Harbored Virulence Genes Research Articles

Molecular characterization of fluoroquinolone and/or cephalosporin resistance in Shigella sonnei isolates from yaks

BackgroundMembers of the genus Shigella are intestinal pathogens and a major cause of seasonal outbreaks of bacterial diarrhea worldwide. Although humans are the conventional hosts of Shigella species, expansion of the Shigella host range to certain animals was recently reported. To investigate the prevalence of Shigella sonnei (S. sonnei) in yaks and perform molecular characterization, we analyzed 1132 fresh yak diarrheal stool samples and collected a total of 44 S. sonnei isolates.ResultsWe performed multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) with XbaI-digested DNA to study genetic relatedness among the 44 isolates, which were differentiated into 4 sequence types (STs) and 32 PFGE types (PTs). All isolates harbored virulence genes, and 87.36% tested positive for invasion plasmid antigen H (ipaH), invasion associated locus (ial) and the Shigella enterotoxin gene sen. According to the results of antimicrobial susceptibility tests, 45.45% (20/44) were resistant to fluoroquinolones and/or cephalosporin. By sequencing the quinolone resistance determining region (QRDR) genes, we identified double mutations in gyrA (Ser83-Leu and Asp87-Asn) and a single mutation in parC (Ser80-Ile). All 12 fluoroquinolone-resistant S. sonnei isolates tested positive for the aac(6′)-Ib-cr gene but negative for qepA. Three isolates harbored qnr genes, including two with qnrS and one with qnrB. In addition, three types of β-lactamase genes, blaTEM-1, blaOXA-1 and blaCTX-M-14/79, were detected in cephalosporin-resistant isolates.ConclusionsThe findings of this study have enriched our knowledge of fluoroquinolone- and/or cephalosporin-resistant S. sonnei isolates from yaks, which has important public health significance.

Proteinaceous Secretory Metabolites of Probiotic Human Commensal Enterococcus hirae 20c, E. faecium 12a and L12b as Antiproliferative Agents Against Cancer Cell Lines.

Cancer is the second leading cause of death worldwide and its incidence is expected to grow by almost 70% in the coming 2 decades. Recent microbiome studies in cancer mice models have shown that certain commensal bacteria play protective roles against cancer. Thus, the use of commensal microflora having anticancer activities for the treatment of cancer appears to be an attractive alternative therapeutic strategy. Lactic acid bacteria (LAB) form an integral component of commensal microflora in healthy individuals. As the vaginal ecosystem is enriched in LAB genera, we screened the vaginal LAB microflora of healthy women for their anti-proliferative abilities against various human cancer cell lines. The secreted metabolites of three enterococcal strains, Enterococcus hirae 20c, Enterococcus faecium 12a and L12b, out of 92 LAB isolates selectively inhibited the in vitro proliferation of various human cancer cell lines in a dose-dependent manner but had no activity against normal human peripheral blood monocytes. Further, proteinase K-treatment of the cell-free supernatant (CS) of all the three enterococci abrogated their anti-proliferative abilities, thereby showing the proteinaceous nature of the secreted metabolites in the CS. The microscopic examination of the cell lines showed that CS-treatment induced apoptosis-like morphological changes in the cancer cells. Further, the probiotic characters of the strains were studied, which showed that all the three strains had broad spectrum antimicrobial activities against various Gram-positive and Gram-negative pathogens, including Mycobacterium smegmatis. All the strains tolerated the gastric acidity and bile juice treatments, and had strong adhesive abilities to the colonic epithelial cell line HCT-15. Furthermore, none of the strains had any known secreted virulence factors or harbored virulence genes. This preliminary study highlights an important functional role of the commensal probiotic enterococcal strains E. hirae and E. faecium for the first time by demonstrating their anticancer properties that should be further tested in the in vivo mammalian models.

Genome Sequencing of Extended-Spectrum β-Lactamase (ESBL)-Producing Klebsiella pneumoniae Isolated from Pigs and Abattoir Workers in Cameroon.

Background and objectives: Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae is a serious public health issue globally. In this study, the antibiotic resistance genes, virulence factors, mobile genetic elements, and genetic lineages of circulating ESBL-producing K. pneumoniae strains isolated from pigs and humans in Cameroonian abattoirs were investigated using whole genome sequencing (WGS), in order to ascertain zoonotic transmission (viz. from animals to humans and/or vice-versa) in the food chain.Methods: During March–October 2016, 288 nasal and rectal pooled samples from 432 pigs as well as nasal and hand swabs from 82 humans were collected from Cameroon and South Africa. Seven ESBL-producing K. pneumoniae circulating in Cameroonian pig abattoirs were selected and their genomic DNA sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using the Qiagen CLC Genomics Workbench and SPAdes. The assembled contigs were annotated using RAST and antibiotic resistance genes, virulence factors, plasmids, and bacteriophages were identified with ResFinder, Virulence Finder, PlasmidFinder, and PHAST, respectively.Results: ESBL-producing K. pneumoniae were detected in pigs (34/158; 21.52%) and exposed workers (8/71; 11.26%) in Cameroon only. The circulating K. pneumoniae strains were dominated principally by the sequence type (ST) 14 and 39. In addition, the “high-risk” ST307 clone and two novel STs assigned ST2958 and ST2959 were detected. Genomic analysis identified various antibiotic resistance genes associated with resistance to β-lactams, aminoglycosides, fluoroquinolones, macrolide, lincosamide and streptogramins, rifampicin, sulfonamides, trimethoprim, phenicols and tetracycline. None of the ESBL-producing K. pneumoniae harbored virulence genes. Intermingled K. pneumoniae populations were observed between pig- and human-source within and across abattoirs in the country.Conclusion: Our study shows that ESBL-producing K. pneumoniae is actively disseminating in pigs and occupationally exposed workers in Cameroonian pig abattoirs and is probably underestimated in the absence of molecular epidemiological studies. It suggests pigs, abattoir workers and food products as potential reservoirs and sources of zoonotic transmission in Cameroon. Our findings underline the existence of a potential unheeded food safety and public health threat associated with these resistant strains and reinforce the crucial importance of implementing appropriate food safety measures and promoting rational antibiotic use.

Pathogenic Characteristics and Antibiotic Resistance of Bacterial Isolates from Farmstead Cheeses.

The objective of this study was to investigate the pathogenicity and antimicrobial resistance of foodborne pathogens isolated from farmstead cheeses. Twenty-seven isolates, including 18 Bacillus cereus, two Escherichia coli, and seven Staphylococcus aureus, were subjected to polymerase chain reaction (PCR) to detect virulence genes and toxin genes, and the antibiotic resistances of the isolates were determined. All E. coli isolates were determined by PCR to be non-pathogenic. Among the 18 B. cereus isolates, 17 isolates (94.4%) were diarrheal type, as indicated by the presence of nheA, entFM, hbIC, cytK and bceT genes, and one isolate (5.6%) was emetic type, based on the presence of the CER gene. Among the seven S. aureus isolates, three (42.9%) had the mecA gene, which is related to methicillin-resistance. Most B. cereus isolates (94.7%) showed antibiotic resistance to oxacillin and penicillin G, and some strains also showed resistance to ampicillin (26.3%), erythromycin (5.3%), tetracycline (10.5%), and vancomycin (5.3%). These results indicate that microbial food safety measures for farmstead cheese must be implemented in Korea because antibiotic resistant foodborne pathogens, with resistance even to vancomycin, harboring virulence genes were found to be present in the final products of farmstead cheese.

Shigella and Enteroinvasive Escherichia Coli.

Shigella and enteroinvasive Escherichia coli (EIEC) are gram-negative bacteria responsible for bacillary dysentery (shigellosis) in humans, which is characterized by invasion and inflammatory destruction of the human colonic epithelium. Different EIEC and Shigella subgroups rose independently from commensal E. coli through patho-adaptive evolution that included loss of functional genes interfering with the virulence and/or with the intracellular lifestyle of the bacteria, as well as acquisition of genetic elements harboring virulence genes. Among the latter is the large virulence plasmid encoding for a type three secretion system (T3SS), which enables translocation of virulence proteins (effectors) from the bacterium directly into the host cell cytoplasm. These effectors enable the pathogen to subvert epithelial cell functions, promoting its own uptake, replication in the host cytosol, and dissemination to adjacent cells while concomitantly inhibiting pro-inflammatory cell death. Furthermore, T3SS effectors are directly involved in Shigella manipulation of immune cells causing their dysfunction and promoting cell death. In the current chapter, we first describe the evolution of the enteroinvasive pathovars and then summarize the overall knowledge concerning the pathogenesis of these bacteria, with a particular focus on Shigella flexneri. Subversion of host cell functions in the human gut, both epithelial and immune cells, by different virulence factors is especially highlighted.

Virulence genotypes and antimicrobial susceptibility patterns of Arcobacter butzleri isolated from seafood and its environment

Arcobacter butzleri is an emerging pathogen isolated from animals, food and the environment. In this study, 147 A. butzleri isolated from seafood and the coastal environment were tested for the presence of ten putative virulence genes (cadF, cj1349, ciaB, mviN, pldA, tlyA, hecA, hecB, irgA, iroE) and antimicrobial susceptibilities. Majority of the isolates harbored mviN (100%), cj1349 (97.2%), ciaB (95.9%), tlyA (91.8%), pldA (91.1%) and cadF (89.7%). Lower detection rates were observed for hecA (10.8%), hecB (19%), iroE (12.9%) and irgA (17.6%). Three A. butzleri isolates harbored all ten virulence genes. The occurrence of cj1349, ciaB, pldA, tlyA and hecA genes was significantly different (P≤0.05) among the isolates from different sources. All (100%) A. butzleri isolates were resistant to vancomycin, cephalothin, cefoxitin and sulphamethizole and susceptible to polymyxin-B, kanamycin, streptomycin, gentamicin, tetracycline and imipenem. Resistance to clinically important antibiotics such as cefotaxime (99.3%), ceftazidime (87.7%), nalidixic acid (70.7%), ampicillin (72.1%), ertapenem and amoxicillin-clavulanic acid (41.9%) was observed in A. butzleri from the environment. The isolates were highly susceptible to norfloxacin (97.9%) and colistin (97.2%), followed by ciprofloxacin (88.4%), meropenem (74.8%), chloramphenicol (72.7%) and erythromycin (69.3%). A. butzleri from different sources were not significantly different with respect to their antimicrobial susceptibility patterns. Multidrug resistance was observed in 66 (81.4%) isolates from fish, 29 (72.5%) isolates from shellfish and 17 (65.3%) isolates from coastal water. A. butzleri harboring virulence genes and resistance to multiple antibiotics found in seafood could be a potential health risk to seafood handlers and consumers. Continuous monitoring of seafood for potentially pathogenic A. butzleri is important to understand the evolution of antibiotic resistance in this emerging food pathogen and to determine the antimicrobial therapy regimen in the event of food-borne A. butzleri infections.

Characterization of pathogenic Enterococcus cecorum from different poultry groups: Broiler chickens, layers, turkeys, and waterfowl.

Enterococcus cecorum (EC) is known as a commensal in the intestines of mammals and birds. However, it has been described as an emerging pathogen in poultry industry worldwide. The aim of this study was to analyze and compare EC isolated from clinical material collected from poultry groups with different production purposes. The genetic diversity among pathogenic EC in relation to each specific poultry type was examined. In total, 148 isolates from independent infection outbreaks (2011–2016) were used: 76 broiler chickens (CB), 37 broiler breeders (BB), 23 layers (CL), 7 waterfowl (W) and 5 turkey (T) flocks (1 isolate/1 flock). We provided age ranges at diagnosis of EC-infection for 5 poultry groups. Isolates obtained from CB were significantly more frequently retrieved from bone marrow, joints, spine, and contrary to BB, CL less frequently retrieved from respiratory system. The study showed differences between EC of various poultry types in relation to 10/32 (31.3%) biochemical parameters. EC isolates from CB were significantly more often positive for βGAL, βNAG, MLZ, and less often positive for PAL and βMAN than isolates from other poultry types. However, BB and W isolates showed higher ability to metabolise mannitol than CB, CL, and T. CB isolates showed lower ability to survive at 60°C. Only chicken EC-isolates harbored virulence genes: CB (8.1%) > BB (3.4%) > CL (2%). No specific pulsotype of EC was associated with a specific poultry. One or several various (up to 6) genetic types of EC may be involved in outbreaks in CB flocks within one year in one region. Outbreaks reported in following years in the same region were usually caused by a distinct set of EC-genetic types. PFGE results indicated at the genetic heterogeneity among pathogenic isolates involved in outbreaks in relation to each poultry type. To our best knowledge, this is the first study which provides a comparison between clinical EC from 5 poultry groups. The study provides a new insight into EC as pathogen of different bird species. The obtained data may be useful in further studies on EC-infections more focused on a specific type of poultry.

Antimicrobial, cytotoxic effect and purification of bacteriocin from vancomycin susceptible Enterococcus faecalis and its safety evaluation for probiotization

A vancomycin susceptible Enterococus faecalis CV7 produced bacteriocin was purified by size exclusion chromatography followed by RP-HPLC C-18 column. Tricine-SDS PAGE unrevealed molecular mass was further confirmed by MALDI-TOF MS as 4.829 kDa. This bacteriocin showed broad spectrum of anti-bacterial activity against important food borne pathogens mainly against Salmonella typhi and this activity also found to be withstood after treatment with different proteolytic enzymes, temperature, pH, solvents and detergents. Cytotoxicity of bacteriocin CV7 by MTT assay showed that lower survival inhibition on HeLa than HT-29 cell line at lower concentrations conclude its potentiality in food biopreservation. Results obtained from PCR amplifications revealed that strain E. faecalis CV7 does not harbor virulence genes esp, ace, cylB, cylA and efaAfs but contains asa1. With this, surviving in the presence of low pH and bile oxgall promising its safety as probiont to control Salmonella infections.

Enterococcus faecium and Enterococcus durans isolated from cheese: Survival in the presence of medications under simulated gastrointestinal conditions and adhesion properties

In this study, we evaluated the survival of Enterococcus faecium and Enterococcus durans, isolated from cheese, in the presence of medications and under simulated in vitro gastrointestinal conditions. The presence of genes encoding virulence factors, the susceptibility to antimicrobial agents, and adhesion properties were also assessed. Enterococcus faecium and E. durans both exhibited resistance to most of the tested medications but showed a large sensitivity to analgesics and antihypertensives; they also showed wide susceptibility to antimicrobial agents. Enterococcus durans SJRP29 had greater resistance to the presence of medications in comparison with the probiotic Lactobacillus acidophilus La-5. The strains, except for E. durans SJRP05, did not harbor virulence genes. Enterococcus durans SJRP14, SJRP17, and SJRP26 were sensitive to all tested antimicrobial agents. Enterococcus faecium was more stable during the simulation of gastrointestinal tract and showed greater viability. At the end of the assay, except for E. durans SJRP17, all strains showed high viability (>7 log cfu/mL). Enterococcus durans SJRP29 stood out from the other strains and was selected for further evaluation; it tolerated up to 3.0% NaCl at 30 and 37°C, besides having good adhesion properties (high values of auto-aggregation, co-aggregation, and hydrophobicity). Additionally, the microorganism did not show bile salt hydrolase activity or mucin degradation. These results encourage carrying out additional tests to evaluate the probiotic features by using in vitro dynamic models and in vivo tests before applying these strains to a food system.

Characterization of Escherichia coli and other Enterobacteriaceae in producer-distributor bulk milk

The current study was undertaken to characterize Escherichia coli and other Enterobacteriaceae in raw and pasteurized producer-distributor bulk milk (PDBM). A total of 258 samples were collected from purchase points in 8 provinces in South Africa. The samples were tested for antibiotic residues, phosphatase, total aerobic bacteria, coliforms, and E. coli counts. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for identification of isolates. Escherichia coli isolates were characterized for virulence factors, antimicrobial resistance, serotypes, and presumptive E. coli O157:H7. Antibiotic residues and alkaline phosphatase were detected in 2% of both raw and pasteurized PDBM (n=258) and 21% pasteurized PDBM (n=104) samples, respectively. A total of 729 isolates belonging to 21 genera and 59 species were identified. Escherichia coli, Enterobacter cloacae, Klebsiella oxytoca, and Raoultella ornithinolytica were the most abundant species. Spoilage Enterobacteriaceae species exceeded 50% of the total isolates. Escherichia coli was detected and isolated from 36% of the milk samples. Thirty-one E. coli isolates harbored virulence genes stx1/stx2 and 38% (n=121) were presumptive O157:H7. The prevalence of samples with presumptive shigatoxin producing E. coli was 10%. Antimicrobial-resistant E. coli isolates were detected in 70% of the milk samples with 36% of stx1/stx2 positive E. coli showing multi-drug resistance. Information obtained from the study will be used for modeling the public health risk posed by milkborne pathogens in PDBM, which in many cases is consumed by poor and vulnerable members of the population.

The Correlation between Resistance to Antimicrobial Agents and Harboring Virulence Factors among Enterococcus Strains Isolated from Clinical Samples

<p class="1Body"><strong>Objectives:</strong> In Iran as well as throughout the world Enterococci have been rated as the important cause of urinary tract and nosocomial infections. The aim of this study was to evaluate the relationship between high antimicrobial resistance activity and harboring the virulence factors among clinical Enterococcus isolates.</p><p class="1Body"><strong>Materials and Methods:</strong> Clinical strains were isolated from hospitalized patients. Prevalence of different virulence genes was evaluated by PCR method and the relation between resistance to antibiotics and harboring virulence genes was evaluated by statistical analysis.</p><p class="1Body"><strong>Results:</strong> The results showed that <em>E. faecalis</em> (60%) is more prevalent than <em>E. faecium </em>(26%) and harboring more virulence factors. The highest resistance was related to gentamicin in both <em>E. faecalis</em> and <em>E. faecium</em> isolates with the rate of 88.7% and 93.5% respectively. Harboring <em>esp</em>, <em>ace</em> and <em>cyl</em>A are significantly related to resistance to different antibiotics.</p><strong>Conclusion:</strong> The antimicrobial resistance and virulence pattern of Enterococcus must be constantly monitored in order to choose the best antimicrobial treat and prevent nosocomial infections.

Molecular characterization and antimicrobial susceptibility of Listeria monocytogenes isolated from foods and humans

A total of 120 Listeria monocytogenes isolates (107 from foods and 13 from humans) in Shanghai, China, were characterized by serogroup typing and virulence genes detection with PCR, antimicrobial susceptibility testing, and molecular subtyping using pulsed-field Gel Electrophoresis (PFGE). Isolates belonged to three L. monocytogenes serogroups, 1/2c, 3c (n = 47, 39.1%) was the most prevalent, followed by 1/2a, 3a (n = 44, 36.7%) and 1/2b, 3b, 7 (n = 29, 24.2%). Although the isolates were all susceptible to most antibiotics tested, 13 isolates were resistant to ceftriaxone and seven to tetracycline. None of the isolates were resistant to multiple antibiotics. The tet (M) genes were detected among tetracycline-resistant isolates. The isolates all harbored virulence genes hly, prfA, plcA, plcB, inlA, inlB, inlI; 116 isolates (96.7%) were positive for inlC; 78 (65.0%) for inlG; 119 (99.2%) for inlJ; 8 (6.67%) for llsX. A total of 74 distinct patterns were generated for the 119 isolates tested using ApaI and AscI with Pulsed Field Gel Electrophoresis (PFGE). Three clinical isolates shared the same PFGE patterns with retail meet (chicken, duck, and beef) isolates. These findings indicated that food of animal origin likely serve as a major vehicle in transmitting human listeriosis in Shanghai. Active surveillance for L. monocytogenes is needed for better understanding the epidemiology of the pathogen, and for effective control and prevention of human listeriosis.

Prevalence of Escherichia coli Virulence Genes in Patients with Diarrhea and a Subpopulation of Healthy Volunteers in Madrid, Spain.

Etiological diagnosis of diarrheal diseases may be complicated by their multi-factorial nature. In addition, Escherichia coli strains present in the gut can occasionally harbor virulence genes (VGs) without causing disease, which complicates the assessment of their clinical significance in particular. The aim of this study was to detect and quantify nine VGs (stx1, stx2, eae, aggR, ehxA, invA, est, elt and bfpA) typically present in five E. coli enteric pathotypes [enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC)] in fecal samples collected from 49 patients with acute diarrhea and 32 healthy controls from Madrid, Spain. In addition, the presence of four serotype-related genes (wzxO104 and fliCH4, rfbO157, and fliCH7) was also determined. Presence of target genes was assessed using a quantitative real-time PCR assay previously developed, and the association of presence and burden of VGs with clinical disease and/or other risk factors was explored. Prevalence of ehxA [typically associated with Shigatoxin producing E. coli (STEC) and (EPEC), invA (EIEC), and the rfbO157+fliCH7 (STEC)] combination were significantly (p < 0.02) higher in the diarrheic group, while the wzxO104+fliCH4 combination was significantly (p = 0.014) more prevalent in the control group. On the other hand, eae was detected in more than 90% of the individuals in both patient and control populations, and it was not associated with bfpA, suggesting the absence of typical EPEC. No significant differences in the quantitative values were detected for any VG among study groups, but the difference in the load of aggR (EAEC) and invA in the patients with respect to the controls was close to the significance, suggesting a potential role of these VGs in the clinical signs observed when they are present at high levels.

New insights about excisable pathogenicity islands in Salmonella and their contribution to virulence

Pathogenicity islands (PAIs) are regions of the chromosome of pathogenic bacteria that harbor virulence genes, which were probably acquired by lateral gene transfer. Several PAIs can excise from the bacterial chromosome by site-specific recombination and in this review have been denominated “excisable PAIs”. Here, the characteristic of some of the excisable PAIs from Salmonella enterica and the possible role and impact of the excision process on bacterial virulence is discussed. Understanding the role of PAI excision could provide important insights relative to the emergence, evolution and virulence of pathogenic enterobacteria.

Virulence Genes among Enterococcus faecalis and Enterococcus faecium Isolated from Coastal Beaches and Human and Nonhuman Sources in Southern California and Puerto Rico.

Most Enterococcus faecalis and E. faecium are harmless to humans; however, strains harboring virulence genes, including esp, gelE, cylA, asa1, and hyl, have been associated with human infections. E. faecalis and E. faecium are present in beach waters worldwide, yet little is known about their virulence potential. Here, multiplex PCR was used to compare the distribution of virulence genes among E. faecalis and E. faecium isolated from beaches in Southern California and Puerto Rico to isolates from potential sources including humans, animals, birds, and plants. All five virulence genes were found in E. faecalis and E. faecium from beach water, mostly among E. faecalis. gelE was the most common among isolates from all source types. There was a lower incidence of asa1, esp, cylA, and hyl genes among isolates from beach water, sewage, septage, urban runoff, sea wrack, and eelgrass as compared to human isolates, indicating that virulent strains of E. faecalis and E. faecium may not be widely disseminated at beaches. A higher frequency of asa1 and esp among E. faecalis from dogs and of asa1 among birds (mostly seagull) suggests that further studies on the distribution and virulence potential of strains carrying these genes may be warranted.

Summer and Winter Prevalence of Shiga Toxin-Producing Escherichia coli (STEC) O26, O45, O103, O111, O121, O145, and O157 in Feces of Feedlot Cattle.

The United States Department of Agriculture Food Safety and Inspection Service has declared seven Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, nonintact beef products. The objective of this study was to determine the prevalence of these seven serogroups and the associated virulence genes (Shiga toxin [stx1, stx2], and intimin [eae]) in cattle feces during summer (June-August 2013) and winter (January-March 2014) months. Twenty-four pen floor fecal samples were collected from each of 24 cattle pens, in both summer and winter months, at a commercial feedlot in the United States. Samples were subjected to culture-based detection methods that included enrichment, serogroup-specific immunomagnetic separation and plating on selective media, followed by a multiplex polymerase chain reaction for serogroup confirmation and virulence gene detection. A sample was considered STEC positive if a recovered isolate harbored an O gene, stx1, and/or stx2, and eae genes. All O serogroups of interest were detected in summer months, and model-adjusted prevalence estimates are as follows: O26 (17.8%), O45 (14.6%), O103 (59.9%), O111 (0.2%), O121 (2.0%), O145 (2.7%), and O157 (41.6%); however, most non-O157 isolates did not harbor virulence genes. The cumulative model-adjusted sample-level prevalence estimates of STEC O26, O103, O145, and O157 during summer (n=576) were 1.0, 1.6, 0.8, and 41.4%, respectively; STEC O45, O111, and O121 were not detected during summer months. In winter, serogroups O26 (0.9%), O45 (1.5%), O103 (40.2%), and O121 (0.2%) were isolated; however, no virulence genes were detected in isolates from cattle feces collected during winter (n=576). Statistically significant seasonal differences in prevalence were identified for STEC O103 and O157 (p<0.05), but data on other STEC were sparse. The results of this study indicate that although non-O157 serogroups were present, non-O157 STEC were rarely detected in feces from the feedlot cattle populations tested in summer and winter months.

Evaluation of the Antibiotic Resistance and Virulence of Escherichia coli Strains Isolated from Chicken Carcasses in 2007 and 2013 from Paraná, Brazil

The frequent use of antimicrobials in commercial poultry production has raised concerns regarding the potential impact of antimicrobials on human health due to selection for resistant bacteria. Several studies have reported similarities between extraintestinal pathogenic Escherichia coli (ExPEC) strains isolated from birds and humans, indicating that these contaminant bacteria in poultry may be linked to human disease. The aim of our study was to analyze the frequency of antimicrobial resistance and virulence factors among E. coli strains isolated from commercial chicken carcasses in Paraná, Brazil, in 2007 and 2013. A total of 84 E. coli strains were isolated from chicken carcasses in 2007, and 121 E. coli strains were isolated in 2013. Polymerase chain reaction was used to detect virulence genes (hlyF, iss, ompT, iron, and iutA) and to determine phylogenetic classification. Antimicrobial susceptibility testing was performed using 15 antimicrobials. The strains were also confirmed as extended-spectrum β-lactamase (ESBL)-producing E. coli with phenotypic and genotypic tests. The results indicated that our strains harbored virulence genes characteristic of ExPEC, with the iutA gene being the most prevalent. The phylogenetic groups D and B1 were the most prevalent among the strains isolated in 2007 and 2013, respectively. There was an increase in the frequency of resistance to a majority of antimicrobials tested. An important finding in this study was the large number of ESBL-producing E. coli strains isolated from chicken carcasses in 2013, primarily for the group 2 cefotaximase (CTX-M) enzyme. ESBL production confers broad-spectrum resistance and is a health risk because ESBL genes are transferable from food-producing animals to humans via poultry meat. These findings suggest that our strains harbored virulence and resistance genes, which are often associated with plasmids that can facilitate their transmission between bacteria derived from different hosts, suggesting zoonotic risks.

Bacteriocin-producing Enterococcus faecalis KT2W2G isolated from mangrove forests in southern Thailand: Purification, characterization and safety evaluation

The aim of this work was to purify and characterize the bacteriocin produced by Enterococcus faecalis KT2W2G isolated from the mangrove forest in southern Thailand, in order to evaluate its potential as a new food protective agent. The active peptide from the cell-free supernatant of Ent. faecalis KT2W2G was purified in 4 steps: (i) precipitation with 70% saturated ammonium sulfate, (ii) elution on a reversed phase cartridge (Sep-Pak C8) using different concentrations of acetonitrile, (iii) cation-exchange chromatography and (iv) final purification by reversed phase-HPLC on a C8 column. Each purification step increased the specific activity and reduced the amount of contaminating non-bacteriocin proteins. The specific activity of purified bacteriocin was 13,470.53 AU/mg of protein, which corresponded to a 48.10-fold increase. Tricine–SDS-PAGE of the purified bacteriocin gave molecular weight ranging between 3.5 and 6.5 kDa. The activity of the partially purified bacteriocin was unaffected by pH (2.0–12.0) and thermostable, but was sensitive to proteolytic enzymes. This bacteriocin maintained full stability after storage at −20, 4 and 37 °C for 2 months. It was stable when incubated for 1 month at 4 °C in 0–30% NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activities against other LAB, food-spoilage and food-borne pathogens. Ent. faecalis KT2W2G was sensitive to kanamycin, tetracycline and vancomycin but resistant to ampicillin, gentamicin and penicillin. PCR amplification demonstrated that Ent. faecalis KT2W2G does not harbor virulence genes cylA, cylB and esp but has virulence genes ace, asa1 and efaAfs. The bacteriocin and its producing strain may find application as bio-preservatives for reduction of food-spoilage and food-borne pathogens in food products.

Epidemic and virulence characteristic of Shigella spp. with extended-spectrum cephalosporin resistance in Xiaoshan District, Hangzhou, China

BackgroundShigellae have become increasingly resistant to the extended-spectrum cephalosporin (ESC) worldwide and pose a great challenge to anti-infection treatment options. The purpose of this study was to determine the resistance, cephalosporin resistance mechanisms, virulence characteristic and genotype of ESC-resistant Shigella.MethodsFrom 2008 to 2012, Shigella isolates collected from diarrhea patients were detected for antibiotics sensitivity by disk diffusion, cephalosporin resistance determinants and virulence genes using polymerase chain reaction (PCR) and genotyping through enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR).ResultsA total of 356 Shigella isolates were gathered, and 198 (55.6%, 58 S. flexneri and 140 S. sonnei) were resistant to ESC. All ESC-resistant isolates were susceptible to imipenem, and only 0.5% isolate was resistant to piperacillin/tazobactam. ESC-resistant S. flexneri showed high degrees of resistance to ampicillin (100%), ampicillin/sulbactam (96.6%), piperacillin (100%), trimethoprim/sulfamethoxazole (74.1%), ciprofloxacin (74.1%), levofloxacin (53.4%), ceftazidime (58.6%) and cefepime (58.6%). ESC-resistant S. sonnei exhibited high resistance rates to ampicillin (100%), piperacillin (100%) and trimethoprim/sulfamethoxazole (96.4%). Cephalosporin resistance genes were confirmed in 184 ESC-resistant isolates. blaCTX-M types (91.8%, mainly blaCTX-M-14, blaCTX-M-15 and blaCTX-M-57) were most prevalent, followed by blaOXA-30 (26.3%). Over 99.0% ESC-resistant isolates harbored virulence genes ial, ipaH, virA and sen. However, set1 were more prevalent in ESC-resistant S. flexneri isolates than in S. sonnei isolates. ERIC-PCR results showed that 2 and 3 main genotypes were detected in ESC-resistant S. flexneri and S. sonnei, respectively.ConclusionOur findings indicated that a high prevalence of ESC-resistant Shigella mediated mainly by blaCTX-M with stronger resistance and virulence, and the existence of specific clones responsible for these Shigella infection in the region studied.

Tu1760 Functional Evidence for a Causative Role of the Intestinal Microbiota in Crohn's Disease-Like Ileitis Using Antibiotic-Treated and Germ-Free TNFDeltaare/+ Mice

BACKGROUND: An increased prevalence of phylogenetic B2 group Escherichia coli (E. coli) has been associated with active disease in IBD patients (IBDEC). Many of the B2 group strains harbor virulence genes present in extraintestinal pathogenic E.coli including hemolysin (hly) and cytotoxic necrotizing factor type 1 (CNF1) the loci of which is often linked. Here we investigated the potential of B2 strains of E.coli isolated from IBD patients to damage the integrity of the intestinal epithelium and the potential contribution of Hly and CNF1. METHODS: E coli strains isolated from 35 patients with IBD were tested for virulence (Petersen et al 2009) and of those isolate C691-04A showed high cytotoxicity on dendritic cells ( submitted) and a rapidly decrease of the trans-epithelial electric resistance (TEER) of a monolayer of intestinal epithelial cell lines. Genome sequencing of C691-04A confirmed the presence of hly cluster. It is known that some E coli have two hly-clusters. In order to investigate the role of hly and CNF1 in disruption of the epithelial integrity the following mutants were created in strain C691-04A; 1: CAS965, hly cluster II upstream of CNF1; 2: CAS954, hly cluster I mutant; 3: CAS956 is a knock out of both loci of hly; 4: CAS964, CNF1 mutant; 5: CAS966, clbA mutant. Caco-2 cells were grown to confluence in 24-well trans-well devices for 14 days to form a polarized cell monolayer and TER measured constantly during exposure to E. coli strains at MOI 10. The wild type and commensal E. coli were included as controls. RESULTS: E. coli C691-04A WT as well as CNF1, clbA and single loci of hly mutants from cluster I and II were all able to damage Caco-2 cell tight junctions in less than 6 hrs co-culture (see figure). Confocal microscopy of occludin stained trans-well sections revealed loss of occludin from the tight junction within 3 hours. However, this phenotype was lost in a mutant with knock out of both hly loci (p<0.001). Commensal E. coli isolated from healthy controls also did not cause loss of TER in 6 h. CONCLUSION: IBD-associated E. coli such as C691-04A can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect is abolished in a mutant with knock out of both loci of the hly genes and diminished in one of the single knockout mutants. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.