Presence Of Shiga Toxin-producing Escherichia Coli Research Articles

Real-time PCR primers and probes for the detection of Shiga toxin genes, including novel subtypes

Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes.A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence.The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.

Absence of Shiga toxin-producing Escherichia coli (STEC) in organic leafy greens from the metropolitan region of São Paulo, Brazil.

Shiga toxin-producing Escherichia coli (STEC) is an important pathogen with public health implications, including its potential association with vegetables. In this study, we investigated the presence of STEC in vegetables obtained from organic producers located in São Paulo city, Brazil. As part of a routine surveillance study conducted over (years of isolation), a total of 200 samples of organic vegetables were screened using biochemical and PCR methods. Among the vegetable samples tested, 30 (15%) were positive for non-Shiga toxin-producing E. coli. While no STEC was detected in the organic vegetables in this study, the presence of non-STEC in vegetables raises concerns about the lack of proper hygiene practices during vegetable handling. This contamination represents a public health risk, particularly considering that these isolates can still be pathogenic, and vegetables are often consumed raw. To address this important issue, continuous monitoring of these farms is recommended to ensure the quality and safety of organic vegetables produced for both domestic consumption and exportation.

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.

Metagenomic survey of agricultural water using long read sequencing: Considerations for a successful analysis

Leafy greens are responsible for nearly half of the produce-related Shiga toxin-producing Escherichia coli (STEC) outbreaks in the United States and recent investigations have implicated agricultural water as a potential source. Current FDA detection protocols require extensive analysis time. We aimed to use Oxford Nanopore rapid sequencing kits for an in-field determination of agricultural water microbiome and possible detection and characterization of STECs strain(s) in these samples. We tested the performance of the nanopore rapid sequencing kit (RAD004) for fast microbiome determination using the well characterized ZymoBIOMICS mock microbial community and the number of reads for each identified species was present in the expected proportion. Rapid sequencing kit (LRK001 and RAD004) library preparation of DNA extracted from agricultural water resulted in poor nanopore sequencing reactions, with low output (0.3–1.7 M reads), a high proportion of failed reads (50–60%), and highly sheared DNA before and after a magnetic bead clean up. To improve performance, we prepared a DNA library with the ligation kit (LSK109), which includes multiple cleaning steps, reducing inherent inhibitors and producing a better outcome (2.2 M reads, 15% failed reads). No definitive presence of STEC could be confirmed in any of the sites. Approximately 100 reads from each site (0.02% of total reads) were identified as Escherichia coli, but the specific strain or their virulence genes could not be detected. Sites 9, 10, and 12 were found to be positive for STEC presence by microbiological techniques after enrichment. The rapid sequencing kits can be appropriate for genus or species level microbial identification, but we recommend the use of the ligation kit for increased sequencing depth and removal of contaminants in agricultural water. However, we were not able to identify any STEC strains in these nanopore microbiome samples, due to low initial concentrations. The results from this pilot study provide preliminary evidence that MinION sequencing of agricultural water using the ligation kit has the potential to be used for rapid microbiome determination in the field with optimal results for water quality surveillance.

Detection of Shiga Toxin-Producing Escherichia coli (STEC) in the Endocervix of Asymptomatic Pregnant Women. Can STEC Be a Risk Factor for Adverse Pregnancy Outcomes?

The presence of Escherichia coli in the vaginal microbiome has been associated with pregnancy complications. In previous works, we demonstrated that Shiga toxin-producing Escherichia coli (STEC) can produce abortion and premature delivery in rats and that Shiga toxin type 2 (Stx2) can impair human trophoblast cell lines. The hypothesis of this work was that STEC may colonize the lower female reproductive tract and be responsible for adverse pregnancy outcomes. Thus, the aim of this work was to evaluate the presence and prevalence of virulence factor genes from STEC in the endocervix of asymptomatic pregnant women. For that purpose, endocervical swabs were collected from pregnant women during their prenatal examination. Swab samples were enriched in a differential medium to select Enterobacteria. Then, positive samples were analyzed by PCR to detect genes characteristic of Escherichia sp. (such as uidA and yaiO), genes specific for portions of the rfb (O-antigen-encoding) regions of STEC O157 (rfb O157), and STEC virulence factor genes (such as stx1, stx2, eae, lpfA O113, hcpA, iha, sab, subAB). The cytotoxic effects of stx2-positive supernatants from E. coli recovered from the endocervix were evaluated in Vero cells. Our results showed that 11.7% of the endocervical samples were positive for E. coli. Additionally, we found samples positive for stx2 and other virulence factors for STEC. The bacterial supernatant from an isolate identified as E. coli O113:NT, carrying the stx2 gene, exhibited cytotoxic activity in Vero, Swan 71 and Hela cells. Our results open a new perspective regarding the presence of STEC during pregnancy.

Risk factors of Shiga toxin-producing Escherichia coli in livestock raised on diversified small-scale farms in California.

The increasing number of diversified small-scale farms (DSSF) that raise outdoor-based livestock in the USA reflects growing consumer demand for sustainably produced food. Diversified farms are small scale and raise a combination of multiple livestock species and numerous produce varieties. This 2015-2016 cross-sectional study aimed to describe the unique characteristics of DSSF in California, estimate the prevalence of Shiga toxin-producing Escherichia coli (STEC) in livestock and evaluate the association between risk factors and the presence of STEC in livestock, using generalised linear mixed models. STEC prevalence was 13.62% (76/558). Significant variables in the mixed-effect logistic regression model included daily maximum temperature (OR 0.95; CI95% 0.91-0.98), livestock sample source (cattle (OR 4.61; CI95% 1.64-12.96) and sheep (OR 5.29; CI95% 1.80-15.51)), multiple species sharing the same barn (OR 6.23; CI95% 1.84-21.15) and livestock having contact with wild areas (OR 3.63; CI95% 1.37-9.62). Identification of STEC serogroups of public health concern (e.g. O157:H7, O26, O103) in this study indicated the need for mitigation strategies to ensure food safety by evaluating risk factors and management practices that contribute to the spread and prevalence of foodborne pathogens in a pre-harvest environment on DSSF.

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

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

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

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

A cluster of Shiga Toxin-producing Escherichia coli O157:H7 highlights raw pet food as an emerging potential source of infection in humans.

In August 2017, a cluster of four persons infected with genetically related strains of Shiga toxin-producing Escherichia coli (STEC) O157:H7 was identified. These strains possessed the Shiga toxin (stx) subtype stx2a, a toxin type known to be associated with severe clinical outcome. One person died after developing haemolytic uraemic syndrome. Interviews with cases revealed that three of the cases had been exposed to dogs fed on a raw meat-based diet (RMBD), specifically tripe. In two cases, the tripe had been purchased from the same supplier. Sampling and microbiological screening of raw pet food was undertaken and indicated the presence of STEC in the products. STEC was isolated from one sample of raw tripe but was different from the strain causing illness in humans. Nevertheless, the detection of STEC in the tripe provided evidence that raw pet food was a potential source of human STEC infection during this outbreak. This adds to the evidence of raw pet food as a risk factor for zoonotic transmission of gastrointestinal pathogens, which is widely accepted for Salmonella, Listeria and Campylobacter spp. Feeding RMBD to companion animals has recently increased in popularity due to the belief that they provide health benefits to animals. Although still rare, an increase in STEC cases reporting exposure to RMBDs was detected in 2017. There has also been an increased frequency of raw pet food incidents in 2017, suggesting an increasing trend in potential risk to humans from raw pet food. Recommendations to reduce the risk of infection included improved awareness of risk and promotion of good hygiene practices among the public when handling raw pet food.

Animal petting zoos as sources of Shiga toxin-producing Escherichia coli, Salmonella and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae.

Animal petting zoos and farm fairs provide the opportunity for children and adults to interact with animals, but contact with animals carries a risk of exposure to zoonotic pathogens and antimicrobial-resistant bacteria. The aim of this study was to assess the occurrence of Shiga toxin-producing Escherichia coli (STEC), Salmonella, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA) in animal faeces from six animal petting zoos and one farm fair in Switzerland. Furthermore, hygiene facilities on the venues were evaluated. Of 163 faecal samples, 75 contained stx1, stx2 or stx1/stx2 genes, indicating the presence of STEC. Samples included faeces from sika deer (100%), sheep (92%), goats (88%), mouflons (80%), camels (62%), llamas (50%), yaks (50%), pigs (29%) and donkeys (6%), whereas no stx genes were isolated from faeces of calves, guinea pigs, hens, ostriches, ponies, zebras or zebus. Salmonella enterica subsp. enterica serovar Stourbridge (S. Stourbridge) was detected in faecal samples from camels. A total of four ESBL-producing E.coli strains were isolated from faeces of goats, camels and pigs. PCR and sequencing identified the presence of blaCTX-M-15 in three and blaCTX-M-65 in one E.coli. Antimicrobial resistance profiling using the disk diffusion method revealed two multidrug-resistant (MDR) E.coli with resistance to ciprofloxacin, gentamicin and azithromycin, all of which are critically important drugs for human medicine. Multilocus sequence typing identified E.coli ST162, E.coli ST2179, extraintestinal high-risk E.coli ST410 and E.coli ST4553, which belongs to the emerging extraintestinal clonal complex (CC) 648. No MRSA was detected. On all animal petting venues, there were inadequacies with regard to access to hygiene information and handwashing hygiene facilities. This study provides data that underscore the importance of hygiene measures to minimize the risk of transmission of zoonotic pathogens and MDR, ESBL-producing E.coli to visitors of animal petting venues.

Investigation of Shiga Toxin-Producing Escherichia coli in Groundwater, River Water, and Fecal Sources in the Kathmandu Valley, Nepal

Shiga toxin-producing Escherichia coli (STEC) is one of the most commonly heard sources of foodborne outbreaks but presently lacks studies of in the Kathmandu Valley. This study explored the presence of STEC in river water (n = 17), groundwater (n = 83), feces (n = 50), and manure (n = 20) in the Kathmandu Valley, Nepal. Samples that were confirmed to be E. coli–positive using the Colilert assay in previous studies were selected to examine the presence of the sfmD, stx1, and stx2 genes using quantitative polymerase chain reaction. Based on Colilert/sfmD gene ratios, the percentage of viable cells of E. coli in the fecal (2.0% ± 2.5%), manure (0.1% ± 0.1%), river water (3.4% ± 3.1%), and groundwater samples (3.9% ± 5.8%) were determined. The presence of the stx1 gene was observed in the fecal (6%), river water (53%), and groundwater (2%) samples, whereas the stx2 gene was detected in the fecal (8%), manure (5%), and river water (71%) samples. Interestingly, the stx/sfmD gene ratios in the groundwater samples were greater than 100%, suggesting the presence of other stx1/stx2-harboring microorganisms. These findings indicated the importance of continuing investigation into waterborne sources of STEC contamination within the Kathmandu Valley.

Analysis of Bacterial Diversity in Relation to the Presence of the Top 7 Shiga Toxin–Producing Escherichia coli throughout Australian Beef Abattoirs

There is increasing evidence that diversity changes in bacterial communities of beef cattle correlate to the presence of Shiga toxin-producing Escherichia coli (STEC). However, studies that found an association between STEC and bacterial diversity have been focused on preslaughter stages in the beef supply chain. This study was designed to test a hypothesis that there are no differences in bacterial diversity between samples with and those without the presence of the top 7 STEC (O26, O45, O103, O111, O121, O145, and O157) throughout processing in an integrated (abattoir A) and a fragmented (abattoir B) Australian beef abattoir. Slaughter and boning room surface samples from each abattoir were analyzed using 16S rRNA amplicon sequencing and tested for the top 7 STEC following the Food Safety and Inspection Service protocol. Potential positives through slaughter were similar between the abattoirs (64 to 81%). However, abattoir B had substantially reduced potential positives in the boning room compared with abattoir A (abattoir A: 23 and 48%; abattoir B: 2 and 7%). Alpha diversity between the sample groups was not significantly different (P > 0.05) regardless of different STEC markers. Nonmetric multidimensional scaling of slaughter samples showed that the bacterial composition in fecal and hide samples shared the least similarity with the communities in carcass and environmental samples. Surface samples from slaughter (carcass and environmental) and boning (carcass, beef trim, and environmental) all appeared randomly plotted on the scale. This indicated that the STEC presence also did not have a significant effect (P > 0.05) on beta diversity. Although presence of STEC appeared to correlate with changes in diversity of fecal and hide bacterial communities in previous studies, it did not appear to have the same effect on other samples throughout processing.

Bacteriophages specific to Shiga toxin-producing Escherichia coli exist in goat feces and associated environments on an organic produce farm in Northern California, USA.

Shiga toxin-producing Escherichia coli (STECs) contamination of produce, as a result of contact with ruminant fecal material, has been associated with serious foodborne illness. Bacteriophages (phages) that infect STECs have primarily been reported to be of cattle origin. However, they likely exist in other environments or in animals that share habitats with cattle, such as goats. To explore the presence and diversity of phages specific to STEC O157 and the top six non-O157 STECs in goat-associated environments, environmental samples consisting of feces (goat and cattle) and soil samples were collected monthly for six months from an organic produce farm. A variety of phages belonging to the Myoviridae, Siphoviridae, and Podoviridae families were isolated from all goat fecal and half of the soil samples. The most commonly isolated phages belonged to Myoviridae and were lytic against STEC O103. The isolated phages had different host ranges, but collectively, showed lytic activity against O157 and the top six non-O157 STEC strains excluding O121. Two non-O157 STECs (O174: H21 and O-antigen-negative: H18) were isolated from soil and cattle feces, respectively. Although prior studies have reported that goats shed STEC into the environment, the findings of the current study suggest that goat feces may also contain lytic STEC-specific phages. The phages of goat origin have the capacity to infect STECs implicated in causing foodborne outbreaks, making them potential candidates for biocontrol pending additional characterization steps. Further work is needed to determine if the addition of goats to the farm environment could potentially reduce the presence of STECs.

Virulence Factors and Antimicrobial Resistance Patterns of Non-O157 Shiga Toxin-producing Escherichia coli Isolated from Different Sources at Sadat City

Aims: A great concern directed to non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes due to their public health importance. Detecting the existence, antimicrobial profiles, and virulence repertoire of different STEC serotypes from animals essential for human food are important.
 Study Design: This study aimed to investigate the presence of STEC in different hosts, the distribution pattern of stx1, stx2, eaeA, and hlyA genes encoding Shiga toxins 1 and 2, intimin, and enterohemolysin, respectively, and the antimicrobial resistance of the detected serotypes.
 Results: A total of 75 samples were collected, 20 fecal samples from broilers, 15 fecal samples from ducks, 20 beef samples, and 20 human urine samples. Escherichia coli was detected at a rate of 60/75 (80%) distributed as;17 (85%), 8 (53.3%), 15(75%), and 20 (100%) from broilers, ducks, human urine, and beef samples, respectively. There was a significant difference between the isolation rates of E. coli from different sources with p<0.05. The prevalent serotypes were O78, O2:H6, O15:H2, and O26: H1. The frequency of stx1 gene was 56/60 (93.3%), stx2 gene was 55/60 (91.6%), the eaeA gene was 35/60 (58.3%), and hlyA gene was 26/60 (43.3%). The most effective antimicrobials were amoxicillin/clavulanic acid and ampicillin with the efficacy of 52/60 (86.6%) for each, while the efficacy of chloramphenicol and doxycycline was 5/60 (8.3%) and 44/60 (73.3%), respectively.
 Conclusion: The high frequency of non-O157 Shiga toxin-Producing Escherichia coli from different samples at Sadat City, high prevalence of virulence factors and resistance to chloramphenicol and doxycycline will help in monitoring the distribution of virulent serotypes and contribute to the establishment of control measures to reduce the spread of infection.

Diagnostic Test Accuracy of Commercial Tests for Detection of Shiga Toxin-Producing Escherichia coli: A Systematic Review and Meta-Analysis.

Rapid detection of Shiga toxin-producing Escherichia coli (STEC) enables appropriate monitoring and treatment. We synthesized available evidence to compare the performance of enzyme immunoassay (EIA) and PCR tests for the detection of STEC. We searched published and gray literature for studies of STEC EIA and/or PCR diagnostic test accuracy relative to reference standards including at least one nucleic acid amplification test. Two reviewers independently screened studies, extracted data, and assessed quality with the second version of the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Bivariate random effects models were used to meta-analyze the clinical sensitivity and specificity of commercial EIA and PCR STEC diagnostic tests, and summary receiver operator characteristic curves were constructed. We evaluated the certainty of evidence with the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. We identified 43 articles reflecting 25 260 specimens. Meta-analysis of EIA and PCR accuracy included 25 and 22 articles, respectively. STEC EIA pooled sensitivity and specificity were 0.681 (95% CI, 0.571-0.773; very low certainty of evidence) and 1.00 (95% CI, 0.998-1.00; moderate certainty of evidence), respectively. STEC PCR pooled sensitivity and specificity were 1.00 (95% CI, 0.904-1.00; low certainty of evidence) and 0.999 (95% CI, 0.997-0.999; low certainty of evidence), respectively. Certainty of evidence was downgraded because of high risk of bias. PCR tests to identify the presence of STEC are more sensitive than EIA tests, with no meaningful loss of specificity. However, given the low certainty of evidence, our results may overestimate the difference in performance.

Shiga toxin-producing Escherichia coli survives storage in wheat flour for two years

Wheat flour has recently been recognised as an exposure vehicle for the foodborne pathogen Shiga toxin-producing Escherichia coli (STEC). Wheat flour milled on two sequential production days in October 2016, and implicated in a Canada wide outbreak of STEC O121:H19, was analysed for the presence of STEC in November 2018. Stored in sealed containers at ambient temperature, the water activity of individual flour samples was below 0.5 at 6 months post-milling and remained static or decreased slightly in individual samples during 18 months of additional storage. STEC O121 was isolated, with the same genotype (stx2a, eae, hlyA) and core genome multilocus sequence type as previous flour and clinical isolates associated with the outbreak. The result of this analysis demonstrates the potential for STEC to persist in wheat flour at levels associated with outbreak infections for periods of up to two years. This has implications for the potential for STEC to survive in other foods with low water activity.

Detection and isolation of Shiga Toxin-producing Escherichia coli (STEC) strains in caecal samples from pigs at slaughter in Italy.

Shiga toxin‐producing Escherichia coli (STEC) strains are food‐borne pathogens of public health concern. Despite ruminants are the most important reservoir, STEC human infections have also been attributed to pigs. We examined for the presence of STEC in 234 samples of swine caecal content collected during the year 2015 at Italian abattoirs in the framework of the harmonized monitoring of antimicrobial resistance (Decision 2013/652/EU). The presence of stx genes was detected in 122 (52.1%) samples, which were subsequently subjected to STEC isolation and characterization. The analysis of the 66 isolated STEC strains showed that the majority of the isolates (74.2%) possessed the stx2a gene subtype, in a few cases (16.7%) in combination with stx2b or stx2c. Only 25.8% of isolates possessed the stx2e subtype, typical of swine‐adapted STEC. None of the isolates possessed the intimin‐coding eae gene and the majority of them did not belong to serogroups commonly associated with human infections. The results of this study suggest that pigs can be considered as potential reservoir of certain STEC types.

Occurrence and antimicrobial resistance of E. coli non-O157 isolated from beef in Mato Grosso, Brazil.

Shiga toxin-producing Escherichia coli (STEC) is an important public health concern pathogen, as it produces two toxins, Stx1 and Stx2, with cytotoxic capacity. In addition, STEC strains are frequently involved in food outbreaks worldwide, leading to public health challenges and economic losses. In this context, the occurrence and antimicrobial resistance profile of the STEC isolated from fresh beef produced in Mato Grosso, Brazil, were estimated. One hundred seven retail beef under vacuum-packaged produced by 13 different slaughterhouses were submitted to microbiological, molecular, and antimicrobial resistance analyses. STEC occurrence in beef was of 4.67%, and five strains presented the stx2 gene. The O111 serogroup, reported in several outbreak cases worldwide, was detected, and other serotypes (O8:H20, O22:H16, and O141:H49) were also isolated. All isolated strains displayed sensitivity to 12 antibiotics, except for two strains, which where streptomycin-resistant. The presence of STEC in retail beef samples indicates public health risks with significant economic impact throughout the retail beef chain.

Prevalence of Salmonella, Campylobacter, and Shiga Toxin–Producing Escherichia coli on the Surfaces of Raw Poultry Packages

Contamination on the exterior surfaces of raw poultry packages can be transmitted to hands and food contact surfaces during shopping and handling. This study compared the level of microbial contamination and prevalence of foodborne pathogens on the surfaces of raw poultry packages as related to the types of products, types of packaging, and packaging conditions. Packages of whole chicken, cut-up chicken (breast and leg quarter), and ground turkey were purchased from retail stores. Aerobic plate counts (APCs) were significantly different ( P < 0.05) among types of products and packaging materials, with ground turkey packages and the heat-sealed, high-walled containers being the lowest. APCs were significantly lower ( P < 0.05) when the packages were intact and tight compared with intact and loose. Of the 105 packages, there were 10 (9.5%) with the presence of either Shiga toxin-producing Escherichia coli (STEC) or Campylobacter; of those packages, 6 (5.7%) were positive for STEC, 7 (6.7%) were positive for Campylobacter, and 3 (2.9%) were positive for both pathogens on the surfaces. Salmonella was not detected on the surfaces of all tested packages. Surfaces of whole chicken packages were significantly ( P < 0.001) more likely to have detectable levels of Campylobacter and STEC than those of cut-up chicken packages. Packages that were positive for Campylobacter and/or STEC had significantly ( P < 0.005) higher APCs than negative packages. The results suggested that STEC is another significant pathogen present on the surfaces of poultry packages in addition to Campylobacter. The presence of STEC on the external packaging of raw poultry raises a concern because consumers may not expect such pathogens on the surfaces of poultry packages.

Occurrence of Escherichia coli carrying Shiga toxin-producing genes in buffaloes on smallholdings in Bangladesh.

Background and Aim:Shiga toxin-producing Escherichia coli (STEC) has emerged as significant foodborne pathogens. Ruminants are the primary reservoir of the zoonotic STEC. In Bangladesh, previous studies reported the presence of STEC in cattle, goat, and sheep; however, there is little information about STEC carriage by buffaloes. This study aimed to determine the occurrence of STEC in healthy (absence of clinical signs and symptoms) buffaloes on smallholdings in Bangladesh and to assess the antimicrobial resistance pattern of identified STEC isolates.Materials and Methods:A total of 100 rectal swab samples were obtained from randomly selected buffaloes on 40 smallholdings in Chittagong Division, Bangladesh. Samples were subjected to bacteriological screening to identify E. coli. All E. coli isolates were examined for the presence of the Shiga toxin-producing genes - Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2) using polymerase chain reaction. The antimicrobial susceptibility of identified STEC isolates was tested using the disk diffusion method.Results:Results show that 71 fecal samples were positive for E. coli in bacteriological screening. The proportion of buffaloes harboring STEC isolates was 11% (11/100) (95% confidence interval [CI] 6.1-18.8], of which 7% (7/100) (95% CI 3.2-13.9) and 4% (4/100) (95% CI 1.2-10.2) carried stx1 and stx2 genes, respectively. Antibiogram revealed that 91% (10/11), 73% (8/11), 55% (6/11), and 55% (6/11) STEC isolates were resistant to tetracycline, sulfamethoxazole-trimethoprim, erythromycin, and ampicillin, respectively. In contrast, 91% (10/11) STEC isolates were sensitive to ciprofloxacin, chloramphenicol, and gentamicin, whereas 73% (8/11) isolates were sensitive to ceftriaxone.Conclusion:This study highlights, for the first time, a significant proportion of fecal samples from healthy buffaloes on smallholdings in Bangladesh harboring antimicrobial-resistant STEC. Transmission of antimicrobial-resistant STEC from buffaloes to humans could pose an added risk to public health in rural Bangladesh.