Shiga Toxin-producing Escherichia Coli Isolates 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.

Added Value of Genomic Surveillance of Virulence Factors in Shiga Toxin-Producing Escherichia coli in New South Wales, Australia.

The disease caused by Shiga toxin-producing Escherichia coli (STEC) remains a significant public health challenge globally, but the incidence of human STEC infections in Australia remains relatively low. This study examined the virulence characteristics and diversity of STEC isolates in the state of New South Wales between December 2017 and May 2020. Utilisation of both whole and core genome multi-locus sequence typing (MLST) allowed for the inference of genomic diversity and detection of isolates that were likely to be epidemiologically linked. The most common STEC serotype and stx subtype detected in this study were O157:H7 and stx1a, respectively. A genomic scan of other virulence factors present in STEC suggested interplay between iron uptake system and virulence factors that mediate either iron release or countermeasures against host defence that could result in a reduction of stx1a expression. This reduced expression of the dominant stx genotype could contribute to the reduced incidence of STEC-related illness in Australia. Genomic surveillance of STEC becomes an important part of public health response and ongoing interrogation of virulence factors in STEC offers additional insights for the public health risk assessment.

Epidemiology and genomic analysis of Shiga toxin-producing Escherichia coli clonal complex 165 in the UK

IntroductionShiga toxin-producing Escherichia coli (STEC) is a zoonotic, foodborne gastrointestinal pathogen that has the potential to cause severe clinical outcomes, including haemolytic uraemic syndrome (HUS). STEC-HUS is the leading cause of renal failure in children and can be fatal. Over the last decade, STEC clonal complex 165 (CC165) has emerged as a cause of STEC-HUS.Gap statementThere is a need to understand the pathogenicity and prevalence of this emerging STEC clonal complex in the UK, to facilitate early diagnosis, improve clinical management, and prevent and control outbreaks.AimThe aim of this study was to characterize CC165 through identification of virulence factors (VFs) and antimicrobial resistance (AMR) determinants in the genome and to integrate the genome data with the available epidemiological data to better understand the incidence and pathogenicity of this clonal complex in the UK.MethodologyAll isolates belonging to CC165 in the archives at the UK public health agencies were sequenced and serotyped, and the virulence gene and AMR profiles were derived from the genome using PHE bioinformatics pipelines and the Centre for Genomic Epidemiology virulence database.ResultsThere were 48 CC165 isolates, of which 43 were STEC, four were enteropathogenic E. coli (EPEC) and one E. coli . STEC serotypes were predominately O80:H2 (n=28), and other serotypes included O45:H2 (n=9), O55:H9 (n=4), O132:H2 (n=1) and O180:H2 (n=1). All but one STEC isolate had Shiga toxin (stx) subtype stx2a or stx2d and 47/48 isolates had the eae gene encoding intimin involved in the intimate attachment of the bacteria to the human gut mucosa. We detected extra-intestinal virulence genes including those associated with iron acquisition (iro) and serum resistance (iss), indicating that this pathogen has the potential to translocate to extra-intestinal sites. Unlike other STEC clonal complexes, a high proportion of isolates (93%, 40/43) were multidrug-resistant, including resistance to aminoglycosides, beta-lactams, chloramphenicol, sulphonamides, tetracyclines and trimethoprim.ConclusionThe clinical significance of this clonal complex should not be underestimated. Exhibiting high levels of AMR and a combination of STEC and extra-intestinal pathogenic E. coli (ExPEC) virulence profiles, this clonal complex is an emerging threat to public health.

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.

Shiga Toxin–Producing Escherichia coli (STEC) and STEC-Associated Virulence Genes in Raw Ground Pork in Canada

Shiga toxin-producing Escherichia coli (STEC) O157:H7/nonmotile and some non-O157 STEC strains are foodborne pathogens. In response to pork-associated O157 STEC outbreaks in Canada, we investigated the occurrence of STEC in Canadian retail raw ground pork during the period of 1 November 2014 to 31 March 2016. Isolated STEC strains were characterized to determine the Shiga toxin gene (stx) subtype and the presence of virulence genes encoding intimin (eae) and enterohemorrhagic E. coli hemolysin (hlyA). O157 STEC and non-O157 STEC strains were isolated from 1 (0.11%) of 879 and 13 (2.24%) of 580 pork samples, respectively. STEC virulence gene profiles containing both eae and hlyA were found only in the O157 STEC (stx2a, eae, hlyA) isolate. The eae gene was absent from all non-O157 STEC isolates. Of the 13 non-O157 STEC isolates, two virulence genes of stx1a and hlyA were found in four (30.8%) O91:H14 STEC isolates, whereas one virulence gene of stx2e, stx1a, and stx2a was identified in five (38.5%), two (15.4%), and one (7.7%) STEC isolates, respectively, of various serotypes. The remaining non-O157 STEC isolate carried stx2, but the subtype is unknown because this isolate could not be recovered for sequencing. O91:H14 STEC (stx1a, hlyA) was previously reported in association with diarrheal illnesses, whereas the other non-O157 STEC isolates identified in this study are not known to be associated with severe human illnesses. Virulence gene profiles identified in this study indicate that the occurrence of non-O157 STEC capable of causing severe human illness is rare in Canadian retail pork. However, O157 STEC in ground pork can occasionally occur; therefore, education regarding the potential risks associated with STEC contamination of pork would be beneficial for the public and those in the food industry to help reduce foodborne illnesses.

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.

Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.

Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.

Antibiotic Susceptibility Profiles and Frequency of Resistance Genes in Clinical Shiga Toxin-Producing Escherichia coli Isolates from Michigan over a 14-Year Period.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that contributes to over 250,000 infections in the United States each year. Because antibiotics are not recommended for STEC infections, resistance in STEC has not been widely researched despite an increased likelihood for the transfer of resistance genes from STEC to opportunistic pathogens residing within the same microbial community. From 2001 to 2014, 969 STEC isolates were collected from Michigan patients. Antibiotic susceptibility profiles to clinically relevant antibiotics were determined using disc diffusion, while epidemiological data were used to identify factors associated with resistance. Whole-genome sequencing was used for serotyping, examining genetic relatedness, and identifying genetic determinants and mechanisms of resistance in the non-O157 isolates. Increasing frequencies of resistance to at least one antibiotic were observed over the 14 years (P = 0.01). While the non-O157 serogroups were more commonly resistant than O157 (odds ratio, 2.4; 95% confidence interval,1.43 to 4.05), the frequency of ampicillin resistance among O157 isolates was significantly higher in Michigan than the national average (P = 0.03). Genomic analysis of 321 non-O157 isolates uncovered 32 distinct antibiotic resistance genes (ARGs). Although mutations in genes encoding resistance to ciprofloxacin and ampicillin were detected in four isolates, most of the horizontally acquired ARGs conferred resistance to aminoglycosides, β-lactams, sulfonamides, and/or tetracycline. This study provides insight into the mechanisms of resistance in a large collection of clinical non-O157 STEC isolates and demonstrates that antibiotic resistance among all STEC serogroups has increased over time, prompting the need for enhanced surveillance.

Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Isolated from Cattle from Central and Southern Chile.

Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), one of the world's most important foodborne pathogens. The pathogen causes severe human diseases and outbreaks. This study aimed to identify and characterize non-O157 STEC isolated from cattle feces from central and southern Chile. We analyzed 446 cattle fecal samples and isolated non-O157 STEC from 12.6% (56/446); a total of 93 different isolates were recovered. Most isolates displayed β-glucuronidase activity (96.8%; 90/93) and fermented sorbitol (86.0%; 80/93), whereas only 39.8% (37/93) were resistant to tellurite. A subgroup of 30 representative non-O157 STEC isolates was selected for whole-genome sequencing and bioinformatics analysis. In silico analysis showed that they grouped into 16 different sequence types and 17 serotypes; the serotypes most frequently identified were O116:H21 and O168:H8 (13% each). A single isolate of serotype O26:H11 was recovered. One isolate was resistant to tetracycline and carried resistance genes tet(A) and tet(R); no other isolate displayed antimicrobial resistance or carried antimicrobial resistance genes. The intimin gene (eae) was identified in 13.3% (4/30) of the genomes and 90% (27/30) carried the stx2 gene. A phylogenetic reconstruction demonstrated that the isolates clustered based on serotypes, independent of geographical origin. These results indicate that cattle in Chile carry a wide diversity of STEC potentially pathogenic for humans based on the presence of critical virulence genes.

Isolation and Characterization of Shiga Toxin-Producing Escherichia coli from Retail Beef Samples from Eight Provinces in China.

While Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen worldwide, data on the molecular and phylogenetic properties of STEC isolates from retail beef samples in China remain scant. Fresh retail beef samples (n = 1062) were collected from eight provinces, and STEC isolates were recovered and characterized. PCR data showed that more than 50% of the samples were stx positive, and 82 STEC isolates were recovered from 14.8% (79/535) stx-positive enriched broths. In contrast, all ciprofloxacin resistant isolates (n = 19) and 13 cefotaxime (CTX) resistant isolates were eae positive and belonged to three serotypes: O111:H8, O26:H11, or O157:H7. Point mutations in quinolone resistance-determining regions and plasmid-mediated quinolone resistance determinants were identified in 16 and 20 isolates, respectively. BlaCTX-M and a point mutation (C-42T) in ampC promoter were detected in 15 and 8 of the CTX resistant isolates, respectively. In addition, macrolide resistance gene mphA was identified in eight azithromycin resistant O111:H8 isolates and one O26:H11 isolate. Single nucleotide polymorphism analysis demonstrated that the O26 and O157 isolates had multiple origins, but the O111 isolates were closely related. Taken together, our data demonstrated that several sequence types associated with hemolytic uremic syndrome from the retail beef samples in China had developed into dangerous multidrug resistant pathogens. The resistant phenotype can facilitate their transmission among the farm animals and human beings when there is an antimicrobial selective pressure.

Zoonotic Potential of Escherichia Coli in Poultry Intestinal Contents in Ismailia City, Egypt with Special Reference to Shiga Toxin-Producing (STEC) Strains

Avian colibacillosis can affect birds of all ages. It is now one of the major causes of economic losses in the poultry industry, as well as a public health risk all over the world.This study aimed to determine the occurrence of E. coli in market-age poultry and the risk of its zoonotic infections in Ismailia city, Egypt. A total of 350 samples were collected from the intestinal contents of the slaughtered poultry (100 farm chickens, 100 backyard chickens, 50 ducks, 50 geese, and 50 turkeys) in Ismailia City. On the other hand, a total of 200 samples were collected from humans at outpatient clinics (100 stool samples and 100 urine samples) from the same investigated area. Samples were examined using bacteriological and molecular examination for genus-specific and virulence gene detection. Results revealed that the isolation rates of E. coli were 70% in farm chickens and turkeys, 88% in backyard chickens, 86% in domestic ducks, 72% in geese. Of avian E. coli isolates, the rates of Shiga toxin-producing E. coli (STEC) strains were 91.4%, 96.6%, 90.9%, 41.7%, and 51.4%% in farm chickens, backyard chickens, ducks, geese, and turkey respectively. In humans, the isolation rate of E. coli was 42% in urine samples and 66% in stool samples. The human STEC isolates were higher in stool samples (26%) than in urine samples (6%). The isolation rates of E. coli were significantly higher in persons who were in contact with poultry than in persons who were not in contact with poultry (p ≤ 0.01). In conclusion, the high isolation rates of STEC and detection with similarities of some E. coli virulence genes (It, St, eaeA, Stx1, and Stx2 genes) from poultry intestinal contents and human samples indicated a significant risk of zoonotic transmission of E. coli via food chain in the investigated area.

Shiga Toxin-Producing Escherichia coli in Wheat Grains: Detection and Isolation by Polymerase Chain Reaction and Culture Methods.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens and seven serogroups, O26, O45, O103, O111, O121, O145, and O157, often called top-7 STEC, account for the majority of the STEC-associated human illnesses in the United States. Two Shiga toxins, Shiga toxins 1 and 2, encoded by stx1 and stx2 genes, are major virulence factors that are involved in STEC infections. Foodborne STEC infections have been linked to a variety of foods of both animal and plant origin, including products derived from cereal grains. In recent years, a few STEC outbreaks have been linked to contaminated wheat flour. The microbiological quality of the wheat grains is a major contributor to the safety of wheat flour. The objective of the study was to utilize polymerase chain reaction (PCR)- and culture-based methods to detect and isolate STEC in wheat grains. Wheat grain samples (n = 625), collected from different regions of the United States, were enriched in modified buffered peptone water with pyruvate (mBPWp) or E. coli (EC) broth, and they were then subjected to PCR- and culture-based methods to detect and isolate STEC. Wheat grains enriched in EC broth yielded more samples positive for stx genes (1.6% vs. 0.32%) and STEC serogroups (5.8% vs. 2.4%) than mBPWp. The four serogroups of top-7 detected and isolated were O26, O45, O103, and O157 and none of the isolates was positive for the Shiga toxin genes. A total of five isolates that carried the stx2 gene were isolated and identified as serogroups O8 (0.6%) and O130 (0.2%). The EC broth was a better medium to enrich wheat grains than mBPWp for the detection and isolation of STEC. The overall prevalence of virulence genes and STEC serogroups in wheat grains was low. The stx2-positive serogroups isolated, O8 and O130, are not major STEC pathogens and have only been implicated in sporadic infections in animals and humans.

Antibiotic-mediated expression analysis of Shiga toxin 1 and 2 in multi-drug-resistant Shiga toxigenic Escherichia coli.

Shiga toxin-producingEscherichia coli(STEC) is an important foodborne pathogens, known to cause enteric infections especially diarrhea, mainly attributed to Shiga toxins (Stxs). The use of certain antibiotics for treating this infection is controversial, owing to an increased risk for producing Stxs (Stx 1 and Stx 2). Increased antibiotic resistance is also thought to be involved in the pathogenesis of STEC diseases. The purpose of this study was to analyze the effects of antibiotics on induction of Stx 1 and Stx 2 in clinical STEC isolates and to investigate the relationships between increased resistance and Stx production. Fifteen clinical isolates were treated with sub minimum inhibitory concentrations (Sub MIC) of clinically used antibiotics (ciprofloxacin, fosfomycin, tigecycline, and meropenem), and the changes in expression levels of stx1 and stx2 genes were estimated using qRT-PCR. The expressions of Shiga toxins were found to be increased up to 6.5- and eightfold under ciprofloxacin and tigecycline Sub MIC, respectively. Fosfomycin had weak induction effect of up to twofold, whereas meropenem had the weakest influence on such expression. Resistant isolates were found to be more prone to increased expression of toxins.

The prevalence and characteristics of Shiga toxin-producing Escherichia coli isolated by the enteric pathogens active surveillance network (Enter-Net) in the Republic of Korea, 2009–2018

Shiga toxin-producing Escherichia coli (STEC) is a water- and food-borne pathogenic agent that causes diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS), and end-stage renal disease. As the annual incidence of STEC increases, disease control is also becoming important in Korea. In this study, we aimed to analyze the incidence trends and characteristics of STEC isolated from diarrheal patients over 10 years. From 2009 to 2018, STECs were collected by the Enteric Pathogens Active Surveillance Network (Enter-Net) and analyzed according to clinical epidemiological information (month of isolation, age, and sex of patient), O serogroup, and shiga toxin type. Shiga toxin genes (stx1 and stx2) and O serogroups of isolates were determined using multiplex PCR and an agglutination method with the available O antisera, respectively. A total of 418 strains were isolated over 10 years. The isolation rate according to age group and season was highest in children ≤4 years old (38.1%) and in the summer season (June to August). Among the 418 isolates, the major serogroups were divided O157 (20.3%), O103 (13.6%), O26 (7.7%), O111 (5.5%), O91 (4.3%), O108 (2.4%), and O8 (2.2%). The most frequently isolated O157 showed a lower isolation rate compared to that isolated from other developed countries. The profiles of stx genes were distinct among serogroups. In O157 and O91, stx1+stx2 was detected more frequently than either stx1 or stx2 alone. Particularly, most of the O157 (98%) isolates harbored the stx2 gene, which is an important factor in severe diseases, including HUS. In O103, O26, O111, and O108, stx1-only was more frequently present than stx2-only or stx1+stx2. As a result of analyzing domestic STECs collected through Enter-Net, it was confirmed that patients ≤4 years of age and in the summer months require attention, and that STEC with a serogroup of O157 is highly likely to cause diseases such as HUS. Therefore, the pathogen active surveillance network for characterization and provision of STEC isolates must be operated continuously.

Insights into the genome architecture and evolution of Shiga toxin encoding bacteriophages of Escherichia coli

BackgroundA total of 179 Shiga toxin-producing Escherichia coli (STEC) complete genomes were analyzed in terms of serotypes, prophage coding regions, and stx gene variants and their distribution. We further examined the genetic diversity of Stx-converting phage genomes (Stx phages), focusing on the lysis-lysogeny decision and lytic cassettes.ResultsWe show that most STEC isolates belong to non-O157 serotypes (73 %), regardless the sources and geographical regions. While the majority of STEC genomes contain a single stx gene (61 %), strains containing two (35 %), three (3 %) and four (1 %) stx genes were also found, being stx2 the most prevalent gene variant. Their location is exclusively found in intact prophage regions, indicating that they are phage-borne. We further demonstrate that Stx phages can be grouped into four clusters (A, B, C and D), three subclusters (A1, A2 and A3) and one singleton, based on their shared gene content. This cluster distribution is in good agreement with their predicted virion morphologies. Stx phage genomes are highly diverse with a vast number of 1,838 gene phamilies (phams) of related sequences (of which 677 are orphams i.e. unique genes) and, although having high mosaicism, they are generally organized into three major transcripts. While the mechanisms that guide lysis–lysogeny decision are complex, there is a strong selective pressure to maintain the stx genes location close to the lytic cassette composed of predicted SAR-endolysin and pin-holin lytic proteins. The evolution of STEC Stx phages seems to be strongly related to acquiring genetic material, probably from horizontal gene transfer events.ConclusionsThis work provides novel insights on the genetic structure of Stx phages, showing a high genetic diversity throughout the genomes, where the various lysis-lysogeny regulatory systems are in contrast with an uncommon, but conserved, lytic system always adjacent to stx genes.

Pathogenic potential assessment of the Shiga toxin–producing Escherichia coli by a source attribution–considered machine learning model

Instead of conventional serotyping and virulence gene combination methods, methods have been developed to evaluate the pathogenic potential of newly emerging pathogens. Among them, the machine learning (ML)-based method using whole-genome sequencing (WGS) data are getting attention because of the recent advances in ML algorithms and sequencing technologies. Here, we developed various ML models to predict the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) isolates using their WGS data. The input dataset for the ML models was generated using distinct gene repertoires from positive (pathogenic) and negative (nonpathogenic) control groups in which each STEC isolate was designated based on the source attribution, the relative risk potential of the isolation sources. Among the various ML models examined, a model using the support vector machine (SVM) algorithm, the SVM model, discriminated between the two control groups most accurately. The SVM model successfully predicted the pathogenicity of the isolates from the major sources of STEC outbreaks, the isolates with the history of outbreaks, and the isolates that cannot be assessed by conventional methods. Furthermore, the SVM model effectively differentiated the pathogenic potentials of the isolates at a finer resolution. Permutation importance analyses of the input dataset further revealed the genes important for the estimation, proposing the genes potentially essential for the pathogenicity of STEC. Altogether, these results suggest that the SVM model is a more reliable and broadly applicable method to evaluate the pathogenic potential of STEC isolates compared with conventional methods.

Microbiological Survey of Wheat Flour Sold at Retail in Canada, 2018 to 2019

Following two O121 Shiga toxin-producing Escherichia coli (STEC) outbreaks linked to wheat flour, this study was conducted to gain baseline information on the occurrence of bacterial pathogens and levels of indicator organisms in wheat flour in Canada. A total of 347 prepackaged wheat flour samples were analyzed for Salmonella species, STEC, Listeria monocytogenes, aerobic colony count (ACC), total coliforms, and Escherichia coli. Salmonella spp. and O157 STEC were not detected in any of the samples. L. monocytogenes was identified in two samples (0.6%) at levels below the limit of detection (<0.7 log CFU/g). Non-O157 STEC were isolated from six samples (1.7%) and were characterized for the presence of STEC virulence genes: stx1, stx2, and their subtypes, eae, hlyA, and aggR. One O103:H25 STEC isolate carried virulence genes (stx1a+eae) that are known to be capable of causing diarrhea and/or bloody diarrhea in humans. Of the five remaining non-O157 STEC isolates, four carried single stx2a or stx2c genes and were considered to have the potential of causing diarrhea. The remaining non-O157 STEC isolate (stx2), while not a priority non-O157 STEC, was not available for sequencing; thus, its potential to cause illness is unknown. ACC, total coliforms, and E. coli were detected (≥0.48 log CFU/g) in 98.8, 72.6, and 0.6% of the flour samples. The mean counts of ACC were greater in whole wheat flour compared with the other flour types tested (P < 0.001). The results of this study suggest that the occurrence of O157 STEC and Salmonella is low but that the occurrence of non-O157 STEC in wheat flour with the potential to cause human illness of diarrhea is relatively common. Therefore, the consumption of raw flour could increase the likelihood of STEC infections. Further research is merited for potential risk mitigation strategies within the food production system and with consumers.

Evaluation of Cattle for Naturally Colonized Shiga Toxin-Producing Escherichia coli Requires Combinatorial Strategies.

Shiga toxin-producing Escherichia coli (STEC) serogroups O157, O26, O103, O111, O121, O145, and O45 are designated as food adulterants by the U.S. Department of Agriculture-Food Safety and Inspection Service. Cattle are the primary reservoir of these human pathogens. In this study, 59 Angus crossbred heifers were tested specifically for these seven STEC serogroups using a combination of standard culture, serological, PCR, and cell cytotoxicity methods to determine if comparable results would be obtained. At the time of fecal sampling, the animals were approximately 2 years old and weighed 1000–1200 lbs. The diet comprised of 37% ground alfalfa hay, 25% ground Sudan hay, and 38% ground corn supplemented with trace minerals and rumensin with ad libitum access to water. Non-O157 STEC were isolated from 25% (15/59) of the animals tested using a combination of EC broth, CHROMagar STECTM, and Rainbow Agar O157. Interestingly, the O157 serogroup was not isolated from any of the animals. Non-O157 STEC isolates were confirmed to be one of the six adulterant serogroups by serology and/or colony PCR in 10/15 animals with the predominant viable, serogroup being O103. PCR using DNA extracted from feces verified most of the colony PCR results but also identified additional virulence and O-antigen genes from samples with no correlating culture results. Shiga toxin- (Stx-) related cytopathic effects on Vero cells with fecal extracts from 55/59 animals could only be associated with the Stx gene profiles obtained by fecal DNA PCR and not culture results. The differences between culture versus fecal DNA PCR and cytotoxicity assay results suggest that the latter two assays reflect the presence of nonviable STEC or infection with STEC not belonging to the seven adulterant serogroups. This study further supports the use of combinatorial culture, serology, and PCR methods to isolate viable STEC that pose a greater food safety threat.

Molecular Detection of Antibiotic Resistance Genes in Shiga Toxin-Producing E. coli Isolated from Different Sources.

Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen associated with human gastroenteritis outbreaks. Extensive use of antibiotics in agriculture selects resistant bacteria that may enter the food chain and potentially causes foodborne illnesses in humans that are less likely to respond to treatment with conventional antibiotics. Due to the importance of antibiotic resistance, this study aimed to investigate the combination of phenotypic and genotypic antibiotic resistance in STEC isolates belonging to serogroups O26, O45, O103, O104, O111, O121, O145, and O157 using disc diffusion and polymerase chain reaction (PCR), respectively. All strains were phenotypically resistant to at least one antibiotic, with 100% resistance to erythromycin, followed by gentamicin (98%), streptomycin (82%), kanamycin (76%), and ampicillin (72%). The distribution of antibiotic resistance genes (ARGs) in the STEC strains was ampC (47%), aadA1 (70%), ere(A) (88%), blaSHV (19%), blaCMY (27%), aac(3)-I (90%), and tet(A) (35%), respectively. The results suggest that most of the strains were multidrug-resistant (MDR) and the most often observed resistant pattern was of aadA1, ere(A), and aac(3)-I genes. These findings indicate the significance of monitoring the prevalence of MDR in both animals and humans around the globe. Hence, with a better understanding of antibiotic genotypes and phenotypes among the diverse STEC strains obtained, this study could guide the administration of antimicrobial drugs in STEC infections when necessary.

Shiga toxin–producing Escherichia coli isolated from pasteurized dairy products from Bahia, Brazil

The presence of pathogenic Shiga toxin-producing Escherichia coli (STEC) in dairy products represents a public health concern because of its ability to produce the toxins Stx1 and Stx2, which cause intestinal diseases. Monitoring the stages of milk production and checking dairy products for contamination are crucial steps to ensure dairy safety. This study aimed to report the occurrence of thermotolerant coliforms, E. coli, and STEC strains in pasteurized dairy products and to evaluate the antibiotic resistance profiles, serotypes, and characterizations of the STEC isolates by pulsed-field gel electrophoresis. We obtained a total of 138 pasteurized dairy products from 15 processing plants in Bahia, Brazil, to examine coliforms, E. coli, and STEC strains. We found that 43% of samples (59/138) contained thermotolerant coliforms, and 30% (42/138) did not comply with Brazilian regulations. Overall, 6% (9/138) were positive for E. coli and 4% (5/138) were positive for STEC. We recovered 9 STEC isolates from pasteurized cream (2/9), Minas Padrão cheese (2/9), Minas Frescal cheese (4/9), and ricotta (1/9). All isolates were stx2-positive, and 2 were eae-positive. All isolates were negative for the "big 6" STEC serogroups, belonging instead to serotypes ONT:HNT, ONT:H12, O148:H-, OR:H40, OR:HNT, and O148:HNT. Pulsed-field gel electrophoresis revealed 100% genetic similarity among 3 isolates from 2 different samples produced in the same production facility, which may suggest cross-contamination. As well, we found isolates that were 98% similar but in samples produced in different production facilities, suggesting a mutual source of contamination or a circulating strain. Two STEC strains exhibited resistance to streptomycin. Although the isolates presented a low resistance profile and no strain belonged to the "big 6" pathogenic group, the circulation of stx2-positive STEC strains in ready-to-eat products highlights the importance of epidemiological surveillance inside the Brazilian dairy chain.