Stx2 Genes Research Articles

Prevalence and patterns of fecal shedding of Shiga toxin–producing Escherichia coli by cattle at a commercial feedlot in South Africa

AbstractHealthy colonized cattle are the major reservoir of Shiga toxin–producing Escherichia coli (STEC) and play a key role in the entry point of the pathogen into the beef chain. Excretion rates and the concentration of the pathogen in feces influence the epidemiology and transmission of the pathogen within herds and to humans. This study evaluated the prevalence and dynamics of fecal shedding of STEC by cattle in a commercial feedlot in Gauteng, South Africa. An initial cross‐sectional survey was conducted; fecal samples were obtained from 106 randomly selected weaned beef calves on arrival at the feedlot using polymerase chain reaction (PCR) to screen by detecting stx1 and stx2 genes. Subsequently, a longitudinal study was conducted, and 15 STEC‐positive and 11 STEC‐negative cattle were sampled monthly and followed to slaughter. STEC O157 and non‐O157 were enumerated in samples using commercial chromogenic agar. Initial prevalence of STEC shedding was 27% (29/106; 95% CI [19, 37%]). All 26 cattle shed STEC intermittently or continuously during the study period, all except one were super‐shedders (≥4 log10 CFU/g) at one or more samplings, and 19 (73%) were persistent or intermittent super‐shedders. Of the 38 STEC isolates recovered, 15 (39%) were serotypeable, representing 11 non‐O157 serogroups, including O101, O168, O178, and O68. The most frequent virulence combination profile was stx1 + eaeA + ehxA (n = 12; 32%). This study confirms the occurrence and variability of STEC super‐shedding in feedlot cattle and highlights that super‐shedding is not limited to STEC O157. It also shows their public health significance.

Validation of the 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) for the Detection of Shiga Toxin Gene (stx1 and/or stx2) in Fresh Raw Ground Beef and Fresh Spinach: AOAC Performance Tested MethodSM 071903.

BackgroundThe 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched foods. The stx assay does not differentiate between stx1 and stx2 but detects the presence of stx1 and/or stx2.ObjectiveThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method was evaluated for AOAC® Performance Tested MethodsSM certification.MethodsMatrix studies, inclusivity/exclusivity, robustness testing, product stability, and lot-to-lot variability testing were conducted to assess the method’s performance.ResultsThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) demonstrated equivalent results to the United States Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 5C.00 reference method for fresh raw ground beef, and the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A reference method for fresh spinach. The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) detected all STEC E. coli strains (E. coli strains with stx1 and/or stx2 genes) and did not detect any of the 45 strains from the exclusivity panel. Robustness testing indicated that small variations in critical test parameters did not adversely affect the assay’s performance. Product consistency and stability testing demonstrated no differences between the lots evaluated.ConclusionThe data collected in these studies demonstrate that the 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of Shiga toxin-producing E. coli in raw ground beef and spinach.HighlightsThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method is suitable for the rapid and specific detection of Shiga toxin-producing E. coli in fresh raw ground beef, and spinach.

Detection of Virulence Genes and Biofilm Forming Capacity of Diarrheagenic E. coli Isolated from Different Water Sources

Diarrheagenic Escherichia coli (DEC) are associated with frequent incidences of waterborne infections and pose health risk to individuals who contact water for domestic or recreational uses. Detection of DEC pathotypes in drinking water can be used as an indicator of fecal contamination. This study aimed to investigate the occurrence of DEC pathotypes and their capacity to form biofilms in drinking water samples collected from different water sources. In this study, PCR analysis was used to determine the occurrence of four clinically significant virulence genes of diarrheagenic E. coli, eaeA (Enteropathogenic E. coli), stx1, stx2 (Enterohemorrhagic E. coli) and sth (Enterotoxigenic E. coli), in drinking water samples (n = 35) by using specific primers and conditions. PCR amplicons were visualized by using agarose gel electrophoresis. A total of 12/35 (34%) samples were detected as positive for at least one of the four DEC virulence genes and 11/12 (91%) E. coli isolates harbored virulence gene while 1/12 (8%) E. coli isolates harbored none. The eaeA and sth genes were the most detected genes (75%), while stx1 and stx2 genes were least detected genes (66%). Biofilm assay confirmed that ETEC pathotypes can cause damage in enteric walls by attaching and effacing to persist diarrheal conditions. This study indicated that drinking water of different sources is contaminated with potential DEC pathotypes and it can be a source of diarrheal diseases. The amplification of four virulence genes associated with DEC pathotypes (EPEC, EHEC and ETEC) in drinking water demonstrates that potentially virulent DEC pathotypes are distributed in water sources and may be a cause of health concern. There is, therefore, an urgent need to monitor DEC pathotypes in drinking water.

Prevalence of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Serogroups Isolated from Fresh Raw Beef Meat Samples in an Industrial Slaughterhouse.

Background The aims of the current study are the identification of O157 and non-O157 Shiga Toxin-Producing Escherichia coli (STEC) serogroups isolated from fresh raw beef meat samples in an industrial slaughterhouse, determination of antimicrobial resistance patterns, and genetic linkage of STEC isolates. Materials and Methods A total of 110 beef samples were collected from the depth of the rump of cattle slaughtered at Hamadan industrial slaughterhouse. After detection of E. coli isolates, STEC strains were identified according to PCR for stx1, stx2, eaeA, and hlyA virulence genes, and STEC serogroups (O157 and non-O157) were identified by PCR. The genetic linkage of STEC isolates was analyzed by the ERIC- (Enterobacterial Repetitive Intergenic Consensus-) PCR method. The antimicrobial susceptibility of STEC isolates was detected by the disk diffusion method according to CLSI guidelines. Results Among 110 collected beef samples, 77 (70%) were positive for E. coli. The prevalence of STEC in E. coli isolates was 8 (10.4%). The overall prevalence of O157 and non-O157 STEC isolates was 12.5% (one isolate) and 87.5% (7 isolates), respectively. The hemolysin gene was detected in 25% (2 isolates) of STEC strains. Evaluation of antibiotic resistance indicated that 100% of STEC isolates were resistant to ampicillin, ampicillin-sulbactam, amoxicillin-clavulanic acid, and cefazolin. Resistance to tetracycline and ciprofloxacin was detected in 62.5% and 12.5% of isolates, respectively. The analysis of the ERIC-PCR results showed five different ERIC types among the STEC isolates. Conclusion The isolation of different clones STECs from beef and the presence of antibiotic-resistant isolates indicate that more attention should be paid to the hygiene of slaughterhouses.

Pathogenicity of Shiga toxin-producing Escherichia coli (STEC) from wildlife: Should we care?

Shiga toxin-producing Escherichia coli (STEC) is one of the most frequent bacterial agents associated with food-borne outbreaks in Europe. In humans, the infection can lead to life-threatening diseases. Domestic and wild animals can harbor STEC, and ruminants are the main STEC reservoirs, although asymptomatic. In the present study we have characterized STEC from wildlife (wild boar (n = 56), red deer (n = 101), red fox (n = 37) and otter (n = 92)). Cultivable STEC (n = 52) were isolated from 17% (n = 49) of the faecal samples. All the isolates were non-O157 STEC encoding stx1 (n = 2; 4%) and/or stx2 genes (n = 51; 98%). Only one strain (2%) isolated from red fox had an antibiotic resistant phenotype. However, when the normalized resistance interpretation of epidemiological cutoffs (NRI ECOFFs) were used, 23% (n = 12) of the strains were non-wildtype to at least one of the antibiotics tested. After analysis by pulsed-field gel electrophoresis (PFGE), 20 strains were selected for whole genome sequencing and belonged to the following serotypes: O27:H30 (n = 15), O146:H28 (n = 2), O146:H21 (n = 1), O178:H19 (n = 1), and O103:H2 (n = 1). In addition to stx, all strains encode several virulence factors such as toxins, adhesins, fimbriae and secretion systems, among others. All sequenced genomes carried several mobile genetic elements (MGEs), such as prophages and/or plasmids. The core genome and the phylogenetic analysis showed close evolutionary relationships between some of the STEC recovered from wildlife and strains of clinical origin, highlighting their pathogenic potential. Overall, our results show the zoonotic potential of STEC strains originating from wildlife, highlighting the importance of monitoring their genomic characteristics following a One Health perspective, in which the health of humans is related to the health of animals, and the environment.

Validation of the 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) for the Detection of Shiga Toxin Gene (stx and eae) in Fresh Raw Beef Trim, Fresh Raw Ground Beef and Fresh Spinach: AOAC Performance Tested MethodSM 071902.

BackgroundThe 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) method is based on gene amplification using real time loop-mediated isothermal amplification with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) and intimin gene (eae) from Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC) in enriched foods.ObjectiveThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) method was evaluated for AOAC® Performance Tested MethodsSM certification.MethodsMatrix studies, inclusivity/exclusivity, robustness, product stability, and lot-to-lot variability testing were conducted to assess the method’s performance.ResultsThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) demonstrated equivalent results to the US Department of Agriculture–Food Safety and Inspection Service Microbiology Laboratory Guidebook 5C.00 for fresh raw beef trim and fresh raw ground beef, and to the US Food and Drug Administration Bacteriological Analytical Manual Chapter 4A for fresh spinach. The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) detected 50 of 50 E. coli strains with stx1 and/or stx2 genes, and the eae gene, and detected zero of 40 strains from the exclusivity panel. Robustness testing indicated that small variations in critical test parameters did not adversely affect the assay’s performance. Product consistency and stability testing demonstrated no differences between the lots evaluated.ConclusionThe data collected demonstrates that the 3M Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) is a reliable method for the rapid and specific detection of STEC in raw beef trim, raw ground beef, and spinach.HighlightsThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) method is suitable for the rapid and specific detection of STECs in raw beef trim, raw ground beef, and spinach.

The predominance of Shiga toxin-producing E. coli in the Southeast Coast of India

In this study, we reported Shiga toxin-producing Escherichia coli (STEC) in 847 samples, including those in coastal waters, sediments, and fish samples in the Southeast Coast of India. A total of 3742 E. coli strains were identified using conventional and molecular identification methods. Of these, 1518 isolates expressed virulent genes Stx1, Stx2, and Eae; effects on these genes on toxicity were examined. Furthermore, 2224 non-STEC isolates caused hemolytic uremic syndrome and played a key role in the persistence of STEC contamination. We conclude that toxin production is not adequate to cause disease, and the pathogenic mechanism of STEC remains poorly defined. Therefore, the present study indicates the status of pollution, highlighting the need for sanitation in public health.

EDL933 Strains of Escherichia coli O157 can Demonstrate Genetic Diversity and Differential Adherence to Bovine Recto-Anal Junction Squamous Epithelial Cells

Background:Differences betweenEscherichia coliO157 (O157) strains are well-established with some of these strains being associated with major outbreaks in the US. EDL933 is one such O157 strain that caused a multistate outbreak in 1982 and has since been used as a prototype in various O157-related experiments.Objective:As O157 can readily acquire genetic mutations, we sought to determine if the genetic and phenotypic profiles of EDL933 strains from different sources would be consistent.Methods:We evaluated wild-type O157 strains stocked as EDL933 from three different laboratories, in the strain typing Polymorphic Amplified Typing Sequence (PATS) and the bovine rectal-anal junction squamous epithelial (RSE) cell- and HEp-2 cell- adherence assays. In addition, we also verified if Shiga toxins (Stx), the Locus of Enterocyte Effacement (LEE) or curli fimbriae contributed to the adherence phenotypes observed using mutant and wild-type EDL933 isolates.Results:Our results showed differences in PATS profiles and RSE cell-adherence phenotype, with no influence from the Stx or LEE genes, between EDL933 from different sources. Interestingly, the EDL933 strain that demonstrated the most contrasting diffuse adherence phenotype on RSE cells, EDL933-T, had decreased curli production that may have contributed to this phenotype.Conclusion:Our observations suggest that a comprehensive characterization of bacterial isolates, even if assigned to the same strain type prior to use in experiments, is warranted to ensure consistency and reproducibility of results.

Mucosa-Associated Escherichia coli in Colorectal Cancer Patients and Control Subjects: Variations in the Prevalence and Attributing Features.

Accumulating evidence indicates that specific strains of mucosa-associated Escherichia coli (E. coli) can influence the development of colorectal carcinoma. This study aimed to investigate the prevalence and characterization of mucosa-associated E. coli obtained from the colorectal cancer (CRC) patients and control group. At two referral university-affiliated hospitals in northwest Iran, 100 patients, 50 with CRC and 50 without, were studied over the course of a year. Fresh biopsy specimens were used to identify mucosa-associated E. coli isolates after dithiothreitol mucolysis. To classify the E. coli strains, ten colonies per sample were typed using enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR). The strains were classified into phylogroups using the quadruplex PCR method. The PCR method was used to examine for the presence of cyclomodulin, bfp, stx1, stx2, and eae-encoding genes. The strains were tested for biofilm formation using the microtiter plate assay. CRC patients had more mucosa-associated E. coli than the control group (p < 0.05). Enteropathogenic Escherichia coli (EPEC) was also found in 23% of CRC strains and 7.1% of control strains (p < 0.05). Phylogroup A was predominant in control group specimens, while E. coli isolates from CRC patients belonged most frequently to phylogroups D and B2. Furthermore, the frequency of cyclomodulin-encoding genes in the CRC patients was significantly higher than the control group. Around 36.9% of E. coli strains from CRC samples were able to form biofilms, compared to 16.6% E. coli strains from the control group (p < 0.05). Noticeably, cyclomodulin-positive strains were more likely to form biofilm in comparison to cyclomodulin-negative strains (p < 0.05). In conclusion, mucosa-associated E. coli especially cyclomodulin-positive isolates from B2 and D phylogroups possessing biofilm-producing capacity colonize the gut mucosa of CRC patients.

Evaluation of the Anti-Shigellosis Activity of Dietary Isothiocyanates in Galleria mellonella Larvae.

Cruciferous vegetables, widely present in daily diets, are a rich source of organosulfur compounds with proven health benefits, especially chemopreventive or antioxidative effects. Isothiocyanate derivatives (ITCs) exhibit a broad spectrum of biological and pharmacological activity and recently, their antibacterial properties have been of particular importance. Here, we have focused on the anti-shigellosis activity of sulforaphane (SFN) and phenethyl ITC (PEITC). The genus Shigella causes gastroenteritis in humans, which constitutes a threat to public health. Production of a potent Stx toxin by S. dysenteriae type 1 results not only in more severe symptoms but also in serious sequela, including the hemolytic uremic syndrome. Here, we present evidence that two aliphatic and aromatic ITCs derivatives, SFN and PEITC, have an effective antibacterial potency against S. dysenteriae, also negatively regulating the stx gene expression. The molecular mechanism of this effect involves induction of the global stress-induced stringent response. ITCs also inhibit bacterial virulence against the Vero and HeLa cells. We present evidence for the therapeutic effect of sulforaphane and phenethyl ITC against a S. dysenteriae infection in the Galleria mellonella larvae model. Thus, our results indicate that isothiocyanates can be effectively used to combat dangerous bacterial infections.

Screening of AmpC-/ESBL-producing Escherichia coli isolates from livestock for STEC/EHEC virulence genes

Livestock is an important reservoir of Shiga toxin-producing Escherichia coli and enterohemorrhagic E. coli (STEC/EHEC) strains and acts as a significant source of transmission to humans. In addition to the virulence of STEC/EHEC isolates, antibiotic resistance is also an escalating problem in these bacteria and increases the risk to public health. Therefore, the present study aimed to explore E. coli O157:H7 serotype and STEC/EHEC virulence genes in AmpC- and extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates from cattle, chicken and sheep. A total of 61 confirmed AmpC- or ESBL-producing E. coli isolates were screened for the virulence genes (stx1, stx2, eae, ehxA, espP, katP and saa) and E. coli O157 (rfbO157) and H7 (fliCH7) genes by polymerase chain reaction (PCR). None of the ESBL-producing E. coli was positive for these genes, but six multidrug-resistant AmpC-producing E. coli were positive for the fliCH7 gene only. When considering the function of the H7 flagellar antigen of E. coli, it may be concluded that the development of ESBL/AmpC beta-lactamase production in the E. coli isolates with H7 flagella, which reside in the chicken intestine, may be potentially important for public health regarding both virulence and antimicrobial resistance.

SEROTYPES, TOXINS AND ANTIBIOTIC RESISTANCE OF Escherichia coli (E.COLI) STRAINS ISOLATED FROM DIARRHEIC RABBITS IN PHU VANG, THUA THIEN HUE

This study was conducted to determine the prevalence of E. coli in rabbits, their biochemical and serological characteristics, common virulence genes, and antibiotic resistance. The diarrhea rabbit feces were collected from households and rabbit farms in Phu Vang - Thua Thien Hue with a total of 250 samples for testing. The results showed that rabbits age from 31 to 45 days old had the highest incidence of diarrhea disease caused by E.coli (92.0%) and the lowest infection rate was observed in rabbits over 60 days old with an infection rate of 30%. Further, the isolated E.coli strains tested biochemical characteristics showed 100% motile, positive for indole and methyl red, fermenting glucose and lactose. Simultaneously these strains were detected belong to 7 serotypes O103, O157, O158, O169, O44, O125, O153 and susceptible to cefuroxime (95.45%), akamicin (86.37%), streptomycin (81.82%), amoxicillin (81.82%), tetracycline (68.18%), colistin (68.18%), ampicillin (63.63%), gentamycin (59.10%) and levofloxacin (50.0%), whilst resistant to doxycycline (100%), sulfamethoxazole-bactrim (95.46%), and neomycin (86.37%). By using PCR assay for detection of virulence genes of the isolated E. coli strains, there were 7 strains carried virulence genes, of which 4/7 E. coli strains carried eaeA and tsh genes (57.14%), 2/7 strains carried stx2 gene (28.57%); 1/7 E. coli strains carried stx1 gene (14.28%) and the F4, F5 and F6 genes were not found in all serotypes in this study.

Shiga-toxin producing Escherichia coli detection in samples from Abruzzo and Molise regions, Italy

Abstract Background Top five serogroups (O157, O111, O26, O103 and O145) of Shiga-toxin producing Escherichia coli (STEC) represent the third most important increasing zoonosis reported in European Union. The ISO methods of analysis, used in foods and environmental samples, quite often does not confirm the molecular detection of STEC genes (stx1 and stx2) in enrichment broth. The aim of this study was to summarize the results of the tests carried out at Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise in the last six years (2016-2021), comparing genes detection in enrichment broth and in colonies grown on agar plates. Methods Along the 2016-2021, a total of 1716 foods and environmental samples from Abruzzo and Molise regions were tested, according to ISO/TS 13136:2012. Results Out of 1716 samples, 16 (0.9%) shown presumptive positive presence of STEC (stx1 and/or stx2) in broth. In spite of that, the presence of STEC (positive colonies for stx1 and/or stx2) was confirmed only in 4 out of 16 samples (one sample of cheese and 3 of minced meat). Then, eae gene was found in 54 samples in broth and only in 3 samples from colonies, during a foodborne outbreak investigation. Serogroup specific targets were detected in 6 broth samples (O26, O145 and O157) and confirmed in colonies only in 2 minced meat samples (O157). Conclusions STEC are a heavy hazard for the consumers and the number of cases is slightly increasing since 2015. Our results highlight a low number of presumptive positive samples and even lower number of STEC detection obtained with the ISO classical method. Furthermore, detection of the target genes in broth was rarely confirmed after plating, highlighting a weak point of the analytical method that needs to be investigated. Efforts are necessary to improve the method and the STEC confirmation from colonies. Immunomagnetic beads targeting the top five serogroups and digital PCR could help in increasing the recovery rate of cultivable STEC. Key messages The presumptive positive samples for STEC genes in foodstuffs from the Abruzzo and Molise regions are quite rare, even lower is the number of confirmed colonies by agar plate isolation. Specific immunomagnetic beads for the top five STEC serogroups could improve the method and the STEC colonies detection, increasing the recovery rate of cultivable strains.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11–6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10–100 PFU for the MNV-1 and 1–10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Molecular characterization and prevalence of virulence factor genes of Shiga toxin-producing Escherichia coli (STEC) isolated from diarrheic children

BackgroundShiga toxin- producing Escherichia coli (STEC), in addition to severe diarrhea, may cause serious and life-threatening complications such as Hemolytic Uremic Syndrome (HUS) in human infections. However, there is no accurate information about the prevalence of these strains in Iran. So this study aimed to investigate the prevalence of serotypes, virulence factors, and antibiotic resistance patterns in STEC isolated from diarrheic children samples. MethodsA total of 315 Stools diarrhea samples were collected, STEC isolates verified by phenotypic and molecular identification methods. Virulence factors, Shiga toxin (Stx), Stx subtyping, serotyping, and antimicrobial resistance genes were detected by PCR methods and sequencing. Cytotoxicity of the Stx positive isolates was examined on Vero cells. ResultsOverall, 150 (47.6%) E. coli were isolated. 10 (6.7%) isolates were confirmed as STEC and Stx1a was identified as the most common Shiga toxin subtype and all of the Stx2 were Stx2a. Serotype O26 was identified in 4 isolates. Iha (70%), lpfA (50%), and Saa (50%) were the predominant virulence factors. The highest toxicity was related to the strain that carried both Stx1 and Stx2 genes simultaneously. The highest rates of resistance were observed against ampicillin and trimethoprim/sulfamethoxazole and the blaCTX-M-15 was the most dominant gene. ConclusionIn this study, indicated other binding virulence factors played a more important role in pathogenicity than intimin in binding. Moreover, virulence factor profile variation causes differences in STEC pathogenesis. Antibiotic resistance with high cytotoxicity in STEC isolates should be considered as a warning to public health.

Multidrug resistance in Shiga toxin-producing Escherichia coli (STEC) isolated from broiler chickens at slaughter

Broiler chickens and derived products are a key source of Shiga toxin-producing Escherichia coli (STEC) in humans. This pathotype is responsible for causing severe episodes of diarrhea, which can progress to systemic complications. A rapid and accurate diagnosis of the disease, and early treatment of the infection with antimicrobials, can prevent it worsening. However, multidrug-resistant strains have potentially negative implications for treatment success. In this context, the aim of the present study was to isolate and identify multidrug-resistant STEC strains from broiler chickens and carcasses. Of 171 E. coli strains, isolated by conventional microbiological techniques and submitted to Polymerase Chain Reaction (PCR), for detection of stx1 and stx2 genes, 21.05% (36/171) were STEC pathotype, and most of them (66.67% - 24/36) carried both stx1 and eae genes. The multidrug resistance pattern was observed in 75% (27/36) of STEC strains. The presence of STEC in broiler chickens and carcasses reinforces that these sources may act as reservoirs for this pathotype. Multidrug-resistant bacteria contaminating animal products represent a public health issue because of the possibility of spread of multidrug-resistant determinants in the food chain and a higher risk of failure in human treatment when antimicrobials are needed.

PSIII-18 Identification of microbial interactions and markers associated with Shiga toxin-producing bacteria colonization in the rectum of beef steers

Abstract This study aimed to identify whether microbial interactions in the rectum contribute to Shiga toxin producing bacteria colonization. In total, 12 rectal digesta samples based on the previously identified Shiga toxin 2 gene (stx2) abundance (DNA) and expression (RNA) in Shiga toxin-producing bacteria (Stx2- group: detectable DNA, n=6; Stx2+ group: detectable DNA and RNA, n = 6) were subjected to microbial profiling using amplicon sequencing. Firmicutes (72.7 ± 2.0 %) and Bacteroidetes (24.6 ± 1.9 %) are the most predominant phyla of rectal microbiota, and no compositional differences were identified between two groups at the phylum level. The Shannon and Chao1 indices weren’t different in rectal digesta microbial communities between two groups. Twenty-four and thirteen taxa were identified to be group-specific genera in microbial communities from Stx2- and Stx2+ group, respectively (2 out of 6, average relative abundance &amp;gt;0.1%). The network analysis indicated 12 and 14 keystone taxa (Generalists, densely connected with other taxa) in microbial communities between Stx2- and Stx2+ groups, respectively. Eight out of 12 and six out of 14 generalists in the Stx2- and Stx2+ group are belonging to group-specific genera, respectively. Generalists belonging to group-specific genera were broadly distributed in Stx2- network while centralized distributed in the Stx2+ network, suggesting the higher stability of the Stx2- network structure in comparison of Stx2+ network computed by the natural connectivity measurement. However, 66 core genera shared by microbial communities between two groups were not classified into network generalists. Overall, our results indicate microbial crosstalks and keystone taxa in microbial communities between two groups differed, suggesting that the microbial interactions rather than the shifts in taxa abundance may be more important affecting host. Moreover, group-specific genera play a vital ecological role in the microbial interactions, indicating the potential for being microbial markers to differentiate Shiga toxin-producing bacteria colonization in beef cattle.

Whole-Genome Sequencing Analysis of a stx-Negative Escherichiacoli O63:H6 Isolate Associated with Hemolytic Uremic Syndrome.

Shiga toxin-encoding genes (stx) of enterohemorrhagic Escherichia coli (EHEC) can be lost during infection or in vitro cultivation, and in clinical practice, it is difficult to distinguish EHEC that have lost stx (EHEC-LST) from enteropathogenic E. coli (EPEC), as both are stx-negative and eae-positive. In this study, we performed whole-genome sequencing (WGS) of a stx-negative, eae-positive E. coli O63:H6 isolate from a child with hemolytic uremic syndrome and compared its genome with those of nine E. coli O63:H6 strains in public databases. Virulence gene profiles were analyzed and core-genome multilocus sequence typing (cgMLST) was conducted. The virulence gene profile of our isolate was consistent with EHEC, except for the absence of stx, and the isolate clustered with seven EHEC strains but was distant from two EPEC strains in cgMLST. In genome alignment, our isolate exhibited a high nucleotide identity with EHEC strain 377323_2f but displayed a gap corresponding to the stx-harboring prophage sequence. Overall, our isolate was genetically closely related to EHEC strains, consistent with this being an EHEC-LST strain. As EHEC-LST may be misdiagnosed as EPEC in routine laboratories, comparative genomic analysis using WGS can be useful to determine whether stx-negative and eae-positive isolates are EHEC-LST or EPEC.

Molecular characterization of enterohemorrhagic Escherichia coli isolated from diarrhea samples from human, livestock, and ground beef in North Jordan.

Background and Aim:Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen with worldwide distribution. Data regarding its presence, distribution, virulence, and antimicrobial susceptibility among various animal species and humans in Jordan are lacking. Therefore, the objectives of this study were to isolate and characterize EHEC from human and animal diarrhea fecal samples and ground beef samples.Materials and Methods:A total of 100 and 270 diarrhea fecal samples from humans and animals, respectively, were collected. In addition, 40 ground beef meat samples were collected from retail markets. EHEC was positively identified by detecting Shiga toxins (stx1 and stx2) genes using multiplex polymerase chain reaction (PCR). Antimicrobial susceptibility patterns were determined using the disk diffusion test. Beta-lactamase production was detected using the double disk diffusion test and the extended-spectrum beta-lactamases (ESBLs) were identified by detection of blaTEM, blaSHV, and OXA-1 genes using multiplex PCR. Pulsed-field gel electrophoresis (PFGE) was used to investigate the relatedness of EHEC isolates from different sources.Results:Out of 410 samples, 194 E. coli isolates were positively identified, of which 57 isolates (29%) were classified as EHEC. Thirty-five (61%) of EHEC isolates were serotyped as O157 (19: O157:H7 and 16: O157:NM). The stx1 gene was detected only among the sheep and goats isolates at a rate of 7.6% and 5.2%, respectively, while the stx2 gene was detected in only one ground beef meat sample. EHEC isolates showed high resistance patterns against amoxicillin, gentamycin, cephalexin, and doxycycline. Twenty-four out of 32 EHEC isolates were determined as ESBL producers, among which 14 isolates expressed the blaSHVgene and 19 isolates expressed the blaTEM while four expressed both genes. PFGE analysis revealed two clusters with high similarity (92%) originated from ground beef meat and cattle fecal samples. No similarities were found between human and animal E. coli isolates.Conclusion:Results of this study indicate widespread ESBL EHEC among humans, animals, and ground beef meat samples. These results represent an important alarm that requires the implementation of appropriate preventative measures by both human and animal health sectors to prevent the transmission of this important foodborne pathogen.

Detection and characterization of Escherichia coli associated Oreochromis niloticus sold in the retail markets at Sohag Governorate, Egypt

Fish is a major vehicle for transmission several bacterial diseases to human. Escherichia coli (E. coli) is one of the main causes of food poisoning outbreaks occurring in Egypt due to consumption of contaminated fish and fish products. This study was performed to investigate prevalence of contamination of Oreochromis niloticus (O. niloticus) sold in the retail markets at Sohag Governorate, Egypt with E. coli and Shiga toxin-producing E. coli (STEC) and to determine the antimicrobial susceptibility of the isolates. Therefore, 110 apparently healthy O. niloticus samples were randomly collected from the fish retail markets at Sohag Governorate cities, Egypt and they were bacteriologically examined for presence of E. coli on the surface and in the gills and muscles. Susceptibility of E. coli isolates were tested to 10 different antibiotics and subsequently representative E. coli isolates (n=12) were serotyped and screened by PCR for presence of stx1 and stx2 genes. 22 E. coli isolates were isolated and identified from surface, gills and muscles of the examined O. niloticus with percentage of (8.2%), (10.0%) and (1.8%) respectively, these isolates were isolated from 12 fish only of the examined O. niloticus with total prevalence of 10.9% (12/110). Serotyping revealed that the investigated E. coli isolates belonging to 5 different O-serogroups comprising O55 (33.4%), O125 (25.0%), O26 (8.3%), O76 (8.3%) and O128 (8.3%) in addition to 2 nontypeable isolates (16.7%).