A National Surveillance of Shiga Toxin‐Producing Escherichia coli in Food‐Producing Animals in Japan

To assess the public health risk, the prevalence and anti‐microbial resistance of Shiga toxin‐producing Escherichia coli (STEC) among food‐producing animals were studied throughout Japan. Faecal samples were collected from healthy animals of 272 cattle, 179 pigs, and 158 broilers on 596 farms in all 47 Japanese prefectures. STEC were isolated from 62 (23%) cattle and 32 (14%) pig samples but from no chicken samples. Of the bovine isolates, 19 belonged to serotypes frequently implicated in human disease (O157:H7/non‐motile (NM)/H not typeable, O26:NM/H11/H21/H not typeable, O113:H21, and O145:NM). The eae genes were observed in 37% of bovine isolates; among them one O145:NM and all four O157 isolates possessed eae‐γ1, and one O145:NM, one O103:H11, and all five O26 isolates possessed eae‐β1 gene. Among the swine isolates, stx2e were dominant, and serotypes frequently implicated in human diseases or eae‐positive isolates were not observed. Bovine isolates showed less anti‐microbial resistance, but six isolates of 26:NM/H11 and O145:NM were multi‐resistant and may need careful monitoring. Swine isolates showed various resistance patterns; chloramphenicol resistance patterns were more common than in bovine isolates. This first national study of STEC in the Japanese veterinary field should aid our understanding of Japan's STEC status.     

Multiple‐Aetiology Enteric Infections Involving Non‐O157 Shiga Toxin‐Producing Escherichia coli – FoodNet, 2001–2010

We describe multiple‐aetiology infections involving non‐O157 Shiga toxin‐producing Escherichia coli (STEC) identified through laboratory‐based surveillance in nine FoodNet sites from 2001 to 2010. A multiple‐aetiology infection (MEI) was defined as isolation of non‐O157 STEC and laboratory evidence of any of the other nine pathogens under surveillance or isolation of >1 non‐O157 STEC serogroup from the same person within a 7‐day period. We compared exposures of patients with MEI during 2001–2010 with those of patients with single‐aetiology non‐O157 STEC infections (SEI) during 2008–2009 and with those of the FoodNet population from a survey conducted during 2006–2007. In total, 1870 non‐O157 STEC infections were reported; 68 (3.6%) were MEI; 60 included pathogens other than non‐O157 STEC; and eight involved >1 serogroup of non‐O157 STEC. Of the 68 MEI, 21 (31%) were part of six outbreaks. STEC O111 was isolated in 44% of all MEI. Of patients with MEI, 50% had contact with farm animals compared with 29% (P < 0.01) of persons with SEI; this difference was driven by infections involving STEC O111. More patients with non‐outbreak‐associated MEI reported drinking well water (62%) than respondents in a population survey (19%) (P < 0.01). Drinking well water and having contact with animals may be important exposures for MEI, especially those involving STEC O111.     

Detection of Shiga Toxin‐Producing Escherichia coli and Enteropathogenic Escherichia coli in Faecal Samples of Healthy Mithun (Bos frontalis) by Multiplex Polymerase Chain Reaction

Faecal samples obtained from 190 healthy mithuns were examined for the presence of Escherichia coli. Total one‐hundred and five E. coli isolates were obtained from these samples, which belonged to 55 different serogroups. These isolates were subjected to multiplex polymerase chain reaction (m‐PCR) for detection of stx1, stx2, eaeA and hlyA genes. Twenty‐three (21.90%) E. coli isolates belonging to 14 serogroups revealed the presence of at least one virulence gene when examined by m‐PCR. Nineteen percent and 2.85% of the mithuns were found to carry Shiga toxin‐producing E. coli (STEC) and enteropathogenic E. coli, respectively. stx1 and stx2 genes were found to be prevalent in 7 (6.67%) and 18 (17.14%) of the isolates respectively, whereas eaeA and hlyA genes were found to be carried by three (2.85% each) isolates. Interestingly, none of the STEC isolates belonged to serogroup O157.     

Non‐O157 Shiga Toxin‐producing Escherichia coli Isolated from Diarrhoeic Calves in Argentina

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin‐producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx₁ gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H‐ (n = 4), O26:H11 (n = 4), O26:H‐ (n = 1), O111:H‐ (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp‐2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H‐ showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non‐typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.     

An investigation of shedding and super‐shedding of Shiga toxigenic Escherichia coli O157 and E. coli O26 in cattle presented for slaughter in the Republic of Ireland

Shiga toxigenic Escherichia coli (STEC) are an important group of pathogens and can be transmitted to humans from direct or indirect contact with cattle faeces. This study investigated the shedding of E. coli O157 and O26 in cattle at the time of slaughter and factors associated with super‐shedding (SS) animals. Rectoanal mucosal swab (RAMS) samples were collected from cattle (n = 1,317) at three large Irish commercial beef abattoirs over an 18 month period, and metadata were collected at the time of sampling regarding farm of origin, animal age, breed and gender. RAMS swabs were examined for the presence and numbers of E. coli O157 and O26 using a previously developed quantitative real‐time PCR protocol. Samples positive by PCR were culturally examined and isolates analysed for the presence of stx subtypes, eae and phylogroup. Any samples with counts >10⁴ CFU/swab of STEC O157 or O26 were deemed to be super‐shedders. Overall, 4.18% (55/1,317) of RAMS samples were positive for STEC O157, and 2.13% (28/1,317) were classified as STEC O157 SS. For STEC O26, 0.76% (10/1,317) of cattle were positive for STEC O26, and 0.23% (3/1,317) were classified as super‐shedders. Fewer STEC shedders and SS were noted among older animals (>37 months). There was a seasonal trend observed for STEC O157, with the highest prevalence of shedding and SS events in the autumn (August to October). The majority of E. coli O157 (50/55) isolates had stx2 and were eae positive, with no significant difference between SS and low shedders (LS). Interestingly, all STEC O26 (n = 10) were eae negative and had varied stx profiles. This study demonstrates that, while the overall shedding rates are relatively low in cattle at slaughter, among positive animals there is a high level of SS, which may pose a higher risk of cross‐contamination during slaughter.     

Development of a PCR‐Based Method for Specific Identification of Genotypic Markers of Shiga Toxin‐Producing Escherichia coli Strains

A simple, rapid and specific PCR‐based method for identification of shiga toxin‐producing Escherichia coli (STEC) was developed. The procedure involves amplification of the E. coli‐specific universal stress protein A (uspA) gene (uspa‐PCR), with the primer pair described by other authors, which allows differentiation of E. coli (STEC and non‐STEC) from other gram‐negative bacteria followed by identification of the main genetic virulence traits of the uspA‐positive isolates. For this purpose, two multiplex PCR assays, based on previously published primer sequences, were established. Assay 1 (mPCR‐1) uses three primer pairs and detects the genes encoding O157 (rfb), enterohemolysin (ehly) and shiga toxin (stx), generating amplification products of 420, 534 and 230 bp, respectively. Assay 2 (mPCR‐2) uses four primer pairs specific for rfb (E. coli O157), eaeA (intimin), stx1 and stx2 (shiga toxin 1 and 2, respectively), generating PCR amplicons of 420, 840, 348 and 584 bp, respectively. These two assays were validated by testing several E. coli reference strains and 202 previously characterized E. coli isolates originating from calves and from children, and 100% agreement with previous results was obtained. The method developed can be used for specific identification of STEC bacteria including those of the O157 serogroup.     

Attribution of human infections with Shiga toxin‐producing Escherichia coli (STEC) to livestock sources and identification of source‐specific risk factors,The Netherlands (2010–2014)

Shiga toxin‐producing Escherichia coli (STEC) is a zoonotic pathogen of public health concern whose sources and transmission routes are difficult to trace. Using a combined source attribution and case–control analysis, we determined the relative contributions of four putative livestock sources (cattle, small ruminants, pigs, poultry) to human STEC infections and their associated dietary, animal contact, temporal and socio‐econo‐demographic risk factors in the Netherlands in 2010/2011–2014. Dutch source data were supplemented with those from other European countries with similar STEC epidemiology. Human STEC infections were attributed to sources using both the modified Dutch model (mDM) and the modified Hald model (mHM) supplied with the same O‐serotyping data. Cattle accounted for 48.6% (mDM) and 53.1% (mHM) of the 1,183 human cases attributed, followed by small ruminants (mDM: 23.5%; mHM: 25.4%), pigs (mDM: 12.5%; mHM: 5.7%) and poultry (mDM: 2.7%; mHM: 3.1%), whereas the sources of the remaining 12.8% of cases could not be attributed. Of the top five O‐serotypes infecting humans, O157, O26, O91 and O103 were mainly attributed to cattle (61%–75%) and O146 to small ruminants (71%–77%). Significant risk factors for human STEC infection as a whole were the consumption of beef, raw/undercooked meat or cured meat/cold cuts. For cattle‐attributed STEC infections, specific risk factors were consuming raw meat spreads and beef. Consuming raw/undercooked or minced meat were risk factors for STEC infections attributed to small ruminants. For STEC infections attributed to pigs, only consuming raw/undercooked meat was significant. Consuming minced meat, raw/undercooked meat or cured meat/cold cuts were associated with poultry‐attributed STEC infections. Consuming raw vegetables was protective for all STEC infections. We concluded that domestic ruminants account for approximately three‐quarters of reported human STEC infections, whereas pigs and poultry play a minor role and that risk factors for human STEC infection vary according to the attributed source.     

Detection and characterisation of Shiga toxin-producing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants

Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this work was to establish the prevalence of non-O157 STEC in free-ranging wild ruminants in the Extremadura region of Spain and to characterise them phenogenotypically. Faecal samples were collected from 243 wild ruminants, including Cervus elaphus, Capreolus capreolus, Dama dama and Ovis musimon and were examined for STEC using both phenotypic (Vero cells) and genotypic (PCR and PFGE) methods.Shiga toxin-producing Escherichia coli were isolated from 58 (23.9%) of the samples and a total of 65 isolates were characterised. A PCR method indicated that 11 (16.9%) strains carried the stx₁ gene, 44 (67.7%) carried the stx₂ gene and 10 (15.4%) carried both these genes. The ehxA gene was detected in 37 (57%) of the isolates but none contained either the eae or saa genes. The isolates were from a total of 12 ‘O’ serogroups, although 80% were restricted to the O2, O8, O128, O146, O166 and O174 serogroups. The most commonly isolated STEC bacteria, which were from the O146 serogroup, exhibited a high degree of polymorphism as indicated by PFGE. Shiga toxin-producing Escherichia coli isolates of serogroups O20, O25, O166, O171, O174 and O176 had not previously been found in wild ruminants. This is the first study to confirm that wild ruminants in Spain are a reservoir of STEC and are thus a potential source of human infection.     

Identification of shiga toxin and intimin coding genes in Escherichia coli isolates from pigeons (Columba livia) in relation to phylotypes and antibiotic resistance patterns

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of human intestinal diseases worldwide. Pigeons are distributed in public areas and are potential reservoirs for pathogenic bacteria. One hundred fifty-four fresh fecal samples were obtained from trapped pigeons in southeast of Iran and were cultured for isolation of E. coli. The isolates were examined to determine the prevalence of stx1, stx2, and eae genes, antimicrobial resistance, and their phylotypes. The confirmed E. coli isolates (138) belong to four phylogenetic groups: A (54.34%), B1 (34.05%), B2 (3.62%), and D (7.79%). Thirteen (9.42%) isolates were positive for one of the examined genes. Eight isolates (5.79%) were positive for eae, four (2.89%) for stx2, and one isolate (1.44%) for stx1 gene. Phylotyping assays showed that eight eae-positive isolates fall into three phylogroups; A (three isolates), B1 (three isolates), and D (two isolates), whereas four stx2-positive isolates belonged to the A (three isolates) and D (one isolate) groups. The stx1-positive isolate belonged to phylogroup A. One hundred six isolates (76.81%) showed resistance to at least one of the selected antibacterial agents. The maximum resistance rate was against oxytetracycline (73.91%), and the minimum was against flumequine (2.17%). Twenty different patterns of drug resistance were observed. According to the results, pigeons could be considered as carriers of STEC strains. However, E. coli isolates of pigeon feces increase the potential of these birds to act as a reservoir of multiple antibiotic resistant bacteria.     

Shiga toxin 2eB‐transgenic lettuce vaccine is effective in protecting weaned piglets from edema disease caused by Shiga toxin‐producing Escherichia coli infection

Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB‐transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB‐lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB‐lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB‐lettuce is a promising first oral vaccine candidate against ED.     

Detection and Analysis of Shiga Toxin-producing Escherichia coli Isolates from Cattle and Sheep Sources in Some Regions of Xinjiang,China

Shiga toxin-producing Escherichia coli(STEC) is a new class of highly pathogenic food-borne pathogens carrying a prephage encoding one or two Shiga toxin genes. It has become an important public health issue that threatens human health. The present work aimed to characterize STEC strains isolated from cattle and sheep at various stages, in parts of Xinjiang, in terms of the presence of prevalence, genetic diversity, and antimicrobial susceptibility to 17 common antibiotics. Through amplification of four virulence genes (stx1, stx2, eae, hlyA)by PCR and ERIC-PCR genotyping to detection STEC isolates. In the present study, a total of 64 STEC strains were isolated from 431 samples from slaughterhouses, farms and markets. Of these, 31 (48.4%) of the isolates harbored stx1 + stx2, and only 29 (45.3%) of the isolates possessed stx1, only 4 (6.3%) of the isolates harbored stx2, and 1 isolates harbored all the 4 virulence genes. Drug sensitivity tests found that STEC strains displayed 7 antimicrobial resistance to midecamycin(61%), cephalothin(4.7%), cefoxitin(4.7%), ampicillin(3.1%), piperacillin(1.6%), tobramycin(1.6%), cefazolin(1.6%). The ERIC-PCR results showed a polymorphic distribution, which was divided into two clusters of A (36 strains) and B (28 strains). STEC strains isolated from cattle and sheep at various stages, in parts of Xinjiang, some of which might have the potential to cause food contamination and human diseases.     

新疆部分地区牛羊源产志贺毒素大肠埃希菌菌株检测与分析

产志贺毒素大肠埃希菌（Shiga toxin-producing Escherichia coli，STEC）是一类携带了前噬菌体编码的一种或两种志贺毒素基因的新发高致病性食源性病原菌，已成为威胁人类健康的重要公共卫生问题。为了解新疆部分地区牛、羊源各个环节产志贺毒素大肠埃希菌的感染情况及其遗传多样性，以及分离株对17种常见抗生素的敏感性，笔者采用PCR方法对STEC分离株进行了4种毒力基因（stx1、stx2、eae、hlyA）的检测和ERIC-PCR基因分型研究。结果表明：从屠宰场、养殖场和市场共431份样品中分离出产志贺毒素的大肠埃希菌64株，其中，编码stx1+stx2的STEC有31株（48.4%），只编码stx1的STEC有29株（45.3%），只编码stx2的STEC有4株（6.3%），4种毒力基因同时存在的有1株。药物敏感性检测发现STEC菌株对麦迪霉素（61%）、头孢噻吩（4.7%）、头孢西丁（4.7%）、氨苄西林（3.1%）、哌拉西林（1.6%）、妥布霉素（1.6%）、头孢唑啉（1.6%）等7种抗生素存在耐药。ERIC-PCR检测结果呈多态性分布，分为A（36株）和B（28株）两个簇。STEC菌株在新疆部分地区牛、羊源各个环节被检出，其中一些菌株可能会增加对食物的污染，从而引起人发病。     

Prevalence,molecular fingerprinting and drug resistance profile of enterovirulent <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from free-ranging yaks of Tawang district,Arunachal Pradesh,India

Of 273 samples (rectal swab) collected from free-ranging yaks of Tawang district, Arunachal Pradesh, 42 Shiga toxin-producing Escherichia coli (STEC), six enteropathogenic E. coli (EPEC) and 27 enterotoxigenic E. coli (ETEC) strains were isolated. All the STEC and EPEC strains were further investigated for respective stx variants (for STEC only) and additional putative virulence factors. The 27 ETEC strains were also screened for characteristic enterotoxin gene(s) and colonization factors. Occurrence of ETEC was significantly (p < 0.05) higher in the diarrheic yaks and yaks of less than 1 year of age. Majority of enterovirulent E. coli isolates were resistant to amikacin, azithromycin, chloramphenicol, colistin, doxycycline, furazolidone, nalidixic acid, nitrofurantoin, streptomycin and tetracycline. Dendrogram, constructed with molecular fingerprinting profiles obtained from RAPD (Randomly Amplified Polymorphic DNA) and ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR, placed the isolates in different clusters irrespective of their serotypes, virulence gene and drug resistance pattern. Collectively, the study indicates that yaks, being a potential reservoir of multidrug resistant STEC and EPEC, may represent significant risk to public health in this region. Higher recovery of ETEC isolates from yaks with diarrhea points out that ETEC may be a major determinant for repeated occurrence of diarrhea in yaks.     

Improved porcine model for Shiga toxin‐producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 10¹¹ colony‐forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum‐deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.     

Prevalence and Genetic Relatedness of Antimicrobial‐Resistant Escherichia coli Isolated From Animals,Foods and Humans in Iceland

The prevalence of resistant bacteria in food products in Iceland is unknown, and little is known of the prevalence in production animals. The aim of this study was to investigate the prevalence and genetic relatedness of antimicrobial‐resistant Escherichia coli from healthy pigs and broiler chicken, pork, broiler meat, slaughterhouse personnel and outpatients in Iceland. A total of 419 E. coli isolates were tested for antimicrobial susceptibility using a microbroth dilution method (VetMIC), and resistant strains were compared using pulsed‐field gel electrophoresis (PFGE). All samples were screened for enrofloxacin‐resistant strains with selective agar plates. The resistance rates among E. coli isolates were moderate to high from caecal and meat samples of pigs (54.1% and 28%), broilers (33.6% and 52%) and slaughterhouse personnel (39.1%), whereas isolates from outpatients showed moderate resistance rates (23.1%). Of notice was resistance to quinolones (minimum inhibitory concentrations: nalidixic acid ≥ 32, ciprofloxacin ≥ 0.12 and enrofloxacin ≥ 0.5), particularly among broiler and broiler meat isolates (18.2% and 36%), as there is no known antimicrobial selection pressure in the broiler production in Iceland. The majority (78.6%) of the resistant E. coli isolates was genotypically different, based on PFGE fingerprint analyses and clustering was limited. However, the same resistance pattern and pulsotype were found among isolates from broiler meat and a slaughterhouse worker, indicating spread of antimicrobial‐resistant E. coli from animals to humans. Diverse resistance patterns and pulsotypes suggest the presence of a large population of resistant E. coli in production animals in Iceland. This study gives baseline information on the prevalence of antimicrobial‐resistant E. coli from production animals, and their food products in Iceland and the moderate to high resistance rates emphasize the need for continuing surveillance. Further studies on the origin of the resistant strains and the genetic relatedness of strains of different origin are needed.     

Genetic Characterization of Antibiotic Resistance in Enteropathogenic Escherichia coli Carrying Extended‐Spectrum β‐Lactamases Recovered from Diarrhoeic Rabbits

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended‐spectrum β‐lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K‐:H2) was observed among 17 EPEC strains, the O92:K‐serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K‐serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim–sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The bla_TEM gene was demonstrated in the majority of ampicillin‐resistant isolates. The aac(3)‐II or aac(3)‐IV genes were detected in the four gentamicin‐resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin‐resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline‐resistant isolates. A total of nine EPEC isolates showed the phenotype SXT‐resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.     

Characterizing the Role of Animal Exposures in Cryptosporidiosis and Shiga Toxin‐producing Escherichia coli Infections: South Dakota, 2012

Cryptosporidium spp. and Shiga toxin‐producing Escherichia coli strains (STEC) are important causes of human illness. Incidence rates of these illnesses are high in South Dakota compared to the USA as a whole. Direct animal contact has been identified as a possible route of exposure for these illnesses. Ruminant animals may carry STEC subclinically, while young ruminants are common sources of zoonotic strains of Cryptosporidium. South Dakota patients with either STEC or cryptosporidiosis during 2012 were interviewed regarding seven categories of animal exposure: (i) petting zoo/fair attendance, (ii) animal event/rodeo attendance, (iii) feed/pet store visits, (iv) farm visits, (v) employment or residence at a farm, (vi) residence with pets and (vii) visiting other households with pets. Of the 50 STEC cases, 78.0% reported animal exposure prior to illness onset, with 23.3% having lived or worked on a farm. Farm visitors in particular had high degrees of animal contact and infrequently practiced personal protective measures. Of the 115 cryptosporidiosis cases, 87.8% reported animal exposures, with 45.6% having lived or worked on a farm and 29.0% having visited a farm prior to illness. Cases with farm exposures reported a high degree of direct animal contact and inconsistent use of personal protective measures. Cryptosporidiosis patients were significantly more likely than STEC patients to have lived or worked on a farm prior to their illness and were older on average. Patients with these illnesses had high rates of animal contact prior to illness. Animal contact on farms emerged as an important exposure route. Educational messages about personal protective measures should be directed at these individuals.     

Detection and characterization of Shiga toxin-producing Escherichia coli in captive non-domestic mammals

Shiga toxin producing-Escherichia coli (STEC) is an important emerging pathogen, and ruminants are recognized as their main natural reservoir. The aim of this work was to establish the frequency of STEC in non-domestic mammals of the Zoo and Botanical Garden of La Plata City, Argentina, and to pheno-genotypically characterize STEC isolates. By polymerase chain reaction (PCR), Shiga toxin (stx) gene sequences were detected in 50.8% of 65 fecal samples. Twenty-five STEC strains were isolated from 38.5% of the Zoo's animals. Ten species of order Cetartiodactyla and one species of order Rodentia were recognized as new STEC carriers. STEC strains belonged to 7 different serotypes including new serotypes O12:H25 and O13:H6. Serotype O146:H28, previously associated with human infections, represented 24% of STEC isolates. The most frequent Shiga toxin identified were type 1c and type 2c. Nineteen strains were positive for iha gene, 8 strains were positive for ehxA gene. Moreover, all strains were positive for lpfAO113 and negative for rfbO157, eae, saa, lpfAO157/OI-141, lpfAO157/OI-154, efa1, and toxB genes. Results obtained by XbaI-pulsed-field gel electrophoresis (XbaI-PFGE) confirmed the transmission of STEC strains among different animal species and suborders. In addition, we observed a potential association between STEC-harboring animal and factors such as belonging to order Cetartiodactyla, living in a pit, and belonging to a non-autochthonous species. This is the first work developed with zoological mammals and STEC in Argentina.     

An outbreak of Shiga toxin‐producing Escherichia coli O157:H7 linked to a mud‐based obstacle course,England, August 2018

In August 2018, Public Health England (PHE) was made aware of five probable cases of Shiga toxin‐producing Escherichia coli (STEC) O157:H7 among individuals reporting participation in a mud‐based obstacle race. An additional four cases, identified via routine whole‐genome sequencing, were subsequently linked to the same event. Two of the nine cases were due to secondary household transmission. Despite an agreement between the event organizers and the local authority, to ensure that all livestock were removed from the site 28 days before the event, sheep were observed grazing on some of the routes taken by the runners 2 days prior to the race taking place. A retrospective review of incidents reported to PHE between 2015 and 2018 identified 41 cases of gastroenteritis associated with muddy assault course events. Of these, 25 cases were due to infection with STEC O157:H7, of which all but one were associated with outbreaks. Due to the environment in which such events take place, it is impossible to entirely remove the risk of exposure to potentially pathogenic zoonoses. However, race organizers should ensure that livestock are removed from the course 28 days before the event. They should also ensure that participants are made aware of the risk of contracting gastrointestinal disease from the environment, and to stress the importance of hand hygiene post‐event and the risk of secondary transmission, particularly to children who are at risk of developing haemolytic uraemic syndrome.     

Identification of putative adhesin genes in shigatoxigenic Escherichia coli isolated from different sources

Shiga toxin-producing Escherichia coli (STEC) is an important pathogen responsible for severe human intestinal and systemic infections. The bacterial factors required for colonization of the hosts are still not well defined. In this study, the prevalence of seven putative adhesive genes that are not encoded in the locus of enterocyte effacement (LEE) in 74 STEC strains isolated from humans (n=39), food (n=6), cattle (n=11), and pigs (n=18) was investigated by PCR. In addition, Shiga toxin (stx) and intimin (eaeA including alpha, beta, gamma, delta, epsilon, zeta variants) genes were tested. The most prevalent adhesin was that encoded by toxB gene (52 of 74 isolates; 70.3%). This marker was found in all 12 strains of O157:H7 serotype and in 23 of 32 (71.9%) isolates of the O157:NM serogroup. Moreover, this gene was also present in other 17 STEC of the non-O157 serogroup. The second most prevalent adhesin was that encoded by the lpfAO157/OI-154 gene (43 isolates; 58.1%). This marker was detected in LEE-positive strains of the O157 serogroup but also in 9 LEE-negative isolates of porcine origin. Several STEC isolates tested (42 strains; 56.7%) had the efa1 gene of the Efa1 putative adhesive marker. This adhesin was almost exclusively found among eaeA-positive strains recovered from humans, food and cattle. On the other hand, iha marker was detected either in LEE-positive (29 isolates) or LEE-negative (12 strains) STEC. Only two eaeA-negative strains had the saa putative adhesive gene. These results show that STEC strains may be able to express several putative adhesins. However, further studies are needed to evaluate the role of the genes identified in the present study in the pathogenesis of human infections.