BioMed Research International: Microbiology The latest articles from Hindawi © 2017 , Hindawi Limited . All rights reserved. Corrigendum to “Application of PK/PD Modeling in Veterinary Field: Dose Optimization and Drug Resistance Prediction” Mon, 06 Nov 2017 06:54:20 +0000 Ijaz Ahmad, Lingli Huang, Haihong Hao, Pascal Sanders, and Zonghui Yuan Copyright © 2017 Ijaz Ahmad et al. All rights reserved. Detection of Cryptosporidium and Cyclospora Oocysts from Environmental Water for Drinking and Recreational Activities in Sarawak, Malaysia Mon, 06 Nov 2017 00:00:00 +0000 Cryptosporidiosis and cyclosporiasis are caused by waterborne coccidian protozoan parasites of the genera Cryptosporidium and Cyclospora, respectively. This study was conducted to detect Cryptosporidium and Cyclospora oocysts from environmental water abstracted by drinking water treatment plants and recreational activities in Sarawak, Malaysia. Water samples (12 each) were collected from Sungai Sarawak Kanan in Bau and Sungai Sarawak Kiri in Batu Kitang, respectively. In addition, 6 water samples each were collected from Ranchan Recreational Park and UNIMAS Lake at Universiti Malaysia Sarawak, Kota Samarahan, respectively. Water physicochemical parameters were also recorded. All samples were concentrated by the iron sulfate flocculation method followed by the sucrose floatation technique. Cryptosporidium and Cyclospora were detected by modified Ziehl-Neelsen technique. Correlation of the parasites distribution with water physicochemical parameters was analysed using bivariate Pearson correlation. Based on the 24 total samples of environmental water abstracted by drinking water treatment plants, all the samples (24/24; 100%) were positive with Cryptosporidium, and only 2 samples (2/24; 8.33%) were positive with Cyclospora. Based on the 12 total samples of water for recreational activities, 4 samples (4/12; 33%) were positive with Cryptosporidium, while 2 samples (2/12; 17%) were positive with Cyclospora. Cryptosporidium oocysts were negatively correlated with dissolved oxygen (DO). Lesley Maurice Bilung, Ahmad Syatir Tahar, Nur Emyliana Yunos, Kasing Apun, Yvonne Ai-Lian Lim, Elexson Nillian, and Hashimatul Fatma Hashim Copyright © 2017 Lesley Maurice Bilung et al. All rights reserved. Antioxidant Potential of Selected Korean Edible Plant Extracts Mon, 06 Nov 2017 00:00:00 +0000 This study aimed to evaluate the antioxidant activity of various plant extracts. A total of 94 kinds of edible plant extracts obtained from the Korea Plant Extract Bank were screened for cytotoxicity, following which the total phenolic content of 24 shortlisted extracts was determined. Of these, extracts from three plants, namely, Castanea crenata (CC) leaf, Camellia japonica (CJ) fruit, and Viburnum dilatatum (VD) leaf, were examined for antioxidant capabilities by measuring radical scavenging activity, ferric reducing/antioxidant power, and lipid peroxidation inhibitory activity. In addition, cellular antioxidant activities of the three extracts were assessed by a cell-based dichlorofluorescein assay and antioxidant response element (ARE) reporter activity assay. The results demonstrated that all three extracts concentration-dependently scavenged free radicals, inhibited lipid peroxidation, reduced the cellular level of reactive oxygen species, and increased ARE-luciferase activity, indicating antioxidant enzyme-inducing potential. In particular, CJ extract showed significantly greater antioxidative activity and antimigratory effect in a breast cancer cell line compared to CC and VD extracts. Hence, CJ extract deserves further study for its in vivo functionality or biologically active constituents. Yaejin Woo, Hyeji Lee, Yong-Seob Jeong, Ga Young Shin, Ja Gyeong Oh, Jong-Sang Kim, and Jisun Oh Copyright © 2017 Yaejin Woo et al. All rights reserved. An Overview of the Biological Effects of Some Mediterranean Essential Oils on Human Health Sun, 05 Nov 2017 00:00:00 +0000 Essential oils (EOs), extracted from aromatic plants, are interesting natural products and represent an important part of the traditional pharmacopeia. The use of some EOs as alternative antimicrobial and pharmaceutical agents has attracted considerable interest recently. Most of the EOs and their single constituents have been reported to inhibit several phytopathogens, human pathogens, and insects as well as their effective uses in food and pharmaceutical industries. The current review discussed the chemical composition and bioactivity of some important EOs extracted from some Mediterranean plants and their principal bioactive single constituents. Information has been furnished on the mechanisms, mode of actions, and factors affecting the bioactivity of some single constituents from different Mediterranean plant EOs. The current review gives an insight into some common plant EOs belonging to Lamiaceae, Apiaceae, Rutaceae, and Verbenaceae families commonly growing in Mediterranean region. Further information has been provided about the medical uses of some EOs for several human diseases covering the pharmacological effects (anti-inflammatory, antioxidant, and anticarcinogenic). The antimicrobial effects have been also considered in the current review. Although plant EOs are considered promising natural alternatives for many chemical drugs, they still need more specific research for wide application especially in food and pharmaceutical industries. Hazem S. Elshafie and Ippolito Camele Copyright © 2017 Hazem S. Elshafie and Ippolito Camele. All rights reserved. Fungicidal, Corrosive, and Mutational Effects of Polyhexamethylene Biguanide Combined with 1-Bromo-3-chloro-5,5-dimethylimidazolidine-2,4-dione Sun, 05 Nov 2017 00:00:00 +0000 Background. The disinfectants polyhexamethylene biguanide (PHMB) and 1-bromo-3-chloro-5,5-dimethylimidazolidine-2,4-dione (BCDMH) each have limitations. So far, their combined usage has not been examined. In this study, the fungicidal activity of combined disinfectant using PHMB and BCDMH, named PB, against Candida albicans was evaluated. Methods. Suspension quantitative fungicidal test and viable fungi count were used to test fungicidal effects against C. albicans. Coupon corrosion testing was used to evaluate disinfectants’ corrosive effects on stainless steel, copper, and aluminum. The mouse lymphoma assay was used to detect mutations induced by PB. Results and Discussion. Fungicidal activity of the combination of 40 mg/L PHMB and 40 mg/L BCDMH was comparable to, or even better than, those of 600 mg/L PHMB or 640 mg/L BCDMH alone. The combination of 400 mg/L PHMB and 400 mg/L BCDMH exhibited good fungicidal effects in field applications. The combination of 100 mg/L PHMB and 100 mg/L BCDMH did not have corrosive effects on stainless steel and no mutagenic effect was observed under the test conditions. Conclusions. The combination of PHMB and BCDMH has strong fungicidal effects and little metal corrosive and mutagenic effect and can be used as one suitable fungicide for wide household and industrial applications, including shipping containers. Bing Niu, Wan Huai, Zhirui Deng, and Qin Chen Copyright © 2017 Bing Niu et al. All rights reserved. In Vitro Activity of Lactobacilli with Probiotic Potential Isolated from Cocoa Fermentation against Gardnerella vaginalis Tue, 31 Oct 2017 10:07:04 +0000 Study of the probiotic potential of microorganisms isolated from fermented foods has been increasing, especially studies related to lactobacilli. In intestinal models, lactobacilli have demonstrated beneficial properties, such as anti-inflammatory activity and increased antibody production, but the molecular mechanisms involving probiotic and antagonistic action as well as their effect on human vaginal cells have not yet been fully elucidated. The aim of this study was to evaluate the functional and antagonistic properties of three strains of lactobacilli isolated from cocoa fermentation (Lactobacillus fermentum 5.2, L. plantarum 6.2, and L. plantarum 7.1) against Gardnerella vaginalis. Our results show that the lactobacilli have potential use as probiotics, since they have high hydrophobicity and autoaggregation properties and effectively adhere to vaginal cells. Metabolites secreted into the culture medium and whole cells of the strains under study are capable of interfering with the growth of G. vaginalis to different degrees. The elucidation of the antagonistic mechanisms as well as their effect on human cells may be useful in the development of a product containing such microorganisms or products secreted by them. Wallace Felipe Blohem Pessoa, Ana Clara Correia Melgaço, Milena Evangelista de Almeida, Louise Pereira Ramos, Rachel Passos Rezende, and Carla Cristina Romano Copyright © 2017 Wallace Felipe Blohem Pessoa et al. All rights reserved. Human Gut Microbiota Associated with Obesity in Chinese Children and Adolescents Sun, 29 Oct 2017 00:00:00 +0000 Objective. To investigate the gut microbiota differences of obese children compared with the control healthy cohort to result in further understanding of the mechanism of obesity development. Methods. We evaluated the 16S rRNA gene, the enterotypes, and quantity of the gut microbiota among obese children and the control cohort and learned the differences of the gut microbiota during the process of weight reduction in obese children. Results. In the present study, we learned that the gut microbiota composition was significantly different between obese children and the healthy cohort. Next we found that functional changes, including the phosphotransferase system, ATP-binding cassette transporters, flagellar assembly, and bacterial chemotaxis were overrepresented, while glycan biosynthesis and metabolism were underrepresented in case samples. Moreover, we learned that the amount of Bifidobacterium and Lactobacillus increased among the obese children during the process of weight reduction. Conclusion. Our results might enrich the research between gut microbiota and obesity and further provide a clinical basis for therapy for obesity. We recommend that Bifidobacterium and Lactobacillus might be used as indicators of healthy conditions among obese children, as well as a kind of prebiotic and probiotic supplement in the diet to be an auxiliary treatment for obesity. Ya-Ping Hou, Qing-Qing He, Hai-Mei Ouyang, Hai-Shan Peng, Qun Wang, Jie Li, Xiao-Fei Lv, Yi-Nan Zheng, Shao-Chuan Li, Hai-Liang Liu, and Ai-Hua Yin Copyright © 2017 Ya-Ping Hou et al. All rights reserved. Self-Conjugation of the Enteropathogenic Escherichia coli Adherence Factor Plasmid of Four Typical EPEC Isolates Thu, 26 Oct 2017 07:29:52 +0000 The enteropathogenic Escherichia coli (EPEC) adherence factor plasmid (pEAF) encodes the proteins involved in the biogenesis of the bundle-forming pilus (BFP), a key virulence factor that mediates microcolony formation and the localized adherence phenotype on the surface of the host enterocytes. The presence or absence of this plasmid defines typical EPEC (tEPEC) and atypical EPEC (aEPEC), respectively. Although lateral transfer of pEAF has been evidenced by phylogenetic studies, conjugal transfer ability has been experimentally established only for two pEAF plasmids from strains isolated in the late 60s. In the present work, we tested the self-conjugation ability of four pEAF plasmids from tEPEC strains isolated between 2007 and 2008 from children in Peru and the potential of aEPEC to receive them. A kanamycin resistance cassette was inserted into donor pEAF plasmids in order to provide a selectable marker in the conjugation experiments. Two aEPEC isolated from the same geographic region were used as recipient strains along with the laboratory E. coli DH5α strain. Here we show that the four pEAF plasmids tested are self-conjugative, with transfer frequencies in the range of 10−6 to 10−9. Moreover, the generation of aEPEC strains harboring pEAF plasmids provides valuable specimens to further perform functional studies. Claudia Silva, Crispín Zavala-Alvarado, and José L. Puente Copyright © 2017 Claudia Silva et al. All rights reserved. In Vitro Antibacterial Activity of Pomegranate Juice and Peel Extracts on Cariogenic Bacteria Wed, 25 Oct 2017 00:00:00 +0000 Aim. To evaluate the antimicrobial activity of hydroalcoholic extracts of pomegranate (Punica granatum L.) peel and juice, against the microorganisms considered the main etiologic agents of dental caries. Methods. The values of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined against Streptococcus mutans Clarke ATCC® 25175™ strain and Rothia dentocariosa clinical isolate. Results. Peel extracts inhibit effectively the growth and survival of S. mutans ATCC 25175 strain and R. dentocariosa clinical isolate with MIC and MBC values of 10 μg/μl and 15 μg/μl, respectively. Furthermore, the pomegranate juice extract showed high inhibitory activity against S. mutans ATCC 25175 strain with a MIC value of 25 μg/μl and a MBC value of 40 μg/μl, whereas, against R. dentocariosa, it has displayed a moderate inhibitory activity, with MIC and MBC values of 20 μg/μl and 140 μg/μl, respectively. Conclusions. In vitro microbiological tests demonstrate that the hydroalcoholic extracts of pomegranate juice and peel are able to contrast the main cariogenic bacteria involved in tooth decay. Although being preliminary data, our results suggest that pomegranate polyphenolic compounds could represent a good adjuvant for the prevention and treatment of dental caries. Gianmaria Fabrizio Ferrazzano, Elisa Scioscia, Daniela Sateriale, Gabiria Pastore, Roberta Colicchio, Chiara Pagliuca, Tiziana Cantile, Brunella Alcidi, Marco Coda, Aniello Ingenito, Elena Scaglione, Annunziata Gaetana Cicatiello, Maria Grazia Volpe, Michele Di Stasio, Paola Salvatore, and Caterina Pagliarulo Copyright © 2017 Gianmaria Fabrizio Ferrazzano et al. All rights reserved. Cucurbitaceae Seed Protein Hydrolysates as a Potential Source of Bioactive Peptides with Functional Properties Tue, 17 Oct 2017 07:20:34 +0000 Seeds from Cucurbitaceae plants (squashes, pumpkins, melons, etc.) have been used both as protein-rich food ingredients and nutraceutical agents by many indigenous cultures for millennia. However, relatively little is known about the bioactive components (e.g., peptides) of the Cucurbitaceae seed proteins (CSP) and their specific effects on human health. Therefore, this paper aims to provide a comprehensive review of latest research on bioactive and functional properties of CSP isolates and hydrolysates. Enzymatic hydrolysis can introduce a series of changes to the CSP structure and improve its bioactive and functional properties, including the enhanced protein solubility over a wide range of pH values. Small-sized peptides in CSP hydrolysates seem to enhance their bioactive properties but adversely affect their functional properties. Therefore, medium degrees of hydrolysis seem to benefit the overall improvement of bioactive and functional properties of CSP hydrolysates. Among the reported bioactive properties of CSP isolates and hydrolysates, their antioxidant, antihypertensive, and antihyperglycaemic activities stand out. Therefore, they could potentially substitute synthetic antioxidants and drugs which might have adverse secondary effects on human health. CSP isolates and hydrolysates could also be implemented as functional food ingredients, thanks to their favorable amino acid composition and good emulsifying and foaming properties. César Ozuna and Ma. Fabiola León-Galván Copyright © 2017 César Ozuna and Ma. Fabiola León-Galván. All rights reserved. A Review of the Combination Therapy of Low Frequency Ultrasound with Antibiotics Mon, 16 Oct 2017 00:00:00 +0000 Single antimicrobial therapy has been unable to resist the global spread of bacterial resistance. Literatures of available in vitro and in vivo studies were reviewed and the results showed that low frequency ultrasound (LFU) has a promising synergistic bactericidal effect with antibiotics against both planktonic and biofilm bacteria. It also can facilitate the release of antibiotics from medical implants. As a noninvasive and targeted therapy, LFU has great potential in treating bacterial infections. However, more in-depth and detailed studies are still needed before LFU is officially applied as a combination therapy in the field of anti-infective treatment. Yun Cai, Jin Wang, Xu Liu, Rui Wang, and Lei Xia Copyright © 2017 Yun Cai et al. All rights reserved. Effectiveness of Chinese Herbal Medicine Combined with Antibiotics for Extensively Drug-Resistant Enterobacteria and Nonfermentative Bacteria Infection: Real-Life Experience in a Retrospective Cohort Wed, 11 Oct 2017 00:00:00 +0000 Chinese herbal medicines (CHMs) have been successfully used in the treatment of infectious diseases, yet the effectiveness of CHMs for extensively drug-resistant enterobacteria (XDRE) infection remains unclear. Herein we developed a retrospective multicenter study including 766 patients with XDRE and nonfermentative bacteria (NFB) infection to investigate the effectiveness of CHMs combined with antibiotics in the treatment of XDRE infections in clinical daily practice in a cohort of patients and compared the regular antibiotics monotherapy. After 14-day treatment, the 547 patients accepted CHMs combined with antibiotics therapy indicating a more desirable effectiveness compared to the 219 patients treated with antibiotics monotherapy. The primary evaluation indexes included white blood cell count (WBC) and percentage of neutrophil (N%) in blood test. Secondary evaluation indexes consisted of body temperature, breath, heart rate, platelets, hemoglobin, red blood cell, albumin, creatinine, glucose, and 28-day survival rates. Briefly speaking, in our experience, CHMs combined with antibiotics therapy achieved more desirable effectiveness in treating XDRE infections compared with antibiotics monotherapy, and CHMs might be a potential huge resource in the field of XDRE infection management and enlighten the new antibiotics research and development. This trial is registered with ChiCTR-ORC-17011760. Yangping Cai, Qing Zhang, Yuefeng Fu, Li Li, Ning Zhao, Aiping Lu, Qingquan Liu, and Miao Jiang Copyright © 2017 Yangping Cai et al. All rights reserved. A Sustained-Release Membrane of Thiazolidinedione-8: Effect on Formation of a Candida/Bacteria Mixed Biofilm on Hydroxyapatite in a Continuous Flow Model Tue, 10 Oct 2017 00:00:00 +0000 Thiazolidinediones (TZDs) have been found to act as effective quorum sensing quenchers, capable of preventing biofilm formation. Our previous studies demonstrated a profound antibiofilm effect of the TZD derivative thiazolidinedione-8 (S-8), either in solution or incorporated into a sustained-release membrane (SRM-S-8) under batch conditions. In the present study, we used a constant depth film fermenter model in order to investigate the impact of SRM-S-8 on mixed C. albicans-S. mutans biofilm development, under flow conditions. We found that essential parameters of cospecies biofilm maintenance and maturation, such as metabolic activity, biofilm thickness, roughness, extracellular polysaccharides production, and morphology of both pathogens, were altered by SRM-S-8 in the flow system. We propose that prolonged and sustained release of S-8 in a flow-through system allows better penetration of the active agent to deeper layers of the mixed biofilm, thereby increasing its activity against both pathogens. In conclusion, the use of a locally applied sustained-release drug delivery system of S-8 can affect the dental polymicrobial biofilm, resulting in clinical improvements and a better patient compliance. Mark Feldman, Julia Shenderovich, Eran Lavy, Michael Friedman, and Doron Steinberg Copyright © 2017 Mark Feldman et al. All rights reserved. “Gold” Pressed Essential Oil: An Essay on the Volatile Fragment from Citrus Juice Industry By-Products Chemistry and Bioactivity Wed, 04 Oct 2017 00:00:00 +0000 Present essay explores the potentials of Citrus juice industry’s by-products as alternative bioactive natural products resources. Four crude Cold Pressed Essential Oils (CPEOs), derived from orange, lemon, grapefruit, and mandarin, were studied. All CPEOs were subjected to water distillation, in order to obtain the volatile fragment, which was further fractionated with respect to distillation period in two parts, concluding to eight samples. These samples along with the four original CPEOs were assessed in relation to their phytochemical content and their repellent and larvicidal properties against Asian Tiger Mosquito. The volatiles recovery rates ranged from 74% to 88% of the CPEO. Limonene presented a significant increase in all samples ranging from 8% to 52% of the respective CPEO’s content and peaked in mandarin’s 2nd volatile fragment which comprised 97% of the essential oil. The refinement process presented clear impacts on both bioassays: a significant increase in larvicidal potency was observed, annotated best by the improvement by 1100% and 1300% of the grapefruit volatile fractions; repellence testing provided only one significant result, the decrease of landings by 50% as a response to mandarin’s second volatile fraction. The applied methodology thus may be considered for the improvement of Citrus juice industry’s by-products chemistry and bioactivity. V. N. Kapsaski-Kanelli, E. Evergetis, A. Michaelakis, D. P. Papachristos, E. D. Myrtsi, S. D. Koulocheri, and S. A. Haroutounian Copyright © 2017 V. N. Kapsaski-Kanelli et al. All rights reserved. Effectiveness of a Lytic Phage SRG1 against Vancomycin-Resistant Enterococcus faecalis in Compost and Soil Sun, 24 Sep 2017 08:46:34 +0000 Nosocomial infections caused by vancomycin-resistant Enterococcus have become a major problem. Bacteriophage therapy is proposed as a potential alternative therapy. Bacteriophages are viruses that infect bacteria and are ubiquitous in nature. Lytic bacteriophage was isolated from sewage water that infects VREF, the causative agent of endocarditis, bacteraemia, and urinary tract infections (UTIs). The phage produced clear plaques with unique clear morphology and well-defined boundaries. TEM results of phage revealed it to be  nm long and  nm wide. The characterization of bacteriophage revealed that infection process of phage was calcium and magnesium dependent and phage titers were highest under optimum conditions for VREF, with an optimal temperature range of 37–50°C. The maximum growth was observed at 37°C, hence having 100% viability. The latent period for phage was small with a burst size of 512 viral particles per bacterial cell. The phage was tested against various clinical strains and results proved it to be host specific. It can be used as a potential therapeutic agent for VREF infections. The phage efficiently eradicated VREF inoculated in cattle compost, poultry compost, and a soil sample which makes it a potential agent for clearing compost and soil sample. Sidra Rahmat Ullah, Saadia Andleeb, Taskeen Raza, Muhsin Jamal, and Khalid Mehmood Copyright © 2017 Sidra Rahmat Ullah et al. All rights reserved. Corrigendum to “Nicotine Enhances Interspecies Relationship between Streptococcus mutans and Candida albicans” Wed, 20 Sep 2017 00:00:00 +0000 Shiyu Liu, Wei Qiu, Keke Zhang, Xuedong Zhou, Biao Ren, Jinzhi He, Xin Xu, Lei Cheng, and Mingyun Li Copyright © 2017 Shiyu Liu et al. All rights reserved. Serological Analysis and Drug Resistance of Chlamydia pneumoniae and Mycoplasma pneumoniae in 4500 Healthy Subjects in Shenzhen, China Tue, 19 Sep 2017 07:08:32 +0000 Objective. To understand the prevalence and distribution of Chlamydia pneumoniae (CP) and Mycoplasma pneumoniae (MP) in the population and to provide a basis for the prevention and treatment of respiratory tract infection. Methods. This study included a total of 4500 healthy subjects who were given physical examination in Shenzhen People’s Hospital from January to December in 2016. Venous blood was drawn from people to detect the MP- and CP-specific IgG and IgM in the serum using chemiluminescence immunoassay (CLIA). The relationship of MP and CP infections with patient age, seasons, and percentage of infections was analyzed. Conclusion. CP and MP cause high rate of asymptomatic infection, which may be associated with the high incidence of CP and MP infection, especially in children and the elderly population. Therefore, the implementation of effective and practical prevention measures has become an urgent need. MP culture and drug sensitivity test should be performed as early as possible in patients with manifested MP infections in order to ensure timely and proper treatment and to reduce the emergence of drug-resistant strains. Dong Du, Shuping Liao, Yan Wu, Yang Jiao, Di Wu, Weiqing Wu, and Dapeng Yu Copyright © 2017 Dong Du et al. All rights reserved. Royal Jelly Inhibits Pseudomonas aeruginosa Adherence and Reduces Excessive Inflammatory Responses in Human Epithelial Cells Sun, 17 Sep 2017 08:25:10 +0000 Pseudomonas aeruginosa is a Gram-negative bacterium and causes respiratory infection especially in elderly patients. Royal jelly has been used worldwide as a traditional remedy and as a nutrient; however, the effect against P. aeruginosa is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against P. aeruginosa. Wild-type strain PAO1 and clinical isolates of P. aeruginosa were used for antibacterial assay and antiadherent assay to abiotic surface and epithelial cells, which are pharynx (Detroit 562) and lung (NCI-H292) epithelial cells. In anti-inflammatory assay, epithelial cells were pretreated with royal jelly before bacterial exposure to investigate its inhibitory effect on interleukin (IL-8) and macrophage inflammatory protein-3α/CCL20 overproduction. Although royal jelly did not have antibacterial activity at concentration of 50% w/v, antiadherent activity was confirmed on the abiotic surface and epithelial cells under concentration of 25%. Pretreatment with royal jelly significantly inhibited overproduction of IL-8 and CCL20 from both cells. These results demonstrated that royal jelly inhibits P. aeruginosa adherence and protects epithelial cells from excessive inflammatory responses against P. aeruginosa infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by P. aeruginosa. Heni Susilowati, Keiji Murakami, Hiromichi Yumoto, Takashi Amoh, Kouji Hirao, Katsuhiko Hirota, Takashi Matsuo, and Yoichiro Miyake Copyright © 2017 Heni Susilowati et al. All rights reserved. Examination of Oral Microbiota Diversity in Adults and Older Adults as an Approach to Prevent Spread of Risk Factors for Human Infections Sun, 10 Sep 2017 08:21:11 +0000 The oral cavity environment may be colonized by polymicrobial communities with complex, poorly known interrelations. The aim of this study was to determine oral microbiota diversity in order to prevent the spread of infectious microorganisms that are risk factors for human health complications in patients requiring treatment due to various disabilities. The study examined Polish adults aged between 40 and 70 years; parasitological, microbiological, and mycological data collected before treatment were analyzed. The diversity of oral microbiota, including relatively high prevalences of some opportunistic, potentially pathogenic strains of bacteria, protozoans, and fungi detected in the patients analyzed, may result in increasing risk of disseminated infections from the oral cavity to neighboring structures and other organs. Increasing ageing of human populations is noted in recent decades in many countries, including Poland. The growing number of older adults with different oral health disabilities, who are more prone to development of oral and systemic pathology, is an increasing medical problem. Results of this retrospective study showed the urgent need to pay more attention to the pretreatment examination of components of the oral microbiome, especially to the strains, which are etiological agents of human opportunistic infections and are particularly dangerous for older adults. Paweł J. Zawadzki, Konrad Perkowski, Marcin Padzik, Elżbieta Mierzwińska-Nastalska, Jacek P. Szaflik, David Bruce Conn, and Lidia Chomicz Copyright © 2017 Paweł J. Zawadzki et al. All rights reserved. Mycochemical Characterization of Agaricus subrufescens considering Their Morphological and Physiological Stage of Maturity on the Traceability Process Sun, 10 Sep 2017 07:27:45 +0000 Agaricus subrufescens Peck is a basidiomycete with immunomodulatory compounds and antitumor activities. This research evaluated the mycochemical composition of A. subrufescens, considering their morphological and physiological stage of maturity, with a particular focus on the development of a traceability process for the formulation of new nutritional products based on fungal foods. The stipes contained a high amount of dry matter (10.33%), total carbohydrate (69.56%), available carbohydrate (63.89%), and energy value (363.97 kcal 100 g−1 DM). The pilei contained a high amount of moisture (90.66%), nitrogen (7.75%), protein (33.96%), ash (8.24), crude fat (2.44%), acid detergent fiber (16.75 g kg−1), neutral detergent fiber (41.82 g kg−1), hemicellulose (25.07 g kg−1), and lignin (9.77 g kg−1). Stipes with mature physiological stage had higher values of dry matter (10.50%), crude fiber (5.94%), total carbohydrate (72.82%), AC (66.88%), and energy value (364.91 kcal 100 g−1 DM). Pilei of the mushrooms in the immature physiological stage had higher values of P (36.83%), N (8.41%), and A (8.44%). Due to the differences between the mycochemical compositions of the morphological parts of mushrooms linked to their physiological stage of maturity, such characteristics have immense potential to be considered for a traceability process. This study can be used for the purpose of providing the consumer with more product diversity, optimizing bioactivities of composts, and allowing farmers an efficient and profitable use of the mushroom biomass. Diego Cunha Zied, Jose E Pardo, Rafael Simões Tomaz, Celso Tadao Miasaki, and Arturo Pardo-Giménez Copyright © 2017 Diego Cunha Zied et al. All rights reserved. Renaissance of Conventional First-Line Antibiotics in Salmonella enterica Clinical Isolates: Assessment of MICs for Therapeutic Antimicrobials in Enteric Fever Cases from Nepal Tue, 05 Sep 2017 00:00:00 +0000 Enteric fever caused by Salmonella enterica is a life-threatening systemic illness of gastrointestinal tract especially in tropical countries. Antimicrobial therapy is generally indicated but resistance towards commonly used antibiotics has limited their therapeutic usefulness. Therefore, we aimed to determine the antimicrobial susceptibility pattern by minimum inhibitory concentration method of common therapeutic regimens against Salmonella enterica from enteric fever clinical cases. Salmonella enterica clinical isolates recovered from the patients with suspected enteric fever whose blood samples were submitted to microbiology laboratory of Manmohan Memorial Community Hospital, Kathmandu, from March 2016 to August 2016, were studied. These isolates were subjected to antimicrobial susceptibility testing against common therapeutic antimicrobials by Kirby-Bauer disk diffusion method. The minimum inhibitory concentration of ciprofloxacin, azithromycin, chloramphenicol, and cefixime was determined by Agar dilution method based on the latest CLSI protocol. A total of 88 isolates of Salmonella enterica were recovered from blood samples of enteric fever cases. Out of them, 74 (84.09%) were Salmonella Typhi and 14 (15.91%) were Salmonella Paratyphi A. On Kirby-Bauer disk diffusion antimicrobial susceptibility testing, entire isolates were susceptible to cotrimoxazole, cefixime, ceftriaxone, azithromycin, and chloramphenicol. Sixty-four (72.7%) Salmonella enterica isolates were nalidixic acid resistant and nonsusceptible to ciprofloxacin and levofloxacin. On MIC determination, four Salmonella isolates were ciprofloxacin resistant with MIC 1 µg/ml and two isolates were ciprofloxacin intermediate with MIC 0.5 µg/ml. The MIC range of azithromycin was from 0.125 µg/ml to 2.0 µg/ml, whereas that for chloramphenicol was 2.0 µg/ml–8.0 µg/ml and for cefixime was 0.0075–0.5 µg/ml, respectively. Despite global surge of antimicrobial resistance among Salmonella enterica clinical isolates, the level of drug resistance in our study was not so high. However, higher level of NARST strains limits therapeutic use of fluoroquinolones and necessitates the routine monitoring of such resistance determinants in order to effectively and rationally manage enteric fever cases. Puspa Raj Khanal, Deepa Satyal, Anjeela Bhetwal, Anjila Maharjan, Shreena Shakya, Snehika Tandukar, and Narayan Prasad Parajuli Copyright © 2017 Puspa Raj Khanal et al. All rights reserved. Effectiveness of the Thermal Treatments Used for Curd Stretching in the Inactivation of Shiga Toxin-Producing O157 and O26 Escherichia coli Tue, 22 Aug 2017 07:17:48 +0000 The kneading treatment of the fresh curd in hot water is a critical control point in the manufacturing of mozzarella. Factors such as the ratio between hot water and curd mass, the rheological properties, and the mixing and kneading activity affect the processing time and the internal temperature of the curd. The aim of this study was to investigate the effect of thermal treatments on the fate of Shiga toxin-producing Escherichia coli (STEC). Nine curd samples (weight 160–270 g) were artificially contaminated with O157 or O26 STEC and stretched in hot water (90–95°C) for 5–10 min. Depending on the heating process and spinning, different nonisothermal profiles were recorded. Observed reductions of O157 and O26 STEC varied between 1.01 and more than 5.38  (Most Probable Number)/g at the end of the temperature treatments. Further, nonisothermal log-linear tail models were developed to compare observed reductions for O157 and O26 VTEC under variable temperature conditions. Results obtained showed that the comparison of predictions provided by the dynamic model with observations described well the linear inactivation pattern since nonsignificant differences were denoted at all profiles tested. The dynamic model developed can be useful to evaluate the effectiveness of the thermal treatments used in the manufacturing of mozzarella in the inactivation of STEC. M. Trevisani, A. Valero, and R. Mancusi Copyright © 2017 M. Trevisani et al. All rights reserved. Effects of Bacillus Serine Proteases on the Bacterial Biofilms Mon, 21 Aug 2017 00:00:00 +0000 Serratia marcescens is an emerging opportunistic pathogen responsible for many hospital-acquired infections including catheter-associated bacteremia and urinary tract and respiratory tract infections. Biofilm formation is one of the mechanisms employed by S. marcescens to increase its virulence and pathogenicity. Here, we have investigated the main steps of the biofilm formation by S. marcescens SR 41-8000. It was found that the biofilm growth is stimulated by the nutrient-rich environment. The time-course experiments showed that S. marcescens cells adhere to the surface of the catheter and start to produce extracellular polymeric substances (EPS) within the first 2 days of growth. After 7 days, S. marcescens biofilms maturate and consist of bacterial cells embedded in a self-produced matrix of hydrated EPS. In this study, the effect of Bacillus pumilus 3-19 proteolytic enzymes on the structure of 7-day-old S. marcescens biofilms was examined. Using quantitative methods and scanning electron microscopy for the detection of biofilm, we demonstrated a high efficacy of subtilisin-like protease and glutamyl endopeptidase in biofilm removal. Enzymatic treatment resulted in the degradation of the EPS components and significant eradication of the biofilms. Olga Mitrofanova, Ayslu Mardanova, Vladimir Evtugyn, Lydia Bogomolnaya, and Margarita Sharipova Copyright © 2017 Olga Mitrofanova et al. All rights reserved. Prevalence of Smear-Positive Tuberculosis among Patients Who Visited Saint Paul’s Specialized Hospital in Addis Ababa, Ethiopia Mon, 21 Aug 2017 00:00:00 +0000 Background. Tuberculosis (TB) continues to be a health problem in both developed and developing countries, including Ethiopia. Objective. In this study, the prevalence of smear-positive tuberculosis among presumptive TB cases who visited the hospital was assessed. Method. Acid fast bacilli (AFB) test was performed on samples collected from 200 presumptive TB cases. Data were analyzed using appropriate statistical tools. Result. Among 200 presumptive TB cases, 10% (20 individuals) (60% were male and 40% were female) were found to be positive for the AFB. Of these AFB positive subjects, 11.2% and 6.3% were from urban and rural areas, respectively. Among 20 AFB positive cases, 45% (9), 45% (9), and 10% (2) were HIV positive, HIV negative, and with HIV status unknown, respectively. The highest AFB positive cases were found within age group between 25 and 44 years (70%) and followed by age above 40 years (30%). It was found out that 75% (15), 15% (3), 5% (1), and 5% (1) were unemployed, government employed, student, and nongovernment employed, accordingly. Conclusion. This study indicated higher level of AFB positive cases within age groups of 25–44 and 65–74 years and also exhibited higher prevalence of TB cases from urban areas. Dinna Abera Nugussie, Getachew Ali Mohammed, and Anteneh Tesfaye Tefera Copyright © 2017 Dinna Abera Nugussie et al. All rights reserved. Transcriptomic Analysis Reveals Genes Mediating Salt Tolerance through Calcineurin/CchA-Independent Signaling in Aspergillus nidulans Sun, 20 Aug 2017 00:00:00 +0000 Adaptation to changes in the environment is crucial for the viability of all organisms. Although the importance of calcineurin in the stress response has been highlighted in filamentous fungi, little is known about the involvement of ion-responsive genes and pathways in conferring salt tolerance without calcium signaling. In this study, high-throughput RNA-seq was used to investigate salt stress-induced genes in the parent, ΔcnaB, and ΔcnaBΔcchA strains of Aspergillus nidulans, which differ greatly in salt adaption. In total, 2,884 differentially expressed genes including 1,382 up- and 1,502 downregulated genes were identified. Secondary transporters, which were upregulated to a greater extent in ΔcnaBΔcchA than in the parent or ΔcnaB strains, are likely to play important roles in response to salt stress. Furthermore, 36 genes were exclusively upregulated in the ΔcnaBΔcchA under salt stress. Functional analysis of differentially expressed genes revealed that genes involved in transport, heat shock protein binding, and cell division processes were exclusively activated in ΔcnaBΔcchA. Overall, our findings reveal that secondary transporters and stress-responsive genes may play crucial roles in salt tolerance to bypass the requirement for the CchA-calcineurin pathway, contributing to a deeper understanding of the mechanisms that influence fungal salt stress adaption in Aspergillus. Sha Wang, Hongchang Zhou, Jun Wu, Jiangyu Han, Shasha Li, and Shengwen Shao Copyright © 2017 Sha Wang et al. All rights reserved. Novel Microbial Diagnostic Methods for Clinical, Environmental, and Food Samples Mon, 31 Jul 2017 00:00:00 +0000 Pratik Banerjee, Irshad M. Sulaiman, György Schneider, Udayan Ray, and Bala Jagadeesan Copyright © 2017 Pratik Banerjee et al. All rights reserved. Mixed Lactobacillus plantarum Strains Inhibit Staphylococcus aureus Induced Inflammation and Ameliorate Intestinal Microflora in Mice Thu, 27 Jul 2017 00:00:00 +0000 Objective. Staphylococcus aureus is an important pathogen that causes intestinal infection. We examined the immunomodulatory function of single and mixed Lactobacillus plantarum strains, as well as their impacts on the structure of the microbiome in mice infected with Staphylococcus aureus. The experiment was divided into three groups: protection, treatment, and control. Serum IFN-γ and IL-4 levels, as well as intestinal sIgA levels, were measured during and 1 week after infection with Staphylococcus aureus with and without Lactobacillus plantarum treatment. We used 16s rRNA tagged sequencing to analyze microbiome composition. IFN-γ/IL-4 ratio decreased significantly from infection to convalescence, especially in the mixed Lactobacillus plantarum group. In the mixed Lactobacillus plantarum group the secretion of sIgA in the intestine of mice (9.4–9.7 ug/mL) was significantly higher than in the single lactic acid bacteria group. The dominant phyla in mice are Firmicutes, Bacteroidetes, and Proteobacteria. Treatment with mixed lactic acid bacteria increased the anti-inflammatory factor and the secretion of sIgA in the intestine of mice infected with Staphylococcus aureus and inhibited inflammation. Dayong Ren, Shengjie Gong, Jingyan Shu, Jianwei Zhu, Fengjun Rong, Zhenye Zhang, Di Wang, Liangfeng Gao, Tianming Qu, Hongyan Liu, and Ping Chen Copyright © 2017 Dayong Ren et al. All rights reserved. Functional Profile Evaluation of Lactobacillus fermentum TCUESC01: A New Potential Probiotic Strain Isolated during Cocoa Fermentation Thu, 20 Jul 2017 07:27:50 +0000 The use of intestinal probiotic bacteria is very common in the food industry and has been the focus of the majority of research in this field. Yet in recent years, research on extraintestinal microorganisms has greatly increased due to their well-known potential as probiotics. Thus, we studied a strain of Lactobacillus fermentum (TCUESC01) extracted from fermenting cocoa. First, we examined the impact of pH on the growth of this strain and studied its survival under conditions similar to those of the human gastrointestinal tract. L. fermentum TCUESC01 demonstrated resistance to conditions mimicking the human stomach and intestines and grew well between pH 5 and pH 7. Next, we subjected L. fermentum TCUESC01 to storage at 4°C in a milk solution and found that it survived well for 28 days. Lastly, we measured the susceptibility of this strain to numerous antibiotics and its tendency to autoaggregate. L. fermentum TCUESC01 showed significant autoaggregation, as well as susceptibility to the majority of antibiotics tested. Overall, our findings support the potential use of this extraintestinal bacterium as a dietary probiotic. Tauá Alves Melo, Thalis Ferreira dos Santos, Lennon Ramos Pereira, Hélic Moreira Passos, Rachel Passos Rezende, and Carla Cristina Romano Copyright © 2017 Tauá Alves Melo et al. All rights reserved. Integron-Mediated Antibiotic Resistance in Acinetobacter baumannii Isolated from Intensive Care Unit Patients, Babol, North of Iran Wed, 19 Jul 2017 08:00:55 +0000 Background. We investigated the integron types and their relation with antibiotic resistance among A. baumannii isolates collected from intensive care unit patients, Babol, north of Iran. Methods. In this cross-sectional study, a total of 73 bronchoalveolar lavage samples were obtained from patients in ICU. Susceptibility testing was performed by disk diffusion method. Types of integrons were identified by an integrase gene PCR. Results. In total, 47.9% A. baumannii isolates were recovered from the BAL samples. All isolates were resistant to ceftazidime. 91.4% and 58.3% of isolates were MDR and XDR, respectively. The rate of colistin resistance with the -test was 5.7%. Molecular analysis of class I, II, and III integrons showed that 25.7%, 88.6%, and 28.6% of the isolates carried the intI, intII, and intIII genes, respectively. Discussion. Our results show that different classes of integrons are commonly spread among A. baumannii strains and these genomic segments can play an important role in the acquisition of MDR and XDR phenotypes. So monitoring drug resistance in A. baumannii isolates with the use of int gene PCR is very important to plan specific infection control measures to prevent the spread of MDR-AB and XDR-AB in Iran’s hospitals. Mitra Deylam Salehi, Elaheh Ferdosi-Shahandashti, Yosef Yahyapour, Soraya Khafri, Abazar Pournajaf, and Ramazan Rajabnia Copyright © 2017 Mitra Deylam Salehi et al. All rights reserved. Comment on “Application of PK/PD Modeling in Veterinary Field: Dose Optimization and Drug Resistance Prediction” Tue, 18 Jul 2017 09:12:53 +0000 Anna Benini and Guido Francesco Fumagalli Copyright © 2017 Anna Benini and Guido Francesco Fumagalli. All rights reserved. Comparative Phenotype and Genome Analysis of Cellvibrio sp. PR1, a Xylanolytic and Agarolytic Bacterium from the Pearl River Mon, 17 Jul 2017 08:52:40 +0000 Cellvibrio sp. PR1 is a xylanolytic and agarolytic bacterium isolated from the Pearl River. Strain PR1 is closely related to Cellvibrio fibrivorans and C. ostraviensis (identity > 98%). The xylanase and agarase contents of strain PR1 reach up to 15.4 and 25.9 U/mL, respectively. The major cellular fatty acids consisted of C16:0 (36.7%), C18:0 (8.8%), C20:0 (6.8%), C15:0 iso 2-OH or/and C16:1ω7c (17.4%), and C18:1ω7c or/and C18:1ω6c (6.7%). A total of 251 CAZyme modules (63 CBMs, 20 CEs, 128 GHs, 38 GTs, and 2 PLs) were identified from 3,730 predicted proteins. Genomic analysis suggested that strain PR1 has a complete xylan-hydrolyzing (5 β-xylanases, 16 β-xylosidases, 17 α-arabinofuranosidases, 9 acetyl xylan esterases, 4 α-glucuronidases, and 2 ferulic acid esterases) and agar-hydrolyzing enzyme system (2 β-agarases and 2 α-neoagarooligosaccharide hydrolases). In addition, the main metabolic pathways of xylose, arabinose, and galactose are established in the genome-wide analysis. This study shows that strain PR1 contains a large number of glycoside hydrolases. Zhangzhang Xie, Weitie Lin, and Jianfei Luo Copyright © 2017 Zhangzhang Xie et al. All rights reserved. Thiocarbamates from Moringa oleifera Seeds Bioactive against Virulent and Multidrug-Resistant Vibrio Species Sun, 09 Jul 2017 08:22:16 +0000 Prospect of antibacterial agents may provide an alternative therapy for diseases caused by multidrug-resistant bacteria. This study aimed to evaluate the in vitro bioactivity of Moringa oleifera seed extracts against 100 vibrios isolated from the marine shrimp Litopenaeus vannamei. Ethanol extracts at low (MOS-E) and hot (MOS-ES) temperature are shown to be bioactive against 92% and 90% of the strains, respectively. The most efficient Minimum Inhibitory Concentration (MIC) levels of MOS-E and MOS-ES against a high percentage of strains were 32 µg mL−1. Bioguided screening of bioactive compounds showed that the ethyl acetate fraction from both extracts was the only one that showed antibacterial activity. Vibriocidal substances, niazirine and niazimicine, were isolated from the aforementioned fraction through chromatographic fractionation. Renata Albuquerque Costa, Oscarina Viana de Sousa, Ernesto Hofer, Jair Mafezoli, Francisco Geraldo Barbosa, and Regine Helena Silva dos Fernandes Vieira Copyright © 2017 Renata Albuquerque Costa et al. All rights reserved. Dissemination and Molecular Characterization of Staphylococcus aureus at a Tertiary Referral Hospital in Xiamen City, China Wed, 05 Jul 2017 00:00:00 +0000 Staphylococcus aureus is a global epidemic pathogen that causes heavy disease burden. The aim of this study was to determine which globally known S. aureus lineages are currently present in a hospital of Xiamen. Therefore, the 426 S. aureus strains were detected by Melting Curve Analysis (MCA) and genotyped by Pulsed Field Gel Electrophoresis (PFGE) as well as Multicolor Melting Curve Analysis-Based Multilocus Melt Typing (MLMT). In addition, Multilocus Sequence Typing (MLST) was used to identify 108 representative strains. In light of eighteen antibiotics except for Vancomycin (by Broth Dilution Method), we used the Kirby-Bauer disc diffusion method to assess antibiotic susceptibility of 426 S. aureus strains. Finally, PFGE analysis revealed 14 different patterns with three major patterns (C10, C8, and C11) that accounted for 69.42% of all S. aureus strains, and MT-1~MT-5 occupied most part of the strains by MLMT. MLST revealed 25 different STs with the predominant types being ST239, ST59, and ST188. There have been 8 antibiotics that showed more than 50% resistance of all S. aureus strains. In summary, we found several of the lineages are predominant in our hospital. And antibiotic resistance is still a severe problem that needs to be controlled in clinic. Yiwen Yu, Yihui Yao, Qinyun Weng, Jingyi Li, Jianwei Huang, Yiqun Liao, Fu Zhu, Qifeng Zhao, Xu Shen, and Jianjun Niu Copyright © 2017 Yiwen Yu et al. All rights reserved. Characterization of Oral Microbiota in Removable Dental Prosthesis Users: Influence of Arterial Hypertension Wed, 21 Jun 2017 09:24:30 +0000 Introduction. Studies have described the possible relation between oral infections and atherosclerotic events. Objective. To characterize the oral microbiota of normotensive and hypertensive users of dental prostheses. Methods. The sample consisted of 41 complete dental prosthesis users, divided into groups: 21 participants with systemic arterial hypertension and 20 normotensive participants. The data collection included the characteristics of the sociodemographic variables and the determination of the microbial load in the saliva. For the descriptive analyses, Statistical Package for the Social Sciences was used. The description of the proportional differences between the groups was based on the application of Mann–Whitney’s statistical test. Statistical significance was set at 5% (p < 0.05). Results. The analysis of the oral microbiota showed the vast growth of aerobic microorganisms in all samples from both groups. The microbial load of streptococci and staphylococci was significantly higher among hypertensive participants. Candida yeasts were detected in the saliva culture of most samples. The hypertensive participants rank in the category of very high colonization index/high risk of infection related to this microorganism. Conclusions. The mouth of dental prosthesis users, especially when hypertensive, can constitute an important reservoir of pathogens, indicating an established inflammatory or infectious condition or risk for developing this condition. Leila Maria Marchi-Alves, Dayana Freitas, Denise de Andrade, Simone de Godoy, Adrielle Naiara Toneti, and Isabel Amélia Costa Mendes Copyright © 2017 Leila Maria Marchi-Alves et al. All rights reserved. Bacterial Community Profile of the Gut Microbiota Differs between Hypercholesterolemic Subjects and Controls Thu, 15 Jun 2017 08:00:18 +0000 The role of gut microbiota in the development of metabolic illnesses has been abundantly demonstrated. Recent studies suggest that gut microbiota alterations may also be related to the development of hypercholesterolemia. Therefore, we aimed to assess differences in the gut bacterial community profiles between hypercholesterolemic subjects and controls. Thirty cases diagnosed with hypercholesterolemia and 27 normocholesterolemic controls were included. A fasting whole blood sample was obtained to determine the lipid profile. In parallel, stool samples were collected and total DNA was isolated to assess the bacterial community profiles by denaturing gradient gel electrophoresis (DGGE). In addition, the Richness, Shannon-Weaver, and Simpson indexes were used to evaluate the richness and diversity of bacterial communities. As expected, serum concentrations of total cholesterol, triglycerides, and LDL-cholesterol were significantly higher in the cases compared with controls. Moreover, DGGE analysis showed a lower richness and diversity of bacterial communities in hypercholesterolemic subjects. In conclusion, our results showed differences in the profiles of bacterial communities between hypercholesterolemic subjects and controls, suggesting a possible role of the gut microbiota in the development of hypercholesterolemia. Camilo Rebolledo, Alejandro Cuevas, Tomás Zambrano, Jacquelinne J. Acuña, Milko A. Jorquera, Kathleen Saavedra, Claudia Martínez, Fernando Lanas, Pamela Serón, Luis A. Salazar, and Nicolás Saavedra Copyright © 2017 Camilo Rebolledo et al. All rights reserved. Purification and Characterization of an ATPase GsiA from Salmonella enterica Mon, 12 Jun 2017 09:05:08 +0000 The coding sequence of Salmonella enterica gsiA was cloned and expressed in E. coli. The protein was purified and ATPase activity was characterized by NADH oxidation method. GsiA exhibited optimum activity at 30°C and at pH 8 in Tris/HCl buffer. GsiA protein was stable at 20°C. 66% and 44% activity remained after incubation at 30°C and 40°C for 30 min. pH 7 and pH 9 incubation would obviously reduce the ATPase activity. In vivo functionality of gsiA was determined by constructing gene deletion strains. gsiA was shown to be essential for GSI mediated glutathione uptake and gsiA deletion could decrease the virulence of Salmonella enterica. Interactions of glutathione import proteins GsiA, GsiB, GsiC, and GsiD were investigated by using bacterial two-hybrid system. GsiA could interact with itself and inner membrane proteins GsiC and GsiD. This report provides the first description of gsiA functions in Salmonella enterica. The results could help elucidating the glutathione uptake mechanism and glutathione functions in bacteria. Zhongshan Wang, Meng Zhang, Xiaodong Shi, and Quanju Xiang Copyright © 2017 Zhongshan Wang et al. All rights reserved. Production of a Functional Frozen Yogurt Fortified with Bifidobacterium spp. Sun, 11 Jun 2017 00:00:00 +0000 Frozen dairy products have characteristics of both yogurt and ice cream and could be the persuasive carriers of probiotics. Functions of the frozen yogurt containing viable bifidobacterial cells are recognized and favored by the people of all ages. We developed a kind of yogurt supplemented by Bifidobacterium species. Firstly, five strains of Bifidobacterium spp. (Bifidobacterium bifidum ATCC 11547, Bifidobacterium longum ATCC 11549, Bifidobacterium infantis ATCC 11551, Bifidobacterium adolescentis ATCC 11550, and Bifidobacterium breve ATCC 11548) were evaluated based on the feasibility criteria of probiotics, comprising acid production, bile tolerance, and adhesion to epithelial cells. Formerly, we combined the optimum strains with yogurt culture (Lactobacillus delbrueckii subsp. bulgaricus EMCC 11102 and Streptococcus salivarius subsp. thermophilus EMCC 11044) for producing frozen yogurt. Finally, physiochemical properties and sensory evaluation of the frozen yogurt were investigated during storage of 60 days at −18°C. Results directed that Bifidobacterium adolescentis ATCC 11550 and Bifidobacterium infantis ATCC 11551 could be utilized with yogurt culture for producing frozen yogurt. Moreover, the frozen yogurt fermented by two bifidobacterial strains and yogurt culture gained the high evaluation in the physiochemical properties and sensory evaluation. In summary, our results revealed that there was no significant difference between frozen yogurt fermented by Bifidobacterium spp. and yogurt culture and that fermented by yogurt culture only. Amro Abdelazez, Zafarullah Muhammad, Qiu-Xue Zhang, Zong-Tao Zhu, Heba Abdelmotaal, Rokayya Sami, and Xiang-Chen Meng Copyright © 2017 Amro Abdelazez et al. All rights reserved. Experimental Germ Tube Induction in Candida albicans: An Evaluation of the Effect of Sodium Bicarbonate on Morphogenesis and Comparison with Pooled Human Serum Mon, 05 Jun 2017 08:10:04 +0000 Objective. The potential of NaHCO3 versus human serum to induce germ tube formation in Candida albicans was investigated. Specimens. A total of 100 isolates were obtained from oral swabs of patients presenting with thrush. Approval for the study was granted by the Joint Research Ethics Committee (JREC/23/08). Method. Confirmed C. albicans isolates by routine methods were tested for germ tube induction using 5 different concentrations of Tris-maleate buffered NaHCO3 and Tris-maleate buffer control. Standard control strains included were C. albicans (ATCC 10231) and C. krusei (ATCC 6258). Microculture was done in 20 μL inoculums on microscope slides for 3 hours at 37°C. The rate of germ tube formation at 10-minute intervals was determined on 100 isolates using the optimum 20 mM Tris-maleate buffered NaHCO3 concentration. Parallel germ tube formation using human serum was done in test tubes. Results. The optimum concentration of NaHCO3 in Tris-maleate buffer for germ tube induction was 20 mM for 67% of isolates. Only 21% of isolates formed germ tubes in Tris-maleate buffer control. There was no significant difference in induction between human serum and Tris-maleate buffered NaHCO3. Conclusion. Tris-maleate buffered NaHCO3 induced germ tube formation in C. albicans isolates at rates similar to human serum. Tapiwa Matare, Pasipanodya Nziramasanga, Lovemore Gwanzura, and Valerie Robertson Copyright © 2017 Tapiwa Matare et al. All rights reserved. Magnetic Separation Methods for the Detection of Mycobacterium avium subsp. paratuberculosis in Various Types of Matrices: A Review Wed, 31 May 2017 00:00:00 +0000 The main reasons to improve the detection of Mycobacterium avium subsp. paratuberculosis (MAP) are animal health and monitoring of MAP entering the food chain via meat, milk, and/or dairy products. Different approaches can be used for the detection of MAP, but the use of magnetic separation especially in conjunction with PCR as an end-point detection method has risen in past years. However, the extraction of DNA which is a crucial step prior to PCR detection can be complicated due to the presence of inhibitory substances. Magnetic separation methods involving either antibodies or peptides represent a powerful tool for selective separation of target bacteria from other nontarget microorganisms and inhibitory sample components. These methods enable the concentration of pathogens present in the initial matrix into smaller volume and facilitate the isolation of sufficient quantities of pure DNA. The purpose of this review was to summarize the methods based on the magnetic separation approach that are currently available for the detection of MAP in a broad range of matrices. Marketa Husakova, Radka Dziedzinska, and Iva Slana Copyright © 2017 Marketa Husakova et al. All rights reserved. Comparison of Antibacterial Activity of Lactobacillus plantarum Strains Isolated from Two Different Kinds of Regional Cheeses from Poland: Oscypek and Korycinski Cheese Wed, 24 May 2017 07:16:56 +0000 Oscypek and korycinski are traditional Polish cheeses, exclusively produced in Tatra and in Podlasie region, respectively, produced from raw, unpasteurized milk. The 29 Lactobacillus plantarum strains were isolated on MRS agar from 12 cheese samples and used as a material for study. The main purpose of the work was to assess the antimicrobial properties and recognition of selected strains for the unique antagonistic activity and preservation role in food. It has been found that the highest antimicrobial activity was observed in the case of L. monocytogenes strains; however, the level of that activity was different depending on the Lb. plantarum strain. Strains from oscypek produced broad spectrum, and a few strains isolated from korycinski cheese produced a narrow spectrum of antimicrobial compounds, other than organic acids and hydrogen peroxide. Moreover, the antagonistic activity shown by Lb. plantarum strains is connected with the source from which a given strain was isolated. Strains isolated from oscypek cheese represented stronger activity against L. monocytogenes, whereas strains isolated from korycinski cheese were more active against E. coli. Strains Lb. plantarum Os13 and Kor14 could be considered as good candidates for protective cultures to extend durability of food products. Aleksandra Ołdak, Dorota Zielińska, Anna Rzepkowska, and Danuta Kołożyn-Krajewska Copyright © 2017 Aleksandra Ołdak et al. All rights reserved. Antimicrobial Activity of Quinazolin Derivatives of 1,2-Di(quinazolin-4-yl)diselane against Mycobacteria Mon, 22 May 2017 00:00:00 +0000 Mycobacterium tuberculosis (M. tuberculosis) is one of the leading causes of morbidity and mortality. Currently, the emergence of drug resistance has an urgent need for new drugs. In previous study, we found that 1,2-di(quinazolin-4-yl)diselane (DQYD), a quinazoline derivative, has anticancer activities against many cancers. However, whether DQYD has the activity of antimycobacterium is still little known. Here our results show that DQYD has a similar value of the minimum inhibitory concentration with clinical drugs against mycobacteria and also has the ability of bacteriostatic activity with dose-dependent and time-dependent manner. Furthermore, the activities of DQYD against M. tuberculosis are associated with intracellular ATP homeostasis. Meanwhile, mycobacterium DNA damage level was increased after DQYD treatment. But there was no correlation between survival of mycobacteria in the presence of DQYD and intercellular reactive oxygen species. This study enlightens the possible benefits of quinazoline derivatives as potential antimycobacterium compounds and furtherly suggests a new strategy to develop new methods for searching antituberculosis drugs. Bikui Tang, Meili Wei, Qun Niu, Yinjiu Huang, Shuo Ru, Xiaofen Liu, Lin Shen, and Qiang Fang Copyright © 2017 Bikui Tang et al. All rights reserved. Acinetobacter baumannii Infection in Transfusion Dependent Thalassemia Patients with Sepsis Wed, 17 May 2017 09:15:30 +0000 Purpose. To identify the Acinetobacter baumannii infection among transfusion dependent thalassemia patients. Methods. A quantitative approach was employed to assess Acinetobacter baumannii infection in transfusion dependent thalassemia patients. Samples were collected from 916 patients, which have shown bacterial growth on MacConkey and blood agar culture media. A. baumannii strains were identified by microbiological methods and Gram’s staining. API 20 E kit (Biomerieux, USA) was used for final identification. Results. From 916 cultured blood specimens, 107 (11.6%) showed growth of A. baumannii. Serum ferritin in thalassemic patients without bacterial infections was  µg/L versus  µg/L in those with bacterial infections (). Acinetobacter baumannii infected patients have shown higher serum ferritin levels (). Serum ferritin in thalassemic patients was  µg/L versus  µg/L in those with bacterial infections (). Acinetobacter baumannii infected patients showed high serum ferritin levels (). The clinical symptoms have been found with A. baumannii +ve with a mean and standard deviation of 47 (5.1%) and A. baumannii −ve with mean and standard deviation of 60 (6.5%). Conclusion. Isolation of asymptomatic A. baumannii from the thalassemia patients shows an alarming situation of bacterial infections. A continuous surveillance of transfusion dependent thalassemia patients is recommended for bacterial sepsis. Muzaheed Abdul Rasheed, Faisal Mousa Alzahrani, and Saeed Sattar Shaikh Copyright © 2017 Muzaheed Abdul Rasheed et al. All rights reserved. Prion Diagnosis: Application of Real-Time Quaking-Induced Conversion Wed, 17 May 2017 00:00:00 +0000 Prions composed of pathogenic scrapie prion protein (PrP) are infectious pathogens that cause progressive neurological conditions known as prion diseases or transmissible spongiform encephalopathies. Although these diseases pose considerable risk to public health, procedures for early diagnosis have not been established. One of the most recent attempts at sensitive and specific detection of prions is the real-time quaking-induced conversion (RT-QuIC) method, which measures the activity of PrP aggregates or amyloid formation triggered by PrP seeds in the presence of recombinant PrP. In this review, we summarize prions, prion diseases, and current approaches to diagnosis, including the principle, conditions for assay performance, and current diagnostic applications of RT-QuIC. Hae-Eun Kang, Youngwon Mo, Raihah Abd Rahim, Hye-Mi Lee, and Chongsuk Ryou Copyright © 2017 Hae-Eun Kang et al. All rights reserved. Helicobacter pylori from Peptic Ulcer Patients in Uganda Is Highly Resistant to Clarithromycin and Fluoroquinolones: Results of the GenoType HelicoDR Test Directly Applied on Stool Sun, 07 May 2017 00:00:00 +0000 Background. Around 70–90% of peptic ulcer disease (PUD) is due to Helicobacter pylori and requires treatment with antimicrobials to which these bacteria are susceptible. Common H. pylori diagnostic tests do not provide drug susceptibility data. Using the GenoType HelicoDR PCR test designed for gastric biopsies for simultaneous detection of H. pylori and its resistance to clarithromycin (CLA)/fluoroquinolones (FLQ), we present evidence for stool as an optional test specimen and also provide data on prevalence of H. pylori resistance to CLA and FLQ in Uganda. Methods. Stool from 142 symptomatic PUD patients at three hospitals in Kampala was screened for H. pylori using a rapid antigen test. The GenoType HelicoDR test was run on all H. pylori antigen positives to determine PCR positivity and resistance to CLA/FLQ. Results. Thirty-one samples (22%) were H. pylori antigen positive, and 21 (68%) of these were H. pylori PCR positive. Six of the 21 (29%) were resistant to CLA and eight to FLQ (42%), while two gave invalid FLQ resistance results. Conclusion. Stool is a possible specimen for the GenoType HelicoDR test for rapid detection of H. pylori and drug resistance. In Uganda, Helicobacter pylori is highly resistant to CLA and FLQ. Denish Calmax Angol, Ponsiano Ocama, Tess Ayazika Kirabo, Alfred Okeng, Irene Najjingo, and Freddie Bwanga Copyright © 2017 Denish Calmax Angol et al. All rights reserved. Identifying the Genes Responsible for Iron-Limited Condition in Riemerella anatipestifer CH-1 through RNA-Seq-Based Analysis Sun, 30 Apr 2017 00:00:00 +0000 One of the important elements for most bacterial growth is iron, the bioavailability of which is limited in hosts. Riemerella anatipestifer (R. anatipestifer, RA), an important duck pathogen, requires iron to live. However, the genes involved in iron metabolism and the mechanisms of iron transport are largely unknown. Here, we investigated the transcriptomic effects of iron limitation condition on R. anatipestifer CH-1 using the RNA-Seq and RNA-Seq-based analysis. Data analysis revealed genes encoding functions related to iron homeostasis, including a number of putative TonB-dependent receptor systems, a HmuY-like protein-dependent hemin (an iron-containing porphyrin) uptake system, a Feo system, a gene cluster related to starch utilization, and genes encoding hypothetical proteins that were significantly upregulated in response to iron limitation. Compared to the number of upregulated genes, more genes were significantly downregulated in response to iron limitation. The downregulated genes mainly encoded a number of outer membrane receptors, DNA-binding proteins, phage-related proteins, and many hypothetical proteins. This information suggested that RNA-Seq-based analysis in iron-limited medium is an effective and fast method for identifying genes involved in iron uptake in R. anatipestifer CH-1. MaFeng Liu, Mi Huang, DeKang Zhu, MingShu Wang, RenYong Jia, Shun Chen, KunFeng Sun, Qiao Yang, Ying Wu, Francis Biville, and AnChun Cheng Copyright © 2017 MaFeng Liu et al. All rights reserved. Evaluation of the Complex Nomenclature of the Clinically and Veterinary Significant Pathogen Salmonella Sun, 30 Apr 2017 00:00:00 +0000 Salmonella encompasses a vast and highly related population of clinically and veterinary significant pathogens. The genus is responsible for an array of diseases such as typhoid fever and salmonellosis (a variety of illnesses including gastroenteritis), which cause public health issues globally. Even with the global recognition of Salmonella as a significant human and veterinary pathogen, the highly complex and evolving nomenclature system of Salmonella is problematic for clinicians, veterinarians, and microbiologists to comprehend. The present paper offers a review of the ever developing nomenclature for this bacterial species. Michael P. Ryan, Jean O’Dwyer, and Catherine C. Adley Copyright © 2017 Michael P. Ryan et al. All rights reserved. In Vitro Evaluations and In Vivo Toxicity and Efficacy Studies of MFM501 against MRSA Thu, 27 Apr 2017 00:00:00 +0000 Previously we have discovered a synthetically derived pyrrolidone alkaloid, MFM501, exhibiting good inhibitory activity against 53 MRSA and MSSA isolates with low cytotoxicity against three normal cell-lines with IC50 values at >625 µg/ml. Time-kill assay, scanning electron microscopy (SEM) analysis, in vivo oral acute toxicity test, and mice peritonitis model were carried out in this study. In the time-kill study, MFM501 showed a less than 3 log10 decrease in bacterial colony concentration value (CFU/ml) which represented a bacteriostatic action while displaying a time-dependent inhibitory mechanism. Following that, SEM analysis suggested that MFM501 may exert its inhibitory activity via cytoplasmic membrane disruption. Moreover, MFM501 showed no toxicity effect on treated mice at an estimated median acute lethal dose (LD50) value of more than 300 mg/kg and less than 2000 mg/kg. For the efficacy test, a mean effective dose (ED50) of 87.16 mg/kg was obtained via a single dose oral administration. Our data demonstrated that MFM501 has the potential to be developed further as a new, safe, and effective oral-delivered antibacterial agent against MRSA isolates. Saiful Azmi Johari, Mastura Mohtar, Sharifah Aminah Syed Mohamad, Mohd Fazli Mohammat, Rohana Sahdan, Azman Mohamed, and Mohamad Jemain Mohamad Ridhwan Copyright © 2017 Saiful Azmi Johari et al. All rights reserved. Novel Treponema pallidum Recombinant Antigens for Syphilis Diagnostics: Current Status and Future Prospects Mon, 24 Apr 2017 00:00:00 +0000 The recombinant protein technology considerably promoted the development of rapid and accurate treponema-specific laboratory diagnostics of syphilis infection. For the last ten years, the immunodominant recombinant inner membrane lipoproteins are proved to be sensitive and specific antigens for syphilis screening. However, the development of an enlarged T. pallidum antigen panel for diagnostics of early and late syphilis and differentiation of syphilis stages or cured syphilis remains as actual goal of multidisciplinary expertise. Current review revealed novel recombinant antigens: surface-exposed proteins, adhesins, and periplasmic and flagellar proteins, which are promising candidates for the improved syphilis serological diagnostics. The opportunities and limitations of diagnostic usage of these antigens are discussed and the criteria for selection of optimal antigens panel summarized. Aleksey Kubanov, Anastassia Runina, and Dmitry Deryabin Copyright © 2017 Aleksey Kubanov et al. All rights reserved. Shin’iseihaito (Xinyiqingfeitang) Suppresses the Biofilm Formation of Streptococcus pneumoniae In Vitro Sun, 16 Apr 2017 00:00:00 +0000 Streptococcus pneumoniae (S. pneumoniae) is the important pathogen that causes otolaryngeal diseases such as sinusitis. S. pneumoniae frequently forms the biofilm to prevent severe circumstances such as antimicrobial agents. Shin’iseihaito (xinyiqingfeitang) is a formula of Japanese traditional Kampo medicine that has 9 crude drugs and provides the medicinal usage for sinusitis. The objective of the present study is to reveal the mechanism of antibiofilm activity by Shin’iseihaito extract (SSHT). SSHT significantly inhibited the formation of biofilm from S. pneumoniae ATCC 49619 in dose- and time-dependent manners. SSHT also significantly suppressed the biofilm formation by other five different cps types of S. pneumoniae clinical isolates. We found that the extracts of 8 out of 9 components in Shin’iseihaito had the inhibitory effects of biofilm formation, and the extract of the root of Scutellaria baicalensis had the strongest effect among the ingredients of Shin’iseihaito. We found that the capsule of SSHT-treated S. pneumoniae was significantly thinner than that of the untreated group and that SSHT reduced the hydrophobicity of bacterial cell surface. Our results suggest that Shin’iseihaito may be a useful agent for the treatment of S. pneumoniae-induced sinusitis because of the inhibition of biofilm formation of S. pneumoniae. Masaaki Minami, Toru Konishi, Hiroshi Takase, and Toshiaki Makino Copyright © 2017 Masaaki Minami et al. All rights reserved. Association Study of Gut Flora in Coronary Heart Disease through High-Throughput Sequencing Sun, 09 Apr 2017 00:00:00 +0000 Objectives. We aimed to explore the impact of gut microbiota in coronary heart disease (CHD) patients through high-throughput sequencing. Methods. A total of 29 CHD in-hospital patients and 35 healthy volunteers as controls were included. Nucleic acids were extracted from fecal samples, followed by α diversity and principal coordinate analysis (PCoA). Based on unweighted UniFrac distance matrices, unweighted-pair group method with arithmetic mean (UPGMA) trees were created. Results. After data optimization, an average of reads in CHD patients and reads in controls was obtained. Reads corresponding to 38 phyla, 90 classes, and 584 genera were detected in CHD patients, whereas 40 phyla, 99 classes, and 775 genera were detected in controls. The proportion of phylum Bacteroidetes (56.12%) was lower and that of phylum Firmicutes was higher (37.06%) in CHD patients than those in the controls (60.92% and 32.06%, ). PCoA and UPGMA tree analysis showed that there were significant differences of gut microbial compositions between the two groups. Conclusion. The diversity and compositions of gut flora were different between CHD patients and healthy controls. The incidence of CHD might be associated with the alteration of gut microbiota. Li Cui, Tingting Zhao, Haibing Hu, Wen Zhang, and Xiuguo Hua Copyright © 2017 Li Cui et al. All rights reserved. Endophytic Actinobacteria Associated with Dracaena cochinchinensis Lour.: Isolation, Diversity, and Their Cytotoxic Activities Thu, 06 Apr 2017 06:32:01 +0000 Dracaena cochinchinensis Lour. is an ethnomedicinally important plant used in traditional Chinese medicine known as dragon’s blood. Excessive utilization of the plant for extraction of dragon’s blood had resulted in the destruction of the important niche. During a study to provide a sustainable way of utilizing the resources, the endophytic Actinobacteria associated with the plant were explored for potential utilization of their medicinal properties. Three hundred and four endophytic Actinobacteria belonging to the genera Streptomyces, Nocardiopsis, Brevibacterium, Microbacterium, Tsukamurella, Arthrobacter, Brachybacterium, Nocardia, Rhodococcus, Kocuria, Nocardioides, and Pseudonocardia were isolated from different tissues of D. cochinchinensis Lour. Of these, 17 strains having antimicrobial and anthracyclines-producing activities were further selected for screening of antifungal and cytotoxic activities against two human cancer cell lines, MCF-7 and Hep G2. Ten of these selected endophytic Actinobacteria showed antifungal activities against at least one of the fungal pathogens, of which three strains exhibited cytotoxic activities with IC50-values ranging between 3 and 33 μg·mL−1. Frequencies for the presence of biosynthetic genes, polyketide synthase- (PKS-) I, PKS-II, and nonribosomal peptide synthetase (NRPS) among these 17 selected bioactive Actinobacteria were 29.4%, 70.6%, and 23.5%, respectively. The results indicated that the medicinal plant D. cochinchinensis Lour. is a good niche of biologically important metabolites-producing Actinobacteria. Nimaichand Salam, Thi-Nhan Khieu, Min-Jiao Liu, Thu-Trang Vu, Son Chu-Ky, Ngoc-Tung Quach, Quyet-Tien Phi, Manik Prabhu Narsing Rao, Angélique Fontana, Samira Sarter, and Wen-Jun Li Copyright © 2017 Nimaichand Salam et al. All rights reserved. Activity of Fosfomycin- and Daptomycin-Containing Bone Cement on Selected Bacterial Species Being Associated with Orthopedic Infections Thu, 06 Apr 2017 00:00:00 +0000 The purpose of this study was to determine activity of fosfomycin/gentamicin and daptomycin/gentamicin-containing PMMA bone-cement against Staphylococcus aureus (MRSA, MSSA), Staphylococcus epidermidis, Enterococcus faecium (VRE), and E. coli (ESBL; only fosfomycin). Test specimens of the bone cement were formed and bacteria in two concentrations were added one time or repeatedly up to 96 h. All fosfomycin-containing cement killed ultimately all MSSA, Staphylococcus epidermidis, and E. coli within 24 h; growth of MRSA was suppressed up to 48 h. Activity of daptomycin-containing cement depended on the concentration; the highest concentrated bone cement used (1.5 g daptomycin/40 g of powder) was active against all one-time added bacteria. When bacteria were added repeatedly to fosfomycin-containing cement, growth was suppressed up to 96 h and that of MRSA and VRE only up to 24 h. The highest concentration of daptomycin suppressed the growth of repeated added bacteria up to 48 h (VRE) until 96 h (MSSA, MRSA). In conclusion, PMMA bone cement with 1.5 g of daptomycin and 0.5 g of gentamicin may be an alternative in treatment of periprosthetic infections caused by Gram-positive bacteria. Sigrun Eick, Kevin Hofpeter, Anton Sculean, Claudia Ender, Susann Klimas, Sebastian Vogt, and Sandor Nietzsche Copyright © 2017 Sigrun Eick et al. All rights reserved. A Robust PCR Protocol for HIV Drug Resistance Testing on Low-Level Viremia Samples Mon, 03 Apr 2017 07:44:26 +0000 The prevalence of drug resistance (DR) mutations in people with HIV-1 infection, particularly those with low-level viremia (LLV), supports the need to improve the sensitivity of amplification methods for HIV DR genotyping in order to optimize antiretroviral regimen and facilitate HIV-1 DR surveillance and relevant research. Here we report on a fully validated PCR-based protocol that achieves consistent amplification of the protease (PR) and reverse transcriptase (RT) regions of HIV-1 pol gene across many HIV-1 subtypes from LLV plasma samples. HIV-spiked plasma samples from the External Quality Assurance Program Oversight Laboratory (EQAPOL), covering various HIV-1 subtypes, as well as clinical specimens were used to optimize and validate the protocol. Our results demonstrate that this protocol has a broad HIV-1 subtype coverage and viral load span with high sensitivity and reproducibility. Moreover, the protocol is robust even when plasma sample volumes are limited, the HIV viral load is unknown, and/or the HIV subtype is undetermined. Thus, the protocol is applicable for the initial amplification of the HIV-1 PR and RT genes required for subsequent genotypic DR assays. Shivani Gupta, Tracy Taylor, Aileen Patterson, Binhua Liang, Jared Bullard, Paul Sandstrom, Gary Van Domselaar, and Hezhao Ji Copyright © 2017 Shivani Gupta et al. All rights reserved. Identification of Streptococcus pneumoniae: Development of a Standardized Protocol for Optochin Susceptibility Testing Using Total Lab Automation Sun, 19 Mar 2017 00:00:00 +0000 Purpose. Optochin susceptibility is one parameter used in the laboratory to identify Streptococcus pneumoniae. However, a single standardized procedure does not exist. Optochin is included neither in the current EUCAST breakpoint tables nor in the CLSI performance standards for antimicrobial susceptibility testing. We wanted to establish an evidence-based protocol for optochin testing for our Total Lab Automation. Methods. We tested seven different agars and four different reading time points (7 h, 12 h, 18 h, and 24 h). To accommodate for serotype diversity, all tests were done with 99 different strains covering 34 different serotypes of S. pneumoniae. We calculated a multivariable linear regression using data from 5544 inhibition zones. Results. Reading was possible for all strains at 12 h. Agar type and manufacturer influenced the size of the inhibition zones by up to 2 mm and they varied considerably depending on serotype (up to 3 mm for serotype 3). Depending on agar and reading time point, up to 38% of inhibition zones were smaller than the cut-off of 14 mm; that is, the result of the test was false-negative. Conclusions. Shortening incubation time from 24 h to 12 h for optochin susceptibility testing is feasible. Agar and incubation time have to be chosen carefully to avoid false-negative results. Irene Burckhardt, Jessica Panitz, Florian Burckhardt, and Stefan Zimmermann Copyright © 2017 Irene Burckhardt et al. All rights reserved. Cytotoxicity and Antimicrobial Activity of Oral Rinses In Vitro Sun, 19 Mar 2017 00:00:00 +0000 While oral rinses used for cosmetic purposes only do not necessarily have to be antiseptic, antimicrobial activity is required for medical indications, including oral and periodontal surgery. So the question arises—is the antimicrobial activity of oral rinses associated with any destructive changes in cell viability in vitro? To answer this question, we examined twelve oral rinses with respect to their antimicrobial and cytotoxic activity. Antimicrobial activity was screened against five bacterial strains using disc diffusion. Cytotoxicity was determined by mitochondrial reductase activity with primary gingival fibroblasts, L929 cells, and HSC-2 epithelial cells. Phase contrast microscopy and trypan blue staining were then performed to reveal cell morphology. Cells remained vital after exposure to oral rinses that were only used for cosmetic purposes. Moderate cytotoxic effects were observed for oral rinses containing 0.05% chlorhexidine, ethanol, or pegylated hydrogenated castor oil and sodium dodecyl sulfate. Other oral rinses containing 0.2% chlorhexidine and cocamidopropyl betaine exhibited strong cytotoxic and antimicrobial activity. Strong cytotoxic but moderate antimicrobial activity was observed in oral rinses containing cetylpyridinium chloride. The in vitro data show that oral rinses are heterogeneous with respect to their cytotoxic and antimicrobial effects. Based on their respective properties, oral rinses can be selected either to reduce the microbial load or for cosmetic purposes. Heinz-Dieter Müller, Sigrun Eick, Andreas Moritz, Adrian Lussi, and Reinhard Gruber Copyright © 2017 Heinz-Dieter Müller et al. All rights reserved. Recent Advances in Biofilmology and Antibiofilm Measures Wed, 15 Mar 2017 00:00:00 +0000 Nithyanand Paramasivam, Shunmugiah Karutha Pandian, Ariel Kushmaro, Supayang Voravuthikunchai, and Aruni Wilson Copyright © 2017 Nithyanand Paramasivam et al. All rights reserved. A Comparative Study of Oral Microbiota in Infants with Complete Cleft Lip and Palate or Cleft Soft Palate Tue, 14 Mar 2017 00:00:00 +0000 Few reports have been published on the early microbiota in infants with various types of cleft palate. We assessed the formation of the oral microbiota in infants with complete cleft lip and palate (CLP ) or cleft soft palate (CSP ) in the neonatal period (T1 time) and again in the gum pad stage (T2 time). Culture swabs from the tongue, palate, and/or cleft margin at T1 and T2 were taken. We analysed the prevalence of the given bacterial species (the percentage) and the proportions in which the palate and tongue were colonised by each microorganism. At T1, Streptococcus mitis (S. mitis) were the most frequently detected in subjects with CLP or CSP (63% and 60%, resp.). A significantly higher frequency of methicillin-sensitive Staphylococcus aureus (S. aureus MSSA) was observed in CLP compared to the CSP group. At T2, significantly higher percentages of S. mitis, S. aureus MSSA, Staphylococcus epidermidis, and members of the Enterobacteriaceae family were noted in CLP infants compared to the CSP. S. mitis and Streptococcus sanguinis appeared with the greatest frequency on the tongue, whereas Streptococcus salivarius was predominant on the palate. The development of the microbiota in CLP subjects was characterised by a significant increase in the prevalence of pathogenic bacteria. Agnieszka Machorowska-Pieniążek, Anna Mertas, Małgorzata Skucha-Nowak, Marta Tanasiewicz, and Tadeusz Morawiec Copyright © 2017 Agnieszka Machorowska-Pieniążek et al. All rights reserved. L-Glutamine Supplementation Alleviates Constipation during Late Gestation of Mini Sows by Modifying the Microbiota Composition in Feces Sun, 12 Mar 2017 00:00:00 +0000 Constipation occurs frequently in both sows and humans, particularly, during late gestation. The microbial community of the porcine gut, the enteric microbiota, plays a critical role in functions that sustain intestinal health. Hence, microbial regulation during pregnancy may be important to prevent host constipation. The present study was conducted to determine whether L-glutamine (Gln) supplementation improved intestinal function and alleviated constipation by regulation of enteric microbiota. 16S rRNA sequences obtained from fecal samples from 9 constipated sows (3 in the constipation group and 6 in the 1.0% Gln group) were assessed from gestational day 70 to 84. Comparative analysis showed that the abundance of intestinal-friendly microbiota, that is, Bacteroidetes () and Actinobacteria (), was comparatively increased in the 1.0% Gln group, while the abundance of pernicious bacteria, Oscillospira () and Treponema (), was decreased. Dietary supplementation with 1.0% Gln may ameliorate constipation of sows by regulated endogenous gut microbiota. Yuanyuan Zhang, Taofeng Lu, Lingxia Han, Lili Zhao, Yinjie Niu, and Hongyan Chen Copyright © 2017 Yuanyuan Zhang et al. All rights reserved. Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water Thu, 09 Mar 2017 00:00:00 +0000 The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks. Lucrecia Acosta Soto, Ana Belén Santísima-Trinidad, Fernando Jorge Bornay-Llinares, Marcos Martín González, José Antonio Pascual Valero, and Margarita Ros Muñoz Copyright © 2017 Lucrecia Acosta Soto et al. All rights reserved. Inhibition of Pseudomonas aeruginosa Biofilm Formation by Traditional Chinese Medicinal Herb Herba patriniae Thu, 09 Mar 2017 00:00:00 +0000 New antimicrobial agents are urgently needed to treat infections caused by drug-resistant pathogens and by pathogens capable of persisting in biofilms. The aim of this study was to identify traditional Chinese herbs that could inhibit biofilm formation of Pseudomonas aeruginosa, an important human pathogen that causes serious and difficult-to-treat infections in humans. A luxCDABE-based reporter system was constructed to monitor the expression of six key biofilm-associated genes in P. aeruginosa. The reporters were used to screen a library of 36 herb extracts for inhibitory properties against these genes. The results obtained indicated that the extract of Herba patriniae displayed significant inhibitory effect on almost all of these biofilm-associated genes. Quantitative analysis showed that H. patriniae extract was able to significantly reduce the biofilm formation and dramatically altered the structure of the mature biofilms of P. aeruginosa. Further studies showed H. patriniae extract decreased exopolysaccharide production by P. aeruginosa and promoted its swarming motility, two features disparately associated with biofilm formation. These results provided a potential mechanism for the use of H. patriniae to treat bacterial infections by traditional Chinese medicines and revealed a promising candidate for exploration of new drugs against P. aeruginosa biofilm-associated infections. Bo Fu, Qiaolian Wu, Minyan Dang, Dangdang Bai, Qiao Guo, Lixin Shen, and Kangmin Duan Copyright © 2017 Bo Fu et al. All rights reserved. Antimicrobial Activity and Chemical Composition of “Kpètè-Kpètè”: A Starter of Benin Traditional Beer Tchoukoutou Mon, 06 Mar 2017 09:40:42 +0000 The aim of this study was to investigate the antibacterial effect of the crude starter “kpètè-kpètè” and lactic acid bacteria used during the production of “tchoukoutou.” To achieve this, a total of 11 lactic acid bacteria and 40 starter samples were collected from four communes. The samples were tested on 29 gram + and − strains by disk diffusion method. The minimum inhibitory and bactericidal concentrations of starter and lactic acid bacteria were determined by conventional methods. Organic acids, sugar, and volatile compounds were determined using the HPLC method. The “kpètè-kpètè” displays a high antibacterial activity against the tested strains. The most sensitive strain was S. epidermidis (12.5 mm) whereas the resistance strain was Proteus mirabilis (8 mm). All the tested ferment has not any inhibitory effect on Enterococcus faecalis. The lactic acid bacteria isolates of Parakou showed the highest (17.48 mm) antibacterial activity whereas the smallest diameter was obtained with the ferment collected from Boukoumbé (9.80 mm). The starters’ chemical screening revealed the presence of tannins, anthocyanin flavonoids, triterpenes, steroids, reducing compounds, and mucilage O-glycosides. These compounds are probably the source of recorded inhibition effect. The lactic acid bacteria of the “kpètè-kpètè” could be used to develop a food ingredient with probiotic property. Christine N’tcha, Haziz Sina, Adéchola Pierre Polycarpe Kayodé, Joachim D. Gbenou, and Lamine Baba-Moussa Copyright © 2017 Christine N’tcha et al. All rights reserved. Oxidative Stress Induced by Polymyxin E Is Involved in Rapid Killing of Paenibacillus polymyxa Wed, 22 Feb 2017 14:12:36 +0000 Historically, the colistin has been thought to kill bacteria through membrane lysis. Here, we present an alternative mechanism that colistin induces rapid Paenibacillus polymyxa death through reactive oxygen species production. This significantly augments our understanding of the mechanism of colistin action, which is critical knowledge toward the yield development of colistin in the future. Zhiliang Yu, Yuyi Zhu, Wangrong Qin, Jianhua Yin, and Juanping Qiu Copyright © 2017 Zhiliang Yu et al. All rights reserved. Novel Sequential Screening and Enhanced Production of Fibrinolytic Enzyme by Bacillus sp. IND12 Using Response Surface Methodology in Solid-State Fermentation Wed, 22 Feb 2017 11:39:00 +0000 Fibrinolytic enzymes have wide applications in clinical and waste treatment. Bacterial isolates were screened for fibrinolytic enzyme producing ability by skimmed milk agar plate using bromocresol green dye, fibrin plate method, zymography analysis, and goat blood clot lysis. After these sequential screenings, Bacillus sp. IND12 was selected for fibrinolytic enzyme production. Bacillus sp. IND12 effectively used cow dung for its growth and enzyme production ( U/g substrate). Further, the optimum bioprocess parameters were found out for maximum fibrinolytic enzyme production using cow dung as a low cost substrate under solid-state fermentation. Two-level full-factorial experiments revealed that moisture, pH, sucrose, peptone, and MgSO4 were the vital parameters with statistical significance (). Three factors (moisture, sucrose, and MgSO4) were further studied through experiments of central composite rotational design and response surface methodology. Enzyme production of optimized medium showed  U/g material, which was more than fourfold the initial enzyme production ( U/g). The analysis of variance showed that the developed response surface model was highly significant (). The fibrinolytic enzyme digested goat blood clot (100%), chicken skin (%), egg white (100%), and bovine serum albumin (%). Ponnuswamy Vijayaraghavan, P. Rajendran, Samuel Gnana Prakash Vincent, Arumugaperumal Arun, Naif Abdullah Al-Dhabi, Mariadhas Valan Arasu, Oh Young Kwon, and Young Ock Kim Copyright © 2017 Ponnuswamy Vijayaraghavan et al. All rights reserved. RNA-Based Stable Isotope Probing Suggests Allobaculum spp. as Particularly Active Glucose Assimilators in a Complex Murine Microbiota Cultured In Vitro Thu, 16 Feb 2017 00:00:00 +0000 RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that “cross-feeding” occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system. Elena Herrmann, Wayne Young, Douglas Rosendale, Verena Reichert-Grimm, Christian U. Riedel, Ralf Conrad, and Markus Egert Copyright © 2017 Elena Herrmann et al. All rights reserved. Nitrogen Removal Characteristics of Pseudomonas putida Y-9 Capable of Heterotrophic Nitrification and Aerobic Denitrification at Low Temperature Wed, 15 Feb 2017 00:00:00 +0000 The cold-adapted bacterium Pseudomonas putida Y-9 was investigated and exhibited excellent capability for nitrogen removal at 15°C. The strain capable of heterotrophic nitrification and aerobic denitrification could efficiently remove ammonium, nitrate, and nitrite at an average removal rate of 2.85 mg, 1.60 mg, and 1.83 mg NL−1 h−1, respectively. Strain Y-9 performed nitrification in preference to denitrification when ammonium and nitrate or ammonium and nitrite coexisted in the solution. Meantime, the presence of nitrate had no effect on the ammonium removal rate of strain Y-9, and yet the presence of high concentration of nitrite would inhibit the cell growth and decrease the nitrification rate. The experimental results indicate that P. putida Y-9 has potential application for the treatment of wastewater containing high concentrations of ammonium along with its oxidation products at low temperature. Yi Xu, Tengxia He, Zhenlun Li, Qing Ye, Yanli Chen, Enyu Xie, and Xue Zhang Copyright © 2017 Yi Xu et al. All rights reserved. Nicotine Enhances Interspecies Relationship between Streptococcus mutans and Candida albicans Thu, 09 Feb 2017 00:00:00 +0000 Streptococcus mutans and Candida albicans are common microorganisms in the human oral cavity. The synergistic relationship between these two species has been deeply explored in many studies. In the present study, the effect of alkaloid nicotine on the interspecies between S. mutans and C. albicans is explored. We developed a dual-species biofilm model and studied biofilm biomass, biofilm structure, synthesis of extracellular polysaccharides (EPS), and expression of glucosyltransferases (Gtfs). Biofilm formation and bacterial and fungal cell numbers in dual-species biofilms increased in the presence of nicotine. More C. albicans cells were present in the dual-species biofilms in the nicotine-treated groups as determined by scanning electron microscopy. The synthesis of EPS was increased by 1 mg/ml of nicotine as detected by confocal laser scanning microscopy. The result of qRT-PCR showed gtfs expression was upregulated when 1 mg/ml of nicotine was used. We speculate that nicotine promoted the growth of S. mutans, and more S. mutans cells attracted more C. albicans cells due to the interaction between two species. Since S. mutans and C. albicans are putative pathogens for dental caries, the enhancement of the synergistic relationship by nicotine may contribute to caries development in smokers. Shiyu Liu, Wei Qiu, Keke Zhang, Xuedong Zhou, Biao Ren, Jinzhi He, Xin Xu, Lei Cheng, and Mingyun Li Copyright © 2017 Shiyu Liu et al. All rights reserved. Significance of Heavy-Ion Beam Irradiation-Induced Avermectin B1a Production by Engineered Streptomyces avermitilis Tue, 24 Jan 2017 07:42:38 +0000 Heavy-ion irradiation technology has advantages over traditional methods of mutagenesis. Heavy-ion irradiation improves the mutation rate, broadens the mutation spectrum, and shortens the breeding cycle. However, few data are currently available regarding its effect on Streptomyces avermitilis morphology and productivity. In this study, the influence of heavy-ion irradiation on S. avermitilis when cultivated in approximately 10 L stirred-tank bioreactors was investigated. The specific productivity of the avermectin (AVM) B1a-producing mutant S. avermitilis 147-G58 increased notably, from 3885 to 5446 μg/mL, approximately 1.6-fold, compared to the original strain. The mycelial morphology of the mutant fermentation processes was microscopically examined. Additionally, protein and metabolite identification was performed by using SDS-PAGE, 2- and 3-dimensional electrophoresis (2DE and 3DE). The results showed that negative regulation gene deletion of mutants led to metabolic process upregulating expression of protein and improving the productivity of an avermectin B1a. The results showed that the heavy-ion beam irradiation dose that corresponded to optimal production was well over the standard dose, at approximately 80 Gy at 220 AMeV (depending on the strain). This study provides reliable data and a feasible method for increasing AVM productivity in industrial processes. Shu-Yang Wang, Yong-Heng Bo, Xiang Zhou, Ji-Hong Chen, Wen-Jian Li, Jian-Ping Liang, Guo-Qing Xiao, Yu-Chen Wang, Jing Liu, Wei Hu, and Bo-Ling Jiang Copyright © 2017 Shu-Yang Wang et al. All rights reserved. Mechanism of Reduced Susceptibility to Fosfomycin in Escherichia coli Clinical Isolates Thu, 19 Jan 2017 00:00:00 +0000 In recent years, multidrug resistance of Escherichia coli has become a serious problem. However, resistance to fosfomycin (FOM) has been low. We screened E. coli clinical isolates with reduced susceptibility to FOM and characterized molecular mechanisms of resistance and reduced susceptibility of these strains. Ten strains showing reduced FOM susceptibility (MIC ≥ 8 μg/mL) in 211 clinical isolates were found and examined. Acquisition of genes encoding FOM-modifying enzyme genes (fos genes) and mutations in murA that underlie high resistance to FOM were not observed. We examined ability of FOM incorporation via glucose-6-phosphate (G6P) transporter and sn-glycerol-3-phosphate transporter. In ten strains, nine showed lack of growth on M9 minimum salt agar supplemented with G6P. Eight of the ten strains showed fluctuated induction by G6P of uhpT that encodes G6P transporter expression. Nucleotide sequences of the uhpT, uhpA, glpT, ptsI, and cyaA shared several deletions and amino acid mutations in the nine strains with lack of growth on G6P-supplemented M9 agar. In conclusion, reduction of uhpT function is largely responsible for the reduced sensitivity to FOM in clinical isolates that have not acquired FOM-modifying genes or mutations in murA. However, there are a few strains whose mechanisms of reduced susceptibility to FOM are still unclear. Yasuo Ohkoshi, Toyotaka Sato, Yuuki Suzuki, Soh Yamamoto, Tsukasa Shiraishi, Noriko Ogasawara, and Shin-ichi Yokota Copyright © 2017 Yasuo Ohkoshi et al. All rights reserved. Antibiotic Resistance of Campylobacter Recovered from Faeces and Carcasses of Healthy Livestock Wed, 18 Jan 2017 09:44:26 +0000 Campylobacter is of major significance in food safety and human and veterinary medicine. This study highlighted resistance situation in the area of veterinary public health in Ghana. Using selective mCCDA agar, isolates were confirmed phenotypically on API CAMPY and genotypically by multiplex PCR of IpxA gene. The susceptibility profile of species to common and relevant antibiotics was determined by the Kirby-Bauer disk diffusion method. Cattle, sheep, goat, and pig faecal samples analysed, respectively, yielded 13.2% (16/121), 18.6% (22/102), 18.5% (25/135), and 28.7% (29/101) Campylobacter species while 34.5% (38/110), 35.9% (42/117), 23.9% (32/134), and 36.3% (37/102) were, respectively, recovered from the carcasses. Species identified in faeces were C. jejuni 35.8% (33/92), C. jejuni subsp. doylei 4.3% (4/92), C. coli 47.8% (44/92), and C. lari 12.0% (11/92). Species discovered in carcasses were C. jejuni 83.9% (125/149), C. jejuni subsp. doylei 2.0% (3/149), C. coli 6.0% (9/149), and C. lari 8.1% (12/149). Resistance ranged from 92 to 97% to the β-lactams, 7 to 69% to the quinolones, 0 to 44% to the aminoglycosides, 97 to 100% to erythromycin, 48 to 94% to tetracycline, 45 to 88% to chloramphenicol, and 42 to 86% to trimethoprim/sulfamethoxazole as 0% resistance was observed against imipenem. Akosua B. Karikari, Kwasi Obiri-Danso, Enoch H. Frimpong, and Karen A. Krogfelt Copyright © 2017 Akosua B. Karikari et al. All rights reserved. Plant Fungal Pathogenesis Tue, 17 Jan 2017 00:00:00 +0000 Jun Yang, Tom Hsiang, Vijai Bhadauria, Xiao-Lin Chen, and Guotian Li Copyright © 2017 Jun Yang et al. All rights reserved. Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications Thu, 12 Jan 2017 13:54:55 +0000 Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60–70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials. Altaf Ahmed Simair, Abdul Sattar Qureshi, Imrana Khushk, Chaudhry Haider Ali, Safia Lashari, Muhammad Aqeel Bhutto, Ghulam Sughra Mangrio, and Changrui Lu Copyright © 2017 Altaf Ahmed Simair et al. All rights reserved. Decolourisation Capabilities of Ligninolytic Enzymes Produced by Marasmius cladophyllus UMAS MS8 on Remazol Brilliant Blue R and Other Azo Dyes Thu, 12 Jan 2017 09:42:07 +0000 Marasmius cladophyllus was examined for its ability to degradatively decolourise the recalcitrant dye Remazol Brilliant Blue R (RBBR) and screened for the production of ligninolytic enzymes using specific substrates. Monitoring dye decolourisation by the decrease in absorbance ratio of shows that the decolourisation of RBBR dye was associated with the dye degradation. Marasmius cladophyllus produces laccase and lignin peroxidase in glucose minimal liquid medium containing RBBR. Both enzyme activities were increased, with laccase activity recorded 70 times higher reaching up to 390 U L−1 on day 12. Further in vitro RBBR dye decolourisation using the culture medium shows that laccase activity was correlated with the dye decolourisation. Fresh RBBR dye continuously supplemented into the decolourised culture medium was further decolourised much faster in the subsequent round of the RBBR dye decolourisation. In vitro dye decolourisation using the crude laccase not only decolourised 76% of RBBR dye in just 19 hours but also decolourised 54% of Orange G and 33% of Congo red at the same period of time without the use of any exogenous mediator. This rapid dye decolourisation ability of the enzymes produced by M. cladophyllus thus suggested its possible application in the bioremediation of dye containing wastewater. Ngieng Ngui Sing, Ahmad Husaini, Azham Zulkharnain, and Hairul Azman Roslan Copyright © 2017 Ngieng Ngui Sing et al. All rights reserved. Emerging Perils of Extended Spectrum β-Lactamase Producing Enterobacteriaceae Clinical Isolates in a Teaching Hospital of Nepal Thu, 29 Dec 2016 06:17:27 +0000 Introduction. Infections due to extended spectrum β-lactamase producing Enterobacteriaceae are on the rise. They pose serious public health problems due to their resistance to large number of antibiotics. However, little is known about the genotypes of ESBL from Nepal. Therefore, the study presents results of phenotypic and molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. isolated from various clinical specimens in a tertiary care teaching hospital of Nepal. Methods. A total of 172 Enterobacteriaceae clinical isolates recovered from various clinical specimens were analyzed for their antibiotic susceptibility test. Detection of ESBLs was carried out using combination disk test and multiplex PCR for their genotypes (CTX-M, SHV, and TEM). Results. Out of 172 clinical isolates, 70 (40.6%) of them were found ESBL producers. The major source of ESBL producers was urinary tract samples and the highest ESBL production was observed in Escherichia coli (46.5%). Among ESBL genotypes, CTX-M (91.4%) was most predominant, followed by TEM (65.7%) and SHV (11.4%) in both of the isolates. Conclusions. High level of drug resistance and ESBL production was observed among the clinical isolates. There is a need for longitudinal and nationwide surveillance for drug resistance in clinical isolates and antimicrobial stewardship is necessary to guide the appropriate and judicious antibiotic use. Narayan Prasad Parajuli, Pooja Maharjan, Govardhan Joshi, and Puspa Raj Khanal Copyright © 2016 Narayan Prasad Parajuli et al. All rights reserved. Enteric Pathogens and Coinfections in Foals with and without Diarrhea Tue, 27 Dec 2016 14:06:31 +0000 Diarrhea is a major clinical problem affecting foals up to 3 months of age. The aim of this study was to identify enteric microorganisms involved in monoinfections and coinfections and the associated virulence factors in healthy and diarrheic foals. Diarrheic (D) () and nondiarrheic (ND) foals () up to three months of age were studied. Fecal samples were analyzed for identification of infectious agents (microbiological culturing, molecular techniques, and microscopic analyses). Escherichia coli fimH (30% versus 25%), Salmonella spp. (25% versus 7%), Strongyloides westeri (25% versus 25%), Clostridium perfringens type A (21% versus 10%), E. coli ag43 (20% versus 35%), Strongylus (11% versus 18%), and vapA-positive Rhodococcus equi (5% versus 2%) were the most frequent enteric pathogens detected in D and ND foals, respectively. The frequency of toxin A-positive C. perfringens was significantly increased in the D () compared with the ND animals. R. equi strains harboring virulent plasmids were also identified (VapA 85-kb type I and VapA 87-kb type I) in D and ND foals. Coinfections were observed in 46% of the D and 33% of the ND foals. Our results demonstrate the great diversity of enteric pathogens, virulence factors, and coinfections involved in enteric infections of foals. Giovane Olivo, Thays Mizuki Lucas, Alexandre Secorun Borges, Rodrigo Otávio Silveira Silva, Francisco Carlos Faria Lobato, Amanda Keller Siqueira, Domingos da Silva Leite, Paulo Eduardo Brandão, Fábio Gregori, José Paes de Oliveira-Filho, Shinji Takai, and Márcio Garcia Ribeiro Copyright © 2016 Giovane Olivo et al. All rights reserved. Methicillin-Resistant Staphylococcus aureus Biofilms and Their Influence on Bacterial Adhesion and Cohesion Sun, 18 Dec 2016 14:35:25 +0000 Twenty-five methicillin-resistant Staphylococcus aureus (MRSA) isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype was spa type t127 (22 out of 25 isolates); the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested. Khulood Hamid Dakheel, Raha Abdul Rahim, Vasantha Kumari Neela, Jameel R. Al-Obaidi, Tan Geok Hun, and Khatijah Yusoff Copyright © 2016 Khulood Hamid Dakheel et al. All rights reserved. Distribution of Suicin Gene Clusters in Streptococcus suis Serotype 2 Belonging to Sequence Types 25 and 28 Sun, 18 Dec 2016 12:41:01 +0000 Recently, we reported the purification and characterization of three distinct lantibiotics (named suicin 90-1330, suicin 3908, and suicin 65) produced by Streptococcus suis. In this study, we investigated the distribution of the three suicin lantibiotic gene clusters among serotype 2 S. suis strains belonging to sequence type (ST) 25 and ST28, the two dominant STs identified in North America. The genomes of 102 strains were interrogated for the presence of suicin gene clusters encoding suicins 90-1330, 3908, and 65. The gene cluster encoding suicin 65 was the most prevalent and mainly found among ST25 strains. In contrast, none of the genes related to suicin 90-1330 production were identified in 51 ST25 strains nor in 35/51 ST28 strains. However, the complete suicin 90-1330 gene cluster was found in ten ST28 strains, although some genes in the cluster were truncated in three of these isolates. The vast majority (101/102) of S. suis strains did not possess any of the genes encoding suicin 3908. In conclusion, this study indicates heterogeneous distribution of suicin genes in S. suis. Taryn B. T. Athey, Katy Vaillancourt, Michel Frenette, Nahuel Fittipaldi, Marcelo Gottschalk, and Daniel Grenier Copyright © 2016 Taryn B. T. Athey et al. All rights reserved. Prevalence and Antimicrobial Resistance of Salmonella Isolated from Animal-Origin Food Items in Gondar, Ethiopia Thu, 15 Dec 2016 14:28:47 +0000 Salmonella has been found to be the major cause of foodborne diseases and a serious public health problem in the world, with an increasing concern for the emergence and spread of antimicrobial-resistant strains. A cross-sectional study was conducted between February 2014 and December 2015 on food items of animal origin to assess the prevalence and antimicrobial resistance profiles of Salmonella isolates using standard bacteriological methods. The overall prevalence rate of 5.5% was recorded from the total analyzed food items of animal origin. Salmonella isolates were detected from 12% of raw meat, 8% of minced meat, 2.9% of burger samples, 18% of raw eggs, and 6% of raw milk. Furthermore, antimicrobial susceptibility test identified 47.6% resistant Salmonella isolates, 28.6% intermediately sensitive isolates, and 23.8% susceptible isolates. Among Salmonella isolates tested, 42.6%, 28.6%, and 14.3% were found to be relatively resistant to tetracycline, sulfamethoxazole-trimethoprim, and ampicillin, respectively, while 9.5%–19% were intermediately resistant to tetracycline, amoxicillin, ampicillin, cephalothin, and nitrofurantoin. Therefore, our findings provide the prevalence and drug resistance of Salmonella from foods of animal origin and contribute information to scientists as well as public health researchers to minimize the prevalent and resistant foodborne Salmonella species in Ethiopia. Mebrat Ejo, Legesse Garedew, Zabishwork Alebachew, and Walelgn Worku Copyright © 2016 Mebrat Ejo et al. All rights reserved. Analysis of Polyphenolic Compounds in Extracts from Leaves of Some Malus domestica Cultivars: Antiradical and Antimicrobial Analysis of These Extracts Wed, 14 Dec 2016 14:20:16 +0000 In this study, methanol, ethyl acetate, water extracts, and precipitate were obtained from leaves of Malus domestica cultivars: Golden delicious, Jonagold, Elstar, Ligol, and Mutsu. Antiradical activity of these extracts was measured using the radical, and antimicrobial activity was measured with the disk-diffusion method. Phenolic compounds were measured with the colorimetric method and identified with high performance liquid chromatography (HPLC). The highest antiradical activity was observed for the Jonagold variety, and in particular strong activity was noted for ethyl acetate extracts. Antimicrobial activity was observed against strains of Staphylococcus aureus, Enterococcus faecalis, and the fungus Candida glabrata. Particularly susceptible to the extracts activity appeared to be Staphylococcus aureus, but the growth of Candida glabrata was inhibited in the presence of ethyl acetate extracts. With the HPLC method we identified a high amount of phloridzin (above 500 mg per g of ethyl acetate extracts), lower amounts of hyperoside, isoquercitrin, and quercitrin, and traces of p-hydroxybenzoic and chlorogenic acids. The contribution of phloridzin to antiradical activity of methanol and ethyl acetate extracts was very high (above 90%). In water extract the contribution of phloridzin was between 38.9 and 55.2%, chlorogenic acid 22.7 and 36.1%, and hyperoside 12.2 and 13.3%. Alina Sowa, Grażyna Zgórka, Aleksandra Szykuła, Roman Franiczek, Beata Żbikowska, Andrzej Gamian, and Zbigniew Sroka Copyright © 2016 Alina Sowa et al. 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.