method for production of artemisinin and its precursor with antimicrobial activities. Suganthi Appalasamy, Kiah Yann Lo, Song Jin Ch'ng, Ku Nornadia, Ahmad Sofiman Othman, and Lai-Keng Chan Copyright © 2014 Suganthi Appalasamy et al. All rights reserved. Optimization of Milk-Based Medium for Efficient Cultivation of Bifidobacterium pseudocatenulatum G4 Using Face-Centered Central Composite-Response Surface Methodology Sun, 12 Jan 2014 12:11:17 +0000 This study was undertaken to optimize skim milk and yeast extract concentration as a cultivation medium for optimal Bifidobacteria pseudocatenulatum G4 (G4) biomass and β-galactosidase production as well as lactose and free amino nitrogen (FAN) balance after cultivation period. Optimization process in this study involved four steps: screening for significant factors using 23 full factorial design, steepest ascent, optimization using FCCD-RSM, and verification. From screening steps, skim milk and yeast extract showed significant influence on the biomass production and, based on the steepest ascent step, middle points of skim milk (6% wt/vol) and yeast extract (1.89% wt/vol) were obtained. A polynomial regression model in FCCD-RSM revealed that both factors were found significant and the strongest influence was given by skim milk concentration. Optimum concentrations of skim milk and yeast extract for maximum biomass G4 and β-galactosidase production meanwhile low in lactose and FAN balance after cultivation period were 5.89% (wt/vol) and 2.31% (wt/vol), respectively. The validation experiments showed that the predicted and experimental values are not significantly different, indicating that the FCCD-RSM model developed is sufficient to describe the cultivation process of G4 using skim-milk-based medium with the addition of yeast extract. Khalilah Abdul Khalil, Shuhaimi Mustafa, Rosfarizan Mohammad, Arbakariya Bin Ariff, Yamin Shaari, Yazid Abdul Manap, Siti Aqlima Ahmad, and Farrah Aini Dahalan Copyright © 2014 Khalilah Abdul Khalil et al. All rights reserved. Control Efficacy of an Endophytic Bacillus amyloliquefaciens Strain BZ6-1 against Peanut Bacterial Wilt, Ralstonia solanacearum Sun, 12 Jan 2014 00:00:00 +0000 We aimed to isolate and identify endophytic bacteria that might have efficacy against peanut bacterial wilt (BW) caused by Ralstonia solanacearum. Thirty-seven endophytic strains were isolated from healthy peanut plants in R. solanacearum-infested fields and eight showed antagonistic effects against R. solanacearum. Strain BZ6-1 with the highest antimicrobial activity was identified as Bacillus amyloliquefaciens based on morphology, biochemistry, and 16S rRNA analysis. Culture conditions of BZ6-1 were optimized using orthogonal test method and inhibitory zone diameter in dual culture plate assay reached 34.2 mm. Furthermore, main antimicrobial substances of surfactin and fengycin A homologues produced by BZ6-1 were analyzed by high performance liquid chromatography electrospray ionization tandem mass spectrometry. Finally, pot experiments were adopted to test the control efficiency of BZ6-1 against peanut BW. Disease incidence decreased significantly from 84.5% in the control to 12.1% with addition of 15 mL (108 cfu mL−1) culture broth for each seedling, suggesting the feasibility of strain BZ6-1 in the biological control of peanut plants BW. Xiaobing Wang and Guobin Liang Copyright © 2014 Xiaobing Wang and Guobin Liang. All rights reserved. Assessment of Genetic Diversity of Zoonotic Brucella spp. Recovered from Livestock in Egypt Using Multiple Locus VNTR Analysis Mon, 06 Jan 2014 16:28:49 +0000 Brucellosis is endemic in most parts of Egypt, where it is caused mainly by Brucella melitensis biovar 3, and affects cattle and small ruminants in spite of ongoing efforts devoted to its control. Knowledge of the predominant Brucella species/strains circulating in a region is a prerequisite of a brucellosis control strategy. For this reason a study aiming at the evaluation of the phenotypic and genetic heterogeneity of a panel of 17 Brucella spp. isolates recovered from domestic ruminants (cattle, buffalo, sheep, and goat) from four governorates during a period of five years (2002–2007) was carried out using microbiological tests and molecular biology techniques (PCR, MLVA-15, and sequencing). Thirteen strains were identified as B. melitensis biovar 3 while all phenotypic and genetic techniques classified the remaining isolates as B. abortus () and B. suis biovar 1 (). MLVA-15 yielded a high discriminatory power (), indicating a high genetic diversity among the B. melitensis strains circulating among domestic ruminants in Egypt. This is the first report of the isolation of B. suis from cattle in Egypt which, coupled with the finding of B. abortus, suggests a potential role of livestock as reservoirs of several zoonotic Brucella species in the region. Ahmed M. S. Menshawy, Marta Perez-Sancho, Teresa Garcia-Seco, Hosein I. Hosein, Nerea García, Irene Martinez, Ashraf E. Sayour, Joaquín Goyache, Ragab A. A. Azzam, Lucas Dominguez, and Julio Alvarez Copyright © 2014 Ahmed M. S. Menshawy et al. All rights reserved. Current Methods in the Molecular Typing of Mycobacterium tuberculosis and Other Mycobacteria Sun, 05 Jan 2014 13:32:39 +0000 In the epidemiology of tuberculosis (TB) and nontuberculous mycobacterial (NTM) diseases, as in all infectious diseases, the key issue is to define the source of infection and to disclose its routes of transmission and dissemination in the environment. For this to be accomplished, the ability of discerning and tracking individual Mycobacterium strains is of critical importance. Molecular typing methods have greatly improved our understanding of the biology of mycobacteria and provide powerful tools to combat the diseases caused by these pathogens. The utility of various typing methods depends on the Mycobacterium species under investigation as well as on the research question. For tuberculosis, different methods have different roles in phylogenetic analyses and person-to-person transmission studies. In NTM diseases, most investigations involve the search for environmental sources or phylogenetic relationships. Here, too, the type of setting determines which methodology is most suitable. Within this review, we summarize currently available molecular methods for strain typing of M. tuberculosis and some NTM species, most commonly associated with human disease. For the various methods, technical practicalities as well as discriminatory power and accomplishments are reviewed. Tomasz Jagielski, Jakko van Ingen, Nalin Rastogi, Jarosław Dziadek, Paweł K. Mazur, and Jacek Bielecki Copyright © 2014 Tomasz Jagielski et al. All rights reserved. Microbial Assessment and Prevalence of Foodborne Pathogens in Natural Cheeses in Japan Tue, 31 Dec 2013 13:45:06 +0000 The production and consumption of domestic natural cheese in Japan is increasing year by year. More than ninety percent of domestic natural cheese is produced in Hokkaido region of Japan, while information on its quality and safety related to foodborne pathogens is limited. To assess the microbiological safety of domestic natural cheese, a total of 126 natural cheese samples produced in Hokkaido were collected from December, 2012, to July, 2013. In addition to standard plate count (SPC) and coliform counts, the prevalence study of three pathogens (Listeria monocytogenes, pathogenic Escherichia coli, and Salmonella spp.) was performed on each sample. Real-time PCR and matrix-assisted laser desorption-ionization time-of-flight mass spectrometer methods were employed for identification of presumptive pathogens. Coliform was detected in 25 samples (19.8%) with a minimum of 25 cfu/g and a maximum of more than 3.0 × 106 cfu/g. Salmonella spp. and L. monocytogenes were not isolated from any of the samples. Only one sample (0.80%) showed positive PCR amplification for ipaH gene suggesting possible contamination of enteroinvasive E. coli or Shigella in this product. Overall results indicate that natural cheeses produced in Hokkaido region were satisfactory microbiological quality according to existing international standards. Firew Kassa Esho, Budbazar Enkhtuya, Akiko Kusumoto, and Keiko Kawamoto Copyright © 2013 Firew Kassa Esho et al. All rights reserved. Antimicrobial Activity, Growth Inhibition of Human Tumour Cell Lines, and Phytochemical Characterization of the Hydromethanolic Extract Obtained from Sapindus saponaria L. Aerial Parts Thu, 26 Dec 2013 14:26:41 +0000 The hydromethanolic extract of Sapindus saponaria L. aerial parts was investigated for antimicrobial activity (against several Gram-positive and Gram-negative bacteria and fungi) and capacity to inhibit the growth of different human tumor cell lines as also nontumor liver cells. The evaluated extract was further characterized in terms of phytochemicals using UV, 1H-NMR, 13C-NMR, and MS spectroscopic tools. The extract has shown a significant antimicrobial activity on all tested bacterial and fungal species. The best activity was achieved against Bacillus cereus and Staphylococcus aureus among bacteria and against all three Penicillium species tested. It also revealed cytotoxicity against human colon (HCT-15), cervical (HeLa), breast (MCF-7), and lung (NCI-H460) carcinoma cell lines, with HeLa being the most susceptible tumor cell line. The extract was not toxic for nontumor liver cells. Chromatographic separation of the extract resulted in the isolation and identification of stigmasterol, oleanolic acid, luteolin, luteolin 8---glucoside (orientin), luteolin 6---glucoside (isoorientin), luteolin 7---glucuronide, and rutin. The results of the present findings may be useful for the discovery of novel antitumor and antimicrobial agents from plant origin. Khaled N. Rashed, Ana Ćirić, Jasmina Glamočlija, Ricardo C. Calhelha, Isabel C. F. R. Ferreira, and Marina Soković Copyright © 2013 Khaled N. Rashed et al. All rights reserved. Genotypically Different Clones of Staphylococcus aureus Are Diverse in the Antimicrobial Susceptibility Patterns and Biofilm Formations Wed, 25 Dec 2013 14:30:16 +0000 This study evaluated whether genotypically different clinical isolates of S. aureus have similar susceptibilities to individual antibiotics. It further aims to check the impact of biofilm on the in vitro activity of vancomycin, daptomycin, linezolid, and tigecycline against S. aureus clones. The study used a total of 60 different clinical MSSA and MRSA isolates. Susceptibilities were performed in planktonic cultures by macrobroth dilution and epsilon-test (E test) system. Biofilm production was determined using an adherent plate assay. The efficacy of antimicrobial activities against biofilms formation was checked using confocal laser scanning microscopy (CLSM). The study found that similar and different spa, MLST, and SCCmec types displayed high variation in their susceptibilities to antibiotics with tigecycline and daptomycin being the most effective. The biofilms were found resistant to high concentrations of most antibiotics tested with daptomycin being the most effective drug used in adhesive biofilms. A considerable difference exists among similar and various clone types against antibiotics tested. This variation could have contributed to the degree of virulence even within the same clonal genotype and enhanced heterogeneity in the infection potential. Thus, the development of a rapid and precise identification profile for each clone in human infections is important. Salman Sahab Atshan, Mariana Nor Shamsudin, Leslie Than Thian Lung, Zamberi Sekawi, Chong Pei Pei, Arunkumar Karunanidhi, Jayakayatri Jeevajothi Nathan, Alreshidi Mateg Ali, Ehsanollah Ghaznavi-Rad, Salwa A. Abduljaleel, and Rukman Awang Hamat Copyright © 2013 Salman Sahab Atshan et al. All rights reserved. Propionibacterium acnes in Human Health and Disease Tue, 24 Dec 2013 15:57:31 +0000 Andrew McDowell, Sheila Patrick, Yoshinobu Eishi, Peter Lambert, and Anne Eady Copyright © 2013 Andrew McDowell et al. All rights reserved. Short Communication: Subtyping of Mycobacterium kansasii by PCR-Restriction Enzyme Analysis of the hsp65 Gene Sun, 22 Dec 2013 18:12:26 +0000 Mycobacterium kansasii is one of the most common causes of pulmonary disease resulting from nontuberculous mycobacteria (NTM). It is also the most frequently isolated NTM species from clinical specimens in Poland. The aim of this study was to investigate the distribution of M. kansasii subtypes among patients suspected of having pulmonary NTM disease. Fifty clinical isolates of M. kansasii recovered from as many patients with suspected mycobacterial lung disease between 2000 and 2010 in Poland were genotyped by PCR-restriction enzyme analysis (PCR-REA) of partial hsp65 gene. Mycobacterium kansasii subtype I was the only genotype to be identified among the isolates, both disease-associated and non-disease-associated. Isolation of M. kansasii subtype I from clinical specimens may be indicative of infection but may also merely represent colonization. Zofia Bakuła, Aleksandra Safianowska, Magdalena Nowacka-Mazurek, Jacek Bielecki, and Tomasz Jagielski Copyright © 2013 Zofia Bakuła et al. All rights reserved. Genotyping of Clinical Mycobacterium tuberculosis Isolates Based on IS6110 and MIRU-VNTR Polymorphisms Tue, 17 Dec 2013 13:45:14 +0000 In this study, 155 clinical Mycobacterium tuberculosis isolates were subject to genotyping with fast ligation-mediated PCR (FLiP). This typing method is a modified mixed-linker PCR, a rapid approach based on the PCR amplification of HhaI restriction fragments of genomic DNA containing the 3′ end of IS6110 and resolving the amplicons by polyacrylamide gel electrophoresis. The results were compared with previous data of the more commonly used methods, 15-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and, to verify combined FLiP/MIRU-VNTR clusters, the reference IS6110 restriction fragment length polymorphism (RFLP). FLiP banding patterns were highly reproducible and polymorphic. This method differentiated 119 types among the study set compared to 108 distinct MIRU-VNTR profiles. The discriminatory power of FLiP was slightly higher than that of MIRU-VNTR analysis (Hunter-Gaston Discriminatory Index = 0.991 and 0.990, resp.). Detailed comparison of the clusters defined by each of the methods revealed, however, a more apparent difference in the discriminatory abilities that favored FLiP. Clustering of strains by using combined results of these two PCR-based methods correlated well with IS6110 RFLP-defined clusters, further confirming high discriminatory potential of FLiP typing. These results indicate that FLiP could be an attractive and valuable secondary typing technique for verification of MIRU-VNTR clusters of M. tuberculosis strains. Anna Żaczek, Anna Brzostek, Arkadiusz Wojtasik, Jarosław Dziadek, and Anna Sajduda Copyright © 2013 Anna Żaczek et al. All rights reserved. Fish Tank Granuloma Caused by Mycobacterium marinum in Two Aquarists: Two Case Reports Thu, 12 Dec 2013 08:23:27 +0000 Mycobacterium marinum, the cause of chronic systemic infections in fish, occasionally causes granulomatous skin and soft tissue lesions in humans. Cutaneous mycobacterial infection in two patients owing to unusual circumstances is presented in this report. The first patient was infected through improper hygienic behavior, while infection in the second patient was previously misdiagnosed as rheumatoid arthritis and treated with methylprednisolone for a period of three months, which resulted in a rare systemic spread of M. marinum into the bones of the hand, testis, and epididymis. Simultaneously, screening for possible sources of M. marinum infection in patients' aquaria revealed positive fish harboring VNTR profiles identical to those obtained for clinical isolates from patients. Michal Slany, Petr Jezek, and Monika Bodnarova Copyright © 2013 Michal Slany et al. All rights reserved. Mutations in the embB Gene and Their Association with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates from Poland Wed, 11 Dec 2013 12:04:30 +0000 Ethambutol (EMB) continues to be used as part of a standard drug regimen for the treatment of tuberculosis (TB). Mutations in the embB gene and those within its conserved EMB resistance determining region (ERDR) in particular have repeatedly been associated with resistance to EMB in Mycobacterium tuberculosis. The aim of this study was to examine the mutational “hot spots” in the embB gene, including the ERDR, among multidrug-resistant (MDR) M. tuberculosis clinical isolates and to find a possible association between embB mutations and resistance to EMB. An 863-bp fragment of the embB gene was sequenced in 17 EMB-resistant and 33 EMB-susceptible MDR-TB isolates. In total, eight embB mutation types were detected in 6 distinct codons of 27 (54%) M. tuberculosis isolates. Mutations in codon 306 were most common, found in both EMB-resistant (9) and EMB-susceptible (11) isolates. Only mutations in codons 406 and 507 were found exclusively in four and one EMB-resistant isolates, respectively. Sequence analysis of the ERDR in the embB gene is not sufficient for rapid detection of EMB resistance, and the codon 306 mutations are not good predictive markers of resistance to EMB. Zofia Bakuła, Agnieszka Napiórkowska, Jacek Bielecki, Ewa Augustynowicz-Kopeć, Zofia Zwolska, and Tomasz Jagielski Copyright © 2013 Zofia Bakuła et al. All rights reserved. Correlation of N-Acetyltransferase 2 Genotype with Isoniazid Acetylation in Polish Tuberculosis Patients Sat, 07 Dec 2013 15:04:06 +0000 Isoniazid (INH), a key agent in the treatment of tuberculosis (TB), is metabolized primarily by the genetically polymorphic N-acetyltransferase 2 (NAT2) enzyme. Patients treated with INH can be classified as fast, intermediate, and slow acetylators. The objective of this study was to explore the relationship between NAT2 genotypes and the serum concentrations of INH. Blood samples from 130 patients were taken for the analysis, and plasma INH concentrations were determined by using the high-performance liquid chromatography (HPLC) technology. Acetylation genotype was determined on genomic DNA by using an allele-specific polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) assay. Once the NAT2 genotypes were established, patients were classified into three categories: fast, intermediate, and slow acetylators. Of the 130 patients studied, 84 (64.6%) were slow, 39 (30%) were intermediate, and 7 (5.4%) were fast acetylators. Analysis of INH concentrations in the blood of patients receiving the approximate doses of the drug revealed that, at the time intervals examined, the average concentration of INH was 2- to 7-fold higher among slow acetylators compared to fast and intermediate acetylators. Conclusion. Determining mutations in the NAT2 gene enabled the identification of the INH acetylation type in patients and the genotyping results were consistent with the phenotype determined by methods of measurement of drug bioavailability. Anna Zabost, Sylwia Brzezińska, Monika Kozińska, Maria Błachnio, Jacek Jagodziński, Zofia Zwolska, and Ewa Augustynowicz-Kopeć Copyright © 2013 Anna Zabost et al. All rights reserved. Molybdate Reduction to Molybdenum Blue by an Antarctic Bacterium Thu, 05 Dec 2013 15:15:08 +0000 A molybdenum-reducing bacterium from Antarctica has been isolated. The bacterium converts sodium molybdate or Mo6+ to molybdenum blue (Mo-blue). Electron donors such as glucose, sucrose, fructose, and lactose supported molybdate reduction. Ammonium sulphate was the best nitrogen source for molybdate reduction. Optimal conditions for molybdate reduction were between 30 and 50 mM molybdate, between 15 and 20°C, and initial pH between 6.5 and 7.5. The Mo-blue produced had a unique absorption spectrum with a peak maximum at 865 nm and a shoulder at 710 nm. Respiratory inhibitors such as antimycin A, sodium azide, potassium cyanide, and rotenone failed to inhibit the reducing activity. The Mo-reducing enzyme was partially purified using ion exchange and gel filtration chromatography. The partially purified enzyme showed optimal pH and temperature for activity at 6.0 and 20°C, respectively. Metal ions such as cadmium, chromium, copper, silver, lead, and mercury caused more than 95% inhibition of the molybdenum-reducing activity at 0.1 mM. The isolate was tentatively identified as Pseudomonas sp. strain DRY1 based on partial 16s rDNA molecular phylogenetic assessment and the Biolog microbial identification system. The characteristics of this strain would make it very useful in bioremediation works in the polar and temperate countries. S. A. Ahmad, M. Y. Shukor, N. A. Shamaan, W. P. Mac Cormack, and M. A. Syed Copyright © 2013 S. A. Ahmad et al. All rights reserved. A New Protocol to Detect Multiple Foodborne Pathogens with PCR Dipstick DNA Chromatography after a Six-Hour Enrichment Culture in a Broad-Range Food Pathogen Enrichment Broth Mon, 02 Dec 2013 15:08:59 +0000 A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5–10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments. Masahiro Hayashi, Tatsuya Natori, Sayoko Kubota-Hayashi, Machiko Miyata, Kiyofumi Ohkusu, Keiko Kawamoto, Hisao Kurazono, Souichi Makino, and Takayuki Ezaki Copyright © 2013 Masahiro Hayashi et al. All rights reserved. α-Ketoglutarate Accumulation Is Not Dependent on Isocitrate Dehydrogenase Activity during Tellurite Detoxification in Escherichia coli Mon, 25 Nov 2013 13:37:06 +0000 Tellurite is toxic to most microorganisms because of its ability to generate oxidative stress. However, the way in which tellurite interferes with cellular processes is not fully understood to date. In this line, it was previously shown that tellurite-exposed cells displayed reduced activity of the α-ketoglutarate dehydrogenase complex (α-KGDH), which resulted in α-ketoglutarate (α-KG) accumulation. In this work, we assessed if α-KG accumulation in tellurite-exposed E. coli could also result from increased isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) activities, both enzymes involved in α-KG synthesis. Unexpectedly both activities were found to decrease in the presence of the toxicant, an observation that seems to result from the decreased transcription of icdA and gdhA genes (encoding ICDH and GDH, resp.). Accordingly, isocitrate levels were found to increase in tellurite-exposed E. coli. In the presence of the toxicant, cells lacking icdA or gdhA exhibited decreased reactive oxygen species (ROS) levels and higher tellurite sensitivity as compared to the wild type strain. Finally, a novel branch activity of ICDH as tellurite reductase is presented. Claudia A. Reinoso, Vasu D. Appanna, and Claudio C. Vásquez Copyright © 2013 Claudia A. Reinoso et al. All rights reserved. Acid Lipase from Candida viswanathii: Production, Biochemical Properties, and Potential Application Sun, 17 Nov 2013 13:33:52 +0000 Influences of environmental variables and emulsifiers on lipase production of a Candida viswanathii strain were investigated. The highest lipase activity (101.1 U) was observed at 210 rpm, pH 6.0, and 27.5°C. Other fermentation parameters analyzed showed considerable rates of biomass yield ( g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield () of 10.061 U/g. Crude lipase presented optimal activity at acid pH of 3.5, suggesting a new lipolytic enzyme for this genus and yeast in general. In addition, crude lipase presented high stability in acid conditions and temperature between 40 and 45°C, after 24 h of incubation in these temperatures. Lipase remained active in the presence of organic solvents maintaining above 80% activity in DMSO, methanol, acetonitrile, ethanol, acetone, 1-propanol, isopropanol, and 2-propanol. Effectiveness for the hydrolysis of a wide range of natural triglycerides suggests that this new acid lipase has high potential application in the oleochemical and food industries for hydrolysis and/or modification of triacylglycerols to improve the nutritional properties. Alex Fernando de Almeida, Sâmia Maria Tauk-Tornisielo, and Eleonora Cano Carmona Copyright © 2013 Alex Fernando de Almeida et al. All rights reserved. Purification and Characterization of Tannin Acyl Hydrolase Produced by Mixed Solid State Fermentation of Wheat Bran and Marigold Flower by Penicillium notatum NCIM 923 Sun, 17 Nov 2013 11:31:58 +0000 Tannin acyl hydrolase produced extracellularly by the fungal strain Penicillium notatum NCIM 923 in mixed solid state fermentation of wheat bran and marigold flower in the ratio 4 : 1 was purified from the cell-free extract broth by ammonium sulphate fractionation followed by diethylaminoethyl-cellulose column chromatography. Tannase was purified by 19.89-fold with yield of 11.77%. The specific activity of crude tannase was found to be 1.31 U/mg protein while that of purified tannase was 22.48 U/mg protein. SDS-PAGE analysis indicated that the enzyme is dimeric with one major band of molecular mass 97 kDa and a very light band of molecular mass 43 kDa. Temperature of 35 to 40°C and pH 5 were optimum for tannase activity. The enzyme retained more than 60% of its stability at 60°C and 40% stability at pH 3 and 8, respectively. was found to be  M and  U/mg. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food processing industry. Saswati Gayen and Uma Ghosh Copyright © 2013 Saswati Gayen and Uma Ghosh. All rights reserved. Use of Frankia and Actinorhizal Plants for Degraded Lands Reclamation Mon, 11 Nov 2013 13:46:09 +0000 Degraded lands are defined by soils that have lost primary productivity due to abiotic or biotic stresses. Among the abiotic stresses, drought, salinity, and heavy metals are the main threats in tropical areas. These stresses affect plant growth and reduce their productivity. Nitrogen-fixing plants such as actinorhizal species that are able to grow in poor and disturbed soils are widely planted for the reclamation of such degraded lands. It has been reported that association of soil microbes especially the nitrogen-fixing bacteria Frankia with these actinorhizal plants can mitigate the adverse effects of abiotic and biotic stresses. Inoculation of actinorhizal plants with Frankia significantly improves plant growth, biomass, shoot and root N content, and survival rate after transplanting in fields. However, the success of establishment of actinorhizal plantation in degraded sites depends upon the choice of effective strains of Frankia. Studies related to the beneficial role of Frankia on the establishment of actinorhizal plants in degraded soils are scarce. In this review, we describe some examples of the use of Frankia inoculation to improve actinorhizal plant performances in harsh conditions for reclamation of degraded lands. Nathalie Diagne, Karthikeyan Arumugam, Mariama Ngom, Mathish Nambiar-Veetil, Claudine Franche, Krishna Kumar Narayanan, and Laurent Laplaze Copyright © 2013 Nathalie Diagne et al. All rights reserved. Development of an Immunochromatographic Test Strip for Detection of Cholera Toxin Thu, 07 Nov 2013 15:32:40 +0000 Because cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC) with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT), which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC) was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae. Eiki Yamasaki, Ryuta Sakamoto, Takashi Matsumoto, Fumiki Morimatsu, Takayuki Kurazono, Toyoko Hiroi, G. Balakrish Nair, and Hisao Kurazono Copyright © 2013 Eiki Yamasaki et al. All rights reserved. Propionibacterium acnes: An Underestimated Pathogen in Implant-Associated Infections Wed, 06 Nov 2013 14:40:30 +0000 The role of Propionibacterium acnes in acne and in a wide range of inflammatory diseases is well established. However, P. acnes is also responsible for infections involving implants. Prolonged aerobic and anaerobic agar cultures for 14 days and broth cultures increase the detection rate. In this paper, we review the pathogenic role of P. acnes in implant-associated infections such as prosthetic joints, cardiac devices, breast implants, intraocular lenses, neurosurgical devices, and spine implants. The management of severe infections caused by P. acnes involves a combination of antimicrobial and surgical treatment (often removal of the device). Intravenous penicillin G and ceftriaxone are the first choice for serious infections, with vancomycin and daptomycin as alternatives, and amoxicillin, rifampicin, clindamycin, tetracycline, and levofloxacin for oral treatment. Sonication of explanted prosthetic material improves the diagnosis of implant-associated infections. Molecular methods may further increase the sensitivity of P. acnes detection. Coating of implants with antimicrobial substances could avoid or limit colonization of the surface and thereby reduce the risk of biofilm formation during severe infections. Our understanding of the role of P. acnes in human diseases will likely continue to increase as new associations and pathogenic mechanisms are discovered. María Eugenia Portillo, Stéphane Corvec, Olivier Borens, and Andrej Trampuz Copyright © 2013 María Eugenia Portillo et al. All rights reserved. Molecular Characterization and In Silico Analysis of Naturally Occurring TEM Beta-Lactamase Variants among Pathogenic Enterobacteriaceae Infecting Indian Patients Mon, 28 Oct 2013 10:06:01 +0000 Cephalosporin resistance, particularly due to encoded -lactamases, among Enterobacteriaceae is, though, an increasing public health problem in India; their circulating genetic variants remain unknown. The present study deals with determination of variants among 134 pathogenic Enterobacteriaceae of Indian origin. Their resistance profile against 3rd generation cephalosporins was determined. The presence of variants among the bacterial plasmids was characterized by PCR followed by sequencing. Intergenic relations among the variants was determined by phylogenetic analysis. protein were modeled by Modeller9v5 and verified. The catalytic pockets were characterized, and their interaction with cephalosporins was analyzed using AutoDock tools. More than 87% of isolates showed cephalosporin resistance with ESBL production among 57.8% of Escherichia coli and 50.6% of klebsiella pneumoniae. (84.21%), like (3.94%), (3.94%), (3.94%), (3.94%), and (7.89%) were detected in 76 isolates. Four variants, namely, like, , , and , coexisted in 3 isolates. The largest catalytic pocket of explained its expanded activity towards -lactam--lactamase inhibitor combinations. Molecular docking indicated differential resistance pattern of variants. Lena Dhara, Anusri Tripathi, and Arijit Pal Copyright © 2013 Lena Dhara et al. All rights reserved. Prevalence and Antibiotic Resistance Pattern of Methicillin-Resistant Staphylococcus aureus from an Orthopaedic Hospital in Nigeria Sun, 27 Oct 2013 18:09:46 +0000 Patients with surgical wounds have been reported to be at high risk of MRSA carriage and infection. The prevalence and antibiotic resistance pattern of this organism in the orthopaedic ward of Ahmadu Bello University Teaching Hospital (ABUTH), Zaria-Nigeria, a 547-bed Nigerian hospital, were thus studied. A total of 185 isolates of Staphylococcus aureus were confirmed from 217 samples taken from the orthopaedic wards of the hospital using standard isolation methods. Out of these, 44 (23.8%) were from the wounds of patients and 70 (37.8%) from the skin. The remaining 65 (35.1%) and 6 (3.2%) were from their beds and the atmospheric air, respectively. Out of these, 33 (75%), 36 (51.4%), and 48 (73.8%) from wounds, skin, and bed, respectively, were found to be methicillin-resistant Staphylococcus aureus (MRSA) using the disc-sensitive test methods. None was detected from the atmosphere. The antibiotic susceptibility pattern results showed the level of resistance to be ampicillin 100% in all the three sites, pefloxacin 90.9%, 72.2%, 66.7%, ceftriaxone 69.7%, 72.2%, 70.8%, gentamicin 54.5%, 52.8%, 37.5%, and ciprofloxacin 51.5%, 47.2%, 35.4% at the wound, skin, and bed sites, respectively. Results confirm that MRSA continues to pose a threat to the hospitalized patients, especially those with bone and wound infections. C. E. Udobi, A. F. Obajuluwa, and J. A. Onaolapo Copyright © 2013 C. E. Udobi et al. All rights reserved. What Is the Best Strategy for Enhancing the Effects of Topically Applied Ozonated Oils in Cutaneous Infections? Sun, 27 Oct 2013 07:44:57 +0000 Owing to diabetes, atherosclerosis, and ageing, there are several million patients undergoing skin lesions degenerated into infected ulcers with very little tendency to heal and implying a huge socioeconomical cost. Previous medical experience has shown that the daily application of ozonated oil eliminates the infection and promotes a rapid healing. The purpose of the study is the optimization of the antimicrobial effect of ozonated oils by testing in vitro four bacterial species and one yeast without or in the presence of different amounts of human serum. The results obtained suggest that a gentle and continuous removal of debris and exudate is an essential condition for the potent bactericidal effect of ozonated oils. In fact, even small amounts of human serum inactivate ozone derivatives and protect bacteria. The application of ozonated oil preparations is very promising in a variety of skin and mucosal infections. Moreover, ozonated oils are far less expensive than antibiotic preparations. I. Zanardi, S. Burgassi, E. Paccagnini, M. Gentile, V. Bocci, and V. Travagli Copyright © 2013 I. Zanardi et al. All rights reserved. In Silico Modeling and Functional Interpretations of Cry1Ab15 Toxin from Bacillus thuringiensis BtB-Hm-16 Tue, 22 Oct 2013 18:43:52 +0000 The theoretical homology based structural model of Cry1Ab15 δ-endotoxin produced by Bacillus thuringiensis BtB-Hm-16 was predicted using the Cry1Aa template (resolution 2.25 Å). The Cry1Ab15 resembles the template structure by sharing a common three-domain extending conformation structure responsible for pore-forming and specificity determination. The novel structural differences found are the presence of β0 and α3, and the absence of α7b, β1a, α10a, α10b, β12, and α11a while α9 is located spatially downstream. Validation by SUPERPOSE and with the use of PROCHECK program showed folding of 98% of modeled residues in a favourable and stable orientation with a total energy Z-score of −6.56; the constructed model has an RMSD of only 1.15 Å. These increments of 3D structure information will be helpful in the design of domain swapping experiments aimed at improving toxicity and will help in elucidating the common mechanism of toxin action. Sudhanshu Kashyap Copyright © 2013 Sudhanshu Kashyap. All rights reserved. Biosynthesis of Osmoregulated Periplasmic Glucans in Escherichia coli: The Phosphoethanolamine Transferase Is Encoded by opgE Tue, 22 Oct 2013 09:29:17 +0000 Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity. Sébastien Bontemps-Gallo, Virginie Cogez, Catherine Robbe-Masselot, Kevin Quintard, Jacqueline Dondeyne, Edwige Madec, and Jean-Marie Lacroix Copyright © 2013 Sébastien Bontemps-Gallo et al. All rights reserved. Fibrinogen-Induced Streptococcus mutans Biofilm Formation and Adherence to Endothelial Cells Tue, 08 Oct 2013 08:50:19 +0000 Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen on S. mutans resistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation by S. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support. S. mutans in biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence of S. mutans to endothelial cells. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells which may contribute to infective endocarditis. Telma Blanca Lombardo Bedran, Jabrane Azelmat, Denise Palomari Spolidorio, and Daniel Grenier Copyright © 2013 Telma Blanca Lombardo Bedran et al. All rights reserved. Gene Expression Profiling of Clostridium botulinum under Heat Shock Stress Mon, 30 Sep 2013 11:46:10 +0000 During growth, C. botulinum is always exposed to different environmental changes, such as temperature increase, nutrient deprivation, and pH change; however, its corresponding global transcriptional profile is uncharacterized. This study is the first description of the genome-wide gene expression profile of C. botulinum in response to heat shock stress. Under heat stress (temperature shift from 37°C to 45°C over a period of 15 min), 176 C. botulinum ATCC 3502 genes were differentially expressed. The response included overexpression of heat shock protein genes (dnaK operon, groESL, hsp20, and htpG) and downregulation of aminoacyl-tRNA synthetase genes (valS, queA, tyrR, and gatAB) and ribosomal and cell division protein genes (ftsZ and ftsH). In parallel, several transcriptional regulators (marR, merR, and ompR families) were induced, suggesting their involvement in reshuffling of the gene expression profile. In addition, many ABC transporters (oligopeptide transport system), energy production and conversion related genes (glpA and hupL), cell wall and membrane biogenesis related genes (fabZ, fabF, and fabG), flagella-associated genes (flhA, flhM, flhJ, flhS, and motAB), and hypothetical genes also showed changed expression patterns, indicating that they may play important roles in survival under high temperatures. Wan-dong Liang, Yun-tian Bi, Hao-yan Wang, Sheng Dong, Ke-shen Li, and Jin-song Li Copyright © 2013 Wan-dong Liang et al. All rights reserved. Antagonistic Activity of Lactobacillus Isolates against Salmonella typhi In Vitro Sun, 29 Sep 2013 15:41:39 +0000 Background. Enteric fever is a global health problem, and rapidly developing resistance to various drugs makes the situation more alarming. The potential use of Lactobacillus to control typhoid fever represents a promising approach, as it may exert protective actions through various mechanisms. Methods. In this study, the probiotic potential and antagonistic activities of 32 Lactobacillus isolates against Salmonella typhi were evaluated. The antimicrobial activity of cell free supernatants of Lactobacillus isolates, interference of Lactobacillus isolates with the Salmonella adherence and invasion, cytoprotective effect of Lactobacillus isolates, and possibility of concurrent use of tested Lactobacillus isolates and antibiotics were evaluated by testing their susceptibilities to antimicrobial agents, and their oxygen tolerance was also examined. Results. The results revealed that twelve Lactobacillus isolates could protect against Salmonella typhi infection through interference with both its growth and its virulence properties, such as adherence, invasion, and cytotoxicity. These Lactobacillus isolates exhibited MIC values for ciprofloxacin higher than those of Salmonella typhi and oxygen tolerance and were identified as Lactobacillus plantarum. Conclusion. The tested Lactobacillus plantarum isolates can be introduced as potential novel candidates that have to be subjected for in vivo and application studies for treatment and control of typhoid fever. Amira Abdel-Daim, Nadia Hassouna, Mohamed Hafez, Mohamed Seif Aldeen Ashor, and Mohammad M. Aboulwafa Copyright © 2013 Amira Abdel-Daim et al. All rights reserved. Antibiogram, Adhesive Characteristics, and Incidence of Class 1 Integron in Aeromonas Species Isolated from Two South African Rivers Sun, 29 Sep 2013 14:15:05 +0000 Aeromonas species are well distributed in freshwater environments, and their natural susceptibility to antimicrobials renders them interesting candidates for the survey of antimicrobial resistance in freshwater milieu. Water samples were collected from Kat and Tyume rivers in the Eastern Cape province of South Africa, and a total of 45 isolates identified as Aeromonas species were recovered from the two rivers. All Aeromonas isolates were resistant to oxacillin, penicillin, clindamycin, cephalothin, vancomycin, and rifamycin, while appreciable susceptibilities (89.3 : 94.1%, 82.1 : 94.1%, 85.7 : 88.2%, and 92.9 : 88.2%) were observed against ciprofloxacin, chloramphenicol, nitrofurantoin, and gentamicin from Kat and Tyume rivers, respectively. Multiple antibiotic resistance (MAR) indices ranged from 0.016 to 0.044 for the two rivers. Class 1 integron was detected in about 20% of the isolates, and all the isolates except one showed ability to produce biofilm in vitro as weak producers (53.33%), moderate producers (15.56%), and strong producers (28.9%). This investigation provides a baseline data on antibiotic resistance as well as the adhesive characteristics of Aeromonas isolates from Tyume and Kat rivers in the Eastern Cape province of South Africa. Isoken H. Igbinosa, Vincent N. Chigor, Etinosa O. Igbinosa, Lawrence C. Obi, and Anthony I. Okoh Copyright © 2013 Isoken H. Igbinosa et al. All rights reserved. Effects of Flavonoids on Rumen Fermentation Activity, Methane Production, and Microbial Population Tue, 24 Sep 2013 08:36:34 +0000 This research was carried out to evaluate the effects of flavone, myricetin, naringin, catechin, rutin, quercetin, and kaempferol at the concentration of 4.5% of the substrate (dry matter basis) on the rumen microbial activity in vitro. Mixture of guinea grass and concentrate (60 : 40) was used as the substrate. The results showed that all the flavonoids except naringin and quercetin significantly () decreased the dry matter degradability. The gas production significantly () decreased by flavone, myricetin, and kaempferol, whereas naringin, rutin, and quercetin significantly () increased the gas production. The flavonoids suppressed methane production significantly (). The total VFA concentration significantly () decreased in the presence of flavone, myricetin, and kaempferol. All flavonoids except naringin and quercetin significantly () reduced the carboxymethyl cellulase, filter paperase, xylanase, and β-glucosidase activities, purine content, and the efficiency of microbial protein synthesis. Flavone, myricetin, catechin, rutin, and kaempferol significantly () reduced the population of rumen microbes. Total populations of protozoa and methanogens were significantly () suppressed by naringin and quercetin. The results of this research demonstrated that naringin and quercetin at the concentration of 4.5% of the substrate (dry matter basis) were potential metabolites to suppress methane production without any negative effects on rumen microbial fermentation. Ehsan Oskoueian, Norhani Abdullah, and Armin Oskoueian Copyright © 2013 Ehsan Oskoueian et al. All rights reserved. Antiadherent and Antibiofilm Activity of Humulus lupulus L. Derived Products: New Pharmacological Properties Mon, 23 Sep 2013 15:42:03 +0000 New antimicrobial properties of products derived from Humulus lupulus L. such as antiadherent and antibiofilm activities were evaluated. The growth of gram-positive but not gram-negative bacteria was inhibited to different extents by these compounds. An extract of hop cones containing 51% xanthohumol was slightly less active against S. aureus strains (MIC range 31.2–125.0 μg/mL) than pure xanthohumol (MIC range 15.6–62.5 μg/mL). The spent hop extract, free of xanthohumol, exhibited lower but still relevant activity (MIC range 1-2 mg/mL). There were positive coactions of hop cone, spent hop extracts, and xanthohumol with oxacillin against MSSA and with linezolid against MSSA and MRSA. Plant compounds in the culture medium at sub-MIC concentrations decreased the adhesion of Staphylococci to abiotic surfaces, which in turn caused inhibition of biofilm formation. The rate of mature biofilm eradication by these products was significant. The spent hop extract at MIC reduced biofilm viability by 42.8%, the hop cone extract by 74.8%, and pure xanthohumol by 86.5%. When the hop cone extract or xanthohumol concentration was increased, almost complete biofilm eradication was achieved (97–99%). This study reveals the potent antibiofilm activity of hop-derived compounds for the first time. Marcin Rozalski, Bartlomiej Micota, Beata Sadowska, Anna Stochmal, Dariusz Jedrejek, Marzena Wieckowska-Szakiel, and Barbara Rozalska Copyright © 2013 Marcin Rozalski et al. All rights reserved. Bioaccumulation Experiments in Mussels Contaminated with the Food-Borne Pathogen Arcobacter butzleri: Preliminary Data for Risk Assessment Thu, 12 Sep 2013 13:50:10 +0000 The aim of this study was to evaluate, at a laboratory scale, the ability of this microorganism to grow in seawater and bioaccumulate in mussels (Mytilus galloprovincialis) maintained in constantly aerated tanks, containing twenty litres of artificial seawater. Three concentrations of A. butzleri LMG were tested (about  CFU/mL,  CFU/mL, and  CFU/mL). Following contamination, enumeration of A. butzleri was performed from water and mussels each day, for up to 96 h. Three contamination experiments with artificial seawater in absence of mussels were also performed in the same manner. In the experiments with mussels, A. butzleri declined in water of approximately 1 log every 24 h from the contamination. In artificial seawater without mussels the concentration of A. butzleri remained on the same logarithmic level in the first 48 h and then decreased of about 1 log every 24 hours. In mussels, the concentration was approximately 2 log lower than the exposition level after 24 h from the contamination, and then it decreased exponentially of 1 log every 24 h. Our findings suggest that in the experimental conditions tested A. butzleri is neither able to effectively grow in seawater nor bioaccumulate in mussels, at least in the free and cultivable form. Donatella Ottaviani, Serena Chierichetti, Elena Rocchegiani, Chiara Bartolini, Laura Masini, Sabrina Santarelli, and Francesca Leoni Copyright © 2013 Donatella Ottaviani et al. All rights reserved. The Assessment of Proteus mirabilis Susceptibility to Ceftazidime and Ciprofloxacin and the Impact of These Antibiotics at Subinhibitory Concentrations on Proteus mirabilis Biofilms Thu, 12 Sep 2013 08:56:06 +0000 Rods of the Proteus genus are commonly isolated from patients, especially from the urinary tracts of the catheterised patients. The infections associated with biomaterials are crucial therapeutic obstacles, due to the bactericidal resistance of the biofilm. The aim of this study was to assess the susceptibility of P. mirabilis planktonic forms to ciprofloxacin and ceftazidime, the ability to form biofilm, and the impact of chosen sub-MIC concentrations of these antibiotics on biofilm at different stages of its formation. The research included 50 P. mirabilis strains isolated from wounds and the urinary tracts from patients of the University Hospital No. 1 in Bydgoszcz. The assessment of susceptibility to ciprofloxacin and ceftazidime was conducted using micromethods. The impact of sub-MIC concentrations of the chosen antibiotics on the biofilm was measured using the TTC method. The resistance to ciprofloxacin was confirmed for 20 strains (40.0%) while to ceftazidime for 32 (64.0%) of the tested P. mirabilis strains. All of the tested strains formed biofilm: 24.0% weakly, 26.0% moderately, and 50.0% strongly. It was determined that ciprofloxacin and ceftazidime caused eradication of the biofilm. Moreover, the connection between origin of the strains, biofilm maturity level, and resistance to antibiotics was proved. Joanna Kwiecińska-Piróg, Krzysztof Skowron, Katarzyna Zniszczol, and Eugenia Gospodarek Copyright © 2013 Joanna Kwiecińska-Piróg et al. All rights reserved. Optimization of Dairy Sludge for Growth of Rhizobium Cells Mon, 09 Sep 2013 11:27:22 +0000 In this study dairy sludge was evaluated as an alternative cultivation medium for Rhizobium. Growth of bacterial strains at different concentrations of Dairy sludge was monitored. Maximum growth of all strains was observed at 60% Dairy sludge concentration. At 60% optical density (OD) values are 0.804 for Rhizobium trifolii (MTCC905), 0.825 for Rhizobium trifolii (MTCC906), and 0.793 for Rhizobium meliloti (MTCC100). Growth pattern of strains was observed at 60% Dairy sludge along with different synthetic media (tryptone yeast, Rhizobium minimal medium and yeast extract mannitol). Growth in 60% Dairy sludge was found to be superior to standard media used for Rhizobium. Media were optimized using 60% dairy sludge along with different concentrations of yeast extract (1–7 g/L) and mannitol (7–13 g/L) in terms of optical density at different time intervals, that is, 24, 48 and 72 hours. Maximum growth was observed in 6 g/L of yeast extract and 12 g/L of mannitol at 48-hour incubation period in all strains. The important environmental parameters such as pH were optimized using 60% dairy sludge, 60% dairy sludge +6 g/L yeast extract, and 60% dairy sludge +12 g/L mannitol. The maximum growth of all strains was found at pH 7.0. The present study recommends the use of 60% dairy sludge as a suitable growth medum for inoculant production. Ashok Kumar Singh, Gauri Singh, Digvijay Gautam, and Manjinder Kaur Bedi Copyright © 2013 Ashok Kumar Singh et al. All rights reserved. Bauhinia variegata Leaf Extracts Exhibit Considerable Antibacterial, Antioxidant, and Anticancer Activities Thu, 05 Sep 2013 09:12:35 +0000 The present study reports the phytochemical profiling, antimicrobial, antioxidant, and anticancer activities of Bauhinia variegata leaf extracts. The reducing sugar, anthraquinone, and saponins were observed in polar extracts, while terpenoids and alkaloids were present in nonpolar and ethanol extracts. Total flavonoid contents in various extracts were found in the range of 11–222.67 mg QE/g. In disc diffusion assays, petroleum ether and chloroform fractions exhibited considerable inhibition against Klebsiella pneumoniae. Several other extracts also showed antibacterial activity against pathogenic strains of E. coli, Proteus spp. and Pseudomonas spp. Minimum bactericidal concentration (MBC) values of potential extracts were found between 3.5 and 28.40 mg/mL. The lowest MBC (3.5 mg/mL) was recorded for ethanol extract against Pseudomonas spp. The antioxidant activity of the extracts was compared with standard antioxidants. Dose dependent response was observed in reducing power of extracts. Polar extracts demonstrated appreciable metal ion chelating activity at lower concentrations (10–40 μg/mL). Many extracts showed significant antioxidant response in beta carotene bleaching assay. AQ fraction of B. variegata showed pronounced cytotoxic effect against DU-145, HOP-62, IGR-OV-1, MCF-7, and THP-1 human cancer cell lines with 90–99% cell growth inhibitory activity. Ethyl acetate fraction also produced considerable cytotoxicity against MCF-7 and THP-1 cell lines. The study demonstrates notable antibacterial, antioxidant, and anticancer activities in B. variegata leaf extracts. Amita Mishra, Amit Kumar Sharma, Shashank Kumar, Ajit K. Saxena, and Abhay K. Pandey Copyright © 2013 Amita Mishra et al. All rights reserved. Probiotic Potential and Safety Properties of Lactobacillus plantarum from Slovak Bryndza Cheese Wed, 04 Sep 2013 15:42:45 +0000 One hundred and twenty-five acid-resistant presumptive lactobacilli were isolated from Slovak Bryndza cheese and screened for their antimicrobial activity against eight bacterial pathogens using spot agar assay. Out of twenty-six Lactobacillus strains with strong inhibition activity, twenty were identified as Lactobacillus plantarum and six as Lactobacillus fermentum. The most active eleven L. plantarum isolates were further characterized in vitro for some probiotic and safety properties. Only three isolates K10, K21, and ZS07 showed the ability to grow over 50% in the presence of 0.3% bile. Strong deconjugation efficiency was determined for CK06 and K21. The highest β-galactosidase activity was shown in isolates ZS11, B01, CK06, and ZS07. Only three of the strains had the ability to produce tyramine: CK06, LM1, and ZS11. Strains K09, K21, ZS11, and ZS15 were susceptible to all tested antibiotics. Analysis of the results confirmed the L. plantarum isolates ZS07 and K21 as the most suitable for probiotic use, due to their desirable probiotic and safety characteristics. Anna Belicová, Mária Mikulášová, and Roman Dušinský Copyright © 2013 Anna Belicová et al. All rights reserved. Screening of a Protein That Interacts with the Matrix Attachment Region-Binding Protein from Dunaliella salina Wed, 04 Sep 2013 14:35:13 +0000 We isolated the matrix attachment region-binding protein (MBP) DMBP-1 from Dunaliella salina in our previous studies. MBPs are part of the cis-acting protein family cluster. The regulatory function possibly works through the interaction of the MBPs with each other. In the present study, DMBP-1 was used as the bait in screening the D. salina cDNA library for DMBP-1 interactors that could potentially mediate the DMBP-1-regulated functions. A novel MBP, namely, DMBP-2, was identified as a DMBP-1 binding partner. The cDNA of DMBP-1 was 823 bp long and contained a 573 bp open reading frame, which encoded a polypeptide of 191 amino acids. The interaction between DMBP-2 and DMBP-1 was further confirmed through glutathione S-transferase pull-down assays. Rui Yang, Zhaoxi Li, Yan Lin, Baosheng Yang, and Tianyun Wang Copyright © 2013 Rui Yang et al. All rights reserved. Staphylococcus aureus Clinical Isolates: Antibiotic Susceptibility, Molecular Characteristics, and Ability to Form Biofilm Sat, 31 Aug 2013 12:29:25 +0000 Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd), PFGE types, accessory gene regulator (agr) groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1–7 (56 isolates) were methicillin resistant (MRSA) and 8–10 (36 isolates) were methicillin sensitive (MSSA). One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq), and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70%) but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant. N. Indrawattana, O. Sungkhachat, N. Sookrung, M. Chongsa-nguan, A. Tungtrongchitr, S. P. Voravuthikunchai, T. Kong-ngoen, H. Kurazono, and W. Chaicumpa Copyright © 2013 N. Indrawattana et al. All rights reserved. Genotypic and Antimicrobial Characterisation of Propionibacterium acnes Isolates from Surgically Excised Lumbar Disc Herniations Wed, 28 Aug 2013 10:56:10 +0000 The anaerobic skin commensal Propionibacterium acnes is an underestimated cause of human infections and clinical conditions. Previous studies have suggested a role for the bacterium in lumbar disc herniation and infection. To further investigate this, five biopsy samples were surgically excised from each of 64 patients with lumbar disc herniation. P. acnes and other bacteria were detected by anaerobic culture, followed by biochemical and PCR-based identification. In total, 24/64 (38%) patients had evidence of P. acnes in their excised herniated disc tissue. Using recA and mAb typing methods, 52% of the isolates were type II (50% of culture-positive patients), while type IA strains accounted for 28% of isolates (42% patients). Type III (11% isolates; 21% patients) and type IB strains (9% isolates; 17% patients) were detected less frequently. The MIC values for all isolates were lowest for amoxicillin, ciprofloxacin, erythromycin, rifampicin, tetracycline, and vancomycin (≤1mg/L). The MIC for fusidic acid was 1-2 mg/L. The MIC for trimethoprim and gentamicin was 2 to ≥4 mg/L. The demonstration that type II and III strains, which are not frequently recovered from skin, predominated within our isolate collection (63%) suggests that the role of P. acnes in lumbar disc herniation should not be readily dismissed. Jess Rollason, Andrew McDowell, Hanne B. Albert, Emma Barnard, Tony Worthington, Anthony C. Hilton, Ann Vernallis, Sheila Patrick, Tom Elliott, and Peter Lambert Copyright © 2013 Jess Rollason et al. All rights reserved. Effects of Gamma Irradiation on Bacterial Microflora Associated with Human Amniotic Membrane Mon, 26 Aug 2013 08:34:36 +0000 Human amniotic membrane is considered a promising allograft material for the treatment of ocular surface reconstruction, burns, and other skin defects. In order to avoid the transmission of any diseases, grafts should be perfectly sterile. Twenty-five amniotic sacs were collected to determine the microbiological quality of human amniotic membrane, to analyze the radiation sensitivity pattern of the microorganism, and to detect the radiation decimal reduction dose () values. All the samples were found to be contaminated, and the bioburden was ranged from to  cfu/g. Initially, a total fifty bacterial isolates were characterized according to their cultural, morphological, and biochemical characteristics and then tested for the radiation sensitivity in an incremental series of radiation doses from 1 to 10 KGy. The results depict gradual decline in bioburden with incline of radiation doses. Staphylococcus spp. were the most frequently isolated bacterial contaminant in tissue samples (44%). The values of the bacterial isolates were ranged from 0.6 to 1.27 KGy. Streptococcus spp. were found to be the highest radioresistant strain with the radiation sterilization dose (RSD) of 11.4 KGy for a bioburden level of 1000. To compare the differences, values were also calculated by graphical evaluations of the data with two of the representative isolates of each bacterial species which showed no significant variations. Findings of this study indicate that lower radiation dose is quite satisfactory for the sterilization of amniotic membrane grafts. Therefore, these findings would be helpful to predict the efficacy of radiation doses for the processing of amniotic membrane for various purposes. Fahmida Binte Atique, Kazi Tahsin Ahmed, S. M. Asaduzzaman, and Kazi Nadim Hasan Copyright © 2013 Fahmida Binte Atique et al. All rights reserved. Thermal Stability of Glucokinases in Thermoanaerobacter tengcongensis Sat, 24 Aug 2013 09:37:12 +0000 In the genome of Thermoanaerobacter tengcongensis, three genes belonging to ROK (Repressor, ORF, and Kinase) family are annotated as glucokinases (GLKs). Using enzyme assays, the three GLKs were identified as ATP-dependent GLK (ATP-GLK), ADP-dependent GLK (ADP-GLK), and N-acetyl-glucosamine/mannosamine kinase (glu/man-NacK). The kinetic properties of the three GLKs such as , , optimal pH, and temperature were characterized, demonstrating that these enzymes performed the specific functions against varied substrates and under different temperatures. The abundance of ATP-GLK was attenuated when culture temperature was elevated and was almost undetectable at 80°C, whereas the ADP-GLK abundance was insensitive to temperature changes. Using degradation assays, ATP-GLK was found to have significantly faster degradation than ADP-GLK at 80°C. Co-immunoprecipitation results revealed that heat shock protein 60 (HSP60) could interact with ATP-GLK and ADP-GLK at 60 and 75°C, whereas at 80°C, the interaction was only effectively with ADP-GLK but not ATP-GLK. The functions of GLKs in T. tengcongensis are temperature dependent, likely regulated through interactions with HSP60. Zhong Qian, Jingjing Zhao, Xue Bai, Wei Tong, Zhen Chen, Hanfu Wei, Quanhui Wang, and Siqi Liu Copyright © 2013 Zhong Qian et al. All rights reserved. Composted versus Raw Olive Mill Waste as Substrates for the Production of Medicinal Mushrooms: An Assessment of Selected Cultivation and Quality Parameters Wed, 21 Aug 2013 12:14:06 +0000 Two-phase olive mill waste (TPOMW, “alperujo”) is a highly biotoxic sludge-like effluent of the olive-oil milling process with a huge seasonal production. One of the treatment approaches that has so far received little attention is the use of TPOMW as substrate for the cultivation of edible mushrooms. Fifteen fungal strains belonging to five species (Basidiomycota), that is, Agrocybe cylindracea, Pleurotus cystidiosus, P. eryngii, P. ostreatus, and P. pulmonarius, were evaluated for their efficacy to colonize media composed of TPOMW, which was used either raw or composted in mixtures with wheat straw in various ratios. Qualified strains exhibited high values of biological efficiency (e.g., 120–135% for Pleurotus spp. and 125% for A. cylindracea) and productivity in subsequent cultivation experiments on substrates supplemented with 20–40% composted TPOMW or 20% raw TPOMW. Only when supplementation exceeded 60% for raw TPOMW, a negative impact was noted on mushroom yields which could be attributed to the effluent's toxicity (otherwise alleviated in the respective composted TPOMW medium). Earliness and mushroom size as well as quality parameters such as total phenolic content and antioxidant activity did not demonstrate significant differences versus the control wheat-straw substrate. The substrates hemicellulose content was negatively correlated with mycelium growth rates and yields and positively with earliness; in addition, cellulose: lignin ratio presented a positive correlation with mycelium growth and mushroom weight for A. cylindracea and with earliness for all species examined. TPOMW-based media revealed a great potential for the substitution of traditional cultivation substrates by valorizing environmentally hazardous agricultural waste. Georgios I. Zervakis, Georgios Koutrotsios, and Panagiotis Katsaris Copyright © 2013 Georgios I. Zervakis et al. All rights reserved. An In Vitro Evaluation of Antioxidant and Colonic Microbial Profile Levels following Mushroom Consumption Tue, 20 Aug 2013 10:31:41 +0000 The biological activity of mushroom consumption is achieved by the antioxidant effect of constituent biomolecules released during digestion. In the following study, the consumption of mushroom fungi was determined to increase the number of Lactobacillus and Bifidobacterium strains within the colon. The main phenolic antioxidant compounds identified were both gentisic and homogentisic acids. Moreover, the flavonoid catechin as well as a significant amount of δ- and γ-tocopherols was determined. The amount of Lactobacillus and Bifidobacterium strains from different sections of the human colon was significantly correlated with levels of antioxidative biomolecules. The experimental data clearly demonstrate a significant impact of mushroom consumption on the fermentative function of microorganisms in the human colon, resulting in the homeostasis of normal physiological colonic functions. Emanuel Vamanu, Diana Pelinescu, Ionela Avram, and Sultana Nita Copyright © 2013 Emanuel Vamanu et al. All rights reserved. Monounsaturated Fatty Acids Are Substrates for Aldehyde Generation in Tellurite-Exposed Escherichia coli Wed, 07 Aug 2013 13:10:17 +0000 Reactive oxygen species (ROS) damage macromolecules and cellular components in nearly all kinds of cells and often generate toxic intracellular byproducts. In this work, aldehyde generation derived from the Escherichia coli membrane oxidation as well as membrane fatty acid profiles, protein oxidation, and bacterial resistance to oxidative stress elicitors was evaluated. Studies included wild-type cells as well as cells exhibiting a modulated monounsaturated fatty acid (MUFA) ratio. The hydroxyaldehyde 4-hydroxy 2-nonenal was found to be most likely produced by E. coli, whose levels are dependent upon exposure to oxidative stress elicitors. Aldehyde amounts and markers of oxidative damage decreased upon exposure to E. coli containing low MUFA ratios, which was paralleled by a concomitant increase in resistance to ROS-generating compounds. MUFAs ratio, lipid peroxidation, and aldehyde generation were found to be directly related; that is, the lower the MUFAs ratio, the lower the peroxide and aldehyde generation levels. These results provide additional evidence about MUFAs being targets for membrane lipid oxidation and their relevance in aldehyde generation. Gonzalo A. Pradenas, Waldo A. Díaz-Vásquez, José M. Pérez-Donoso, and Claudio C. Vásquez Copyright © 2013 Gonzalo A. Pradenas et al. All rights reserved. Secretion of Biologically Active Heterologous Oxalate Decarboxylase (OxdC) in Lactobacillus plantarum WCFS1 Using Homologous Signal Peptides Thu, 18 Jul 2013 11:45:07 +0000 Current treatment options for patients with hyperoxaluria and calcium oxalate stone diseases are limited and do not always lead to sufficient reduction in urinary oxalate excretion. Oxalate degrading bacteria have been suggested for degrading intestinal oxalate for the prevention of calcium oxalate stone. Here, we reported a recombinant Lactobacillus plantarum WCFS1 (L. plantarum) secreting heterologous oxalate decarboxylase (OxdC) that may provide possible therapeutic approach by degrading intestinal oxalate. The results showed secretion and functional expression of OxdC protein in L. plantarum driven by signal peptides Lp_0373 and Lp_3050. Supernatant of the recombinant strain containing pLp_0373sOxdC and pLp_3050sOxdC showed OxdC activity of 0.05 U/mg and 0.02 U/mg protein, while the purified OxdC from the supernatant showed specific activity of 18.3 U/mg and 17.5 U/mg protein, respectively. The concentration of OxdC protein in the supernatant was 8–12 μg/mL. The recombinant strain showed up to 50% oxalate reduction in medium containing 10 mM oxalate. In conclusion, the recombinant L. plantarum harboring pLp_0373sOxdC and pLp_3050sOxdC can express and secrete functional OxdC and degrade oxalate up to 50% and 30%, respectively. Ponnusamy Sasikumar, Sivasamy Gomathi, Kolandaswamy Anbazhagan, and Govindan Sadasivam Selvam Copyright © 2013 Ponnusamy Sasikumar et al. All rights reserved. Microbial Biofilms and Breast Tissue Expanders Tue, 16 Jul 2013 13:10:55 +0000 We previously developed and validated a vortexing-sonication technique for detection of biofilm bacteria on the surface of explanted prosthetic joints. Herein, we evaluated this technique for diagnosis of infected breast tissue expanders and used it to assess colonization of breast tissue expanders. From April 2008 to December 2011, we studied 328 breast tissue expanders at Mayo Clinic, Rochester, MN, USA. Of seven clinically infected breast tissue expanders, six (85.7%) had positive cultures, one of which grew Propionibacterium species. Fifty-two of 321 breast tissue expanders (16.2%, 95% CI, 12.3–20.7%) without clinical evidence of infection also had positive cultures, 45 growing Propionibacterium species and ten coagulase-negative staphylococci. While vortexing-sonication can detect clinically infected breast tissue expanders, 16 percent of breast tissue expanders appear to be asymptomatically colonized with normal skin flora, most commonly, Propionibacterium species. Melissa J. Karau, Kerryl E. Greenwood-Quaintance, Suzannah M. Schmidt, Nho V. Tran, Phyllis A. Convery, Steven R. Jacobson, Uldis Bite, Ricky P. Clay, Paul M. Petty, Craig H. Johnson, Jayawant Mandrekar, and Robin Patel Copyright © 2013 Melissa J. Karau et al. All rights reserved. Isolation and Molecular Identification of Keratinophilic Fungi from Public Parks Soil in Shiraz, Iran Mon, 15 Jul 2013 11:45:35 +0000 Introduction. Keratinophilic fungi are an important group of fungi that live in soil. The aim of this study was to isolate and identify keratinophilic fungi from the soil of different parks in Shiraz. Materials and Methods. A total of 196 soil samples from 43 parks were collected. Isolation of the fungi was performed by hair bait technique. The isolated colonies were identified by morphologic feature of macro- and microconidia and molecular method, using DNA sequence analysis. ITS region of ribosomal DNA was amplified and the PCR products were sequenced. Results. 411 isolates from 22 genera were identified. Fusarium (23.8%), Chrysosporium (13.13%), Acremonium (12.65%), Penicillium (12.39%), Microsporum gypseum (1.94%), Bionectria ochroleuca (1.21%), Bipolaris spicifera (1.21%), Scedosporium apiospermum (0.82%), Phialophora reptans (0.82%), Cephalosporium curtipes (0.49%), Scedosporium dehoogii (0.24%), Ochroconis constricta (0.24%), Nectria mauritiicola (0.49%), Chaetomium (0.49%), Scopulariopsis (0.24%), Malbranchea (0.24%), and Tritirachium (0.24%) were the most important isolates. Most of the fungi were isolated from the soils with the PH range of 7 to 8. Conclusion. Our study results showed that many keratinophilic fungi isolated from the parks soil are important for public health and children are an important group at a high risk of being exposed to these fungi. Keyvan Pakshir, Moosa Rahimi Ghiasi, Kamiar Zomorodian, and Ali Reza Gharavi Copyright © 2013 Keyvan Pakshir et al. All rights reserved. Characterization of Legionella pneumophila Isolated from Environmental Water and Ashiyu Foot Spa Thu, 11 Jul 2013 14:01:15 +0000 Hot springs are the most common infectious source of Legionella pneumophila in Japan. However, little is known about the association between L. pneumophila and environmental waters other than hot springs. In this study, water samples from 22 environmental water sites were surveyed; of the 22 samples, five were L. pneumophila positive (23%). L. pneumophila was mainly isolated from ashiyu foot spas, a type of hot spring for the feet (3/8, 38%). These isolates had genetic loci or genes that encoded the virulence factors of L. pneumophila. Moreover, these isolates showed higher intracellular growth and stronger cytotoxicity compared with the reference strain. These results suggest that ashiyu foot spa can be the original source for L. pneumophila infection. Masato Tachibana, Masaya Nakamoto, Yui Kimura, Takashi Shimizu, and Masahisa Watarai Copyright © 2013 Masato Tachibana et al. All rights reserved. Serotype Distribution of Salmonella Isolates from Turkey Ground Meat and Meat Parts Wed, 10 Jul 2013 10:21:08 +0000 The aim of the study was to find out the serotype distribution of 169 Salmonella colonies recovered from 112 Salmonella positive ground turkey (115 colonies) and 52 turkey meat parts (54 colonies). Out of 15 Salmonella serotypes: S. Corvallis, S. Kentucky, S. Bredeney, S. Virchow, S. Saintpaul and S. Agona were identified as the predominant serovars at the rates of 27%, 13%, 12%, 12%, 11%, and 10%, respectively. Other serotypes were below 6% of the total isolates. All S. Kentucky and S. Virchow and most of the S. Corvallis (39/46) and S. Heidelberg (9/9) serotypes were recovered from ground turkey. The results indicate that turkey ground meat and meat parts were contaminated with quite distinct Salmonella serotypes. This is the first study reporting Salmonella serotype distribution in turkey meat and S. Corvallis as predominant serotype in poultry meat in Turkey. Irfan Erol, Muammer Goncuoglu, Naim Deniz Ayaz, Lüppo Ellerbroek, Fatma Seda Bilir Ormanci, and Ozlem Iseri Kangal Copyright © 2013 Irfan Erol et al. All rights reserved. Characterization and Complete Sequence of Lactonase Enzyme from Bacillus weihenstephanensis Isolate P65 with Potential Activity against Acyl Homoserine Lactone Signal Molecules Tue, 09 Jul 2013 10:48:18 +0000 Acyl homoserine lactones (AHLs) are the most common class of quorum sensing signal molecules (autoinducers) that have been reported to be essential for virulence of many relevant pathogenic bacteria such as Pseudomonas aeruginosa. New approach for controlling infections of such bacteria is through quorum quenching. In this study, the acyl homoserine lactone inhibitory activity of the crude enzyme from a Bacillus weihenstephanensis-isolate P65 was characterized. The crude enzyme was found to have relatively high thermal stability and was stable in pH range 6 to 9. The crude enzyme extract was found to have lactonase activity of 36.3 U/mg total protein. Maximum enzyme activity was achieved within a range of 28–50°C and pH 6–9. None of the metals used enhanced the activity neither did EDTA inhibit it. However, a concentration of 10 mM Fe+2 reduced the activity to 73.8%. Catalytic activity and kinetic constants were determined using hexanoyl homoserine lactone as a substrate. Studying enzyme substrate specificity using synthetic standard signals displayed broad spectrum of activity. The enzyme was found to be constitutive. Isolation and complete nucleotide sequence of the respective lactonase gene were done and submitted to the Genbank database under accession code KC823046. Masarra Mohammed Sakr, Khaled Mohamed Anwar Aboshanab, Mohammad Mabrouk Aboulwafa, and Nadia Abdel-Haleem Hassouna Copyright © 2013 Masarra Mohammed Sakr et al. All rights reserved. Correlation between Phylogroups and Intracellular Proteomes of Propionibacterium acnes and Differences in the Protein Expression Profiles between Anaerobically and Aerobically Grown Cells Wed, 26 Jun 2013 11:18:52 +0000 Propionibacterium acnes is one of the dominant commensals on the human skin and also an opportunistic pathogen in relation to acne, sarcoidosis, prostate cancer, and various infections. Recent investigations using housekeeping and virulence genes have revealed that the species consists of three major evolutionary clades (types I, II, and III). In order to investigate protein expression differences between these phylogroups, proteomic profiles of 21 strains of P. acnes were investigated. The proteins extracted from cells cultured under anaerobic and aerobic conditions were analysed using a SELDI-TOF mass spectrometer, high-resolution capillary gel electrophoresis, and LC-MS/ MS. The SELDI spectral profiles were visualised as a heat map and a dendrogram, which resulted in four proteomic groups. Strains belonging to type I were represented in the proteome Group A, while Group B contained type III strains. Groups C and D contained mixtures of types I and II. Each of these groups was not influenced by differences in culture conditions. Under anoxic growth conditions, a type IB strain yielded high expressions of some proteins, such as methylmalonyl-CoA epimerase and the Christie-Atkins-Munch-Petersen (CAMP) factor. The present study revealed good congruence between genomic and proteomic data suggesting that the microenvironment of each subtype may influence protein expression. Itaru Dekio, Renata Culak, Min Fang, Graham Ball, Saheer Gharbia, and Haroun N. Shah Copyright © 2013 Itaru Dekio et al. All rights reserved. Candida Infections, Causes, Targets, and Resistance Mechanisms: Traditional and Alternative Antifungal Agents Wed, 26 Jun 2013 11:03:41 +0000 The genus Candida includes about 200 different species, but only a few species are human opportunistic pathogens and cause infections when the host becomes debilitated or immunocompromised. Candida infections can be superficial or invasive. Superficial infections often affect the skin or mucous membranes and can be treated successfully with topical antifungal drugs. However, invasive fungal infections are often life-threatening, probably due to inefficient diagnostic methods and inappropriate initial antifungal therapies. Here, we briefly review our current knowledge of pathogenic species of the genus Candida and yeast infection causes and then focus on current antifungal drugs and resistance mechanisms. An overview of new therapeutic alternatives for the treatment of Candida infections is also provided. Claudia Spampinato and Darío Leonardi Copyright © 2013 Claudia Spampinato and Darío Leonardi. All rights reserved. A Simple Assay to Screen Antimicrobial Compounds Potentiating the Activity of Current Antibiotics Thu, 20 Jun 2013 13:15:51 +0000 Antibiotic resistance continues to pose a significant problem in the management of bacterial infections, despite advances in antimicrobial chemotherapy and supportive care. Here, we suggest a simple, inexpensive, and easy-to-perform assay to screen antimicrobial compounds from natural products or synthetic chemical libraries for their potential to work in tandem with the available antibiotics against multiple drug-resistant bacteria. The aqueous extract of Juglans regia tree bark was tested against representative multiple drug-resistant bacteria in the aforementioned assay to determine whether it potentiates the activity of selected antibiotics. The aqueous extract of J. regia bark was added to Mueller-Hinton agar, followed by a lawn of multiple drug-resistant bacteria, Salmonella typhi or enteropathogenic E. coli. Next, filter paper discs impregnated with different classes of antibiotics were placed on the agar surface. Bacteria incubated with extract or antibiotics alone were used as controls. The results showed a significant increase (>30%) in the zone of inhibition around the aztreonam, cefuroxime, and ampicillin discs compared with bacteria incubated with the antibiotics/extract alone. In conclusion, our assay is able to detect either synergistic or additive action of J. regia extract against multiple drug-resistant bacteria when tested with a range of antibiotics. Junaid Iqbal, Ruqaiyyah Siddiqui, Shahana Urooj Kazmi, and Naveed Ahmed Khan Copyright © 2013 Junaid Iqbal et al. All rights reserved. Changes of PBP5 Gene Expression in Enterococcal Isolates from Renal Transplantation Recipients Wed, 19 Jun 2013 12:07:06 +0000 The aim of the study was to evaluate changes in expression of PBP5 gene associated with immunosuppression. A linear locked nucleic acid (LNA) probe was used to measure resistance gene expression by the Flow-FISH method. Expression of the PBP5 gene measured by Flow-FISH was higher in enterococcal strains isolated from renal transplantation (RTx) recipients than in commensal strains. Additionally, in contrast to commensal strains in isolates from RTx patients, PBP5 gene expression was 17.45% higher in biofilms than in planktonic cells. Detailed comparison also showed that cyclosporine seemed to induce higher expression of PBP5 as compared to tacrolimus. T. Jarzembowski, A. Daca, J. Witkowski, B. Rutkowski, J. Gołębiewska, and A. Dębska-Ślizień Copyright © 2013 T. Jarzembowski et al. All rights reserved. Antioxidant Capacity and Antimutagenic Potential of Murraya koenigii Tue, 18 Jun 2013 16:24:15 +0000 It is well known that the intake of antioxidants with increased consumption of fruits and vegetables and medicinal herbs contributes towards reduced risk of certain diseases including cancers. This study aims to evaluate the broad-spectrum antioxidant and antimutagenic activities as well as to elucidate phytochemical profile of an Indian medicinal plant Murraya koenigii (curry) leaves. Leaves of the plant were successively fractionated in various organic solvents. Benzene fraction demonstrated the highest phenolic content followed by petroleum ether. The benzene fraction showed maximum antioxidant activity in all tested assays, namely, phosphomolybdenum, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, ferric reducing antioxidant power (FRAP) and cupric reducing antioxidant capacity (CUPRAC) assays. Based on the promising broad-spectrum antioxidant activity, benzene fraction was further evaluated for antimutagenic activity and showed a dose-dependent antimutagenic response in Ames Salmonella mutagenicity assay. It inhibited 72–86% mutagenicity induced by sodium azide, methyl methanesulfonate, benzo(a)pyrene, and 2-aminoflourene at the maximum tested concentration (100 μg/mL) in Salmonella typhimurium tester strains. At least 21 compounds were detected by GC/MS. The findings clearly demonstrated that phenolic-rich benzene fraction has promising broad-spectrum antioxidant and antimutagenic property and needs further evaluation to exploit its therapeutic potential. Maryam Zahin, Farrukh Aqil, Fohad Mabood Husain, and Iqbal Ahmad Copyright © 2013 Maryam Zahin et al. All rights reserved. Effects of Chitosan on Candida albicans: Conditions for Its Antifungal Activity Mon, 17 Jun 2013 09:44:17 +0000 The effects of low molecular weight (96.5 KDa) chitosan on the pathogenic yeast Candida albicans were studied. Low concentrations of chitosan, around 2.5 to 10 μg·mL−1 produced (a) an efflux of K+ and stimulation of extracellular acidification, (b) an inhibition of Rb+ uptake, (c) an increased transmembrane potential difference of the cells, and (d) an increased uptake of Ca2+. It is proposed that these effects are due to a decrease of the negative surface charge of the cells resulting from a strong binding of the polymer to the cells. At higher concentrations, besides the efflux of K+, it produced (a) a large efflux of phosphates and material absorbing at 260 nm, (b) a decreased uptake of Ca2+, (c) an inhibition of fermentation and respiration, and (d) the inhibition of growth. The effects depend on the medium used and the amount of cells, but in YPD high concentrations close to 1 mg·mL−1 are required to produce the disruption of the cell membrane, the efflux of protein, and the growth inhibition. Besides the findings at low chitosan concentrations, this work provides an insight of the conditions required for chitosan to act as a fungistatic or antifungal and proposes a method for the permeabilization of yeast cells. Antonio Peña, Norma Silvia Sánchez, and Martha Calahorra Copyright © 2013 Antonio Peña et al. All rights reserved. Etiologic Aspect of Sarcoidosis as an Allergic Endogenous Infection Caused by Propionibacterium acnes Sun, 16 Jun 2013 10:08:53 +0000 Sarcoidosis is a systemic granulomatous disease of unknown etiology. Propionibacterium acnes is the only microorganism that has been isolated from sarcoid lesions. Many P. acnes have been detected in sarcoid lymph nodes using quantitative PCR and in sarcoid granulomas by in situ hybridization. P. acnes trigger factor protein causes a cellular immune response only in sarcoid patients and induces pulmonary granulomas in mice sensitized with the protein and adjuvant, but only those with latent P. acnes infection in their lungs. Eradication of P. acnes by antibiotics prevents the development of granulomas in this experimental model. Although P. acnes is the most common commensal bacterium in the lungs and lymph nodes, P. acnes-specific antibody detected the bacterium within sarcoid granulomas of these organs. P. acnes can cause latent infection in the lung and lymph node and persist in a cell-wall-deficient form. The dormant form is activated endogenously under certain conditions and proliferates at the site of latent infection. In patients with P. acnes hypersensitivity, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Proliferating bacteria may escape granulomatous isolation, spreading to other organs. Latent P. acnes infection in systemic organs can be reactivated by another triggering event, leading to systemic sarcoidosis. Yoshinobu Eishi Copyright © 2013 Yoshinobu Eishi. All rights reserved. Deciphering the Intracellular Fate of Propionibacterium acnes in Macrophages Wed, 05 Jun 2013 15:36:54 +0000 Propionibacterium acnes is a Gram-positive bacterium that colonizes various niches of the human body, particularly the sebaceous follicles of the skin. Over the last years a role of this common skin bacterium as an opportunistic pathogen has been explored. Persistence of P. acnes in host tissue has been associated with chronic inflammation and disease development, for example, in prostate pathologies. This study investigated the intracellular fate of P. acnes in macrophages after phagocytosis. In a mouse model of P. acnes-induced chronic prostatic inflammation, the bacterium could be detected in prostate-infiltrating macrophages at 2 weeks postinfection. Further studies performed in the human macrophage cell line THP-1 revealed intracellular survival and persistence of P. acnes but no intracellular replication or escape from the host cell. Confocal analyses of phagosome acidification and maturation were performed. Acidification of P. acnes-containing phagosomes was observed at 6 h postinfection but then lost again, indicative of cytosolic escape of P. acnes or intraphagosomal pH neutralization. No colocalization with the lysosomal markers LAMP1 and cathepsin D was observed, implying that the P. acnes-containing phagosome does not fuse with lysosomes. Our findings give first insights into the intracellular fate of P. acnes; its persistency is likely to be important for the development of P. acnes-associated inflammatory diseases. Natalie Fischer, Tim N. Mak, Debika Biswal Shinohara, Karen S. Sfanos, Thomas F. Meyer, and Holger Brüggemann Copyright © 2013 Natalie Fischer et al. All rights reserved. A Honey Trap for the Treatment of Acne: Manipulating the Follicular Microenvironment to Control Propionibacterium acnes Tue, 14 May 2013 09:01:39 +0000 Today, as 40 years ago, we still rely on a limited number of antibiotics and benzoyl peroxide to treat inflammatory acne. An alternative way of suppressing the growth of Propionibacterium acnes is to target the environment in which it thrives. We conjecture that P. acnes colonises a relatively “extreme” habitat especially in relation to the availability of water and possibly related factors such as ionic strength and osmolarity. We hypothesise that the limiting “nutrient” within pilosebaceous follicles is water since native sebum as secreted by the sebaceous gland contains none. An aqueous component must be available within colonised follicles, and water may be a major factor determining which follicles can sustain microbial populations. One way of preventing microbial growth is to reduce the water activity () of this component with a biocompatible solute of very high water solubility. For the method to work effectively, the solute must be small, easily diffusible, and minimally soluble in sebaceous lipids. Xylose and sucrose, which fulfil these criteria, are nonfermentable by P. acnes and have been used to reduce water activity and hence bacterial colonisation of wounds. A new follicularly targeted topical treatment for acne based on this approach should be well tolerated and highly effective. E. Anne Eady, Alison M. Layton, and Jonathan H. Cove Copyright © 2013 E. Anne Eady et al. All rights reserved.