non-O157 Shiga Toxin-producing Escherichia Coli Serotypes Research Articles

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.

CPHLN recommendations for the laboratory detection of Shiga toxin-producing Escherichia coli (O157 and non-O157).

Shiga toxin-producing Escherichia coli (STEC) are important enteric pathogens responsible for sporadic cases and outbreaks of gastroenteritis. E.coli O157:H7/NM (STEC O157) are the most commonly known STEC serotypes but it is now increasingly apparent that non-O157 STEC serotypes have been underreported in the past because they were not part of routine screening in many front-line laboratories. The Canadian Public Health Laboratory Network (CPHLN) has identified the need for improved detection and surveillance of non-O157 STEC and has developed the following recommendations to assist in the decision-making process for clinical and reference microbiology laboratories. These recommendations should be followed to the best of a laboratory's abilities based on the availability of technology and resources. The CPHLN recommends that when screening for the agents of bacterial gastroenteritis from a stool sample, front-line laboratories use either a chromogenic agar culture or a culture-independent diagnostic test (CIDT). CIDT options include nucleic acid amplification tests (NAATs) to detect Shiga toxin genes or enzyme immunoassays (EIAs) to detect Shiga toxins. If either CIDT method is positive for possible STEC, laboratories must have a mechanism to culture and isolate STEC in order to support both provincial and national surveillance as well as outbreak investigations and response. These CPHLN recommendations should result in improved detection of STEC in patients presenting with diarrhea, especially when due to the non-O157 serotypes. These measures should enhance the overall quality of healthcare and food safety, and provide better protection of the public via improved surveillance and outbreak detection and response.

Persistence of Non-O157 Shiga Toxin–Producing Escherichia coli in Dairy Compost during Storage

Dairy compost with 20, 30, or 40% moisture content (MC) was inoculated with a mixture of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serovars at a final concentration of 5.1 log CFU/g and then stored at 22 and 4°C for 125 days. Six storage conditions-4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC-were investigated for the persistence of non-O157 STEC in the dairy compost. During the entire storage, fluctuations in indigenous mesophilic bacterial levels were observed within the first 28 days of storage. After inoculation, the non-O157 STEC population increased 0.69 and 0.79 log CFU/g in the dairy compost with 30 and 40% MC at 22°C within the first day, respectively; for all other storage conditions, the pathogen population decreased rapidly. After the 125-day storage, the reductions of non-O157 STEC for 4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC storage conditions were >4.52, >4.55, 3.89, >4.61, 3.60, and 3.17 log CFU/g, respectively. All the survival curves showed an extensive tail, indicating non-O157 STEC can survive at least for 125 days in the dairy compost. The survival data were analyzed with log-linear with tailing and Weibull models. Compared with the log-linear with tailing model, the Weibull model was found to be a better choice for predicting the survival of non-O157 STEC in dairy compost owing to a high overall R2 value (0.8738 to 0.9909). The decay rate of non-O157 STEC was higher in dairy compost stored at 4°C compared with at 22°C, and the same trend was found for the compost with 40% MC versus 20% MC. In addition, two non-O157 STEC serotypes (STEC O145 and O45) were detected on the last day of the longitudinal study and may deserve special attention in the Big 6 STEC group. Our results have provided scientific data for risk assessment of the microbiological safety of dairy compost to control non-O157 STEC during subsequent storage of dairy compost.

Occurrence of virulent and antibiotic-resistant Shiga toxin-producing Escherichia coli in some food products and human stool in Egypt.

Aim:Shiga toxin-producing Escherichia coli (STEC) represent a severe public health issue worldwide, causing life-threatening diseases in the human gastrointestinal tract. This study aimed to determine the occurrence of virulent and antibiotic-resistant STEC in retail meat and milk products and human stool samples and to characterize the genes encoding for virulence and antibiotic resistance among the identified STEC isolates.Materials and Methods:A total of 260 food samples were randomly collected from retail markets in different localities of El Giza Governorate, Egypt. 50 stool specimens were obtained from children that had diarrhea at Embaba Fever Hospital. All collected samples were initially subjected to bacteriological examination and serotyping, and then subsequently, the isolates were exposed to polymerase chain reaction application and sequencing for the identification of the virulence-related genes. Finally, the virulent STEC isolates were tested for antibiotic susceptibility.Results:Serotyping of the 76 biochemically identified isolates showed that 18 were STEC with a predominance of non-O157 (16) while 2 O157:K-serotype was detected only in one food and one human isolate. Molecular identification of the virulence genes illustrated that the minced meat showed the highest prevalence of STEC (8%) as compared to the other food products. In the humans, the O157 was the only serotype that expresses the Shiga toxin-associated gene (eaeA). Antibiotic susceptibility test displayed that 13 of the 17 food and human isolates (76.47%) were resistant to cephalothin (KF30). 9 of the 13 cephalothin-resistant isolates harbor the β lactamase (blaTEM)-resistant gene. All isolates were sensitive to chloramphenicol, ciprofloxacin, amikacin, and gentamicin. DNA sequencing and phylogenetic analysis of the stx2-positive minced meat isolate revealed a high genetic relatedness with beef minced meat from the USA and Australia.Conclusion:This study showed the predominance of non-O157 among the identified isolates. Minced meat showed the highest prevalence of STEC as compared to the other food products, and this work illustrates the necessity to consider the food products as a potential source of the non-O157 STEC serotypes. DNA sequencing and phylogenetic analysis revealed a high genetic relatedness with beef minced meat from the USA and Australia. This highlights the high probability of worldwide spread of such serotypes, signifying the importance of the one world concept.

Persistence and reduction of Shiga toxin-producing Escherichia coli serotype O26:H11 in different types of raw fermented sausages

Fermented sausages have been identified as source of several outbreaks of Shiga toxin-producing Escherichia coli (STEC). Illnesses linked to non-O157 STEC serotypes appear to be on the rise worldwide, and serogroup O26 is the second most reported in Europe after O157. However, data on the behavior of serogroup O26 in food are rare, so that the aim of this study was to investigate the survival of STEC O26:H11 in different types of fermented sausages (“Teewurst”, fast-ripened and long-fermented salami). Challenge studies were performed with an inoculation cocktail which consisted of three STEC O26:H11 strains isolated from human, cattle and food sources. In the short-ripened spreadable sausage type “Teewurst” STEC counts decreased by only 0.5 log10 within 28days. In contrast, STEC reductions from 2.2 to 2.6 log10 units were observed in the different salami products, while the most pronounced decrease of 1.0 log10 unit within one day was detected in fast-ripened sausages with glucono delta-lactone (GdL). Moreover, numbers of the food-associated E. coli O26:H11 strain were significantly higher (p<0.001) than those of the human and cattle STEC O26:H11 strains in all types of fermented sausages. Approximately 60% of all STEC isolates from GdL salami shared the genotypic virulence profile of the food-associated E. coli O26:H11 strain. In summary, hurdles of acidification and drying during salami ripening resulted in reductions of STEC O26:H11 counts. However, our results also indicate that STEC O26:H11 can persist in the environment of “Teewurst” and might therefore pose a risk to public health.

Isolation and characterization of non-O157 Shiga toxin-producing Escherichia coli from beef carcasses, cuts and trimmings of abattoirs in Argentina.

Several foods contaminated with Shiga toxin-producing Escherichia coli (STEC) are associated with human diseases. Some countries have established microbiological criteria for non-O157 STEC, thus, the absence of serogroups O26, O45, O103, O104, O111, O121, and O145 in sprouts from the European Union or ground beef and beef trimmings from the United States is mandatory. While in Argentina screening for O26, O103, O111, O145 and O121 in ground beef, ready-to-eat food, sausages and vegetables is mandatory, other countries have zero-tolerance for all STEC in chilled beef. The aim of this study was to provide data on the prevalence of non-O157 STEC isolated from beef processed in eight Argentinean cattle slaughterhouses producing beef for export and local markets, and to know the non-O157 STEC profiles through strain characterization and genotypic analysis. Samples (n = 15,965) from 3,205 beef carcasses, 9,570 cuts and 3,190 trimmings collected between March and September 2014 were processed in pools of five samples each. Pools of samples (n = 3,193) from 641 carcasses, 1,914 cuts and 638 trimming were analyzed for non-O157 STEC isolation according to ISO/CEN 13136:2012. Of these, 37 pools of carcasses (5.8%), 111 pools of cuts (5.8%) and 45 pools of trimmings (7.0%) were positive for non-O157 STEC. STEC strains (n = 200) were isolated from 193 pools of samples. The most prevalent serotypes were O174:H21, O185:H7, O8:H19, O178:H19 and O130:H11, and the most prevalent genotypes were stx2c(vh-b) and stx2a/saa/ehxA. O103:H21 strain was eae-positive and one O178:H19 strain was aggR/aaiC-positive. The prevalence of non-O157 STEC in beef carcasses reported here was low. None of the non-O157 STEC strains isolated corresponded to the non-O157 STEC serotypes and virulence profiles isolated from human cases in Argentina in the same study period. The application of microbiological criteria for each foodstuff should be determined by risk analysis in order to have a stringent monitoring system. Likewise, zero-tolerance intervention measures should be applied in beef, together with GMP and HACCP. Further, collaborative efforts for risk assessment, management and communication are extremely important to improve the safety of foodstuffs.

Characterization and Molecular Subtyping of Shiga Toxin-Producing Escherichia coli Strains in Butcher Shops.

Shiga toxin-producing Escherichia coli (STEC) are important emerging foodborne human pathogens. Ruminants are the main animal reservoir of STEC currently known, and meat can become contaminated at different stages of the production chain. The aim of this work was to subtype and establish the epidemiological relatedness of non-O157 STEC strains isolated from ground beef and the environment in butcher shops before (evaluation stage, 2010-2011 period) and after (verification stage, 2013) implementing improvement actions. Sixty-eight non-O157 STEC strains were tested for eae, saa, ehxA, iha, efa1, toxB, subAB, cdt-V, astA, aggR, and aaiC genes, and stx1 and stx2 variants were determined. Pulsed-field gel electrophoresis (PFGE) was carried out with XbaI and XmaJI. From the 68 strains, 92.6%, 75.0%, 58.8%, 53.5%, 10.3%, 7.3%, and 4.4% were positive for iha, ehxA, subAB, saa, cdt-V, astA, and eae, respectively. All strains were aggR/aaiC-negative. PFGE showed that 19 strains grouped in 9 clusters and 41 showed unique XbaI patterns. During the evaluation stage (2010-2011), we identified clonal strains in different samples, circulating clones in different butcher shops, and more than one different strain in the same butcher shop. The bovine origin of meat and its manufacturing process could not ensure the total absence of all non-O157 STEC serotypes in this foodstuff. Most strains isolated during the evaluation (2010-2011) and verification (2013) stages did not exhibit a genotypic profile associated with human disease. It is necessary to conduct periodic reviews of the new epidemiological information and verify that the analyses of non-O157 STEC in food are appropriate to identify strains affecting the population.

Improving the Enrichment and Plating Methods for Rapid Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in Dairy Compost

A culture method to detect non-O157 Shiga toxin-producing Escherichia coli (STEC) was optimized in this study. The finished dairy compost with 30% moisture content was inoculated with a cocktail of six non-O157 STEC serovars at initial concentrations of 1 to 100 CFU/g. Afterward, non-O157 STEC cells in the inoculated dairy compost were enriched by four methods, followed by plating onto cefixime-tellurite sorbitol MacConkey agar supplemented with 5 mg/liter novobiocin (CTNSMAC) and modified Rainbow agar containing 5 mg/liter novobiocin, 0.05 mg/liter cefixime trihydrate, and 0.15 mg/liter potassium tellurite (mRBA). Immunomagnetic bead separation (IMS) was used to compare the cell concentration of individual non-O157 STEC serotypes after enrichment. There was no significant difference (P > 0.05) between CTN-SMAC and mRBA for non-O157 STEC enumeration. The single-step selective enrichment recovered ca. 0.54 log CFU/g more cells (ca. 0.41 log CFU/g for compost-adapted cells) (P < 0.05) compared with the two-step enrichment. Furthermore, the duration of the process to detect non-O157 STEC from dairy compost by selective enrichment, followed by IMS, was optimized. Among six non-O157 STEC serotypes, serotypes O111, O45, and O145 reached the highest cell density after enrichment in dairy compost, and the cell populations reached 7.3, 7.4, and 7.8 log CFU/g within 16 h of incubation, respectively. In contrast, without an enrichment step, the IMS detection limit of individual non-O157 STEC serovars ranged from 3.15 to 4.15 log CFU/g in dairy compost. These results demonstrate that low levels of non-O157 STEC can be detected within 2 days from dairy compost by using a culture method with an optimized enrichment procedure followed by IMS.

Enteric Human Pathogens of Wild Boar, Feral Swine, and Javelina (Order: Artiodactyla)

Artiodactyls are even-toed ungulates that include domestic and wild ruminant herbivores (cattle, sheep, goat, deer, elk), pigs, and javelinas. There is increasing evidence that some wild ungulate species are important reservoirs of enteric human pathogens, and they may contribute to foodborne disease transmission directly through ingestion of undercooked game meat or indirectly via fecal contamination of fresh fruit and vegetable crops or agricultural water sources. To better understand zoonotic risks from feral swine and javelina in the U.S. southwestern desert, we conducted a prevalence survey of Shiga toxin-producing Escherichia coli (STEC) and Salmonella in colonic-fecal samples from animals collected through hunter-harvest or depredation. STEC O157:H7 was detected in feral swine, but not in javelina samples. Salmonella and non-O157 STEC serotypes of potential public health importance were found in both species. The findings underscore the importance of continued public health education efforts to protect hunters from exposure to these pathogens when handling game animals. Fresh fruit and vegetable growers should continue to prevent intrusions by wild and feral animals in the produce production environment to minimize transport of fecal-borne pathogens to crops intended for human consumption.

A Polyclonal Antibody Based Immunoassay Detects Seven Subtypes of Shiga Toxin 2 Produced by Escherichia coli in Human and Environmental Samples

BackgroundShiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains.Methods and FindingsHere we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity.ConclusionsThe sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

Characterization of Shiga toxin-producing Escherichia coli isolated from dairy cows in Argentina

To feno-genotypically characterize the Shiga toxin-producing Escherichia coli (STEC) population in Argentinean dairy cows. From 540 STEC positive samples, 170 isolates were analyzed by multiplex PCR and serotyping. Of these, 11% carried stx1, 52% stx2 and 37% stx1/stx2. The ehxA, saa and eae were detected in 77%, 66% and 3%, respectively. Thirty-five per cent of strains harboured the profile stx1, stx2, saa, ehxA and 29% stx2, saa, ehxA. One hundred and fifty-six strains were associated with 29 different O serogroups, and 19 H antigens were distributed among 157 strains. STEC O113:H21, O130:H11 and O178:H19 were the most frequently found serotypes. The STEC O157:H7 were detected in low rate and corresponded to the stx2(+) , eae(+) , ehxA(+) virulence pattern. We detected a diversity of STEC strains in dairy cattle from Argentina, most of them carrying genes linked to human disease. The non-O157 STEC serotypes described in this study are associated worldwide with disease in humans and represent a risk for the public health. For this, any microbiological control in dairy farms should be targeted not only to the search of O157:H7 serotype.