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

Diversity of antimicrobial resistance, stress resistance, and virulence factors of Salmonella, Shiga toxin-producing Escherichia coli, and Listeria monocytogenes from produce, spices, and tree nuts by whole genome sequencing

IntroductionThe objective of this study was to analyze antimicrobial resistance (AMR), stress resistance, and virulence factors through whole genome sequencing (WGS) of 192 isolates comprising 164 Salmonella isolates, 8 non-O157 Shiga toxin-producing Escherichia coli (STEC) isolates, and 20 Listeria monocytogenes isolates.MethodsThese isolates were sourced from a national survey conducted between 2010 and 2017, involving 31,322 samples of produce (31 isolates), nuts (43 isolates), and spices (118 isolates).ResultsThe findings yielded several key insights: (1) Within all Salmonella isolates studied, the most prevalent Salmonella serotypes included Give, Kentucky, Senftenberg, Mbandaka, Anatum, Newport, and Weltevreden. (2) All eight non-O157 STEC isolates were found to carry the genes blaEC, acrF, and mdtM, while all 20 L. monocytogenes isolates possessed fosX and lin genes. The Salmonella isolates displayed diverse AMR gene profiles, with 3.65% exhibiting multi-drug resistance. (3) Both Salmonella and non-O157 STEC isolates were discovered to carry stress genes associated with acid resistance, but none of the L. monocytogenes isolates carried an acid resistance gene. Salmonella isolates were found to carry multiple metal-resistance genes. The non-O157 STEC isolates universally exhibited acid resistance genes, and 4 out of the 20 L. monocytogenes isolates were equipped with resistance genes against biocides. (4) All of the STEC isolates (100%) carried stx1 and stx2 genes, while none of them carried eae and wyz genes. Most L. monocytogenes isolates were found to contain 29 virulence genes and 1 pathogenicity island. All Salmonella isolates carried SPI-9, but lacked SPI-7, SPI-10, SPI-11, SPI-12, mig-5, prefA, tviA, and viaB genes.ConclusionThese findings on AMR, stress resistance, and virulence factors among the investigated isolates highlight the potential risks they pose to public health and provide the scientific foundation for the development of preventative and control strategies and guidance pertaining to these major foodborne pathogens.

Prevalence of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Serogroups Isolated from Fresh Raw Beef Meat Samples in an Industrial Slaughterhouse.

Background The aims of the current study are the identification of O157 and non-O157 Shiga Toxin-Producing Escherichia coli (STEC) serogroups isolated from fresh raw beef meat samples in an industrial slaughterhouse, determination of antimicrobial resistance patterns, and genetic linkage of STEC isolates. Materials and Methods A total of 110 beef samples were collected from the depth of the rump of cattle slaughtered at Hamadan industrial slaughterhouse. After detection of E. coli isolates, STEC strains were identified according to PCR for stx1, stx2, eaeA, and hlyA virulence genes, and STEC serogroups (O157 and non-O157) were identified by PCR. The genetic linkage of STEC isolates was analyzed by the ERIC- (Enterobacterial Repetitive Intergenic Consensus-) PCR method. The antimicrobial susceptibility of STEC isolates was detected by the disk diffusion method according to CLSI guidelines. Results Among 110 collected beef samples, 77 (70%) were positive for E. coli. The prevalence of STEC in E. coli isolates was 8 (10.4%). The overall prevalence of O157 and non-O157 STEC isolates was 12.5% (one isolate) and 87.5% (7 isolates), respectively. The hemolysin gene was detected in 25% (2 isolates) of STEC strains. Evaluation of antibiotic resistance indicated that 100% of STEC isolates were resistant to ampicillin, ampicillin-sulbactam, amoxicillin-clavulanic acid, and cefazolin. Resistance to tetracycline and ciprofloxacin was detected in 62.5% and 12.5% of isolates, respectively. The analysis of the ERIC-PCR results showed five different ERIC types among the STEC isolates. Conclusion The isolation of different clones STECs from beef and the presence of antibiotic-resistant isolates indicate that more attention should be paid to the hygiene of slaughterhouses.

Shiga Toxin–Producing Escherichia coli (STEC) and STEC-Associated Virulence Genes in Raw Ground Pork in Canada

Shiga toxin-producing Escherichia coli (STEC) O157:H7/nonmotile and some non-O157 STEC strains are foodborne pathogens. In response to pork-associated O157 STEC outbreaks in Canada, we investigated the occurrence of STEC in Canadian retail raw ground pork during the period of 1 November 2014 to 31 March 2016. Isolated STEC strains were characterized to determine the Shiga toxin gene (stx) subtype and the presence of virulence genes encoding intimin (eae) and enterohemorrhagic E. coli hemolysin (hlyA). O157 STEC and non-O157 STEC strains were isolated from 1 (0.11%) of 879 and 13 (2.24%) of 580 pork samples, respectively. STEC virulence gene profiles containing both eae and hlyA were found only in the O157 STEC (stx2a, eae, hlyA) isolate. The eae gene was absent from all non-O157 STEC isolates. Of the 13 non-O157 STEC isolates, two virulence genes of stx1a and hlyA were found in four (30.8%) O91:H14 STEC isolates, whereas one virulence gene of stx2e, stx1a, and stx2a was identified in five (38.5%), two (15.4%), and one (7.7%) STEC isolates, respectively, of various serotypes. The remaining non-O157 STEC isolate carried stx2, but the subtype is unknown because this isolate could not be recovered for sequencing. O91:H14 STEC (stx1a, hlyA) was previously reported in association with diarrheal illnesses, whereas the other non-O157 STEC isolates identified in this study are not known to be associated with severe human illnesses. Virulence gene profiles identified in this study indicate that the occurrence of non-O157 STEC capable of causing severe human illness is rare in Canadian retail pork. However, O157 STEC in ground pork can occasionally occur; therefore, education regarding the potential risks associated with STEC contamination of pork would be beneficial for the public and those in the food industry to help reduce foodborne illnesses.

Antibiotic Susceptibility Profiles and Frequency of Resistance Genes in Clinical Shiga Toxin-Producing Escherichia coli Isolates from Michigan over a 14-Year Period.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that contributes to over 250,000 infections in the United States each year. Because antibiotics are not recommended for STEC infections, resistance in STEC has not been widely researched despite an increased likelihood for the transfer of resistance genes from STEC to opportunistic pathogens residing within the same microbial community. From 2001 to 2014, 969 STEC isolates were collected from Michigan patients. Antibiotic susceptibility profiles to clinically relevant antibiotics were determined using disc diffusion, while epidemiological data were used to identify factors associated with resistance. Whole-genome sequencing was used for serotyping, examining genetic relatedness, and identifying genetic determinants and mechanisms of resistance in the non-O157 isolates. Increasing frequencies of resistance to at least one antibiotic were observed over the 14 years (P = 0.01). While the non-O157 serogroups were more commonly resistant than O157 (odds ratio, 2.4; 95% confidence interval,1.43 to 4.05), the frequency of ampicillin resistance among O157 isolates was significantly higher in Michigan than the national average (P = 0.03). Genomic analysis of 321 non-O157 isolates uncovered 32 distinct antibiotic resistance genes (ARGs). Although mutations in genes encoding resistance to ciprofloxacin and ampicillin were detected in four isolates, most of the horizontally acquired ARGs conferred resistance to aminoglycosides, β-lactams, sulfonamides, and/or tetracycline. This study provides insight into the mechanisms of resistance in a large collection of clinical non-O157 STEC isolates and demonstrates that antibiotic resistance among all STEC serogroups has increased over time, prompting the need for enhanced surveillance.

Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Isolated from Cattle from Central and Southern Chile.

Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), one of the world's most important foodborne pathogens. The pathogen causes severe human diseases and outbreaks. This study aimed to identify and characterize non-O157 STEC isolated from cattle feces from central and southern Chile. We analyzed 446 cattle fecal samples and isolated non-O157 STEC from 12.6% (56/446); a total of 93 different isolates were recovered. Most isolates displayed β-glucuronidase activity (96.8%; 90/93) and fermented sorbitol (86.0%; 80/93), whereas only 39.8% (37/93) were resistant to tellurite. A subgroup of 30 representative non-O157 STEC isolates was selected for whole-genome sequencing and bioinformatics analysis. In silico analysis showed that they grouped into 16 different sequence types and 17 serotypes; the serotypes most frequently identified were O116:H21 and O168:H8 (13% each). A single isolate of serotype O26:H11 was recovered. One isolate was resistant to tetracycline and carried resistance genes tet(A) and tet(R); no other isolate displayed antimicrobial resistance or carried antimicrobial resistance genes. The intimin gene (eae) was identified in 13.3% (4/30) of the genomes and 90% (27/30) carried the stx2 gene. A phylogenetic reconstruction demonstrated that the isolates clustered based on serotypes, independent of geographical origin. These results indicate that cattle in Chile carry a wide diversity of STEC potentially pathogenic for humans based on the presence of critical virulence genes.

Evaluation of Cattle for Naturally Colonized Shiga Toxin-Producing Escherichia coli Requires Combinatorial Strategies.

Shiga toxin-producing Escherichia coli (STEC) serogroups O157, O26, O103, O111, O121, O145, and O45 are designated as food adulterants by the U.S. Department of Agriculture-Food Safety and Inspection Service. Cattle are the primary reservoir of these human pathogens. In this study, 59 Angus crossbred heifers were tested specifically for these seven STEC serogroups using a combination of standard culture, serological, PCR, and cell cytotoxicity methods to determine if comparable results would be obtained. At the time of fecal sampling, the animals were approximately 2 years old and weighed 1000–1200 lbs. The diet comprised of 37% ground alfalfa hay, 25% ground Sudan hay, and 38% ground corn supplemented with trace minerals and rumensin with ad libitum access to water. Non-O157 STEC were isolated from 25% (15/59) of the animals tested using a combination of EC broth, CHROMagar STECTM, and Rainbow Agar O157. Interestingly, the O157 serogroup was not isolated from any of the animals. Non-O157 STEC isolates were confirmed to be one of the six adulterant serogroups by serology and/or colony PCR in 10/15 animals with the predominant viable, serogroup being O103. PCR using DNA extracted from feces verified most of the colony PCR results but also identified additional virulence and O-antigen genes from samples with no correlating culture results. Shiga toxin- (Stx-) related cytopathic effects on Vero cells with fecal extracts from 55/59 animals could only be associated with the Stx gene profiles obtained by fecal DNA PCR and not culture results. The differences between culture versus fecal DNA PCR and cytotoxicity assay results suggest that the latter two assays reflect the presence of nonviable STEC or infection with STEC not belonging to the seven adulterant serogroups. This study further supports the use of combinatorial culture, serology, and PCR methods to isolate viable STEC that pose a greater food safety threat.

Microbiological Survey of Wheat Flour Sold at Retail in Canada, 2018 to 2019

Following two O121 Shiga toxin-producing Escherichia coli (STEC) outbreaks linked to wheat flour, this study was conducted to gain baseline information on the occurrence of bacterial pathogens and levels of indicator organisms in wheat flour in Canada. A total of 347 prepackaged wheat flour samples were analyzed for Salmonella species, STEC, Listeria monocytogenes, aerobic colony count (ACC), total coliforms, and Escherichia coli. Salmonella spp. and O157 STEC were not detected in any of the samples. L. monocytogenes was identified in two samples (0.6%) at levels below the limit of detection (<0.7 log CFU/g). Non-O157 STEC were isolated from six samples (1.7%) and were characterized for the presence of STEC virulence genes: stx1, stx2, and their subtypes, eae, hlyA, and aggR. One O103:H25 STEC isolate carried virulence genes (stx1a+eae) that are known to be capable of causing diarrhea and/or bloody diarrhea in humans. Of the five remaining non-O157 STEC isolates, four carried single stx2a or stx2c genes and were considered to have the potential of causing diarrhea. The remaining non-O157 STEC isolate (stx2), while not a priority non-O157 STEC, was not available for sequencing; thus, its potential to cause illness is unknown. ACC, total coliforms, and E. coli were detected (≥0.48 log CFU/g) in 98.8, 72.6, and 0.6% of the flour samples. The mean counts of ACC were greater in whole wheat flour compared with the other flour types tested (P < 0.001). The results of this study suggest that the occurrence of O157 STEC and Salmonella is low but that the occurrence of non-O157 STEC in wheat flour with the potential to cause human illness of diarrhea is relatively common. Therefore, the consumption of raw flour could increase the likelihood of STEC infections. Further research is merited for potential risk mitigation strategies within the food production system and with consumers.

Genomic Insights Into Clinical Shiga Toxin-Producing Escherichia coli Strains: A 15-Year Period Survey in Jönköping, Sweden.

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens that can cause human infections ranging from asymptomatic carriage to bloody diarrhea (BD) and fatal hemolytic uremic syndrome (HUS). However, the molecular mechanism of STEC pathogenesis is not entirely known. Here, we demonstrated a large scale of molecular epidemiology and in-depth genomic study of clinical STEC isolates utilizing clinical and epidemiological data collected in Region Jönköping County, Sweden, over a 15-year period. Out of 184 STEC isolates recovered from distinct patients, 55 were from patients with BD, and 129 were from individuals with non-bloody stools (NBS). Five individuals developed HUS. Adults were more associated with BD. Serotypes O157:H7, O26:H11, O103:H2, O121:H19, and O104:H4 were more often associated with BD. The presence of Shiga toxin-encoding gene subtypes stx2a, stx2a + stx2c, and stx1a + stx2c was associated with BD, while stx1a was associated with milder disease. Multiplex virulence and accessory genes were correlated with BD; these genes encode toxins, adhesion, autotransporters, invasion, and secretion system. A number of antimicrobial resistance (AMR) genes, such as aminoglycoside, aminocoumarin, macrolide, and fluoroquinolone resistance genes, were prevalent among clinical STEC isolates. Whole-genome phylogeny revealed that O157 and non-O157 STEC isolates evolved from distinct lineages with a few exceptions. Isolates from BD showed more tendency to cluster closely. In conclusion, this study unravels molecular trait of clinical STEC strains and identifies genetic factors associated with severe clinical outcomes, which could contribute to management of STEC infections and disease progression if confirmed by further functional validation.

Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Cultured from Cattle Farms in Xinjiang Uygur Autonomous Region, China, During 2016-2017.

Most outbreaks of Shiga toxin-producing Escherichia coli (STEC) are attributed to consumption of contaminated foodstuffs including beef and dairy products. In this study, we evaluated the prevalence of non-O157 STEC cultured from beef and dairy cattle and collected in Xinjiang Uygur Autonomous Region in China. Results identified 67 non-O157 STEC recovered from the 793 samples including beef cattle (10.28%, 43/418) and dairy cattle (6.40%, 24/375). A total of 67 non-O157 STEC was sequenced allowing for in silico analyses of their serotypes, virulence genes, and identification of the corresponding multilocus sequence types (STs). Twenty-one O serogroups and nine H serotypes were identified and the dominant serotype identified was O22:H8. One stx1 subtype (stx1a) and four stx2 subtypes (2a, 2b, 2c, and 2d) were found in the 67 non-O157 STEC isolates. The results revealed that stx1a+stx2a-positive STEC isolates were predominant (32.83%, 22/67), followed by stx1a+stx2d (29.85%, 20/67) and stx2a alone (17.91%, 12/67). Non-O157 STEC isolates carried virulence genes ehxA (98.51%), subA (53.73%), and cdtB (17.91%). Of the four adherence-associated genes tested, eaeA was absent, whereas lpfA and iha were present in 67 and 55 non-O157 STEC isolates, respectively. The STEC isolates were divided into 48 pulsed-field gel electrophoresis patterns and 10 STs, and ST446 (O22:H8) was the dominant clone (22.38%). Our results revealed that there was a high genetic diversity among non-O157 STEC isolated from beef and dairy cattle, some of which have potential to cause human diseases.

Antimicrobial Resistance of Non-O157 Shiga Toxin-Producing Escherichia coli Isolated from Humans and Domestic Animals.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) is an important pathogen that can cause zoonotic diseases. To investigate the antimicrobial resistance of STEC in China, non-O157 STEC isolates, recovered from domestic animals and humans from 12 provinces, were analyzed using antimicrobial susceptibility testing and whole genome characterization. Out of the 298 isolates tested, 115 strains showed resistance to at least one antimicrobial and 85 strains showed multidrug resistance. The highest resistance rate was to tetracycline (32.6%), followed by nalidixic acid (25.2%) and chloramphenicol and azithromycin (both 18.8%). However, imipenem and meropenem were effective against all isolates. Antimicrobial resistance patterns varied among strains from different sources. Strains from pig, sheep, humans, and cattle showed resistance rates of 100.0%, 46.9%, 30.3%, and 6.3% to one or more antimicrobials, respectively. Forty-three genes related to 11 antimicrobial classes were identified among these strains. The colistin-resistance gene mcr was only carried by strains from pigs. A new fosfomycin-resistant gene, fosA7, was detected in strains from humans, cattle, and sheep. Whole genome phylogenetic analysis showed that strains from the four sources were genetically diverse and scattered throughout the phylogenetic tree; however, some strains from the same source had a tendency to cluster closely. These results provide a reference to monitor the emergence and spread of multidrug resistant STEC strains among animals and humans. Furthermore, with a better understanding of antimicrobial genotypes and phenotypes among the diverse STEC strains obtained, this study could guide the administration of antimicrobial drugs in STEC infections when necessary.

Isolation and Characterization of Non-O157 Shiga Toxin–Producing Escherichia coli in Foods Sold at Retail Markets in China

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are significant foodborne pathogens that can cause acute diarrhea in humans. This study was conducted to investigate the contamination by non-O157 STEC in different types of food sold at retail markets in the People's Republic of China and to characterize non-O157 STEC strains. From May 2012 to April 2014, 1,200 retail food samples were collected from markets in 24 cities in China. Forty-four non-O157 isolates were recovered from 43 STEC-positive samples. Of the isolates, 22 and 19 carried the stx1 and stx2 genes, respectively, and 3 harbored both stx1 and stx2. stx1a and stx2a were the most prevalent stx subtypes. Other virulence genes, ent, hlyA, astA, eae, espB, iha, subAB, and tia, were commonly detected. Diverse O serogroups were identified among these isolates. Multilocus sequence typing indicated the high genetic diversity of the isolates. Thirty-two sequence types (STs) were identified among the 44 isolates, with ST383 (9.09%), ST134 (6.82%), and ST91 (6.82%) the most prevalent. Nine new STs were found. The isolates had a high prevalence of resistance to cephalothin, ampicillin, tetracycline, trimethoprim-sulfamethoxazole, nalidixic acid, streptomycin, and chloramphenicol. Twenty isolates (45.45%) were resistant to at least three antibiotics. This study provides updated surveillance data for non-O157 STEC isolates from foods sold at retail markets. Virulent and multidrug-resistant non-O57 STEC strains were isolated from all types of food. Our findings highlight the need for increased monitoring of non-O157 STEC in retail foods.

Characterization of Non-O157 STEC Infecting Bacteriophages Isolated from Cattle Faeces in North-West South Africa

Non-O157 Shiga toxin-producing Escherichia coli (STEC) E. coli are emerging pathotypes that are frequently associated with diseases in humans around the world. The consequences of these serogroups for public health is a concern given the lack of effective prevention and treatment measures. In this study, ten bacteriophages (phages; SA20RB, SA79RD, SA126VB, SA30RD, SA32RD, SA35RD, SA21RB, SA80RD, SA12KD and SA91KD) isolated from cattle faeces collected in the North-West of South Africa were characterized. Activity of these phages against non-O157 STEC isolates served as hosts for these phages. All of the phages except SA80RD displayed lytic against non-O157 E. coli isolates. Of 22 non-O157 E. coli isolates, 14 were sensitive to 9 of the 10 phages tested. Phage SA35RD was able to lyse 13 isolates representing a diverse group of non-O157 E. coli serotypes including a novel O-antigen Shiga toxigenic (wzx-Onovel5:H19) strain. However, non-O157 E. coli serotypes O76:H34, O99:H9, O129:H23 and O136:H30 were insensitive to all phages. Based on transmission electron microscopy, the non-O157 STEC phages were placed into Myoviridae (n = 5) and Siphoviridae (n = 5). Genome of the phage ranged from 44 to 184.3 kb. All but three phages (SA91KD, SA80RD and SA126VB) were insensitive to EcoRI-HF and HindIII nucleases. This is the first study illustrating that cattle from North-West South Africa harbour phages with lytic potentials that could potentially be exploited for biocontrol against a diverse group of non-O157 STEC isolated from the same region.

Characterizing the Multidrug Resistance of non-O157 Shiga Toxin-Producing Escherichia coli Isolates from Cattle Farms and Abattoirs.

Non-O157 Shiga toxin-producing Escherichia coli (STECs) are not as well characterized as O157 STEC cases, despite their similar prevalence in many countries. Hence, the objective of this study was to investigate the phenotypic and genotypic basis of multidrug resistance (MDR) in non-O157 STEC farm- and abattoir-sourced isolates and assess the potential dissemination of these MDR profiles in vitro. Susceptibility testing to 20 antimicrobials was performed on 146 non-O157 STECs isolated from farm and abattoir environments. Eighty-seven percent of non-O157 STEC isolates were multidrug resistant to antimicrobials used during veterinary and agricultural practice. Antimicrobial resistance was significantly higher in abattoir isolates compared with the farm isolates (p < 0.05). Corresponding resistance determinants and integrons were investigated by polymerase chain reaction, with the predominant resistance determinants detected being floR, ampC, tet(A), blaTEM, and sul1. This is the first report of tet(G) in a non-O157 STEC isolate. Class 1 integrons were detected in 17 isolates. Resistance to ampicillin, cephalothin, chloramphenicol, kanamycin, neomycin, sulfonamides, trimethoprim, and tetracycline was associated with transferable plasmids belonging to incompatibility groups IncP, IncB/O, and IncFIB. Most MDR non-O157 STECs (90%) isolated in this study belong to phylogenetic groups A and B1. These findings suggest that MDR non-O157 STECs are emerging as a result of nonpathogenic E. coli acquiring virulence and resistance genes. This may convey a certain competitive advantage in the colonization of cattle when antimicrobial selective pressures are present, thereby leading to an increase in contamination of food with MDR non-O157 STECs.

Molecular and Phylogenetic Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Strains in China.

Shiga toxin-producing Escherichia coli (STEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. The aim of this study was to assess the molecular epidemiologic features of non-O157 STEC strains from different resources in China and illustrate the role of animal reservoirs or animal-derived foodstuffs in human STEC infections. A collection of 301 non-O157 STEC isolates from domestic and wild animals (i.e., cattle, goat, pig, yak, pika, and antelope), raw meats (i.e., beef, pork, mutton, chicken, and duck), diarrheal patients, and healthy carriers in different regions of China were selected in this study. Of the 301 analyzed STEC isolates, 67 serogroups, and 118 serotypes were identified; this included some predominant serogroups associated with human disease, such as O26, O45, O103, O111, and O121. Eighteen different combinations of stx subtypes were found. Eleven isolates carried the intimin gene eae, 93 isolates contained ehxA, and 73 isolates carried astA. The prevalence of other putative adhesion genes saa, paa, efa1, and toxB was 28.90% (87), 6.98% (21), 2.31% (7), and 1% (3), respectively. The phylogenetic distribution of isolates was analyzed by multilocus sequence typing (MLST). Ninety-four sequence types were assigned across the 301 isolates. A subset of isolates recovered from yak and pika residing in the similar wild environments, Qinghai-Tibetan plateau, showed similar genetic profiles and more tendencies to cluster together. Isolates from goat and mutton exhibited close genetic relatedness with those from human-derived isolates, providing evidence that transmission may have occurred locally within intraspecies or interspecies, and importantly, from animal reservoirs, or raw meats to humans. Comparing isolates in this study with highly virulent strains by MLST, along with serotyping and virulence profiles, it is conceivable that some of isolates from goat, yak, or raw meats may have potential to cause human diseases.

An in vitro combined antibiotic-antibody treatment eliminates toxicity from Shiga toxin-producing Escherichia coli.

Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the "big six" serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments.

Epidemiology and microbiology of Shiga toxin-producing Escherichia coli other than serogroup O157 in England, 2009-2013.

The implementation of direct testing of clinical faecal specimens for gastrointestinal (GI) pathogens by PCR offers a sensitive and comprehensive approach for the detection of Shiga toxin-producing Escherichia coli (STEC). The introduction of a commercial PCR assay, known as GI PCR, for the detection of GI pathogens at three frontline hospital laboratories in England between December 2012 and December 2013 led to a significant increase in detection of STEC other than serogroup O157 (non-O157 STEC). In 2013, 47 isolates were detected in England, compared with 57 in the preceding 4 years (2009-2012). The most common non-O157 STEC serogroup detected was O26 (23.2 %). A total of 47 (47.5 %) STEC isolates had stx2 only, 28 (28.3 %) carried stx1 and stx2, and the remaining 24 (24.2 %) had stx1 only. Stx2a (64.0 %) was the most frequently detected Stx2 subtype. The eae (intimin) gene was detected in 52 (52.5 %) non-O157 STEC isolates. Six strains of STEC O104 had aggR, but this gene was not detected in any other STEC serogroups in this study. Haemolytic ureamic syndrome was significantly associated with STEC strains possessing eae [odds ratio (OR) 5.845, P = 0.0235] and/or stx2a (OR 9.56, P = 0.0034) subtypes. A matched case-control analysis indicated an association between non-O157 STEC cases and contact with farm animals. Widespread implementation of the PCR approach in England will determine the true incidence of non-O157 STEC infection, highlight the burden in terms of morbidity and mortality, and facilitate the examination of risk factors to indicate whether there are niche risk exposures for particular strains.

Isolation and Characterization of Shiga Toxin-Producing Escherichia coli Serogroups O26, O45, O103, O111, O113, O121, O145, and O157 Shed from Range and Feedlot Cattle from Postweaning to Slaughter

Cattle are the main reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains. E. coli O26, O45, O103, O111, O121, O145, and O157 are among the STEC serogroups that cause severe foodborne illness and have been declared as adulterants by the U.S. Department of Agriculture, Food Safety and Inspection Service. The objectives of this study were (i) to estimate the prevalence of non-O157 STEC and E. coli O157 in naturally infected beef cows and in steer calves at postweaning, during finishing, and at slaughter and (ii) to test non-O157 STEC isolates for the presence of virulence genes stx1, stx2, eaeA, and ehlyA. Samples were collected from study animals during multiple sampling periods and included fecal grabs, rectal swabs, and midline sponge samples. Laboratory culture, PCR, and multiplex PCR were performed to recover and identify E. coli and the virulence genes. The prevalence of non-O157 STEC (serogroups O26, O45, O103, O111, O121, O113, and O145) fecal shedding ranged from 8% (4 of48 samples) to 39% (15 of 38 samples) in cows and 2% (1 of 47 samples) to 38% (9 of 24 samples) in steer calves. The prevalence of E. coli O157 fecal shedding ranged from 0% (0 of 38 samples) to 52% (25 of 48 samples) in cows and 2% (1 of 47 samples) to 31% (15 of 48 samples) in steer calves. In steer calves, the prevalence of non-O157 STEC and E. coli O157 was highest at postweaning, at 16% (15 of 96 samples) and 23% (22 of 96 samples), respectively. Among the 208 non-O157 STEC isolates, 79% (164 isolates) had stx1, 79% (165 isolates) had stx2, and 58% (121 isolates) had both stx1 and stx2 genes. The percentage of non-O157 STEC isolates encoding the eaeA gene was low; of the 165 isolates tested, 8 (5%) were positive for eaeA and 135 (82%) were positive for ehlyA. Findings from this study provide further evidence of non-O157 STEC shedding in beef cows and steer calves particularly at the stage of postweaning and before entry into the feedlot.

Non-O157 Shiga Toxin-Producing Escherichia coli in U.S. Retail Ground Beef

Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are the leading cause of STEC-associated infections in humans in the United States. In the United States, these organisms are considered adulterants in raw nonintact beef products and in intact beef destined to be made into or used in nonintact raw beef products. The objective of this study was to provide an estimate of the burden of the six serogroups of non-O157 STEC in ground beef obtained from retail stores across the United States. A convenience sample of commercial ground beef products (n = 1,129) were purchased from retail stores in 24 states from October 2011 to May 2012. The samples had various lean/fat proportions, muscle group of origin (chuck, round, sirloin, or not specified), and packaging types. For each ground beef sample, 25 g was inoculated in 225 ml of modified tryptic soy broth, stomached for 1 min, and then incubated at 41°C for 18 ± 2 h. These enrichment cultures were then screened for stx, eae, and O group genes using a commercially available, closed-platform PCR-based method. The potential positive samples were subjected to immunomagnetic separation and plated on modified Rainbow agar. Morphologically typical colonies were subjected to latex agglutination and PCR determination of stx and eae genes. Nine (0.8%) of the ground beef samples were potentially positive for at least one STEC serogroup after PCR screening. The serogroups detected by PCR assay were O26 (four samples), O103 (four samples), O145 (three samples), O45 (two samples), and O121 (one sample). No STEC isolates belonging to these serogroups were recovered from the sample cultures. The current research provides updated surveillance data for non-O157 STEC isolates among commercial ground beef products and information regarding the potential sources of contamination from different parts of beef trims destined for ground beef production.

Prevalence of shiga toxin producing Escherichia coli, Salmonella enterica, and Listeria monocytogenes at public access watershed sites in a California Central Coast agricultural region

Produce contaminated with enteric pathogens is a major source of foodborne illness in the United States. Lakes, streams, rivers, and ponds were sampled with Moore swabs bi-monthly for over 2 years at 30 locations in the vicinity of a leafy green growing region on the Central California Coast and screened for Shiga toxin producing Escherichia coli (STEC), Salmonella enterica, and Listeria monocytogenes to evaluate the prevalence and persistence of pathogen subtypes. The prevalence of STEC from 1386 samples was 11%; 110 samples (8%) contained E. coli O157:H7 with the highest prevalence occurring close to cattle operations. Non-O157 STEC isolates represented major clinical O-types and 57% contained both shiga toxin types 1 and 2 and intimin. Multiple Locus Variable Number Tandem Repeat Analysis of STEC isolates indicated prevalent strains during the period of study. Notably, Salmonella was present at high levels throughout the sampling region with 65% prevalence in 1405 samples resulting in 996 isolates with slightly lower prevalence in late autumn. There were 2, 8, and 14 sites that were Salmonella-positive over 90, 80, and 70% of the time, respectively. The serotypes identified most often were 6,8:d:-, Typhimurium, and Give. Interestingly, analysis by Pulsed Field Gel Electrophoresis indicated persistence and transport of pulsotypes in the region over several years. In this original study of L. monocytogenes in the region prevalence was 43% of 1405 samples resulting in 635 individual isolates. Over 85% of the isolates belonged to serotype 4b with serotypes 1/2a, 1/2b, 3a, 4d with 4e representing the rest, and there were 12 and 2 sites that were positive over 50 and 80% of the time, respectively. Although surface water is not directly used for irrigation in this region, transport to the produce can occur by other means. This environmental survey assesses initial contamination levels toward an understanding of transport leading to produce recalls or outbreaks.

Comparison between ImmunoCard STAT!® and real-time PCR as screening tools for both O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in Southern Alberta, Canada

An increasing number of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections and outbreaks have been reported. In this study, we evaluated the performance of ImmunoCard STAT!® (Meridian Bioscience, Inc., Cincinnati, OH, USA) as a method to screen stool specimens for STEC (O157 and non-O157). An in-house real-time PCR method was used as the “gold standard”. We also evaluated the prevalence and clinical characteristics of STEC infections in the Alberta South West Zone. From July to November 2011, 819 stool specimens submitted for routine stool culture were tested. With our in-house real-time PCR, 7 O157:H7 and 10 non-O157 STEC isolates were identified for a total of 17 STECs. In comparison, ImmunoCard STAT!® identified a total of 6, resulting in a sensitivity and specificity of 35% and 99%, respectively (P < 0.05). Because of the low sensitivity, ImmunoCard STAT!® cannot be recommended as a routine screening test for STEC from enriched stool specimens. The rate of STEC positivity as detected by PCR was 2.08%, of which 0.86% was O157:H7 and 1.22% non-O157 STEC. Five of the 7 cases of STEC O157 infection experienced bloody diarrhea, and 1 developed hemolytic uremic syndrome.