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Raw cow-milk has essentially role inbacteria transferring, causing diseases like Escherichia coli (E. coli).The presentstudy has aimed to delineate E. coli virulence gene heat labile (LT) and heat stable (ST) by using Polymerase Chain Reaction (PCR). In terms of methodology, 125 raw cow-milk samples from four suburbs distinct of karbala as Al-Hussainiya, Al-Zebeliya, Al-hindiya and Al-hurr have been gathered and examined from December 2018 to March 2019.Accordingly, E. colihas been distinguished in 51 samples(40.8%), also the study has shown that the E. coli isolated from raw cow-milk is resistant to Ticarcillin (%92.1) followed by TicarcillinClavulanicacid (90.1%), Topramycin (25.4%),Aztreonam (25.4%) and sensitive to Meropenem (100%),Imepenem (100%) and Colistin (%100). It also sensitive to Ciprofloxacin (94.1%) followed byPipracillin / Tazobactam (88.2%) Pipracillin(%82.3), Trimethoprim / Sulfamethoxazole(80.3%),Pefloxacin (78.4%),Cefepime (76.4%), Gentamicin (76.4%), Minocycline (74.5%) and Amicacin (%72.5).As a result, the molecular features of isolatedE. colihas beenperformed by PCR through the use of proper primers. Virulence profile of genes reveals that 2/51(4%)isolates of E. coli harbored LT genes and 7/51 (14%)isolates of E. coliharbored ST genes. The results have also highlighted poor hygienic practices in the study regions, bringing microbiological risks from raw cow-milk, therefore, the study has proposed an improved hygienic practices on processing, production and distribution to ensure the safety of dairy products, safeguarding public health and to emphasis veterinarians or stakeholders’ role in production and distribution. [ABSTRACT FROM AUTHOR]

Highlights • Novel SCC mec type XII MRSA isolates were prevalent in the swine farms and slaughterhouse. • All SCC mec XII MRSA were multiresistant and carried MGEs (lsa(E) cluster and Tn 558). • All MRSA of SCC mec XII carried multiple virulence genes and clotted ruminant plasma. • PFGE showed cases of swine farm-to-slaughterhouse transmission of SCC mec XII MRSA. Abstract Methicillin-resistant Staphylococcus aureus (MRSA) has been a major public health concern. In this study, a total of 1485 samples from three swine farms, one slaughterhouse and one indoor market in Xiamen, China were collected in 2015, and the prevalence and profiles of MRSA were assessed. All the MRSA isolates were characterized by molecular typing, antibiotic susceptibility, coagulation activity, as well as PCR screening for 38 antibiotic resistance genes, two mobile genetic elements (lsa (E)-containing multiresistance gene cluster and Tn 558), and 36 virulence genes. During the study, 54 of 1485 (3.6%) samples from the swine production, processing and retail chain were found positive for MRSA. A relatively rare SCC mec XII genotype was prevalent in swine farm (84.6%, 11/13) and slaughterhouse isolates (80.6%, 25/31), but absent in the market isolates (0%, 0/10). Notably, all staphylococcal cassette chromosome mec (SCC mec) type XII MRSA isolates were resistant to at least 6 classes of antibiotics, carried two mobile genetic elements (lsa (E)-containing multiresistance gene cluster and Tn 558) and harbored multiple virulence genes. These multidrug resistant MRSA isolates could also coagulate both bovine and caprine plasma. Our results on the SCC mec XII MRSA isolates, particularly their profiles of related genotypes, antibiotic resistance and virulence determinants, illustrated the evolvement of livestock-associated (LA)-MRSA in the swine production environment and spread along the processing chain. The dominance of the SCC mec XII in MRSA isolates found in this study, differed from previous reports from China, indicated potential contribution associated with the production process. [ABSTRACT FROM AUTHOR]

Since 2015, an emerging infectious disease of inclusion body hepatitis and hydropericardium syndrome (IBH-HPS) has been occurred in China, which caused economic loss in poultry farming. In this study, we isolated four fowl adenovirus strains from flocks with an outbreak of HPS. The complete nucleotide sequence of SC-Neijiang was determined and its pathogenicity was evaluated. Phylogenetic analysis based on hexon gene revealed that all the isolates belonged to fowl adenovirus serotype 4. The full genome sequence of SC-Neijiang has a size of 43,719 bp, with 54.85% G + C content. Compared with JSJ13, 11-amino-acid deletion at the ORF29 was appeared on SC-Neijiang. In infectious experiments, 80% (16/20) birds died in intramuscular route and lesions characteristic for Hydropericardium Syndrome (HPS), while 5% (1/20) birds died in nasal route. The viral DNA was further detected by real-time PCR in several chicken organs. The highest titers were recorded in all the organs at day 5 post-infection. To our knowledge, this is first report on the prevalence of fowl adenovirus in Southwest China. This research elucidated the characteristics of genome sequence and pathogenicity of Chinese FAdV-4 strain and provided theoretical support for the prevention and control of the disease. [ABSTRACT FROM AUTHOR]

Objectives: The criteria for identification of Enterococcus faecium (Efm) with the ability to cause human infections are currently being debated by the European Food Safety Authority (EFSA). Strains that have an MIC of ampicillin of ≤ 2 mg/L and lack IS16/esp/hyl genes should be regarded as safe for use as feed additives in animal nutrition, despite the lack of knowledge about putative virulence marker (PVM) distribution in community Efm. We analysed the distribution of major PVM and ampicillin phenotypes in large Efm collections to investigate further the safety of strains from a public health perspective.Methods: Thirty-three PVM were assessed by PCR/sequencing among clonally disparate Efm (n = 328; 1986-2015) from different origins. We analysed ampicillin susceptibility (Etest/broth microdilution) according to EUCAST guidelines, clonal relationship (MLST) and genomic location of PVM (S1-PFGE/hybridization).Results: Infection-derived Efm were more enriched in PVM and the increase in ampicillin MIC was positively correlated with an enrichment in different PVM. PVM coding for surface (esp/sgrA/ecbA/complete acm) and pili proteins, or others enhancing colonization (hyl/ptsD/orf1481) or plasticity (IS16), were strongly associated with clinical Efm (mostly clade A1), but also observed in clades A2/B at different rates. ptsD was a good marker of ampicillin-resistant Efm. ptsD, IS16, orf1481, sgrA and hospital variants of complete pili gene clusters are proposed as markers to assess the safety of Efm strains.Conclusions: Our study expands on the distribution of PVM in diverse Efm lineages and demonstrates the enrichment in infection-derived strains of PVM not previously included in EFSA's list of Efm safety criteria. The evidence of relevant Efm infection markers can impact the risk assessment of Efm strains in different public health contexts. [ABSTRACT FROM AUTHOR]

The current study was aimed to investigate the prevalence of ureC, rsbA, zapA and mrpA virulence genes using polymerase chain reaction (PCR) in Proteus spp. isolated from 5 commercially popular species of pet turtles and comparison of the mrpA gene sequences of Proteus mirabilis isolates with human clinical isolates. A total of 24 isolates in pet turtles were identified, comprised of P. mirabilis (15), Proteus vulgaris (7) and Proteus hauseri (2). The prevalence of ureC, rsbA, zapA and mrpA genes among all identified Proteus spp. isolates were 91.7%, 50%, 45.8% and 45.8%, respectively. The average percentage similarities of mrpA gene sequence of pet turtle P. mirabilis isolates to human urinary and respiratory isolates were 96.35% and 94.85%, respectively. The prevalence of virulence genes and high similarity of mrpA gene sequences between pet turtles and human P. mirabilis isolates revealed that though pet turtles are healthy, these animals may pose a potential risk of urinary and respiratory infections to humans. [ABSTRACT FROM AUTHOR]

Background Enterococcus spp. is the common of intestinal micro flora in humans but nowadays this gram-positive bacterium causes the variety of nosocomial infections. Resistance to antibiotic and also, presence of different virulence genes in the enterococcus spp. can change it to problematic microorganisms in the health care centers. The aim of this study was determined the genotyping, antimicrobial resistance and virulence factor gene profiles of vancomycin resistance Enterococcus faecalis isolated from blood culture. Methods In this study, enterococcus isolated from BACTEC was collected and antibiotic susceptibility testing was done according to CLSI recommendation. Important virulence genes and vancomyci resistance genes were detected by PCR and molecular typing was performed by RAPD PCR assay. Results Nine enterococcus collected from 194 positive BACTEC and seven out of nine were vancomycin-resistant enterococcus (VRE). vanA gene observed in all VRE and none of strains carried vanB and vanC genes. efbaA and gelE virulence factors have been detected in all strains. ace , esp , and cyl virulence factors genes harbored in two, seven and eight isolates respectively. asaI was not detected in any strains. All seven VRE isolates were related to the one specific molecular type and two different molecular types observed in the two vancomycin susceptible enterococci according to molecular epidemiology results. Conclusion More prevalence of the VRE in enterococcus isolated from BACTEC is so important and on the other hand high genetic relationship in the isolated VRE can be very considerable for nosocomial infection committee in the hospital. [ABSTRACT FROM AUTHOR]

In this study, we collected 565 fecal samples from free-range Tibetan yaks to examine whether these animals can serve as a reservoir for pathogenic and antimicrobial-resistant Escherichia coli. The antimicrobial sensitivity testing of each resulting isolate was evaluated according to the disk diffusion method, whereas PCR analysis was performed for detection of resistance and virulence genes. Additionally, strains containing extended-spectrum beta-lactamases (ESBLs) genes were initially identified via the double-disk synergy method and confirmed by PCR analysis. Of the 488 E. coli isolates examined, approximately 31.1% were multidrug resistant. OmpA (30.2%) and etrA (23.1%), and blaCTX-M (27.6%) and blaTEM (14.4%) were the mainly prevalent virulence and drug resistance genes, respectively. Notably, we detected the CTX-M type but not the SHV type among the ESBL-producing strains and there was a significant association between resistance and virulence genes for aac(3)-lla, blaTEM/ompA, blaCTX-M/etrA, and cmlA/cnf1 (P<0.05). Lastly, the majority of the strains belonged to phylogroup A (72.7%). This is the first report of the occurrence of pathogenic and antimicrobialresistant ESBL-producing E. coli strains in free-range Tibetan yaks. [ABSTRACT FROM AUTHOR]

Variants of the Helicobacter pylori cag pathogenicity island ( cag PAI) and certain virulence genotypes have been proposed to be associated with different gastric disorders. In the present study, we designed a new PCR-based strategy to investigate the intactness of cag PAI in Iranian patients using highly specific primer sets spanning the cag PAI region. The possible relationship between the cag PAI status of the strains and clinical outcomes was also determined. We also characterized virulence genotypes ( cagL , cagA , vacA , babA2 and sabA ) and variants of CagA EPIYA motifs in these strains. H. pylori was detected in 61 out of 126 patients with various gastroduodenal diseases. The cagL , cagA , vacA s1m1, vacA s1m2, vacA s2m2, babA2 , and sabA genotypes were detected in 96.7%, 85.2%, 29.5%, 45.9%, 24.6%, 96.7%, and 83.6% of the strains, respectively. Among the 52 cagA -positive strains, EPIYA motifs ABC, ABCC, ABCCC, and mixed types were orderly detected in the 39, 7, 1, and 5 strains. The cag PAI positivity included both intact and partially deleted, with the overall frequencies of 70.5% and 26.2%, respectively. The majority of the strains from patients with PUD (87.5%), gastric erosion (83.3%) and cancer (80%) presented an intact cag PAI, while a lower frequency of cag PAI intactness was detected in gastritis patients (61.1%). However, no significant relationship was found between the possession of intact cag PAI and clinical outcomes. Furthermore, we found that cagA and vacA s1m1 genotypes were significantly correlated with intact cag PAI ( P = 0.015 and P = 0.012). A significant correlation was also found between EPIYA-ABC and intact cag PAI ( P = 0.010). The proposed PCR-based scheme was found to be useful for determining the intactness of cag PAI. Our findings also indicate that the cag PAI appears to be intact and rather conserved in majority of Iranian strains. Finally, our study proposed that H. pylori strains with partially deleted cag PAI were less likely to cause severe diseases in comparison with those carrying intact cag PAI. [ABSTRACT FROM AUTHOR]

Escherichia coli O26 has been identified as the most common non-O157 Shiga toxin-producing E. coli (STEC) serogroup to cause human illnesses in the United States and has been implicated in outbreaks around the world. E. coli has high genomic plasticity, which facilitates the loss or acquisition of virulence genes. Attaching and effacing E. coli (AEEC) O26 strains have frequently been isolated from bovine feces, and there is a need to better characterize the relatedness of these strains to defined molecular pathotypes and to describe the extent of their genetic diversity. High-throughput real-time PCR was used to screen 178 E. coli O26 isolates from a single U.S. cattle feedlot, collected from May to July 2011, for the presence or absence of 25 O26 serogroupspecific and virulence-associated markers. The selected markers were capable of distinguishing these strains into molecularly defined groups (yielding 18 unique marker combinations). Analysis of the clustered regularly interspaced short palindromic repeat 1 (CRISPR1) and CRISPR2a loci further discriminated isolates into 24 CRISPR types. The combination of molecular markers and CRISPR typing provided 20.8% diversity. The recent CRISPR PCR target SP-O26-E, which was previously identified only in stx2-positive O26:H11 human clinical strains, was identified in 96.4% (161/167 [95% confidence interval, 99.2 to 93.6%]) of the stx-negative AEEC O26:H11 bovine fecal strains. This supports that these stx-negative strains may have previously contained a prophage carrying stx or could acquire this prophage, thus possibly giving them the potential to become pathogenic to humans. These results show that investigation of specific genetic markers may further elucidate our understanding of the genetic diversity of AEEC O26 strains in bovine feces. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was (i) To attempt isolation and identification of Salmonella species from samples. (ii) Serotyping of Salmonella isolates. (iii) Detection of virulence factor associated genes by polymerase chain reaction (PCR). Materials and Methods: A total of 284 samples comprised of chevon and mutton (112 samples each) as well as 60 samples (20 each of retail meat shops environment samples viz. Butchers' hands, knives and log swabs) were collected from the retail meat shops in and around Anand City under aseptic precautions. Rappaport-vassiliadis soy bean meal broth and tetrathionate broth was used for the enrichment of all the samples and inoculation was done on brilliant green agar and xylose lysine deoxycholate agar. This was followed by the confirmation of isolates using biochemical tests. For the serotyping, isolates were sent to the National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, Himachal Pradesh. Detection of virulence genes was performed by PCR technique using previously reported primer. Result: Of 284 meats and retail meat shops environment samples, 13 (4.58%) samples were found positive for Salmonella. It was interesting to know that incidence of Salmonella was more in mutton (6.25%) than chevon (3.57%). In case of meat shop environmental samples 1 (5.00%) sample observed positive for Salmonella separately among the butchers' hands and knives swabs (Each of 20 samples) examined. Out of 13, eleven isolates detected as Salmonella Typhimurium, whereas only two isolates were detected as Salmonella Enteritidis. All Salmonella isolates possess invA and stn genes, whereas nine isolates had a presence of spvR gene while only five of the isolates revealed the presence of spvC gene as shown by in vitro detection of virulence genes by PCR. Conclusion: Therefore, might be suggested that the good hygiene practices and effective control measures should be taken to encourage clean meat production with prolonged shelf-life. [ABSTRACT FROM AUTHOR]

Introduction: Aeromonas are food- and water-borne bacteria that are considered to be zoonotic human pathogens. This study aimed to investigate the presence of genes associated with virulence in human and animal Aeromonas isolates and the potential role of animal isolates with regards to human Aeromonas infections. Methodology: The presence of aerA, hlyA, alt, ast, laf, ascF-G, stx1 and stx2 putative virulence genes in 40 human and animal Aeromonas isolates (16 human and 24 animal isolates) were examined by polymerase chain reaction (PCR). DNA fragments of expected sizes were purified and sequenced. BLAST in the NCBI was used to verify any amplified products. Results: PCR screening showed that hlyA, alt, and laf genes were determined at ratios of 6.25%, 50%, and 6.25%, respectively, in human isolates. The ratios of hlyA, alt, ascF-G, laf, stx2, and stx1 genes in animal isolates were 58.3%, 20.83%, 33.3%, 20.83%, 8.33%, and 4.17%, respectively. Neither aerA nor ast genes were detected in any isolates. Any one of eight putative virulence genes was not detected in seven human and eight animal isolates in the study. Conclusions: The current study is the first to investigate the presence of the virulence gene in gull Aeromonas isolates. The manifestation of the presence of the virulence gene and gene combinations was considerable, especially in fish and gull isolates when compared with clinical human isolates. The current study demonstrates the potential importance of fish and gulls in terms of human Aeromonas infections. [ABSTRACT FROM AUTHOR]

The objective of this study was to analyze the antibiotic resistance phenotype and genotype of Salmonella isolated from broiler production facilities. A total of 193 Salmonella isolates recovered from commercial farms in British Columbia, Canada, were evaluated. Susceptibility to antibiotics was determined with the Sensititre system. Virulence and antibiotic resistance genes were detected by PCR assay. Genetic diversity was determined by pulse-field gel electrophoresis (PFGE) typing. Seventeen serovars of Salmonella were identified. The most prevalent Salmonella serovars were Kentucky (29.0% of isolates), Typhimurium (23.8%), Enteritidis (13.5%), and Hadar (11.9%); serovars Heidelberg, Brandenburg, and Thompson were identified in 7.7, 4.1, and 3.6% of isolates, respectively. More than 43% of the isolates were simultaneously resistant to ampicillin, amoxicillin-clavulanic acid, ceftiofur, cefoxitim, and ceftriaxone. This β-lactam resistance pattern was observed in 33 (58.9%) of the Salmonella Kentucky isolates; 2 of these isolates were also resistant to chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Genes associated with resistance to aminoglycosides (aadA1, aadA2, and strA), β-lactams (blaCMY-2, blaSHV, and blaTEM), tetracycline (tetA and tetB), and sulfonamide (sul1) were detected among corresponding resistant isolates. The invasin gene (invA) and the Salmonella plasmid virulence gene (spvC) were found in 97.9 and 25.9% of the isolates, respectively, with 33 (71.7%) of the 46 Salmonella Typhimurium isolates and 17 (65.4%) of the 26 Salmonella Enteritidis isolates carrying both invA and spvC. PGFE typing revealed that the antibiotic-resistant serovars were genetically diverse. These data confirm that broiler chickens can be colonized by genetically diverse antibiotic-resistant Salmonella isolates harboring virulence determinants. The presence of such strains is highly relevant to food safety and public health. [ABSTRACT FROM AUTHOR]

Presently, the understanding of bacterial enteric diseases in the community and their virulence factors relies almost exclusively on clinical disease reporting and examination of clinical pathogen isolates. This study aimed to investigate the feasibility of an alternative approach that monitors potential enteropathogenic Escherichia colt (EPEC) and enterohemorrhagic E. coli (EHEC) prevalence and intimin gene (eae) diversity in a community by directly quantifying and characterizing target virulence genes in the sanitary sewage. The quantitative PCR (qPCR) quantification of the eae, stx1 and stx2 genes in sanitary sewage samples collected over a 13-month period detected eae in all 13 monthly sewage samples at significantly higher abundance (93 to 7,240 calibrator cell equivalents [CCE]/100 ml) than stx1 and stx2, which were detected sporadically. The prevalence level of potential EPEC in the sanitary sewage was estimated by calculating the ratio of eae to uidA, which averaged 1.0% (σ = 0.4%) over the 13-month period. Cloning and sequencing of the eae gene directly from the sewage samples covered the majority of the eae diversity in the sewage and detected 17 unique eae alleles belonging to 14 subtypes. Among them, eae-β2 was identified to be the most prevalent subtype in the sewage, with the highest detection frequency in the clone libraries (41.2%) and within the different sampling months (85.7%). Additionally, sewage and environmental E. coli isolates were also obtained and used to determine the detection frequencies of the virulence genes as well as eae genetic diversity for comparison. [ABSTRACT FROM AUTHOR]

Most illnesses caused by E scherichia coli O157: H7 have been associated with eating undercooked, contaminated meat. In this study, a number of 200 cooked beef doner kebab samples from different restaurants across cities of Tehran and Mashhad were analyzed. After enrichment, selective plating and biochemical tests, the DNA from suspected colonies were extracted. Three multiplex polymerase chain reaction ( PCR) assays to confirm the isolates as E . coli O157: H7 and the presence of virulence factors were performed. Specific primers for cH7 and rfbO157 genes in the first and for stx1 and stx2 genes in the second m- PCR were used. The third m- PCR performed using specific primers for eaeA and EhlyA genes. From 25 nonsorbitol-fermenting isolates, 21 isolates confirmed as E . coli O157: H7. The results of third m- PCR showed 12 isolates harboring the EhlyA gene, three isolates the eaeA gene and three isolates harboring both the EhlyA and eaeA genes. Practical Applications The presence of E scherichia coli O157: H7 in cooked beef doner kebabs could be considered a serious public health hazard. These three multiplex PCR assay are a suitable method for rapid identification of E . coli O157: H7 virulence genes. [ABSTRACT FROM AUTHOR]

Salmonella can contaminate finished products of butcher shops, mainly through cross-contamination of utensils exposed to raw materials. To identify the main sources of contamination with this foodbome pathogen in four butcher shop environments, surface samples were obtained from employees' hands, cutting boards, knives, floor of the refrigeration room, meat grinders, and meat tenderizers (32 samples per area) and analyzed for Salmonella using the International Organization for Standardization method 6579, with modifications. Suspect isolates were identified by PCR (targeting ompC), and confirmed Salmonella isolates were subjected to pulsed-field gel electrophoresis (after treatment with restriction enzyme XbaI), analyzed for the presence of virulence genes (invA, sefA, and spvC), and screened for resistance to 12 antimicrobials. Salmonella isolates was identified only on cutting boards (five samples) from three butcher shops. Fifteen isolates were confirmed as Salmonella belonging to four pulse types (similarity of 71.1 to 100%). The invA gene was detected in 13 isolates, and the sefA was found in 8 isolates; no isolate carried spvC. All tested isolates were resistant to clindamycin and sensitive to amikacin and cefotaxine, and all isolates were resistant to at least 3 of the 12 antimicrobials tested. The results indicate the importance of cutting boards as a source of Salmonella contamination in butcher shops. The presence of multidrug-resistant Salmonella strains possessing virulence genes highlights the health risks for consumers. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) are important human pathogens, and attention to non-O157 serogroups has increased in recent years. Although cattle are normally considered the primary reservoir for STEC, recent illnesses associated with goat contact have indicated that these animals are important potential reservoirs for the organisms. The prevalence of STEC, particularly non-O157 serogroups, in U.S. goats has not been well described. Our objective was to determine the prevalence of six major non-O157 STEC serogroups in the feces of meat goats. Rectal contents from 296 goats were collected postevisceration at a slaughter plant in the southeastern United States over 9 days during a 12-week period from August through October 2012. Samples were enriched in E. coli broth, and DNA was extracted and used as template in an 1 l-gene multiplex PCR that detected six non-O157 serogroups (O26, O45, O103, O121, O111, and O145) and virulence genes. Samples were considered positive when at least one non-O157 STEC serotype was present with either stx1 or stx2. All six non-O157 serogroups were detected by PCR in our samples, and 14.5% of samples were positive for at least one serogroup. Prevalence of O26 was highest, with 6.4% of goat fecal samples positive. The prevalence of 045 was 3.4%, O103 was 4.4%, O111 was 4.1%, O121 was 1.4%, and O145 was 3.0%. Twenty-two (7.4%) of 296 fecal samples had more than one non-O157 serogroup detected in the feces. Two samples had evidence of three non-O157 STEC serogroups. Goats appear to be an important reservoir for non-O157 STEC, and further work to understand the characteristics, epidemiology, and ecology of STEC in these animals is warranted. [ABSTRACT FROM AUTHOR]

Yersinia enterocolitica is a foodborne pathogen that causes illness in humans and animals. The biotype 4/O:3 has been commonly associated with yersiniosis and is characterized by the presence of chromosomal and extra-chromosomal virulence genes. Molecular typing methods have been successfully used to characterize Y. enterocolitica genetic heterogeneity and to study the epidemiology of the bacteria from different origins. In this study, 320 Y. enterocolitica biotype 4/O:3 isolates originating in pigs and slaughterhouses were characterized according to the virulence profile, and 61 isolates were typified through SE-AFLP, ERIC-PCR, and PFGE techniques. The majority of the isolates originated from pigs, and the predominant virulence profile was ail+ virF+ rfbC+ ystA+, representing 83.4% of the tested isolates. All of the Y. enterocolitica 4/O:3 isolates were positive for at least ystA gene. The SE-AFLP and ERIC-PCR patterns were highly homogeneous. The SE-AFLP was more discriminative than the ERIC-PCR and tended to cluster isolates according to the slaughterhouse. Despite the limited genetic diversity of Y. enterocolitica 4/O:3, PFGE was shown to be the most discriminative technique considering one band of difference. Fattening pigs proved to be an important reservoir of Y. enterocolitica biotype 4/O:3 carrying virulence genes. [ABSTRACT FROM AUTHOR]

Aim: To know the prevalence of gram positive isolates and their virulence genes obtained from respiratory tract of apparently healthy as well as sick goats. Material and Methods: Nasal swabs and tissue samples were screened for the presence of microbial pathogens by cultural isolation, biochemical confirmation. Further, these isolates were subjected to the virulence gene detection by PCR. Results: Out of 144 isolates, 57 isolates of gram positive pathogens were obtained and confirmed as Staphylococcus spp. (43 isolates) and Streptococcus spp. (14 isolates) on the basis of Gram staining, morphology, cultural identification and biochemical characters. Five isolates (11.6%) were found to be positive for Coagulase gene; 11 isolates (25.6%) were found to be positive for clfA gene; and 14 isolates (32.6%) were found to be positive for Spa gene. Conclusion: The presence of these genes confirmed the pathogenic potential of gram positive pathogens and their association with clinical manifestations in respiratory tract infections of goats. [ABSTRACT FROM AUTHOR]

Aims To detect putative virulence genes in clinical strains of Yersinia enterocolitica biovar 1 A by suppression subtractive hybridization between two closely related strains of clinical and nonclinical origin having the same serotype ( O:6,30-6,31). Methods and Results Suppression Subtractive Hybridization ( SSH) was used to identify genomic differences between clinical (serotype O:6,30-6,31, from diarrhoeic human stools) and nonclinical (serotype O:6,30-6,31, from wastewater) strains of Y. enterocolitica biovar 1 A. Following genomic subtraction and DNA sequencing, nine DNA sequences that were present only in clinical biovar 1 A strains were identified. The sequences identified using SSH showed similarity to conserved hypothetical proteins, proteins related to iron acquisition and haemin storage, type 1 secretion proteins, flagellar hook proteins, exported protein and ABC transport system. All these sequences showed high similarity with Y. enterocolitica 8081 (biovar 1 B). The distribution of these genes was further analysed using PCR in 26 clinical strains of Y. enterocolitica biovar 1 A. The results revealed that the distribution of these genes was not uniform. Conclusions Genes related to iron acquisition and storage, and flagellar proteins might be responsible for virulence of some of the clinical strains of Y. enterocolitica biovar 1A. Significance and Impact of the Study Genes identified in this study might be useful in understanding the pathogenic potential of clinical strains of Y. enterocolitica biovar 1 A. [ABSTRACT FROM AUTHOR]

Abstract: Enteric pathogens such as Salmonella enterica serovar Typhimurium and Enterohaemorrhagic Escherichia coli require an initial indispensable step of attachment or invasion of enterocytes before they can produce systemic disease and translocate to their target organs. Prevention of either of these steps will result in an avirulent state and limit their pathogenicity. In vitro tests demonstrated that molecules secreted by Bifidobacterium bifidum interfere with both attachment and invasion. The main regulatory genes controlling the virulence factors essential for these pathogenicity steps were efficiently down-regulated when treated with chromatographically separated B. bifidum cell free fractions as measured by reporter constructs and confirmed by RT-PCR. Moreover, the ability of both pathogens to colonize eukaryotic cells was significantly reduced, and the capacity of Salmonella to survive and multiply within macrophages was also diminished upon treatment with these bioactive molecules. These results indicate that probiotic Bifidobacteria strains may represent an effective alternative approach to control food-borne enteric pathogens. [Copyright &y& Elsevier]

Enterococcus faecalis is a major cause of nosocomial infections in humans and has been linked to severe extra-intestinal infections in poultry. A zoonotic potential has been suggested and the aim of the present study was to investigate similarities in virulence gene profiles of E. faecalis originating from infections in humans and poultry respectively. A total of 106 isolates of E. faecalis [26 human clinical isolates, 60 poultry clinical isolates (including two small-colony variants (SCVs) and 20 poultry cloacal isolates] were investigated for presence of seven virulence-associated genes: ace, asa1, cylA, efaA, EF0591, esp and gelE. For each gene, the PCR-amplification product was sequenced from one isolate in each group to explore intragenic variations between genes of human and poultry origin. Haemolytic and protease activities were assessed and isolates were assigned a sequence type (ST). Three of the seven genes investigated ( ace, efaA and gelE) were present in all isolates. The a sa1 was detected in 63/80 and 13/26 isolates of poultry and human origin respectively. For cylA, the numbers were 46/80 and 14/26 respectively. Among poultry isolates, esp and EF0591 were the least frequently observed genes (1/80 and 20/80 respectively); the prevalences among human isolates were 1/26 and 18/26 respectively. A high degree of similarity between genes in human and poultry isolates were confirmed by sequencing of amplification products. None of the cylA-positive isolates demonstrated haemolytic activity, while the phenotypic expression of gelatinase varied. The ST16 was the only ST shared by human and poultry isolates. The SCV isolates did not show a unique virulence profile or phylogeny. In conclusion, regardless of the distinct phylogenetic background of most E. faecalis isolates of human and poultry origin, we found major similarities in virulence gene profile and gene sequences in isolates from the two sources, supporting the zoonotic risk associated with this organism. [ABSTRACT FROM AUTHOR]

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Urinary tract infections (UTIs) are among the most common bacterial infections and are responsible for significant morbidity and health care costs worldwide. The main bacterial cause of uncomplicated UTI is Escherichia coli, which possesses numerous virulence factors (VFs). Many studies of the pathogenesis of E. coli UTI have centered on VF genes. Hence, the development of better molecular assays to study YF genes would facilitate these studies. We developed a highly sensitive and specific multiplex PCR-based reverse line blot (mPCRJRLB) assay to simultaneously detect 22 VF genes of uropathogenic E. coli and then used it to characterize 180 isolates from nonpregnant women of child-bearing age with cystitis and 153 fecal isolates from similar-age healthy women, in regional New South Wales, Australia. The assay accurately identified all VF genes (of the 22 under study) known to be present in 30 previously characterized control strains. The detection limits were 28 ng of DNA from E. coli isolates and 50 CFU/ml in mock-infected urine specimens containing known concentrations of E. coli. Cystitis isolates (compared to the fecal isolates) showed a significantly higher prevalence of 18 individual VF genes and contained significantly more VF genes per isolate (median number, 18.5 versus 6.5 [P = 0.001]). Discordance between paired probes for a given VF gene occurred in several clinical test isolates but no reference strains and among the test isolates was associated with fecal source (10% of VF genes versus 2% for cystitis isolates [P < 0.001]). This novel mPCR/RLB method is a potentially powerful tool for investigating the prevalence and distribution of VFs in E. coli. [ABSTRACT FROM AUTHOR]

Summary: Objectives: Little is known about Escherichia coli Orthopaedic Implant Infections (OII) pathogenesis. Thus, we compared 30 clinical strains isolated in this context with 30 clinical strains of faecal origin, in order to identify phenotypic and genetic features related to E. coli OII. Methods: Phylogenetic analysis and detection of 19 virulence genes were performed by PCR. Ability to form biofilm was studied using the crystal violet reference method and the innovative BioFilm Ring Test®. Results: Most of the OII isolates (56.7%) belonged to the virulence-associated phylogenetic group B2, but did not present a specific set of virulence factors. S fimbriae was the only adhesin significantly associated with OII isolates. Isolates varied greatly in their ability to form biofilm but OII isolates did not produce significantly more biofilm in vitro than isolates of faecal origin, whatever the method used. Conclusions: Neither a specific pathogenic signature nor an increased ability to form biofilm in vitro was detected in E. coli strains isolated from OII. Nevertheless, genetic properties of these isolates could provide a clue to their origin. Hence, we found that virulence factors of uropathogenic strains and urological disorders were frequently detected among our OII cohort. [Copyright &y& Elsevier]

Background: Escherichia coli O157:H7 have emerged as pathogens that can cause food-borne infections and severe illnesses in humans, such as hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS). Since this bacterium is transferred by the foodstuff, especially hamburger it is called "hamburger bacteria". The aim of this study was isolation and identification of E.coli O157:H7 from hamburger samples in Shiraz, Isfahan. Methods: In this cross- sectional study, 320 samples of Hamburger were collected and enriched in TSB with novobiocin medium in 37ºC temperature. Fermentation of sorbitol and lactose and activities of β-glucoronidase of separated bacteria were examined by using the SMAC and VRBA media and choromoagar medium. Then the existence of E.coli O157:H7 was confirmed with the specific antiserum. At the end existence of virulence genes and antibiotic resistance strains were tested with multiplex PCR and disk diffusion methods. Findings: Out of all examined samples, 259 (80.93%) sorbitol negative bacteria were separated. From which 227 (87.64%) had ability of lactose fermentation. Then by using biochemical tests, 192 samples (84.58%) were identified as E.coli. Also the rate of recognition of MUG negative and E.coli O157:H7 were 50.00% and 3.43% respectively. By evaluation of virulence markers, eae and stx1 genes were identified in 3 samples and eae, stx1 and stx2 identified in 1 sample. Also in 1 sample only hly gene was detected. All isolated strains were resistant to most antibiotics. Conclusion: Since this bacterium is an high risk pathogene and hamburger was a common source in recent outbreak, monitoring of all kinds of meat products is recommended. [ABSTRACT FROM AUTHOR]

Aims: The present study aimed to develop a colony hybridization method for the exhaustive detection and isolation of diarrhoeagenic Escherichia coli (DEC) from samples containing numerous coliform bacteria. Methods and Results: Digoxigenin-labelled DNA probes were designed to detect seven pathotypes of DEC based on type-specific genes. A total of 615 meat, food and faeces samples identified as DEC-positive by multiple real-time PCR for the virulence genes ( eae, stx, elt, est, virB, aggR, afaB and astA) were analysed by a colony hybridization method, which involved filtering enrichment cultures through hydrophobic grid-membrane filters. DEC were isolated from 72·5% (446/615) of samples by the colony hybridization method but were only detected in 26·3% (162/615) of samples by a conventional culture method. The hybridization method was particularly effective for isolating low-level contaminants, such as enterotoxigenic and Shiga toxin-producing E. coli, which were isolated from 51·8% (58/112) of samples identified as positive by PCR for the enterotoxin genes, in contrast to only 4·5% (5/112) of samples analysed by the conventional method. Conclusions: The developed colony hybridization system allows for the efficient and simultaneous isolation of all DEC pathotypes. Significance and Impact of the Study: The colony hybridization system described here permits the sensitive isolation of DEC and represents a suitable tool for ecological investigations of DEC. [ABSTRACT FROM AUTHOR]

O157:H7 is considered among the most important recently emerged food-borne bacteria causing severe hemorrhagic diarrhea. Antibiotic treatment is not recommended as a prospective curative agent against this pathogen. Therefore, potency assessment of the local lytic phage isolates infecting E. coli O157:H7 as an alternate remedy to antibiotics was the principal concern of this study. Phage isolates against E. coli O157:H7 were checked by polymerase chain reaction for the presence of the virulence genes stx1 and stx2, and the safe phages were further screened in vitro for their capacity as biocontrol agents. Two bacteriophage strains, namely PAH6 and P2BH2, that had expressed potential antibacterial activity ( P < 0.05) in vitro were selected for in vivo testing in ligated rabbit ileal loop models. Both phage isolates were capable of decreasing fluid accumulation in rabbit ileal loops along with reducing bacterial growth ( r = 0.992). Combined application of the phages was found most satisfactory, reducing seven log cycles of bacterial growth. Consistent results in both in vivo and in vitro experiments demonstrate the applicability of bacteriophages as a rapid response tool against E. coli O157:H7. To our knowledge, this is the first successful application of the rabbit ileal loop test for therapeutic evaluation of bacteriophages. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study was to isolate and identify Escherichia coli O157:H7 from pigs, cattle, humans, beef, pork and water samples and to determine their putative virulence genes by PCR analysis. A total of 220 samples were analysed; 5600 presumptive E. coli O157:H7 were screened for the presence of rfb O157 and fliC H7 gene fragments by PCR and 130 isolates were confirmed. The prevalence of E. coli O157:H7 was higher in pigs and pork 88(67.7%) than in cattle and beef 36(27.7%), water 3(2.3%) or humans 1 (0.77%). Moreover, the pathogen was more frequently isolated from faecal (16.9%–43.1%) than from meat samples (10.8%–24.6%). A large proportion—73 (56.2%)—of the isolates possessed the hlyA gene, while 48 (36.9%) harboured the eaeA gene. Although there were no major differences in the number of isolates harbouring the stx 1 and stx 2 genes, respectively, only a small proportion 13(10%) harboured both shiga toxin genes. Despite this, the proportion of isolates that possessed the stx 1 29(22.3%) was higher than those possessing the stx 2 gene. None of the E. coli O157:H7 isolates harboured all four shiga-toxin producing E. coli (STEC) virulence genes investigated. When comparing the proportion of isolates obtained from the different sample sources and/or stations, significant positive correlations were observed between isolates from Mafikeng and Lichtenburg (r = 0.981, p < 0.05) and those from Mafikeng and Rustenburg (r = 0.991, p < 0.05). These results therefore indicate that meat and faeces samples obtained from major cities in the northwest province were contaminated with E. coli O157:H7. We suggest that there is a need for improving the sanitary conditions of farms, abattoirs and butcher shops. This could reduce transmission of E. coli O157:H7 to humans. [Copyright &y& Elsevier]

The incidence of the virulence-associated genes cdtA, cdtB, cdtC, cadF, dnaJ, racR, and pldA has been investigated in Campylobacter jejuni and Campylobacter coli collected from raw chicken and beef from retailers in Tehran, Iran, and from hospitalized children (age, ≤14 years) suffering from diarrhea. Campylobacter spp. were collectively identified by morphological and biochemical methods. Campylobacter jejuni and C. coli were discriminated from other Campylobacter spp. by amplification of a specific conserved fragment of the 16S rRNA gene. The distinction between C. jejuni and C. coli was subsequently made by molecular determination of the presence of the hipO gene in C. jejuni or the ask gene in C. coli. Fragments of the studied virulence-associated genes, cdtA, cdtB, cdtC, cadF, racR, dnaJ, and pldA, were amplified by PCR and subjected to horizontal gel electrophoresis. A total of 71 isolates of C. jejuni and 24 isolates of C. coli from meat were analyzed, while the numbers of isolates from the hospitalized children were 28 and 9, respectively. The unequal distribution of C. jejuni and C. coli in the samples has also been reported in other studies. Statistical analyses by the use of the two-tailed Fisher's exact test of the occurrence of the virulence genes in the isolates of different origins showed that the occurrence of the dnaJ gene was consistently significantly higher in all C. jejuni isolates than in C. coli. The occurrence of the other virulence markers did not differ significantly between species in the majority of the isolates. The PCR results also showed that the occurrence of the virulence markers in the analyzed isolates was much lower than in other studies, which may be caused by a divergent genomic pool of our isolates in comparison with others. L'incidence des gènes associés à la virulence cdtA, cdtB, cdtC, cadF, dnaJ, racR et pldA a été examinée chez Campylobacter jejuni et Campylobacter coli recueillis de poulet cru et de viande de bœuf vendus par des détaillants à Téhéran, Iran, ou prélevés chez des enfants hospitalisés (≤14 ans d'âge) souffrant de diarrhée. Les souches de Campylobacter ont été collectivement identifiées par des méthodes morphologiques et biochimiques. Campylobacter jejuni et C. coli ont été discriminées des autres espèces de Campylobacter par l'amplification d'un segment conservé spécifique du gène de l'ARNr 16S. La distinction entre C. jejuni et C. coli a été subséquemment réalisée par la détermination moléculaire de la présence de hipO chez C. jejuni et de ask chez C. coli. Des fragments des gènes associés à la virulence cdtA, cdtB, cdtC, cadF, dnaJ, racR et pldA ont été amplifiés par PCR et soumis à une électrophorèse sur gel horizontal. Un total de 71 isolats de C. jejuni et de 24 isolats de C. coli dérivés de la viande ont été analysés, alors que le nombre d'isolats recueillis chez les enfants hospitalisés était de 28 et 9, respectivement. La distribution inégale de C. jejuni et de C. coli dans les échantillons a déjà été rapportée dans d'autres études. Des analyses statistiques de l'occurrence des gènes de virulence des isolats d'origines différentes par le test de Fisher exact bilatéral ont montré que l'occurrence du gène dnaJ était systématiquement plus élevée chez tous les isolats de C. jejuni relativement à C. coli. Dans la majorité des isolats restants, l'occurrence des marqueurs de virulence ne différait pas de façon significative. Les résultats en PCR ont aussi montré que l'occurrence des marqueurs de virulence des isolats analysés était beaucoup plus faible comparativement à d'autres études, ce qui pourrait être causé par un pool génomique divergent de nos isolats, comparativement aux autres. [ABSTRACT FROM AUTHOR]

Background: The cag pathogenicity island (cag-PAI) is one of the major virulence factors of Helicobacter pylori, showing considerable geographic variation.Aim: We investigated the prevalence of cagA, cagE, and cagT genes of cag-PAI and their association with proinflammatory IL-1B-511/-31/+3954 polymorphisms in Venezuelan chronic gastritis patients from a high-risk gastric cancer region.Methods: Presence of cag-PAI genes and IL-1B polymorphisms in 121 biopsy specimens was evaluated by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP), respectively.Results: cagA (+) and triple-positive (cagAET (+)) strains were detected in 79.3% and 70.2% of patients, respectively. We found that infection with cagA (+) and cagAET (+) strains was associated (P < 0.05) with hosts harboring both IL-1B +3954C allele and IL-1B-511T/-31C/+3954C haplotype (TCC (+)). The frequency of gastric atrophy was significantly higher (P < 0.020) among cagAET (+)/IL-1B-TCC (+) combined genotype carriers.Conclusion: Carriage of IL-1B +3954C allele and IL-1B-TCC (+) haplotype could favor colonization of bacterial cagAET (+) strains, and the combination of these bacterial and host haplotypes could play a synergistic role in development of premalignant gastric lesions. This work contributes to understanding of the complex interaction between H. pylori virulence factors and cytokine genotypes involved in gastrointestinal diseases. [ABSTRACT FROM AUTHOR]

Vibrios belonging to the Harveyi clade are pathogenic marine bacteria affecting both vertebrates and invertebrates, thereby causing a severe threat to the aquaculture industry. In this study, the expression of haemolysin, metalloprotease, serine protease, the quorum sensing master regulator LuxR and the virulence regulator ToxR in different Harveyi clade isolates was measured with reverse transcriptase real-time PCR with specific primers. There was relatively low variation in the in vitro expression levels of the quorum sensing master regulator luxR (sevenfold), whereas for the other genes, the difference in expression between the isolates showing lowest and highest expression levels was over 25-fold. Furthermore, there was a significant correlation between expression levels of toxR and luxR and between the expression levels of these regulators and the protease genes. The expression levels of luxR, toxR and haemolysin were negatively correlated with the survival of brine shrimp larvae challenged with the isolates. Finally, a non-virulent, a moderately virulent and a strongly virulent isolate were selected to study in vivo expression of the virulence genes during infection of gnotobiotic brine shrimp larvae. The in vivo gene expression study showed a clear difference in virulence gene expression between both virulent isolates and the non-virulent isolate. [ABSTRACT FROM AUTHOR]

The aim of this study was to detect Shiga toxins ( stx1 and stx2), enterohaemolysin ( EhlyA) and intimin ( eaeA) virulence genes of 11 Escherichia coli O157:H7 strains isolated from faecal samples of 300 clinically healthy Anatolian water buffaloes by PCR. Multiplex PCR was used for the detection of stx1 and stx2, and singleplex PCRs were used for the detection of EhlyA and eaeA virulence genes respectively. A total of three (27.3%) strains were determined to harbour both of the stx1 and stx2 genes, of these, one (9.1%) only harboured these two genes alone, one (9.1%) also contained the EhlyA gene and one (9.1%) additionally contained the EhlyA and the eaeA genes. EhlyA gene was obtained from eight (72.7%) strains, six (54.5%) of these were alone. eaeA gene was positive in only one (9.1%) strain. Only one (9.1%) of the 11 E. coli O157:H7 strains harboured all the four virulence genes. Two (18.2%) of the isolates had none of the virulence genes. Enterohaemolysin was found to be the most common virulence factor. In conclusion, the virulence factors of E. coli O157:H7 strains isolated from the faeces of Anatolian water buffaloes were investigated and detected for the first time in Turkey. [ABSTRACT FROM AUTHOR]

Abstract: Cholera diarrhea is still a major health challenge for a large part of globe. Global replacement of Vibrio cholerae classical biotype by El Tor biotype, emergence of O139 serogroup and rapid spread of antibiotic resistant strains indicate the continuous evolution in V. cholerae. In this study, 114 V. cholerae O1 serotype Ogawa isolates, collected from different cholera outbreaks in different regions of India between 2004 and 2007 were subjected to biochemical, immunological and molecular characterization. All the isolates were PCR positive for various toxigenic, pathogenic and regulatory genes, viz. ompW, ctxB, rfbO1, tcp, zot, rtxC, ace, hlyA, ompU and toxR. The antibiogram studies of isolates revealed the resistance towards several antibiotics including nalidixic acid, co-trimoxazole, streptomycin, nitrofurantoin and polymyxin B. However, antibiogram of the strains confirmed susceptibility to tetracycline and chloramphenicol in all the isolates. This study also substantiated the wide spread of class 1 integrons and SXT elements (mobile genetic elements for antibiotic resistance) in clinical isolates in India. Sequencing of cholera toxin (ctxB) gene revealed the replacement of traditional ctxB of El Tor biotype with ctxB of classical biotype in all the O1 El Tor strains collected since 2004. In addition, in 2007, a new variant of O1 El Tor strain with further modifications in ctxB of classical biotype is discovered among the Indian isolates. [Copyright &y& Elsevier]

The organs of 191 water buffalo calves died with clinical symptoms of diarrhoea were tested for the presence of Salmonella spp., E. coli, Criptosporidium spp., Giardia spp., coronavirus, rotavirus and C. perfringens. Fifty-eight strains of Salmonella spp. were isolated by cultural method and most frequently identified as typhimurium (16%), muenster (12%) and give (10%). All the strains were further characterized by PCR detection of 19 loci, including prophage genes and virulence factors, with the twofold to differentiate the isolated salmonellae and to describe their virulence. The 58 strains displayed 26 different PCR profiles, depending on the amplified genes. The 9 strains of S. typhimurium exhibited 7 different PCR profiles, carrying at least 5 out of 9 virulence factors. Among the other serotypes, 6 exhibited a high number of virulence genes (=5), while all the other strains had either a lower number or no virulence loci. This PCR characterization proved to be discriminating and informative about Salmonella virulence; it might contribute to a better understanding of its role in the aetiology of diarrhoea in water buffalo calves. [ABSTRACT FROM AUTHOR]

Abstract: Listeria monocytogenes is a foodborne pathogen which is able to cause serious disease both in humans and in animals. Several studies have demonstrated variations in the levels of virulence among L. monocytogenes strains. Invasion and growth ability of L. monocytogenes into cultured cells have been used to evaluate its pathogenicity. In particular, invasiveness and growth ability have been typically investigated using HeLa cell line. This study aimed to provide further insights on the virulence potential as well as on the molecular and phenotypic characteristics of L. monocytogenes isolated both from food sources and food environments. Thirty-eight isolates were tested for cell invasion and intracellular growth. Among the latter, 15 strains exhibited a high invasion index (I.I.); 18 strains showed intermediate II and 5 isolates revealed a low II. Regarding intracellular growth, all tested isolates had a replication time between 2 and 6h. Furthermore, nine virulence-associated genes (hlyA, actA, inlA, inlB, iap, plcA, plcB, mpl, prfA) were investigated by the multiplex PCR assay. All tested virulence genes were detected in all strains. Interestingly, a polymorphism was observed in the actA gene. However, the polymorphism could not be related to a different level of invasion or intracellular growth. In conclusion, data presented in this study have revealed considerable differences in the ability of L. monocytogenes strains to invade host cells and suggest the presence of additional factors that may contribute to adhesion and invasion. Virulence of L. monocytogenes is still not fully understood in some respects. Further studies focused on the mechanisms of L. monocytogenes pathogenicity together with the development of more reliable and efficient methods for virulence determination in this species are still required. [Copyright &y& Elsevier]

Background: In 2005, the first disease-specific Helicobacter pylori virulence factor that induced duodenal ulcer and had a suppressive action on gastric cancer has been identified, and was named duodenal ulcer promoting gene (dupA). However, the importance of the dupA gene on clinical outcomes is conflicting in subsequent studies. The aim of this study was to estimate the magnitude of the risk for clinical outcomes associated with dupA gene. Methods: A meta-analysis of case-control studies which provided raw data on the infection rates with the dupApositive H. pylori detected by polymerase chain reaction was performed. Results: Seventeen studies with a total of 2,466 patients were identified in the search. Infection with the dupApositive H. pylori increased the risk for duodenal ulcer by 1.41-fold (95% confidence interval [CI], 1.12-1.76) overall. Subgroup analysis showed that the summary odds ratio (OR) was 1.57 (95% CI, 1.19-2.06) in Asian countries and 1.09 (95% CI, 0.73-1.62) in Western countries. There was no association between the presence of the dupA gene and gastric cancer and gastric ulcer. Publication bias did not exist. Conclusion: Our meta-analysis confirmed the importance of the presence of the dupA gene for duodenal ulcer, especially in Asian countries. [ABSTRACT FROM AUTHOR]

We carried out the first study of Enterococcus faecalisclinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalisisolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6′)-aph(2″), aph(3′), ant(6), ant(3″)(9), aph(2″)-Id, aph(2″)-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6′)-Ie-aph(2″)-Ia, aph(3′), ant(6), ant(3″)(9)were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cylgene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agggenes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalisin Cuba, that is, most Cuban strains were related to European strains, whereas others to U.S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits. [ABSTRACT FROM AUTHOR]

The article presents a study on the contribution of (p)ppGpp synthetase ReIA in the virulence and stress adaptation of Enterococcus faecalis. Two reIA mutants by allelic replacement were constructed with the use of thermosensitive conditional replication pMAD system. The mutants were subjected to microarray experiments and real time quantitative polymerase chain reaction (RT qPCR). Results reveal that ef1974 locus is one of the cause of the production of (p)ppGpp.

Abstract: The present study was to investigate antimicrobial resistance profiles of Escherichia coli O26 and O111 from cattle and to characterize the virulence genes of the resistant isolates. This paper reports the high prevalence of antimicrobial resistant E. coli O26 and O111 from cattle. Among 37 E. coli O26 and 25 E. coli O111 isolates from the fecal specimens obtained from cattle, 26 (70%) and 15 (60%) were resistant to at least one antibiotic, respectively. Forty (98%) of the 41 resistant isolates were resistant to two or more antibiotics. Among the 22 antibiotics tested in this study, ampicillin was the most common antibiotic that the isolates were resistant to, followed by tetracycline and streptomycin. None of the isolates were resistant to fluoroquinolones, such as ciprofloxacin, ofloxacin and norfloxacin, and to ceftriaxone, amikacin and imipenem. Eighteen different resistant types among the 41 isolates were observed by the cluster analysis. The most frequent antibiotic-resistance type was ampicillin-tetracycline-streptomycin-cephalothin-sulfisoxazole-ticarcillin-kanamycin-minocycline-piperacillin-chloramphenicol, which accounted for 9 (22%) of the resistant isolates. The observation of frequent and multiple resistances to antibiotics highlights the need for their careful use if their benefits are to be preserved. PCR analysis of the EHEC virulence markers showed that 25 of the resistant E. coli O26 and O111 isolates tested positive for stx2 or both stx1 and stx2. This suggests that the majority of these isolates can cause serious diseases in humans and may complicate the future therapeutic options under development. [Copyright &y& Elsevier]

Abstract: A variety of environmental factors, such as oxygen, pH, osmolarity and antimicrobial agents, modulate the expression of Salmonella pathogenicity islands (SPI) genes. This study investigated SPI-1 gene expression and the pathogenicity of quinolone-resistant Salmonella. mRNA expression levels of the invA and avrA genes, located in SPI-1, in quinolone-susceptible and quinolone-resistant Salmonella strains were determined using real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). Twenty-five quinolone-resistant Salmonella mutants were derived from quinolone-susceptible strains by multiple-passage selection through increasing concentrations of ciprofloxacin in vitro, while an additional 15 strains were quinolone-resistant Salmonella clinical isolates. Sequence analysis showed no gene deletion or point mutations of nine SPI-1 genes (including invA and avrA) occurred in either the selected or clinical quinolone-resistant strains, while a single gyrA point mutation (S83F) was observed in all 40 quinolone-resistant strains. The mRNA expression levels of invA and avrA were significantly decreased (P <0.005) in quinolone-resistant strains (clinically acquired or experimentally selected in vitro), compared to the quinolone-susceptible strains. The resistant strains also had a slower growth rate combined with decreased epithelial cell invasion and intracellular replication in epithelial cells and macrophages. The results suggest that quinolone-resistance may be associated with lower virulence and pathogenicity than in quinolone-susceptible strains. [Copyright &y& Elsevier]

A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively from thoroughbred horses, and were submitted for screening prior to breeding programmes. Most, including a reference strain isolated in 1955, belonged to a cluster of genetically similar isolates of sequence type (ST) 60. K1 isolates, all from humans, belonged to a previously identified cluster of ST 23. [ABSTRACT FROM AUTHOR]

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified o five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients. [ABSTRACT FROM AUTHOR]

Abstract: The gram-negative bacterium Pasteurella multocida constitutes a heterogeneous species associated with wide range of disease in many animals. Isolates are classified into five groups based on capsular antigen (capA, B, D, E and F). Recently, a new valuable PCR-based method was introduced to determine the epidemiological correlation between P. multocida infection and existence of virulence genes including tbpA, pfhA, toxA and hgbB. However, this method is tedious and laborious. Thus, in the current study, we designed a reliable multiplex PCR method for rapid detection of virulence genes in P. multocida. Eighty seven strains of P. multocida isolated from various clinically healthy and infected hosts were examined by uniplex PCR method for each virulence associated genes. Based on our improved and simplified multiplex PCR method, rapid detection of four virulence genes was accomplished. It is proposed that its implementation may benefit the epidemiological investigations. [Copyright &y& Elsevier]

The article presents a study on the fatal case of Yersinia pseudotuberculosis 7 infection in a captive male Bolivian squirrel monkey (Saimiri boliviensis) after suffering from diarrhea and debilitation. It discloses that multiple yellowish-white nodules, mesenteric lymph nodes and solitary lymphatic nodules, as well as necrotizing enteritis with enlarged Peyer's patches containing Gram-negative bacilli, were found in the spleen and liver during necropsy. It uses polymerase chain reaction (PCR) and slide agglutination with rabbit immune sera to isolate the Y. pseudotuberculosis stereotype 7 from the spleen and liver. It states that VirF genes, which is used as marker of virulence plasimid (pYV) and ypmA genes in Y. pseudotoberculosis, were demonstrated using the PCR analysis.

Objectives: The aetiology of several human diarrhoeas has been increasingly associated with the presence of virulence factors rather than with the bacterial species hosting the virulence genes, exemplified by the sporadic emergence of new bacterial hosts. Two important virulence factors are the Shiga toxin (Stx) and the E. coli outer membrane protein (Eae) or intimin, encoded by the stx and eae genes, respectively. Although several polymerase chain reaction (PCR) protocols target these virulence genes, few aim at detecting all variants or have an internal amplification control (IAC) included in a multiplex assay. The objective of this work was to develop a simple multiplex PCR assay in order to detect all stx and eae variants, as well as to detect bacteria belonging to the Enterobacteriaceae, also used as an IAC. Results: The wecA gene coding for the production of the Enterobacterial Common Antigen was used to develop an Enterobacteriaceae specific qPCR. Universal primers for the detection of stx and eae were developed and linked to a wecA primer pair in a robust triplex PCR. In addition, subtyping of the stx genes was achieved by subjecting the PCR products to restriction digestion and semi-nested duplex PCR, providing a simple screening assay for human diarrhoea diagnostic. [ABSTRACT FROM AUTHOR]