Shiga Toxin-producing Escherichia Coli Isolates Research Articles

Biofilm formation, antibiotic resistance and genotyping of Shiga toxin-producing Escherichia coli isolated from retail chicken meats

ABSTRACT 1. The Shiga toxin-producing Escherichia coli (STEC) is a hazardous zoonotic agent for chicken meat consumers. This study determined the serogroups and evaluated the virulence genes, antibiotic resistance, biofilm-forming profiles and genetic relationships of STEC isolates in chicken meat. 2. A total of 100 samples belonging to dressed-whole chicken and different parts of the chicken (wing, breast, thigh, drumstick) were collected between September and November 2019 from different retail markets in Kayseri, Türkiye. 3. Phenotypic (identification, disc diffusion test, Congo red agar and microtitre plate tests) and molecular tests (identification, serogrouping, virulence factors, biofilm, antibiotic susceptibility, 16S rRNA sequencing and enterobacterial repetitive intergenic consensus-PCR for typing of the isolates) were carried out. 4. E. coli was isolated from 35% of the samples and 35% of the samples harboured at least one STEC. Among 35 STEC isolates, 3 (8.5%), 6 (17.1%), 2 (5.7%) and 3 (8.5%) were found to be positive for fliCH2, fliCH8, fliCH11, fliCH19 genes, respectively. Out of 35 STEC positive isolates, 4 (11.4%) were identified as E. coli O157, from which 2 (5.7%) were E. coli O157:H7. E. coli O157 was detected in two (10%), one (5%), one (5%) of the thigh, drumstick and whole chicken samples, respectively. 5. Biofilm-forming ability was reported in 33 (94.2%) of 35 E. coli isolates, whilst the biofilm-associated genes detected among 35 STEC isolates included csgA (88.5%), fimH (88.5%), bcsA (85.7%), agn43 (14.2%) and papC (8.5%). The STEC strains showed resistance against ampicillin (88.5%) and erythromycin (88.5%), followed by tetracycline (74.2%) and gentamicin (25.7%). However, the distribution of isolates harbouring bla CMY, ere(A), tet(A) and aac(3)-IV antibiotic resistance genes was found to be 17.1%, 11.4%, 85.7% and 5.7%, respectively. 6. ERIC-PCR showed that E. coli strains obtained from different parts and whole of chicken samples had genetic diversities. ERIC-PCR patterns grouped strains of 35 STEC into eight clusters designated A-H, with 73% similarity. Proper hygiene measures and staff training are essential for public health during poultry processing and in retail stores to control STEC.

Molecular Characterisation and Antimicrobial Resistance Patterns of Shiga Toxin Producing Escherichia coli Isolated from Farm Water Samples

Shiga toxin-producing Escherichia coli (STEC) strains are considered the most common food-borne zoonotic pathogen and are highly pathogenic to humans in low infectious doses, causing food-borne diseases through consumption of contaminated water or food. Resistance against antibiotics by STEC is a big concern nowadays. Two hundred farm water samples (Cattle-40, Buffaloes-40, Sheep-30, Goat-20, Pig-20 and poultry-50) were collected aseptically from different livestock farms in and around Proddatur, Andhra Pradesh, India and Processed for E. coli isolation, identification with culture method and molecular characterization by PCR. E. coli was characterized as STEC with two genes i.e. stx1 and stx2. All the STEC isolates were subjected to an antibiotic sensitivity test by disc diffusion method against ten antibiotics. Results showed that out of 200 farm water samples, 196 were positive for E. coli with an overall prevalence of 98% (196/200) and 62.2% (122/196) for STEC by PCR. Antimicrobial susceptibility test by disc diffusion method against ten antibiotics revealed the higher resistance to Cepahlothin (100%) followed by Tetracyclin (98.4%), Ampicillin (96.7%), Streptomycin (95%), Sulphonamides (91.8%), Trimethoprim (84.4%), Kanamycin (34.4%), Chloromphenicol (17.2%), Colistin (9.0%) and least resistance to Gentamycin (4.9%).

Occurrence of Shiga toxin-producing Escherichia colicarrying antimicrobial resistance genes in sheep on smallholdings in Bangladesh.

Shiga toxin-producingEscherichia coli(STEC) are zoonotic foodborne pathogens and a significant concern with the emergence of antibiotic resistance. Close human contact might have a higher chance of being transmitted to humans from sheep if the sheep population is a potential reservoir of antimicrobial-resistant STEC. Therefore, this study aimed to examine the sheep population in rural Bangladesh for antimicrobial-resistant STEC. We screened 200 faecal samples collected from sheep in three Upazilas from the Chattogram district. Randomisation of sampling was not performed due to the smaller flock size (two to six animals per smallholding). Phenotypically positiveE. coliisolates were examined for two Shiga toxin-producing genes -stx1 andstx2. PCR-positive STEC isolates were investigated for the presence of antimicrobial resistance (AMR) genes -blaTEM ,sul1andsul2. In total, 123 of the 200 tested samples were confirmed positiveE. coliusing culture-based methods. PCR results show 17 (13.8%)E. coliisolates harboured ≥ one virulent gene (stx1or/andstx2) of STEC. The AMR profile of STEC isolates was determined utilising the disc diffusion method. Of the STEC isolates, 82, 76, 71 and 71% were susceptible tochloramphenicol, gentamicin, ciprofloxacin and ampicillin. In contrast, 47% of isolates were resistant to trimethoprim-sulfamethoxazole, and 41% were resistant to amoxicillin. In addition, six of the tested STEC isolates exhibitedthe blaTEM gene; eight STEC isolates hadthe sul1gene, andthe sul2gene was detected in ten STEC isolates. To our knowledge, this study is the first to reveal a substantial percentage of STEC isolated from sheep in rural Bangladesh harbouring AMR genes.

Shiga toxin-producing Escherichia coli clonal complex 32, including serotype O145:H28, in the UK and Ireland.

Introduction. Shiga toxin-producing Escherichia coli (STEC) O157:H7 has been the most clinically significant STEC serotype in the UK for over four decades. Over the last 10 years we have observed a decrease in STEC O157:H7 and an increase in non-O157 STEC serotypes, such as O145:H28.Gap Statement. Little is known about the microbiology and epidemiology of STEC belonging to CC32 (including O145:H28) in the UK. The aim of this study was to integrate genomic data with patient information to gain a better understanding of the virulence, disease severity, epidemic risk assessment and population structure of this clinically significant clonal complex.Methodology. Isolates of E. coli belonging to CC32 (n=309) in the archives of public health agencies in the UK and Ireland were whole-genome-sequenced, virulence-profiled and integrated with enhanced surveillance questionnaire (ESQ) data, including exposures and disease severity.Results. Overall, diagnoses of STEC belonging to CC32 (290/309, 94 %) in the UK have increased every year since 2014. Most cases were female (61 %), and the highest proportion of cases belonged to the 0-4 age group (53/211,25 %). The frequency of symptoms of diarrhoea (92 %), abdominal pain (84 %), blood in stool (71 %) and nausea (51 %) was similar to that reported in cases of STEC O157:H7, although cases of STEC CC32 were more frequently admitted to hospital (STEC CC32 48 % vs O157:H7 34 %) and/or developed haemolytic uraemic syndrome (HUS) (STEC CC32 9 % vs O157:H7 4 %).The majority of STEC isolates (268/290, 92 %) had the stx2a/eae virulence gene combination, most commonly associated with progression to STEC HUS. There was evidence of person-to-person transmission and small, temporally related, geographically dispersed outbreaks, characteristic of foodborne outbreaks linked to nationally distributed products.Conclusion. We recommend more widespread use of polymerase chain reaction (PCR) for the detection of all STEC serogroups, the development of consistent strategies for the follow-up testing of PCR-positive faecal specimens, the implementation of more comprehensive and standardized collection of epidemiological data, and routine sharing of sequencing data between public health agencies worldwide.

Survival of Salmonella and Shiga toxin–producing Escherichia coli during tempering of wheat berries

Wheat flour contaminated with Salmonella or Shiga toxin–producing Escherichia coli (STEC) has been linked to foodborne outbreaks and recalls. The survival of five strains of rifampicin-resistant Salmonella and STEC on wheat berries was evaluated during tempering (hydration). Wheat berries were inoculated with Salmonella (cocktail) or STEC (individual strains or cocktail) and dried at ambient conditions for 24 h. Sterile water (control), lactic acid (LA; 5.4 or 10.9%) or peracetic acid (PAA; 100 or 500 ppm) was added and then samples were held at 15, 23, or 30 °C for up to 24 h. Significant population reductions were observed after addition of LA or PAA (time 0 h), with small but significant additional declines occurring between 0 and 12 h but not between 12 and 24 h. Salmonella populations declined 0.43 and 1.79 log CFU/g on wheat berries tempered at 23 °C for 12 h with sterile water and 5.4% LA, respectively. Similar 12-h reductions of 0.45–0.80 log CFU/g (water) and 1.43–1.68 log CFU/g (5.4% LA) were observed for the STEC cocktail. Increasing LA to 10.9% during tempering resulted in significantly greater reductions (1.87–2.03 log CFU/g) of STEC after 12 h; similar reductions were observed for individual STEC isolates (1.74–2.14 log CFU/g). Reductions of STEC during 12 h of tempering were 1.70 and 1.78 log CFU/g with 10.9% LA at 15 °C and 30 °C, respectively, and 0.73 and 1.21 log CFU/g with 100 and 500 PAA, respectively at ambient conditions. Addition of LA or PAA during tempering may provide a tool to reduce Salmonella or STEC in wheat.

Multidrug-Resistant Shiga Toxin-Producing Escherichia coli Isolated from Philippine Native Swine (Sus scrofa L.)

The interest in the use of Philippine native swine (PNS) as a product alternative to the white hybrid has been growing in the Philippines. However, PNS raised in low-cost production systems are prone to infection by zoonotic pathogens, such as the Shiga toxin-producing Escherichia coli (STEC) that causes diarrhea and life-threatening complications. The multidrug-resistant (MDR) strains of STEC further aggravate the problem as they serve as reservoir of antibiotic resistance genes. This study aimed to detect the occurrence of MDR STEC in 57 fecal samples of weaning- and market-age PNS. In addition, the class 1 integrons of the MDR STEC were characterized due to their ability to capture antibiotic resistance genes and hitchhike in mobile genetic elements, thereby spreading resistance. Fifty-three out of fifty-seven PNS or 93% were found to be positive for the presence of STEC by amplifying the stx1 gene. Disk diffusion assay against 12 antibiotic agents showed that the STEC isolates are most resistant to tetracycline (73.58%), ampicillin (37.74%), trimethoprim/sulfamethoxazole (32.08%), and streptomycin (32.08%). Seventeen STEC isolates exhibited the MDR phenotype and contained class 1 integron (intI1). Four aadA (adenylyltransferase, streptomycin resistance) and dfrA (dihydrofolate reductase, trimethoprim resistance) gene variants were identified in the variable region of the class 1 integrons. The results of this study will be useful in devising effective management practices that would control the presence and spread of this pathogen in the farm.

Molecular characterization and phylogeny of Shiga toxin-producing Escherichia coli derived from cattle farm.

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen, which can cause diseases such as diarrhea, hemorrhagic enteritis, and hemolytic uremic syndrome in humans. Twelve STEC isolates were collected from beeves and feces of commercial animals in China between 2019 and 2020 for this study. In addition to the determination of serotype and Shiga toxin subtype, whole-genome sequencing (WGS) was used for determining phylogenetic relationships, antimicrobial resistance (AMR), virulence genes, and sequence type (ST) of isolates. A total of 27 AMR genes were detected, and each STEC isolate carried more than 10 AMR genes. Eight STEC isolates from ground beef and four STEC isolated from feces were screened. A total of seven serotypes were identified, and one isolate ONT:H10 was undetermined by SeroTypeFinder. Three O157:H7 strains were confirmed and the remaining five serogroups were confirmed as O26:H11, O81:H31, O105:H8, O178:H19, and O136:H12. The phylogenetic analysis showed that STEC isolates of the same serotype or ST were clustered together based on cgMLST. The comparison of the genomes of 157 STEC reference isolates worldwide with our local STEC isolates showed that STEC isolates screened in China represented various collections and could not form a separate cluster but were interspersed among the STEC reference collection, which suggested that several STEC isolates shared a common ancestor irrespective of STEC serotype isolates. cgMLST revealed that isolates of the same O serotype clustered irrespective of their H type. Further investigation is required to determine the pathogenic potential of other serotypes of STEC, particularly in regard to these rare serotypes.

Virulence and antimicrobial-resistance of shiga toxin-producing E. coli (STEC) Isolated from edible shellfish and its public health significance

Shiga toxin-producing E. coli (STEC) are an important cause of foodborne illness in humans with infections ranging from mild non-bloody diarrhea to bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). This study aimed to investigate the distribution of STEC in shellfish from coastal shores of Lake Timsah in Ismailia Governorate, Egypt and its probable hazard to seafood consumers. Samples from the external surface and tissues of shrimp (n = 45), crabs (n = 45), and oysters (n = 45) batches were examined bacteriologically for the presence of STEC and tested for their antibiotic sensitivity. Moreover, occurrence of virulence genes was determined via detection of stx1, stx2 and eaeA genes using PCR. Overall, E. coli and presumptive STEC isolates (from CHROMagar) were identified from the surface (55.6 and 5.9%) and tissues (42.2 and 8.9%) of the examined shellfish batches, respectively. Five STEC isolates had been confirmed and found belonging to O26:H11, O125:H6, O146:H21, and O159 serogroups, those were 4 isolates from tissues of the three shellfish species and one isolate from the crab surface. The STEC isolates were multi-drug resistant, showing complete resistance to; penicillins, amoxycillin/clavulanic acid, colistin, fosfomycin, ceftriaxone, ciprofloxacin, and tetracycline, however, they were sensitive to gentamycin except O159 serogroup. The current study revealed low level of contamination of shellfish from coastal shores of Lake Timsah with STEC, however, it also highlights the extreme level of antimicrobial resistance exhibited by the presumptive and confirmed STEC isolates which is very hazardous for seafood consumers in the study area.

Relationship between virulence factors and antimicrobial resistance genes of pathogenic Escherichia coli from diarrheic weaned piglets

A total of 690 pathogenic Escherichia (E.) coli isolates from weaned piglets were examined for antimicrobial resistance phenotypes, resistance genes, and virulence gene profiles. Also, 29 enterotoxigenic E. coli (ETEC) and 35 Shiga-toxin producing E. coli (STEC) isolates were analyzed using multi-locus sequence typing (MLST). Comparisons of the associations between antimicrobial resistance phenotypes, resistance genes, and virulence genes were performed separately by assessing odds ratio (OR). Although majorities of associations were not confirmed however, we found that associations between specific virulence factors-antimicrobial resistance. F18 encoding isolates showed association with resistance to cefazolin (OR = 3.08) and cefoxitin (OR = 3.65), and also with antimicrobial resistance gene mcr-3 (OR = 4.58). There was a high correlation between F4-STb (OR = 13.56), F4-LT (OR = 8.77), F4-EAST-I (OR = 4.97), and F18-Stx2e (OR = 3.83). Most of ETEC (21 of 29, 72.4%) isolates were assigned to ST100, and 20 of 35 STEC isolates (57.1%) were ST1. There were 5 clusters, and each cluster showed specific antimicrobial resistance patterns. Cluster I showed resistance to gentamicin, streptomycin, neomycin, nalidixic acid, ciprofloxacin, norfloxacin, trimethoprim / sulfamethoxazole, and tetracyclines whereas, cluster V showed resistance to ampicillin, amoxicillin / clavulanic acid, cephalothin, cefoxitin, cefazolin, norfloxacin, and colistin. Although there is need to do more experiments to clarify why certain virulence factors showed relationship with antimicrobial resistance, it is clear that there is a significant association between specific virulence genes and antimicrobial resistance in E. coli from weaned piglets with enteric colibacillosis in Korea.

Microbial Antagonism in Food-Enrichment Culture: Inhibition of Shiga Toxin-Producing Escherichia coli and Shigella Species.

Bacterial pathogens, such as Shiga toxin-producing Escherichia coli (STEC) and Shigella spp., are important causes of foodborne illness internationally. Recovery of these organisms from foods is critical for food safety investigations to support attribution of illnesses to specific food commodities; however, isolation of bacterial cultures can be challenging. Methods for the isolation of STEC and Shigella spp. from foods typically require enrichment to amplify target organisms to detectable levels. Yet, during enrichment, target organisms can be outcompeted by other bacteria in food matrices due to faster growth rates, or through production of antimicrobial agents such as bacteriocins or bacteriophages. The purpose of this study was to evaluate the occurrence of Shigella and STEC inhibitors produced by food microbiota. The production of antimicrobial compounds in cell-free extracts from 200 bacterial strains and 332 food-enrichment broths was assessed. Cell-free extracts produced by 23 (11.5%) of the strains tested inhibited growth of at least one of the five Shigella and seven STEC indicator strains used in this study. Of the 332 enrichment broths tested, cell-free extracts from 25 (7.5%) samples inhibited growth of at least one of the indicator strains tested. Inhibition was most commonly associated with E. coli recovered from meat products. Most of the inhibiting compounds were determined to be proteinaceous (34 of the 48 positive samples, 71%; including 17 strains, 17 foods) based on inactivation by proteolytic enzymes, indicating presence of bacteriocins. The cell-free extracts from 13 samples (27%, eight strains, five foods) were determined to contain bacteriophages based on the observation of plaques in diluted extracts and/or resistance to proteolytic enzymes. These results indicate that the production of inhibitors by food microbiota may be an important challenge for the recovery of foodborne pathogens, particularly for Shigella sonnei. The performance of enrichment media for recovery of Shigella and STEC could be improved by mitigating the impact of inhibitors produced by food microbiota during the enrichment process.

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.

Draft Genome Sequences of Tetracycline-Resistant Shiga Toxin-Producing Escherichia coli Isolates from Food.

ABSTRACTShiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen transmitted from animal to humans through contaminated food. Here, we report the draft genome sequences of six STEC isolates (six serotypes) from food (cheese, coriander, and pea protein pellets) in different countries; these isolates were resistant to tetracycline, with MIC values ranging from <1.5 to 256 μg/mL.

Prevalence and Molecular Characterisation of Extended-Spectrum Beta-Lactamase-Producing Shiga Toxin-Producing Escherichia coli, from Cattle Farm to Aquatic Environments.

Extended-spectrum beta-lactamase (ESBL)-producing bacteria are a major problem for public health worldwide because of limited treatment options. Currently, only limited information is available on ESBL-producing Shiga toxin-producing Escherichia coli (STEC) in cattle farms and the surrounding aquatic environment. This study sought to track and characterise ESBL-producing STEC disseminating from a cattle farm into the water environment. Animal husbandry soil (HS), animal manure (AM), animal drinking water (ADW), and nearby river water (NRW) samples were collected from the cattle farm. Presumptive ESBL-producing STEC were isolated and identified using chromogenic media and mass spectrophotometry methods (MALDI-TOF-MS), respectively. The isolates were subjected to molecular analysis, and all confirmed ESBL-producing STEC isolates were serotyped for their O serogroups and assessed for antibiotic resistance genes (ARGs) and for the presence of selected virulence factors (VFs). A phylogenetic tree based on the multilocus sequences was constructed to determine the relatedness among isolates of ESBL-producing STEC. The highest prevalence of ESBL-producing STEC of 83.33% was observed in HS, followed by ADW with 75%, NRW with 68.75%, and the lowest was observed in AM with 64.58%. Out of 40 randomly selected isolates, 88% (n = 35) belonged to the serogroup O45 and 13% (n = 5) to the serogroup O145. The multilocus sequence typing (MLST) analysis revealed four different sequence types (STs), namely ST10, ST23, ST165, and ST117, and the predominant ST was found to be ST10. All 40 isolates carried sul1 (100%), while blaOXA, blaCTX-M, sul2, blaTEM, and qnrS genes were found in 98%, 93%, 90%, 83%, and 23% of the 40 isolates, respectively. For VFs, only stx2 was detected in ESBL-producing STEC isolates. The results of the present study indicated that a cattle environment is a potential reservoir of ESBL-producing STEC, which may disseminate into the aquatic environment through agricultural runoff, thus polluting water sources. Therefore, continual surveillance of ESBL-producing STEC non-O157 would be beneficial for controlling and preventing STEC-related illnesses originating from livestock environments.

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.

Presence and Virulence Characteristics of Shiga Toxin Escherichia coli and Non-Shiga Toxin-Producing Escherichia coli O157 in Products from Animal Protein Supply Chain Enterprises in South Africa.

Consumption of food that is contaminated with Shiga toxin–producing Escherichia coli (STEC) has been linked to serious foodborne disease outbreaks. Our aim was to provide a descriptive study on the presence and virulence factors of STEC and non-STEC O157 isolates recovered from 2017 diverse meat and meat product samples from all provinces of South Africa (n = 1758) and imported meat from South Africa's major ports of entry (n = 259). A cross-sectional study was undertaken to analyze raw intact meat, raw processed (nonintact) meat, and ready-to-eat (RTE) meat from cattle, game, sheep, pork, and poultry. Isolation was performed using International Organization for Standardization-based microbiological techniques, while detection and characterization were performed using real-time PCR (RT-PCR) and conventional PCR targeting the stx1, stx2, eae, and ehxA genes. A total of 28 of 1758 (1.59%; confidence interval [CI] 1.1–2) samples from the domestic market tested positive (n = 10 Escherichia coli O157:H7; n = 14 Escherichia coli O157: non-H7; and n = 4 non-O157 STEC), while 4/259 (1.54%; CI 0.4–4) samples from ports of entry tested positive for Escherichia coli O157:H7 based on RT-PCR. On average, diverse samples from domestic meat and meat products from cattle showed the highest number of positive samples (22/1758; 1.3%; CI 0.8–2). RT-PCR detected more positive samples (n = 32) compared with culture (n = 17). Sixteen different virulence factor combinations were observed. Our findings demonstrate a relatively low presence of diverse STEC strains along the meat value chain. To our knowledge, this is the first extensive report in South Africa to analyze STEC and non-STEC O157 from local and imported samples from many animal species. This is important as it reveals virulence factors in STEC strains circulating in meat and meat products in South Africa, which contribute to the risk of infection.

"FAMILY CLUSTER ASSOCIATED WITH SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O26 SEROGROUP"

"Introduction: Shiga toxin-producing Escherichia coli (STEC) can cause severe human infections especially in the pediatric population who are at risk of progressing to life threatening systemic complications such as hemolytic uremic syndrome (HUS). In Romania, enhanced monitoring of STEC-associated HUS was implemented in year 2016 after a cluster of infections caused by STEC O26:H11 serotype heightened public awareness of their consequences. Objectives: This study describes the microbiological investigation initiated to identify a STEC-associated HUS pediatric case and determine additional infections among his household contacts. Methods: Fecal specimens collected from a small child hospitalized with HUS and his asymptomatic parents and brother were investigated using a culture-based approach which targeted STEC isolation, identification, and typing. PCR-based assays for key virulence genes (i.e., stx1 and stx2 types/subtypes, eae, and ehxA) were combined with traditional methods such as serogroup determination by slide agglutination. Also, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for strain genotyping. Results: STEC was identified only in the stool samples of the siblings, one who developed HUS and the other who remained asymptomatic. Both strains belonged to O26 E. coli serogroup and displayed an identical virulence gene profile (presence of stx2a, eae, and ehxA genes). Molecular typing by PFGE showed the STEC strains as highly related genetically and MLST assigned them to sequence type ST21 lineage. Conclusion: A family cluster of O26 STEC infections was detected by using a culture- based molecular approach. Further analysis at whole genome sequencing resolution level is needed for a detailed characterization of such pathogens of significant public health importance. Keywords: HUS, Shiga toxin-producing Escherichia coli, virulence genes, PCR, molecular typing"

Assessment of the Microbiological Quality and Safety of Unpasteurized Milk Cheese for Sale in England between 2019 and 2020

Cheese made with unpasteurized milk has been associated with outbreaks of illness. However, there are limited data on the prevalence of Shiga toxin-producing Escherichia coli (STEC) in these products and a lack of clarity over the significance of E. coli as a general indicator of hygiene in raw milk cheeses. The aim of this study was to provide further data to address both of these issues, as well as assessing the overall microbiological quality of raw milk cheeses available to consumers in England. A total of 629 samples of cheese were collected from retailers, catering premises, and manufacturers throughout England. The majority (80%) were made using cow's milk, with 14% made from sheep's milk and 5% from goat's milk. Samples were from 18 different countries of origin, with the majority originating from either the United Kingdom (40%) or France (35%). When interpreted against European Union microbiological criteria and United Kingdom guidance, 82% were considered to be of satisfactory microbiological quality, 5% were borderline, and 12% were unsatisfactory. Four samples (0.6%) were potentially injurious to health due to the isolation of STEC from one, >104 CFU/g of coagulase-positive staphylococci in two, and >100 CFU/g of Listeria monocytogenes in the fourth sample. Indicator E. coli and Listeria species were detected more frequently in soft compared with hard cheese. Higher levels of indicator E. coli were significantly associated with a greater likelihood of detecting Shiga toxin genes (stx1 and/or stx2).

Prevalence, Antimicrobial Resistance, and Whole Genome Sequencing Analysis of Shiga Toxin-Producing Escherichia coli (STEC) and Enteropathogenic Escherichia coli (EPEC) from Imported Foods in China during 2015-2021.

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are foodborne pathogens that cause hemolytic uremic syndrome and fatal infant diarrhea, respectively, but the characterization of these bacteria from imported food in China are unknown. A total of 1577 food samples from various countries during 2015–2021 were screened for STEC and EPEC, and the obtained isolates were tested for antimicrobial resistance and whole genome sequencing analysis was performed. The prevalence of STEC and EPEC was 1.01% (16/1577) and 0.51% (8/1577), respectively. Antimicrobial resistances to tetracycline (8%), chloramphenicol (8%), ampicillin (4%), ceftazidime (4%), cefotaxime (4%), and trimethoprim-sulfamethoxazole (4%) were observed. The antimicrobial resistance phenotypes corresponded with genotypes for most strains, and some resistance genes were related to mobile genetic elements. All 16 STEC isolates were eae negative, two solely contained stx1 (stx1a or stx1c), 12 merely carried stx2 (stx2a, stx2d, or stx2e), and two had both stx1 and stx2 (stx1c + stx2b, stx1a + stx2a + stx2c). Although they were eae negative, several STEC isolates carried other adherence factors, such as iha (5/16), sab (1/16), and lpfA (8/16), and belonged to serotypes (O130:H11, O8:H19, and O100:H30) or STs (ST297, ST360), which have caused human infections. All the eight EPEC isolates were atypical EPEC; six serotypes and seven STs were found, and clinically relevant EPEC serotypes O26:H11, O103:H2, and O145:H28 were identified. Two STEC/ETEC (enterotoxigenic E. coli) hybrids and one EPEC/ETEC hybrid were observed, since they harbored sta1 and/or stb. The results revealed that food can act as a reservoir of STEC/EPEC with pathogenic potential, and had the potential ability to transfer antibiotic resistance and virulence genes.

Improved Genomic Identification, Clustering, and Serotyping of Shiga Toxin-Producing Escherichia coli Using Cluster/Serotype-Specific Gene Markers.

Shiga toxin-producing Escherichia coli (STEC) have more than 470 serotypes. The well-known STEC O157:H7 serotype is a leading cause of STEC infections in humans. However, the incidence of non-O157:H7 STEC serotypes associated with foodborne outbreaks and human infections has increased in recent years. Current detection and serotyping assays are focusing on O157 and top six (“Big six”) non-O157 STEC serogroups. In this study, we performed phylogenetic analysis of nearly 41,000 publicly available STEC genomes representing 460 different STEC serotypes and identified 19 major and 229 minor STEC clusters. STEC cluster-specific gene markers were then identified through comparative genomic analysis. We further identified serotype-specific gene markers for the top 10 most frequent non-O157:H7 STEC serotypes. The cluster or serotype specific gene markers had 99.54% accuracy and more than 97.25% specificity when tested using 38,534 STEC and 14,216 non-STEC E. coli genomes, respectively. In addition, we developed a freely available in silico serotyping pipeline named STECFinder that combined these robust gene markers with established E. coli serotype specific O and H antigen genes and stx genes for accurate identification, cluster determination and serotyping of STEC. STECFinder can assign 99.85% and 99.83% of 38,534 STEC isolates to STEC clusters using assembled genomes and Illumina reads respectively and can simultaneously predict stx subtypes and STEC serotypes. Using shotgun metagenomic sequencing reads of STEC spiked food samples from a published study, we demonstrated that STECFinder can detect the spiked STEC serotypes, accurately. The cluster/serotype-specific gene markers could also be adapted for culture independent typing, facilitating rapid STEC typing. STECFinder is available as an installable package (https://github.com/LanLab/STECFinder) and will be useful for in silico STEC cluster identification and serotyping using genome data.

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.