BioMed Research International: Microbiology The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . 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. Short Communication: 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/g), lipase yield ( U/g), and biomass productivity ( 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. Analysis of Complete Genomes of Propionibacterium acnes Reveals a Novel Plasmid and Increased Pseudogenes in an Acne Associated Strain Mon, 13 May 2013 08:17:47 +0000 The human skin harbors a diverse community of bacteria, including the Gram-positive, anaerobic bacterium Propionibacterium acnes. P. acnes has historically been linked to the pathogenesis of acne vulgaris, a common skin disease affecting over 80% of all adolescents in the US. To gain insight into potential P. acnes pathogenic mechanisms, we previously sequenced the complete genome of a P. acnes strain HL096PA1 that is highly associated with acne. In this study, we compared its genome to the first published complete genome KPA171202. HL096PA1 harbors a linear plasmid, pIMPLE-HL096PA1. This is the first described P. acnes plasmid. We also observed a five-fold increase of pseudogenes in HL096PA1, several of which encode proteins in carbohydrate transport and metabolism. In addition, our analysis revealed a few island-like genomic regions that are unique to HL096PA1 and a large genomic inversion spanning the ribosomal operons. Together, these findings offer a basis for understanding P. acnes virulent properties, host adaptation mechanisms, and its potential role in acne pathogenesis at the strain level. Furthermore, the plasmid identified in HL096PA1 may potentially provide a new opportunity for P. acnes genetic manipulation and targeted therapy against specific disease-associated strains. Gabriela Kasimatis, Sorel Fitz-Gibbon, Shuta Tomida, Marthew Wong, and Huiying Li Copyright © 2013 Gabriela Kasimatis et al. All rights reserved. Bacteriophages Infecting Propionibacterium acnes Thu, 11 Apr 2013 14:48:50 +0000 Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of the Propionibacterium acnes bacteriophage research and point out how bacteriophage research has been an important part of the research on P. acnes itself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs). Finally, the potential to use P. acnes bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases will be discussed. Holger Brüggemann and Rolf Lood Copyright © 2013 Holger Brüggemann and Rolf Lood. All rights reserved. Estimation of Ion Competition via Correlated Responsivity Offset in Linear Ion Trap Mass Spectrometry Analysis: Theory and Practical Use in the Analysis of Cyanobacterial Hepatotoxin Microcystin-LR in Extracts of Food Additives Tue, 26 Mar 2013 15:06:25 +0000 Responsivity is a conversion qualification of a measurement device given by the functional dependence between the input and output quantities. A concentration-response-dependent calibration curve represents the most simple experiment for the measurement of responsivity in mass spectrometry. The cyanobacterial hepatotoxin microcystin-LR content in complex biological matrices of food additives was chosen as a model example of a typical problem. The calibration curves for pure microcystin and its mixtures with extracts of green alga and fish meat were reconstructed from the series of measurement. A novel approach for the quantitative estimation of ion competition in ESI is proposed in this paper. We define the correlated responsivity offset in the intensity values using the approximation of minimal correlation given by the matrix to the target mass values of the analyte. The estimation of the matrix influence enables the approximation of the position of a priori unknown responsivity and was easily evaluated using a simple algorithm. The method itself is directly derived from the basic attributes of the theory of measurements. There is sufficient agreement between the theoretical and experimental values. However, some theoretical issues are discussed to avoid misinterpretations and excessive expectations. Jan Urban, Pavel Hrouzek, Dalibor Štys, and Harald Martens Copyright © 2013 Jan Urban et al. All rights reserved. A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes Thu, 21 Mar 2013 11:20:14 +0000 A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM. Guo-Qing Zhang, Ying-Ying Wu, Tzi-Bun Ng, Qing-Jun Chen, and He-Xiang Wang Copyright © 2013 Guo-Qing Zhang et al. All rights reserved. Anti-Inflammatory and Antiapoptotic Responses to Infection: A Common Denominator of Human and Bovine Macrophages Infected with Mycobacterium avium Subsp. paratuberculosis Sun, 20 Jan 2013 07:40:49 +0000 Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of a chronic intestinal inflammation in ruminants named Johne's disease or paratuberculosis and a possible etiopathological agent of human Crohn's disease (CD). Analysis of macrophage transcriptomes in response to Map infection is expected to provide key missing information in the understanding of the role of this pathogen in establishing an inappropriate and persistent infection in a susceptible host and of the molecular mechanisms that might underlie the early phases of CD. In this paper we summarize transcriptomic studies of human and bovine peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDMs), and macrophages-like cell lines in vitro infected with Map. Most studies included in this paper consistently reported common gene expression signatures of bovine and human macrophages in response to Map such as enhanced expression of the anti-inflammatory cytokines IL-10 and IL-6, which promote bacterial survival. Overexpression of IL-10 could be responsible for the Map-associated reduction in the expression of the proapoptotic TNF-α gene observed in bovine and human macrophages. Naiara Abendaño, Ramon A. Juste, and Marta Alonso-Hearn Copyright © 2013 Naiara Abendaño et al. All rights reserved. Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Staphylococcus aureus Tue, 15 Jan 2013 11:54:30 +0000 Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP) assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 102 CFU/mL when compared to 12.5 ng/μL and 103 CFU/mL for PCR (spa and arcC). Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV), and 100% negative predictive value (NPV). When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control), the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields. King Ting Lim, Cindy Shuan Ju Teh, and Kwai Lin Thong Copyright © 2013 King Ting Lim et al. All rights reserved. Improved Growth of Lactobacillus bulgaricus and Streptococcus thermophilus as well as Increased Antioxidant Activity by Biotransforming Litchi Pericarp Polysaccharide with Aspergillus awamori Thu, 03 Jan 2013 14:29:38 +0000 This study was conducted to increase the bioactivity of litchi pericarp polysaccharides (LPPs) biotransformed by Aspergillus awamori. Compared to the non-A. awamori-fermented LPP, the growth effects of A. awamori-fermented LPP on Lactobacillus bulgaricus and Streptococcus thermophilus were four and two times higher after 3 days of fermentation, respectively. Increased 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and DNA protection activity of litchi pericarp polysaccharides were also achieved after A. awamori fermentation. Moreover, the relative content of glucose and arabinose in LPP after fermentation decreased from 58.82% to 22.60% and from 18.82% to 10.09%, respectively, with a concomitant increase in the relative contents of galactose, rhamnose, xylose, and mannose. Furthermore, lower molecular weight polysaccharides were obtained after A. awamori fermentation. It can be concluded that A. awamori was effective in biotransforming LPP into a bioactive mixture with lower molecular weight polysaccharides and higher antioxidant activity and relative galactose content. Sen Lin, Lingrong Wen, Bao Yang, Guoxiang Jiang, John Shi, Feng Chen, and Yueming Jiang Copyright © 2013 Sen Lin et al. All rights reserved. Transcriptional Response of Bovine Monocyte-Derived Macrophages after the Infection with Different Argentinean Mycobacterium bovis Isolates Tue, 01 Jan 2013 10:21:47 +0000 Infection of bovines with Mycobacterium bovis causes important financial hardship in many countries presenting also a risk for humans. M. bovis is known to be adapted to survive and thrive within the intramacrophage environment. In spite of its relevance, at present the information about macrophage expression patterns is scarce, particularly regarding the bovine host. In this study, transcriptomic analysis was used to detect genes differentially expressed in macrophages derived from peripheral blood mononuclear cells at early stages of infection with two Argentinean strains of M. bovis, a virulent and an attenuated strains. The results showed that the number of differentially expressed genes in the cells infected with the virulent strain (5) was significantly lower than those in the cells infected with the attenuated strain (172). Several genes were more strongly expressed in infected macrophages. Among them, we detected encoding transcription factors, anthrax toxin receptor, cell division and apoptosis regulator, ankyrin proteins, cytoskeleton proteins, protein of cell differentiation, and regulators of endocytic traffic of membrane. Quantitative real-time PCR of a selected group of differentially expressed genes confirmed the microarrays results. Altogether, the present results contribute to understanding the mechanisms involved in the early interaction of M. bovis with the bovine macrophage. Karina Caimi, Federico Blanco, Marcelo Soria, and Fabiana Bigi Copyright © 2012 Karina Caimi et al. All rights reserved. Synthesis and Antimicrobial Activity of Silver-Doped Hydroxyapatite Nanoparticles Sun, 30 Dec 2012 15:14:02 +0000 The synthesis of nanosized particles of Ag-doped hydroxyapatite with antibacterial properties is of great interest for the development of new biomedical applications. The aim of this study was the evaluation of (PO4)6(OH)2 nanoparticles (Ag:HAp-NPs) for their antibacterial and antifungal activity. Resistance to antimicrobial agents by pathogenic bacteria has emerged in the recent years and became a major health problem. Here, we report a method for synthesizing Ag doped nanocrystalline hydroxyapatite. A silver-doped nanocrystalline hydroxyapatite was synthesized at 100°C in deionised water. Also, in this paper Ag:HAp-NPs are evaluated for their antimicrobial activity against Gram-positive and Gram-negative bacteria and fungal strains. The specific antimicrobial activity revealed by the qualitative assay is demonstrating that our compounds are interacting differently with the microbial targets, probably due to the differences in the microbial wall structures. Carmen Steluta Ciobanu, Simona Liliana Iconaru, Mariana Carmen Chifiriuc, Adrian Costescu, Philippe Le Coustumer, and Daniela Predoi Copyright © 2013 Carmen Steluta Ciobanu et al. All rights reserved. Formation and Resuscitation of Viable but Nonculturable Salmonella typhi Wed, 26 Dec 2012 08:52:25 +0000 Salmonella typhi is a pathogen that causes the human disease of typhoid fever. The aim of this study was to investigate the viable but nonculturable (VBNC) state of S. typhi. Some samples were stimulated at 4°C or −20°C, while others were induced by different concentrations of CuSO4. Total cell counts remained constant throughout several days by acridine orange direct counting; however, plate counts declined to undetectable levels within 48 hours by plate counting at −20°C. The direct viable counts remained fairly constant at this level by direct viable counting. Carbon and nitrogen materials slowly decreased which indicated that a large population of cells existed in the VBNC state and entered the VBNC state in response to exposure to 0.01 or 0.015 mmol/L CuSO4 for more than 14 or 12 days, respectively. Adding 3% Tween 20 or 1% catalase enabled cells to become culturable again, with resuscitation times of 48 h and 24 h, respectively. The atomic force microscope results showed that cells gradually changed in shape from short rods to coccoids, and decreased in size when they entered the VBNC state. Further animal experiments suggested that resuscitated cells might regain pathogenicity. Bin Zeng, Guozhong Zhao, Xiaohong Cao, Zhen Yang, Chunling Wang, and Lihua Hou Copyright © 2013 Bin Zeng et al. All rights reserved. In Vitro Antibacterial and Antibiofilm Activities of Chlorogenic Acid against Clinical Isolates of Stenotrophomonas maltophilia including the Trimethoprim/Sulfamethoxazole Resistant Strain Thu, 20 Dec 2012 09:42:43 +0000 The in vitro antibacterial and antibiofilm activity of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia was investigated through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill and biofilm assays. A total of 9 clinical S. maltophilia isolates including one isolate resistant to trimethoprim/sulfamethoxazole (TMP/SMX) were tested. The inhibition zone sizes for the isolates ranged from 17 to 29 mm, while the MIC and MBC values ranged from 8 to 16 μg mL−1 and 16 to 32 μg mL−1. Chlorogenic acid appeared to be strongly bactericidal at 4x MIC, with a 2-log reduction in viable bacteria at 10 h. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC compared to 1x MIC values ( A 490 nm) of chlorogenic acid. The data from this study support the notion that the chlorogenic acid has promising in vitro antibacterial and antibiofilm activities against S. maltophilia. Arunkumar Karunanidhi, Renjan Thomas, Alex van Belkum, and Vasanthakumari Neela Copyright © 2013 Arunkumar Karunanidhi et al. All rights reserved. Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus in Dairy Cow Suffering from Mastitis Mon, 19 Nov 2012 16:57:48 +0000 To develop a rapid detection method of Staphylococcus aureus using loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of the nuc gene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was  CFU/mL and that of PCR was  CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection of Staphylococcus aureus has many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection of Staphylococcus aureus. Zhang Tie, Wang Chunguang, Wei Xiaoyuan, Zhao Xinghua, and Zhong Xiuhui Copyright © 2012 Zhang Tie et al. All rights reserved. Cytotoxicity of Bacterial Metabolic Products, including Listeriolysin O, on Leukocyte Targets Wed, 03 Oct 2012 10:16:40 +0000 Bacterial toxins can exhibit anticancer activities. Here we investigated the anticancer effects of the listeriolysin O toxin produced by Listeria monocytogenes. We found that supernatants of Listeria monocytogenes strains (wild type, 1189, and 1190) were cytotoxic to the Jurkat cell line and human peripheral blood mononuclear cells (PBMC) in a concentration-dependent manner. The supernatant of strain 1044, not producing listeriolysin O, was inactive. The supernatants of Listeria strains were also cytotoxic toward B cells of chronic leukemia patients, with no significant differences in activities between strains. We also tested supernatants of Bacillus subtilis strains BR1-90, BR1-S, and BR1-89 producing listeriolysin O. BR1-S and BR1-89 were cytotoxic to PBMC and to Jurkat cells, the latter being more sensitive to the supernatants. BR1-90 was not hemolytic or cytotoxic to PBMC, but was cytotoxic to Jurkat cells in the concentration range of 10–30%, suggesting that listeriolysin O is selectively effective against T cells. Overall, the response of human peripheral blood mononuclear and human leukemia cell lines to bacteria supernatants containing listeriolysin O depended on the bacteria strain, target cell type, and supernatant concentration. R. Stachowiak, M. Lyzniak, B. K. Budziszewska, K. Roeske, J. Bielecki, G. Hoser, and J. Kawiak Copyright © 2012 R. Stachowiak et al. All rights reserved. Characterization of Flavonol Inhibition of DnaB Helicase: Real-Time Monitoring, Structural Modeling, and Proposed Mechanism Tue, 02 Oct 2012 13:38:16 +0000 DnaB helicases are motor proteins essential for DNA replication, repair, and recombination and may be a promising target for developing new drugs for antibiotic-resistant bacteria. Previously, we established that flavonols significantly decreased the binding ability of Klebsiella pneumoniae DnaB helicase (KpDnaB) to dNTP. Here, we further investigated the effect of flavonols on the inhibition of the ssDNA binding, ATPase activity, and dsDNA-unwinding activity of KpDnaB. The ssDNA-stimulated ATPase activity of KpDnaB was decreased to 59%, 75%, 65%, and 57%, in the presence of myricetin, quercetin, kaempferol, and galangin, respectively. The ssDNA-binding activity of KpDnaB was only slightly decreased by flavonols. We used a continuous fluorescence assay, based on fluorescence resonance energy transfer (FRET), for real-time monitoring of KpDnaB helicase activity in the absence and presence of flavonols. Using this assay, the flavonol-mediated inhibition of the dsDNA-unwinding activity of KpDnaB was observed. Modeled structures of bound and unbound DNA showed flavonols binding to KpDnaB with distinct poses. In addition, these structural models indicated that L214 is a key residue in binding any flavonol. On the basis of these results, we proposed mechanisms for flavonol inhibition of DNA helicase. The resulting information may be useful in designing compounds that target K. pneumoniae and other bacterial DnaB helicases. Hsin-Hsien Lin and Cheng-Yang Huang Copyright © 2012 Hsin-Hsien Lin and Cheng-Yang Huang. All rights reserved. Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin Tue, 02 Oct 2012 13:33:59 +0000 A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant. Cord C. Uphoff, Sabine-A. Denkmann, and Hans G. Drexler Copyright © 2012 Cord C. Uphoff et al. All rights reserved. Strain-Specific Transfer of Antibiotic Resistance from an Environmental Plasmid to Foodborne Pathogens Wed, 27 Jun 2012 14:13:10 +0000 Pathogens resistant to multiple antibiotics are rapidly emerging, entailing important consequences for human health. This study investigated if the broad-host-range multiresistance plasmid pB10, isolated from a wastewater treatment plant, harbouring amoxicillin, streptomycin, sulfonamide, and tetracycline resistance genes, was transferable to the foodborne pathogens Salmonella spp. or E. coli O157:H7 and how this transfer alters the phenotype of the recipients. The transfer ratio was determined by both plating and flow cytometry. Antibiotic resistance profiles were determined for both recipients and transconjugants using the disk diffusion method. For 14 of the 15 recipient strains, transconjugants were detected. Based on plating, transfer ratios were between 6.8×10−9 and 3.0×10−2 while using flow cytometry, transfer ratios were between <1.0×10−5 and 1.9×10−2. With a few exceptions, the transconjugants showed phenotypically increased resistance, indicating that most of the transferred resistance genes were expressed. In summary, we showed that an environmental plasmid can be transferred into foodborne pathogenic bacteria at high transfer ratios. However, the transfer ratio seemed to be recipient strain dependent. Moreover, the newly acquired resistance genes could turn antibiotic susceptible strains into resistant ones, paving the way to compromise human health. Eva Van Meervenne, Els Van Coillie, Frederiek-Maarten Kerckhof, Frank Devlieghere, Lieve Herman, Leen S. P. De Gelder, Eva M. Top, and Nico Boon Copyright © 2012 Eva Van Meervenne et al. All rights reserved. Prevalence of Adhesion and Regulation of Biofilm-Related Genes in Different Clones of Staphylococcus aureus Sun, 03 Jun 2012 11:47:11 +0000 Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the same spa type were found to have similar properties in adheringto thepolystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes). icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC) was confirmed by RT-PCR. Salman Sahab Atshan, Mariana Nor Shamsudin, Zamberi Sekawi, Leslie Than Thian Lung, Rukman Awang Hamat, Arunkumar Karunanidhi, Alreshidi Mateg Ali, Ehsanollah Ghaznavi-Rad, Hamed Ghasemzadeh-Moghaddam, Johnson Shueh Chong Seng, Jayakayatri Jeevajothi Nathan, and Chong Pei Pei Copyright © 2012 Salman Sahab Atshan et al. All rights reserved. Comparative Characterisation of Genotypically Different Clones of MRSA in the Production of Biofilms Wed, 14 Mar 2012 10:46:28 +0000 The ability to adhere and produce biofilms is characteristic of enhanced virulence among isolates of methicillin-resistant Staphylococcus aureus (MRSA). The aim of the study is to find out whether these characteristics are consistently similar among isolates variations of MRSA. The study used 30 various isolates of MRSA belong to 13 spa types and 5 MLST types and determined the aggregation, the adherence, and the production of biofilms and slime for each isolate. The methods used to evaluate these characteristics were a modified Congo red agar assay (MCRA), a microtiter plate assay (MPA), high-magnification light microscopy, scanning electron microscopy (SEM), and PCR. The study found that isolates belonging to similar Spa, SCCmec, and ST types have similar abilities to produce biofilms; however, their ability to produce slime on CRA was found to be different. Moreover, isolates that have different Spa types showed high variation in their ability to produce biofilms. The results of light microscope revealed the isolates that produced strong and weak biofilms and formed similar aggregation on the glass surfaces. SEM results showed that all 30 MRSA isolates that were tested were 100% positive for biofilm formation, although to varying degrees. Further testing using PCR confirmed that 100% of the 30 isolates tested were positive for the presence of the icaADBC, fnbA, eno, ebps, clfA, and clfB genes. The prevalence of fib, cna, fnbB, and bbp in MRSA clones was 90, 93.33, 53.33, and 10%, respectively. This study indicate that differences in biofilm production capacities are caused by the differences in surface protein A (Spa) type and are not due to differences in MLST and SCCmec types. Salman Sahab Atshan, Mariana Nor Shamsudin, Leslie Than Thian Lung, Zamberi Sekawi, Ehsanollah Ghaznavi-Rad, and Chong Pei Pei Copyright © 2012 Salman Sahab Atshan et al. All rights reserved. Functional Foods: Towards Improving Oral Health Fri, 24 Feb 2012 16:40:44 +0000 Itzhak Ofek, Carla Pruzzo, and David Spratt Copyright © 2012 Itzhak Ofek et al. All rights reserved. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line Tue, 21 Feb 2012 13:27:24 +0000 Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed. Michał Arabski, Aneta Węgierek-Ciuk, Grzegorz Czerwonka, Anna Lankoff, and Wiesław Kaca Copyright © 2012 Michał Arabski et al. All rights reserved. Evaluation of Plant and Fungal Extracts for Their Potential Antigingivitis and Anticaries Activity Sun, 19 Feb 2012 14:09:48 +0000 The link between diet and health has lead to the promotion of functional foods which can enhance health. In this study, the oral health benefits of a number of food homogenates and high molecular mass and low molecular mass fractions were investigated. A comprehensive range of assays were performed to assess the action of these foods on the development of gingivitis and caries using bacterial species associated with these diseases. Both antigingivitis and anticaries effects were investigated by assays examining the prevention of biofilm formation and coaggregation, disruption of preexisting biofilms, and the foods' antibacterial effects. Assays investigating interactions with gingival epithelial cells and cytokine production were carried out to assess the foods' anti- gingivitis properties. Anti-caries properties such as interactions with hydroxyapatite, disruption of signal transduction, and the inhibition of acid production were investigated. The mushroom and chicory homogenates and low molecular mass fractions show promise as anti-caries and anti-gingivitis agents, and further testing and clinical trials will need to be performed to evaluate their true effectiveness in humans. D. A. Spratt, M. Daglia, A. Papetti, M. Stauder, D. O'Donnell, L. Ciric, A. Tymon, B. Repetto, C. Signoretto, Y. Houri-Haddad, M. Feldman, D. Steinberg, S. Lawton, P. Lingström, J. Pratten, E. Zaura, G. Gazzani, C. Pruzzo, and M. Wilson Copyright © 2012 D. A. Spratt et al. All rights reserved. The Anticaries Effect of a Food Extract (Shiitake) in a Short-Term Clinical Study Tue, 07 Feb 2012 15:25:16 +0000 The main objective was to investigate whether low-molecular-weight fraction of edible mushroom shiitake extract (Lentinus edodes) possesses caries-preventive properties. The study was designed as a double-blind, three-leg, cross-over, randomized, controlled clinical trial carried out on two series of volunteers at the University of Gothenburg, and the Academic Centre for Dentistry Amsterdam. Volunteers rinsed twice daily with a solution containing low-molecular-weight fraction of edible mushroom, placebo (negative control without active ingredients), or Meridol (positive control, AmF-SnF2) for two weeks, with a two-week washout period between each rinsing period. Changes in the acidogenicity of dental plaque before and after a sucrose challenge, shifts in microbial composition, and plaque scores were determined. Frequent rinses with shiitake reduced the metabolic activity of dental plaque. No reduction of plaque scores and no inhibition of the production of organic acids in plaque was found. Minor differences in microbial composition between test sessions were found. To conclude, the results indicate that shiitake extract has anticariogenic potential, but not to the same extent as the positive control. Peter Lingström, Egija Zaura, Haidar Hassan, Mark J. Buijs, Pamie Hedelin, Jonathan Pratten, David Spratt, Maria Daglia, Aneta Karbowiak, Caterina Signoretto, Martijn Rosema, Fridus van der Weijden, and Michael Wilson Copyright © 2012 Peter Lingström et al. All rights reserved. Good Oral Health and Diet Thu, 26 Jan 2012 07:30:27 +0000 An unhealthy diet has been implicated as risk factors for several chronic diseases that are known to be associated with oral diseases. Studies investigating the relationship between oral diseases and diet are limited. Therefore, this study was conducted to describe the relationship between healthy eating habits and oral health status. The dentistry has an important role in the diagnosis of oral diseases correlated with diet. Consistent nutrition guidelines are essential to improve health. A poor diet was significantly associated with increased odds of oral disease. Dietary advice for the prevention of oral diseases has to be a part of routine patient education practices. Inconsistencies in dietary advice may be linked to inadequate training of professionals. Literature suggests that the nutrition training of dentists and oral health training of dietitians and nutritionists is limited. G. A. Scardina and P. Messina Copyright © 2012 G. A. Scardina and P. Messina. All rights reserved. Inhibition of Streptococcus gordonii Metabolic Activity in Biofilm by Cranberry Juice High-Molecular-Weight Component Wed, 18 Jan 2012 09:54:25 +0000 Previous studies demonstrated that a cranberry high-molecular-mass, nondialyzable material (NDM) can inhibit adhesion of numerous species of bacteria and prevents bacterial coaggregation of bacterial pairs. Bacterial coaggregation leads to plaque formation leading to biofilm development on surfaces of oral cavity. In the present study, we evaluated the effect of low concentrations of NDM on Streptococcus gordonii metabolic activity and biofilm formation on restorative dental surfaces. We found that the NDM selectively inhibited metabolic activity of S. gordonii, without affecting bacterial viability. Inhibiting the metabolic activity of bacteria in biofilm may benefit the health of the oral cavity. Jegdish Babu, Cohen Blair, Shiloah Jacob, and Ofek Itzhak Copyright © 2012 Jegdish Babu et al. All rights reserved. Nutritional Requirements for the Improvement of Growth and Sporulation of Several Strains of Monascus purpureus on Solid State Cultivation Thu, 29 Dec 2011 12:00:08 +0000 This paper describes the nutritional requirements for the improvement of growth and sporulation of several strains of Monascus purpureus on solid state cultivation. The findings revealed that glucose enhanced growth of all M. purpureus strains tested but inhibited the sporulation rate. On the other hand, sucrose induced sporulation but inhibited production of cell mass. A combination of glucose and sucrose greatly enhanced sporulation and cell mass production of M. purpureus. Although growth and sporulation rate were related to the ratio of carbon to nitrogen (C/N ratio), the types and concentrations of carbon and nitrogen sources also greatly influenced the growth kinetics. Among the media tested, Hiroi-PDA medium was the most preferred medium for all M. purpureus strains tested for the enhancement of radial growth rate, sporulation, and cell production. Hence, Hiroi-PDA could be suggested as the generic basal medium for the cultivation of M. purpureus. However, individual medium optimization is required for significant enhancement in growth and sporulation of each strain of M. purpureus. Zahra Ajdari, Afshin Ebrahimpour, Musaalbakri Abdul Manan, Muhajir Hamid, Rosfarizan Mohamad, and Arbakariya B. Ariff Copyright © 2011 Zahra Ajdari et al. All rights reserved. The Fur Transcription Regulator and Fur-Regulated Genes in Clostridium botulinum A ATCC 3502 Mon, 12 Dec 2011 08:39:32 +0000 Clostridium botulinum is a spore-forming bacterium that can produce a very powerful neurotoxin that causes botulism. In this study, we have investigated the Fur transcription regulators in Clostridium botulinum and Fur-regulated genes in Clostridium botulinum A ATCC 3502. We found that gene loss may be the main cause leading to the different numbers of Fur transcription regulators in different Clostridium botulinum strains. Meanwhile, 46 operons were found to be regulated by the Fur transcription regulator in Clostridium botulinum A ATCC 3502, involved in several functional classifications, including iron acquisition, iron utilization, iron transport, and transcription regulator. Under an iron-restricted medium, we experimentally found that a Fur transcription regulator (CBO1372) and two operons (DedA, CBO2610–CBO2614 and ABC transporter, CBO0845–CBO0847) are shown to be differentially expressed in Clostridium botulinum A ATCC 3502. This study has provided-us novel insights into the diversity of Fur transcription regulators in different Clostridium botulinum strains and diversity of Fur-targeted genes, as well as a better understanding of the dynamic changes in iron restriction occurring in response to this stress. Weibin Zhang, Junhua Ma, Chengyuan Zang, Yingying Song, and Peipei Liu Copyright © 2011 Weibin Zhang et al. All rights reserved. Plant and Fungal Food Components with Potential Activity on the Development of Microbial Oral Diseases Mon, 17 Oct 2011 13:19:36 +0000 This paper reports the content in macronutrients, free sugars, polyphenols, and inorganic ions, known to exert any positive or negative action on microbial oral disease such as caries and gingivitis, of seven food/beverages (red chicory, mushroom, raspberry, green and black tea, cranberry juice, dark beer). Tea leaves resulted the richest material in all the detected ions, anyway tea beverages resulted the richest just in fluoride. The highest content in zinc was in chicory, raspberry and mushroom. Raspberry is the richest food in strontium and boron, beer in selenium, raspberry and mushroom in copper. Beer, cranberry juice and, especially green and black tea are very rich in polyphenols, confirming these beverages as important sources of such healthy substances. The fractionation, carried out on the basis of the molecular mass (MM), of the water soluble components occurring in raspberry, chicory, and mushroom extracts (which in microbiological assays revealed the highest potential action against oral pathogens), showed that both the high and low MM fractions are active, with the low MM fractions displaying the highest potential action for all the fractionated extracts. Our findings show that more compounds that can play a different active role occur in these foods. Maria Daglia, Adele Papetti, Dora Mascherpa, Pietro Grisoli, Giovanni Giusto, Peter Lingström, Jonathan Pratten, Caterina Signoretto, David A. Spratt, Michael Wilson, Egija Zaura, and Gabriella Gazzani Copyright © 2011 Maria Daglia et al. All rights reserved. The Role of Synthetic Biology in the Design of Microbial Cell Factories for Biofuel Production Sat, 15 Oct 2011 14:11:30 +0000 Insecurity in the supply of fossil fuels, volatile fuel prices, and major concerns regarding climate change have sparked renewed interest in the production of fuels from renewable resources. Because of this, the use of biodiesel has grown dramatically during the last few years and is expected to increase even further in the future. Biodiesel production through the use of microbial systems has marked a turning point in the field of biofuels since it is emerging as an attractive alternative to conventional technology. Recent progress in synthetic biology has accelerated the ability to analyze, construct, and/or redesign microbial metabolic pathways with unprecedented precision, in order to permit biofuel production that is amenable to industrial applications. The review presented here focuses specifically on the role of synthetic biology in the design of microbial cell factories for efficient production of biodiesel. Verónica Leticia Colin, Analía Rodríguez, and Héctor Antonio Cristóbal Copyright © 2011 Verónica Leticia Colin et al. All rights reserved. In Vitro Assessment of Shiitake Mushroom (Lentinula edodes) Extract for Its Antigingivitis Activity Wed, 28 Sep 2011 09:36:36 +0000 Gingivitis is a preventable disease characterised by inflammation of the gums due to the buildup of a microbial biofilm at the gingival margin. It is implicated as a precursor to periodontitis, a much more serious problem which includes associated bone loss. Unfortunately, due to poor oral hygiene among the general population, gingivitis is prevalent and results in high treatment costs. Consequently, the option of treating gingivitis using functional foods, which promote oral health, is an attractive one. Medicinal mushrooms, including shiitake, have long been known for their immune system boosting as well as antimicrobial effects; however, they have not been employed in the treatment of oral disease. In the current study, the effectiveness of shiitake mushroom extract was compared to that of the active component in the leading gingivitis mouthwash, containing chlorhexidine, in an artificial mouth model (constant depth film fermenter). The total bacterial numbers as well as numbers of eight key taxa in the oral community were investigated over time using multiplex qPCR. The results indicated that shiitake mushroom extract lowered the numbers of some pathogenic taxa without affecting the taxa associated with health, unlike chlorhexidine which has a limited effect on all taxa. Lena Ciric, Anna Tymon, Egija Zaura, Peter Lingström, Monica Stauder, Adele Papetti, Caterina Signoretto, Jonathan Pratten, Michael Wilson, and David Spratt Copyright © 2011 Lena Ciric et al. All rights reserved. Production of a Solvent, Detergent, and Thermotolerant Lipase by a Newly Isolated Acinetobacter sp. in Submerged and Solid-State Fermentations Tue, 27 Sep 2011 11:49:16 +0000 The lipase production ability of a newly isolated Acinetobacter sp. in submerged (SmF) and solid-state (SSF) fermentations was evaluated. The results demonstrated this strain as one of the rare bacterium, which is able to grow and produce lipase in SSF even more than SmF. Coconut oil cake as a cheap agroindustrial residue was employed as the solid substrate. The lipase production was optimized in both media using artificial neural network. Multilayer normal and full feed forward backpropagation networks were selected to build predictive models to optimize the culture parameters for lipase production in SmF and SSF systems, respectively. The produced models for both systems showed high predictive accuracy where the obtained conditions were close together. The produced enzyme was characterized as a thermotolerant lipase, although the organism was mesophile. The optimum temperature for the enzyme activity was 45°C where 63% of its activity remained at 70°C after 2 h. This lipase remained active after 24 h in a broad range of pH (6–11). The lipase demonstrated strong solvent and detergent tolerance potentials. Therefore, this inexpensive lipase production for such a potent and industrially valuable lipase is promising and of considerable commercial interest for biotechnological applications. Anahita Khoramnia, Afshin Ebrahimpour, Boon Kee Beh, and Oi Ming Lai Copyright © 2011 Anahita Khoramnia et al. All rights reserved. Effects of Fruit and Vegetable Low Molecular Mass Fractions on Gene Expression in Gingival Cells Challenged with Prevotella intermedia and Actinomyces naeslundii Sun, 18 Sep 2011 14:04:31 +0000 Low molecular mass (LMM) fractions obtained from extracts of raspberry, red chicory, and Shiitake mushrooms have been shown to be an useful source of specific antibacterial, antiadhesion/coaggregation, and antibiofilm agent(s) that might be used for protection towards caries and gingivitis. In this paper, the effects of such LMM fractions on human gingival KB cells exposed to the periodontal pathogens Prevotella intermedia and Actinomyces naeslundii were evaluated. Expression of cytokeratin 18 (CK18) and β4 integrin (β4INT) genes, that are involved in cell proliferation/differentiation and adhesion, and of the antimicrobial peptide β2 defensin (HβD2) in KB cells was increased upon exposure to either live or heat-killed bacteria. All LMM fractions tested prevented or reduced the induction of gene expression by P. intermedia and A. naeslundii depending on the experimental conditions. Overall, the results suggested that LMM fractions could modulate the effects of bacteria associated with periodontal disease in gingival cells. Laura Canesi, Cristina Borghi, Monica Stauder, Peter Lingström, Adele Papetti, Jonathan Pratten, Caterina Signoretto, David A. Spratt, Mike Wilson, Egija Zaura, and Carla Pruzzo Copyright © 2011 Laura Canesi et al. All rights reserved. The Effects of Fractions from Shiitake Mushroom on Composition and Cariogenicity of Dental Plaque Microcosms in an In Vitro Caries Model Wed, 14 Sep 2011 17:51:41 +0000 The aim of the current study was to investigate the anticariogenic potential of the (sub)fractions obtained from the edible mushroom shiitake (Lentinula edodes) in in vitro caries model. We used a modified constant depth film fermentor (CDFF) with pooled saliva as the inoculum and bovine dentin as a substratum. The test compounds were low molecular weight fraction (MLMW) of the shiitake extract and subfractions 4 and 5 (SF4 and SF5) of this fraction. Chlorhexidine (CHX) and water served as a positive and a negative control, respectively. Dentin mineral loss was quantified (TMR), microbial shifts within the microcosms were determined (qPCR), and the acidogenicity of the microcosms was assessed (CIA). From the compounds tested, the SF4 of shiitake showed strong inhibiting effect on dentin demineralization and induced microbial shifts that could be associated with oral health. The acid producing potential was increased, suggesting uncoupling of the glycolysis of the microbiota by the exposure to SF4. In conclusion, the results suggest that SF4 of shiitake has an anticariogenic potential. Egija Zaura, Mark J. Buijs, Michel A. Hoogenkamp, Lena Ciric, Adele Papetti, Caterina Signoretto, Monica Stauder, Peter Lingström, Jonathan Pratten, David A. Spratt, and Michael Wilson Copyright © 2011 Egija Zaura et al. All rights reserved. Effects of Mushroom and Chicory Extracts on the Physiology and Shape of Prevotella intermedia, a Periodontopathogenic Bacterium Sun, 11 Sep 2011 13:57:40 +0000 Contrary to the common assumption that food has a negative impact on oral health, research has shown that several foods contain a number of components with antibacterial and antiplaque activity. These natural compounds may be useful for improving daily oral hygiene. In this study we evaluate the mode of antimicrobial action of fractions of mushroom and red chicory extracts on Prevotella intermedia, a periodontopathogenic bacterium. The minimal inhibitory concentration corresponded to 0.5x compared to the natural food concentration for both extracts. This concentration resulted in a bacteriostatic effect in mushroom extract and in a slightly bactericidal effect in chicory extract. Cell mass continued to increase even after division stopped. As regards macromolecular synthesis, DNA was almost totally inhibited upon addition of either mushroom or chicory extract, and RNA to a lesser extent, while protein synthesis continued. Cell elongation occurred after septum inhibition as documented by scanning electron microscopy and cell measurement. The morphogenetic effects are reminiscent of the mode of action of antibiotics such as quinolones or 𝛽-lactams. The discovery of an antibiotic-like mode of action suggests that these extracts can be advantageously employed for daily oral hygiene in formulations of cosmetic products such as mouthwashes and toothpastes. Caterina Signoretto, Anna Marchi, Anna Bertoncelli, Gloria Burlacchini, Francesco Tessarolo, Iole Caola, Elisabetta Pezzati, Egija Zaura, Adele Papetti, Peter Lingström, Jonathan Pratten, David A. Spratt, Michael Wilson, and Pietro Canepari Copyright © 2011 Caterina Signoretto et al. All rights reserved. Testing a Low Molecular Mass Fraction of a Mushroom (Lentinus edodes) Extract Formulated as an Oral Rinse in a Cohort of Volunteers Thu, 08 Sep 2011 08:23:27 +0000 Although foods are considered enhancing factors for dental caries and periodontitis, laboratory researches indicate that several foods and beverages contain components endowed with antimicrobial and antiplaque activities. A low molecular mass (LMM) fraction of an aqueous mushroom extract has been found to exert these activities in in vitro experiments against potential oral pathogens. We therefore conducted a clinical trial in which we tested an LMM fraction of shiitake mushroom extract formulated in a mouthrinse in 30 young volunteers, comparing the results with those obtained in two identical cohorts, one of which received water (placebo) and the other Listerine. Plaque index, gingival index and bacterial counts in plaque samples were determined in all volunteers over the 11 days of the clinical trial. Statistically significant differences (𝑃<0.05) were obtained for the plaque index on day 12 in subjects treated with mushroom versus placebo, while for the gingival index significant differences were found for both mushroom versus placebo and mushroom versus Listerine. Decreases in total bacterial counts and in counts of specific oral pathogens were observed for both mushroom extract and Listerine in comparison with placebo. The data suggest that a mushroom extract may prove beneficial in controlling dental caries and/or gingivitis/periodontitis. Caterina Signoretto, Gloria Burlacchini, Anna Marchi, Marcello Grillenzoni, Giacomo Cavalleri, Lena Ciric, Peter Lingström, Elisabetta Pezzati, Maria Daglia, Egija Zaura, Jonathan Pratten, David A. Spratt, Michael Wilson, and Pietro Canepari Copyright © 2011 Caterina Signoretto et al. All rights reserved. In Vitro Culture Conditions for Maintaining a Complex Population of Human Gastrointestinal Tract Microbiota Sun, 24 Jul 2011 13:45:57 +0000 A stable intestinal microbiota is important in maintaining human physiology and health. Although there have been a number of studies using in vitro and in vivo approaches to determine the impact of diet and xenobiotics on intestinal microbiota, there is no consensus for the best in vitro culture conditions for growth of the human gastrointestinal microbiota. To investigate the dynamics and activities of intestinal microbiota, it is important for the culture conditions to support the growth of a wide range of intestinal bacteria and maintain a complex microbial community representative of the human gastrointestinal tract. Here, we compared the bacterial community in three culture media: brain heart infusion broth and high- and low-carbohydrate medium with different growth supplements. The bacterial community was analyzed using denaturing gradient gel electrophoresis (DGGE), pyrosequencing and real-time PCR. Based on the molecular analysis, this study indicated that the 3% fecal inoculum in low-concentration carbohydrate medium with 1% autoclaved fecal supernatant provided enhanced growth conditions to conduct in vitro studies representative of the human intestinal microbiota. Bong-Soo Kim, Jong Nam Kim, and Carl E. Cerniglia Copyright © 2011 Bong-Soo Kim et al. All rights reserved. Dampening Host Sensing and Avoiding Recognition in Pseudomonas aeruginosa Pneumonia Thu, 14 Jul 2011 15:14:33 +0000 Pseudomonas aeruginosa is an opportunistic pathogen and causes a wide range of acute and chronic infections. P. aeruginosa infections are kept in check by an effective immune surveillance in the healthy host, while any imbalance or defect in the normal immune response can manifest in disease. Invasive acute infection in the immunocompromised patients is mediated by potent extracellular and cell bound bacterial virulence factors. Life-threatening chronic infection in cystic fibrosis patients is maintained by pathogenic variants that contribute to evade detection and clearance by the immune system. Here, we reviewed the molecular basis of receptor-mediated recognition of P. aeruginosa and their role in initiating inflammation and the colonization. In addition, the consequence of the P. aeruginosa genetic adaptation for the antibacterial defence and the maintaining of chronic infection are discussed. Cristina Cigana, Nicola Ivan Lorè, Maria Lina Bernardini, and Alessandra Bragonzi Copyright © 2011 Cristina Cigana et al. All rights reserved. The Gut Microbiota and Human Health with an Emphasis on the Use of Microencapsulated Bacterial Cells Sat, 02 Jul 2011 15:57:53 +0000 The gut microbiota plays a crucial role in maintaining health. Alterations of the gut bacterial population have been associated with a number of diseases. Past and recent studies suggest that one can positively modify the contents of the gut microbiota by introducing prebiotics, probiotics, synbiotics, and other therapeutics. This paper focuses on probiotic modulation of the gut microbiota by their delivery to the lower gastrointestinal tract (GIT). There are numerous obstacles to overcome before microorganisms can be utilized as therapeutics. One important limitation is the delivery of viable cells to the lower GIT without a significant loss of cell viability and metabolic features through the harsh conditions of the upper GIT. Microencapsulation has been shown to overcome this, with various types of microcapsules available for resolving this limitation. This paper discusses the gut microbiota and its role in disease, with a focus on microencapsulated probiotics and their potentials and limitations. Satya Prakash, Catherine Tomaro-Duchesneau, Shyamali Saha, and Arielle Cantor Copyright © 2011 Satya Prakash et al. All rights reserved. Manipulation of pH Shift to Enhance the Growth and Antibiotic Activity of Xenorhabdus nematophila Mon, 23 May 2011 08:54:40 +0000 To evaluate the effects of pH control strategy on cell growth and the production of antibiotic (cyclo(2-Me-BABA-Gly)) by Xenorhabdus nematophila and enhance the antibiotic activity. The effects of uncontrolled- (different initial pH) and controlled-pH (different constant pH and pH-shift) operations on cell growth and antibiotic activity of X. nematophila YL00I were examined. Experiments showed that the optimal initial pH for cell growth and antibiotic production of X. nematophila YL001 occurred at 7.0. Under different constant pH, a pH level of 7.5 was found to be optimal for biomass and antibiotic activity at 23.71 g/L and 100.0 U/mL, respectively. Based on the kinetic information relating to the different constant pH effects on the fermentation of X. nematophila YL001, a two-stage pH control strategy in which pH 6.5 was maintained for the first 24 h, and then switched to 7.5 after 24 h, was established to improve biomass production and antibiotic activity. By applying this pH-shift strategy, the maximal antibiotic activity and productivity were significantly improved and reaching 185.0 U/mL and 4.41 U/mL/h, respectively, compared to values obtained from constant pH operation (100.0 U/mL and 1.39 U/mL/h). Yonghong Wang, Xiangling Fang, Yongpeng Cheng, and Xing Zhang Copyright © 2011 Yonghong Wang et al. All rights reserved. Molecular Breeding of Advanced Microorganisms for Biofuel Production Mon, 17 Jan 2011 10:36:43 +0000 Large amounts of fossil fuels are consumed every day in spite of increasing environmental problems. To preserve the environment and construct a sustainable society, the use of biofuels derived from different kinds of biomass is being practiced worldwide. Although bioethanol has been largely produced, it commonly requires food crops such as corn and sugar cane as substrates. To develop a sustainable energy supply, cellulosic biomass should be used for bioethanol production instead of grain biomass. For this purpose, cell surface engineering technology is a very promising method. In biobutanol and biodiesel production, engineered host fermentation has attracted much attention; however, this method has many limitations such as low productivity and low solvent tolerance of microorganisms. Despite these problems, biofuels such as bioethanol, biobutanol, and biodiesel are potential energy sources that can help establish a sustainable society. Hiroshi Sakuragi, Kouichi Kuroda, and Mitsuyoshi Ueda Copyright © 2011 Hiroshi Sakuragi et al. All rights reserved. In Situ Biodiesel Production from Fast-Growing and High Oil Content Chlorella pyrenoidosa in Rice Straw Hydrolysate Mon, 17 Jan 2011 09:31:38 +0000 Rice straw hydrolysate was used as lignocellulose-based carbon source for Chlorella pyrenoidosa cultivation and the feasibility of in situ biodiesel production was investigated. 13.7 g/L sugar was obtained by enzymatic hydrolyzation of rice straw. Chlorella pyrenoidosa showed a rapid growth in the rice straw hydrolysate medium, the maximum biomass concentration of 2.83 g/L was obtained in only 48 hours. The lipid content of the cells reached as high as 56.3%. In situ transesterification was performed for biodiesel production. The optimized condition was 1 g algal powder, 6 mL n-hexane, and 4 mL methanol with 0.5 M sulfuric acid at the temperature of 90°C in 2-hour reaction time, under which over 99% methyl ester content and about 95% biodiesel yield were obtained. The results suggested that the method has great potential in the production of biofuels with lignocellulose as an alternative carbon source for microalgae cultivation. Penglin Li, Xiaoling Miao, Rongxiu Li, and Jianjiang Zhong Copyright © 2011 Penglin Li et al. All rights reserved. Enhancement of 2,3-Butanediol Production by Klebsiella oxytoca PTCC 1402 Thu, 13 Jan 2011 09:29:13 +0000 Optimal operating parameters of 2,3-Butanediol production using Klebsiella oxytoca under submerged culture conditions are determined by using Taguchi method. The effect of different factors including medium composition, pH, temperature, mixing intensity, and inoculum size on 2,3-butanediol production was analyzed using the Taguchi method in three levels. Based on these analyses the optimum concentrations of glucose, acetic acid, and succinic acid were found to be 6, 0.5, and 1.0 (% w/v), respectively. Furthermore, optimum values for temperature, inoculum size, pH, and the shaking speed were determined as 37°C, 8 (g/L), 6.1, and 150 rpm, respectively. The optimal combinations of factors obtained from the proposed DOE methodology was further validated by conducting fermentation experiments and the obtained results revealed an enhanced 2,3-Butanediol yield of 44%. Maesomeh Anvari and Mohammad Reza Safari Motlagh Copyright © 2011 Maesomeh Anvari and Mohammad Reza Safari Motlagh. All rights reserved. Herpesvirus BACs: Past, Present, and Future Wed, 27 Oct 2010 14:15:57 +0000 The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy. Charles Warden, Qiyi Tang, and Hua Zhu Copyright © 2011 Charles Warden et al. All rights reserved. Constitutive High Level Expression of an Endoxylanase Gene from the Newly Isolated Bacillus subtilis AQ1 in Escherichia coli Tue, 21 Sep 2010 14:18:59 +0000 A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted as Bacillus subtilis (B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed in E. coli. In E. coli the recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely, 2173.6±51.4 and 2745.3±11 U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the native B. subtilis AQ1 endoxylanase and that of 95% homologous recombinant one from B. subtilis DB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression in E. coli and hence made the production in E. coli feasible. Is Helianti, Niknik Nurhayati, Maria Ulfah, Budiasih Wahyuntari, and Siswa Setyahadi Copyright © 2010 Is Helianti et al. All rights reserved. The Structure and Function of Serum Opacity Factor: A Unique Streptococcal Virulence Determinant That Targets High-Density Lipoproteins Thu, 08 Jul 2010 11:28:44 +0000 Serum opacity factor (SOF) is a virulence determinant expressed by a variety of streptococcal and staphylococcal species including both human and animal pathogens. SOF derives its name from its ability to opacify serum where it targets and disrupts the structure of high-density lipoproteins resulting in formation of large lipid vesicles that cause the serum to become cloudy. SOF is a multifunctional protein and in addition to its opacification activity, it binds to a number of host proteins that mediate adhesion of streptococci to host cells, and it plays a role in resistance to phagocytosis in human blood. This article will provide an overview of the structure and function of SOF, its role in the pathogenesis of streptococcal infections, its vaccine potential, its prevalence and distribution in bacteria, and the molecular mechanism whereby SOF opacifies serum and how an understanding of this mechanism may lead to therapies for reducing high-cholesterol concentrations in blood, a major risk factor for cardiovascular disease. Harry S. Courtney and Henry J. Pownall Copyright © 2010 Harry S. Courtney and Henry J. Pownall. All rights reserved. Role of Antimicrobial Selective Pressure and Secondary Factors on Antimicrobial Resistance Prevalence in Escherichia coli from Food-Producing Animals in Japan Wed, 02 Jun 2010 08:00:08 +0000 The use of antimicrobial agents in the veterinary field affects the emergence, prevalence, and dissemination of antimicrobial resistance in bacteria isolated from food-producing animals. To control the emergence, prevalence, and dissemination of antimicrobial resistance, it is necessary to implement appropriate actions based on scientific evidence. In Japan, the Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM) was established in 1999 to monitor the antimicrobial susceptibility of foodborne and commensal bacteria from food-producing animals. The JVARM showed that the emergence and prevalence of resistant Escherichia coli were likely linked to the therapeutic antimicrobial use in food-producing animals through not only direct selection of the corresponding resistance but also indirect selections via cross-resistance and coresistance. In addition, relevant factors such as host animals and bacterial properties might affect the occurrence and prevalence of antimicrobial-resistant E. coli under the selective pressure from antimicrobial usage. This paper reviews the trends in antimicrobial resistance in E. coli and consumption of antimicrobials agents in Japan and introduces the relationship between antimicrobial usage and prevalence of antimicrobial-resistant bacteria, from food-producing animals under the JVARM program. In this paper, we will provide the underlying information about the significant factors that can help control antimicrobial resistance in bacteria in veterinary medicine. Kazuki Harada and Tetsuo Asai Copyright © 2010 Kazuki Harada and Tetsuo Asai. All rights reserved. High Prevalence of Arcobacter Carriage in Older Subjects with Type 2 Diabetes Mon, 24 May 2010 08:47:55 +0000 Arcobacters are potential pathogens related to diarrheic infections and, rarely, septicaemia. This study evaluated the prevalence of arcobacters in stool samples of subjects with (𝑛=38) and without (𝑛=61) type 2 diabetes by using cultural and molecular techniques. Three Arcobacter positive cultures were found, all among diabetic subjects, whereas molecular analysis showed a carriage rate of 79% and 26.2% in subjects with and without type 2 diabetes (𝑃<.001), respectively. The multivariate analysis showed that type 2 diabetes (𝛽=1.913; 95%CI: 2.378–19.285; 𝑃<.0001) and age (𝛽=1.744; 95%CI: 2.077–15.766; 𝑃=.001) were the only factors independently associated with arcobacters colonization in this population. Our study demonstrated a high prevalence of arcobacters colonization in type 2 diabetic and older subjects. The clinical significance and the potential health risk associated with these emerging species remain to be determined. Maria Teresa Fera, Giuseppina T. Russo, Antonino Di Benedetto, Erminia La Camera, Angelo Orlando, Annalisa Giandalia, Vincenzo F. Ruffa, Giulia Lanza, Valeria Lentini, Giuseppa Perdichizzi, and Domenico Cucinotta Copyright © 2010 Maria Teresa Fera et al. All rights reserved. Factorial Design to Optimize Biosurfactant Production by Yarrowia lipolytica Tue, 23 Mar 2010 15:01:45 +0000 In order to improve biosurfactant production by Yarrowia lipolytica IMUFRJ 50682, a factorial design was carried out. A 24 full factorial design was used to investigate the effects of nitrogen sources (urea, ammonium sulfate, yeast extract, and peptone) on maximum variation of surface tension (ΔST) and emulsification index (EI). The best results (67.7% of EI and 20.9 mN m−1 of ΔST) were obtained in a medium composed of 10 g 1−1 of ammonium sulfate and 0.5 g 1−1 of yeast extract. Then, the effects of carbon sources (glycerol, hexadecane, olive oil, and glucose) were evaluated. The most favorable medium for biosurfactant production was composed of both glucose (4% w/v) and glycerol (2% w/v), which provided an EI of 81.3% and a ΔST of 19.5 mN m−1. The experimental design optimization enhanced ΔEI by 110.7% and ΔST by 108.1% in relation to the standard process. Gizele Cardoso Fontes, Priscilla Filomena Fonseca Amaral, Marcio Nele, and Maria Alice Zarur Coelho Copyright © 2010 Gizele Cardoso Fontes et al. All rights reserved. Cloning, Identification, and Characterization of the rpoS-Like Sigma Factor rpoX from Vibrio alginolyticus Tue, 29 Dec 2009 13:40:08 +0000 Vibrio alginolyticus ZJ-51 displays phase variation between opaque/rugose colonies (Op) and translucent/smooth colonies (Tr). These colony variants show great differences in biofilm formation and motility. In this study, a gene encoding for an rpoS-like sigma factor, rpoX, has been cloned and characterized. The absence of rpoX did not affect colony switching rate but did decrease biofilm formation in both the Op and the Tr variants. When challenged with hydrogen peroxide, the ΔrpoX in the Op background showed a slightly higher survival rate compared with the wild type, whereas survival was decreased in the Tr background. Deletion of rpoX in the Tr background resulted in a higher ability to resist ethanol challenges and to survive hyperosmolarity challenges, and in the Op background the opposite phenotype was observed. This indicates that the rpoX gene is involved in biofilm formation and stress response but the effects are controlled by colony phase variation in V. alginolyticus. Jing-jing Zhao, Chang Chen, Lv-ping Zhang, and Chao-qun Hu Copyright © 2009 Jing-jing Zhao et al. All rights reserved. Modelling the Survival of Escherichia coli O157:H7 on Raw Portioned Tomatoes, Inoculated with Aspergillus fumigatus and Emericella nidulans Wed, 16 Dec 2009 08:58:01 +0000 The metabiotic interactions occurring among two fungi (Aspergillus fumigatus and Emericella nidulans) and Escherichia coli O157:H7 on raw portioned tomatoes were studied. Tomatoes, preinoculated with the moulds and inoculated with the pathogen, were packaged in air and stored at 4, 8 and 12∘C for 9 days; pathogen cell number and pH were monitored throughout the storage and the data were modeled using three different equations (Geeraerd, Weibull, and modified Weibull), to assess the shoulder length, the 1-log reduction time, and the death time. Both A. fumigatus and E. nidulans increased the survival of E. coli O157:H7 through the prolongation of the shoulder length; in contrast, the death time was significantly increased. The results of this paper suggested that the metabiotic interactions aspergilli/E. coli O 157:H7 could be of public concern, as the consumption of tomatoes (or other fruits and vegetables) contaminated both by the moulds and the pathogen is a possible scenario. Daniela Cardillo, Antonio Bevilacqua, Francesca Cibelli, Clelia Altieri, and Milena Sinigaglia Copyright © 2009 Daniela Cardillo et al. All rights reserved. Nitric Oxide Production by the Human Intestinal Microbiota by Dissimilatory Nitrate Reduction to Ammonium Sun, 01 Nov 2009 16:14:45 +0000 The free radical nitric oxide (NO) is an important signaling molecule in the gastrointestinal tract. Besides eukaryotic cells, gut microorganisms are also capable of producing NO. However, the exact mechanism of NO production by the gut microorganisms is unknown. Microbial NO production was examined under in vitro conditions simulating the gastrointestinal ecosystem using L-arginine or nitrate as substrates. L-arginine did not influence the microbial NO production. However, NO concentrations in the order of 90 ng NO-N per L feed medium were produced by the fecal microbiota from nitrate. 15N tracer experiments showed that nitrate was mainly reduced to ammonium by the dissimilatory nitrate reduction to ammonium (DNRA) pathway. To our knowledge, this is the first study showing that gastrointestinal microbiota can generate substantial amounts of NO by DNRA and not by the generally accepted denitrification or L-arginine pathway. Further work is needed to elucidate the exact role between NO produced by the gastrointestinal microbiota and host cells. Joan Vermeiren, Tom Van de Wiele, Willy Verstraete, Pascal Boeckx, and Nico Boon Copyright © 2009 Joan Vermeiren et al. All rights reserved. Adding to Yersinia enterocolitica Gene Pool Diversity: Two Cryptic Plasmids from a Biotype 1A Isolate Sun, 11 Oct 2009 09:28:09 +0000 We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs) carried by a Yersinia enterocolitica biotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs) encoding the first CcdA/CcdB-like antitoxin/toxin system described for a Yersinia plasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described in Salmonella (CcdA/B), Klebsiella (RepA), and Plesiomonas (MobA/C) indicating genomic fluidity among members of the Enterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9%) was similar to that of pYe4449-1 (53.7%) and differed from that of the Y. enterocolitica genome (47.3%). Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at 4∘C but not at or above 27∘C suggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing the DxxGN(x)nDxxGN motif. Our findings illustrate the exceptional gene pool diversity within the species Y. enterocolitica driven by horizontal gene transfer events. Daniela Lepka, Tobias Kerrinnes, Evelyn Skiebe, Birgitt Hahn, Angelika Fruth, and Gottfried Wilharm Copyright © 2009 Daniela Lepka et al. All rights reserved. Chlamydia trachomatis Alters Iron-Regulatory Protein-1 Binding Capacity and Modulates Cellular Iron Homeostasis in HeLa-229 Cells Sun, 16 Aug 2009 10:22:07 +0000 Chlamydia trachomatis (CT) is the leading cause of diseases related to reproductive health and iron plays important role in chlamydial pathogenesis. Iron homeostasis in chlamydia-infected cells is not clear thus far. This study shows that expression of the transferrin receptor (TfR) is downregulated, whereas expression of the ferritin heavy chain is upregulated in CT-infected HeLa-229 cells. Expression of iron-regulatory protein (IRP)-1 predominates over IRP-2 in infected cells. In infected cells, attenuated binding activity of IRP-iron responsive elements (IREs) is observed using the electrophoretic mobility-shift assay. These results suggest that iron homeostasis is modulated in CT-infected HeLa cells at the interface of acquisition and commensal use of iron. Harsh Vardhan, Apurb R. Bhengraj, Rajneesh Jha, and Aruna Singh Mittal Copyright © 2009 Harsh Vardhan et al. All rights reserved. Characterization of Multidrug Resistant ESBL-Producing Escherichia coli Isolates from Hospitals in Malaysia Wed, 05 Aug 2009 15:12:08 +0000 The emergence of Escherichia coli that produce extended spectrum 𝛽-lactamases (ESBLs) and are multidrug resistant (MDR) poses antibiotic management problems. Forty-seven E. coli isolates from various public hospitals in Malaysia were studied. All isolates were sensitive to imipenem whereas 36 were MDR (resistant to 2 or more classes of antibiotics). PCR detection using gene-specific primers showed that 87.5% of the ESBL-producing E. coli harbored the 𝑏𝑙𝑎TEM gene. Other ESBL-encoding genes detected were 𝑏𝑙𝑎OXA, 𝑏𝑙𝑎SHV, and 𝑏𝑙𝑎CTX-M. Integron-encoded integrases were detected in 55.3% of isolates, with class 1 integron-encoded intI1 integrase being the majority. Amplification and sequence analysis of the 5CS region of the integrons showed known antibiotic resistance-encoding gene cassettes of various sizes that were inserted within the respective integrons. Conjugation and transformation experiments indicated that some of the antibiotic resistance genes were likely plasmid-encoded and transmissible. All 47 isolates were subtyped by PFGE and PCR-based fingerprinting using random amplified polymorphic DNA (RAPD), repetitive extragenic palindromes (REPs), and enterobacterial repetitive intergenic consensus (ERIC). These isolates were very diverse and heterogeneous. PFGE, ERIC, and REP-PCR methods were more discriminative than RAPD in subtyping the E. coli isolates. King-Ting Lim, Rohani Yasin, Chew-Chieng Yeo, Savithri Puthucheary, and Kwai-Lin Thong Copyright © 2009 King-Ting Lim et al. All rights reserved. Functional Expression of a DNA-Topoisomerase IB from Cryptosporidium parvum Mon, 27 Jul 2009 14:53:05 +0000 Cryptosporidium parvum, one of the most important causative organisms of human diarrheas during childhood, contains a monomeric DNA-topoisomerase IB (CpTopIB) in chromosome 7. Heterologous expression of CpTopIB gene in a budding yeast strain lacking this activity proves that the cryptosporidial enzyme is functional in vivo. The enzymatic activity is comprised in a single polypeptide, which contains all the structural features defining a fully active TopIB. Relaxation activity of the yeast extracts was detected only when CpTopIB ORF was expressed in a yeast expression system showing time and protein dependence under steady state kinetic conditions. The susceptibility of CpTopIB-transformed yeast to the irreversible inhibitor camptothecin and its water-soluble derivatives (topotecan and SN-38) was assessed. César Ordóñez, Javier Alfonso, Rafael Balaña-Fouce, and David Ordóñez Copyright © 2009 César Ordóñez et al. All rights reserved. Integrative Gene Cloning and Expression System for Streptomyces sp. US 24 and Streptomyces sp. TN 58 Bioactive Molecule Producing Strains Mon, 15 Jun 2009 09:16:48 +0000 Streptomyces sp. US 24 and Streptomyces sp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed using E. coli ET12567 (pUZ8002), as a conjugal donor, carrying the integrative plasmid pSET152. For the Streptomyces sp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas for Streptomyces sp. TN 58, the integration was in single copy at the attB site. Plasmid pSET152 was inherited every time for all analysed Streptomyces sp. US 24 and Streptomyces sp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA) from Streptomyces sp. SK was expressed in strain Streptomyces sp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of the Streptomyces sp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch. Samiha Sioud, Bertrand Aigle, Ines Karray-Rebai, Slim Smaoui, Samir Bejar, and Lotfi Mellouli Copyright © 2009 Samiha Sioud et al. All rights reserved. Identification of IbeR as a Stationary-Phase Regulator in Meningitic Escherichia coli K1 that Carries a Loss-of-Function Mutation in rpoS Thu, 12 Mar 2009 14:08:25 +0000 IbeR is a regulator present in meningitic Escherichia coli strain E44 that carries a loss-of-function mutation in the stationary-phase (SP) regulatory gene rpoS. In order to determine whether IbeR is an SP regulator in E44, two-dimensional gel electrophoresis and LC-MS were used to compare the proteomes of a noninvasive ibeR deletion mutant BR2 and its parent strain E44 in the SP. Four up-regulated (TufB, GapA, OmpA, AhpC) and three down-regulated (LpdA, TnaA, OpmC) proteins in BR2 were identified when compared to E44. All these proteins contribute to energy metabolism or stress resistance, which is related to SP regulation. One of the down-regulated proteins, tryptophanase (TnaA), which is regulated by RpoS in other E. coli strains, is associated with SP regulation via production of a signal molecule indole. Our studies demonstrated that TnaA was required for E44 invasion, and that indole was able to restore the noninvasive phenotype of the tnaA mutant. The production of indole was significantly reduced in BR2, indicating that ibeR is required for the indole production via tnaA. Survival studies under different stress conditions indicated that IbeR contributed to bacteria stress resistance in the SP. Taken together, IbeR is a novel regulator contributing to the SP regulation. Feng Chi, Ying Wang, Timothy K. Gallaher, Chun-Hua Wu, Ambrose Jong, and Sheng-He Huang Copyright © 2009 Feng Chi et al. All rights reserved. Competition of Lactobacillus paracasei with Salmonella enterica for Adhesion to Caco-2 Cells Mon, 24 Mar 2008 00:00:00 +0000 Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. In this study, we examined the ability of Lactobacillus paracasei to inhibit the adhesion of pathogenic Salmonella enterica to human colon adenocarcinoma Caco-2 cells. Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively. In adhesion experiments, bacteria were added to the cells for 2 or 4 hours. The number of attached bacteria was expressed as colony-forming units (CFUs), Caco-2 cells were counted in hematocytometer. Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to 𝐿. paracasei and almost 27-fold higher after 4 hours of contact. Two types of experiments were done: coincubation (both bacteria were added to Caco-2 cells simultaneously), and preincubation (𝐿. paracasei was incubated with Caco-2 cells first, and then 𝑆. enterica was added). In coincubation experiment, the presence of 𝐿. paracasei decreased 𝑆. enterica adhesion by 4-fold and in preincubation experiment even 7-fold. Generally, Lactobacillus spent culture supernatants (SCSs) acted weaker as inhibitors of Salmonella adhesion in comparison to the whole 𝐿. paracasei culture in coincubation experiment. In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation. Alicja Jankowska, Daniel Laubitz, Hanna Antushevich, Romuald Zabielski, and Elżbieta Grzesiuk Copyright © 2008 Alicja Jankowska et al. All rights reserved. Distinction between Pore Assembly by Staphylococcal α-Toxin versus Leukotoxins Wed, 28 Feb 2007 00:00:00 +0000 The staphylococcal bipartite leukotoxins and the homoheptameric α-toxin belong to the same family of β-barrel pore-forming toxins despite slight differences. In the α-toxin pore, the N-terminal extremity of each protomer interacts as a deployed latch with two consecutive protomers in the vicinity of the pore lumen. N-terminal extremities of leukotoxins as seen in their three-dimensional structures are heterogeneous in length and take part in the β-sandwich core of soluble monomers. Hence, the interaction of these N-terminal extremities within structures of adjacent monomers is questionable. We show here that modifications of their N-termini by two different processes, using fusion with glutathione S-transferase (GST) and bridging of the N-terminal extremity to the adjacent β-sheet via disulphide bridges, are not deleterious for biological activity. Therefore, bipartite leukotoxins do not need a large extension of their N-terminal extremities to form functional pores, thus illustrating a microheterogeneity of the structural organizations between bipartite leukotoxins and α-toxin. Olivier Joubert, Joëlle Voegelin, Valérie Guillet, Samuel Tranier, Sandra Werner, Didier A. Colin, Mauro Dalla Serra, Daniel Keller, Henri Monteil, Lionel Mourey, and Gilles Prévost Copyright © 2007 Olivier Joubert et al. All rights reserved. Site-Directed Mutagenesis to Assess the Binding Capacity of Class S Protein of Staphylococcus aureus Leucotoxins to the Surface of Polymorphonuclear Cells Thu, 23 Feb 2006 00:00:00 +0000 Staphylococcal leucotoxins result from the association of class S components and class F component inducing the activation and the permeabilization of the target cells. Like α-toxin, the leucotoxins are pore-forming toxins with more than 70% β-sheet. This was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. In addition, threonine 28 of a predicted and conserved β-sheet at the N-terminal extremity of class S proteins composing leucotoxins aligns with histidine 35 of α-toxin, which has a key role in oligomerization of the final pore. Flow cytometry was used to study different aminoacid substitutions of the threonine 28 in order to evaluate its role in the biological activity of these class S proteins. Finally, results show that threonine 28 of the leucotoxin probably plays a role similar to that of histidine 35 of α-toxin. Mutations on this threonin largely influenced the secondary interaction of the class F component and led to inactive toxin. L. Baba Moussa, S. Werner, M. Coraiola, D. A. Colin, D. Keller, A. Sanni, M. Dalla Serra, H. Monteil, and G. Prévost Copyright © 2006 L. Baba Moussa et al. All rights reserved. Plasmid-Mediated Dimethoate Degradation by Bacillus licheniformis Isolated From a Fresh Water Fish Labeo rohita Mon, 01 Jan 1900 00:00:00 +0000 The Bacillus licheniformis strain isolated from the intestine of Labeo rohita by an enrichment technique showed capability of utilizing dimethoate as the sole source of carbon. The bacterium rapidly utilized dimethoate beyond 0.6 mg/mL and showed prolific growth in a mineral salts medium containing 0.45 mg/mL dimethoate. The isolated B licheniformis exhibited high level of tolerance of dimethoate (3.5 mg/mL) in nutrient broth, while its cured mutant did not tolerate dimethoate beyond 0.45 mg/mL and it was unable to utilize dimethoate. The wild B licheniformis strain transferred dimethoate degradation property to E coli C600 (Nar, F−) strain. The transconjugant harbored a plasmid of the same molecular size (approximately 54 kb) as that of the donor plasmid; the cured strain was plasmid less. Thus a single plasmid of approximately 54 kb was involved in dimethoate degradation. Genes encoding resistance to antibiotic and heavy metal were also located on the plasmid. Manisha Deb Mandal, Shyamapada Mandal, and Nishith Kumar Pal Copyright © 2005 Hindawi Publishing Corporation. All rights reserved. Response of Escherichia Coli Containing Mycobacterial Carotene Genes to UV Radiation Mon, 01 Jan 1900 00:00:00 +0000 The plasmid pC5, which encodes biogenesis of lycopene in Mycobacterium aurum A +, was partially digested by restriction endonucleases and generated fragments were cloned. After transformation of Escherichia coli (colorless bacteria) with the plasmids so constructed, seven orange clones were detected and found to carry the same recombinant plasmid (pC51). E. coli cells containing this plasmid synthesize neurosporene and lycopene, and were more resistant to ultraviolet irradiation than non pigmented strain. Mohamed Houssaini-Iraqui, Naima Khamlichi, Jamal El Yamani, and Nalin Rastogi Copyright © 2001 Hindawi Publishing Corporation. All rights reserved.