BioMed Research International: Microbiology The latest articles from Hindawi Publishing Corporation © 2016 , Hindawi Publishing Corporation . All rights reserved. Transferrin Impacts Bacillus thuringiensis Biofilm Levels Tue, 29 Nov 2016 14:31:53 +0000 The present study examined the impact of transferrin on Bacillus thuringiensis biofilms. Three commercial strains, an environmental strain (33679), the type strain (10792), and an isolate from a diseased insect (700872), were cultured in iron restricted minimal medium. All strains produced biofilm when grown in vinyl plates at 30°C. B. thuringiensis 33679 had a biofilm biomass more than twice the concentration exhibited by the other strains. The addition of transferrin resulted in slightly increased growth yields for 2 of the 3 strains tested, including 33679. In contrast, the addition of 50 μg/mL of transferrin resulted in an 80% decrease in biofilm levels for strain 33679. When the growth temperature was increased to 37°C, the addition of 50 μg/mL of transferrin increased culture turbidity for only strain 33679. Biofilm levels were again decreased in strain 33679 at 37°C. Growth of B. thuringiensis cultures in polystyrene resulted in a decrease in overall growth yields at 30°C, with biofilm levels significantly decreased for 33679 in the presence of transferrin. These findings demonstrate that transferrin impacts biofilm formation in select strains of B. thuringiensis. Identification of these differences in biofilm regulation may be beneficial in elucidating potential virulence mechanisms among the differing strains. Bianca Garner, Elrica Brown, Martha Taplin, Angel Garcia, and Baracka Williams-Mapp Copyright © 2016 Bianca Garner et al. All rights reserved. Enhanced Performance and Mode of Action of a Novel Antibiofilm Hydrofiber® Wound Dressing Sun, 20 Nov 2016 14:31:44 +0000 Biofilm development in wounds is now acknowledged to be a precursor to infection and a cause of delayed healing. A next-generation antibiofilm carboxymethylcellulose silver-containing wound dressing (NGAD) has been developed to disrupt and kill biofilm microorganisms. This in vitro study aimed to compare its effectiveness against various existing wound dressings and examine its mode of action. A number of biofilm models of increasing complexity were used to culture biofilms of wound-relevant pathogens, before exposure to test dressings. Confocal microscopy, staining, and imaging of biofilm constituents, total viable counting, and elemental analysis were conducted to assess dressing antibiofilm performance. Live/dead staining and viable counting of biofilms demonstrated that the NGAD was more effective at killing biofilm bacteria than two other standard silver dressings. Staining of biofilm polysaccharides showed that the NGAD was also more effective at reducing this protective biofilm component than standard silver dressings, and image analyses confirmed the superior biofilm killing and removal performance of the NGAD. The biofilm-disruptive and silver-enhancing modes of action of the NGAD were supported by significant differences () in biofilm elemental markers and silver donation. This in vitro study improves our understanding of how antibiofilm dressing technology can be effective against the challenge of biofilm. David Parsons, Kate Meredith, Victoria J. Rowlands, Darryl Short, Daniel G. Metcalf, and Philip G. Bowler Copyright © 2016 David Parsons et al. All rights reserved. The Use of Two Culturing Methods in Parallel Reveals a High Prevalence and Diversity of Arcobacter spp. in a Wastewater Treatment Plant Thu, 17 Nov 2016 13:44:41 +0000 The genus Arcobacter includes species considered emerging food and waterborne pathogens. Despite Arcobacter has been linked to the presence of faecal pollution, few studies have investigated its prevalence in wastewater, and the only isolated species were Arcobacter butzleri and Arcobacter cryaerophilus. This study aimed to establish the prevalence of Arcobacter spp. at a WWTP using in parallel two culturing methods (direct plating and culturing after enrichment) and a direct detection by m-PCR. In addition, the genetic diversity of the isolates was established using the ERIC-PCR genotyping method. Most of the wastewater samples (96.7%) were positive for Arcobacter and a high genetic diversity was observed among the 651 investigated isolates that belonged to 424 different ERIC genotypes. However, only few strains persisted at different dates or sampling points. The use of direct plating in parallel with culturing after enrichment allowed recovering the species A. butzleri, A. cryaerophilus, Arcobacter thereius, Arcobacter defluvii, Arcobacter skirrowii, Arcobacter ellisii, Arcobacter cloacae, and Arcobacter nitrofigilis, most of them isolated for the first time from wastewater. The predominant species was A. butzleri, however, by direct plating predominated A. cryaerophilus. Therefore, the overall predominance of A. butzleri was a bias associated with the use of enrichment. Arturo Levican, Luis Collado, and Maria José Figueras Copyright © 2016 Arturo Levican et al. All rights reserved. The Gut Microbial Community of Antarctic Fish Detected by 16S rRNA Gene Sequence Analysis Sun, 13 Nov 2016 09:10:04 +0000 Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and a range of fitness in fish, but little is known about the gut microbial community in Antarctic fish. In this study, the composition of intestinal microbial community in four species of Antarctic fish was detected based on 16S rRNA gene sequencing. As a result, 1 004 639 sequences were obtained from 13 samples identified into 36 phyla and 804 genera, in which Proteobacteria, Actinobacteria, Firmicutes, Thermi, and Bacteroidetes were the dominant phyla, and Rhodococcus, Thermus, Acinetobacter, Propionibacterium, Streptococcus, and Mycoplasma were the dominant genera. The number of common OTUs (operational taxonomic units) varied from 346 to 768, while unique OTUs varied from 84 to 694 in the four species of Antarctic fish. Moreover, intestinal bacterial communities in individuals of each species were not really similar, and those in the four species were not absolutely different, suggesting that bacterial communities might influence the physiological characteristics of Antarctic fish, and the common bacterial communities might contribute to the fish survival ability in extreme Antarctic environment, while the different ones were related to the living habits. All of these results could offer certain information for the future study of Antarctic fish physiological characteristics. Wei Song, Lingzhi Li, Hongliang Huang, Keji Jiang, Fengying Zhang, Xuezhong Chen, Ming Zhao, and Lingbo Ma Copyright © 2016 Wei Song et al. All rights reserved. β-Lactoglobulin Influences Human Immunity and Promotes Cell Proliferation Sun, 13 Nov 2016 07:39:31 +0000 β-Lactoglobulin (LG) is suspected to enhance or modulate human immune responses. Moreover, LG is also hypothesized to increase human cell proliferation. However, these potential functions of LG have not been directly or thoroughly addressed. In this study, we demonstrated that LG is a potent stimulator of cell proliferation using a hybridoma cell (a splenocyte fused with a myeloma cell) model. LG’s ability to promote cell proliferation was lost when the protein is denatured. To further investigate the influence of LG’s conformation on cell proliferation, we chemically modified LG by either carboxymethylation (CM) or acetylation and observed significantly reduced cell proliferation when the protein structure was altered. Furthermore, we proved that LG enhances cell proliferation via receptor-mediated membrane IgM receptor. These data indicated that nondenatured LG is the major component in milk that modulates cell proliferation. Collectively, our study showed that LG plays a key role in enhancing immune responses by promoting cell proliferation through IgM receptor. Chun San Tai, Yi Yun Chen, and Wen Liang Chen Copyright © 2016 Chun San Tai et al. All rights reserved. Identification of Species of Nontuberculous Mycobacteria Clinical Isolates from 8 Provinces of China Wed, 02 Nov 2016 06:31:19 +0000 Pulmonary diseases caused by nontuberculous mycobacteria (NTM) are increasing in incidence and prevalence worldwide. In this study, we identified NTM species of the clinical isolates from 8 provinces in China, in order to preliminarily provide some basic scientific data in the different species and distribution of NTM related to pulmonary disease in China. A total of 523 clinical isolates from patients with tuberculosis (TB) diagnosed clinically from 2005 to 2012 were identified to the species using conventional and molecular methods, including multilocus PCR, rpoB and hsp65 PCR-PRA, hsp65, rpoB, and 16S-23S internal transcribed spacer region sequencing. The isolates were identified into 3 bacterium genera, including NTM, Gordonia bronchialis, and Nocardia farcinica, and, for the 488 NTM isolates, 27 species were identified. For all the 27 species of NTM which were found to cause pulmonary infections in humans, the most prevalent species was M. intracellulare, followed by M. avium and M. abscessus. And seven other species were for the first time identified in patients with TB in China. NTM species identification is very important for distinguishing between tuberculosis and NTM pulmonary diseases, and the species diversity drives the creation of diverse and integrated identification methods with higher accuracy and efficacy. Haican Liu, Lulu Lian, Yi Jiang, Mingxiang Huang, Yunhong Tan, Xiuqin Zhao, Jingrui Zhang, Qin Yu, Jiao Liu, Haiyan Dong, Bing Lu, Yimou Wu, and Kanglin Wan Copyright © 2016 Haican Liu et al. All rights reserved. Genomic Analysis of a Marine Bacterium: Bioinformatics for Comparison, Evaluation, and Interpretation of DNA Sequences Tue, 01 Nov 2016 10:54:48 +0000 A total of five highly related strains of an unidentified marine bacterium were analyzed through their short genome sequences (AM260709–AM260713). Genome-to-Genome Distance (GGDC) showed high similarity to Pseudoalteromonas haloplanktis (X67024). The generated unique Quick Response (QR) codes indicated no identity to other microbial species or gene sequences. Chaos Game Representation (CGR) showed the number of bases concentrated in the area. Guanine residues were highest in number followed by cytosine. Frequency of Chaos Game Representation (FCGR) indicated that CC and GG blocks have higher frequency in the sequence from the evaluated marine bacterium strains. Maximum GC content for the marine bacterium strains ranged 53-54%. The use of QR codes, CGR, FCGR, and GC dataset helped in identifying and interpreting short genome sequences from specific isolates. A phylogenetic tree was constructed with the bootstrap test (1000 replicates) using MEGA6 software. Principal Component Analysis (PCA) was carried out using EMBL-EBI MUSCLE program. Thus, generated genomic data are of great assistance for hierarchical classification in Bacterial Systematics which combined with phenotypic features represents a basic procedure for a polyphasic approach on unambiguous bacterial isolate taxonomic classification. Bhagwan N. Rekadwad, Juan M. Gonzalez, and Chandrahasya N. Khobragade Copyright © 2016 Bhagwan N. Rekadwad et al. All rights reserved. Recent Nanotechnology Approaches for Prevention and Treatment of Biofilm-Associated Infections on Medical Devices Mon, 31 Oct 2016 14:29:46 +0000 Bacterial colonization in the form of biofilms on surfaces causes persistent infections and is an issue of considerable concern to healthcare providers. There is an urgent need for novel antimicrobial or antibiofilm surfaces and biomedical devices that provide protection against biofilm formation and planktonic pathogens, including antibiotic resistant strains. In this context, recent developments in the material science and engineering fields and steady progress in the nanotechnology field have created opportunities to design new biomaterials and surfaces with anti-infective, antifouling, bactericidal, and antibiofilm properties. Here we review a number of the recently developed nanotechnology-based biomaterials and explain underlying strategies used to make antibiofilm surfaces. Mohankandhasamy Ramasamy and Jintae Lee Copyright © 2016 Mohankandhasamy Ramasamy and Jintae Lee. All rights reserved. Changes in the Expression of Biofilm-Associated Surface Proteins in Staphylococcus aureus Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants Thu, 27 Oct 2016 07:14:43 +0000 Sublethal concentrations (sub-MICs) of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three Staphylococcus aureus biofilm formers (strong B+++, moderate B++, and weak B+) that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25–2.5% ethanol and 2500 μg/mL chloramine T significantly enhanced S. aureus biofilm formation. To visualize differences in biofilm compactness between S. aureus biofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors. Lenka Cincarova, Ondrej Polansky, Vladimir Babak, Pavel Kulich, and Petr Kralik Copyright © 2016 Lenka Cincarova et al. All rights reserved. Typing Method for the QUB11a Locus of Mycobacterium tuberculosis: IS6110 Insertions and Tandem Repeat Analysis Wed, 12 Oct 2016 09:31:48 +0000 QUB11a is used as a locus for variable number of tandem repeats (VNTR) analysis of Mycobacterium tuberculosis Beijing lineage. However, amplification of QUB11a occasionally produces large fragments (>1,400 bp) that are not easily measured by capillary electrophoresis because of a lack of the typical stutter peak patterns that are used for counting repeat numbers. IS6110 insertion may complicate VNTR analysis of large QUB11a fragments in M. tuberculosis. We established a method for determining both tandem repeat numbers and IS6110 insertion in the QUB11a locus of M. tuberculosis using capillary electrophoresis analysis and BsmBI digestion. All 29 large QUB11a fragments (>1,200 bp) investigated contained IS6110 insertions and varied in the number of repeats (18 patterns) and location of IS6110 insertions. This method allows VNTR analysis with high discrimination. Eriko Maeda-Mitani, Koichi Murakami, Akira Oishi, Yoshiki Etoh, Nobuyuki Sera, and Shuji Fujimoto Copyright © 2016 Eriko Maeda-Mitani et al. All rights reserved. Synthesis and Antibacterial Activity of Quaternary Ammonium 4-Deoxypyridoxine Derivatives Wed, 05 Oct 2016 13:45:07 +0000 A series of novel quaternary ammonium 4-deoxypyridoxine derivatives was synthesized. Two compounds demonstrated excellent activity against a panel of Gram-positive methicillin-resistant S. aureus strains with MICs in the range of 0.5–2 μg/mL, exceeding the activity of miramistin. At the same time, both compounds were inactive against the Gram-negative E. coli and P. aeruginosa strains. Cytotoxicity studies on human skin fibroblasts and embryonic kidney cells demonstrated that the active compounds possessed similar toxicity with benzalkonium chloride but were slightly more toxic than miramistin. SOS-chromotest in S. typhimurium showed the lack of DNA-damage activity of both compounds; meanwhile, one compound showed some mutagenic potential in the Ames test. The obtained results make the described chemotype a promising starting point for the development of new antibacterial therapies. Nikita V. Shtyrlin, Sergey V. Sapozhnikov, Albina S. Galiullina, Airat R. Kayumov, Oksana V. Bondar, Elena P. Mirchink, Elena B. Isakova, Alexander A. Firsov, Konstantin V. Balakin, and Yurii G. Shtyrlin Copyright © 2016 Nikita V. Shtyrlin et al. All rights reserved. Bioactive Natural Products 2016 Thu, 29 Sep 2016 14:03:12 +0000 Yiannis Kourkoutas, Nikos Chorianopoulos, Kimon A. G. Karatzas, and Ibrahim M. Banat Copyright © 2016 Yiannis Kourkoutas et al. All rights reserved. In Vitro and In Vivo Biofilm Characterization of Methicillin-Resistant Staphylococcus aureus from Patients Associated with Pharyngitis Infection Wed, 28 Sep 2016 15:31:41 +0000 The present investigation was deliberately aimed at evaluating the biofilm-forming ability of 63 clinical MRSA isolates recovered from pharyngitis patients through different phenotypic assays. The molecular detection of adhesion (icaA/icaD/icaB/icaC), adhesins (fnbA/fnbB, clfA, and cna), staphylococcal accessory regulator (sarA), and α-toxin (hla) genes was done by employing polymerase chain reaction (PCR). Out of 63 isolates, 49 (77.8%) were found slime positive by the Congo red agar (CRA) method and 44 (69.8%) as biofilm positive by the quantitative microtitre plate assays. The results of MATH assay showed that most of the test pathogens are hydrophilic in nature. The molecular investigation of biofilm-associated genes revealed that 84.13% () of isolates were found positive for icaADBC genes. The fnbA and fnbB genes were present in 49 (77.8%) and 51 (81%) MRSA isolates, respectively. In addition, 58.7% (), 73% (), and 69.8% () of the isolates harboured the clfA, cna, and hla genes, respectively. Further, nearly 81% () of the isolates were found positive for the gene sarA and all the ica negative isolates were also negative for the gene. Furthermore, the results of in vivo adherence assay unveiled the factual commonness in the in vitro adherence method. Shanmugaraj Gowrishankar, Arumugam Kamaladevi, Krishnaswamy Balamurugan, and Shunmugiah Karutha Pandian Copyright © 2016 Shanmugaraj Gowrishankar et al. All rights reserved. An Ash1-Like Protein MoKMT2H Null Mutant Is Delayed for Conidium Germination and Pathogenesis in Magnaporthe oryzae Mon, 26 Sep 2016 13:49:09 +0000 Ash1 is a known H3K36-specific histone demethylase that is required for normal Hox gene expression and fertility in Drosophila and mammals. However, little is known about the expression and function of the fungal ortholog of Ash1 in phytopathogenic fungus Magnaporthe oryzae. Here we report that MoKMT2H, an Ash1-like protein, is required for conidium germination and virulence in rice. We obtained MoKMT2H null mutant (MoKMT2H) using a target gene replacement strategy. In the MoKMT2H null mutants, global histone methyltransferase modifications (H3K4me3, H3K9me3, H3K27me3, and H3K36me2/3) of the genome were unaffected. The MoKMT2H mutants showed no defect in vegetative hyphal growth, conidium morphology, conidiation, or disease lesion formation on rice leaves. However, the MoKMT2H deletion mutants were delayed for conidium germination and consequently had decreased virulence. Taken together, our results indicated that MoKMT2H plays an important role in conidium germination during appressorium formation in the rice blast fungus and perhaps other pathogenic plant fungi. Zhaojun Cao, Yue Yin, Xuan Sun, Jun Han, Qing peng Sun, Min Lu, Jinbao Pan, and Weixiang Wang Copyright © 2016 Zhaojun Cao et al. All rights reserved. Use of Plant Extracts as an Effective Manner to Control Clostridium perfringens Induced Necrotic Enteritis in Poultry Sun, 25 Sep 2016 09:00:30 +0000 Necrotic enteritis (NE) is an important concern in poultry industry since it causes economic losses, increased mortality, reduction of bird welfare, and contamination of chicken products for human consumption. For decades, the use of in-feed antimicrobial growth promoters (AGPs) has been the main strategy to control intestinal pathogens including Clostridium perfringens (CP), the causative agent of NE. However, the use of AGPs in animal diet has been linked to the emergence and transmission of antimicrobial resistance through food-borne microorganisms, which has led to the ban of AGPs in many countries. This scenario has challenged the poultry industry to search for safer alternative products in order to prevent NE. In this context, the utilization of natural plant extracts with antimicrobial properties appears as a promising and feasible tool to control NE in chicken. In this paper, we review the scientific studies analyzing the potential of plant extracts as alternative feed additives to reduce NE in poultry, with focus on two types of plant products that arise as promising candidates: tannins and essential oils. Some of these products showed antimicrobial activity against CP and coccidia in vitro and in vivo and are able to increase productive performance, emulating the bioactive properties of AGPs. J. M. Diaz Carrasco, L. M. Redondo, E. A. Redondo, J. E. Dominguez, A. P. Chacana, and M. E. Fernandez Miyakawa Copyright © 2016 J. M. Diaz Carrasco et al. All rights reserved. Black Currant (Ribes nigrum L.) and Bilberry (Vaccinium myrtillus L.) Fruit Juices Inhibit Adhesion of Asaia spp. Sun, 25 Sep 2016 08:17:11 +0000 The aim of the study was to evaluate the activity of high-polyphenolic black currant (Ribes nigrum L.) and bilberry (Vaccinium myrtillus L.) juices against bacterial strains Asaia lannensis and Asaia bogorensis isolated as spoilage of commercial soft drinks. The composition of fruit juices was evaluated using chromatographic techniques HPLC and LC-MS. The adhesion to glass, polystyrene, and polyethylene terephthalate in two different culture media was evaluated by luminometry and the plate count method. The major anthocyanins in the V. myrtillus were petunidin-3-glucoside, malvidin-3-glucoside, cyanidin-3-glucoside, and delphinidin-3-glucoside, while in R. nigrum delphinidin-3-rutinoside and cyanidin-3-rutinoside were detected. The LC-MS analysis showed presence of anthocyanins (delphinidin, cyanidin, petunidin, and malvidin derivatives), phenolic acids (chlorogenic and neochlorogenic acids), flavonols (quercetin-3-glucoside, quercetin-3-rutinoside), and flavanols (procyanidin B2 and procyanidin type A2). Additionally, in the bilberry juice A type procyanidin trimer was detected. The adhesion of Asaia spp. cells depended on the type of medium, carbon sources, and the type of abiotic surfaces. We noted that the adhesion was significantly stronger in minimal medium containing sucrose. The addition of bilberry and black currant juices notably reduced bacterial growth as well as cell adhesion to polyethylene terephthalate surfaces. Hubert Antolak, Agata Czyzowska, and Dorota Kregiel Copyright © 2016 Hubert Antolak et al. All rights reserved. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay Tue, 20 Sep 2016 08:03:37 +0000 Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields. Hua-Ying Fu, Sheng-Ren Sun, Jin-Da Wang, Kashif Ahmad, Heng-Bo Wang, Ru-Kai Chen, and San-Ji Gao Copyright © 2016 Hua-Ying Fu et al. All rights reserved. Experimental Models of Oral Biofilms Developed on Inert Substrates: A Review of the Literature Tue, 06 Sep 2016 08:37:36 +0000 The oral ecosystem is a very complex environment where more than 700 different bacterial species can be found. Most of them are organized in biofilm on dental and mucosal surfaces. Studying this community is important because a rupture in stability can lead to the preeminence of pathogenic microorganisms, causing dental decay, gingivitis, or periodontitis. The multitude of species complicates biofilm analysis so its reproduction, collection, and counting are very delicate. The development of experimental models of dental biofilms was therefore essential and multiple in vitro designs have emerged, each of them especially adapted to observing biofilm formation of specific bacteria within specific environments. The aim of this review is to analyze oral biofilm models. Lopez-Nguyen Darrene and Badet Cecile Copyright © 2016 Lopez-Nguyen Darrene and Badet Cecile. All rights reserved. Effects of Betaine Aldehyde Dehydrogenase-Transgenic Soybean on Phosphatase Activities and Rhizospheric Bacterial Community of the Saline-Alkali Soil Sun, 04 Sep 2016 09:46:15 +0000 The development of transgenic soybean has produced numerous economic benefits; however the potential impact of root exudates upon soil ecological systems and rhizospheric soil microbial diversity has also received intensive attention. In the present study, the influence of saline-alkali tolerant transgenic soybean of betaine aldehyde dehydrogenase on bacterial community structure and soil phosphatase during growth stages was investigated. The results showed that, compared with nontransgenic soybean as a control, the rhizospheric soil pH of transgenic soybean significantly decreased at the seedling stage. Compared to HN35, organic P content was 13.5% and 25.4% greater at the pod-filling stage and maturity, respectively. The acid phosphatase activity of SRTS was significantly better than HN35 by 12.74% at seedling, 14.03% at flowering, and 59.29% at podding, while alkaline phosphatase achieved maximum activity in the flowering stage and was markedly lower than HN35 by 13.25% at pod-filling. The 454 pyrosequencing technique was employed to investigate bacterial diversity, with a total of 25,499 operational taxonomic units (OTUs) obtained from the 10 samples. Notably, the effect of SRTS on microbial richness and diversity of rhizospheric soil was marked at the stage of podding and pod-filling. Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla among all samples. Compared with HN35, the relative abundance of Proteobacteria was lower by 2.01%, 2.06%, and 5.28% at the stage of seedling, at pod-bearing, and at maturity. In genus level, the relative abundance of Gp6, Sphingomonas sp., and GP4 was significantly inhibited by SRTS at the stage of pod-bearing and pod-filling. Ying Nie, Da-qing Wang, Guang Zhao, Song Yu, and Hong-yan Wang Copyright © 2016 Ying Nie et al. All rights reserved. Roles of Rack1 Proteins in Fungal Pathogenesis Tue, 30 Aug 2016 08:00:28 +0000 Pathogenic fungi cause diseases on various organisms. Despite their differences in life cycles, fungal pathogens use well-conserved proteins and pathways to regulate developmental and infection processes. In this review, we focus on Rack1, a multifaceted scaffolding protein involved in various biological processes. Rack1 is well conserved in eukaryotes and plays important roles in fungi, though limited studies have been conducted. To accelerate the study of Rack1 proteins in fungi, we review the functions of Rack1 proteins in model and pathogenic fungi and summarize recent progress on how Rack1 proteins are involved in fungal pathogenesis. Xue Zhang, Rashmi Jain, and Guotian Li Copyright © 2016 Xue Zhang et al. All rights reserved. Polyprenols of Ginkgo biloba Enhance Antibacterial Activity of Five Classes of Antibiotics Tue, 23 Aug 2016 14:24:04 +0000 Polyprenol (GBP) from Ginkgo biloba Leaves (GBL) is an important lipid with many bioactive effects. The effect of GBP on antibacterial properties of five antibiotics belonging to different classes was through analysis of inhibition halos, MIC, and FIC index. And we studied the time-killing curves and Ca2+ mobilization assay in Staphylococcus aureus cells treated with GBP microemulsion and gentamicin sulfate under MIC/2 conditions. These results showed that the GBP microemulsion (average diameter 90.2 nm) combining with gentamicin sulfate had the highest enhancing antibacterial effect against Staphylococcus aureus, and the MIC value was 33.0 μg/mL. The increase of the antibacterial effect of tested antibiotics was positively correlated with the decrease of the average diameter of GBP microemulsion. Moreover, GBP microemulsion enhanced antibacterial effect and prolonged antibacterial time of GBP combining with gentamicin sulfate against Staphylococcus aureus. GBP microemulsion could enhance the ability of gentamicin inducing an increase in intracellular calcium concentrations to Staphylococcus aureus. GBP microemulsion could help some classes of antibiotics to inhibit or kill bacteria. This study supports the fact that GBP microemulsion obviously can not only reduce the dosage of some classes of antibiotics, but also reduce the frequency of the antibiotic use in vitro. Ran Tao, Chengzhang Wang, Jianzhong Ye, Hao Zhou, and Hongxia Chen Copyright © 2016 Ran Tao et al. All rights reserved. Growing Menace of Antibacterial Resistance in Clinical Isolates of Pseudomonas aeruginosa in Nepal: An Insight of Beta-Lactamase Production Tue, 23 Aug 2016 06:22:59 +0000 Introduction. Pseudomonas aeruginosa is the most frequently isolated organism as it acts as the opportunistic pathogen and can cause infections in immunosuppressed patients. The production of different types of beta-lactamases renders this organism resistant to many commonly used antimicrobials. Therefore, the aim of this study was to document the antibiotic resistance rate in Pseudomonas aeruginosa isolated from different clinical specimens. Methods. Pseudomonas aeruginosa recovered was identified by standard microbiological methods. Antibiotic susceptibility testing was performed by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines and all the suspected isolates were tested for the production of ESBLs, MBLs, and AmpC. Results. Out of total (178) isolates, 83.1% were recovered from the inpatient department (IPD). Majority of the isolates mediated resistance towards the beta-lactam antibiotics, while nearly half of the isolates were resistant to ciprofloxacin. Most of the aminoglycosides used showed resistance rate up to 75% but amikacin proved to be better option. No resistance to polymyxin was observed. ESBLs, MBLs, and AmpC mediated resistance was seen in 33.1%, 30.9%, and 15.7% isolates, respectively. Conclusions. Antibiotic resistance rate and beta-lactamase mediated resistance were high. Thus, regular surveillance of drug resistance is of utmost importance. Shamshul Ansari, Rabindra Dhital, Sony Shrestha, Sangita Thapa, Ram Puri, Niraj Chaudhary, Suresh Khatiwada, and Rajendra Gautam Copyright © 2016 Shamshul Ansari et al. All rights reserved. Roles of Peroxisomes in the Rice Blast Fungus Tue, 16 Aug 2016 13:00:07 +0000 The rice blast fungus, Magnaporthe oryzae, is a model plant pathogenic fungus and is a severe threat to global rice production. Over the past two decades, it has been found that the peroxisomes play indispensable roles during M. oryzae infection. Given the importance of the peroxisomes for virulence, we review recent advances of the peroxisomes roles during M. oryzae infection processes. We firstly introduce the molecular mechanisms and life cycles of the peroxisomes. And then, metabolic functions related to the peroxisomes are also discussed. Finally, we provide an overview of the relationship between peroxisomes and pathogenicity. Xiao-Lin Chen, Zhao Wang, and Caiyun Liu Copyright © 2016 Xiao-Lin Chen et al. All rights reserved. Microbial and Natural Metabolites That Inhibit Splicing: A Powerful Alternative for Cancer Treatment Tue, 16 Aug 2016 07:49:40 +0000 In eukaryotes, genes are frequently interrupted with noncoding sequences named introns. Alternative splicing is a nuclear mechanism by which these introns are removed and flanking coding regions named exons are joined together to generate a message that will be translated in the cytoplasm. This mechanism is catalyzed by a complex machinery known as the spliceosome, which is conformed by more than 300 proteins and ribonucleoproteins that activate and regulate the precision of gene expression when assembled. It has been proposed that several genetic diseases are related to defects in the splicing process, including cancer. For this reason, natural products that show the ability to regulate splicing have attracted enormous attention due to its potential use for cancer treatment. Some microbial metabolites have shown the ability to inhibit gene splicing and the molecular mechanism responsible for this inhibition is being studied for future applications. Here, we summarize the main types of natural products that have been characterized as splicing inhibitors, the recent advances regarding molecular and cellular effects related to these molecules, and the applications reported so far in cancer therapeutics. Nancy Martínez-Montiel, Nora Hilda Rosas-Murrieta, Mónica Martínez-Montiel, Mayra Patricia Gaspariano-Cholula, and Rebeca D. Martínez-Contreras Copyright © 2016 Nancy Martínez-Montiel et al. All rights reserved. Microtubule Polymerization Functions in Hypersensitive Response and Accumulation of H2O2 in Wheat Induced by the Stripe Rust Tue, 16 Aug 2016 07:47:51 +0000 The plant cytoskeleton, including microtubules and microfilaments, is one of the important factors in determining the polarity of cell division and growth, as well as the interaction of plants with invading pathogens. In defense responses of wheat against the stripe rust (Puccinia striiformis f. sp. tritici) infection, hypersensitive response is the most crucial event to prevent the spread of pathogens. In order to reveal the effect of microtubules on the hypersensitive cell death and H2O2 accumulation in the interaction of wheat (Triticum aestivum) cv. Suwon 11 with an incompatible race, CYR23, wheat leaves were treated with microtubule inhibitor, oryzalin, before inoculation. The results showed that the frequency of infection sites with hypersensitive response occurrence was significantly reduced, and hypersensitive cell death in wheat leaves was suppressed compared to the control. In addition, the frequency and the incidence of infected cells with H2O2 accumulation were also reduced after the treatment with oryzalin. Those results indicated that microtubules are related to hypersensitive response and H2O2 accumulation in wheat induced by the stripe rust, and depolymerization of microtubules reduces the resistance of plants to pathogen infection in incompatible interaction, suggesting that microtubules play a potential role in the expression of resistance of wheat against the stripe rust fungus. Juan Wang, Yang Wang, Xinjie Liu, Yuanliu Xu, and Qing Ma Copyright © 2016 Juan Wang et al. All rights reserved. In Vitro Antimicrobial Bioassays, DPPH Radical Scavenging Activity, and FTIR Spectroscopy Analysis of Heliotropium bacciferum Thu, 11 Aug 2016 06:06:15 +0000 The present study deals with the antimicrobial, antioxidant, and functional group analysis of Heliotropium bacciferum extracts. Disc diffusion susceptibility method was followed for antimicrobial assessment. Noteworthy antimicrobial activities were recorded by various plant extracts against antibiotic resistant microorganisms. Plant flower extracts antioxidant activity was investigated against 2, 2-diphenyl-1-picryl hydrazyl radical by ultraviolet spectrophotometer (517 nm). Plant extracts displayed noteworthy radical scavenging activities at all concentrations (25–225 μg/mL). Notable activities were recorded by crude, chloroform and ethyl acetate extracts up to 88.27% at 225 μg/mL concentration. Compounds functional groups were examined by Fourier transform infrared spectroscopic studies. Alkanes, alkenes, alkyl halides, amines, carboxylic acids, amides, esters, alcohols, phenols, nitrocompounds, and aromatic compounds were identified by FTIR analysis. Thin layer chromatography bioautography was carried out for all plant extracts. Different bands were separated by various solvent systems. The results of the current study justify the use of Heliotropium bacciferum in traditional remedial herbal medicines. Sohail Ahmad, Naser M. AbdEl-Salam, and Riaz Ullah Copyright © 2016 Sohail Ahmad et al. All rights reserved. The Influence of Ethanolic Extract of Brazilian Green Propolis Gel on Hygiene and Oral Microbiota in Patients after Mandible Fractures Wed, 10 Aug 2016 11:29:01 +0000 Maintenance of proper oral hygiene by dental plaque elimination is one of the most important factors affecting the healing process in postoperative oral wounds. Propolis is a substance produced by bees. Ethanolic extract of propolis has bactericidal, fungicidal, anti-inflammatory, and antioxidative properties. Moreover, it can scavenge free radicals. The purpose of this paper is to demonstrate the efficacy of a gel containing 3% of ethanolic extract of Brazilian green propolis (EEP-B) when used for maintaining oral hygiene in patients with postoperative oral mucosal wounds. The hygiene was assessed using API, OHI, and SBI followed by microbiological examinations. The patients were divided into two groups. Group 1 consisted of those who used a gel containing EEP-B for oral hygiene, and group 2 consisted of those who used a gel without EEP-B. Although improved oral hygiene was noted in both groups, the improvement was markedly greater in the group using gel containing EEP-B. Summing up the results of microbiological examinations, EEP-B has beneficial effect on mouth microflora in postoperative period. Propolis preparations used for oral hygiene allow eliminating microorganisms of pathogenic character and physiological flora microorganisms considered as being opportunistic, with no harmful influence on physiological microflora in oral ecosystem. Iwona Niedzielska, Zbigniew Puszczewicz, Anna Mertas, Damian Niedzielski, Bartosz Różanowski, Stefan Baron, Tomasz Konopka, Agnieszka Machorowska-Pieniążek, Małgorzata Skucha-Nowak, Marta Tanasiewicz, Jarosław Paluch, Jarosław Markowski, Bogusława Orzechowska-Wylęgała, Wojciech Król, and Tadeusz Morawiec Copyright © 2016 Iwona Niedzielska et al. All rights reserved. ChSte7 Is Required for Vegetative Growth and Various Plant Infection Processes in Colletotrichum higginsianum Wed, 03 Aug 2016 07:54:36 +0000 Colletotrichum higginsianum is an important hemibiotrophic phytopathogen that causes crucifer anthracnose in various regions of the world. In many plant-pathogenic fungi, the Ste11-Ste7-Fus3/Kss1 kinase pathway is essential to pathogenicity and various plant infection processes. To date, the role of ChSte7 in C. higginsianum encoding a MEK orthologue of Ste7 in Saccharomyces cerevisiae has not been elucidated. In this report, we investigated the function of ChSte7 in the pathogen. The ChSte7 is predicted to encode a 522-amino-acid protein with a S_TKc conserved domain that shares 44% identity with Ste7 in S. cerevisiae. ChSte7 disruption mutants showed white colonies with irregularly shaped edges and extremely decreased growth rates and biomass productions. The ChSte7 disruption mutants did not form appressoria and showed defects in pathogenicity on leaves of Arabidopsis thaliana. When inoculated onto wounded leaf tissues, the ChSte7 disruption mutants grew only on the surface of host tissues but failed to cause lesions beyond the wound site. In contrast, both the wild-type and complementation strains showed normal morphology, produced appressoria, and caused necrosis on leaves of Arabidopsis. Analysis with qRT-PCR suggested that ChSte7 was highly expressed during the late stages of infection. Taken together, our results demonstrate that ChSte7 is involved in regulation of vegetative growth, appressorial formation of C. higginsianum, and postinvasive growth in host tissues. Qinfeng Yuan, Meijuan Chen, Yaqin Yan, Qiongnan Gu, Junbin Huang, and Lu Zheng Copyright © 2016 Qinfeng Yuan et al. All rights reserved. Detection of Extended Spectrum Beta-Lactamases Resistance Genes among Bacteria Isolated from Selected Drinking Water Distribution Channels in Southwestern Nigeria Wed, 03 Aug 2016 06:54:59 +0000 Extended Spectrum Beta-Lactamases (ESBL) provide high level resistance to beta-lactam antibiotics among bacteria. In this study, previously described multidrug resistant bacteria from raw, treated, and municipal taps of DWDS from selected dams in southwestern Nigeria were assessed for the presence of ESBL resistance genes which include , , and by PCR amplification. A total of 164 bacteria spread across treated (33), raw (66), and municipal taps (68), belonging to -Proteobacteria, -Proteobacteria, -Proteobacteria, Flavobacteriia, Bacilli, and Actinobacteria group, were selected for this study. Among these bacteria, the most commonly observed resistance was for ampicillin and amoxicillin/clavulanic acid (61 isolates). Sixty-one isolates carried at least one of the targeted ESBL genes with being the most abundant (50/61) and being detected least (3/61). Klebsiella was the most frequently identified genus (18.03%) to harbour ESBL gene followed by Proteus (14.75%). Moreover, combinations of two ESBL genes, or , were observed in 11 and 1 isolate, respectively. In conclusion, classic ESBL gene was present in multiple bacterial strains that were isolated from DWDS sources in Nigeria. These environments may serve as foci exchange of genetic traits in a diversity of Gram-negative bacteria. Ayodele T. Adesoji and Adeniyi A. Ogunjobi Copyright © 2016 Ayodele T. Adesoji and Adeniyi A. Ogunjobi. All rights reserved. Antidermatophytic Activity of Ethanolic Extract from Croton tiglium Sun, 03 Jul 2016 07:45:49 +0000 Dermatophytosis, which is caused mainly by genera of Trichophyton, Epidermophyton, and Microsporum, is a frequent dermatological problem in tropical and subtropical countries. Investigations were carried out in this study to evaluate the antidermatophytic activity of the stems, leaves, and seeds of Croton tiglium, one of the traditional medicine plants indigenous to Asia. Ethanolic extracts of the stems, leaves, and seeds of C. tiglium were prepared by cold soak or heat reflux methods. The antidermatophytic activities of the extracts were evaluated by disc diffusion and microdilution susceptibility assays against Trichophyton mentagrophytes, T. rubrum, and Epidermophyton floccosum. The active components in the extracts were analyzed and identified by GC-MS. All ethanolic extracts of C. tiglium showed some antifungal activities against the three dermatophytes. The ethanolic stem extract had the greatest inhibitory activities against T. mentagrophytes and E. floccosum with MICs at 0.16 mg/mL and had a lower activity against T. rubrum (MIC: 0.31 mg/mL). Oleic acid and hexadecanoic acid were found to be the major constituents in the stem extract that demonstrated strong antidermatophytic activities. The ethanolic extracts of stem or seed of C. tiglium exhibit strong antidermatophytic activities and, thus, could be considered for application on treating skin fungal infections after appropriate processing. Han Chien Lin, Yu-Liang Kuo, Wen-Ju Lee, Hui-Yi Yap, and Shao-Hung Wang Copyright © 2016 Han Chien Lin et al. All rights reserved. Immunomodulatory Effects of Lactobacillus plantarum Lp62 on Intestinal Epithelial and Mononuclear Cells Thu, 30 Jun 2016 13:06:35 +0000 Probiotic lactic acid bacteria are known for their ability to modulate the immune system. They have been shown to inhibit inflammation in experiments with animal models, cell culture, and clinical trials. The objective of this study was to elucidate the anti-inflammatory potential of Lactobacillus plantarum Lp62, isolated from cocoa fermentation, in a cell culture model. Lp62 inhibited IL-8 production by Salmonella Typhi-stimulated HT-29 cells and prevented the adhesion of pathogens to these epithelial cells. The probiotic strain was able to modulate TNF-α, IL1-β, and IL-17 secretion by J774 macrophages. J774 activation was reduced by coincubation with Lp62. PBMC culture showed significantly higher levels of CD4+CD25+ T lymphocytes following treatment with Lp62. Probiotics also induced increased IL-10 secretion by mononuclear cells. L. plantarum Lp62 was able to inhibit inflammatory stimulation in epithelial cells and macrophages and activated a tolerogenic profile in mononuclear cells of healthy donors. These results indicate this strain for a possible application in the treatment or prevention of inflammatory diseases. Thalis Ferreira dos Santos, Tauá Alves Melo, Milena Evangelista Almeida, Rachel Passos Rezende, and Carla Cristina Romano Copyright © 2016 Thalis Ferreira dos Santos et al. All rights reserved. Conjugated Linoleic Acid Production by Bifidobacteria: Screening, Kinetic, and Composition Mon, 27 Jun 2016 14:27:11 +0000 Conjugated linoleic acids (CLA) are positional and geometric isomers of linoleic acid involved in a number of health aspects. In humans, CLA production is performed by gut microbiota, including some species of potential probiotic bifidobacteria. 128 strains of 31 Bifidobacterium species were screened with a spectrophotometric assay to identify novel CLA producers. Most species were nonproducers, while producers belonged to B. breve and B. pseudocatenulatum. GC-MS revealed that CLA producer strains yielded 9cis,11trans-CLA and 9trans,11trans-CLA, without any production of other isomers. Hydroxylated forms of LA were absent in producer strains, suggesting that the myosin-cross-reactive antigen (MCRA) protein that exerts hydratase activity is not involved in LA isomerization. Moreover, both CLA producer and nonproducer species bear a MCRA homologue. The strain B. breve WC 0421 was the best CLA producer, converting LA into 68.8% 9cis,11trans-CLA and 25.1% 9trans,11trans-CLA. Production occurred mostly during the lag and the exponential phase. For the first time, production and incorporation of CLA in biomass were assessed. B. breve WC 0421 stored CLA in the form of free fatty acids, without changing the composition of the esterified fatty acids, which mainly occurred in the plasmatic membrane. Stefano Raimondi, Alberto Amaretti, Alan Leonardi, Andrea Quartieri, Caterina Gozzoli, and Maddalena Rossi Copyright © 2016 Stefano Raimondi et al. All rights reserved. Synergistic Effects of Sulfated Polysaccharides from Mexican Seaweeds against Measles Virus Wed, 22 Jun 2016 11:56:44 +0000 Sulfated polysaccharides (SPs) extracted from five seaweed samples collected or cultivated in Mexico (Macrocystis pyrifera, Eisenia arborea, Pelvetia compressa, Ulva intestinalis, and Solieria filiformis) were tested in this study in order to evaluate their effect on measles virus in vitro. All polysaccharides showed antiviral activity (as measured by the reduction of syncytia formation) and low cytotoxicity (MTT assay) at inhibitory concentrations. SPs from Eisenia arborea and Solieria filiformis showed the highest antiviral activities (confirmed by qPCR) and were selected to determine their combined effect. Their synergistic effect was observed at low concentrations (0.0274 μg/mL and 0.011 μg/mL of E. arborea and S. filiformis SPs, resp.), which exhibited by far a higher inhibitory effect (96% syncytia reduction) in comparison to the individual SP effects (50% inhibition with 0.275 μg/mL and 0.985 μg/mL of E. arborea and S. filiformis, resp.). Time of addition experiments and viral penetration assays suggest that best activities of these SPs occur at different stages of infection. The synergistic effect would allow reducing the treatment dose and toxicity and minimizing or delaying the induction of antiviral resistance; sulfated polysaccharides of the tested seaweed species thus appear as promising candidates for the development of natural antiviral agents. Karla Morán-Santibañez, Lucia Elizabeth Cruz-Suárez, Denis Ricque-Marie, Daniel Robledo, Yolanda Freile-Pelegrín, Mario A. Peña-Hernández, Cristina Rodríguez-Padilla, and Laura M. Trejo-Avila Copyright © 2016 Karla Morán-Santibañez et al. All rights reserved. Comparative Analysis of Human B Cell Epitopes Based on BCG Genomes Sun, 12 Jun 2016 12:05:58 +0000 Background. Tuberculosis is a huge global health problem. BCG is the only vaccine used for about 100 years against TB, but the reasons for protection variability in populations remain unclear. To improve BCG efficacy and develop a strategy for new vaccines, the underlying genetic differences among BCG subtypes should be understood urgently. Methods and Findings. Human B cell epitope data were collected from the Immune Epitope Database. Epitope sequences were mapped with those of 15 genomes, including 13 BCGs, M. bovis AF2122/97, and M. tuberculosis H37Rv, to identify epitopes distribution. Among 398 experimentally verified B cell epitopes, 321 (80.7%) were conserved, while the remaining 77 (19.3%) were lost to varying degrees in BCGs. The variable protective efficacy of BCGs may result from the degree of B cell epitopes deficiency. Conclusions. Here we firstly analyzed the genetic characteristics of BCGs based on B cell epitopes and found that B cell epitopes distribution may contribute to vaccine efficacy. Restoration of important antigens or effective B cell epitopes in BCG could be a useful strategy for vaccine development. Machao Li, Haican Liu, Xiuqin Zhao, and Kanglin Wan Copyright © 2016 Machao Li et al. All rights reserved. Prevalence of Extended Spectrum β-Lactamase and Antimicrobial Susceptibility Pattern of Clinical Isolates of Pseudomonas from Patients of Khyber Pakhtunkhwa, Pakistan Sun, 05 Jun 2016 12:33:16 +0000 Majority of gram negative pathogenic bacteria are responsible for extended spectrum β-lactamases (ESBLs) production, which show resistance to some newer generation of antibiotics. The study was aimed at evaluating the prevalence of ESBL and antibiotic susceptibility pattern of Pseudomonas isolates collected during 2010 to 2014 from tertiary care hospitals of Peshawar, Pakistan. Out of 3450 samples, 334 Pseudomonas spp. isolates comprised of 232 indoor and 102 outdoor patients were obtained from different specimens and their susceptibility pattern was determined against 20 antibiotics. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer agar diffusion method and ESBL production was detected by Synergy Disc Diffusion technique. The mean age group of the patients was 29.9 + 9.15 years. Meronem showed best activity (91.02%) from class carbapenem, β-lactam and β-lactamase inhibitors exhibited 69.16% activity, and doxycycline had a diminished activity (10.18%) to Pseudomonas spp. Outdoor isolates were more resistant than the indoor and during the course of the study the sensitivity rate of antibiotics was gradually reducing. ESBL production was observed in 44.32% while the remaining was non-ESBL. The moderate active antibiotics were amikacin (50.7%), SCF (51.4%), TZP (52.7%), and MXF (54.1%) among ESBL producing isolates. Lack of antibiotic policy, irrational uses (3GCs particularly), and the emergence of antibiotic resistant organisms in hospitals may be causes of high antibiotic resistance. Manzoor Ahmad, Mukhtiar Hassan, Anwar Khalid, Imran Tariq, Muhammad Hassham Hassan Bin Asad, Abdul Samad, Qaisar Mahmood, and Ghulam Murtaza Copyright © 2016 Manzoor Ahmad et al. All rights reserved. Cockroaches as a Source of High Bacterial Pathogens with Multidrug Resistant Strains in Gondar Town, Ethiopia Thu, 02 Jun 2016 09:18:52 +0000 Background. Cockroaches are source of bacterial infections and this study was aimed to assess bacterial isolates and their antimicrobial profiles from cockroaches in Gondar town, Ethiopia. Methods. A total of 60 cockroaches were collected from March 1 to May 30, 2014, in Gondar town. Bacterial species were isolated from external and internal parts of cockroaches. Disk diffusion method was used to determine antibiotic susceptibility patterns. Data were entered and analyzed by using SPSS version 20; values <0.005 were considered as statistically significant. Results. Of 181 identified bacteria species, 110 (60.8%) and 71 (39.2%) were identified from external and internal parts of cockroaches, respectively. Klebsiella pneumoniae 32 (17.7%), Escherichia coli 29 (16%), and Citrobacter spp. 27 (15%) were the predominant isolates. High resistance rate was observed to cotrimoxazole, 60 (33.1%), and least resistance rate was noted to ciprofloxacin, 2 (1.1%). Additionally, 116 (64.1%) of the isolates were MDR strains; Salmonella spp. were the leading MDR isolates (100%) followed by Enterobacter (90.5%) and Shigella spp. (76.9%). Conclusion. Cockroaches are the potential source of bacteria pathogens with multidrug resistant strains and hence effective preventive and control measures are required to minimize cockroach related infections. Feleke Moges, Setegn Eshetie, Mengistu Endris, Kahsay Huruy, Dagnachew Muluye, Tigist Feleke, Fisha G/Silassie, Getenet Ayalew, and Raja Nagappan Copyright © 2016 Feleke Moges et al. All rights reserved. Fecal Colonization with Extended-Spectrum Beta-Lactamase and AmpC-Producing Escherichia coli Tue, 31 May 2016 09:34:18 +0000 Background. Extended-spectrum β-lactamases (ESβLs) and AmpC β-lactamases cause β-lactam resistance in Escherichia coli. Fecal colonization by ESβL- and/or AmpC-positive E. coli is a source of nosocomial infections. Methods. In order to investigate inpatient fecal colonization by ESβLs and AmpC, antibiotic sensitivity tests were conducted and minimum inhibitory concentrations (MICs) were determined using the disk diffusion method and E-test, respectively. Characterization of ESβL and AmpC was performed using E-test strips, and a set of PCRs and DNA sequence analyses were used to characterize the ESβL and AmpC genes. Results. The whole collection of E. coli isolates () was sensitive to imipenem, tigecycline, colistin, and fosfomycin, while 26% of the isolates showed reduced susceptibility to ceftazidime (MIC ≥ 4 μg/mL). ESβL was phenotypically identified in 26% (13/50) of cases, while AmpC activity was detected in two ESβL-producing E. coli isolates. All ESβL-producing E. coli were positive for the CTX-M gene, eleven isolates carried , and two isolates carried gene. Two CTX-M-positive E. coli isolates carried . Conclusions. The alimentary tract is a significant reservoir for ESβL- and/or AmpC-producing E. coli, which may lead to nosocomial infection. Mohamed H. Al-Agamy, Taghrid S. El Mahdy, and Atef M. Shibl Copyright © 2016 Mohamed H. Al-Agamy et al. All rights reserved. Effect of LongZhang Gargle on Biofilm Formation and Acidogenicity of Streptococcus mutans In Vitro Wed, 25 May 2016 13:16:50 +0000 Streptococcus mutans, with the ability of high-rate acid production and strong biofilm formation, is considered the predominant bacterial species in the pathogenesis of human dental caries. Natural products which may be bioactive against S. mutans have become a hot spot to researches to control dental caries. LongZhang Gargle, completely made from Chinese herbs, was investigated for its effects on acid production and biofilm formation by S. mutans in this study. The results showed an antimicrobial activity of LongZhang Gargle against S. mutans planktonic growth at the minimum inhibitory concentration (MIC) of 16% and minimum bactericidal concentration (MBC) of 32%. Acid production was significantly inhibited at sub-MIC concentrations. Biofilm formation was also significantly disrupted, and 8% was the minimum concentration that resulted in at least 50% inhibition of biofilm formation (MBIC50). A scanning electron microscopy (SEM) showed an effective disruption of LongZhang Gargle on S. mutans biofilm integrity. In addition, a confocal laser scanning microscopy (CLSM) suggested that the extracellular polysaccharides (EPS) synthesis could be inhibited by LongZhang Gargle at a relatively low concentration. These findings suggest that LongZhang Gargle may be a promising natural anticariogenic agent in that it suppresses planktonic growth, acid production, and biofilm formation against S. mutans. Yutao Yang, Shiyu Liu, Yuanli He, Zhu Chen, and Mingyun Li Copyright © 2016 Yutao Yang et al. All rights reserved. Biological Activities of Aerial Parts Extracts of Euphorbia characias Tue, 24 May 2016 10:36:32 +0000 The aim of the present study was to evaluate antioxidant, antimicrobial, anti-HIV, and cholinesterase inhibitory activities of aqueous and alcoholic extracts from leaves, stems, and flowers of Euphorbia characias. The extracts showed a high antioxidant activity and were a good source of total polyphenols and flavonoids. Ethanolic extracts from leaves and flowers displayed the highest inhibitory activity against acetylcholinesterase and butyrylcholinesterase, showing potential properties against Alzheimer’s disease. Antimicrobial assay showed that leaves and flowers extracts were active against all Gram-positive bacteria tested. The ethanolic leaves extract appeared to have the strongest antibacterial activity against Bacillus cereus with MIC value of 312.5 μg/mL followed by Listeria monocytogenes and Staphylococcus aureus that also exhibited good sensitivity with MIC values of 1250 μg/mL. Moreover, all the extracts possessed anti-HIV activity. The ethanolic flower extract was the most potent inhibitor of HIV-1 RT DNA polymerase RNA-dependent and Ribonuclease H with IC50 values of 0.26 and 0.33 μg/mL, respectively. The LC-DAD metabolic profile showed that ethanolic leaves extract contains high levels of quercetin derivatives. This study suggests that Euphorbia characias extracts represent a good source of natural bioactive compounds which could be useful for pharmaceutical application as well as in food system for the prevention of the growth of food-borne bacteria and to extend the shelf-life of processed foods. Maria Barbara Pisano, Sofia Cosentino, Silvia Viale, Delia Spanò, Angela Corona, Francesca Esposito, Enzo Tramontano, Paola Montoro, Carlo Ignazio Giovanni Tuberoso, Rosaria Medda, and Francesca Pintus Copyright © 2016 Maria Barbara Pisano et al. All rights reserved. Etiologic Agents of Bacterial Sepsis and Their Antibiotic Susceptibility Patterns among Patients Living with Human Immunodeficiency Virus at Gondar University Teaching Hospital, Northwest Ethiopia Mon, 23 May 2016 12:10:03 +0000 Background. Bacterial sepsis is a major cause of illness in human immunodeficiency virus infected patients. There is scarce evidence about sepsis among HIV patients in Ethiopia. This study aimed to determine the etiologic agents of bacterial sepsis and their antibiotic susceptibility patterns among HIV infected patients. Methods. A cross-sectional study was carried out from March 1 to May 2, 2013. One hundred patients infected with HIV and suspected of having sepsis were included. Sociodemographic data were collected by interview and blood sample was aseptically collected from study participants. All blood cultures were incubated aerobically at 35°C and inspected daily for 7 days. The positive blood cultures were identified following the standard procedures and antimicrobial susceptibility testing was performed using disk diffusion technique. Data was entered by Epi-info version 3.5.1 and analysis was done using SPSS version 20. Results. Of the study participants, 31 (31%) confirmed bacterial sepsis. The major isolates were 13 (13%) Staphylococcus aureus, 8 (8%) coagulates negative staphylococci, and 3 (3%) viridans streptococci. Majority of the isolates, 25 (80.6%), were multidrug resistant to two or more antimicrobial agents. Conclusions. Bacterial sepsis was a major cause of admission for HIV infected patients predominated by Staphylococcus aureus and coagulase negative staphylococci species and most of the isolates were multidrug resistant. Gelila Alebachew, Brhanu Teka, Mengistu Endris, Yitayal Shiferaw, and Belay Tessema Copyright © 2016 Gelila Alebachew et al. All rights reserved. Effects of Nutrients in Substrates of Different Grains on Aflatoxin B1 Production by Aspergillus flavus Tue, 17 May 2016 08:58:00 +0000 The current study was to better understand the potential factors affecting aflatoxin B1 (AFB1) accumulation varies between different grains. The nutrient composition and contents of defatted substrates were determined; additionally, according to the nutrient content of the substrates, the effects of starch, soluble sugars, amino acids, and trace elements on AFB1 production and mycelial growth in Czapek-Dox medium were examined. These results verified that removal of lipids from ground substrates significantly reduced the substrate’s potential for AFB1 production by Aspergillus flavus. Maltose, glucose, sucrose, arginine, glutamic acid, aspartic acid, and zinc significantly induced AFB1 production up to 1.7- to 26.6-fold. And stachyose more significantly promoted A. flavus growth than the other nutrients. Thus, this study demonstrated that, combined with the nutrients content of grains, in addition to lipids, sucrose, stachyose, glutamic acid, and zinc might play key roles in various grains that are differentially infected by A. flavus. Particularly, two new nutrients (arginine and stachyose) of the grains we found significantly stimulate AFB1 production and A. flavus growth, respectively. The results provide new concepts for antifungal methods to protect food and animal feed from AFB1 contamination. Jie Liu, Lvhui Sun, Niya Zhang, Jiacai Zhang, Jiao Guo, Chong Li, Shahid Ali Rajput, and Desheng Qi Copyright © 2016 Jie Liu et al. All rights reserved. Improvement of the Texture of Yogurt by Use of Exopolysaccharide Producing Lactic Acid Bacteria Sun, 15 May 2016 08:02:38 +0000 19 Streptococcus thermophilus with high exopolysaccharide production were isolated from traditional Chinese fermented dairy products. The exopolysaccharide and viscosity of milk fermented by these 19 isolates were assayed. The strains of Streptococcus thermophilus zlw TM11 were selected because its fermented milk had the highest exopolysaccharide content (380 mg/L) and viscosity (7716 mpa/s). Then Streptococcus thermophilus zlw TM11 was combined with Lactobacillus delbrueckii subsp. bulgaricus 3 4.5 and the combination was named SH-1. The quality of the yogurt fermented by SH-1 and two commercial starter cultures (YO-MIX 465, YF-L711) were compared. It was shown that the exopolysaccharide content of yogurt fermented by SH-1 was similar to that of yogurt fermented by YF-L711 and significantly higher than YO-MIX 465 (). In addition, the yogurt fermented by SH-1 had the lowest syneresis (8.5%) and better texture and sensory than the samples fermented by YO-MIX 465 and YF-L711. It manifested that the selected higher exopolysaccharide production starter SH-1 could be used as yogurt starter and reduce the amount of adding stabilizer, which can compare with the imported commercial starter culture. Xue Han, Zhe Yang, Xueping Jing, Peng Yu, Yingchun Zhang, Huaxi Yi, and Lanwei Zhang Copyright © 2016 Xue Han et al. All rights reserved. Antifungal Activity of Lactobacillus sp. Bacteria in the Presence of Xylitol and Galactosyl-Xylitol Thu, 12 May 2016 17:25:27 +0000 Lactic acid fermentation is a natural method of antimicrobial food protection. Antagonistic activity of Lactobacillus sp. bacteria, taking part in this process, is directed mainly against the same or other microorganisms. In this work we determine the impact of the presence of xylitol and galactosyl-xylitol on the antagonistic activity of 60 Lactobacillus sp. strains against indicator molds (Alternaria alternata, Alternaria brassicicola, Aspergillus niger, Fusarium latenicum, Geotrichum candidum, and Mucor hiemalis) and yeasts (Candida vini). We used double-layer method to select antifungal strains of Lactobacillus bacteria and poisoned medium method to confirm their fungistatic properties. Additionally, we examined the inhibition of Alternaria brassicicola by Lactobacillus paracasei ŁOCK 0921 cultivated with xylitol or galactosyl-xylitol directly on wild cherries. The presence of xylitol and its galactosyl derivative led to increase of spectrum of antifungal activity in most of the studied plant-associated lactobacilli strains. However, no single strain exhibited activity against all the indicator microorganisms. The antifungal activity of Lactobacillus bacteria against molds varied considerably and depended on both the indicator strain and the composition of the medium. The presence of xylitol and galactosyl-xylitol in the growth medium is correlated with the antifungal activity of the studied Lactobacillus sp. bacteria against selected indicator molds. Lidia Lipińska, Robert Klewicki, Elżbieta Klewicka, Krzysztof Kołodziejczyk, Michał Sójka, and Adriana Nowak Copyright © 2016 Lidia Lipińska et al. All rights reserved. Vector Potential of Blattella germanica (L.) (Dictyoptera: Blattidae) for Medically Important Bacteria at Food Handling Establishments in Jimma Town, Southwest Ethiopia Wed, 11 May 2016 13:50:30 +0000 Cockroaches have been regarded as possible vectors of human enteropathogens. Their presence and crawl particularly in food handling establishments could be risky for human health. Therefore, this study was done to determine the vector potential of cockroach for medically important bacterial pathogens in restaurants and cafeterias. A cross-sectional study was conducted on cockroaches from restaurants and cafeterias in Jimma town from May to September 2014. Standard taxonomic keys and microbiological techniques were applied for species identification and isolation. Data was analyzed in SPSS version 16.0. All cockroaches trapped were the German cockroach, Blattella germanica (L.) (Dictyoptera: Blattidae). Escherichia coli was the most frequently isolated followed by Salmonella species (serogroups B, D, E, C1, and NG), Bacillus cereus, and Shigella flexneri. Wide varieties of bacteria of medical relevance were also identified. Of which, Klebsiella spp. 49(40.8%), Bacillus spp., and Staphylococcus saprophyticus were predominant. Blattella germanica (L.) (Dictyoptera: Blattidae) could serve as a potential vector for the dissemination of foodborne pathogens such as Salmonella spp., Shigella flexneri, E. coli, S. aureus, and B. cereus and these bacteria could be a major threat to public health. Therefore, environmental sanitation and standard hygiene need to be applied in the food handling establishments in that locality. Fithamlak Solomon, Fanuel Belayneh, Gebre Kibru, and Solomon Ali Copyright © 2016 Fithamlak Solomon et al. All rights reserved. Diversity of Virulence Factors Associated with West Australian Methicillin-Sensitive Staphylococcus aureus Isolates of Human Origin Mon, 09 May 2016 07:51:01 +0000 An extensive array of virulence factors associated with S. aureus has contributed significantly to its success as a major nosocomial pathogen in hospitals and community causing variety of infections in affected patients. Virulence factors include immune evading capsular polysaccharides, poly-N-acetyl glucosamine, and teichoic acid in addition to damaging toxins including hemolytic toxins, enterotoxins, cytotoxins, exfoliative toxin, and microbial surface components recognizing adhesive matrix molecules (MSCRAMM). In this investigation, 31 West Australian S. aureus isolates of human origin and 6 controls were analyzed for relative distribution of virulence-associated genes using PCR and/or an immunoassay kit and MSCRAMM by PCR-based typing. Genes encoding MSCRAMM, namely, Spa, ClfA, ClfB, SdrE, SdrD, IsdA, and IsdB, were detected in >90% of isolates. Gene encoding α-toxin was detected in >90% of isolates whereas genes encoding β-toxin and SEG were detectable in 50–60% of isolates. Genes encoding toxin proteins, namely, SEA, SEB, SEC, SED, SEE, SEH, SEI, SEJ, TSST, PVL, ETA, and ETB, were detectable in >50% of isolates. Use of RAPD-PCR for determining the virulence factor-based genetic relatedness among the isolates revealed five cluster groups confirming genetic diversity among the MSSA isolates, with the greatest majority of the clinical S. aureus (84%) isolates clustering in group IIIa. Charlene Babra Waryah, Jully Gogoi-Tiwari, Kelsi Wells, Karina Yui Eto, Elnaz Masoumi, Paul Costantino, Michael Kotiw, and Trilochan Mukkur Copyright © 2016 Charlene Babra Waryah et al. All rights reserved. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens Thu, 05 May 2016 12:03:18 +0000 The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are the leading cause of nosocomial infections throughout the world. Most of them are multidrug resistant isolates, which is one of the greatest challenges in clinical practice. Multidrug resistance is amongst the top three threats to global public health and is usually caused by excessive drug usage or prescription, inappropriate use of antimicrobials, and substandard pharmaceuticals. Understanding the resistance mechanisms of these bacteria is crucial for the development of novel antimicrobial agents or other alternative tools to combat these public health challenges. Greater mechanistic understanding would also aid in the prediction of underlying or even unknown mechanisms of resistance, which could be applied to other emerging multidrug resistant pathogens. In this review, we summarize the known antimicrobial resistance mechanisms of ESKAPE pathogens. Sirijan Santajit and Nitaya Indrawattana Copyright © 2016 Sirijan Santajit and Nitaya Indrawattana. All rights reserved. Isolation and Characterization of a Broad Spectrum Bacteriocin from Bacillus amyloliquefaciens RX7 Thu, 28 Apr 2016 14:20:45 +0000 We isolated a Bacillus strain, RX7, with inhibitory activity against Listeria monocytogenes from soil and identified it as Bacillus amyloliquefaciens based on 16S rRNA gene sequencing. The inhibitory activity was stable over a wide range of pH and was fully retained after 30 min at 80°C, after which it decreased gradually at higher temperatures. The activity was sensitive to the proteolytic action of α-chymotrypsin, proteinase-K, and trypsin, indicating its proteinaceous nature. This bacteriocin was active against a broad spectrum of bacteria and the fungus Candida albicans. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of approximately 5 kDa. Ammonium sulfate precipitation and anion-exchange and gel permeation chromatography integrated with reverse phase-high-performance liquid chromatography were used for bacteriocin purification. Automated N-terminal Edman degradation of the purified RX7 bacteriocin recognized the first 15 amino acids as NH2-X-Ala-Trp-Tyr-Asp-Ile-Arg-Lys-Leu-Gly-Asn-Lys-Gly-Ala, where the letter X in the sequence indicates an unknown or nonstandard amino acid. Based on BLAST similarity search and multiple alignment analysis, the obtained partial sequence showed high homology with the two-peptide lantibiotic haloduracin (HalA1) from Bacillus halodurans, although at least two amino acids differed between the sequences. A time-kill study demonstrated a bactericidal mode of action of RX7 bacteriocin. Kong Boon Lim, Marilen P. Balolong, Sang Hoon Kim, Ju Kyoung Oh, Ji Yoon Lee, and Dae-Kyung Kang Copyright © 2016 Kong Boon Lim et al. All rights reserved. Antibacterial Efficacy of Calcium Hypochlorite with Vibringe Sonic Irrigation System on Enterococcus faecalis: An In Vitro Study Wed, 27 Apr 2016 12:05:58 +0000 Aim. The purpose of this study was to compare the in vitro efficacy of calcium hypochlorite (Ca[OCl]2) and sodium hypochlorite (NaOCl) associated with sonic (Vibringe) irrigation system in root canals which were contaminated with Enterococcus faecalis. Material and Methods. The root canals of 84 single-rooted premolars were enlarged up to a file 40, autoclaved, inoculated with Enterococcus faecalis, and incubated for 21 days. The samples were divided into 7 groups according to the irrigation protocol: G0: no treatment; G1: distilled water; G2: 2.5% NaOCl; G3: 2.5% Ca(OCl)2; G4: distilled water with sonic activation; G5: 2.5% NaOCl with sonic activation; and G6: 2.5% Ca(OCl)2 with sonic activation. Before and after decontamination procedures microbiological samples were collected and the colony-forming units were counted and the percentages of reduction were calculated. Results. Distilled water with syringe irrigation and sonic activation groups demonstrated poor antibacterial effect on Enterococcus faecalis compared to other experimental groups (). There was no statistically significant difference between syringe and sonic irrigation systems with Ca(OCl)2 and NaOCl. Conclusion. The antimicrobial property of Ca(OCl)2 has been investigated and compared with that of NaOCl. Both conventional syringe irrigation and sonic irrigation were found effective at removing E. faecalis from the root canal of extracted human teeth. Aysin Dumani, Hatice Korkmaz Guvenmez, Sehnaz Yilmaz, Oguz Yoldas, and Zeliha Gonca Bek Kurklu Copyright © 2016 Aysin Dumani et al. All rights reserved. Phytochemical Composition and Antibacterial Activity of Hydroalcoholic Extracts of Pterospartum tridentatum and Mentha pulegium against Staphylococcus aureus Isolates Thu, 14 Apr 2016 13:10:04 +0000 Pterospartum tridentatum and Mentha pulegium are largely used in Portuguese folk medicine to treat several human disorders and inflammatory processes but without any consistent evidence for those beneficial pointed properties. Thus, the aim of the current work is to evaluate its benefits and phytochemicals related to those beneficial properties. A distinct polyphenol profile between P. tridentatum and M. pulegium was found. Taxifolin, myricetin, ginestin, ginestein, and ginestein derivatives, biochanin A-glucoside, and biochanin A were identified in P. tridentatum, whilst in M. pulegium the luteolin-7-rutinoside, diosmin, and apigenin and respective derivatives were most representative polyphenols. These variations had implications in the antiradical and antibacterial activity and the P. tridentatum exhibited the highest antibacterial activity against methicillin-resistant and methicillin-sensitive Staphylococcus aureus MSSA, which was mainly dose-dependent. This antibacterial activity seems to be related to high content of flavonols, flavones, and isoflavones, which can act synergistically with each other against this type of bacteria. Our results showed consistent evidence that Pterospartum tridentatum and Mentha pulegium are an important reservoir of phytochemicals with antiradical activity and antibacterial capacity and thus they might be used in a preventive way or in a combined pharmaceutical and antibiotic therapy against pathogenic bacteria. Alfredo Aires, Eduardo Marrinhas, Rosa Carvalho, Carla Dias, and Maria José Saavedra Copyright © 2016 Alfredo Aires et al. All rights reserved. The First Outbreak Caused by Acinetobacter baumannii ST208 and ST195 in China Sun, 10 Apr 2016 15:57:27 +0000 This study aimed to analyze the clinical characteristics of patients and molecular mechanisms of the first outbreak mainly caused by sequence types (STs) 208 multidrug resistant (MDR) Acinetobacter baumannii in China. A total of 10 clinical samples were collected from 5 patients who were involved in the outbreak. Bacterial identification and antibiotic sensitivity tests were performed by the VITEK-2 COMPACT automated system. MICs of tigecycline for clinical isolates were determined using broth microdilution. The clonal relatedness of A. baumannii clinical isolates in our local settings was determinated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 7 A. baumannii strains were isolated and all were MDR strains; two of them were carbapenem-nonsusceptible strains. blaOXA-23 was the only acquired carbapenemase gene in the isolates. The isolates belonged to a single clonal pulsotype determined by PFGE and two sequences types (STs) determined by MLST. The isolates belonged to the globally disseminated clonal complex 92, among which ST195 and ST208 were the most common sequence types (71.43% and 28.57%). The outbreak was successfully controlled by stringent infection control measures, especially improving the hand hygiene compliance and enhancing antimicrobial stewardship. In conclusion, this is the first description of an outbreak caused mainly by A. baumannii of ST208 in China. Infection control measures should be strengthened when infection outbreaks in hospital. Junyan Qu, Yu Du, Rujia Yu, and Xiaoju Lü Copyright © 2016 Junyan Qu et al. All rights reserved. Polyphenols from Chilean Propolis and Pinocembrin Reduce MMP-9 Gene Expression and Activity in Activated Macrophages Mon, 28 Mar 2016 08:58:03 +0000 Polyphenols from diverse sources have shown anti-inflammatory activity. In the context of atherosclerosis, macrophages play important roles including matrix metalloproteinases synthesis involved in degradation of matrix extracellular components affecting the atherosclerotic plaque stability. We prepared a propolis extract and pinocembrin in ethanol solution. Propolis extract was chemically characterized using LC-MS. The effect of treatments on gene expression and proteolytic activity was measured in vitro using murine macrophages activated with LPS. Cellular toxicity associated with both treatments and the vehicle was determined using MTT and apoptosis/necrosis detection assays. MMP-9 gene expression and proteolytic activity were measured using qPCR and zymography, respectively. Thirty-two compounds were identified in the propolis extract, including pinocembrin among its major components. Treatment with either ethanolic extract of propolis or pinocembrin inhibits MMP-9 gene expression in a dose-dependent manner. Similarly, an inhibitory effect was observed in proteolytic activity. However, the effect showed by ethanolic extract of propolis was higher than the effect of pinocembrin, suggesting that MMP-9 inhibition results from a joint contribution between the components of the extract. These data suggest a potential role of polyphenols from Chilean propolis in the control of extracellular matrix degradation in atherosclerotic plaques. Nicolás Saavedra, Alejandro Cuevas, Marcela F. Cavalcante, Felipe A. Dörr, Kathleen Saavedra, Tomás Zambrano, Dulcineia S. P. Abdalla, and Luis A. Salazar Copyright © 2016 Nicolás Saavedra et al. All rights reserved. Dietary Flavonoid Hyperoside Induces Apoptosis of Activated Human LX-2 Hepatic Stellate Cell by Suppressing Canonical NF-κB Signaling Sun, 27 Mar 2016 13:38:49 +0000 Hyperoside, an active compound found in plants of the genera Hypericum and Crataegus, is reported to exhibit antioxidant, anticancer, and anti-inflammatory activities. Induction of hepatic stellate cell (HSC) apoptosis is recognized as a promising strategy for attenuation of hepatic fibrosis. In this study, we investigated whether hyperoside treatment can exert antifibrotic effects in human LX-2 hepatic stellate cells. We found that hyperoside induced apoptosis in LX-2 cells and decreased levels of α-smooth muscle actin (α-SMA), type I collagen, and intracellular reactive oxygen species (ROS). Remarkably, hyperoside also inhibited the DNA-binding activity of the transcription factor NF-κB and altered expression levels of NF-κB-regulated genes related to apoptosis, including proapoptotic genes Bcl-Xs, DR4, Fas, and FasL and anti-apoptotic genes A20, c-IAP1, Bcl-, and RIP1. Our results suggest that hyperoside may have potential as a therapeutic agent for the treatment of liver fibrosis. Liwen Wang, Zhiwei Yue, Mengzheng Guo, Lianying Fang, Liang Bai, Xinyu Li, Yongqing Tao, Suying Wang, Qiang Liu, Dexian Zhi, and Hui Zhao Copyright © 2016 Liwen Wang et al. All rights reserved. Advantages and Limitations of Direct PCR Amplification of Bacterial 16S-rDNA from Resected Heart Tissue or Swabs Followed by Direct Sequencing for Diagnosing Infective Endocarditis: A Retrospective Analysis in the Routine Clinical Setting Thu, 24 Mar 2016 13:48:11 +0000 Infective endocarditis (IE) is a life-threatening disease that is associated with high morbidity and mortality. Its long-term prognosis strongly depends on a timely and optimized antibiotic treatment. Therefore, identification of the causative pathogen is crucial and currently based on blood cultures followed by characterization and susceptibility testing of the isolate. However, antibiotic treatment starting prior to blood sampling or IE caused by fastidious or intracellular microorganisms may cause negative culture results. Here we investigate the additional diagnostic value of broad-range PCR in combination with direct sequencing on resected heart tissue or swabs in patients with tissue or swab culture-negative IE in a routine clinical setting. Sensitivity, specificity, and positive and negative predictive values of broad-range PCR from diagnostic material in our patients were 33.3%, 76.9%, 90.9%, and 14.3%, respectively. We identified a total of 20 patients (21.5%) with tissue or culture-negative IE who profited by the additional application of broad-range PCR. We conclude that broad-range PCR on resected heart tissue or swabs is an important complementary diagnostic approach. It should be seen as an indispensable new tool for both the therapeutic and diagnostic management of culture-negative IE and we thus propose its possible inclusion in Duke’s diagnostic classification scheme. Daniela Maneg, Janina Sponsel, Iris Müller, Benedikt Lohr, John Penders, Katharina Madlener, and Klaus-Peter Hunfeld Copyright © 2016 Daniela Maneg et al. All rights reserved. Polyphenol-Rich Extract from Propolis Reduces the Expression and Activity of Streptococcus mutans Glucosyltransferases at Subinhibitory Concentrations Wed, 23 Mar 2016 12:56:22 +0000 Tooth decay is an infectious disease, whose main causative agent identified is Streptococcus mutans (S. mutans). Diverse treatments have been used to eradicate this microorganism, including propolis. To date, it has been shown that polyphenols from Chilean propolis inhibit S. mutans growth and biofilm formation. However, the molecular mechanisms underlying this process are unclear. In the present study, we assessed the effect of Chilean propolis on the expression and activity of the glycosyltransferases enzymes and their related genes. Polyphenol-rich extract from propolis inhibited gene expression of glycosyltransferases (GtfB, GtfC, and GtfD) and their related regulatory genes, for example, VicK, VicR, and CcpA. Moreover, the treatment inhibited glucosyltransferases activity measured by the formation of sucrose-derived glucans. Additionally, an inhibitory effect was observed in the expression of SpaP involved in sucrose-independent virulence of S. mutans. In summary, our results suggest that Chilean propolis has a dose-dependent effect on the inhibition of genes involved in S. mutans virulence and adherence through the inhibition of glucosyltransferases, showing an anticariogenic potential of polyphenols from propolis beyond S. mutans growth inhibition. Jorge Jesús Veloz, Nicolás Saavedra, Marysol Alvear, Tomás Zambrano, Leticia Barrientos, and Luis A. Salazar Copyright © 2016 Jorge Jesús Veloz et al. All rights reserved. vacA Genotype Status of Helicobacter pylori Isolated from Foods with Animal Origin Mon, 07 Mar 2016 14:19:18 +0000 According to controversial theories and results of studies, foods with animal origins play an important role in the transmission of H. pylori to human. The aim of this study was to determine the distribution of vacA genotypes of H. pylori, isolated from milk and meat samples of cow, sheep, goat, camel, and buffalo. Eight hundred and twenty raw milk and meat samples were collected from various parts of Iran. Samples were cultured and those found positive for H. pylori were analyzed for the presence of various genotypes of vacA gene. Out of 420 milk and 400 meat samples, 92 (21.90%) and 105 (26.25%) were positive for H. pylori, respectively. The most commonly detected genotypes in the vacA gene were s1a (86.80%), m1a (79.18%), s1b (69.54%), and m1b (63.45%) and detected combined genotypes were mostly m1as1a (68.52%), m1as1b (60.40%), m1bs1b (55.83%), and m1bs1a (53.29%). High presence of bacteria in the milk and meat samples of sheep represents that sheep may be the natural host of H. pylori. High presence of H. pylori strains in milk and meat samples similar to vacA genotypes in human being suggests that milk and meat samples could be the sources of bacteria for human. Elnaz Saeidi and Amirhossein Sheikhshahrokh Copyright © 2016 Elnaz Saeidi and Amirhossein Sheikhshahrokh. All rights reserved. Dissemination of Extended-Spectrum β-Lactamase- and AmpC β-Lactamase-Producing Escherichia coli within the Food Distribution System of Ho Chi Minh City, Vietnam Wed, 17 Feb 2016 08:49:39 +0000 To investigate the dissemination of ESBL/pAmpC-producing E. coli within the food distribution system of Ho Chi Minh City (HCMC), Vietnam, the prevalence of ESBL/pAmpC-producing E. coli strains in chicken meat, pork, beef, and fish/shrimp samples obtained from slaughterhouses, a wholesale market, and supermarkets was examined. Among the total of 330 collected food samples, ESBL/pAmpC-producing E. coli was detected in 150 samples (45.5%). The highest prevalence of these isolates was in chicken meat (76/82, 92.7%), followed by pork (32/92, 34.8%), beef (18/74, 34.3%), and fish/shrimp (24/82, 29.3%). A total of 342 strains of ESBL/pAmpC-producing E. coli were isolated from 150 positive food samples. The most prevalent genes responsible for ESBL or pAmpC activity belonged to the CTX-M-9 (110/342, 31.2%), CTX-M-1 (102/342, 29.8%), and CIT (118/342, 34.5%) groups. To our knowledge, this is the first report of the high occurrence of pAmpC (37.1%) in animal-based food in Vietnam. Among the 342 total ESBL/pAmpC-producing E. coli isolates, 276 (80.7%) were resistant to at least 6 antibiotic agents. Notably, high percentages of resistance to ciprofloxacin and fosfomycin were found in isolates from chicken (80.5% and 50.8%, resp.). These findings demonstrate that animal-based food products in HCMC represent a major reservoir of ESBL/pAmpC-producing E. coli. Do Phuc Nguyen, Thi Anh Dao Nguyen, Thi Hien Le, Nguyen Minh Doan Tran, Thanh Phong Ngo, Van Chinh Dang, Takao Kawai, Masashi Kanki, Ryuji Kawahara, Michio Jinnai, Shinya Yonogi, Yuji Hirai, Yoshimasa Yamamoto, and Yuko Kumeda Copyright © 2016 Do Phuc Nguyen et al. All rights reserved. Application of PK/PD Modeling in Veterinary Field: Dose Optimization and Drug Resistance Prediction Tue, 16 Feb 2016 06:52:45 +0000 Among veterinary drugs, antibiotics are frequently used. The true mean of antibiotic treatment is to administer dose of drug that will have enough high possibility of attaining the preferred curative effect, with adequately low chance of concentration associated toxicity. Rising of antibacterial resistance and lack of novel antibiotic is a global crisis; therefore there is an urgent need to overcome this problem. Inappropriate antibiotic selection, group treatment, and suboptimal dosing are mostly responsible for the mentioned problem. One approach to minimizing the antibacterial resistance is to optimize the dosage regimen. PK/PD model is important realm to be used for that purpose from several years. PK/PD model describes the relationship between drug potency, microorganism exposed to drug, and the effect observed. Proper use of the most modern PK/PD modeling approaches in veterinary medicine can optimize the dosage for patient, which in turn reduce toxicity and reduce the emergence of resistance. The aim of this review is to look at the existing state and application of PK/PD in veterinary medicine based on in vitro, in vivo, healthy, and disease model. Ijaz Ahmad, Lingli Huang, Haihong Hao, Pascal Sanders, and Zonghui Yuan Copyright © 2016 Ijaz Ahmad et al. All rights reserved. Antibacterial Action of a Condensed Tannin Extracted from Astringent Persimmon as a Component of Food Addictive Pancil PS-M on Oral Polymicrobial Biofilms Mon, 15 Feb 2016 11:47:26 +0000 The purpose of this study was to evaluate the antibacterial activity against polymicrobial (PM) biofilms of a condensed tannin extracted from astringent persimmon (PS-M), which is contained in refreshing beverages commercially available in Japan. Salivary PM biofilms were formed anaerobically on glass coverslips for 24 and 72 h and were treated for 5 min with sterilized deionized water (DW), 0.05 and 0.2 wt% chlorhexidine digluconate (CHX), and 0.5–4.0 wt% PS-M solution. The colony forming units (CFU/mL) were determined and morphological changes of the biofilms were observed by scanning electron microscopy (SEM). The CFUs were lower in all PS-M and CHX groups compared to the DW group. PS-M exerted a dose-dependent effect. PS-M (1.53 × 107) at a dose of 4.0 wt% had the same effect as 0.2 wt% CHX (2.03 × 107), regardless of the culture period. SEM revealed the biofilm structures were considerably destroyed in the 4.0 wt% PS-M and 0.2 wt% CHX. These findings indicate that the antibacterial effects of PS-M, a naturally derived substance, are comparable to those of CHX. PS-M may keep the oral cavity clean and prevent dental caries and periodontal disease related to dental plaque, as well as systemic disease such as aspiration pneumonitis. Kiyoshi Tomiyama, Yoshiharu Mukai, Masahiro Saito, Kiyoko Watanabe, Hidefumi Kumada, Tomotaro Nihei, Nobushiro Hamada, and Toshio Teranaka Copyright © 2016 Kiyoshi Tomiyama et al. All rights reserved. The Comparative Evaluation of the Antimicrobial Effect of Propolis with Chlorhexidine against Oral Pathogens: An In Vitro Study Tue, 02 Feb 2016 08:19:28 +0000 This study aimed to compare the antimicrobial effectiveness of ethanolic extract of propolis (EEP) to chlorhexidine gluconate (CHX) on planktonic Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus salivarius subsp. salivarius, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Staphylococcus aureus, Enterococcus faecalis, Actinomyces israelii, Candida albicans, and their single-species biofilms by agar dilution and broth microdilution test methods. Both agents inhibited the growth of all planktonic species. On the other hand, CHX exhibited lower minimum bactericidal concentrations than EEP against biofilms of A. actinomycetemcomitans, S. aureus, and E. faecalis whereas EEP yielded a better result against Lactobacilli and P. intermedia. The bactericidal and fungicidal concentrations of both agents were found to be equal against biofilms of Streptecocci, P. gingivalis, A. israelii, and C. albicans. The results of this study revealed that propolis was more effective in inhibiting Gram-positive bacteria than the Gram-negative bacteria in their planktonic state and it was suggested that EEP could be as effective as CHX on oral microorganisms in their biofilm state. A. Eralp Akca, Gülçin Akca, Fulya Toksoy Topçu, Enis Macit, Levent Pikdöken, and I. Şerif Özgen Copyright © 2016 A. Eralp Akca et al. All rights reserved. Evaluation of Verigene Blood Culture Test Systems for Rapid Identification of Positive Blood Cultures Thu, 21 Jan 2016 11:32:55 +0000 The performance of molecular tests using the Verigene Gram-Positive and Gram-Negative Blood Culture nucleic acid tests (BC-GP and BC-GN, resp.; Naosphere, Northbrook, IL, USA) was evaluated for the identification of microorganisms detected from blood cultures. Ninety-nine blood cultures containing Gram-positive bacteria and 150 containing Gram-negative bacteria were analyzed using the BC-GP and BC-GN assays, respectively. Blood cultures were performed using the Bactec blood culture system (BD Diagnostic Systems, Franklin Lakes, NJ, USA) and conventional identification and antibiotic-susceptibility tests were performed using a MicroScan system (Siemens, West Sacramento, CA, USA). When a single strain of bacteria was isolated from the blood culture, Verigene assays correctly identified 97.9% (94/96) of Gram-positive bacteria and 93.8% (137/146) of Gram-negative bacteria. Resistance genes mecA and vanA were correctly detected by the BC-GP assay, while the extended-spectrum β-lactamase CTX-M and the carbapenemase OXA resistance gene were detected from 30 cases cultures by the BC-GN assay. The BC-GP and BC-GN assays showed high agreement with conventional identification and susceptibility tests. These tests are useful for rapid identification of microorganisms and the detection of clinically important resistance genes from positive Bactec blood cultures. Jae-Seok Kim, Go-Eun Kang, Han-Sung Kim, Hyun Soo Kim, Wonkeun Song, and Kyu Man Lee Copyright © 2016 Jae-Seok Kim et al. All rights reserved. Characteristics of Salmonella spp. Isolated from Wild Birds Confiscated in Illegal Trade Markets, Rio de Janeiro, Brazil Mon, 11 Jan 2016 05:57:26 +0000 The prevalence of Salmonella spp. was investigated in 109 wild birds poached in the illegal wildlife trade in Rio de Janeiro; most of them are passerines from Thraupidae family and three from Psittacidae. One strain of Salmonella ser. Typhimurium and two strains of Salmonella ser. Panama were isolated from passerine species and all of them showed resistance to multiple antimicrobial drugs, like ampicillin, ceftriaxone, ceftiofur, tetracycline, gentamicin, nalidixic acid, ciprofloxacin, and enrofloxacin. PFGE showed 100% similarity among the Salmonella ser. Typhimurium strain isolated from a Temminck’s seedeater (Sporophila falcirostris) and the strains isolated from a human outbreak, in southern Brazil. The two Salmonella ser. Panama strains isolated from two chestnut-capped blackbirds (Chrysomus ruficapillus) present in the same catch showed the same clonal origin and have never been associated with epizooties and human outbreaks. Potential for dissemination of resistant Salmonella through situations offered by captive management and the isolation of the same strain from wild birds and human sources may become a problem for the conservation of natural populations and to public health. Carlos Alexandre Rey Matias, Ingrid Annes Pereira, Maiara dos Santos de Araújo, André Felipe Mercês Santos, Rudi Pereira Lopes, Sandra Christakis, Dália dos Prazeres Rodrigues, and Salvatore Siciliano Copyright © 2016 Carlos Alexandre Rey Matias et al. All rights reserved. Effect of Tyrosol and Farnesol on Virulence and Antibiotic Resistance of Clinical Isolates of Pseudomonas aeruginosa Wed, 30 Dec 2015 07:12:10 +0000 Mixed-species biofilms could create a protected environment that allows for survival to external antimicrobials and allows different bacterial-fungal interactions. Pseudomonas aeruginosa-Candida albicans coexistence is an example for such mixed-species community. Numerous reports demonstrated how P. aeruginosa or its metabolites could influence the growth, morphogenesis, and virulence of C. albicans. In this study, we investigated how the C. albicans quorum sensing compounds, tyrosol and farnesol, might affect Egyptian clinical isolates of P. aeruginosa regarding growth, antibiotic sensitivity, and virulence. We could demonstrate that tyrosol possesses an antibacterial activity against P. aeruginosa (10 µM inhibited more than 50% of growth after 16 h cultivation). Moreover, we could show for the first time that tyrosol strongly inhibits the production of the virulence factors hemolysin and protease in P. aeruginosa, whereas farnesol inhibits, to lower extent, hemolysin production in this bacterial pathogen. Cumulatively, tyrosol is expected to strongly affect P. aeruginosa in mixed microbial biofilm. Shaymaa Hassan Abdel-Rhman, Areej Mostafa El-Mahdy, and Mohammed El-Mowafy Copyright © 2015 Shaymaa Hassan Abdel-Rhman et al. All rights reserved. New Derivatives of Pyridoxine Exhibit High Antibacterial Activity against Biofilm-Embedded Staphylococcus Cells Tue, 29 Dec 2015 13:38:55 +0000 Opportunistic bacteria Staphylococcus aureus and Staphylococcus epidermidis often form rigid biofilms on tissues and inorganic surfaces. In the biofilm bacterial cells are embedded in a self-produced polysaccharide matrix and thereby are inaccessible to biocides, antibiotics, or host immune system. Here we show the antibacterial activity of newly synthesized cationic biocides, the quaternary ammonium, and bisphosphonium salts of pyridoxine (vitamin B6) against biofilm-embedded Staphylococci. The derivatives of 6-hydroxymethylpyridoxine were ineffective against biofilm-embedded S. aureus and S. epidermidis at concentrations up to 64 μg/mL, although all compounds tested exhibited low MICs (2 μg/mL) against planktonic cells. In contrast, the quaternary ammonium salt of pyridoxine (N,N-dimethyl-N-((2,2,8-trimethyl-4H-dioxino[4,5-c]pyridin-5-yl)methyl)octadecan-1-aminium chloride (3)) demonstrated high biocidal activity against both planktonic and biofilm-embedded bacteria. Thus, the complete death of biofilm-embedded S. aureus and S. epidermidis cells was obtained at concentrations of 64 and 16 μg/mL, respectively. We suggest that the quaternary ammonium salts of pyridoxine are perspective to design new synthetic antibiotics and disinfectants for external application against biofilm-embedded cells. Airat R. Kayumov, Aliya A. Nureeva, Elena Yu. Trizna, Guzel R. Gazizova, Mikhail I. Bogachev, Nikita V. Shtyrlin, Mikhail V. Pugachev, Sergey V. Sapozhnikov, and Yurii G. Shtyrlin Copyright © 2015 Airat R. Kayumov et al. All rights reserved. Performances and Reliability of Bruker Microflex LT and VITEK MS MALDI-TOF Mass Spectrometry Systems for the Identification of Clinical Microorganisms Thu, 17 Dec 2015 06:40:21 +0000 In clinical microbiology laboratories, routine microbial identification is mostly performed using culture based methodologies requiring 24 to 72 hours from culturing to identification. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology has been established as a cost effective, reliable, and faster alternative identification platform. In this study, we evaluated the reliability of the two available MALDI-TOF MS systems for their routine clinical level identification accuracy and efficiency in a clinical microbiology laboratory setting. A total of 1,341 routine phenotypically identified clinical bacterial and fungal isolates were selected and simultaneously analyzed using VITEK MS (bioMérieux, France) and Microflex LT (Bruker Diagnostics, Germany) MALDI-TOF MS systems. For any isolate that could not be identified with either of the systems and for any discordant result, 16S rDNA gene or ITS1/ITS2 sequencing was used. VITEK MS and Microflex LT correctly identified 1,303 (97.17%) and 1,298 (96.79%) isolates to the species level, respectively. In 114 (8.50%) isolates initial phenotypic identification was inaccurate. Both systems showed a similar identification efficiency and workflow robustness, and they were twice as more accurate compared to routine phenotypic identification in our sample pool. MALDITOF systems with their accuracy and robustness offer a good identification platform for routine clinical microbiology laboratories. Kivanc Bilecen, Gorkem Yaman, Ugur Ciftci, and Yahya Rauf Laleli Copyright © 2015 Kivanc Bilecen et al. All rights reserved. Identification of Extended-Spectrum β-Lactamases Escherichia coli Strains Isolated from Market Garden Products and Irrigation Water in Benin Mon, 07 Dec 2015 08:42:57 +0000 The present study aimed at biochemical and molecular characterization of Escherichia coli strains isolated from horticultural products and irrigation water of Cotonou. The samples were collected from 12 market gardeners of 4 different sites. Rapid’ E. coli medium was used for identification of E. coli strains and the antimicrobial susceptibility was performed by the agar disk diffusion method. The β-lactamases production was sought by the liquid acidimetric method. The genes coding for β-lactamases and toxins were identified by PCR method. The results revealed that about 34.95% of the analyzed samples were contaminated by E. coli. Cabbages were the most contaminated by E. coli (28.26%) in dry season. All isolated strains were resistant to amoxicillin. The penicillinase producing E. coli carried (67.50%), (10%), and (22.50%) genes. The study revealed that the resistance genes such as SLTI (35.71%), SLTII (35.71%), ETEC (7.15%), and VTEC (21.43%) were carried. Openly to the found results and considering the importance of horticultural products in Beninese food habits, it is important to put several strategies aiming at a sanitary security by surveillance and sensitization of all the actors on the risks of some practices. Wassiyath Moussé, Haziz Sina, Farid Baba-Moussa, Pacôme A. Noumavo, Nadège A. Agbodjato, Adolphe Adjanohoun, and Lamine Baba-Moussa Copyright © 2015 Wassiyath Moussé et al. All rights reserved. Epidemiology, Detection, and Control of Foodborne Microbial Pathogens Mon, 16 Nov 2015 06:36:04 +0000 Miguel Prieto, Pierre Colin, Pablo Fernández-Escámez, and Avelino Alvarez-Ordóñez Copyright © 2015 Miguel Prieto et al. All rights reserved. Regulated Control of the Assembly and Diversity of LPS by Noncoding sRNAs Thu, 05 Nov 2015 14:38:30 +0000 The outer membrane (OM) of Gram-negative bacteria is asymmetric due to the presence of lipopolysaccharide (LPS) facing the outer leaflet of the OM and phospholipids facing the periplasmic side. LPS is essential for bacterial viability, since it provides a permeability barrier and is a major virulence determinant in pathogenic bacteria. In Escherichia coli, several steps of LPS biosynthesis and assembly are regulated by the RpoE sigma factor and stress responsive two-component systems as well as dedicated small RNAs. LPS composition is highly heterogeneous and dynamically altered upon stress and other challenges in the environment because of the transcriptional activation of RpoE regulon members and posttranslational control by RpoE-regulated Hfq-dependent RybB and MicA sRNAs. The PhoP/Q two-component system further regulates Kdo2-lipid A modification via MgrR sRNA. Some of these structural alterations are critical for antibiotic resistance, OM integrity, virulence, survival in host, and adaptation to specific environmental niches. The heterogeneity arises following the incorporation of nonstoichiometric modifications in the lipid A part and alterations in the composition of inner and outer core of LPS. The biosynthesis of LPS and phospholipids is tightly coupled. This requires the availability of metabolic precursors, whose accumulation is controlled by sRNAs like SlrA, GlmZ, and GlmY. Gracjana Klein and Satish Raina Copyright © 2015 Gracjana Klein and Satish Raina. All rights reserved. Comparative Genomic Hybridization Analysis of Yersinia enterocolitica and Yersinia pseudotuberculosis Identifies Genetic Traits to Elucidate Their Different Ecologies Wed, 28 Oct 2015 14:11:35 +0000 Enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis are both etiological agents for intestinal infection known as yersiniosis, but their epidemiology and ecology bear many differences. Swine are the only known reservoir for Y. enterocolitica 4/O:3 strains, which are the most common cause of human disease, while Y. pseudotuberculosis has been isolated from a variety of sources, including vegetables and wild animals. Infections caused by Y. enterocolitica mainly originate from swine, but fresh produce has been the source for widespread Y. pseudotuberculosis outbreaks within recent decades. A comparative genomic hybridization analysis with a DNA microarray based on three Yersinia enterocolitica and four Yersinia pseudotuberculosis genomes was conducted to shed light on the genomic differences between enteropathogenic Yersinia. The hybridization results identified Y. pseudotuberculosis strains to carry operons linked with the uptake and utilization of substances not found in living animal tissues but present in soil, plants, and rotting flesh. Y. pseudotuberculosis also harbors a selection of type VI secretion systems targeting other bacteria and eukaryotic cells. These genetic traits are not found in Y. enterocolitica, and it appears that while Y. pseudotuberculosis has many tools beneficial for survival in varied environments, the Y. enterocolitica genome is more streamlined and adapted to their preferred animal reservoir. Kaisa Jaakkola, Panu Somervuo, and Hannu Korkeala Copyright © 2015 Kaisa Jaakkola et al. All rights reserved. Comparison of Antibiotic Resistance and Virulence Factors among Escherichia coli Isolated from Conventional and Free-Range Poultry Thu, 22 Oct 2015 13:43:00 +0000 Microbiological contamination in commercial poultry production has caused concerns for human health because of both the presence of pathogenic microorganisms and the increase in antimicrobial resistance in bacterial strains that can cause treatment failure of human infections. The aim of our study was to analyze the profile of antimicrobial resistance and virulence factors of E. coli isolates from chicken carcasses obtained from different farming systems (conventional and free-range poultry). A total of 156 E. coli strains were isolated and characterized for genes encoding virulence factors described in extraintestinal pathogenic E. coli (ExPEC). Antimicrobial susceptibility testing was performed for 15 antimicrobials, and strains were confirmed as extended spectrum of β-lactamases- (ESBLs-) producing E. coli by phenotypic and genotypic tests. The results indicated that strains from free-range poultry have fewer virulence factors than strains from conventional poultry. Strains from conventionally raised chickens had a higher frequency of antimicrobial resistance for all antibiotics tested and also exhibited genes encoding ESBL and AmpC, unlike free-range poultry isolates, which did not. Group 2 CTX-M and CIT were the most prevalent ESBL and AmpC genes, respectively. The farming systems of poultries can be related with the frequency of virulence factors and resistance to antimicrobials in bacteria. Vanessa L. Koga, Sara Scandorieiro, Eliana C. Vespero, Alexandre Oba, Benito G. de Brito, Kelly C. T. de Brito, Gerson Nakazato, and Renata K. T. Kobayashi Copyright © 2015 Vanessa L. Koga et al. All rights reserved. Efficient and Specific Detection of Salmonella in Food Samples Using a stn-Based Loop-Mediated Isothermal Amplification Method Mon, 12 Oct 2015 10:17:04 +0000 The Salmonella enterotoxin (stn) gene exhibits high homology among S. enterica serovars and S. bongori. A set of 6 specific primers targeting the stn gene were designed for detection of Salmonella spp. using the loop-mediated isothermal amplification (LAMP) method. The primers amplified target sequences in all 102 strains of 87 serovars of Salmonella tested and no products were detected in 57 non-Salmonella strains. The detection limit in pure cultures was 5 fg DNA/reaction when amplified at 65°C for 25 min. The LAMP assay could detect Salmonella in artificially contaminated food samples as low as 220 cells/g of food without a preenrichment step. However, the sensitivity was increased 100-fold (~2 cells/g) following 5 hr preenrichment at 35°C. The LAMP technique, with a preenrichment step for 5 and 16 hr, was shown to give 100% specificity with food samples compared to the reference culture method in which 67 out of 90 food samples gave positive results. Different food matrixes did not interfere with LAMP detection which employed a simple boiling method for DNA template preparation. The results indicate that the LAMP method, targeting the stn gene, has great potential for detection of Salmonella in food samples with both high specificity and high sensitivity. Mevaree Srisawat and Watanalai Panbangred Copyright © 2015 Mevaree Srisawat and Watanalai Panbangred. All rights reserved. Contribution of Avian Salmonella enterica Isolates to Human Salmonellosis Cases in Constantine (Algeria) Mon, 12 Oct 2015 09:18:16 +0000 An epidemiological investigation was carried out on one hundred Salmonella isolates from broiler farms, slaughterhouses, and human patients in the Constantine region of Algeria, in order to explore the contribution of avian strains to human salmonellosis cases in this region over the same period of time. The isolates were characterized by phenotypic as well as genotypic methods. A large variety of antimicrobial resistance profiles was found among human isolates, while only seven profiles were found among avian isolates. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), Insertion Sequence 200-PCR (IS200-PCR), and Pulsed Field Gel Electrophoresis (PFGE) resulted in the allocation of the isolates to 16, 20, and 34 different profiles, respectively. The 3 genotyping methods led to complementary results by underlining the clonality of some serovars with the diffusion and persistence of a single clone in the Constantine area as well as stressing the polymorphism present in isolates belonging to other serovars, indicating the diversity of potential reservoirs of nontyphoidal Salmonella. Altogether, our results seem to indicate that nontyphoidal avian Salmonella may play an important role in human salmonellosis in the Constantine region. Rachid Elgroud, Sophie A. Granier, Muriel Marault, Annaëlle Kerouanton, Abdesslem Lezzar, Chafia Bouzitouna-Bentchouala, Anne Brisabois, and Yves Millemann Copyright © 2015 Rachid Elgroud et al. All rights reserved. Occurrence, Persistence, and Virulence Potential of Listeria ivanovii in Foods and Food Processing Environments in the Republic of Ireland Mon, 12 Oct 2015 08:51:41 +0000 The aim of this study was to assess the occurrence of L. ivanovii in foods and food processing environments in Ireland, to track persistence, and to characterize the disease causing potential of the isolated strains. A total of 2,006 samples (432 food samples and 1,574 environmental swabs) were collected between March 2013 and March 2014 from 48 food business operators (FBOs) belonging to different production sectors (dairy, fish, meat, and fresh-cut vegetable). Six of the forty-eight FBOs had samples positive for L. ivanovii on at least one sampling occasion. L. ivanovii was present in fifteen samples (fourteen environmental samples and one food sample). All but one of those positive samples derived from the dairy sector, where L. ivanovii prevalence was 1.7%. Six distinguishable pulsotypes were obtained by PFGE analysis, with one pulsotype being persistent in the environment of a dairy food business. Sequence analysis of the sigB gene showed that fourteen isolates belonged to L. ivanovii subsp. londoniensis, while only one isolate was L. ivanovii subsp. ivanovii. Cell invasion assays demonstrated that the majority of L. ivanovii strains were comparable to L. monocytogenes EGDe in their ability to invade CACO-2 epithelial cells whilst four isolates had significantly higher invasion efficiencies. Avelino Alvarez-Ordóñez, Dara Leong, Ciara A. Morgan, Colin Hill, Cormac G. M. Gahan, and Kieran Jordan Copyright © 2015 Avelino Alvarez-Ordóñez et al. All rights reserved. Population Diversity of Campylobacter jejuni in Poultry and Its Dynamic of Contamination in Chicken Meat Mon, 12 Oct 2015 06:09:07 +0000 This study aimed to analyse the diversity of the Campylobacter jejuni population in broilers and to evaluate the major source of contamination in poultry meat. Eight rearing cycles over one year provided samples from three different broiler farms processed at the same slaughterhouse. A total of 707  C. jejuni were isolated from cloacal swabs before slaughter and from the breast skin of carcasses after slaughter and after chilling. All suspected Campylobacter colonies were identified with PCR assays and C. jejuni was genotyped by sequence analysis of the flaA short variable region (SVR) and by pulsed-field gel electrophoresis (PFGE) using SmaI enzyme. Phenotypic antibiotic resistance profiles were also assayed using minimal inhibitory concentration (MIC). The flocks carried many major C. jejuni clones possibly carrying over the rearing cycles, but cross contamination between farms may happen. Many isolates were resistant to fluoroquinolones, raising an issue of high public concern. Specific Campylobacter populations could be harboured within each poultry farm, with the ability to contaminate chickens during each new cycle. Thus, although biosecurity measures are applied, with a persistent source of contamination, they cannot be efficient. The role of the environment needs further investigation to better address strategies to control Campylobacter. Francesca Marotta, Giuliano Garofolo, Guido Di Donato, Giuseppe Aprea, Ilenia Platone, Silvia Cianciavicchia, Alessandra Alessiani, and Elisabetta Di Giannatale Copyright © 2015 Francesca Marotta et al. All rights reserved. Genetic Diversity and Incidence of Virulence-Associated Genes of Arcobacter butzleri and Arcobacter cryaerophilus Isolates from Pork, Beef, and Chicken Meat in Poland Sun, 11 Oct 2015 14:09:26 +0000 Incidence of 9 virulence-associated genes and genetic diversity was determined in 79 A. butzleri and 6 A. cryaerophilus isolates from pork, beef, and chicken meat. All A. butzleri isolates harboured the tlyA gene, and most of them carried ciaB, mviN, pldA, cadF, and cj1349 genes. ciaB was found to occur with higher frequency in poultry if compared with pork (), while irgA was more frequent in poultry than in beef (). All 6 A. cryaerophilus isolates harboured the ciaB gene, while mviN and tlyA were detected in 3 out of these isolates. Only one isolate carried the cadF gene. All beef-derived A. cryaerophilus isolates carried ciaB, mviN, and tlyA genes. A. cryaerophilus isolates from chicken meat harboured ciaB gene only. The pork-derived isolate harboured ciaB and cadF genes. Seventy-four genotypes were distinguished within 79 A. butzleri isolates. Nineteen from 21 isolates derived from beef and pork were found to be closely related to A. butzleri from chicken meat. Each of the 6 A. cryaerophilus isolates was found to have unique genotype. We demonstrated that closely related genotypes can spread within pork, beef, and chicken meat populations of A. butzleri but not A. cryaerophilus. Iwona Zacharow, Jarosław Bystroń, Ewa Wałecka-Zacharska, Magdalena Podkowik, and Jacek Bania Copyright © 2015 Iwona Zacharow et al. All rights reserved. Management of Food-Related Diarrhea Outbreak in the Emergency Department: Lessons Learned from the German STEC O104:H4 Epidemic Sun, 11 Oct 2015 13:50:33 +0000 Emergency department (ED) management of the German STEC O104:H4 outbreak in 2011 was not limited to patients being truly infected with STEC. In parallel to spread of alarming news in public media, patients suffering from diarrhea due to other reasons fearfully presented, equally. We retrospectively characterized these two cohorts for anamnestic, clinical, and laboratory findings at their first ED contact. From 15th of May to July 2011, 302 adult patients with diarrheal complaint presented at the EDs of two tertiary hospitals in Lubeck, northern Germany. Fecal testing for STEC was obtained in 245 (81%) patients: 105 were STEC-positive and 140 were STEC-negative. Anamnestic characteristics (defecation rate, visible bloody diarrhea, and lower abdominal pain), abdominal tenderness, and some laboratory findings were significantly different between both cohorts but not reliable to exclude STEC. In >90% of STEC-positive patients diarrheal symptoms had started in May, reflecting the retrospective nationwide peak of infections, whereas the majority of STEC-negative patients became symptomatic in June 2011. During the German STEC O104:H4 outbreak a definite distinction at initial ED contact between STEC-positive versus STEC-negative patients by clinical judgment alone was not reliable. Fecal testing in the ED, however, might survey the outbreak of foodborne infections with the utmost precision. Friedhelm Sayk, Niels Henrik Asselborn, Nora Eisemann, Alexander Katalinic, Jörg Metzner, Sebastian Wolfrum, Klaus Fellermann, Johannes Knobloch, and Martin Nitschke Copyright © 2015 Friedhelm Sayk et al. All rights reserved. UV-Heat Treatments for the Control of Foodborne Microbial Pathogens in Chicken Broth Sun, 11 Oct 2015 13:36:56 +0000 This investigation established the process criteria for using UV-C light and mild heat (UV-H treatment) to inactivate 5-Log10 cycles (performance criterion) of common foodborne pathogen populations, Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus, when inoculated in chicken broth. To define the target microorganism and the proper UV-H treatment conditions (including UV dose, treatment time, and temperature) that would achieve the stated performance criterion, mathematical equations based on Geeraerd’s model were developed for each microorganism. For the sake of comparison, inactivation equations for heat treatments were also performed on the same chicken broth and for the same microorganisms. L. monocytogenes was the most UV-H resistant microorganism at all temperatures, requiring a UV dose between 6.10 J/mL (5.6 min) and 2.26 J/mL (2.09 min) to achieve 5-Log10 reductions. In comparison with UV treatments at room temperatures, the combination of UV and mild heat allowed both the UV dose and treatment time to be reduced by 30% and 63% at 55°C and 60°C, respectively. Compared to heat treatments, the UV-H process reduced the heating time for 5-Log10 reductions of all the investigated microorganisms in chicken broth from 20-fold to 2-fold when the operating temperature varied from 53 to 60°C. M. Gouma, E. Gayán, J. Raso, S. Condón, and I. Álvarez Copyright © 2015 M. Gouma et al. All rights reserved. Salmonella Typhimurium and Salmonella Sofia: Growth in and Persistence on Eggs under Production and Retail Conditions Sun, 11 Oct 2015 13:34:55 +0000 Salmonellosis in Australia has been linked to eggs and egg products with specific serotypes associated with outbreaks. We compared attachment to and survival on egg shells and growth in eggs of two Salmonella serotypes, an egg outbreak associated Salmonella Typhimurium and a non-egg-associated Salmonella enterica ssp. II 1,4,12,27:b:[e,n,x] (S. Sofia). Experiments were conducted at combinations of 4, 15, 22, 37 and 42°C. No significant differences occurred between the serotypes in maximum growth rates, which were significantly greater () in egg yolk (0.427 log10 CFU/mL/h) compared to whole egg (0.312 log10 CFU/mL/h) and egg white (0.029 log10 CFU/mL/h). Attachment to egg shells varied by time (1 or 20 min) and temperature (4, 22 and 42°C), with S. Typhimurium isolates attaching at higher levels () than S. Sofia after 1 min at 4°C and S. Typhimurium ATCC 14028 attaching at higher () levels at 22°C. Survival on egg shells was not significantly different across isolates. Salmonella serotypes behaved similarly regarding growth in egg contents, attachment to egg shells and survival on eggs, indicating that other factors more likely contributed to reasons for S. Typhimurium being implicated in multiple egg-associated outbreaks. Catherine M. McAuley, Lesley L. Duffy, Nela Subasinghe, Geoff Hogg, John Coventry, and Narelle Fegan Copyright © 2015 Catherine M. McAuley et al. All rights reserved. Survival of Unstressed and Acid-, Cold-, and Starvation-Stress-Adapted Listeria monocytogenes in Ham Extract with Hops Beta Acids and Consumer Acceptability of HBA on Ready-to-Eat Ham Sun, 11 Oct 2015 13:24:18 +0000 The efficacy of hops beta acids (HBA) against unstressed and stress-adapted Listeria monocytogenes in ham extract and the consumers’ acceptability of HBA on ready-to-eat (RTE) hams were investigated. Unstressed or acid-, cold-, or starvation-stress-adapted L. monocytogenes was inoculated (1.3–1.5 log CFU/mL) into 10% ham extract, without (control) or with HBA (4.44 or 10.0 µg/mL). Survival/growth of the pathogen during storage (7.2°C, 26 days) was monitored periodically. Sensory evaluation (30 participants, 9-point hedonic scale) was performed with hams dipped into 0.05, 0.11, and 0.23% HBA solution. Ham extracts without HBA supported rapid growth of unstressed and stress-adapted cells with growth rates of 0.39–0.71 log CFU/mL/day and lag phases of 0–3.26 days. HBA inhibited growth of unstressed L. monocytogenes by slowing () growth rate (0.24–0.29 log CFU/mL/day) and increasing () length of the lag phase (3.49–12.98 days) compared to control. Acid-, cold-, or starvation-stress-adapted cells showed cross protection against HBA with greater () growth rates (0.44–0.66 log CFU/mL/day) and similar or shorter lag phases (0–5.44 days) than unstressed cells. HBA did not () affect sensory attributes of RTE ham. These results are useful for RTE meat processors to develop operational protocols using HBA to control L. monocytogenes. Li Wang and Cangliang Shen Copyright © 2015 Li Wang and Cangliang Shen. All rights reserved. Molecular Epidemiology of Invasive Listeriosis due to Listeria monocytogenes in a Spanish Hospital over a Nine-Year Study Period, 2006–2014 Sun, 11 Oct 2015 12:11:24 +0000 We investigated the pathogenicity, invasiveness, and genetic relatedness of 17 clinical Listeria monocytogenes stains isolated over a period of nine years (2006–2014). All isolates were phenotypically characterised and growth patterns were determined. The antimicrobial susceptibility of L. monocytogenes isolates was determined in E-tests. Invasion assays were performed with epithelial HeLa cells. Finally, L. monocytogenes isolates were subtyped by PFGE and MLST. All isolates had similar phenotypic characteristics (β-haemolysis and lecithinase activity), and three types of growth curve were observed. Bacterial recovery rates after invasion assays ranged from 0.09% to 7.26% (1.62 ± 0.46). MLST identified 11 sequence types (STs), and 14 PFGE profiles were obtained, indicating a high degree of genetic diversity. Genetic studies unequivocally revealed the occurrence of one outbreak of listeriosis in humans that had not previously been reported. This outbreak occurred in October 2009 and affected three patients from neighbouring towns. In conclusion, the molecular epidemiological analysis clearly revealed a cluster (three human cases, all ST1) of not previously reported listeriosis cases in northwestern Spain. Our findings indicate that molecular subtyping, in combination with epidemiological case analysis, is essential and should be implemented in routine diagnosis, to improve the tracing of the sources of outbreaks. Jaime Ariza-Miguel, María Isabel Fernández-Natal, Francisco Soriano, Marta Hernández, Beatrix Stessl, and David Rodríguez-Lázaro Copyright © 2015 Jaime Ariza-Miguel et al. All rights reserved. Comparison of Primary Models to Predict Microbial Growth by the Plate Count and Absorbance Methods Sun, 11 Oct 2015 11:16:29 +0000 The selection of a primary model to describe microbial growth in predictive food microbiology often appears to be subjective. The objective of this research was to check the performance of different mathematical models in predicting growth parameters, both by absorbance and plate count methods. For this purpose, growth curves of three different microorganisms (Bacillus cereus, Listeria monocytogenes, and Escherichia coli) grown under the same conditions, but with different initial concentrations each, were analysed. When measuring the microbial growth of each microorganism by optical density, almost all models provided quite high goodness of fit () for all growth curves. The growth rate remained approximately constant for all growth curves of each microorganism, when considering one growth model, but differences were found among models. Three-phase linear model provided the lowest variation for growth rate values for all three microorganisms. Baranyi model gave a variation marginally higher, despite a much better overall fitting. When measuring the microbial growth by plate count, similar results were obtained. These results provide insight into predictive microbiology and will help food microbiologists and researchers to choose the proper primary growth predictive model. María-Leonor Pla, Sandra Oltra, María-Dolores Esteban, Santiago Andreu, and Alfredo Palop Copyright © 2015 María-Leonor Pla et al. All rights reserved. Twenty Years of Listeria in Brazil: Occurrence of Listeria Species and Listeria monocytogenes Serovars in Food Samples in Brazil between 1990 and 2012 Sun, 11 Oct 2015 09:30:43 +0000 Listeria spp. isolated from different food products and collected from 12 Brazilian states were sent to the Laboratory of Bacterial Zoonoses (Oswaldo Cruz Institute, Brazil) for identification. The aims of this study were to characterize these isolates, from 1990 to 2012, by using biochemical, morphological, and serotyping tests, and to analyze the distribution of L. monocytogenes serotypes on different food products and geographical locations. Serotyping was performed using polyclonal somatic and flagellar antisera. Of 5953 isolates, 5770 were identified as Listeria spp., from which 3429 (59.4%) were L. innocua, 2248 (38.9%) were L. monocytogenes, and 93 (1.6%) were other Listeria spp. L. innocua was predominantly isolated from 1990 to 2000, while L. monocytogenes was from 2001 to 2012. Regarding the serotype distribution in the foods, serotypes 1/2a and 4b were most common in processed meat and ready-to-eat products, respectively; serotypes 1/2a, 1/2b, and 4b were the most common in nonprocessed meat. The results above confirm the presence of the main serotypes of L. monocytogenes in different parts of the food chain from three regions of the country and emphasize the importance of improving the control measures, as tolerance zero policy and microbiological surveillance in Brazil. Deyse Christina Vallim, Cristina Barroso Hofer, Rodrigo de Castro Lisbôa, André Victor Barbosa, Leonardo Alves Rusak, Cristhiane Moura Falavina dos Reis, and Ernesto Hofer Copyright © 2015 Deyse Christina Vallim et al. All rights reserved. Surveillance of Food- and Smear-Transmitted Pathogens in European Soldiers with Diarrhea on Deployment in the Tropics: Experience from the European Union Training Mission (EUTM) Mali Sun, 11 Oct 2015 08:43:30 +0000 Introduction. Since 2013, European soldiers have been deployed on the European Union Training Mission (EUTM) in Mali. From the beginning, diarrhea has been among the most “urgent” concerns. Diarrhea surveillance based on deployable real-time PCR equipment was conducted between December 2013 and August 2014. Material and Methods. In total, 53 stool samples were obtained from 51 soldiers with acute diarrhea. Multiplex PCR panels comprised enteroinvasive bacteria, diarrhea-associated Escherichia coli (EPEC, ETEC, EAEC, and EIEC), enteropathogenic viruses, and protozoa. Noroviruses were characterized by sequencing. Cultural screening for Enterobacteriaceae with extended-spectrum beta-lactamases (ESBL) with subsequent repetitive sequence-based PCR (rep-PCR) typing was performed. Clinical information was assessed. Results. Positive PCR results for diarrhea-associated pathogens were detected in 43/53 samples, comprising EPEC (), ETEC (), EAEC (), Norovirus (), Shigella spp./EIEC (), Cryptosporidium parvum (), Giardia duodenalis (), Salmonella spp. (), Astrovirus (), Rotavirus (), and Sapovirus (). ESBL-positive Enterobacteriaceae were grown from 13 out of 48 samples. Simultaneous infections with several enteropathogenic agents were observed in 23 instances. Symptoms were mild to moderate. There were hints of autochthonous transmission. Conclusions. Multiplex real-time PCR proved to be suitable for diarrhea surveillance on deployment. Etiological attribution is challenging in cases of detection of multiple pathogens. Hagen Frickmann, Philipp Warnke, Claudia Frey, Salvatore Schmidt, Christian Janke, Kay Erkens, Ulrich Schotte, Thomas Köller, Winfried Maaßen, Andreas Podbielski, Alfred Binder, Rebecca Hinz, Benjamin Queyriaux, Dorothea Wiemer, Norbert Georg Schwarz, and Ralf Matthias Hagen Copyright © 2015 Hagen Frickmann et al. All rights reserved. Sampling and Homogenization Strategies Significantly Influence the Detection of Foodborne Pathogens in Meat Sun, 11 Oct 2015 08:29:29 +0000 Efficient preparation of food samples, comprising sampling and homogenization, for microbiological testing is an essential, yet largely neglected, component of foodstuff control. Salmonella enterica spiked chicken breasts were used as a surface contamination model whereas salami and meat paste acted as models of inner-matrix contamination. A systematic comparison of different homogenization approaches, namely, stomaching, sonication, and milling by FastPrep-24 or SpeedMill, revealed that for surface contamination a broad range of sample pretreatment steps is applicable and loss of culturability due to the homogenization procedure is marginal. In contrast, for inner-matrix contamination long treatments up to 8 min are required and only FastPrep-24 as a large-volume milling device produced consistently good recovery rates. In addition, sampling of different regions of the spiked sausages showed that pathogens are not necessarily homogenously distributed throughout the entire matrix. Instead, in meat paste the core region contained considerably more pathogens compared to the rim, whereas in the salamis the distribution was more even with an increased concentration within the intermediate region of the sausages. Our results indicate that sampling and homogenization as integral parts of food microbiology and monitoring deserve more attention to further improve food safety. Alexander Rohde, Jens Andre Hammerl, Bernd Appel, Ralf Dieckmann, and Sascha Al Dahouk Copyright © 2015 Alexander Rohde et al. All rights reserved. The Use of Plant Antimicrobial Compounds for Food Preservation Sun, 11 Oct 2015 08:28:51 +0000 Foodborne disease is a global issue with significant impact on human health. With the growing consumer demand for natural preservatives to replace chemical compounds, plant antimicrobial compounds must be thoroughly investigated for their potential to serve as biopreservatives. This review paper will focus on the plant-derived products as antimicrobial agents for use in food preservation and to control foodborne pathogens in foods. Structure, modes of action, stability, and resistance to these plant compounds will be discussed as well as their application in food industries and possible technologies by which they can be delivered. Benefits as well as challenges, such as the need for further research for implementation and governmental regulation, will be highlighted. Tana Hintz, Karl K. Matthews, and Rong Di Copyright © 2015 Tana Hintz et al. All rights reserved. Impact of Moderate Heat, Carvacrol, and Thymol Treatments on the Viability, Injury, and Stress Response of Listeria monocytogenes Sun, 11 Oct 2015 08:01:31 +0000 The microbial safety and stability of minimally processed foods are based on the application of combined preservative factors. Since microorganisms are able to develop adaptive networks to survive under conditions of stress, food safety may be affected, and therefore understanding of stress adaptive mechanisms plays a key role in designing safe food processing conditions. In the present study, the viability and the sublethal injury of Listeria monocytogenes exposed to moderate heat (55°C) and/or essential oil compounds (carvacrol and thymol, 0.3 mM) treatments were studied. Synergistic effects were obtained when combining mild heat (55°C) with one or both essential oil compounds, leading to inactivation kinetics values three to four times lower than when using heat alone. All the treatments applied caused some injury in the population. The injury levels ranged from around 20% of the surviving population under the mildest conditions to more than 99.99% under the most stringent conditions. Protein extracts of cells exposed to these treatments were analysed by two-dimensional gel electrophoresis. The results obtained revealed that stressed cells exhibited differential protein expression to control cells. The proteins upregulated under these stressing conditions were implicated, among other functions, in stress response, metabolism, and protein refolding. L. Guevara, V. Antolinos, A. Palop, and P. M. Periago Copyright © 2015 L. Guevara et al. All rights reserved. Bayesian Estimation of the True Prevalence and of the Diagnostic Test Sensitivity and Specificity of Enteropathogenic Yersinia in Finnish Pig Serum Samples Sun, 11 Oct 2015 07:57:29 +0000 Bayesian analysis was used to estimate the pig’s and herd’s true prevalence of enteropathogenic Yersinia in serum samples collected from Finnish pig farms. The sensitivity and specificity of the diagnostic test were also estimated for the commercially available ELISA which is used for antibody detection against enteropathogenic Yersinia. The Bayesian analysis was performed in two steps; the first step estimated the prior true prevalence of enteropathogenic Yersinia with data obtained from a systematic review of the literature. In the second step, data of the apparent prevalence (cross-sectional study data), prior true prevalence (first step), and estimated sensitivity and specificity of the diagnostic methods were used for building the Bayesian model. The true prevalence of Yersinia in slaughter-age pigs was 67.5% (95% PI 63.2–70.9). The true prevalence of Yersinia in sows was 74.0% (95% PI 57.3–82.4). The estimates of sensitivity and specificity values of the ELISA were 79.5% and 96.9%. M. J. Vilar, J. Ranta, S. Virtanen, and H. Korkeala Copyright © 2015 M. J. Vilar et al. All rights reserved. Characterization of Shiga Toxigenic Escherichia coli O157 and Non-O157 Isolates from Ruminant Feces in Malaysia Sun, 11 Oct 2015 07:46:59 +0000 Shiga toxigenic Escherichia coli (STEC) O157 and several other serogroups of non-O157 STEC are causative agents of severe disease in humans world-wide. The present study was conducted to characterize STEC O157 and non-O157 serogroups O26, O103, O111, O121, O45, and O145 in ruminants in Malaysia. A total of 136 ruminant feces samples were collected from 6 different farms in Peninsular Malaysia. Immunomagnetic beads were used to isolate E. coli O157 and non-O157 serogroups, while PCR was used for the detection and subtyping of STEC isolates. STEC O157:H7 was isolated from 6 (4%) feces samples and all isolates obtained carried ,  eaeA-γ1, and ehxA. Non-O157 STEC was isolated from 2 (1.5%) feces samples with one isolate carrying , , , and ehxA and the other carrying alone. The presence of STEC O157 and non-O157 in a small percentage of ruminants in this study together with their virulence characteristics suggests that they may have limited impact on public health. Asanthi Perera, Charles M. Clarke, Gary A. Dykes, and Narelle Fegan Copyright © 2015 Asanthi Perera et al. All rights reserved. Variations of Tongue Coating Microbiota in Patients with Gastric Cancer Thu, 17 Sep 2015 12:05:33 +0000 The physical status of humans can be estimated by observing the appearance of the tongue coating, known as tongue diagnosis. The goals of this study were to reveal the relationship between tongue coating appearance and the oral microbiota in patients with gastric cancer and to open a novel research direction supporting tongue diagnosis. We used a tongue manifestation acquisition instrument to analyse the thickness of the tongue coating of patients with gastric cancer and that of healthy controls, and high-throughput sequencing was used to describe the microbial community of the tongue coating by sequencing the V2–V4 region of the 16S rDNA. The tongue coatings of 74 patients with gastric cancer were significantly thicker than those of 72 healthy controls (343.11 ± 198.22 versus 98.42 ± 48.25, ); 51.35% of the patients were assessed as having thick tongue coatings, whereas all healthy controls were assessed as having thin tongue coatings. Thick tongue coatings presented lower microbial community diversity than thin tongue coatings. The tongue coating bacterial community is associated with the appearance of the tongue coating. The tongue coating may be a potential source for diagnosing gastric cancer, but its sensitivity needs to be further improved. Jie Hu, Shuwen Han, Yan Chen, and Zhaoning Ji Copyright © 2015 Jie Hu et al. All rights reserved. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell Sun, 06 Sep 2015 07:31:09 +0000 The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells. Yumei Luo, Zikai Chen, Detu Zhu, Haitao Tu, and Shen Quan Pan Copyright © 2015 Yumei Luo et al. All rights reserved. Molecular Detection of Equine Herpesvirus Types 1 and 4 Infection in Healthy Horses in Isfahan Central and Shahrekord Southwest Regions, Iran Tue, 01 Sep 2015 05:46:51 +0000 This study was undertaken to investigate molecularly the occurrence of EHV-1 and EHV-4 infection among equine population in regions, Iran. Blood samples from 53 and 37 randomly selected horses settled in Isfahan and Shahrekord, Iran, respectively, were collected. Detection of EHV-1 and EHV-4 genes in the blood samples was done using polymerase chain reaction (PCR). Out of 53 and 37 samples from Isfahan and Shahrekord, 4 (18.18%) and 3 (8.10%) were positive for PCR of EHV-1, respectively. Nine (16.98%) and 6 (16.21%) were positive for PCR of EHV-4, while 6 (11.32%) and 3 (8.10%) were positive for PCR of both EHV-1 and EHV-4, in Isfahan and Shahrekord, respectively. Of the 7 blood samples positive for EHV-1, 4 (16.66%) and 3 (8.10%) were from horses >3 years old while 2 (18.18%) and 1 (16.66%) were from 2-3 years old horses, in Isfahan and Shahrekord, respectively. Out of the 7 and 3 samples positive for PCR of EHV-1 in Isfahan and Shahrekord, 4 (22.2%) and 1 (7.69%) were Standardbred, while 3 (14.28%) and 2 (13.33%) were Thoroughbreds, respectively. EHV-4 was detected in blood of 4 (22.22%) and 2 (15.83%) Standardbreds and from 4 (19.04%) and 4 (26.66%) Thoroughbred horses in Isfahan and Shahrekord, respectively. This study has shown that horses settled in Isfahan central and Shahrekord southwest regions, Iran, are infected by EHV-1 and EHV-4 and thus serve as potential reservoirs and disseminators of the viruses. Taghi Taktaz Hafshejani, Shahin Nekoei, Behnam Vazirian, Abbas Doosti, Faham Khamesipour, and Madubuike Umunna Anyanwu Copyright © 2015 Taghi Taktaz Hafshejani et al. All rights reserved. Mutations Found in embCAB, embR, and ubiA Genes of Ethambutol-Sensitive and -Resistant Mycobacterium tuberculosis Clinical Isolates from China Mon, 31 Aug 2015 12:01:32 +0000 To better understand the molecular mechanisms of Ethambutol (EMB) resistance, the mutant hot spot region of five genes (embB, embA, embC, embR, and ubiA) was amplified and sequenced in 109 EMB-resistant and 153 EMB-susceptible clinical isolates from China. Twenty-seven EMB-susceptible isolates were found to have nonsynonym mutations, 23 of which were in embB. The mutations occurred most frequently in embB (85.3%, 93) and were seldom in embC (2.8%, 3), embA (3.7%, 4), embR (3.7%, 4), and ubiA (8.3%, 9) in EMB-resistant isolates. For the embB gene, 63 isolates showed mutations at embB306, 20 at embB406, nine at embB497, and five at embB354 in EMB-resistant isolates. In addition, the particular mutants at embB406 and embB497 indicated both high levels of EMB resistance ( μg/mL) and broad anti-TB drug resistance spectrums. Our data supported the facts that embB306 could be used as a marker for EMB resistance with a sensitivity of 57.8% and a specificity of 78.8%. Yuhui Xu, Hongyan Jia, Hairong Huang, Zhaogang Sun, and Zongde Zhang Copyright © 2015 Yuhui Xu et al. All rights reserved. Expression of the Genes Encoding the Trk and Kdp Potassium Transport Systems of Mycobacterium tuberculosis during Growth In Vitro Mon, 17 Aug 2015 11:44:06 +0000 Two potassium (K+)-uptake systems, Trk and Kdp, are operative in Mycobacterium tuberculosis (Mtb), but the environmental factors triggering their expression have not been determined. The current study has evaluated the expression of these genes in the Mtb wild-type and a trk-gene knockout strain at various stages of logarithmic growth in relation to extracellular K+ concentrations and pH. In both strains, mRNA levels of the K+-uptake encoding genes were relatively low compared to those of the housekeeping gene, sigA, at the early- and mid-log phases, increasing during late-log. Increased gene expression coincided with decreased K+ uptake in the context of a drop in extracellular pH and sustained high extracellular K+ concentrations. In an additional series of experiments, the pH of the growth medium was manipulated by the addition of 1N HCl/NaOH. Decreasing the pH resulted in reductions in both membrane potential and K+ uptake in the setting of significant induction of genes encoding both K+ transporters. These observations are consistent with induction of the genes encoding the active K+ transporters of Mtb as a strategy to compensate for loss of membrane potential-driven uptake of K+ at low extracellular pH. Induction of these genes may promote survival in the acidic environments of the intracellular vacuole and granuloma. Moloko C. Cholo, Elizabeth J. van Rensburg, Ayman G. Osman, and Ronald Anderson Copyright © 2015 Moloko C. Cholo et al. All rights reserved. Qat Chewing and Periodontal Pathogens in Health and Disease: Further Evidence for a Prebiotic-Like Effect Sun, 16 Aug 2015 11:20:55 +0000 Aim. Qat chewing has been reported to induce subgingival microbial shifts suggestive of prebiotic-like properties. The objective here was to assess the effect of qat chewing on a panel of classical and new putative periopathogens in health and periodontitis. Materials and Methods. 40 qat chewers and 40 nonchewers, equally stratified by periodontal health status, were recruited. Taqman, real-time PCR was used to quantify total bacteria, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Parvimonas micra, Filifactor alocis, Synergistetes, and TM7s in pooled subgingival biofilm samples. Differences in microbial parameters between the study groups were analysed using ordinal regression. Results. In health, the qat chewers harboured significantly lower relative counts of P. gingivalis, T. forsythia, Synergistetes, and TM7s after adjustment for multiple comparisons (). At nominal significance level, they also carried lower counts of TM7s and P. micra (). In periodontitis, the chewers had lower counts of all taxa; however, only T. denticola withstood correction for multiple comparisons (). Conclusions. Qat chewing is associated with lower proportions of periopathogens, particularly in subjects with healthy periodontium, which supports previous reports of its prebiotic-like properties. This potentially beneficial biological effect can be exploited by attempting to isolate the active fraction. Abdulrahman Al-Alimi, Tara Taiyeb-Ali, Nasruddin Jaafar, and Nezar Noor Al-hebshi Copyright © 2015 Abdulrahman Al-Alimi et al. All rights reserved. Drug Susceptibility Testing of 31 Antimicrobial Agents on Rapidly Growing Mycobacteria Isolates from China Thu, 13 Aug 2015 12:09:14 +0000 Objectives. Several species of rapidly growing mycobacteria (RGM) are now recognized as human pathogens. However, limited data on effective drug treatments against these organisms exists. Here, we describe the species distribution and drug susceptibility profiles of RGM clinical isolates collected from four southern Chinese provinces from January 2005 to December 2012. Methods. Clinical isolates (73) were subjected to in vitro testing with 31 antimicrobial agents using the cation-adjusted Mueller-Hinton broth microdilution method. The isolates included 55 M. abscessus, 11 M. fortuitum, 3 M. chelonae, 2 M. neoaurum, and 2 M. septicum isolates. Results. M. abscessus (75.34%) and M. fortuitum (15.07%), the most common species, exhibited greater antibiotic resistance than the other three species. The isolates had low resistance to amikacin, linezolid, and tigecycline, and high resistance to first-line antituberculous agents, amoxicillin-clavulanic acid, rifapentine, dapsone, thioacetazone, and pasiniazid. M. abscessus and M. fortuitum were highly resistant to ofloxacin and rifabutin, respectively. The isolates showed moderate resistance to the other antimicrobial agents. Conclusions. Our results suggest that tigecycline, linezolid, clofazimine, and cefmetazole are appropriate choices for M. abscessus infections. Capreomycin, sulfamethoxazole, tigecycline, clofazimine, and cefmetazole are potentially good choices for M. fortuitum infections. Our drug susceptibility data should be useful to clinicians. Hui Pang, Guilian Li, Xiuqin Zhao, Haican Liu, Kanglin Wan, and Ping Yu Copyright © 2015 Hui Pang et al. All rights reserved. Antibiotic Resistance of Bacteria Tue, 04 Aug 2015 10:37:52 +0000 Madhab K. Chattopadhyay, Ranadhir Chakraborty, Hans-Peter Grossart, Gundlapally S. Reddy, and Medicharla V. Jagannadham Copyright © 2015 Madhab K. Chattopadhyay et al. All rights reserved. First Report of Klebsiella pneumoniae-Carbapenemase-3-Producing Escherichia coli ST479 in Poland Mon, 03 Aug 2015 09:45:26 +0000 An increase in the antibiotic resistance among members of the Enterobacteriaceae family has been observed worldwide. Multidrug-resistant Gram-negative rods are increasingly reported. The treatment of infections caused by Escherichia coli and other Enterobacteriaceae has become an important clinical problem associated with reduced therapeutic possibilities. Antimicrobial carbapenems are considered the last line of defense against multidrug-resistant Gram-negative bacteria. Unfortunately, an increase of carbapenem resistance due to the production of Klebsiella pneumoniae carbapenemase (KPC) enzymes has been observed. In this study we describe the ability of E. coli to produce carbapenemase enzymes based on the results of the combination disc assay with boronic acid performed according to guidelines established by the European Community on Antimicrobial Susceptibility Testing (EUCAST) and the biochemical Carba NP test. Moreover, we evaluated the presence of genes responsible for the production of carbapenemases (, , , ) and genes encoding other β-lactamases (, , ) among E. coli isolate. The tested isolate of E. coli that possessed the and genes was identified. The tested strain exhibited susceptibility to colistin (0.38 μg/mL) and tigecycline (1 μg/mL). This is the first detection of in an E. coli ST479 in Poland. Dominika Ojdana, Paweł Sacha, Dorota Olszańska, Piotr Majewski, Piotr Wieczorek, Jadwiga Jaworowska, Anna Sieńko, Anna Jurczak, and Elżbieta Tryniszewska Copyright © 2015 Dominika Ojdana et al. All rights reserved. Microbiological Analysis of Necrosols Collected from Urban Cemeteries in Poland Sun, 02 Aug 2015 08:59:51 +0000 Decomposition of organic matter is the primary function in the soil ecosystem, which involves bacteria and fungi. Soil microbial content depends on many factors, and secondary biological and chemical contaminations change and affect environmental feedback. Little work has been done to estimate the microbiological risk for cemetery employees and visitors. The potential risk of infection for people in the cemetery is primarily associated with injury and wound contamination during performing the work. The aim of this study was to analyze the microbiota of cemetery soil obtained from cemeteries and bacterial composition in selected soil layers encountered by gravediggers and cemetery caretakers. The most common bacterial pathogens were Enterococcus spp. (80.6%), Bacillus spp. (77.4%), and E. coli (45.1%). The fungi Penicillium spp. and Aspergillus spp. were isolated from 51% and 6.4% of samples, respectively. Other bacterial species were in the ground cemetery relatively sparse. Sampling depth was not correlated with bacterial growth (), but it was correlated with several differences in microbiota composition (superficial versus deep layer). Ireneusz Całkosiński, Katarzyna Płoneczka-Janeczko, Magda Ostapska, Krzysztof Dudek, Andrzej Gamian, and Krzysztof Rypuła Copyright © 2015 Ireneusz Całkosiński et al. All rights reserved. Absence of the mecA Gene in Methicillin Resistant Staphylococcus aureus Isolated from Different Clinical Specimens in Shendi City, Sudan Tue, 28 Jul 2015 06:30:01 +0000 Absolute dependence on mecA gene as the defining standard in determining the resistance of S. aureus to methicillin became the subject of distrust by many researchers. The present study aimed to determine the frequency of mecA gene in methicillin resistant S. aureus (MRSA) isolates using polymerase chain reaction and to correlate its presence to conventional method. In this regard, two hundred S. aureus isolates were collected from patients with different diseases attending different hospitals in Shandi City, Sudan. Phenotypic Kirby-Bauer method confirmed the existence of methicillin resistant S. aureus in 61.5% of the subjected isolates with MICs ranging from 4 μg/mL to 256 μg/mL when using E-test. However, when amplifying a 310 bp fragment of the mecA gene by PCR, twelve out of the 123 MRSA isolates (9.8%) were mecA negative, whereas all the 77 methicillin sensitive S. aureus (MSSA) were mecA negative. In conclusion, this study drew attention to the credibility of the mecA gene and its usefulness in the detection of all MRSA strains without referring to the traditional methods. Hence, it is highly recommended to consider alternative mechanisms for β-lactam resistance that may compete with mecA gene in the emergence of MRSA phenomenon in the community. Mogahid M. Elhassan, Hani A. Ozbak, Hassan A. Hemeg, Miskelyemen A. Elmekki, and Leila M. Ahmed Copyright © 2015 Mogahid M. Elhassan et al. All rights reserved. Induction Murine Models of Chronic Fatigue Syndrome by Brucella abortus Antigen Injections: Is Anemia Induced or Not? Thu, 11 Jun 2015 06:46:42 +0000 To investigate whether Brucella abortus (BA) antigen injections lead to anemia, and to establish an appropriate Chronic Fatigue Syndrome (CFS) animal model by BA injections, 6 repeated injections of BA antigen were fulfilled every 2 weeks. At a high dose of particles/mouse, anemia was induced within 2 weeks and then recovered a lot at the end of the research, while at a moderate dose of (3 injections) shifting to /mouse (3 injections) anemia was absent. In both groups running wheel activity remained very low even 6 weeks after the last injection. Junji Moriya, Qiang He, Hiroaki Uenishi, Sumiyo Akazawa, Jun-ichi Yamakawa, Junji Kobayashi, and Yasuhito Ishigaki Copyright © 2015 Junji Moriya et al. All rights reserved. Impact of Infection Dose and Previous Serum Antibodies against the Locus of Enterocyte Effacement Proteins on Escherichia coli O157:H7 Shedding in Calves following Experimental Infection Mon, 08 Jun 2015 08:01:34 +0000 Escherichia coli O157:H7 is the main causative agent of haemolytic uremic syndrome. Cattle are the main reservoir of these bacteria, and have been shown to develop immune response to colonization. Our aim was to investigate the faecal shedding pattern of E. coli O157:H7 in calves challenged intragastrically with either 108 or 1010 CFU, as well as the ability of specific preexisting antibodies to reduce shedding of the pathogen. Shedding was analysed by direct counting as well as enrichment of rectoanal mucosal swabs. Statistical analysis was performed using a linear model for repeated measures with and without the inclusion of preexisting antibodies against the carboxy-terminal fraction of intimin-γ (γ-intimin C280) as a covariable. Results suggest that there is a statistical difference in the area under the shedding curves between both doses for 14 as well as 28 days after challenge (p = 0.0069 and 0.0209, resp.). This difference is increased when the prechallenge antibodies are taken into account (p = 0.0056 and 0.0185). We concluded that the bacterial dose influences shedding on calves experimentally challenged and that preexisting antibodies against E. coli O157:H7 γ-intimin C280 could partially reduce faecal excretion. L. Martorelli, C. J. Hovde, D. A. Vilte, A. Albanese, E. Zotta, C. Ibarra, R. J. C. Cantet, E. C. Mercado, and A. Cataldi Copyright © 2015 L. Martorelli et al. All rights reserved. Could Histoplasma capsulatum Be Related to Healthcare-Associated Infections? Wed, 27 May 2015 09:13:02 +0000 Healthcare-associated infections (HAI) are described in diverse settings. The main etiologic agents of HAI are bacteria (85%) and fungi (13%). Some factors increase the risk for HAI, particularly the use of medical devices; patients with severe cuts, wounds, and burns; stays in the intensive care unit, surgery, and hospital reconstruction works. Several fungal HAI are caused by Candida spp., usually from an endogenous source; however, cross-transmission via the hands of healthcare workers or contaminated devices can occur. Although other medically important fungi, such as Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Histoplasma capsulatum, have never been considered nosocomial pathogens, there are some factors that point out the pros and cons for this possibility. Among these fungi, H. capsulatum infection has been linked to different medical devices and surgery implants. The filamentous form of H. capsulatum may be present in hospital settings, as this fungus adapts to different types of climates and has great dispersion ability. Although conventional pathogen identification techniques have never identified H. capsulatum in the hospital environment, molecular biology procedures could be useful in this setting. More research on H. capsulatum as a HAI etiologic agent is needed, since it causes a severe and often fatal disease in immunocompromised patients. Laura Elena Carreto-Binaghi, Lisandra Serra Damasceno, Nayla de Souza Pitangui, Ana Marisa Fusco-Almeida, Maria José Soares Mendes-Giannini, Rosely Maria Zancopé-Oliveira, and Maria Lucia Taylor Copyright © 2015 Laura Elena Carreto-Binaghi et al. All rights reserved. In Vitro Properties of Potential Probiotic Indigenous Lactic Acid Bacteria Originating from Traditional Pickles Tue, 26 May 2015 06:56:10 +0000 The suitable properties of potential probiotic lactic acid bacteria (LAB) strains (preselected among 153 strains on the basis of their potential technological properties) isolated from traditional Çubuk pickles were examined in vitro. For this purpose, these strains (21 Lactobacillus plantarum, 11 Pediococcus ethanolidurans, and 7 Lactobacillus brevis) were tested for the ability to survive at pH 2.5, resistance to bile salts, viability in the presence of pepsin-pancreatin, ability to deconjugate bile salts, cholesterol assimilation, and surface hydrophobicity properties. Most of the properties tested could be assumed to be strain-dependent. However, L. plantarum and L. brevis species were found to possess desirable probiotic properties to a greater extent compared to P. ethanolidurans. In contrast to P. ethanolidurans strains, the tested L. plantarum and L. brevis strains exhibited bile salt tolerance, albeit to different extent. All tested strains showed less resistance to intestinal conditions than gastric juice environment. Based on the survival under gastrointestinal conditions, 22 of the 39 strains were selected for further characterization. The eight strains having the highest cholesterol assimilation and surface hydrophobicity ratios could be taken as promising probiotic candidates for further in vivo studies, because of the strongest variations found among the tested strains with regard to these properties. Mehmet Tokatlı, Gökşen Gülgör, Simel Bağder Elmacı, Nurdan Arslankoz İşleyen, and Filiz Özçelik Copyright © 2015 Mehmet Tokatlı et al. All rights reserved. Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes Thu, 21 May 2015 15:59:05 +0000 Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab), L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes. Zhiliang Yu, Chenglin Guo, and Juanping Qiu Copyright © 2015 Zhiliang Yu et al. All rights reserved. Immunoendocrine Interactions during HIV-TB Coinfection: Implications for the Design of New Adjuvant Therapies Thu, 14 May 2015 11:28:43 +0000 Worldwide, around 14 million individuals are coinfected with both tuberculosis (TB) and human immunodeficiency virus (HIV). In coinfected individuals, both pathogens weaken immunological system synergistically through mechanisms that are not fully understood. During both HIV and TB infections, there is a chronic state of inflammation associated to dramatic changes in immune cytokine and endocrine hormone levels. Despite this, the relevance of immunoendocrine interaction on both the orchestration of an effective immune response against both pathogens and the control of the chronic inflammation induced during HIV, TB, or both infections is still controversial. The present study reviews immunoendocrine interactions occurring during HIV and TB infections. We also expose our own findings on immunoendocrine cross talk in HIV-TB coinfection. Finally, we evaluate the use of adrenal hormones and their derivatives in immune-therapy and discuss the use of some of these compounds like the adjuvant for the prevention and treatment of TB in HIV patients. Guadalupe Veronica Suarez, Maria Belen Vecchione, Matias Tomas Angerami, Omar Sued, Andrea Claudia Bruttomesso, Oscar Adelmo Bottasso, and Maria Florencia Quiroga Copyright © 2015 Guadalupe Veronica Suarez et al. All rights reserved. Effects of Arsenite Resistance on the Growth and Functional Gene Expression of Leptospirillum ferriphilum and Acidithiobacillus thiooxidans in Pure Culture and Coculture Tue, 12 May 2015 13:36:15 +0000 The response of iron-oxidizing Leptospirillum ferriphilum YSK and sulfur-oxidizing Acidithiobacillus thiooxidans A01 to arsenite under pure culture and coculture was investigated based on biochemical characterization (concentration of iron ion and pH value) and related gene expression. L. ferriphilum YSK and At. thiooxidans A01 in pure culture could adapt up to 400 mM and 800 mM As(III) after domestication, respectively, although arsenite showed a negative effect on both strains. The coculture showed a stronger sulfur and ferrous ion oxidation activity when exposed to arsenite. In coculture, the pH value showed no significant difference when under 500 mM arsenite stress, and the cell number of At. thiooxidans was higher than that in pure culture benefiting from the interaction with L. ferriphilum. The expression profile showed that the arsenic efflux system in the coculture was more active than that in pure culture, indicating that there is a synergetic interaction between At. thiooxidans A01 and L. ferriphilum YSK. In addition, a model was proposed to illustrate the interaction between arsenite and the ars operon in L. ferriphilum YSK and At. thiooxidans A01. This study will facilitate the effective application of coculture in the bioleaching process by taking advantage of strain-strain communication and coordination. Huidan Jiang, Yili Liang, Huaqun Yin, Yunhua Xiao, Xue Guo, Ying Xu, Qi Hu, Hongwei Liu, and Xueduan Liu Copyright © 2015 Huidan Jiang et al. All rights reserved. Emergence of Carbapenem-Resistant Klebsiella pneumoniae: Progressive Spread and Four-Year Period of Observation in a Cardiac Surgery Division Mon, 04 May 2015 06:18:43 +0000 Frequent use of carbapenems has contributed to the increase to K. pneumoniae strains resistant to this class of antibiotics (CRKP), causing a problem in the clinical treatment of patients. This investigation reports the epidemiology, genetic diversity, and clinical implication of the resistance to drugs mediated by CRKP in our hospital. A total of 280 K. pneumoniae strains were collected; in particular 98/280 (35%) were CRKP. Sequencing analysis of CRKP isolated strains showed that 9/98 of MBL-producing strains carried the gene and 89/98 of the isolates were positive for . Antimicrobial susceptibility tests revealed a complete resistance to third-generation cephalosporins and a moderate resistance to tigecycline, gentamicin, and fluoroquinolones with percentages of resistance of 61%, 64%, and 98%, respectively. A resistance of 31% was shown towards trimethoprim-sulfamethoxazole. Colistin was the most active agent against CRKP with 99% of susceptibility. Clonality was evaluated by PFGE and MLST: MLST showed the same clonal type, ST258, while PFGE analysis indicated the presence of a major clone, namely, pulsotype A. This finding indicates that the prevalent resistant isolates were genetically related, suggesting that the spread of these genes could be due to clonal dissemination as well as to genetic exchange between different clones. Fortunata Lombardi, Paola Gaia, Rea Valaperta, Maria Cornetta, Milvana Rosa Tejada, Luca Di Girolamo, Alessandra Moroni, Federica Ramundo, Alessio Colombo, Massimiliano Valisi, and Elena Costa Copyright © 2015 Fortunata Lombardi et al. All rights reserved. Antimicrobial Resistance Profile and Genotypic Characteristics of Streptococcus suis Capsular Type 2 Isolated from Clinical Carrier Sows and Diseased Pigs in China Mon, 04 May 2015 06:18:11 +0000 Streptococcus suis serotype 2 is an important zoonotic pathogen. Antimicrobial resistance phenotypes and genotypic characterizations of S. suis 2 from carrier sows and diseased pigs remain largely unknown. In this study, 96 swine S. suis type 2, 62 from healthy sows and 34 from diseased pigs, were analyzed. High frequency of tetracycline resistance was observed, followed by sulfonamides. The lowest resistance of S. suis 2 for β-lactams supports their use as the primary antibiotics to treat the infection of serotype 2. In contrast, 35 of 37 S. suis 2 with ML phenotypes were isolated from healthy sows, mostly encoded by the ermB and/or the mefA genes. Significantly lower frequency of mrp+/epf+/sly+ was observed among serotype 2 from healthy sows compared to those from diseased pigs. Furthermore, isolates from diseased pigs showed more homogeneously genetic patterns, with most of them clustered in pulsotypes A and E. The data indicate the genetic complexity of S. suis 2 between herds and a close linkage among isolates from healthy sows and diseased pigs. Moreover, many factors, such as extensive use of tetracycline or diffusion of Tn916 with tetM, might have favored for the pathogenicity and widespread dissemination of S. suis serotype 2. Chunping Zhang, Zhongqiu Zhang, Li Song, Xuezheng Fan, Fang Wen, Shixin Xu, and Yibao Ning Copyright © 2015 Chunping Zhang et al. All rights reserved. In Vitro Activity of Imipenem and Colistin against a Carbapenem-Resistant Klebsiella pneumoniae Isolate Coproducing SHV-31, CMY-2, and DHA-1 Sun, 03 May 2015 15:23:46 +0000 We investigated the synergism of colistin and imipenem against a multidrug-resistant K. pneumoniae isolate which was recovered from a severe hip infection. PCR and DNA sequencing were used to characterize the outer membrane porin genes and the resistance genes mediating the common β-lactamases and carbapenemases. Synergism was evaluated by time-kill studies. The , , and were detected. Outer membrane porin genes analysis revealed loss of ompK36 and frame-shift mutation of ompK35. The common carbapenemase genes were not found. Time-kill studies demonstrated that a combination of 1x MIC of colistin (2 mg/L) and 1x MIC of imipenem (8 mg/L) was synergistic and bactericidal but with inoculum effect. Bactericidal activity without inoculum effect was observed by concentration of 2x MIC of colistin alone or plus 2x MIC of imipenem. In conclusion, colistin plus imipenem could be an alternative option to treat carbapenem-resistant K. pneumoniae infections. Hung-Jen Tang, Yee-Huang Ku, Mei-Feng Lee, Yin-Ching Chuang, and Wen-Liang Yu Copyright © 2015 Hung-Jen Tang et al. All rights reserved. Epidemiological Characterization of Drug Resistance among Mycobacterium tuberculosis Isolated from Patients in Northeast of Iran during 2012-2013 Sun, 03 May 2015 14:08:31 +0000 Introduction. Tuberculosis is still one of the most important health problems in developing countries and increasing drug resistance is the main concern for its treatment. This study was designed to characterize the drug resistant Mycobacterium tuberculosis isolated from patients suffering from pulmonary tuberculosis in northeast of Iran. Method. In this cross-sectional study during 2012-2013, drug susceptibility testing was performed on Mycobacterium tuberculosis isolated in northeast of Iran using proportional method. Epidemiological data concerning these strains were also analyzed. Results. Among 125 studied isolates, 25 mycobacteria (20%) were diagnosed as nontuberculosis mycobacteria. Among the remaining 100 Mycobacterium tuberculosis isolates, the resistance rates were 7%, 7%, 3%, and 9% against isoniazid, rifampin, ethambutol, and streptomycin, respectively. Four isolates were resistant against both isoniazid and rifampin (MDR tuberculosis). The highest resistance rate was observed among 15–45-year-old patients. The MDR tuberculosis was much more prevalent among those who had previous history of treatment. Conclusion. Considering these findings, DOTS strategy should be emphasized and promptly used in order to prevent further resistance. Regarding the high rate of nontuberculosis mycobacteria, it is recommended that confirmatory tests were performed before any therapeutic decision. Ashraf Tavanaee Sani, Abolfazl Shakiba, Maryam Salehi, Hamid Reza Bahrami Taghanaki, Seiedeh Fatemeh Ayati Fard, and Kiarash Ghazvini Copyright © 2015 Ashraf Tavanaee Sani et al. All rights reserved. Population Structure and Oxacillin Resistance of Staphylococcus aureus from Pigs and Pork Meat in South-West of Poland Sun, 03 May 2015 13:03:12 +0000 The genotypes and oxacillin resistance of 420 S. aureus isolates from pigs () and pork () were analyzed. Among 18 spa types detected in S. aureus from pig t011, t021, t034, t091, t318, t337, and t1334 were the most frequent. Among 30 spa types found in S. aureus isolates from pork t084, t091, t499, t4309, t12954, and t13074 were dominant. The animal S. aureus isolates were clustered into MLST clonal complexes CC7, CC9, CC15, CC30, and CC398 and meat-derived isolates to CC1, CC7, and CC15. Thirty-six MRSA were isolated exclusively from pigs. All MRSA were classified to spa t011 SCCmecV. BORSA phenotype was found in 14% S. aureus isolates from pigs and 10% isolates from pork meat. spa t034 dominated among BORSA from pigs and t091 among meat-derived BORSA. This is the first report on spa types and oxacillin resistance of S. aureus strains from pigs and pork meat in Poland. Besides S. aureus CC9, CC30, and CC398 known to be distributed in pigs, the occurrence of genotype belonging to CC7 in this species has been reported for the first time. To our knowledge it is also the first report concerning CC398 BORSA isolates from pigs and pork meat. Paweł Krupa, Jarosław Bystroń, Magdalena Podkowik, Joanna Empel, Aneta Mroczkowska, and Jacek Bania Copyright © 2015 Paweł Krupa et al. All rights reserved. An Activity of Thioacyl Derivatives of 4-Aminoquinolinium Salts towards Biofilm Producing and Planktonic Forms of Coagulase-Negative Staphylococci Sun, 03 May 2015 12:54:57 +0000 Microorganisms present in different environments have developed specific mechanisms of settling on various abiotic and biotic surfaces by forming a biofilm. It seems to be well justified to search for new compounds enabling biofilm reduction, which is highly resistant to antibiotics. This study was thus an initial assessment of the antibacterial activity of two new quinoline derivatives of a structure of 3-thioacyl 1-methyl 4-arylaminoquinolinium salts against coagulase-negative staphylococci (CoNS) isolated from a hospital environment, in a form of both biofilms and in planktonic form. Thirty-three stains of CoNS isolated from the hospital environment (air, surfaces) and seven reference strains from the ATCC collection were selected for the study. The mean MIC value for 1-methyl-3-benzoylthio-4-(4-chlorophenylamino)quinolinum chloride (4-chlorophenylamino derivative) was 42.60 ± 19.91 μg/mL, and in the case of strains subjected to 1-methyl-3-benzoylthio-4-(4-fluorophenylamino)quinolinum chloride (4-fluorophenylamino derivative) activity, the mean MIC value was 43.20 ± 14.30 μg/mL. The mean concentration of 4-chlorophenylamino derivative that inhibited biofilm formation was 86.18 ± 30.64 μg/mL. The mean concentration of 4-fluorophenylamino derivatives that inhibited biofilm formation was higher and amounted to 237.09 ± 160.57 μg/mL. Based on the results, both derivatives of the examined compounds exhibit high antimicrobial activity towards strains growing both in planktonic and biofilm form. Robert D. Wojtyczka, Andrzej Zięba, Arkadiusz Dziedzic, Małgorzata Kępa, and Danuta Idzik Copyright © 2015 Robert D. Wojtyczka et al. All rights reserved. Occurrence of Multidrug Resistant Extended Spectrum Beta-Lactamase-Producing Bacteria on Iceberg Lettuce Retailed for Human Consumption Sun, 03 May 2015 12:21:20 +0000 Antibiotic resistance in bacteria is a global problem exacerbated by the dissemination of resistant bacteria via uncooked food, such as green leafy vegetables. New strains of bacteria are emerging on a daily basis with novel expanded antibiotic resistance profiles. In this pilot study, we examined the occurrence of antibiotic resistant bacteria against five classes of antibiotics on iceberg lettuce retailed in local convenience stores in Rochester, Michigan. In this study, 138 morphologically distinct bacterial colonies from 9 iceberg lettuce samples were randomly picked and tested for antibiotic resistance. Among these isolates, the vast majority (86%) demonstrated resistance to cefotaxime, and among the resistant bacteria, the majority showed multiple drug resistance, particularly against cefotaxime, chloramphenicol, and tetracycline. Three bacterial isolates (2.17%) out of 138 were extended spectrum beta-lactamase (ESBL) producers. Two ESBL producers (T1 and T5) were identified as Klebsiella pneumoniae, an opportunistic pathogen with transferable sulfhydryl variable- (SHV-) and TEM-type ESBLs, respectively. The DNA sequence analysis of the detected in K. pneumoniae isolate T1 revealed 99% relatedness to genes found in clinical isolates. This implies that iceberg lettuce is a potential reservoir of newly emerging and evolving antibiotic resistant bacteria and its consumption poses serious threat to human health. Natasha Bhutani, Chithra Muraleedharan, Deepa Talreja, Sonia Walia Rana, Sandeep Walia, Ashok Kumar, and Satish K. Walia Copyright © 2015 Natasha Bhutani et al. All rights reserved. Discrepancies in Drug Susceptibility Test for Tuberculosis Patients Resulted from the Mixed Infection and the Testing System Sun, 03 May 2015 12:08:30 +0000 To find the potential reasons for the discrepancies in the drug susceptibility test (DST) of M. tuberculosis isolates, twenty paired isolates with disputed drug susceptibilities to isoniazid (INH) were selected according to the MGIT960 testing and Löwenstein-Jensen (L-J) proportion methods. Their MICs were confirmed again by broth microdilution method and by L-J proportion method. The spoligotyping results showed that, of all the 20 paired strains, 11 paired isolates belonged to the Beijing genotype and 6 paired isolates belonged to SIT1634, and that each of the remaining 3 paired isolates had two genotypes, namely, SIT1 and SIT1634. Those 3 paired isolates with different intrapair spoligotypes were further confirmed as mixed infection by the results that those three pairs of isolates with different 12 locus MIRU intrapair types and one pair carried different base pair at codon 315 (AGC versus AAC). Totally mutations in the katG gene were identified in 13 paired isolates. No mutations were found in the regulatory sequences and open reading frames (ORF) of the inhA and ahpC genes in any of the tested isolates. Those results showed that the different test systems and the mixed infection with particular genotypes of M. tuberculosis strains contributed to the drug susceptibility discrepancies. Zaoxian Mei, Zhaogang Sun, Dapeng Bai, Yuhui Xu, Zhiling Li, Hairong Huang, Chuanyou Li, Shaofa Xu, and Li Li Copyright © 2015 Zaoxian Mei et al. All rights reserved. Characterization of Multidrug Resistant Extended-Spectrum Beta-Lactamase-Producing Escherichia coli among Uropathogens of Pediatrics in North of Iran Sun, 03 May 2015 10:19:42 +0000 Escherichia coli remains as one of the most important bacteria causing infections in pediatrics and producing extended-spectrum beta-lactamases (ESBLs) making them resistant to beta-lactam antibiotics. In this study we aimed to genotype ESBL-producing E. coli isolates from pediatric patients for ESBL genes and determine their association with antimicrobial resistance. One hundred of the E. coli isolates were initially considered ESBL producing based on their MIC results. These isolates were then tested by polymerase chain reaction (PCR) for the presence or absence of CTX, TEM, SHV, GES, and VEB beta-lactamase genes. About 30.5% of isolated E. coli was ESBL-producing strain. The TEM gene was the most prevalent (49%) followed by SHV (44%), CTX (28%), VEB (8%), and GES (0%) genes. The ESBL-producing E. coli isolates were susceptible to carbapenems (66%) and amikacin (58%) and showed high resistance to cefixime (99%), colistin (82%), and ciprofloxacin (76%). In conclusion, carbapenems were the most effective antibiotics against ESBl-producing E. coli in urinary tract infection in North of Iran. The most prevalent gene is the TEM-type, but the other resistant genes and their antimicrobial resistance are on the rise. Mohammad Sadegh Rezai, Ebrahim Salehifar, Alireza Rafiei, Taimour Langaee, Mohammadreza Rafati, Kheironesa Shafahi, and Gohar Eslami Copyright © 2015 Mohammad Sadegh Rezai et al. All rights reserved. Neem (Azadirachta indica A. Juss) Oil to Tackle Enteropathogenic Escherichia coli Sun, 03 May 2015 08:53:07 +0000 Neem (Azadirachta indica A. Juss) oil (NO) was assayed against forty-eight isolates of Escherichia coli by standardised disc diffusion test and microdilution test. By molecular biology characterization, fourteen isolates resulted in diarrheagenic E. coli with sixteen primer pairs that specifically amplify unique sequences of virulence genes and of 16S rRNA. The NO showed biological activity against all isolates. The bacterial growth inhibition zone by disc diffusion method (100 µL NO) ranged between 9.50 ± 0.70 and 30.00 ± 1.00 mm. The antibacterial activity was furthermore determined at lower NO concentrations (1 : 10–1 : 10,000). The percent of growth reduction ranged between 23.71 ± 1.00 and 99.70 ± 1.53. The highest bacterial growth reduction was 1 : 10 NO concentration with 50 µL of bacterial suspension (ca. 1 × 106 CFU/mL). There is significant difference between the antibacterial activities against pathogenic and nonpathogenic E. coli, as well as NO and ciprofloxacin activities. Viable cells after the different NO concentration treatments were checked by molecular biology assay using PMA dye. On the basis of the obtained results, NO counteracts E. coli and also influences the virulence of E. coli viable cells after NO treatment. The NO metabolomic composition was obtained using fingerprint HPTLC. Paola Del Serrone, Chiara Toniolo, and Marcello Nicoletti Copyright © 2015 Paola Del Serrone et al. All rights reserved. Diffusion and Persistence of Multidrug Resistant Salmonella Typhimurium Strains Phage Type DT120 in Southern Italy Tue, 28 Apr 2015 09:13:47 +0000 Sixty-two multidrug resistant Salmonella enterica serovar Typhimurium strains isolated from 255 clinical strains collected in Southern Italy in 2006–2008 were characterised for antimicrobial resistance genes, pulsotype, and phage type. Most strains (83.9%) were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT) encoded in 88.5% by the Salmonella genomic island (SGI1) and in 11.5% by the InH-like integron (–aadA1) and catA1, sul1, and tet(B) genes. STYMXB.0061 (75%) and DT120 (84.6%) were the prevalent pulsotype and phage type identified in these strains, respectively. Five other resistance patterns were found either in single or in a low number of isolates. The pandemic clone DT104 (ACSSuT encoded by SGI1) has been identified in Italy since 1992, while strains DT120 (ACSSuT encoded by SGI1) have never been previously reported in Italy. In Europe, clinical strains DT120 have been reported from sporadic outbreaks linked to the consumption of pork products. However, none of these strains were STYMXB.0061 and SGI1 positive. The prevalent identification and persistence of DT120 isolates would suggest, in Southern Italy, a phage type shifting of the pandemic DT104 clone pulsotype STYMXB.0061. Additionally, these findings raise epidemiological concern about the potential diffusion of these emerging multidrug resistant (SGI linked) DT120 strains. Danila De Vito, Rosa Monno, Federica Nuccio, Marilisa Legretto, Marta Oliva, Maria Franca Coscia, Anna Maria Dionisi, Carla Calia, Carmen Capolongo, and Carlo Pazzani Copyright © 2015 Danila De Vito et al. All rights reserved. Identification and Characterization of Chlamydia abortus Isolates from Yaks in Qinghai, China Tue, 28 Apr 2015 06:39:55 +0000 Recently, the yak population has exhibited reproductive disorders, which are considered to be associated with Chlamydia abortus (C. abortus) in Qinghai, China. In this study, a total of 9 aborted fetuses (each from a different herd) and 126 vaginal swab samples from the 9 herds were collected and analyzed. C. abortus DNA was detected from all of the 9 aborted fetuses and 30 of the 126 vaginal swab samples (23.81%) from yak cows in the selected herds. Four C. abortus strains were isolated from embryonated egg yolk sacs inoculated with foetal organ suspensions. The isolated C. abortus strains were further identified, which showed identical restriction profiles with the C. abortus reference strain using AluI restriction enzyme in the RFLP test. Moreover, the isolated C. abortus strains and C. abortus-positive vaginal swab samples were genotyped by multiple loci variable number tandem repeat analysis and all belonged to the genotype 2 group. These findings suggested that C. abortus played a substantial role in yak abortion in Qinghai, China. Zhaocai Li, Xiaoan Cao, Baoquan Fu, Yilin Chao, Jinshan Cai, and Jizhang Zhou Copyright © 2015 Zhaocai Li et al. All rights reserved. Recent Advances and Future Perspective in Microbiota and Probiotics Thu, 23 Apr 2015 07:59:27 +0000 Haruki Kitazawa, Susana Alvarez, Alexander Suvorov, Vyacheslav Melnikov, Julio Villena, and Borja Sánchez Copyright © 2015 Haruki Kitazawa et al. All rights reserved. Intrinsic Immunomodulatory Effects of Low-Digestible Carbohydrates Selectively Extend Their Anti-Inflammatory Prebiotic Potentials Tue, 21 Apr 2015 13:34:02 +0000 The beneficial effects of carbohydrate-derived fibers are mainly attributed to modulation of the microbiota, increased colonic fermentation, and the production of short-chain fatty acids. We studied the direct immune responses to alimentary fibers in in vitro and in vivo models. Firstly, we evaluated the immunomodulation induced by nine different types of low-digestible fibers on human peripheral blood mononuclear cells. None of the fibers tested induced cytokine production in baseline conditions. However, only one from all fibers almost completely inhibited the production of anti- and proinflammatory cytokines induced by bacteria. Secondly, the impact of short- (five days) and long-term (three weeks) oral treatments with selected fibers was assessed in the trinitrobenzene-sulfonic acid colitis model in mice. The immunosuppressive fiber significantly reduced levels of inflammatory markers over both treatment periods, whereas a nonimmunomodulatory fiber had no effect. The two fibers did not differ in terms of the observed fermentation products and colonic microbiota after three weeks of treatment, suggesting that the anti-inflammatory action was not related to prebiotic properties. Hence, we observed a direct effect of a specific fiber on the murine immune system. This intrinsic, fiber-dependent immunomodulatory potential may extend prebiotic-mediated protection in inflammatory bowel disease. Jérôme Breton, Coline Plé, Laetitia Guerin-Deremaux, Bruno Pot, Catherine Lefranc-Millot, Daniel Wils, and Benoit Foligné Copyright © 2015 Jérôme Breton et al. All rights reserved. Impact of a Complex Food Microbiota on Energy Metabolism in the Model Organism Caenorhabditis elegans Sun, 19 Apr 2015 14:37:35 +0000 The nematode Caenorhabditis elegans is widely used as a model system for research on aging, development, and host-pathogen interactions. Little is currently known about the mechanisms underlying the effects exerted by foodborne microbes. We took advantage of C. elegans to evaluate the impact of foodborne microbiota on well characterized physiological features of the worms. Foodborne lactic acid bacteria (LAB) consortium was used to feed nematodes and its composition was evaluated by 16S rDNA analysis and strain typing before and after colonization of the nematode gut. Lactobacillus delbrueckii, L. fermentum, and Leuconostoc lactis were identified as the main species and shown to display different worm gut colonization capacities. LAB supplementation appeared to decrease nematode lifespan compared to the animals fed with the conventional Escherichia coli nutrient source or a probiotic bacterial strain. Reduced brood size was also observed in microbiota-fed nematodes. Moreover, massive accumulation of lipid droplets was revealed by BODIPY staining. Altered expression of nhr-49, pept-1, and tub-1 genes, associated with obesity phenotypes, was demonstrated by RT-qPCR. Since several pathways are evolutionarily conserved in C. elegans, our results highlight the nematode as a valuable model system to investigate the effects of a complex microbial consortium on host energy metabolism. Elena Zanni, Chiara Laudenzi, Emily Schifano, Claudio Palleschi, Giuditta Perozzi, Daniela Uccelletti, and Chiara Devirgiliis Copyright © 2015 Elena Zanni et al. All rights reserved. The Effect of LAB as Probiotic Starter Culture and Green Tea Extract Addition on Dry Fermented Pork Loins Quality Sun, 19 Apr 2015 12:05:31 +0000 The objective of this study was to evaluate the microbiological, physicochemical, and sensory quality of dry fermented pork loin produced with the addition of Lb. rhamnosus LOCK900 probiotic strain, 0.2% glucose, and 1.5% green tea extract. Three loins were prepared: control sample (P0: no additives), sample supplemented with glucose and probiotic strain (P1), and sample with glucose, green tea extract, and probiotic (P2). The samples were analyzed after 21 days of ripening and 180 days of storage. The results indicated that the highest count of LAB was observed both in the samples: with probiotic and with probiotic and green tea extract (7.00 log cfu/g after ripening; 6.00 log cfu/g after storage). The oxidation-reduction potential values were lower in the probiotic loin samples. Probiotic and green tea extract have not caused color changes of study loins during storage. The study demonstrated that an addition of probiotic and green tea extract to dry fermented loins is possible and had no impact on sensory quality after product storage. Katarzyna Neffe-Skocińska, Danuta Jaworska, Danuta Kołożyn-Krajewska, Zbigniew Dolatowski, and Luiza Jachacz-Jówko Copyright © 2015 Katarzyna Neffe-Skocińska et al. All rights reserved. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two β-Glycoside Hydrolases Sun, 19 Apr 2015 11:35:21 +0000 Probiotics, prebiotics, and combinations thereof, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro- and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE) in the acidic (pH 4–7) and the alkaline (pH 6–11) regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5–13.9-fold or decreasing 1.5–7.8-fold in relative abundance. Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho--glucosidase (LBA0881) and phospho--galactosidase II (LBA0726). The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium NCFM. Several of the upregulated or downregulated identified proteins associated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involved in carbohydrate metabolism. Gabriella C. van Zanten, Nadja Sparding, Avishek Majumder, Sampo J. Lahtinen, Birte Svensson, and Susanne Jacobsen Copyright © 2015 Gabriella C. van Zanten et al. All rights reserved. Detection of Tropical Fungi in Formalin-Fixed, Paraffin-Embedded Tissue: Still an Indication for Microscopy in Times of Sequence-Based Diagnosis? Sun, 19 Apr 2015 08:53:44 +0000 Introduction. The aim of the study was the evaluation of panfungal PCR protocols with subsequent sequence analysis for the diagnostic identification of invasive mycoses in formalin-fixed, paraffin-embedded tissue samples with rare tropical mycoses. Materials and Methods. Five different previously described panfungal PCR/sequencing protocols targeting 18S and 28S ribosomal RNA gene fragments as well as internal transcribed spacer 1 and 2 fragments were evaluated with a collection of 17 formalin-fixed, paraffin-embedded tissue samples of patients with rare and/or tropical invasive mycoses, comprising chromoblastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, mucormycosis, mycetoma/maduromycosis, and rhinosporidiosis, in a proof-of-principle analysis. Results. The primers of the panfungal PCRs readily and predominantly reacted with contaminating environmental fungi that had deposited on the paraffin blocks. Altogether three sequence results of histoplasmosis and mycetoma samples that matched the histological assessment were associated with sample age <10 years and virtually without PCR inhibition. Conclusions. The high risk of amplifying environmental contaminants severely reduces the usefulness of the assessed panfungal PCR/sequencing protocols for the identification of rare and/or tropical mycoses in stored formalin-fixed, paraffin-embedded tissues. Histological assessment remains valuable for such indications if cultural differentiation is impossible from inactivated sample material. Hagen Frickmann, Ulrike Loderstaedt, Paul Racz, Klara Tenner-Racz, Petra Eggert, Alexandra Haeupler, Ralf Bialek, and Ralf Matthias Hagen Copyright © 2015 Hagen Frickmann et al. All rights reserved. Phenotypic Characteristics Associated with Virulence of Clinical Isolates from the Sporothrix Complex Sun, 19 Apr 2015 08:30:18 +0000 The Sporothrix complex members cause sporotrichosis, a subcutaneous mycosis with a wide spectrum of clinical manifestations. Several specific phenotypic characteristics are associated with virulence in many fungi, but studies in this field involving the Sporothrix complex species are scarce. Melanization, thermotolerance, and production of proteases, catalase, and urease were investigated in 61 S. brasiliensis, one S. globosa, and 10 S. schenckii strains. The S. brasiliensis strains showed a higher expression of melanin and urease compared with S. schenckii. These two species, however, presented similar thermotolerances. Our S. globosa strain had low expression of all studied virulence factors. The relationship between these phenotypes and clinical aspects of sporotrichosis was also evaluated. Strains isolated from patients with spontaneous regression of infection were heavily melanized and produced high urease levels. Melanin was also related to dissemination of internal organs and protease production was associated with HIV-coinfection. A murine sporotrichosis model showed that a S. brasiliensis strain with high expression of virulence factors was able to disseminate and yield a high fungal burden in comparison with a control S. schenckii strain. Our results show that virulence-related phenotypes are variably expressed within the Sporothrix complex species and might be involved in clinical aspects of sporotrichosis. Rodrigo Almeida-Paes, Luã Cardoso de Oliveira, Manoel Marques Evangelista Oliveira, Maria Clara Gutierrez-Galhardo, Joshua Daniel Nosanchuk, and Rosely Maria Zancopé-Oliveira Copyright © 2015 Rodrigo Almeida-Paes et al. All rights reserved. Bioactive Natural Products: Facts, Applications, and Challenges Wed, 15 Apr 2015 09:07:32 +0000 Yiannis Kourkoutas, Kimon A. G. Karatzas, Vasilis P. Valdramidis, and Nikos Chorianopoulos Copyright © 2015 Yiannis Kourkoutas et al. All rights reserved. Helicobacter pylori and Pathogenesis Wed, 08 Apr 2015 11:22:59 +0000 Akio Tomoda, Shigeru Kamiya, and Hidekazu Suzuki Copyright © 2015 Akio Tomoda et al. All rights reserved. Transcriptome and Proteome Expression Analysis of the Metabolism of Amino Acids by the Fungus Aspergillus oryzae in Fermented Soy Sauce Tue, 07 Apr 2015 12:51:13 +0000 Amino acids comprise the majority of the flavor compounds in soy sauce. A portion of these amino acids are formed from the biosynthesis and metabolism of the fungus Aspergillus oryzae; however, the metabolic pathways leading to the formation of these amino acids in A. oryzae remain largely unknown. We sequenced the transcriptomes of A. oryzae 100-8 and A. oryzae 3.042 under similar soy sauce fermentation conditions. 2D gel electrophoresis was also used to find some differences in protein expression. We found that many amino acid hydrolases (endopeptidases, aminopeptidases, and X-pro-dipeptidyl aminopeptidase) were expressed at much higher levels (mostly greater than double) in A. oryzae 100-8 than in A. oryzae 3.042. Our results indicated that glutamate dehydrogenase may activate the metabolism of amino acids. We also found that the expression levels of some genes changed simultaneously in the metabolic pathways of tyrosine and leucine and that these conserved genes may modulate the function of the metabolic pathway. Such variation in the metabolic pathways of amino acids is important as it can significantly alter the flavor of fermented soy sauce. Guozhong Zhao, Yunping Yao, Chunling Wang, Fengwei Tian, Xiaoming Liu, Lihua Hou, Zhen Yang, Jianxin Zhao, Hao Zhang, and Xiaohong Cao Copyright © 2015 Guozhong Zhao et al. All rights reserved. Gastric Carcinogenesis and Underlying Molecular Mechanisms: Helicobacter pylori and Novel Targeted Therapy Tue, 07 Apr 2015 09:44:33 +0000 The oxygen-derived free radicals that are released from activated neutrophils are one of the cytotoxic factors of Helicobacter pylori-induced gastric mucosal injury. Increased cytidine deaminase activity in H. pylori-infected gastric tissues promotes the accumulation of various mutations and might promote gastric carcinogenesis. Cytotoxin-associated gene A (CagA) is delivered into gastric epithelial cells via bacterial type IV secretion system, and it causes inflammation and activation of oncogenic pathways. H. pylori infection induces epigenetic transformations, such as aberrant promoter methylation in tumor-suppressor genes. Aberrant expression of microRNAs is also reportedly linked to gastric tumorogenesis. Moreover, recent advances in molecular targeting therapies provided a new interesting weapon to treat advanced gastric cancer through anti-human epidermal growth factor receptor 2 (HER-2) therapies. This updated review article highlights possible mechanisms of gastric carcinogenesis including H. pylori-associated factors. Toshihiro Nishizawa and Hidekazu Suzuki Copyright © 2015 Toshihiro Nishizawa and Hidekazu Suzuki. All rights reserved. Interplay of the Gastric Pathogen Helicobacter pylori with Toll-Like Receptors Mon, 06 Apr 2015 12:05:29 +0000 Toll-like receptors (TLRs) are crucial for pathogen recognition and downstream signaling to induce effective immunity. The gastric pathogen Helicobacter pylori is a paradigm of persistent bacterial infections and chronic inflammation in humans. The chronicity of inflammation during H. pylori infection is related to the manipulation of regulatory cytokines. In general, the early detection of H. pylori by TLRs and other pattern recognition receptors (PRRs) is believed to induce a regulatory cytokine or chemokine profile that eventually blocks the resolution of inflammation. H. pylori factors such as LPS, HSP-60, NapA, DNA, and RNA are reported in various studies to be recognized by specific TLRs. However, H. pylori flagellin evades the recognition of TLR5 by possessing a conserved N-terminal motif. Activation of TLRs and resulting signal transduction events lead to the production of pro- and anti-inflammatory mediators through activation of NF-κB, MAP kinases, and IRF signaling pathways. The genetic polymorphisms of these important PRRs are also implicated in the varied outcome and disease progression. Hence, the interplay of TLRs and bacterial factors highlight the complexity of innate immune recognition and immune evasion as well as regulated processes in the progression of associated pathologies. Here we will review this important aspect of H. pylori infection. Suneesh Kumar Pachathundikandi, Judith Lind, Nicole Tegtmeyer, Emad M. El-Omar, and Steffen Backert Copyright © 2015 Suneesh Kumar Pachathundikandi et al. All rights reserved. Helicobacter pylori Outer Membrane Protein 18 (Hp1125) Is Involved in Persistent Colonization by Evading Interferon-γ Signaling Mon, 06 Apr 2015 11:34:45 +0000 Outer membrane proteins (OMPs) can induce an immune response. Omp18 (HP1125) of H. pylori is a powerful antigen that can induce significant interferon-γ (IFN-γ) levels. Previous studies have suggested that IFN-γ plays an important role in H. pylori clearance. However, H. pylori has multiple mechanisms to avoid host immune surveillance for persistent colonization. We generated an omp18 mutant (H. pylori 26695 and H. pylori SS1) strain to examine whether Omp18 interacts with IFN-γ and is involved in H. pylori colonization. qRT-PCR revealed that IFN-γ induced Omp18 expression. qRT-PCR and western blot analysis revealed reduced expressions of virulence factors CagA and NapA in H. pylori 26695 with IFN-γ treatment, but they were induced in the Δomp18 strain. In C57BL/6 mice infected with H. pylori SS1 and the Δomp18 strain, the Δomp18 strain conferred defective colonization and activated a stronger inflammatory response. Signal transducer phosphorylation and transcription 1 (STAT1) activator was downregulated by the wild-type strain but not the Δomp18 strain in IFN-γ-treated macrophages. Furthermore, Δomp18 strain survival rates were poor in macrophages compared to the wild-type strain. We concluded that H. pylori Omp18 has an important function influencing IFN-γ-mediated immune response to participate in persistent colonization. Yuqun Shan, Xingxiao Lu, Yingnan Han, Xinpeng Li, Xiao Wang, Chunhong Shao, Lixiang Wang, Zhifang Liu, Wei Tang, Yundong Sun, and Jihui Jia Copyright © 2015 Yuqun Shan et al. All rights reserved. Comment on “Helicobacter pylori Outer Membrane Protein 18 (Hp1125) Is Involved in Persistent Colonization by Evading Interferon-γ Signaling” Mon, 06 Apr 2015 08:57:19 +0000 Amin Talebi Bezmin Abadi and Enzo Ierardi Copyright © 2015 Amin Talebi Bezmin Abadi and Enzo Ierardi. All rights reserved. Nordihydroguaiaretic Acid Disrupts the Antioxidant Ability of Helicobacter pylori through the Repression of SodB Activity In Vitro Mon, 06 Apr 2015 08:15:17 +0000 Iron-cofactored superoxide dismutase (SodB) of Helicobacter pylori plays an indispensable role in the bacterium’s colonization of the stomach. Previously, we demonstrated that FecA1, a Fe3+-dicitrate transporter homolog, contributes to SodB activation by supplying ferrous iron (Fe2+) to SodB, and fecA1-deletion mutant strains have reduced gastric mucosal-colonization ability in Mongolian gerbils, suggesting that FecA1 is a possible target for the development of a novel eradication therapy. This study aimed to identify novel FecA1-binding compounds in silico and then examined the effect of a predicted FecA1-binding compound on H. pylori SodB activity in vitro. Specifically, we demonstrated that nordihydroguaiaretic acid (NDGA) is a predicted FecA1-binding compound. NDGA reduced intracellular Fe2+ levels in H. pylori and reduced SodB activity. Additionally, NDGA increased H2O2 sensitivity of H. pylori and increased the metronidazole (Mtz) sensitivity. The present study demonstrated that NDGA repressed SodB activity associated with the gastric mucosal-colonization via inhibition of intracellular Fe2+ uptake by FecA1, suggesting that NDGA might be effective for the development of a novel eradication therapy. Hitoshi Tsugawa, Hideki Mori, Juntaro Matsuzaki, Tatsuhiro Masaoka, Tasuku Hirayama, Hideko Nagasawa, Yasubumi Sakakibara, Makoto Suematsu, and Hidekazu Suzuki Copyright © 2015 Hitoshi Tsugawa et al. All rights reserved. Prevalence of Helicobacter pylori vacA, cagA, and iceA Genotypes in Cuban Patients with Upper Gastrointestinal Diseases Mon, 06 Apr 2015 06:58:22 +0000 Virulence factors of Helicobacter pylori can predict the development of different gastroduodenal diseases. There are scarce reports in Cuba about H. pylori isolates genotyping. The aim of the present investigation was to identify allelic variation of the virulence genes vacA, cagA, and iceA in sixty-eight patients diagnosed as H. pylori positive by culture. In seven out of 68 patients, strains from both gastric regions were obtained and considered independent. DNA was extracted from all the H. pylori strains and evaluated by PCR-genotyping. The vacA s1 allele, cagA gene, and iceA2 allele were the most prevalent (72.0%, 56.0%, and 57.3%, respectively). Alleles from m-region showed a similar frequency as s1a and s1b subtypes. The presence of multiple H. pylori genotypes in a single biopsy and two gastric region specimens were found. Significant statistical association was observed between iceA2 allele and patients with non-peptic ulcer dyspepsia (NUD) () as well as virulence genotypes (s1, s1m2) and patients over 40 years old (). In conclusion, the results demonstrated a high prevalence of H. pylori virulent genotypes in Cuban patients over 40 years old while iceA2 alleles demonstrated a good specificity in patients with NUD. Onelkis Feliciano, Oderay Gutierrez, Lidunka Valdés, Trini Fragoso, Ana Maria Calderin, Antonio Eduardo Valdes, and Rafael Llanes Copyright © 2015 Onelkis Feliciano et al. All rights reserved. Proposal of a Screening MIRU-VNTR Panel for the Preliminary Genotyping of Mycobacterium bovis in Mexico Sun, 05 Apr 2015 11:57:24 +0000 Mycobacterium bovis is the major causative agent of bovine tuberculosis, one of the most relevant zoonoses in the world, and affects a wide range of wild and domesticated animals. Development of screening panels in mycobacterial genotyping, according to specific geographical regions, is strongly needed. The aim of this study is to select a panel, constituted by highly polymorphic MIRU-VNTR loci, to discriminate clinical isolates of M. bovis in Mexico. In this study, 65 isolates of M. bovis obtained from clinical bovine samples proceeding from different geographic regions of Mexico were identified by phenotypic and genotypic tests and subsequently genotyped by a 24-locus MIRU-VNTR panel. The most polymorphic loci were selected to build a panel with a high discriminatory power similar to the 24-locus panel results. A panel of seven elements (QUB 11a, MIRU 26, ETR-A, QUB 26, MIRU 16, MIRU 27, and MIRU 39) with the highest allelic diversity showed an appropriate differentiation. The selected MIRU-VNTR elements, according to the regional allelic variability, may be used in the preliminary genotyping of Mycobacterium bovis isolates in Mexico. Enrique Bolado-Martínez, Iliana Benavides-Dávila, Maria del Carmen Candia-Plata, Moisés Navarro-Navarro, Magali Avilés-Acosta, and Gerardo Álvarez-Hernández Copyright © 2015 Enrique Bolado-Martínez et al. All rights reserved. Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker for Mycobacterium Species Identification Sun, 05 Apr 2015 09:29:58 +0000 Objectives. To evaluate the resolution and reliability of the rpsA gene, encoding ribosomal protein S1, as a novel biomarker for mycobacteria species identification. Methods. A segment of the rpsA gene (565 bp) was amplified by PCR from 42 mycobacterial reference strains, 172 nontuberculosis mycobacteria clinical isolates, and 16 M. tuberculosis complex clinical isolates. The PCR products were sequenced and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Results. Comparative sequence analysis of the rpsA gene provided the basis for species differentiation within the genus Mycobacterium. Slow- and rapid-growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. The sequences discrepancy was obvious between M. kansasii and M. gastri, M. chelonae and M. abscessus, M. avium and M. intracellulare, and M. szulgai and M. malmoense, which cannot be achieved by 16S ribosomal DNA (rDNA) homologue genes comparison. 183 of the 188 (97.3%) clinical isolates, consisting of 8 mycobacterial species, were identified correctly by rpsA gene blast. Conclusions. Our study indicates that rpsA sequencing can be used effectively for mycobacteria species identification as a supplement to 16S rDNA sequence analysis. Hongfei Duan, Guan Liu, Xiaobo Wang, Yuhong Fu, Qian Liang, Yuanyuan Shang, Naihui Chu, and Hairong Huang Copyright © 2015 Hongfei Duan et al. All rights reserved. Comparative Evaluation of Different Test Combinations for Diagnosis of Mycobacterium avium Subspecies paratuberculosis Infecting Dairy Herds in India Sun, 05 Apr 2015 07:26:24 +0000 A total of 355 cows were sampled (serum, ; faeces, ; milk, ) from dairy farms located in the Punjab state of India. Faeces and serum/milk samples were screened by acid fast staining and “indigenous ELISA,” respectively. IS900 PCR was used to screen faeces and milk samples. Bio-load of MAP in dairy cows was 36.9, 15.6, 16.3, and 14.4%, using microscopy, serum ELISA, milk ELISA and milk PCR, respectively. Estimated kappa values between different test combinations: serum and milk ELISA, faecal microscopy and faecal PCR, milk ELISA and milk PCR, faecal PCR and serum ELISA were 0.325, 0.241, 0.682, and 0.677, respectively. Estimation of the relative sensitivity and specificity of different tests in the present study indicated that “serum ELISA” and “milk ELISA” were good screening tests, add “milk PCR” was “confirmatory test” for MAP infection. Combination of milk ELISA with milk PCR may be adopted as a model strategy for screening and diagnosis of JD in lactating/dairy cattle herds in Indian conditions. Rajni Garg, Prasanna Kumar Patil, Shoor Vir Singh, Shukriti Sharma, Ravi Kumar Gandham, Ajay Vir Singh, Gurusimiran Filia, Pravin Kumar Singh, Sujata Jayaraman, Saurabh Gupta, Kundan Kumar Chaubey, Ruchi Tiwari, Mani Saminathan, Kuldeep Dhama, and Jagdip Singh Sohal Copyright © 2015 Rajni Garg et al. All rights reserved. Microbiological Quality of Ready-to-Eat Vegetables Collected in Mexico City: Occurrence of Aerobic-Mesophilic Bacteria, Fecal Coliforms, and Potentially Pathogenic Nontuberculous Mycobacteria Mon, 30 Mar 2015 12:44:05 +0000 The aims of this study were to evaluate the microbiological quality and the occurrence of nontuberculous mycobacteria (NTM) in a variety of salads and sprouts from supermarkets and street vendors in Mexico City. Aerobic-mesophilic bacteria (AMB) were present in 100% of RTE-salads samples; 59% of samples were outside guidelines range (>5.17 log10 CFU per g). Although fecal coliforms (FC) were present in 32% of samples, only 8% of them exceeded the permissible limit (100 MPN/g). Regarding the 100 RTE-sprouts, all samples were also positive for AMB and total coliforms (TC) and 69% for FC. Seven NTM species were recovered from 7 salad samples; they included three M. fortuitum, two M. chelonae, one M. mucogenicum, and one M. sp. Twelve RTE-sprouts samples harbored NTM, which were identified as M. porcinum (five), M. abscessus (two), M. gordonae (two), M. mucogenicum (two), and M. avium complex (one). Most RTE-salads and RTE-sprouts had unsatisfactory microbiological quality and some harbored NTM associated with illness. No correlation between the presence of coliforms and NTM was found. Overall, these results suggest that RTE-salads and RTE-sprouts might function as vehicles for NTM transmission in humans; hence, proper handling and treatment before consumption of such products might be recommendable. Jorge Francisco Cerna-Cortes, Nancy Leon-Montes, Ana Laura Cortes-Cueto, Laura P. Salas-Rangel, Addy Cecilia Helguera-Repetto, Daniel Lopez-Hernandez, Sandra Rivera-Gutierrez, Elizabeth Fernandez-Rendon, and Jorge Alberto Gonzalez-y-Merchand Copyright © 2015 Jorge Francisco Cerna-Cortes et al. All rights reserved. Antibiotic-Resistant Vibrios in Farmed Shrimp Mon, 30 Mar 2015 11:22:44 +0000 Antimicrobial susceptibility pattern was determined in 100 strains of Vibrio isolated from the Litopenaeus vannamei shrimp and identified phenotypically. A high antibiotic-resistance index (75%) was observed, with the following phenotypic profiles: monoresistance (), cross-resistance to β-lactams () and multiple resistance (). Plasmid resistance was characterized for penicillin (), penicillin + ampicillin (), penicillin + aztreonam (), and ampicillin (). Resistance to antimicrobial drugs by the other strains () was possibly mediated by chromosomal genes. The findings of this study support the conclusion that the cultured shrimps can be vehicles of vibrios resistant to β-lactam and tetracycline. Renata Albuquerque Costa, Rayza Lima Araújo, Oscarina Viana Souza, and Regine Helena Silva dos Fernandes Vieira Copyright © 2015 Renata Albuquerque Costa et al. All rights reserved. The Water Cycle, a Potential Source of the Bacterial Pathogen Bacillus cereus Mon, 30 Mar 2015 09:19:27 +0000 The behaviour of the sporulating soil-dwelling Bacillus cereus sensu lato (B. cereus sl) which includes foodborne pathogenic strains has been extensively studied in relation to its various animal hosts. The aim of this environmental study was to investigate the water compartments (rain and soil water, as well as groundwater) closely linked to the primary B. cereus sl reservoir, for which available data are limited. B. cereus sl was present, primarily as spores, in all of the tested compartments of an agricultural site, including water from rain to groundwater through soil. During rain events, leachates collected after transfer through the soil eventually reached the groundwater and were loaded with B. cereus sl. In groundwater samples, newly introduced spores of a B. cereus model strain were able to germinate, and vegetative cells arising from this event were detected for up to 50 days. This first B. cereus sl investigation in the various types of interrelated environments suggests that the consideration of the aquatic compartment linked to soil and to climatic events should provide a better understanding of B. cereus sl ecology and thus be relevant for a more accurate risk assessment of food poisoning caused by B. cereus sl pathogenic strains. Julien Brillard, Christian M. S. Dupont, Odile Berge, Claire Dargaignaratz, Stéphanie Oriol-Gagnier, Claude Doussan, Véronique Broussolle, Marina Gillon, Thierry Clavel, and Annette Bérard Copyright © 2015 Julien Brillard et al. All rights reserved. Listeriosis Outbreaks in British Columbia, Canada, Caused by Soft Ripened Cheese Contaminated from Environmental Sources Mon, 30 Mar 2015 09:17:07 +0000 Soft ripened cheese (SRC) caused over 130 foodborne illnesses in British Columbia (BC), Canada, during two separate listeriosis outbreaks. Multiple agencies investigated the events that lead to cheese contamination with Listeria monocytogenes (L.m.), an environmentally ubiquitous foodborne pathogen. In both outbreaks pasteurized milk and the pasteurization process were ruled out as sources of contamination. In outbreak A, environmental transmission of L.m. likely occurred from farm animals to personnel to culture solutions used during cheese production. In outbreak B, birds were identified as likely contaminating the dairy plant’s water supply and cheese during the curd-washing step. Issues noted during outbreak A included the risks of operating a dairy plant in a farm environment, potential for transfer of L.m. from the farm environment to the plant via shared toilet facilities, failure to clean and sanitize culture spray bottles, and cross-contamination during cheese aging. L.m. contamination in outbreak B was traced to wild swallows defecating in the plant’s open cistern water reservoir and a multibarrier failure in the water disinfection system. These outbreaks led to enhanced inspection and surveillance of cheese plants, test and release programs for all SRC manufactured in BC, improvements in plant design and prevention programs, and reduced listeriosis incidence. Lorraine McIntyre, Lynn Wilcott, and Monika Naus Copyright © 2015 Lorraine McIntyre et al. All rights reserved. Resistance Determinants and Their Association with Different Transposons in the Antibiotic-Resistant Streptococcus pneumoniae Thu, 26 Mar 2015 11:34:44 +0000 Multiple resistance of Streptococcus pneumoniae is generally associated with their unique recombination-mediated genetic plasticity and possessing the mobile genetic elements. The aim of our study was to detect antibiotic resistance determinants and conjugative transposons in 138 antibiotic-resistant pneumococcal strains isolated from nasopharynx of healthy young children from Lublin, Poland. These strains resistant to tetracycline and/or to chloramphenicol/erythromycin/clindamycin were tested by PCR using the specific genes as markers. The presence of Tn916 family transposons, carrying tet(M) and int/xisTn916, was observed in all of the tested strains. Tn916 was detected in 16 strains resistant only to tetracycline. Tn6002 and Tn3872-related element were found among 99 erm(B)-carrying strains (83.8% and 3.0%, resp.). Eight strains harbouring mef(E) and erm(B) genes were detected, suggesting the presence of Tn2010 and Tn2017 transposons. Among 101 chloramphenicol-resistant strains, two variants of Tn5252-related transposon were distinguished depending on the presence of int/xis5252 genes specific for cat gene-containing Tn5252 (75.2% of strains) or gene, specific for cat-containing ICESp23FST81 element (24.8% of strains). In 6 strains Tn916-like and Tn5252-like elements formed a Tn5253-like structure. Besides clonal dissemination of resistant strains of pneumococci in the population, horizontal transfer of conjugative transposons is an important factor of the high prevalence of antibiotic resistance. Izabela Korona-Glowniak, Radoslaw Siwiec, and Anna Malm Copyright © 2015 Izabela Korona-Glowniak et al. All rights reserved. BIODESERT: Exploring and Exploiting the Microbial Resource of Hot and Cold Deserts Thu, 26 Mar 2015 07:25:05 +0000 Ameur Cherif, George Tsiamis, Stéphane Compant, and Sara Borin Copyright © 2015 Ameur Cherif et al. All rights reserved. Vector-Borne Viral Diseases Tue, 24 Mar 2015 12:38:27 +0000 Penghua Wang, Fengwei Bai, Gong Cheng, Jianfeng Dai, and Michael J. Conway Copyright © 2015 Penghua Wang et al. All rights reserved. Evaluation of the Efficacy of a Bacteriophage in the Treatment of Pneumonia Induced by Multidrug Resistance Klebsiella pneumoniae in Mice Tue, 24 Mar 2015 06:18:12 +0000 Multidrug-resistant Klebsiella pneumoniae (MRKP) has steadily grown beyond antibiotic control. However, a bacteriophage is considered to be a potential antibiotic alternative for treating bacterial infections. In this study, a lytic bacteriophage, phage 1513, was isolated using a clinical MRKP isolate KP 1513 as the host and was characterized. It produced a clear plaque with a halo and was classified as Siphoviridae. It had a short latent period of 30 min, a burst size of 264 and could inhibit KP 1513 growth in vitro with a dose-dependent pattern. Intranasal administration of a single dose of 2 × 109 PFU/mouse 2 h after KP 1513 inoculation was able to protect mice against lethal pneumonia. In a sublethal pneumonia model, phage-treated mice exhibited a lower level of K. pneumoniae burden in the lungs as compared to the untreated control. These mice lost less body weight and exhibited lower levels of inflammatory cytokines in their lungs. Lung lesion conditions were obviously improved by phage therapy. Therefore, phage 1513 has a great effect in vitro and in vivo, which has potential to be used as an alternative to an antibiotic treatment of pneumonia that is caused by the multidrug-resistant K. pneumoniae. Fang Cao, Xitao Wang, Linhui Wang, Zhen Li, Jian Che, Lili Wang, Xiaoyu Li, Zhenhui Cao, Jiancheng Zhang, Liji Jin, and Yongping Xu Copyright © 2015 Fang Cao et al. All rights reserved. Vector Borne Infections in Italy: Results of the Integrated Surveillance System for West Nile Disease in 2013 Sun, 22 Mar 2015 09:20:25 +0000 The epidemiology of West Nile disease (WND) is influenced by multiple ecological factors and, therefore, integrated surveillance systems are needed for early detecting the infection and activating consequent control actions. As different animal species have different importance in the maintenance and in the spread of the infection, a multispecies surveillance approach is required. An integrated and comprehensive surveillance system is in place in Italy aiming at early detecting the virus introduction, monitoring the possible infection spread, and implementing preventive measures for human health. This paper describes the integrated surveillance system for WND in Italy, which incorporates data from veterinary and human side in order to evaluate the burden of infection in animals and humans and provide the public health authorities at regional and national levels with the information needed for a fine tune response. Christian Napoli, Simona Iannetti, Caterina Rizzo, Antonino Bella, Daria Di Sabatino, Rossana Bruno, Francesca Sauro, Vanessa Martini, Vincenzo Ugo Santucci, Silvia Declich, and Paolo Calistri Copyright © 2015 Christian Napoli et al. All rights reserved. Sialic Acid Expression in the Mosquito Aedes aegypti and Its Possible Role in Dengue Virus-Vector Interactions Sun, 22 Mar 2015 09:03:44 +0000 Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission. Jorge Cime-Castillo, Philippe Delannoy, Guillermo Mendoza-Hernández, Verónica Monroy-Martínez, Anne Harduin-Lepers, Humberto Lanz-Mendoza, Fidel de la Cruz Hernández-Hernández, Edgar Zenteno, Carlos Cabello-Gutiérrez, and Blanca H. Ruiz-Ordaz Copyright © 2015 Jorge Cime-Castillo et al. All rights reserved. Occurrence of West Nile Virus Antibodies in Wild Birds, Horses, and Humans in Poland Thu, 19 Mar 2015 14:35:51 +0000 Serum samples of 474 wild birds, 378 horses, and 42 humans with meningitis and lymphocytic meningitis were collected between 2010 and 2014 from different areas of Poland. West Nile virus (WNV) antibodies were detected using competition enzyme linked immunosorbent assays: ELISA-1 ID Screen West Nile Competition, IDvet, ELISA-2 ID Screen West Nile IgM Capture, and ELISA-3 Ingezim West Nile Compac. The antibodies were found in 63 (13.29%) out of 474 wild bird serum samples and in one (0.26%) out of 378 horse serum samples. Fourteen (33.33%) out of 42 sera from patients were positive against WNV antigen and one serum was doubtful. Positive samples obtained in birds were next retested with virus microneutralisation test to confirm positive results and cross-reactions with other antigens of the Japanese encephalitis complex. We suspect that positive serological results in humans, birds, and horses indicate that WNV can be somehow closely related with the ecosystem in Poland. Jowita Samanta Niczyporuk, Elżbieta Samorek-Salamonowicz, Sylvie Lecollinet, Sławomir Andrzej Pancewicz, Wojciech Kozdruń, and Hanna Czekaj Copyright © 2015 Jowita Samanta Niczyporuk et al. All rights reserved. The Date Palm Tree Rhizosphere Is a Niche for Plant Growth Promoting Bacteria in the Oasis Ecosystem Thu, 19 Mar 2015 14:32:03 +0000 In arid ecosystems environmental factors such as geoclimatic conditions and agricultural practices are of major importance in shaping the diversity and functionality of plant-associated bacterial communities. Assessing the influence of such factors is a key to understand (i) the driving forces determining the shape of root-associated bacterial communities and (ii) the plant growth promoting (PGP) services they provide. Desert oasis environment was chosen as model ecosystem where agriculture is possible by the microclimate determined by the date palm cultivation. The bacterial communities in the soil fractions associated with the root system of date palms cultivated in seven oases in Tunisia were assessed by culture-independent and dependent approaches. According to 16S rRNA gene PCR-DGGE fingerprinting, the shapes of the date palm rhizosphere bacterial communities correlate with geoclimatic features along a north-south aridity transect. Despite the fact that the date palm root bacterial community structure was strongly influenced by macroecological factors, the potential rhizosphere services reflected in the PGP traits of isolates screened in vitro were conserved among the different oases. Such services were exerted by the 83% of the screened isolates. The comparable numbers and types of PGP traits indicate their importance in maintaining the plant functional homeostasis despite the different environmental selection pressures. Raoudha Ferjani, Ramona Marasco, Eleonora Rolli, Hanene Cherif, Ameur Cherif, Maher Gtari, Abdellatif Boudabous, Daniele Daffonchio, and Hadda-Imene Ouzari Copyright © 2015 Raoudha Ferjani et al. All rights reserved. Genetic and Technological Characterisation of Vineyard- and Winery-Associated Lactic Acid Bacteria Thu, 19 Mar 2015 13:16:01 +0000 Vineyard- and winery-associated lactic acid bacteria (LAB) from two major PDO regions in Greece, Peza and Nemea, were surveyed. LAB were isolated from grapes, fermenting musts, and winery tanks performing spontaneous malolactic fermentations (MLF). Higher population density and species richness were detected in Nemea than in Peza vineyards and on grapes than in fermenting musts. Pediococcus pentosaceus and Lactobacillus graminis were the most abundant LAB on grapes, while Lactobacillus plantarum dominated in fermenting musts from both regions. No particular structure of Lactobacillus plantarum populations according to the region of origin was observed, and strain distribution seems random. LAB species diversity in winery tanks differed significantly from that in vineyard samples, consisting principally of Oenococcus oeni. Different strains were analysed as per their enological characteristics and the ability to produce biogenic amines (BAs). Winery-associated species showed higher resistance to low pH, ethanol, SO2, and CuSO4 than vineyard-associated isolates. The frequency of BA-producing strains was relatively low but not negligible, considering that certain winery-associated Lactobacillus hilgardii strains were able to produce BAs. Present results show the necessity of controlling the MLF by selected starters in order to avoid BA accumulation in wine. Aspasia A. Nisiotou, Dimitra Dourou, Maria-Evangelia Filippousi, Ellie Diamantea, Petros Fragkoulis, Chryssoula Tassou, and Georgios Banilas Copyright © 2015 Aspasia A. Nisiotou et al. All rights reserved. The Formation of Biofilms by Pseudomonas aeruginosa: A Review of the Natural and Synthetic Compounds Interfering with Control Mechanisms Thu, 19 Mar 2015 12:30:24 +0000 P. aeruginosa is an opportunistic pathogenic bacterium responsible for both acute and chronic infections. Beyond its natural resistance to many drugs, its ability to form biofilm, a complex biological system, renders ineffective the clearance by immune defense systems and antibiotherapy. The objective of this report is to provide an overview (i) on P. aeruginosa biofilm lifestyle cycle, (ii) on the main key actors relevant in the regulation of biofilm formation by P. aeruginosa including QS systems, GacS/GacA and RetS/LadS two-component systems and C-di-GMP-dependent polysaccharides biosynthesis, and (iii) finally on reported natural and synthetic products that interfere with control mechanisms of biofilm formation by P. aeruginosa without affecting directly bacterial viability. Concluding remarks focus on perspectives to consider biofilm lifestyle as a target for eradication of resistant infections caused by P. aeruginosa. Tsiry Rasamiravaka, Quentin Labtani, Pierre Duez, and Mondher El Jaziri Copyright © 2015 Tsiry Rasamiravaka et al. All rights reserved. Identification of the Alternative Splicing of the UL49 Locus of Human Cytomegalovirus Thu, 19 Mar 2015 10:14:57 +0000 The UL49 ORF of human cytomegalovirus (HCMV) is essential for viral replication; conserved among all herpes viruses; however, the function is unclear. Once the UL49 ORF was precisely deleted from the start to stop codon, the mutant did not yield infectious progeny. In this study, we find out many alternatively processed ESTs in UL49 locus in HCMV-infected cells, in which there are two novel transcription termination sites in UL49 locus. Most of these ESTs are rare transcripts that contain directed repeat sequences in the intron splicing regions. There is a typical GU-AG intron splicing site in UL49Y transcripts. The 1847 bp UL49Y cDNA spans an ORF from 335 to 1618 and encodes a putative protein of 427 amino acids with a predicted molecular mass of 47.1 kDa. All the new EST sequences and UL49Y cDNA sequence have been deposited in the GenBank database (GenBank Accession nos. GW314860-GW314900 and GU376796). This study provides us with very important clues for revealing the importance of the UL49 locus alternative splicing. Guang Yang, Wei Li, Wenzhen Liao, Xin Zhang, Yi Zou, Jianfeng Dai, Yueqin Li, Chunxia Jing, and Tianhong Zhou Copyright © 2015 Guang Yang et al. All rights reserved. The Global Ecology and Epidemiology of West Nile Virus Thu, 19 Mar 2015 08:58:25 +0000 Since its initial isolation in Uganda in 1937 through the present, West Nile virus (WNV) has become an important cause of human and animal disease worldwide. WNV, an enveloped virus of the genus Flavivirus, is naturally maintained in an enzootic cycle between birds and mosquitoes, with occasional epizootic spillover causing disease in humans and horses. The mosquito vectors for WNV are widely distributed worldwide, and the known geographic range of WNV transmission and disease has continued to increase over the past 77 years. While most human infections with WNV are asymptomatic, severe neurological disease may develop resulting in long-term sequelae or death. Surveillance and preventive measures are an ongoing need to reduce the public health impact of WNV in areas with the potential for transmission. Caren Chancey, Andriyan Grinev, Evgeniya Volkova, and Maria Rios Copyright © 2015 Caren Chancey et al. All rights reserved. Role of Microorganisms Present in Dairy Fermented Products in Health and Disease Thu, 19 Mar 2015 07:11:00 +0000 Clara G. de los Reyes-Gavilán, María Fernández, John Andrew Hudson, and Riitta Korpela Copyright © 2015 Clara G. de los Reyes-Gavilán et al. All rights reserved. Evaluation of the Inflammatory Response in Macrophages Stimulated with Exosomes Secreted by Mycobacterium avium-Infected Macrophages Mon, 16 Mar 2015 12:49:45 +0000 Exosomes secreted from Mycobacterium avium-infected macrophages contain numerous antigens of both M. avium and the host cell and are involved in the induction and expression of the inflammatory responses in macrophages. The interaction between exosomes secreted from M. avium-infected macrophages and macrophage phagocytosis, cytokine secretion, immunostimulation, and apoptosis was analyzed. Upon stimulation with exosomes secreted from M. avium-infected macrophages, the phagocytosis of dextran by treated macrophages was increased. Furthermore, the expression of CD40, CD80, CD81, CD86, HLA-DR, and most notably CD195 was enhanced. Additionally, the secretion of IL-6, IL-8, IL-10, IFN-γ, and TNF-α was increased by stimulated macrophages. Exosome stimulation did not induce macrophage apoptosis when compared with macrophages infected with M. avium. Caspase expression, including that of caspases 3, 6, and 8, was also not altered in exosome stimulated macrophages. Thus exosomes trigger the inflammatory response in macrophages owing to the presence of bacterial antigens but have no effect on macrophage viability. Jianjun Wang, Yongliang Yao, Jing Xiong, Jianhong Wu, Xin Tang, and Guangxin Li Copyright © 2015 Jianjun Wang et al. All rights reserved. Antimicrobial Activity of Lactic Acid Bacteria in Dairy Products and Gut: Effect on Pathogens Mon, 16 Mar 2015 12:09:17 +0000 The food industry seeks alternatives to satisfy consumer demands of safe foods with a long shelf-life able to maintain the nutritional and organoleptic quality. The application of antimicrobial compounds-producing protective cultures may provide an additional parameter of processing in order to improve the safety and ensure food quality, keeping or enhancing its sensorial characteristics. In addition, strong evidences suggest that certain probiotic strains can confer resistance against infection with enteric pathogens. Several mechanisms have been proposed to support this phenomenon, including antimicrobial compounds secreted by the probiotics, competitive exclusion, or stimulation of the immune system. Recent research has increasingly demonstrated the role of antimicrobial compounds as protective mechanism against intestinal pathogens and therefore certain strains could have an effect on both the food and the gut. In this aspect, the effects of the combination of different strains keep unknown. The development of multistrain probiotic dairy products with good technological properties and with improved characteristics to those shown by the individual strains, able to act not only as protective cultures in foods, but also as probiotics able to exert a protective action against infections, has gained increased interest. Juan L. Arqués, Eva Rodríguez, Susana Langa, José María Landete, and Margarita Medina Copyright © 2015 Juan L. Arqués et al. All rights reserved. Biotechnological and Agronomic Potential of Endophytic Pink-Pigmented Methylotrophic Methylobacterium spp. Tue, 10 Mar 2015 11:08:38 +0000 The genus Methylobacterium is composed of pink-pigmented facultative methylotrophic (PPFM) bacteria, which are able to synthesize carotenoids and grow on reduced organic compounds containing one carbon (C1), such as methanol and methylamine. Due to their high phenotypic plasticity, these bacteria are able to colonize different habitats, such as soil, water, and sediment, and different host plants as both endophytes and epiphytes. In plant colonization, the frequency and distribution may be influenced by plant genotype or by interactions with other associated microorganisms, which may result in increasing plant fitness. In this review, different aspects of interactions with the host plant are discussed, including their capacity to fix nitrogen, nodule the host plant, produce cytokinins, auxin and enzymes involved in the induction of systemic resistance, such as pectinase and cellulase, and therefore plant growth promotion. In addition, bacteria belonging to this group can be used to reduce environmental contamination because they are able to degrade toxic compounds, tolerate high heavy metal concentrations, and increase plant tolerance to these compounds. Moreover, genome sequencing and omics approaches have revealed genes related to plant-bacteria interactions that may be important for developing strains able to promote plant growth and protection against phytopathogens. Manuella Nóbrega Dourado, Aline Aparecida Camargo Neves, Daiene Souza Santos, and Welington Luiz Araújo Copyright © 2015 Manuella Nóbrega Dourado et al. All rights reserved. The High PMNs Phagocytosis Resistance of Enterococcal Isolates from RTx Patients Tue, 10 Mar 2015 09:56:24 +0000 Infections caused by opportunistic pathogens such as enterococci remain difficult to manage, especially in immunocompromised patients. Because of infections’ limited symptoms in such patients the additional problems are to find proper diagnostic criteria and the management of infection. Here we aimed to compare the resistance of commensal enterococcal strains and RTx patients’ isolates, to PMNs phagocytosis. Thirty-six enterococcal urine and faecal isolates from RTx patients and 17 faecal isolates from healthy volunteers were cultured in planktonic and biofilm forms in 37°C or 42°C. Another tested variable was the addition of immunosuppressant to the culture media. Bacterial cells were stained with fluorescent reporter (CFDA, PI) and incubated with PMNs. Results of phagocytosis were estimated as a mean fluorescence intensity (MFI) of PMNs using flow cytometry. Commensal enterococci cultured in all abovementioned (37°C and 42°C/the addition of immunosuppressant) conditions were less resistant to phagocytosis compared to RTx isolates. Observed significant difference in phagocytosis resistance suggests that patients in immunosuppression are colonized with high risk strains which may lead to the development of infection. Tomasz Jarzembowski, Agnieszka Daca, Jacek M. Witkowski, Ewa Bryl, and Bolesław Rutkowski Copyright © 2015 Tomasz Jarzembowski et al. All rights reserved. Delivery of Antibiotics from Cementless Titanium-Alloy Cubes May Be a Novel Way to Control Postoperative Infections Tue, 10 Mar 2015 06:17:12 +0000 Bacterial colonisation and biofilm formation onto orthopaedic devices are difficult to eradicate. In most cases infection is treated by surgical removal of the implant and cleaning of the infected area, followed by extensive treatment with broad-spectrum antibiotics. Such treatment causes great discomfort, is expensive, and is not always successful. In this study we report on the release of vancomycin through polyethersulfone membranes from channels in cementless titanium-alloy cubes. The cubes were constructed with LaserCUSING from Ti6Al4V ELI powder. Vancomycin was released by non-Fickian anomalous (constraint) diffusion. Approximately 50% of the vancomycin was released within the first 17 h. However, sustained delivery of vancomycin for 100 h was possible by reinjecting the channels. Refillable implants may be a novel way to control postoperative infections. Martin B. Bezuidenhout, Anton D. van Staden, Gert A. Oosthuizen, Dimitar M. Dimitrov, and Leon M. T. Dicks Copyright © 2015 Martin B. Bezuidenhout et al. All rights reserved. Impact on Human Health of Microorganisms Present in Fermented Dairy Products: An Overview Mon, 09 Mar 2015 09:56:51 +0000 Fermented dairy products provide nutrients in our diet, some of which are produced by the action of microorganisms during fermentation. These products can be populated by a diverse microbiota that impacts the organoleptic and physicochemical characteristics foods as well as human health. Acidification is carried out by starter lactic acid bacteria (LAB) whereas other LAB, moulds, and yeasts become dominant during ripening and contribute to the development of aroma and texture in dairy products. Probiotics are generally part of the nonstarter microbiota, and their use has been extended in recent years. Fermented dairy products can contain beneficial compounds, which are produced by the metabolic activity of their microbiota (vitamins, conjugated linoleic acid, bioactive peptides, and gamma-aminobutyric acid, among others). Some microorganisms can also release toxic compounds, the most notorious being biogenic amines and aflatoxins. Though generally considered safe, fermented dairy products can be contaminated by pathogens. If proliferation occurs during manufacture or storage, they can cause sporadic cases or outbreaks of disease. This paper provides an overview on the current state of different aspects of the research on microorganisms present in dairy products in the light of their positive or negative impact on human health. María Fernández, John Andrew Hudson, Riitta Korpela, and Clara G. de los Reyes-Gavilán Copyright © 2015 María Fernández et al. All rights reserved. Antimicrobial Peptides: Current and Potential Applications in Biomedical Therapies Thu, 05 Mar 2015 13:59:46 +0000 Joel E. López-Meza, Alejandra Ochoa-Zarzosa, José E. Barboza-Corona, and Dennis K. Bideshi Copyright © 2015 Joel E. López-Meza et al. All rights reserved. Incidence and Diversity of Antimicrobial Multidrug Resistance Profiles of Uropathogenic Bacteria Thu, 05 Mar 2015 13:56:21 +0000 The aim of this study was to assess the most frequent multidrug resistant (MDR) profiles of the main bacteria implicated in community-acquired urinary tract infections (UTI). Only the MDR profiles observed in, at least, 5% of the MDR isolates were considered. A quarter of the bacteria were MDR and the most common MDR profile, including resistance to penicillins, quinolones, and sulfonamides (antibiotics with different mechanisms of action, all mainly recommended by the European Association of Urology for empirical therapy of uncomplicated UTI), was observed, alone or in association with resistance to other antimicrobial classes, in the main bacteria implicated in UTI. The penicillin class was included in all the frequent MDR profiles observed in the ten main bacteria and was the antibiotic with the highest prescription during the study period. The sulfonamides class, included in five of the six more frequent MDR profiles, was avoided between 2000 and 2009. The results suggest that the high MDR percentage and the high diversity of MDR profiles result from a high prescription of antibiotics but also from antibiotic-resistant genes transmitted with other resistance determinants on mobile genetic elements and that the UTI standard treatment guidelines must be adjusted for the community of Aveiro District. Inês Linhares, Teresa Raposo, António Rodrigues, and Adelaide Almeida Copyright © 2015 Inês Linhares et al. All rights reserved. The Relationship of CSF and Plasma Cytokine Levels in HIV Infected Patients with Neurocognitive Impairment Tue, 03 Mar 2015 09:19:15 +0000 Although HAD is now rare due to HAART, the milder forms of HAND persist in HIV-infected patients. HIV-induced systemic and localized inflammation is considered to be one of the mechanisms of HAND. The levels of cytokines in CSF were associated with neurocognitive impairment in HIV infection. However, the changes of cytokines involved in cognition impairment in plasma have not been shown, and their relationships between CSF and plasma require to be addressed. We compared cytokine levels in paired CSF and plasma samples from HIV-infected individuals with or without neurocognitive impairment. Cytokine concentrations were measured by Luminex xMAP. In comparing the expression levels of cytokines in plasma and CSF, IFN-α2, IL-8, IP-10, and MCP-1 were significantly higher in CSF. Eotaxin was significantly higher in plasma, whereas G-CSF showed no difference between plasma and CSF. G-CSF , IL-8 , IP-10 , and MCP-1 in CSF showed significant difference between HIV-CI and HIV-NC group, which may indicate their relationship to HIV associated neurocognitive impairment. In addition, G-CSF and IP-10 in plasma were significantly higher in HIV-CI than HIV-NC. The consistent changes of G-CSF and IP-10 in paired plasma and CSF samples might enhance their potential for predicting HAND. Lin Yuan, An Liu, Luxin Qiao, Bo Sheng, Meng Xu, Wei Li, and Dexi Chen Copyright © 2015 Lin Yuan et al. All rights reserved. cspA Influences Biofilm Formation and Drug Resistance in Pathogenic Fungus Aspergillus fumigatus Tue, 03 Mar 2015 06:09:26 +0000 The microbial cell wall plays a crucial role in biofilm formation and drug resistance. cspA encodes a repeat-rich glycophosphatidylinositol-anchored cell wall protein in the pathogenic fungus Aspergillus fumigatus. To determine whether cspA has a significant impact on biofilm development and sensitivity to antifungal drugs in A. fumigatus, a ΔcspA mutant was constructed by targeted gene disruption, and we then reconstituted the mutant to wild type by homologous recombination of a functional cspA gene. Deletion of cspA resulted in a rougher conidial surface, reduced biofilm formation, decreased resistance to antifungal agents, and increased internalization by A549 human lung epithelial cells, suggesting that cspA not only participates in maintaining the integrity of the cell wall, but also affects biofilm establishment, drug response, and invasiveness of A. fumigatus. Zhongqi Fan, Zhe Li, Zongge Xu, Hongyan Li, Lixiang Li, Cong Ning, Lin Ma, Xiangli Xie, Guangyi Wang, and Huimei Yu Copyright © 2015 Zhongqi Fan et al. All rights reserved. Proteomic Analysis of Human Brain Microvascular Endothelial Cells Reveals Differential Protein Expression in Response to Enterovirus 71 Infection Mon, 02 Mar 2015 12:49:57 +0000 2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC), to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications. Wenying Luo, Jiayu Zhong, Wei Zhao, Jianjun Liu, Renli Zhang, Liang Peng, Wenxu Hong, Sheng He Huang, and Hong Cao Copyright © 2015 Wenying Luo et al. All rights reserved. Cytotoxicity of Cyclodipeptides from Pseudomonas aeruginosa PAO1 Leads to Apoptosis in Human Cancer Cell Lines Mon, 02 Mar 2015 12:26:15 +0000 Pseudomonas aeruginosa is an opportunistic pathogen of plants and animals, which produces virulence factors in order to infect or colonize its eukaryotic hosts. Cyclodipeptides (CDPs) produced by P. aeruginosa exhibit cytotoxic properties toward human tumor cells. In this study, we evaluated the effect of a CDP mix, comprised of cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) that were isolated from P. aeruginosa, on two human cancer cell lines. Our results demonstrated that the CDP mix promoted cell death in cultures of the HeLa cervical adenocarcinoma and Caco-2 colorectal adenocarcinoma cell lines in a dose-dependent manner, with a 50% inhibitory concentration (IC50) of 0.53 and 0.66 mg/mL, for HeLa and Caco-2 cells, respectively. Flow cytometric analysis, using annexin V and propidium iodide as apoptosis and necrosis indicators, respectively, clearly showed that HeLa and Caco-2 cells exhibited apoptotic characteristics when treated with the CDP mix at a concentration <0.001 mg/mL. IC50 values for apoptotic cells in HeLa and Caco-2 cells were 6.5 × 10−5 and 1.8 × 10−4 mg/mL, respectively. Our results indicate that an apoptotic pathway is involved in the inhibition of cell proliferation caused by the P. aeruginosa CDP mix. Dolores Vázquez-Rivera, Omar González, Jaquelina Guzmán-Rodríguez, Alma L. Díaz-Pérez, Alejandra Ochoa-Zarzosa, José López-Bucio, Víctor Meza-Carmen, and Jesús Campos-García Copyright © 2015 Dolores Vázquez-Rivera et al. All rights reserved. Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris Mon, 02 Mar 2015 07:12:26 +0000 We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC). However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins. Sara Arbulu, Juan J. Jiménez, Loreto Gútiez, Luis M. Cintas, Carmen Herranz, and Pablo E. Hernández Copyright © 2015 Sara Arbulu et al. All rights reserved. Dengue Patients Exhibit Higher Levels of PrM and E Antibodies Than Their Asymptomatic Counterparts Sun, 01 Mar 2015 14:03:38 +0000 Dengue virus infection is a common tropical disease which often occurs without being detected. These asymptomatic cases provide information in relation to the manifestation of immunological aspects. In this study, we developed an ELISA method to compare neutralizing effects of dengue prM and E antibodies between dengue patients and their asymptomatic household members. Recombinant D2 premembrane (prM) was constructed, cloned, and tested for antigenicity. The recombinant protein was purified and tested with controls by using an indirect ELISA method. Positive dengue serum samples with their asymptomatic pair were then carried out onto the developed ELISA. In addition, commercially available recombinant envelope (E) protein was used to develop an ELISA which was tested with the same set of serum samples in the prM ELISA. Asymptomatic individuals showed preexisting heterotypic neutralizing antibodies. The recombinant prM was antigenically reactive in the developed ELISA. Dengue patients had higher prM and E antibodies compared to their household members. Our study highlights the neutralizing antibodies levels with respect to dengue prM and E between dengue patients and asymptomatic individuals. Adeline Syin Lian Yeo, Anusyah Rathakrishnan, Seok Mui Wang, Sasheela Ponnampalavanar, Rishya Manikam, Jameela Sathar, Santha Kumari Natkunam, and Shamala Devi Sekaran Copyright © 2015 Adeline Syin Lian Yeo et al. All rights reserved. Current and Potential Applications of Host-Defense Peptides and Proteins in Urology Sun, 01 Mar 2015 13:05:52 +0000 The use of antibiotics has become increasingly disfavored as more multidrug resistant pathogens are on the rise. A promising alternative to the use of these conventional drugs includes antimicrobial peptides or host-defense peptides. These peptides typically consist of short amino acid chains with a net cationic charge and a substantial portion of hydrophobic residues. They mainly target the bacterial cell membrane but are also capable of translocating through the membrane and target intracellular components, making it difficult for bacteria to gain resistance as multiple essential cellular processes are being targeted. The use of these peptides in the field of biomedical therapies has been examined, and the different approaches to using them under various settings are constantly being discovered. In this review, we discuss the current and potential applications of these host-defense peptides in the field of urology. Besides the use of these peptides as antimicrobial agents, the value of these biological molecules has recently been expanded to their use as antitumor and anti-kidney-stone agents. Joey Chor Yee Lo and Dirk Lange Copyright © 2015 Joey Chor Yee Lo and Dirk Lange. All rights reserved. Molecular Detection and Sensitivity to Antibiotics and Bacteriocins of Pathogens Isolated from Bovine Mastitis in Family Dairy Herds of Central Mexico Sun, 01 Mar 2015 12:39:47 +0000 Thirty-two farms ( cows) located in the state of Guanajuato, Mexico, were sampled. Pathogens from bovine subclinical mastitis (SCM) and clinical mastitis (CLM) were identified by 16S rDNA and the sensitivity to both antibiotics and bacteriocins of Bacillus thuringiensis was tested. Forty-six milk samples were selected for their positive California Mastitis Test (CMT) (≥3) and any abnormality in the udder or milk. The frequency of SCM and CLM was 39.1% and 9.3%, respectively. Averages for test day milk yield (MY), lactation number (LN), herd size (HS), and number of days in milk (DM) were 20.6 kg, 2.8 lactations, 16.7 animals, and 164.1 days, respectively. MY was dependent on dairy herd (DH), LN, HS, and DM , and correlations between udder quarters from the CMT were around 0.49 . Coagulase-negative staphylococci were mainly identified, as well as Staphylococcus aureus, Streptococcus uberis, Brevibacterium stationis, B. conglomeratum, and Staphylococcus agnetis. Bacterial isolates were resistant to penicillin, clindamycin, ampicillin, and cefotaxime. Bacteriocins synthesized by Bacillus thuringiensis inhibited the growth of multiantibiotic resistance bacteria such as S. agnetis, S. equorum, Streptococcus uberis, Brevibacterium stationis, and Brachybacterium conglomeratum, but they were not active against S. sciuri, a microorganism that showed an 84% resistance to antibiotics tested in this study. Ma. Fabiola León-Galván, José E. Barboza-Corona, A. Arianna Lechuga-Arana, Mauricio Valencia-Posadas, Daniel D. Aguayo, Carlos Cedillo-Pelaez, Erika A. Martínez-Ortega, and Abner J. Gutierrez-Chavez Copyright © 2015 Ma. Fabiola León-Galván et al. All rights reserved. Biologically Active and Antimicrobial Peptides from Plants Sun, 01 Mar 2015 12:31:25 +0000 Bioactive peptides are part of an innate response elicited by most living forms. In plants, they are produced ubiquitously in roots, seeds, flowers, stems, and leaves, highlighting their physiological importance. While most of the bioactive peptides produced in plants possess microbicide properties, there is evidence that they are also involved in cellular signaling. Structurally, there is an overall similarity when comparing them with those derived from animal or insect sources. The biological action of bioactive peptides initiates with the binding to the target membrane followed in most cases by membrane permeabilization and rupture. Here we present an overview of what is currently known about bioactive peptides from plants, focusing on their antimicrobial activity and their role in the plant signaling network and offering perspectives on their potential application. Carlos E. Salas, Jesus A. Badillo-Corona, Guadalupe Ramírez-Sotelo, and Carmen Oliver-Salvador Copyright © 2015 Carlos E. Salas et al. All rights reserved. Antibacterial Activity of Synthetic Peptides Derived from Lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 Sun, 01 Mar 2015 12:13:25 +0000 Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4–33 μM) and E. faecalis (MIC 10–33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield. María A. León-Calvijo, Aura L. Leal-Castro, Giovanni A. Almanzar-Reina, Jaiver E. Rosas-Pérez, Javier E. García-Castañeda, and Zuly J. Rivera-Monroy Copyright © 2015 María A. León-Calvijo et al. All rights reserved. Ascorbic Acid, Ultraviolet C Rays, and Glucose but not Hyperthermia Are Elicitors of Human β-Defensin 1 mRNA in Normal Keratinocytes Sun, 01 Mar 2015 10:06:07 +0000 Hosts’ innate defense systems are upregulated by antimicrobial peptide elicitors (APEs). Our aim was to investigate the effects of hyperthermia, ultraviolet A rays (UVA), and ultraviolet C rays (UVC) as well as glucose and ascorbic acid (AA) on the regulation of human β-defensin 1 (DEFB1), cathelicidin (CAMP), and interferon-γ (IFNG) genes in normal human keratinocytes (NHK). The indirect in vitro antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes of these potential APEs was tested. We found that AA is a more potent APE for DEFB1 than glucose in NHK. Glucose but not AA is an APE for CAMP. Mild hypo- (35°C) and hyperthermia (39°C) are not APEs in NHK. AA-dependent DEFB1 upregulation below 20 mM predicts in vitro antimicrobial activity as well as glucose- and AA-dependent CAMP and IFNG upregulation. UVC upregulates CAMP and DEFB1 genes but UVA only upregulates the DEFB1 gene. UVC is a previously unrecognized APE in human cells. Our results suggest that glucose upregulates CAMP in an IFN-γ-independent manner. AA is an elicitor of innate immunity that will challenge the current concept of late activation of adaptive immunity of this vitamin. These results could be useful in designing new potential drugs and devices to combat skin infections. Luis Antonio Cruz Díaz, María Guadalupe Flores Miramontes, Paulina Chávez Hurtado, Kirk Allen, Marisela Gonzalez Ávila, and Ernesto Prado Montes de Oca Copyright © 2015 Luis Antonio Cruz Díaz et al. All rights reserved. Plant Antimicrobial Peptides as Potential Anticancer Agents Sun, 01 Mar 2015 09:57:02 +0000 Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms and are promising candidates to treat infections caused by pathogenic bacteria to animals and humans. AMPs also display anticancer activities because of their ability to inactivate a wide range of cancer cells. Cancer remains a cause of high morbidity and mortality worldwide. Therefore, the development of methods for its control is desirable. Attractive alternatives include plant AMP thionins, defensins, and cyclotides, which have anticancer activities. Here, we provide an overview of plant AMPs anticancer activities, with an emphasis on their mode of action, their selectivity, and their efficacy. Jaquelina Julia Guzmán-Rodríguez, Alejandra Ochoa-Zarzosa, Rodolfo López-Gómez, and Joel E. López-Meza Copyright © 2015 Jaquelina Julia Guzmán-Rodríguez et al. All rights reserved. Effect of Recombinant Prophenin 2 on the Integrity and Viability of Trichomonas vaginalis Sun, 01 Mar 2015 08:12:34 +0000 Trichomonas vaginalis is the causal agent of trichomoniasis, which is associated with preterm child delivery, low birth weight, and an increased risk of infection by human papilloma virus and human immunodeficiency virus following exposure. Several reports have established increasing numbers of trichomoniasis cases resistant to metronidazole, the agent used for treatment, and it is therefore important to identify new therapeutic alternatives. Previously, our group reported the effect of tritrpticin, a synthetic peptide derived from porcine prophenin, on T. vaginalis; however, the hemolytic activity of this small peptide complicates its possible use as a therapeutic agent. In this study, we report that the propeptide and the processed peptide of prophenin 2 (cleaved with hydroxylamine) affected the integrity and growth of T. vaginalis and that pro-prophenin 2 displays some resistance to proteolysis by T. vaginalis proteinases at 1 h. Its effect on T. vaginalis as well as its low hemolytic activity and short-time stability to parasite proteinases makes prophenin 2 an interesting candidate for synergistic or alternative treatment against T. vaginalis. J. L. Hernandez-Flores, M. C. Rodriguez, A. Gastelum Arellanez, A. Alvarez-Morales, and E. E. Avila Copyright © 2015 J. L. Hernandez-Flores et al. All rights reserved. Bioaccessible Antioxidants in Milk Fermented by Bifidobacterium longum subsp. longum Strains Mon, 23 Feb 2015 13:44:54 +0000 Bifidobacterium longum subsp. longum is among the dominant species of the human gastrointestinal microbiota and could thus have potential as probiotics. New targets such as antioxidant properties have interest for beneficial effects on health. The objective of this study was to evaluate the bioaccessibility of antioxidants in milk fermented by selected B. longum subsp. longum strains during in vitro dynamic digestion. The antioxidant capacity of cell extracts from 38 strains, of which 32 belong to B. longum subsp. longum, was evaluated with the ORAC (oxygen radical absorbance capacity) method. On the basis of screening and gene sequence typing by multilocus locus sequence analysis (MLSA), five strains were chosen for fermenting reconstituted skim milk. Antioxidant capacity varied among the strains tested (). Two strains of B. longum subsp. longum (CUETM 172 and 171) showed significantly higher ORAC values than the other bifidobacteria strains. However, there does not appear to be a relationship between gene sequence types and antioxidant capacity. The milk fermented by each of the five strains selected (CUETM 268, 172, 245, 247, or PRO 16-10) did not have higher initial ORAC values compared to the nonfermented milk samples. However, higher bioaccessibility of antioxidants in fermented milk (175–358%) was observed during digestion. Mérilie Gagnon, Patricia Savard, Audrey Rivière, Gisèle LaPointe, and Denis Roy Copyright © 2015 Mérilie Gagnon et al. All rights reserved. Biodiversity and γ-Aminobutyric Acid Production by Lactic Acid Bacteria Isolated from Traditional Alpine Raw Cow’s Milk Cheeses Mon, 23 Feb 2015 13:34:17 +0000 “Nostrano-cheeses” are traditional alpine cheeses made from raw cow’s milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of “Nostrano-cheeses” and evaluated their potential to produce γ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters () were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated were Lactobacillus paracasei, Streptococcus thermophilus, and Leuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers were Lactobacillus paracasei but other GABA producing species included Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosaceus, and Streptococcus thermophilus. No Enterococcus faecalis or Sc. macedonicus isolates produced GABA. The isolate producing the highest amount of GABA ( mg/kg) was a Sc. thermophilus. Elena Franciosi, Ilaria Carafa, Tiziana Nardin, Silvia Schiavon, Elisa Poznanski, Agostino Cavazza, Roberto Larcher, and Kieran M. Tuohy Copyright © 2015 Elena Franciosi et al. All rights reserved. Biocheese: A Food Probiotic Carrier Mon, 23 Feb 2015 09:23:54 +0000 This review describes some aspects related to the technological barriers encountered in the development and stability of probiotic cheeses. Aspects concerning the viability of probiotic cultures in this matrix are discussed and the potential of cheese as a biofunctional food carrier is analyzed, outlying some points related to health and safety. In general, the manufacture of probiotic cheese should have little change when compared with the elaboration of cheese in the traditional way. The physicochemical and technological parameters influencing the quality of these products have also to be measured so as to obtain a process optimization. J. M. Castro, M. E. Tornadijo, J. M. Fresno, and H. Sandoval Copyright © 2015 J. M. Castro et al. All rights reserved. Clostridium butyricum Combined with Bifidobacterium infantis Probiotic Mixture Restores Fecal Microbiota and Attenuates Systemic Inflammation in Mice with Antibiotic-Associated Diarrhea Mon, 23 Feb 2015 08:08:00 +0000 Antibiotic-associated diarrhea (AAD) is one of the most common complications of most types of antibiotics. Our aim was to determine the efficacy of Clostridium butyricum, Bifidobacterium infantis, and their mixture for AAD treatment in mice. AAD models were administered with single probiotic strain and probiotic mixture for short term and long term to evaluate the changes of the composition and diversity of intestinal microbiota, histopathology of the colon, and the systemic inflammation. Our data indicated that long-term probiotic therapy, but not short-term course, exerted beneficial effects on the restoration of the intestinal microbiota, the recovery of the tissue architecture, and attenuation of systemic inflammation. All predominant fecal bacteria reached normal level after the long-term probiotic mixture treatment, while IL-10, IFN-γ, and TNF-α also returned to normal level. However, the efficacy for AAD was time dependent and probiotic strain specific. Short-term administration of probiotic strains or mixture showed no apparent positive effects for AAD. In addition, the beneficial effects of C. butyricum combined with B. infantis probiotic mixture were superior to their single strain. This research showed that supplementation with C. butyricum combined with B. infantis probiotic mixture may be a simple and effective method for AAD treatment. Zongxin Ling, Xia Liu, Yiwen Cheng, Yueqiu Luo, Li Yuan, Lanjuan Li, and Charlie Xiang Copyright © 2015 Zongxin Ling et al. All rights reserved. Bioavailability of Dietary Polyphenols and Gut Microbiota Metabolism: Antimicrobial Properties Mon, 23 Feb 2015 07:01:38 +0000 Polyphenolic compounds are plant nutraceuticals showing a huge structural diversity, including chlorogenic acids, hydrolyzable tannins, and flavonoids (flavonols, flavanones, flavan-3-ols, anthocyanidins, isoflavones, and flavones). Most of them occur as glycosylated derivatives in plants and foods. In order to become bioactive at human body, these polyphenols must undergo diverse intestinal transformations, due to the action of digestive enzymes, but also by the action of microbiota metabolism. After elimination of sugar tailoring (generating the corresponding aglycons) and diverse hydroxyl moieties, as well as further backbone reorganizations, the final absorbed compounds enter the portal vein circulation towards liver (where other enzymatic transformations take place) and from there to other organs, including behind the digestive tract or via blood towards urine excretion. During this transit along diverse tissues and organs, they are able to carry out strong antiviral, antibacterial, and antiparasitic activities. This paper revises and discusses these antimicrobial activities of dietary polyphenols and their relevance for human health, shedding light on the importance of polyphenols structure recognition by specific enzymes produced by intestinal microbial taxa. Laura Marín, Elisa M. Miguélez, Claudio J. Villar, and Felipe Lombó Copyright © 2015 Laura Marín et al. All rights reserved. Isolation and Molecular Characterization of Brucella Isolates in Cattle Milk in Uganda Sun, 22 Feb 2015 11:48:15 +0000 Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR) loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections. Denis Rwabiita Mugizi, Shaman Muradrasoli, Sofia Boqvist, Joseph Erume, George William Nasinyama, Charles Waiswa, Gerald Mboowa, Markus Klint, and Ulf Magnusson Copyright © 2015 Denis Rwabiita Mugizi et al. All rights reserved. The Role of Probiotic Lactic Acid Bacteria and Bifidobacteria in the Prevention and Treatment of Inflammatory Bowel Disease and Other Related Diseases: A Systematic Review of Randomized Human Clinical Trials Sun, 22 Feb 2015 08:46:39 +0000 Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic inflammation of the small intestine and colon caused by a dysregulated immune response to host intestinal microbiota in genetically susceptible subjects. A number of fermented dairy products contain lactic acid bacteria (LAB) and bifidobacteria, some of which have been characterized as probiotics that can modify the gut microbiota and may be beneficial for the treatment and the prevention of IBD. The objective of this review was to carry out a systematic search of LAB and bifidobacteria probiotics and IBD, using the PubMed and Scopus databases, defined by a specific equation using MeSH terms and limited to human clinical trials. The use of probiotics and/or synbiotics has positive effects in the treatment and maintenance of UC, whereas in CD clear effectiveness has only been shown for synbiotics. Furthermore, in other associated IBD pathologies, such as pouchitis and cholangitis, LAB and bifidobacteria probiotics can provide a benefit through the improvement of clinical symptoms. However, more studies are needed to understand their mechanisms of action and in this way to understand the effect of probiotics prior to their use as coadjuvants in the therapy and prevention of IBD conditions. Maria Jose Saez-Lara, Carolina Gomez-Llorente, Julio Plaza-Diaz, and Angel Gil Copyright © 2015 Maria Jose Saez-Lara et al. All rights reserved. A Survey of Modulation of Gut Microbiota by Dietary Polyphenols Sun, 22 Feb 2015 08:12:46 +0000 Dietary polyphenols present in a broad range of plant foods have been related to beneficial health effects. This review aims to update the current information about the modulation of the gut microbiota by dietary phenolic compounds, from a perspective based on the experimental approaches used. After referring to general aspects of gut microbiota and dietary polyphenols, studies related to this topic are presented according to their experimental design: batch culture fermentations, gastrointestinal simulators, animal model studies, and human intervention studies. In general, studies evidence that dietary polyphenols may contribute to the maintenance of intestinal health by preserving the gut microbial balance through the stimulation of the growth of beneficial bacteria (i.e., lactobacilli and bifidobacteria) and the inhibition of pathogenic bacteria, exerting prebiotic-like effects. Combination of in vitro and in vivo models could help to understand the underlying mechanisms in the polyphenols-microbiota-host triangle and elucidate the implications of polyphenols on human health. From a technological point of view, supplementation with rich-polyphenolic stuffs (phenolic extracts, phenolic-enriched fractions, etc.) could be an effective option to improve health benefits of functional foods such as the case of dairy fermented foods. Montserrat Dueñas, Irene Muñoz-González, Carolina Cueva, Ana Jiménez-Girón, Fernando Sánchez-Patán, Celestino Santos-Buelga, M. Victoria Moreno-Arribas, and Begoña Bartolomé Copyright © 2015 Montserrat Dueñas et al. All rights reserved. The Effects of Bifidobacterium breve on Immune Mediators and Proteome of HT29 Cells Monolayers Sun, 22 Feb 2015 06:20:57 +0000 The use of beneficial microorganisms, the so-called probiotics, to improve human health is gaining popularity. However, not all of the probiotic strains trigger the same responses and they differ in their interaction with the host. In spite of the limited knowledge on mechanisms of action some of the probiotic effects seem to be exerted through maintenance of the gastrointestinal barrier function and modulation of the immune system. In the present work, we have addressed in vitro the response of the intestinal epithelial cell line HT29 to the strain Bifidobacterium breve IPLA20004. In the array of 84 genes involved in inflammation tested, the expression of 12 was modified by the bifidobacteria. The genes of chemokine CXCL6, the chemokine receptor CCR7, and, specially, the complement component C3 were upregulated. Indeed, HT29 cells cocultivated with B. breve produced significantly higher levels of protein C3a. The proteome of HT29 cells showed increased levels of cytokeratin-8 in the presence of B. breve. Altogether, it seems that B. breve IPLA20004 could favor the recruitment of innate immune cells to the mucosa reinforcing, as well as the physical barrier of the intestinal epithelium. Borja Sánchez, Irene González-Rodríguez, Silvia Arboleya, Patricia López, Ana Suárez, Patricia Ruas-Madiedo, Abelardo Margolles, and Miguel Gueimonde Copyright © 2015 Borja Sánchez et al. All rights reserved. Determination of the In Vitro and In Vivo Antimicrobial Activity on Salivary Streptococci and Lactobacilli and Chemical Characterisation of the Phenolic Content of a Plantago lanceolata Infusion Thu, 12 Feb 2015 09:30:30 +0000 Introduction. Plant extracts may be suitable alternative treatments for caries. Aims. To investigate the in vitro and in vivo antimicrobial effects of Plantago lanceolata herbal tea (from flowers and leaves) on cariogenic bacteria and to identify the major constituents of P. lanceolata plant. Materials and Methods. The MIC and MBC against cariogenic bacteria were determined for P. lanceolata tea. Subsequently, a controlled random clinical study was conducted. Group A was instructed to rinse with a P. lanceolata mouth rinse, and Group B received a placebo mouth rinse for seven days. The salivary colonisation by streptococci and lactobacilli was investigated prior to treatment and on the fourth and seventh days. Finally, the P. lanceolata tea was analysed for its polyphenolic content, and major phenolics were identified. Results and Discussion. P. lanceolata teas demonstrate good in vitro antimicrobial activity. The in vivo test showed that Group A subjects presented a significant decrease in streptococci compared to Group B. The phytochemical analysis revealed that flavonoids, coumarins, lipids, cinnamic acids, lignans, and phenolic compounds are present in P. lanceolata infusions. Conclusions. P. lanceolata extract could represent a natural anticariogenic agent via an antimicrobial effect and might be useful as an ancillary measure to control the proliferation of cariogenic flora. Gianmaria Fabrizio Ferrazzano, Tiziana Cantile, Lia Roberto, Aniello Ingenito, Maria Rosaria Catania, Emanuela Roscetto, Giuseppe Palumbo, Armando Zarrelli, and Antonino Pollio Copyright © 2015 Gianmaria Fabrizio Ferrazzano et al. All rights reserved. Evaluation of Hemagglutination Activity of Chitosan Nanoparticles Using Human Erythrocytes Wed, 11 Feb 2015 14:10:20 +0000 Chitosan is a polysaccharide composed of randomly distributed chains of β-(1-4) D-glucosamine and N-acetyl-D-glucosamine. This compound is obtained by partial or total deacetylation of chitin in acidic solution. The chitosan-based hemostatic agents have been gaining much attention in the management of bleeding. The aim of this study was to evaluate in vitro hemagglutination activity of chitosan nanoparticles using human erythrocytes. The preparation of nanoparticles was achieved by ionotropic gelification technique followed by neutralization with NaOH 1 mol/L−1. The hemagglutination activity was performed on a solution of 2% erythrocytes (pH 7.4 on PBS) collected from five healthy volunteers. The hemolysis determination was made by spectrophotometric analysis. Chitosan nanoparticle solutions without NaOH addition changed the reddish colour of the wells into brown, suggesting an oxidative reaction of hemoglobin and possible cell lysis. All neutralized solutions of chitosan nanoparticles presented positive haemagglutination, without any change in reaction color. Chitosan nanoparticles presented hemolytic activity ranging from 186.20 to 223.12%, while neutralized solutions ranged from 2.56 to 72.54%, comparing to distilled water. Results highlight the need for development of new routes of synthesis of chitosan nanoparticles within human physiologic pH. Jefferson Muniz de Lima, Ronaldo Rodrigues Sarmento, Joelma Rodrigues de Souza, Fábio André Brayner, Ana Paula Sampaio Feitosa, Rafael Padilha, Luiz Carlos Alves, Isaque Jerônimo Porto, Roberta Ferreti Bonan Dantas Batista, Juliano Elvis de Oliveira, Eliton Souto de Medeiros, Paulo Rogério Ferreti Bonan, and Lúcio Roberto Castellano Copyright © 2015 Jefferson Muniz de Lima et al. All rights reserved. Endophytic Bacillus subtilis Strain E1R-J Is a Promising Biocontrol Agent for Wheat Powdery Mildew Wed, 11 Feb 2015 06:01:49 +0000 In this study, the biocontrol efficacies of 14 endophytic bacterial strains were tested against Blumeria graminis f. sp. tritici (Bgt) in pot experiments under greenhouse conditions. Bacillus subtilis strain E1R-j significantly reduced disease index and exhibited the best control (90.97%). When different formulations of E1R-j were sprayed 24 h before Bgt inoculation, fermentation liquid without bacterial cell and crude protein suspension displayed the similar effects; and they reduced disease index more than bacterial cell suspension (109 cfu mL−1) and fermentation liquid without protein. The control effects were not significantly different between 1011 and 109 cfu mL−1 of bacterial cell suspension but were higher than 107 cfu mL−1. Further observations showed that conidial germination and appressorial formation of Bgt were retarded by spraying E1R-j 24 h before Bgt inoculation. Compared with the water check, conidial germination and appressorial formation were decreased by 43.3% and 42.7%, respectively. In the treatment with E1R-j, the number of houstoria significantly reduced and the speed of mycelial extension was slowed down in the wheat leaves. Scanning electron microscopy observation revealed that E1R-j significantly suppressed the conidial germination and caused rupture and deformation of germ tubes. On the surface of wheat leaves, mycelia and conidiophores became shrinking. Xiaoning Gao, Yufei Gong, Yunxia Huo, Qingmei Han, Zhensheng Kang, and Lili Huang Copyright © 2015 Xiaoning Gao et al. All rights reserved. Comparative Analysis of Quinolone Resistance in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli from Chinese Children and Adults Tue, 10 Feb 2015 12:54:23 +0000 The objective of this study was to compare quinolone resistance and gyrA mutations in clinical isolates of Klebsiella pneumoniae and Escherichia coli from Chinese adults who used quinolone in the preceding month and children without any known history of quinolone administration. The antimicrobial susceptibilities of 61 isolates from children and 79 isolates from adults were determined. The mutations in the quinolone resistance-determining regions in gyrA gene were detected by PCR and DNA sequencing. Fluoroquinolone resistance and types of gyrA mutations in isolates from children and adults were compared and statistically analyzed. No significant differences were detected in the resistance rates of ciprofloxacin and levofloxacin between children and adults among isolates of the two species (all ). The double mutation Ser83Leu + Asp87Asn in the ciprofloxacin-resistant isolates occurred in 73.7% isolates from the children and 67.9% from the adults, respectively (). Children with no known history of quinolone administration were found to carry fluoroquinolone-resistant Enterobacteriaceae isolates. The occurrence of ciprofloxacin resistance and the major types of gyrA mutations in the isolates from the children were similar to those from adults. The results indicate that precautions should be taken on environmental issues resulting from widespread transmission of quinolone resistance. Ying Huang, James O. Ogutu, Jiarui Gu, Fengshu Ding, Yuhong You, Yan Huo, Hong Zhao, Wenjing Li, Zhiwei Zhang, Wenli Zhang, Xiaobei Chen, Yingmei Fu, and Fengmin Zhang Copyright © 2015 Ying Huang et al. All rights reserved. Evaluation of Antibacterial Activity of Some Traditionally Used Medicinal Plants against Human Pathogenic Bacteria Mon, 09 Feb 2015 06:28:10 +0000 The worldwide increase of multidrug resistance in both community- and health-care associated bacterial infections has impaired the current antimicrobial therapy, warranting the search for other alternatives. We aimed to find the in vitro antibacterial activity of ethanolic extracts of 16 different traditionally used medicinal plants of Nepal against 13 clinical and 2 reference bacterial species using microbroth dilution method. The evaluated plants species were found to exert a range of in vitro growth inhibitory action against the tested bacterial species, and Cynodon dactylon was found to exhibit moderate inhibitory action against 13 bacterial species including methicillin-resistant Staphylococcus aureus, imipenem-resistant Pseudomonas aeruginosa, multidrug-resistant Salmonella typhi, and S. typhimurium. The minimum inhibitory concentration (MIC) values of tested ethanolic extracts were found from 31 to >25,000 μg/mL. Notably, ethanolic extracts of Cinnamomum camphora, Curculigo orchioides, and Curcuma longa exhibited the highest antibacterial activity against S. pyogenes with a MIC of 49, 49, and 195 μg/mL, respectively; whereas chloroform fraction of Cynodon dactylon exhibited best antibacterial activity against S. aureus with a MIC of 31 μg/mL. Among all, C. dactylon, C. camphora, C. orchioides, and C. longa plant extracts displayed a potential antibacterial activity of MIC < 100 μg/mL. Bishnu P. Marasini, Pankaj Baral, Pratibha Aryal, Kashi R. Ghimire, Sanjiv Neupane, Nabaraj Dahal, Anjana Singh, Laxman Ghimire, and Kanti Shrestha Copyright © 2015 Bishnu P. Marasini et al. All rights reserved. The Influence of Tea Tree Oil (Melaleuca alternifolia) on Fluconazole Activity against Fluconazole-Resistant Candida albicans Strains Wed, 04 Feb 2015 13:14:16 +0000 The aim of this study was to evaluate the activity of fluconazole against 32 clinical strains of fluconazole-resistant Candida albicans, and C. albicans ATCC 10231 reference strain, after their exposure to sublethal concentrations of tea tree oil (TTO) or its main bioactive component terpinen-4-ol. For all tested fluconazole-resistant C. albicans strains TTO and terpinen-4-ol minimal inhibitory concentrations (MICs) were low, ranging from 0.06% to 0.5%. The 24-hour exposure of fluconazole-resistant C. albicans strains to fluconazole with sublethal dose of TTO enhanced fluconazole activity against these strains. Overall, 62.5% of isolates were classified as susceptible, 25.0% exhibited intermediate susceptibility, and 12.5% were resistant. For all of the tested clinical strains the fluconazole MIC decreased from an average of 244.0 μg/mL to an average of 38.46 μg/mL, and the fluconazole minimal fungicidal concentrations (MFC) decreased from an average of 254.67 μg/mL to an average of 66.62 μg/mL. Terpinen-4-ol was found to be more active than TTO, and strongly enhanced fluconazole activity against fluconazole-resistant C. albicans strains. The results of this study demonstrate that combining natural substances such as TTO and conventional drug such as fluconazole, may help treat difficult yeast infections. Anna Mertas, Aleksandra Garbusińska, Ewelina Szliszka, Andrzej Jureczko, Magdalena Kowalska, and Wojciech Król Copyright © 2015 Anna Mertas et al. All rights reserved. Genetic Variability of Candida albicans Sap8 Propeptide in Isolates from Different Types of Infection Wed, 04 Feb 2015 06:33:53 +0000 The secreted aspartic proteases (Saps) are among the most studied virulence determinants in Candida albicans. These proteins are translated as pre-pro-enzymes consisting of a signal sequence followed by a propeptide and the mature enzyme. The propeptides of secreted proteinases are important for the correct processing, folding/secretion of the mature enzyme. In this study, the DNA sequences of C. albicans Saps were screened and a microsatellite was identified in SAP8 propeptide region. The genetic variability of the repetitive region of Sap8 propeptide was determined in 108 C. albicans independent strains isolated from different types of infection: oral infection (OI), oral commensal (OC), vulvovaginal candidiasis (VVC), and bloodstream infections (BSI). Nine different propeptides for Sap8 processing were identified whose frequencies varied with the type of infection. OC strains presented the highest gene diversity while OI isolated the lowest. The contribution of the Saps to mucosal and systemic infections has been demonstrated and recently Sap8 has been implicated in the cleavage of a signalling glycoprotein that leads to Cek1-MAPK pathway activation. This work is the first to identify a variable microsatellite in the propeptide of a secreted aspartic protease and brings new insights into the variability of Sap8. Joana Carvalho-Pereira, Catarina Vaz, Catarina Carneiro, Célia Pais, and Paula Sampaio Copyright © 2015 Joana Carvalho-Pereira et al. All rights reserved. Guidelines for Optimisation of a Multiplex Oligonucleotide Ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array Tue, 03 Feb 2015 13:54:34 +0000 With multiplex oligonucleotide ligation-PCR (MOL-PCR) different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in high-throughput format. As such, MOL-PCR is a convenient solution for microbial detection and identification assays where many markers should be analysed, including for routine further characterisation of an identified microbial pathogenic isolate. For an assay aimed at routine use, optimisation in terms of differentiation between positive and negative results and of cost and effort is indispensable. As MOL-PCR includes a multiplex ligation step, followed by a singleplex PCR and analysis with microspheres on a Luminex device, several parameters are accessible for optimisation. Although MOL-PCR performance may be influenced by the markers used in the assay and the targeted bacterial species, evaluation of the method of DNA isolation, the probe concentration, the amount of microspheres, and the concentration of reporter dye is advisable in the development of any MOL-PCR assay. Therefore, we here describe our observations made during the optimisation of a 20-plex MOL-PCR assay for subtyping of Salmonella Typhimurium with the aim to provide a possible workflow as guidance for the development and optimisation of a MOL-PCR assay for the characterisation of other microbial pathogens. Véronique Wuyts, Nancy H. C. Roosens, Sophie Bertrand, Kathleen Marchal, and Sigrid C. J. De Keersmaecker Copyright © 2015 Véronique Wuyts et al. All rights reserved. Locus of Enterocyte Effacement: A Pathogenicity Island Involved in the Virulence of Enteropathogenic and Enterohemorragic Escherichia coli Subjected to a Complex Network of Gene Regulation Mon, 02 Feb 2015 07:15:57 +0000 The locus of enterocyte effacement (LEE) is a 35.6 kb pathogenicity island inserted in the genome of some bacteria such as enteropathogenic Escherichia coli, enterohemorrhagic E.coli, Citrobacter rodentium, and Escherichia albertii. LEE comprises the genes responsible for causing attaching and effacing lesions, a characteristic lesion that involves intimate adherence of bacteria to enterocytes, a signaling cascade leading to brush border and microvilli destruction, and loss of ions, causing severe diarrhea. It is composed of 41 open reading frames and five major operons encoding a type three system apparatus, secreted proteins, an adhesin, called intimin, and its receptor called translocated intimin receptor (Tir). LEE is subjected to various levels of regulation, including transcriptional and posttranscriptional regulators located both inside and outside of the pathogenicity island. Several molecules were described being related to feedback inhibition, transcriptional activation, and transcriptional repression. These molecules are involved in a complex network of regulation, including mechanisms such as quorum sensing and temporal control of LEE genes transcription and translation. In this mini review we have detailed the complex network that regulates transcription and expression of genes involved in this kind of lesion. Fernanda M. Franzin and Marcelo P. Sircili Copyright © 2015 Fernanda M. Franzin and Marcelo P. Sircili. All rights reserved. In Vitro Inhibitory and Cytotoxic Activity of MFM 501, a Novel Codonopsinine Derivative, against Methicillin-Resistant Staphylococcus aureus Clinical Isolates Sun, 01 Feb 2015 12:14:04 +0000 28 new pyrrolidine types of compounds as analogues for natural polyhydroxy alkaloids of codonopsinine were evaluated for their anti-MRSA activity using MIC and MBC value determination assay against a panel of S. aureus isolates. One pyrrolidine compound, MFM 501, exhibited good inhibitory activity with MIC value of 15.6 to 31.3 μg/mL against 55 S. aureus isolates (43 MRSA and 12 MSSA isolates). The active compound also displayed MBC values between 250 and 500 μg/mL against 58 S. aureus isolates (45 MRSA and 13 MSSA isolates) implying that MFM 501 has a bacteriostatic rather than bactericidal effect against both MRSA and MSSA isolates. In addition, MFM 501 showed no apparent cytotoxicity activity towards three normal cell lines (WRL-68, Vero, and 3T3) with IC50 values of >625 µg/mL. Selectivity index (SI) of MFM 501 gave a value of >10 suggesting that MFM 501 is significant and suitable for further in vivo investigations. These results suggested that synthetically derived intermediate compounds based on natural products may play an important role in the discovery of new anti-infective agents against MRSA. Saiful Azmi Johari, Mastura Mohtar, Sharifah Aminah Syed Mohammad, Rohana Sahdan, Zurina Shaameri, Ahmad Sazali Hamzah, and Mohd Fazli Mohammat Copyright © 2015 Saiful Azmi Johari et al. All rights reserved. Antibiotic Therapies in Maxillofacial Surgery in the Context of Prophylaxis Sun, 01 Feb 2015 11:54:04 +0000 Objectives. There is no single pattern for preventive action as to the duration and type of antibiotic therapy in maxillofacial surgery. In these circumstances, it appears reasonable to set relevant standards for prophylactic procedures after such surgeries. Methods. Retrospective analysis of bacteriological tests has been carried out as well as a susceptibility evaluation of cultured bacterial and fungal strains to antibiotics over a five-year period in subjects treated at the Cranio-Maxillo-Facial Clinic in Katowice. A total of 726 bacterial and fungal strains were cultured in 484 patients (200 women and 284 males). The age of the patients was 40.2 on average. Results. The most frequent bacteria isolated from the patients were Gram-positive 541 (74.5%). Gram-negative bacteria were present in 177 (24.4%) cases. Fungi of the Candida genus were isolated in eight cases (1.1%). Conclusions. The most often isolated bacteria were Streptococcus mitis and Streptococcus oralis, whose number has grown over the last two years. Empiric therapies should be based on ciprofloxacin and gentamicin. It has been observed that all the Gram-positive bacteria are becoming more resistant to all antibiotics. Ampicillin and imipenem were antibiotics with the steepest resistance reduction while vancomycin showed the lowest resistance drop. Bogusława Orzechowska-Wylęgała, Adam Wylęgała, Michał Buliński, and Iwona Niedzielska Copyright © 2015 Bogusława Orzechowska-Wylęgała et al. All rights reserved. Studies on Molecular Characterizations of the Outer Membrane Proteins, Lipids Profile, and Exopolysaccharides of Antibiotic Resistant Strain Pseudomonas aeruginosa Sun, 01 Feb 2015 11:24:38 +0000 Susceptibility of the tested Pseudomonas aeruginosa strain to two different antibiotics, tetracycline (TE) and ciprofloxacin (CIP), was carried out using liquid dilution method. Minimum inhibitory concentrations of TE and CIP were 9.0 and 6.0 mg/100 mL, respectively. Some metabolic changes due to both, the mode of action of TE and CIP on P. aeruginosa and its resistance to high concentrations of antibiotics (sub-MIC) were detected. The total cellular protein contents decreased after antibiotic treatment, while outer membrane protein (OMP) contents were approximately constant for both treated and untreated cells. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the OMPs for untreated and TE and CIP treated cells indicated that the molecular changes were achieved as; lost in, induction and stability of some protein bands as a result of antibiotics treatment. Five bands (with mol. wt. 71.75, 54.8, 31.72, 28.63, and 20.33 KDa) were stable in both treated and untreated tested strains, while two bands (with mol. wt. 194.8 and 118.3 KDa) were induced and the lost of only one band (with mol. wt. 142.5 KDa) after antibiotics treatment. On the other hand, total lipids and phospholipids increased in antibiotic treated cells, while neutral lipids decreased. Also, there was observable stability in the number of fatty acids in untreated and treated cells (11 fatty acids). The unsaturation index was decreased to 56% and 17.6% in both TE and CIP treatments, respectively. The produced amount of EPSs in untreated cultures of P. aeruginosa was relatively higher than in treated cultures with sub-MICs of TE and CIP antibiotics. It was also observed that the amounts of exopolysaccharides (EPSs) increased by increasing the incubation period up to five days of incubation in case of untreated and antibiotic treated cultures. Hany M. Yehia, Wesam A. Hassanein, and Shimaa M. Ibraheim Copyright © 2015 Hany M. Yehia et al. All rights reserved. Influence of Culture Media on Biofilm Formation by Candida Species and Response of Sessile Cells to Antifungals and Oxidative Stress Sun, 01 Feb 2015 10:03:38 +0000 The aims of the study were to evaluate the influence of culture media on biofilm formation by C. albicans, C. glabrata, C. krusei, and C. parapsilosis and to investigate the responses of sessile cells to antifungals and reactive oxygen species (ROS) as compared to planktonic cells. For biofilm formation, the Candida species were grown at different periods of time in YP or YNB media supplemented or not with 0.2 or 2% glucose. Sessile and planktonic cells were exposed to increasing concentrations of antifungals, H2O2, menadione or silver nanoparticles (AgNPs). Biofilms were observed by scanning electron microscopy (SEM) and quantified by the XTT assay. C. albicans formed biofilms preferentially in YPD containing 2% glucose (YPD/2%), C. glabrata in glucose-free YNB or supplemented with 0.2% glucose (YNB/0.2%), while C. krusei and C. parapsilosis preferred YP, YPD/0.2%, and YPD/2%. Interestingly, only C. albicans produced an exopolymeric matrix. This is the first report dealing with the in vitro effect of the culture medium and glucose on the formation of biofilms in four Candida species as well as the resistance of sessile cells to antifungals, AgNPs, and ROS. Our results suggest that candidiasis in vivo is a multifactorial and complex process where the nutritional conditions, the human immune system, and the adaptability of the pathogen should be considered altogether to provide an effective treatment of the patient. Isela Serrano-Fujarte, Everardo López-Romero, Georgina Elena Reyna-López, Ma. Alejandrina Martínez-Gámez, Arturo Vega-González, and Mayra Cuéllar-Cruz Copyright © 2015 Isela Serrano-Fujarte et al. All rights reserved. Biofilm-Forming Staphylococcus epidermidis Expressing Vancomycin Resistance Early after Adhesion to a Metal Surface Sat, 31 Jan 2015 07:10:11 +0000 We investigated biofilm formation and time of vancomycin (VCM) resistance expression after adhesion to a metal surface in Staphylococcus epidermidis. Biofilm-forming Staphylococcus epidermidis with a VCM MIC of 1 μg/mL was used. The bacteria were made to adhere to a stainless steel washer and treated with VCM at different times and concentrations. VCM was administered 0, 2, 4, and 8 hours after adhesion. The amount of biofilm formed was evaluated based on the biofilm coverage rates (BCRs) before and after VCM administration, bacterial viability in biofilm was visually observed using the fluorescence staining method, and the viable bacterial count in biofilm was measured. The VCM concentration required to decrease BCR significantly compared with that of VCM-untreated bacteria was 4 μg/mL, even in the 0 hr group. In the 4 and 8 hr groups, VCM could not inhibit biofilm growth even at 1,024 μg/mL. In the 8 hr group, viable bacteria remained in biofilm at a count of 104 CFU even at a high VCM concentration (1,024 μg/mL). It was suggested that biofilm-forming Staphylococcus epidermidis expresses resistance to VCM early after adhesion to a metal surface. Resistance increased over time after adhesion as the biofilm formed, and strong resistance was expressed 4–8 hours after adhesion. Toshiyuki Sakimura, Shiro Kajiyama, Shinji Adachi, Ko Chiba, Akihiko Yonekura, Masato Tomita, Hironobu Koseki, Takashi Miyamoto, Toshiyuki Tsurumoto, and Makoto Osaki Copyright © 2015 Toshiyuki Sakimura et al. All rights reserved. Health Safety of Soft Drinks: Contents, Containers, and Microorganisms Wed, 28 Jan 2015 13:51:37 +0000 Soft drinks consumption is still a controversial issue for public health and public policy. Over the years, numerous studies have been conducted into the possible links between soft drink intake and medical problems, the results of which, however, remain highly contested. Nevertheless, as a result, increasing emphasis is being placed on the health properties of soft drinks, by both the industry and the consumers, for example, in the expanding area of functional drinks. Extensive legislation has been put in place to ensure that soft drinks manufacturers conform to established national and international standards. Consumers trust that the soft drinks they buy are safe and their quality is guaranteed. They also expect to be provided with information that can help them to make informed decisions about the purchase of products and that the information on product labels is not false or misleading. This paper provides a broad overview of available scientific knowledge and cites numerous studies on various aspects of soft drinks and their implications for health safety. Particular attention is given to ingredients, including artificial flavorings, colorings, and preservatives and to the lesser known risks of microbiological and chemical contamination during processing and storage. Dorota Kregiel Copyright © 2015 Dorota Kregiel. All rights reserved. Small Molecule Inhibitor of Type Three Secretion Suppresses Acute and Chronic Chlamydia trachomatis Infection in a Novel Urogenital Chlamydia Model Wed, 28 Jan 2015 06:47:16 +0000 Previously, we reported that a compound from a group of thiohydrazides of oxamic acids, CL-55, possessed antichlamydial activity in vitro that was accompanied by a decreased translocation of the type three secretion effector, IncA, into the host cell. In this study, the antichlamydial activity of CL-55 was tested in vivo in DBA/2 mice infected with C. trachomatis serovar D. We found that intravaginal inoculation of DBA/2 mice with the clinically relevant strain, C. trachomatis serovar D, results in a course of infection and pathology similar to that observed in humans. The early stage of infection in this model was characterized by a shedding of Chlamydia in vaginal secretions followed by an ascending infection and inflammation in the upper genital tract. We found that CL-55 possessed antibacterial activity in vivo and was able to control C. trachomatis vaginal shedding, ascending infection, and inflammation in the upper genital organs in DBA/2 mice. Our data provide a proof of concept for the protective effect of the thiadiazinon, CL-55, against chlamydial infection in vivo and support the feasibility of further studies of its potential therapeutic applications. Ekaterina A. Koroleva, Natalia V. Kobets, Egor S. Zayakin, Sergei I. Luyksaar, Ludmila A. Shabalina, and Naylia A. Zigangirova Copyright © 2015 Ekaterina A. Koroleva et al. All rights reserved. Phylogeny and Taxonomical Investigation of Trichoderma spp. from Indian Region of Indo-Burma Biodiversity Hot Spot Region with Special Reference to Manipur Wed, 28 Jan 2015 06:43:44 +0000 Towards assessing the genetic diversity and occurrence of Trichoderma species from the Indian region of Indo-Burma Biodiversity hotspot, a total of 193 Trichoderma strains were isolated from cultivated soils of nine different districts of Manipur comprising 4 different agroclimatic zones. The isolates were grouped based on the morphological characteristics. ITS-RFLP of the rDNA region using three restriction digestion enzymes: Mob1, Taq1, and Hinf1, showed interspecific variations among 65 isolates of Trichoderma. Based on ITS sequence data, a total of 22 different types of representative Trichoderma species were reported and phylogenetic analysis showed 4 well-separated main clades in which T. harzianum was found to be the most prevalent spp. among all the Trichoderma spp. Combined molecular and phenotypic data leads to the development of a taxonomy of all the 22 different Trichoderma spp., which was reported for the first time from this unique region. All these species were found to produce different extrolites and enzymes responsible for the biocontrol activities against the harmful fungal phytopathogens that hamper in food production. This potential indigenous Trichoderma spp. can be targeted for the development of suitable bioformulation against soil and seedborne pathogens in sustainable agricultural practice. Th. Kamala, S. Indira Devi, K. Chandradev Sharma, and K. Kennedy Copyright © 2015 Th. Kamala et al. All rights reserved.