Sample Type and Processing Plant Differences in the Proportion of Enterohemorrhagic Escherichia coli O157 and Non-O157 Serogroups in Feces and on Hides of Cull Dairy Cattle at Slaughter.

The study was conducted to determine the proportion and concentration of enterohemorrhagic Escherichia coli (EHEC) O157 and six non-O157 (O26, O45, O103, O111, O121, and O145) serogroups and identify seasonal and processing plant differences in feces and on hides of cull dairy cattle processed in commercial slaughterhouses in the United States. Approximately 60 rectal and 60 hide-on samples from matched carcasses were collected in each of three processing plants, in two periods; summer of 2017 and spring of 2018. Samples before enrichment were spiral plated to quantify EHEC, and postenriched samples underwent culture methods that included immuno-magnetic separation, plating on selective media, and PCR assays for identification and serogroup confirmation of putative isolates. An isolate was considered EHEC O157 positive if it harbored serogroup-specific (rfbE), Shiga toxin (stx1 and/or stx2), and intimin (eae) genes and EHEC non-O157 positive if at least one of the non-O157 serogroup-specific, stx1 and/or stx2, and eae genes was identified. Generalized linear mixed models were fitted to estimate overall proportion of positives for EHEC O157 and non-O157 EHEC serogroups, as well as seasonal and processing plant differences in fecal and hide-on proportion of positives. The fecal EHEC proportion at the sample level was 1.8% (95% CI = 0.0-92.2%) and 4.2% (95% CI = 0.0-100.0%) for EHEC O157 and EHEC non-O157, respectively. Hide sample level proportion of positives was 3.0% (95% CI = 0.0-99.9%) for EHEC O157 and 1.6% (95% CI = 0.0-100.0%) for EHEC non-O157. The proportion of EHEC O157 and non-O157 significantly differed by processing plant and sample type (hide vs. feces), but not by season. The association between proportion of EHEC serogroups in feces with the proportion on hides collected from matched cattle was 7.8% (95% CI = 0.6-53.3%) and 3.8% (95% CI = 0.3-30.8%) for EHEC O157 and non-O157, respectively. Taken together, our findings provide evidence of a low proportion of EHEC serogroups in the feces and on hides of cull dairy cattle and that their proportion varies across processing plants.

Shiga Toxin-Producing Escherichia coli Isolated from Wild Ruminants in Liguria, North-West Italy.

Wildlife may represent an important source of infectious diseases for humans and other wild and domestic animals. Wild ruminants can harbour and transmit Shiga toxin-producing Escherichia coli (STEC) to humans, and some strains even carry important antimicrobial resistance. In this study, 289 livers of wild roe deer, fallow deer, red deer and chamois collected in Liguria, north-west Italy, from 2019 to 2023 were analysed. Overall, 44 STEC strains were isolated from 28 samples. The characterisation of serogroups showed the presence of O104, O113, O145 and O146 serogroups, although for 28 colonies, the serogroup could not be determined. The most prevalent Shiga toxin gene in isolated strains was Stx2, and more specifically the subtype Stx2b. The other retrieved subtypes were Stx1a, Stx1c, Stx1d and Stx2g. The isolated strains generally proved to be susceptible to the tested antimicrobials. However, multi-drug resistances against highly critical antimicrobials were found in one strain isolated from a roe deer. This study highlights the importance of wildlife monitoring in the context of a "One Health" approach.

A challenging STEC strain isolation from patients' stools: an O166:H15 STEC strain with the stx2 gene.

Two patients with acute gastroenteritis tested positive for Shiga toxin-producing Escherichia coli (STEC) by polymerase chain reaction (PCR), and both strains carried the Shiga toxin 2 encoding gene. Since routine culture using CHROMagar STEC failed to recover these isolates, immunomagnetic separation (IMS) targeting the top six non-O157:H7 serotypes was used for isolate recovery. After two subsequent IMS runs, the STEC strains were isolated from trypticase soy broth with and without overnight enrichment for runs 1 and 2, respectively. Serotyping based on whole-genome sequencing revealed that both patients carried the strain O166:H15 STEC with the stx2 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other E. coli serotypes. When the STEC isolates from both stools were cultured on CHROMagar STEC and sheep blood agar (BAP), two distinct colony sizes were apparent after overnight incubation. The small and large colonies were picked and separately cultured on both media, and colony growth was observed for 2 weeks at room temperature after an initial overnight incubation at 37°C. After 1 week, the colonies showed concentric ring structures with a darker center and a lighter surrounding on CHROMagar STEC and a "fried egg"-resembling structure with a raised circular center and a flat surrounding on BAP. Both colony types remained morphologically different on CHROMagar STEC throughout the 15 days. However, on BAP, their appearance was comparable by day 7. IMPORTANCE: Shiga toxin-producing E. coli (STEC) infections can lead to severe complications such as bloody diarrhea and hemolytic uremic syndrome (HUS), especially in young children and the elderly. Strains that carry the shiga toxin 2 gene (stx2), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. E. coli O166:H15 has been previously reported causing a gastroenteritis outbreak in 1996 as a non-STEC strain, however the O166:H15 serotype we recovered carried the stx2 gene. It was particularly challenging to isolate this strain from stools by culture. Consequently, we tested immunomagnetic separation for the STEC recovery, which was a novel approach on clinical stools. Virulence genes were included for the characterization of these isolates.

Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) of non-O157:H7 serotypes are responsible for global and widespread human food-borne disease. Among these serogroups, O26, O45, O103, O111, O121, and O145 account for the majority of clinical infections and are colloquially referred to as the "Big Six." The "Big Six" strain panel we sequenced and analyzed in this study are reference type cultures comprised of six strains representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC) as a resource to the research community under panel number ATCC MP-9. The application of long- and short-read hybrid sequencing yielded closed chromosomes and a total of 14 plasmids of diverse functions. Through high-resolution comparative phylogenomics, we cataloged the shared and strain-specific virulence and resistance gene content and established the close relationship of serogroup O26 and O103 strains featuring flagellar H-type 11. Virulence phenotyping revealed statistically significant differences in the Stx-production capabilities that we found to be correlated to the strain's individual stx-status. Among the carried Stx1a, Stx2a, and Stx2d phages, the Stx2a phage is by far the most responsive upon RecA-mediated phage mobilization, and in consequence, stx2a + isolates produced the highest-level of toxin in this panel. The availability of high-quality closed genomes for this "Big Six" reference set, including carried plasmids, along with the recorded genomic virulence profiles and Stx-production phenotypes will provide a valuable foundation to further explore the plasticity in evolutionary trajectories in these emerging non-O157 STEC lineages, which are major culprits of human food-borne disease.

Shiga Toxin-Producing Escherichia coli Testing in New York 2011-2022 Reveals Increase in Non-O157 Identifications.

Shiga toxin-producing Escherichia coli (STEC) are an important cause of bacterial enteric infection. STEC strains cause serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic uremic syndrome. They have the potential to impact public health due to diagnostic challenges of identifying non-O157 strains in the clinical laboratory. The Wadsworth Center (WC), the public health laboratory of the New York State Department of Health, has isolated and identified non-O157 STEC for decades. A shift from initially available enzyme immunoassay testing to culture-independent diagnostic tests (CIDTs) has increased the uptake of testing at clinical microbiology laboratories. This testing change has resulted in an increased number of specimen submissions to WC. During a 12-year period between 2011 and 2022, WC received 5037 broths and/or stool specimens for STEC confirmation from clinical microbiology laboratories. Of these, 3992 were positive for Shiga toxin genes (stx1 and/or stx2) by real-time PCR. Furthermore, culture methods were utilized to isolate, identify, and characterize 2925 STEC from these primary specimens. Notably, WC observed a >200% increase in the number of STEC specimens received in 2021-2022 compared with 2011-2012 and an 18% increase in the number of non-O157 STEC identified using the same methodologies. During the past decade, the WC testing algorithm has been updated to manage the increase in specimens received, while also navigating the novel COVID-19 pandemic, which took priority over other testing for a period of time. This report summarizes updated methods for confirmation, surveillance, and outbreak detection of STEC and describes findings that may be related to our algorithm updates and the increased use of CIDTs, which is starting to elucidate the true incidence of non-O157 STEC.

Clinical and public health implications of increasing notifications of LEE-negative Shiga toxin-producing Escherichia coli in England, 2014-2022.

Introduction. Shiga toxin-producing Escherichia coli (STEC) belong to a diverse group of gastrointestinal pathogens. The pathogenic potential of STEC is enhanced by the presence of the pathogenicity island called the Locus of Enterocyte Effacement (LEE), including the intimin encoding gene eae.Gap statement. STEC serotypes O128:H2 (Clonal Complex [CC]25), O91:H14 (CC33), and O146:H21 (CC442) are consistently in the top five STEC serotypes isolated from patients reporting gastrointestinal symptoms in England. However, they are eae/LEE-negative and perceived to be a low risk to public health, and we know little about their microbiology and epidemiology.Aim. We analysed clinical outcomes and genome sequencing data linked to patients infected with LEE-negative STEC belonging to CC25 (O128:H2, O21:H2), CC33 (O91:H14) and, and CC442 (O146:H21, O174:H21) in England to assess the risk to public health.Results. There was an almost ten-fold increase between 2014 and 2022 in the detection of all STEC belonging to CC25, CC33 and CC442 (2014 n=38, 2022 n=336), and a total of 1417 cases. There was a higher proportion of female cases (55-70 %) and more adults than children, with patients aged between 20-40 and >70 most at risk across the different serotypes. Symptoms were consistent across the three dominant serotypes O91:H14 (CC33), O146:H21 (CC442) and O128:H2 (CC25) (diarrhoea >75 %; bloody diarrhoea 25-32 %; abdominal pain 64-72 %; nausea 37-45 %; vomiting 10-24 %; and fever 27-30 %). Phylogenetic analyses revealed multiple events of acquisition and loss of different stx-encoding prophage. Additional putative virulence genes were identified including iha, agn43 and subA.Conclusions. Continued monitoring and surveillance of LEE-negative STEC infections is essential due to the increasing burden of infectious intestinal disease, and the risk that highly pathogenic strains may emerge following acquisition of the Shiga toxin subtypes associated with the most severe clinical outcomes.

Predictive modeling of thermal inactivation of non-O157 Shiga toxin-producing Escherichia coli in ground beef with varying fat contents.

A mathematical model to predict the thermal inactivation of non-O157 Shiga toxin-producing Escherichia coli (STEC) in ground beef was developed, with temperature and fat content of ground beef as controlling factors. Survival curves for a cocktail of non-O157 STEC strains in ground beef at four temperatures (55, 60, 65, and 68 °C) and six fat levels (5, 10, 15, 20, 25, and 30%) were generated. Nine primary models-log-linear, log-linear with tail, biphasic, sigmoidal, four-factor sigmoidal, Baranyi, Weibull, mixed Weibull, and Gompertz-were tested for fitting the survival curves. Primary modeling analysis showed the Weibull model had the highest accuracy factor and Akaike's weight, making it the best-fitting model. The parameters of the Weibull model were estimated using a nonlinear mixed, and response surface modeling was used to develop a second-order polynomial regression to estimate the impact of fat in ground beef and cooking temperature on the heat resistance of non-O157 STEC strains. The secondary model was successfully validated by comparing predicted lethality (log10 CFU/g) with the observed values for ground beef containing 10 and 27% fat at 58 and 62 °C. Process lethality obtained from experimental data was within the prediction interval of the predictive model. The developed model will assist the food industry in estimating the appropriate time and temperature required for cooking ground beef to provide adequate protection against STEC contaminants.

Clonal spread of non-O157 Shiga toxigenic Escherichia coli O21:H25 in raw water buffalo milks.

AIMS: This study was conducted to investigate the presence of Shiga toxin-producing O157 and non-O157 E. coli in raw water buffalo milk, as well as to determine the virulence gene profiles, phylogroups, sequence types, and serotypes of the isolated strains. METHODS AND RESULTS: A total of 200 hand-milked raw water buffalo milk samples were collected from 200 different water buffaloes over a period of three months from 20 different farms. Isolation of STEC was performed using CHROMagar STEC. Presence of stx1, stx2, and eaeA genes were investigated by mPCR. Phylogroups and sequence types of E. coli strains were determined by Clermont phylotyping and MLST. Serotyping was performed using PCR or WGS. According to the results, two milk samples obtained from two different farms were found as STEC-positive. All Stx-positive E. coli isolates belonged to phylogenetic group A and were assigned to ST10. WGS results indicated that serotype of two isolates was O21:H25 and average nucleotide identity was detected at 99.99%. Thirteen additional registered E. coli O21:H25 assembled WGS data were obtained from EnteroBase and a phylogenetic tree was constructed. CONCLUSIONS: With this study, the presence of stx2 harboring E. coli O21:H25 in milk was identified for the first time. Although the identified serotype is considered a non-pathogen seropathotype, we conclude it could play an important role in the environmental circulation of Stx-phages and consequently contribute to the emergence of new STEC-related outbreaks.

Draft genome sequences of nine non-O157 Shiga toxin-producing Escherichia coli in ready-to-eat food from supermarkets in Argentina.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are recognized as an important group of bacterial enteropathogens. Here, we report the draft genome sequence of nine strains of non-O157 STEC isolated from ready-to-eat foods in Argentina. The whole-genome sequence data provide a better understanding of these isolates and will aid epidemiological investigation during outbreaks.

Detection of multidrug-resistant Shiga toxin-producing Escherichia coli in some food products and cattle faeces in Al-Sharkia, Egypt: one health menace.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen, that is transmitted from a variety of animals, especially cattle to humans via contaminated food, water, feaces or contact with infected environment or animals. The ability of STEC strains to cause gastrointestinal complications in human is due to the production of Shiga toxins (sxt). However, the transmission of multidrug-resistance STEC strains are linked with a severity of disease outcomes and horizontal spread of resistance genes in other pathogens. The result of this has emerged as a significant threat to public health, animal health, food safety, and the environment. Therefore, the purpose of this study is to investigate the antibiogram profile of enteric E. coli O157 isolated from food products and cattle faeces samples in Zagazig City, Al-Sharkia, Egypt, and to reveal the presence of Shiga toxin genes stx1 and stx2 as virulence factors in multidrug-resistant isolates. In addition to this, the partial 16S rRNA sequencing was used for the identification and genetic recoding of the obtained STEC isolates. RESULTS: There was a total of sixty-five samples collected from different geographical regions at Zagazig City, Al-Sharkia-Egypt, which were divided into: 15 chicken meat (C), 10 luncheon (L), 10 hamburgers (H), and 30 cattle faeces (CF). From the sixty-five samples, only 10 samples (one from H, and 9 from CF) were identified as suspicious E. coli O157 with colourless colonies on sorbitol MacConkey agar media with Cefixime- Telurite supplement at the last step of most probable number (MPN) technique. Eight isolates (all from CF) were identified as multidrug-resistant (MDR) as they showed resistance to three antibiotics with multiple antibiotic resistance (MAR) index ≥ 0.23, which were assessed by standard Kirby-Bauer disc diffusion method. These eight isolates demonstrated complete resistance (100%) against amoxicillin/clavulanic acid, and high frequencies of resistance (90%, 70%, 60%,60%, and 40%) against cefoxitin, polymixin, erythromycin, ceftazidime, and piperacillin, respectively. Those eight MDR E. coli O157 underwent serological assay to confirm their serotype. Only two isolates (CF8, and CF13), both from CF, were showed strong agglutination with antisera O157 and H7, as well as resistance against 8 out of 13 of the used antibiotics with the highest MAR index (0.62). The presence of virulence genes Shiga toxins (stx1 and stx2) was assessed by PCR technique. CF8 was confirmed for carrying stx2, while CF13 was carrying both genes stx1, and stx2. Both isolates were identified by partial molecular 16S rRNA sequencing and have an accession number (Acc. No.) of LC666912, and LC666913 on gene bank. Phylogenetic analysis showed that CF8, and CF13 were highly homologous (98%) to E. coli H7 strain, and (100%) to E. coli DH7, respectively. CONCLUSION: The results of this study provides evidence for the occurrence of E. coli O157:H7 that carries Shiga toxins stx1 and/or stx2, with a high frequency of resistance to antibiotics commonly used in human and veterinary medicine, in Zagazig City, Al-Sharkia, Egypt. This has a high extent of public health risk posed by animal reservoirs and food products with respect to easy transmission causing outbreaks and transfer resistance genes to other pathogens in animal, human, and plants. Therefore, environmental, animal husbandry, and food product surveillance, as well as, clinical infection control, must be strengthened to avoid the extra spread of MDR pathogens, especially MDR STEC strains.

Association of resistance to quaternary ammonium compounds and organic acids with genetic markers and their relationship to Escherichia coli serogroup.

Sanitizer resistance is being extensively investigated due to the potential for bacterial survival and cross-resistance with other antimicrobials. Similarly, organic acids are being used due to their microbial inactivation potential as well as being generally recognized as safe (GRAS). However, little is known about associations of genetic and phenotypic factors in Escherichia coli related to resistance to sanitizers and organic acids as well as differences between "Top 7" serogroups. Therefore, we investigated 746 E. coli isolates for resistance to lactic acid and two commercial sanitizers based on quaternary ammonium and peracetic acid. Furthermore, we correlated resistance to several genetic markers and investigated 44 isolates using Whole Genome Sequencing. Results indicate that factors related to motility, biofilm formation, and Locus of Heat Resistance played a role in resistance to sanitizers and lactic acid. In addition, Top 7 serogroups significantly differed in sanitizer and acid resistance, with O157 being the most consistently resistant to all treatments. Finally, mutations in rpoA, rpoC, and rpoS genes were observed, in addition to presence of a Gad gene with alpha-toxin formation in all O121 and O145 isolates, which may be related to increased resistance of these serogroups to the acids used in the present study.

Occurrence and genetic characterization of Shiga toxin-producing Escherichia coli on bovine and pork carcasses and the environment from transport trucks.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens causing severe diseases. The ability of STEC to produce disease is associated with Shiga toxin (Stx) production. We investigated the occurrence of STEC on bovine and pork carcasses and walls of trucks where they were transported, and we characterized virulence genes and serotypes of STEC strains. We compared the whole genomic sequencing of a STEC O157:H7 strain isolated from a bovine carcass in this work and a STEC O157:H7 strain isolated from a child with HUS, both isolated in 2019. We studied the relationship between these isolates and others collected in the database. The results show a 40% of STEC and two different serogroups were identified (O130 and O157). STEC O157:H7 were isolated from bovine carcasses and harbored stx2, eae, ehxA, katP, espP, stcE, ECSP_0242/1773/2687/2870/2872/3286/3620 and were classified as lineage I/II. In STEC non-O157 isolates, three isolates were isolated from bovine carcasses and harbored the serogroup O130 and one strain isolated from pork carcasses was O-non-typeable. All STEC non-O157 harbored sxt1 gene. The analysis from the whole genome showed that both STEC O157:H7 strains belonged to the hypervirulent clade 8, ST11, phylogroup E, carried the allele tir 255 T > A T, and they were not clonal. The analysis of information allows us to conclude that the STEC strains circulate in pork and bovine carcasses arriving in transport. This situation represents a risk for the consumers and the need to implement an integrated STEC control in the food chain.

Universal hydrolysis probe-based approach for specific detection and genotyping of foodborne pathogens.

Real-time PCR assays are the method of choice for the specific detection of DNA targets. Multiple real-time PCR chemistries are used for developing pathogen detection assays. Among them, a hydrolysis probe is a preferred choice for pathogen detection assays. Two known limitations of hydrolysis probes are high cost and limited storage life. Therefore, this study aimed to develop and validate a universal hydrolysis probe (UHP)-based approach with high-resolution melt (HRM) analysis capabilities. The approach can be used for the detection and genotyping of target DNA. The approach described in this study was validated by standardizing nine UHP assays for detecting seven Shiga toxin-producing Escherichia coli serogroups, Listeria monocytogenes, and Salmonella strains. These nine assays were validated with 141 pure culture bacterial strains. Additionally, the HRM capability of the developed approach was validated for three UHP assays targeting E. coli O26, O111, and O121 using 96 DNAs isolated from enriched food samples. The nine assays specifically detected the target bacterial strains, and the three assays showed single nucleotide polymorphism (SNP) identification capability and no cross-reactivity with non-target strains. The developed approach can be performed in singleplex or multiplex format and combined with HRM analysis. The data from this study demonstrate that the UHP real-time PCR approach is a robust method for detecting any deoxyribonucleic acid target.

The "Big Six": Hidden Emerging Foodborne Bacterial Pathogens.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are emerging serogroups that often result in diseases ranging from diarrhea to severe hemorrhagic colitis in humans. The most common non-O157 STEC are O26, O45, O103, O111, O121, and O145. These serogroups are known by the name "big six" because they cause severe illness and death in humans and the United States Department of Agriculture declared these serogroups as food contaminants. The lack of fast and efficient diagnostic methods exacerbates the public impact of the disease caused by these serogroups. Numerous outbreaks have been reported globally and most of these outbreaks were caused by ingestion of contaminated food or water as well as direct contact with reservoirs. Livestock harbor a variety of non-O157 STEC serovars that can contaminate meat and dairy products, or water sources when used for irrigation. Hence, effective control and prevention approaches are required to safeguard the public from infections. This review addresses the disease characteristics, reservoirs, the source of infections, the transmission of the disease, and major outbreaks associated with the six serogroups ("big six") of non-O157 STEC encountered all over the globe.

A systematic review and meta-analysis of published literature on prevalence of non-O157 Shiga toxin-producing Escherichia coli serogroups (O26, O45, O103, O111, O121, and O145) and virulence genes in feces, hides, and carcasses of pre- and peri-harvest cattle worldwide.

OBJECTIVE: The objective of this study was to summarize peer-reviewed literature on the prevalence and concentration of non-O157 STEC (O26, O45, O103, O111, O121, and O145) serogroups and virulence genes (stx and eae) in fecal, hide, and carcass samples in pre- and peri-harvest cattle worldwide, using a systematic review of the literature and meta-analyses. DATA SYNTHESIS: Seventy articles were eligible for meta-analysis inclusion; data from 65 articles were subjected to random-effects meta-analysis models to yield fecal prevalence estimates. Meta-regression models were built to explore variables contributing to the between-study heterogeneity. RESULTS: Worldwide pooled non-O157 serogroup, STEC, and EHEC fecal prevalence estimates (95% confidence interval) were 4.7% (3.4-6.3%), 0.7% (0.5-0.8%), and 1.0% (0.8-1.1%), respectively. Fecal prevalence estimates significantly differed by geographic region (P < 0.01) for each outcome classification. Meta-regression analyses identified region, cattle type, and specimen type as factors that contribute to heterogeneity for worldwide fecal prevalence estimates. CONCLUSIONS: The prevalence of these global foodborne pathogens in the cattle reservoir is widespread and highly variable by region. The scarcity of prevalence and concentration data for hide and carcass matrices identifies a large data gap in the literature as these are the closest proxies for potential beef contamination at harvest.

Characterisation and epidemiological subtyping of Shiga toxin-producing Escherichia coli isolated from the beef production chain in Gauteng, South Africa.

In South Africa, there is a shortage of epidemiologic data on Shiga toxin-producing Escherichia coli (STEC) in the beef production chain. This study was conducted to characterise STEC isolates originating from three studies conducted in a cattle feedlot, beef abattoirs and retail outlets in Gauteng province, South Africa. Polymerase chain reaction was used to detect virulence genes, the Epsilometer test to assess antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE) to investigate genetic relatedness of isolates, and conventional serotyping for phenotypic identification. Amongst the 86 STEC isolates, the eaeA gene was detected in 20 (23%), and 26 different serogroups were identified, including the clinically important O8, O174, O2, 020 and O117. The majority of the isolates (95%; 82/86) exhibited resistance to one or more antimicrobial agents, and 30 of the isolates (35%) exhibited multi-drug resistance (MDR), being resistant to at least three antimicrobial classes. The PFGE patterns showed a highly diverse but related STEC population, with 45 distinct patterns and evidence of horizontal transmission along the beef production chain. This is significant because it demonstrates continual environmental contamination and risk of contamination along the beef production chain and the food chain. To our knowledge, this is the first study that provides evidence of horizontal transmission of STEC along the beef production chain in South Africa. This epidemiological information could facilitate the development of a proactive strategy for reducing potential foodborne outbreaks and transmission of antimicrobial resistant pathogens in the food chain.

Whole-genome sequencing analysis of Shiga toxin-producing Escherichia coli O22:H8 isolated from cattle prediction pathogenesis and colonization factors and position in STEC universe phylogeny.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen capable of causing illness in humans. In a previous study, our group showed that a STEC isolate belonging to O22:H8 serotype (strain 154) can interfere with STEC O157:H7 colonization both in vitro and in vivo. Using whole-genome sequencing and genomic comparative, we predicted a subset of genes acquired by O22:H8 strain 154 through horizontal gene transfer that might be responsible for the phenotype previously described by our group. Among them were identified genes related to the pathogenesis of non-LEE (locus of enterocyte effacement) STEC, specific metabolic processes, antibiotic resistance and genes encoding for the T6SS-1 that is related to inter-bacterial competition. In addition, we showed that this strain carries stx1c and stx2dact, a mucus-inducible variant. The results obtained in this study provide insights into STEC genomic plasticity and the importance of genomic islands in the adaptation and pathogenesis of this pathogen.

Behavior of Shiga-toxin-producing Escherichia coli in ewe milk stored at different temperatures and during the manufacture and ripening of a raw milk sheep cheese (Zamorano style).

This study was conducted to assess the survival of 2 wild Shiga toxin-producing Escherichia coli strains (one serotype O157:H7 and one non-O157:H7) in ewe milk stored at different conditions and to examine the fate of the O157 strain during the manufacture and ripening of a Spanish sheep hard variety of raw milk cheese (Zamorano). The strains were selected among a population of 50 isolates, which we obtained from ewe milk, because of their high resistance to 0.3% lactic acid. Both strains were inoculated (approximately 2 log10 cfu/mL) in raw and heat-treated (low-temperature holding, LTH; 63°C/30 min) ewe milk and stored for 5 d at 6, 8, and 10°C and also according to a simulation approach for assessing the effects of failures in the cold chain. The minimum growth temperature for the O157:H7 strain in LTH and raw ewe milk was 8°C. For the non-O157:H7 strain, the lowest temperature showing bacterial growth in LTH ewe milk was 6°C, but it did not grow at any of the tested conditions in raw milk. It appears that the O157 strain was more susceptible to cold stress but was likely a better competitor than the non-O157 strain against the milk autochthonous microbiota. For manufacture of Zamorano cheese, raw milk was inoculated with approximately 3 log10 cfu/mL, and after 2 mo of ripening at 10 to 12°C, the cheeses showed the expected general characteristics for this variety. The O157:H7 strain increased 0.9 log10 cfu/g after whey drainage and during ripening and storage decreased by 2.9 log10 cfu/g. Nevertheless, its detectable level (estimated at 6.2 cfu/g) after 2 mo of ripening suggests that Zamorano cheese manufactured from raw ewe milk contaminated with E. coli O157:H7 could represent a public health concern.

The Characterisation of Diarrhoeagenic Verotoxin Producing Non-O157 Escherichia coli among Young Children in Kuantan, Malaysia.

Background: Diarrhoeagenic verotoxin producing non-O157 Escherichia coli (VTEC) are associated with endemic infantile diarrhoea-causing morbidity and mortality worldwide. VTEC can also cause severe illness and has an impact on outbreaks, especially in developing countries. This study aims to investigate the prevalence and characterisation of VTEC and their association in causing infectious diarrhoea among Malaysian children. Methods: Standard microbiological techniques identified a total of 137 non-repeated, clinically significant E. coli isolates. Serological assays discerned non-O157 E. coli serogroup, subjected to virulence screen (VT1 and VT2) by a polymerase chain reaction (PCR). Results: Different PCR sets characterised the 49 clinical isolates of sorbitol positive non-O157 E. coli. Twenty-nine isolates harboured verotoxin genes associated with diarrhoea among children (≤ 5 years old). Among the 29 (59.18%) strains of verotoxin producing E. coli, genotypes VT1 and VT2 were detected in 21 (42.85%) and 5 (10.20%) isolates respectively, while both VT1 and VT2 genes were confirmed in 3 (6.12%) isolates. Conclusion: This study evaluates on the prevalence, serological characteristics and antimicrobial susceptibility patterns of VTEC diarrhoea affected children (≤ 5 years old). Besides, the prevalence of verotoxin gene was determined as a root cause of diarrhoea among Malaysian children.

Epidemiology of Non-O157 Shiga Toxin-Producing Escherichia coli in the Province of Alberta, Canada, from 2018 to 2021.

Non-O157 serogroups contribute significantly to the burden of disease caused by Shiga toxin-producing Escherichia coli (STEC) and have been underrecognized by traditional detection algorithms. We described the epidemiology of non-O157 STEC in Alberta, Canada for the period of 2018 to 2021. All non-O157 STEC isolated from clinical samples were submitted for serotyping and qPCR targeting the stx1 and stx2 genes. A total of 729 isolates were identified. Increased detection occurred over the summer months, peaking in July. Patients 18 years and younger made up 42.4% of cases, with 31.1% in those 0-9 years of age. There was a slight female predominance (399/729, 54.7%) A total of 50 different serogroups were detected; the most common were O26 (30.3%), O103 (15.9%), O111 (12.8%), O121 (11.0%), O118 (3.3%) and O71 (2.9%). These six serogroups made up 76.2% of all isolates. In total, 567 (77.8%) were positive for stx1, 114 (15.6%) were positive for stx2 and 48 (6.6%) were positive for both stx1 and stx2. A wide variety of non-O157 serogroups have been detected in Alberta, with the most frequent serogroups differing from other locations. These results highlight the need for further characterization of their virulence factors and clinical impact.