Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates.
ABSTRACT: A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar aadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII, aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in Citrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coli O157:H7.
Project description:The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the "big six" serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ?7,000-year span, spacer deletion was the primary force generating CRISPR diversity.
Project description:Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-?1 (4.8%; 19 samples), stx-wzx(O103)-eae-? (1.3%; five samples), stx-ihp1(O145)-eae-?1 (0.8%; three samples), and stx-rfbE(O157)-eae-?1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eae?1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eae?1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.
Project description:A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx(1) genes, 278 (54%) possessed stx(2) genes, and 135 (26%) possessed both stx(1) and stx(2). Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx(2) eae-gamma1 ehxA (46 isolates) was the most common, followed by O157:H7 stx(1) stx(2) eae-gamma1 ehxA (34 isolates), O113:H21 stx(2) (25 isolates), O22:H8 stx(1) stx(2) ehxA (15 isolates), O26:H11 stx(1) eae-beta1 ehxA (14 isolates), and O77:H41 stx(2) ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intimin beta1, 82 O157:H7 isolates possessed intimin gamma1, three O111:H- isolates had intimin type gamma2, one O49:H- strain showed intimin type delta, 13 (six of serotype O103:H2) isolates had intimin type epsilon and eight (four of serotype O156:H-) isolates had intimin zeta. We have identified a new variant of the eae intimin gene designated xi (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.
Project description:Pathogenic strains of Escherichia coli, such as E. coli O157:H7, have a low infectious dose and an ability to survive in acidic foods. These bacteria have evolved at least three distinct mechanisms of acid resistance (AR), including two amino acid decarboxylase-dependent systems (arginine and glutamate) and a glucose catabolite-repressed system. We quantified the survival rates for each AR mechanism separately in clinical isolates representing three groups of Shiga toxin-producing E. coli (STEC) clones (O157:H7, O26:H11/O111:H8, and O121:H19) and six commensal strains from ECOR group A. Members of the STEC clones were not significantly more acid resistant than the commensal strains when analyzed using any individual AR mechanism. The glutamate system provided the best protection in a highly acidic environment for all groups of isolates (<0.1 log reduction in CFU/ml per hour at pH 2.0). Under these conditions, there was notable variation in survival rates among the 30 O157:H7 strains, which depended in part on Mg(2+) concentration. The arginine system provided better protection at pH 2.5, with a range of 0.03 to 0.41 log reduction per hour, compared to the oxidative system, with a range of 0.13 to 0.64 log reduction per hour. The average survival rate for the O157:H7 clonal group was significantly less than that of the other STEC clones in the glutamate and arginine systems and significantly less than that of the O26/O111 clone in the oxidative system, indicating that this clonal group is not exceptionally acid resistant with these specific mechanisms.
Project description:Objectives: The aim of this study was to characterize a collection of 95 Shigatoxin-producing E.coli (STEC) isolated from human patients in Switzerland during 2010-2014. Methods: We performed O and H serotyping and molecular subtyping. Results: The five most common serogroups were O157, O145, O26, O103, and O146. Of the 95 strains, 35 (36.8%) carried stx1 genes only, 43 strains (45.2%) carried stx2 and 17 (17.9%) harbored combinations of stx1 and stx2 genes. Stx1a (42 strains) and stx2a (32 strains) were the most frequently detected stx subtypes. Genes for intimin (eae), hemolysin (hly), iron-regulated adhesion (iha), and the subtilase cytotoxin subtypes subAB1, subAB2-1, subAB2-2, or subAB2-3 were detected in 70.5, 83.2, 74.7, and 20% of the strains, respectively. Multilocus sequence typing assigned the majority (58.9%) of the isolates to five different clonal complexes (CC), 11, 32, 29, 20, and 165, respectively. CC11 included all O157:[H7] and O55:[H7] isolates. CC32 comprised O145:[H28] isolates, and O145:[H25] belonged to sequence type (ST) 342. CC29 contained isolates of the O26:[H11], O111:[H8] and O118:[Hnt] serogroups, and CC20 encompassed isolates of O51:H49/[Hnt] and O103:[H2]. CC165 included isolates typed O80:[H2]-ST301, all harboring stx2d, eae-?, hly, and 66.7% additionally harboring iha. All O80:[H2]-ST301 strains harbored at least 7 genes carried by pS88, a plasmid associated with extraintestinal virulence. Compared to data from Switzerland from the years 2000-2009, an increase of the proportion of non-O157 STEC infections was observed as well as an increase of infections due to STEC O146. By contrast, the prevalence of the highly virulent German clone STEC O26:[H11]-ST29 decreased from 11.3% during 2000-2009 to 1.1% for the time span 2010-2014. The detection of O80:[H2]-ST301 harboring stx2d, eae-?, hly, iha, and pS88 related genes suggests an ongoing emergence in Switzerland of an unusual, highly pathogenic STEC serotype. Conclusions: Serotyping and molecular subtyping of clinical STEC demonstrate that although STEC O157 predominates among STEC isolated from diseased humans, non-O157 STEC infections are increasing in Switzerland, including those due to STEC O146:[H2/H21/H28]-ST442/ST738 harboring subAB variants, and the recently emerged STEC O80:[H2]-ST301 harboring eae-? and pS88 associated extraintestinal pathogenic virulence genes.
Project description:Rapid and specific detection of Shiga toxin-producing Escherichia coli (STEC) strains with a high level of virulence for humans has become a priority for public health authorities. This study reports on the development of a low-density macroarray for simultaneously testing the genes stx1, stx2, eae, and ehxA and six different nle genes issued from genomic islands OI-122 (ent, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). Various strains of E. coli isolated from the environment, food, animals, and healthy children have been compared with clinical isolates of various seropathotypes. The eae gene was detected in all enteropathogenic E. coli (EPEC) strains as well as in enterohemorrhagic E. coli (EHEC) strains, except in EHEC O91:H21 and EHEC O113:H21. The gene ehxA was more prevalent in EHEC (90%) than in STEC (42.66%) strains, in which it was unequally distributed. The nle genes were detected only in some EPEC and EHEC strains but with various distributions, showing that nle genes are strain and/or serotype specific, probably reflecting adaptation of the strains to different hosts or environmental niches. One characteristic nle gene distribution in EHEC O157:[H7], O111:[H8], O26:[H11], O103:H25, O118:[H16], O121:[H19], O5:H-, O55:H7, O123:H11, O172:H25, and O165:H25 was ent/espL2, nleB, nleE, nleF, nleH1-2, nleA. (Brackets indicate genotyping of the flic or rfb genes.) A second nle pattern (ent/espL2, nleB, nleE, nleH1-2) was characteristic of EHEC O103:H2, O145:[H28], O45:H2, and O15:H2. The presence of eae, ent/espL2, nleB, nleE, and nleH1-2 genes is a clear signature of STEC strains with high virulence for humans.
Project description:Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha, terD, and hlyA) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers (iha, terD, hlyA, and espP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.
Project description:Shiga toxin-producing Escherichia coli (STEC) can cause severe gastrointestinal disease and colonization among food handlers. In Japan, STEC infection is a notifiable disease, and food handlers are required to undergo routine stool examination for STEC. However, the molecular epidemiology of STEC is not entirely known. We investigated the genomic characteristics of STEC from patients and asymptomatic food handlers in Miyagi Prefecture, Japan. Whole-genome sequencing (WGS) was performed on 65 STEC isolates obtained from 38 patients and 27 food handlers by public health surveillance in Miyagi Prefecture between April 2016 and March 2017. Isolates of O157:H7 ST11 and O26:H11 ST21 were predominant (n = 19, 29%, respectively). Non-O157 isolates accounted for 69% (n = 45) of all isolates. Among 48 isolates with serotypes found in the patients (serotype O157:H7 and 5 non-O157 serotypes, O26:H11, O103:H2, O103:H8, O121:H19 and O145:H28), adhesion genes eae, tir, and espB, and type III secretion system genes espA, espJ, nleA, nleB, and nleC were detected in 41 to 47 isolates (85-98%), whereas isolates with other serotypes found only in food handlers were negative for all of these genes. Non-O157 isolates were especially prevalent among patients younger than 5 years old. Shiga-toxin gene stx1a, adhesion gene efa1, secretion system genes espF and cif, and fimbrial gene lpfA were significantly more frequent among non-O157 isolates from patients than among O157 isolates from patients. The most prevalent resistance genes among our STEC isolates were aminoglycoside resistance genes, followed by sulfamethoxazole/trimethoprim resistance genes. WGS revealed that 20 isolates were divided into 9 indistinguishable core genomes (<5 SNPs), demonstrating clonal expansion of these STEC strains in our region, including an O26:H11 strain with stx1a+stx2a. Non-O157 STEC with multiple virulence genes were prevalent among both patients and food handlers in our region of Japan, highlighting the importance of monitoring the genomic characteristics of STEC.
Project description:Evolutionary analyses of enterohemorrhagic Escherichia coli (EHEC) have identified two distantly related clonal groups: EHEC 1, including serotype O157:H7 and its inferred ancestor O55:H7; and EHEC 2, comprised of several serogroups (O26, O111, O118, etc.). These two clonal groups differ in their virulence and global distribution. Although several fully annotated genomic sequences exist for strains of serotype O157:H7, much less is known about the genomic composition of EHEC 2. In this study, we analyzed a set of 24 clinical EHEC 2 strains representing serotypes O26:H11, O111:H8/H11, O118:H16, O153:H11 and O15:H11 from humans and animals by comparative genomic hybridization (CGH) on an oligoarray based on the O157:H7 Sakai genome.Backbone genes, defined as genes shared by Sakai and K-12, were highly conserved in EHEC 2. The proportion of Sakai phage genes in EHEC 2 was substantially greater than that of Sakai-specific bacterial (non-phage) genes. This proportion was inverted in O55:H7, reiterating that a subset of Sakai bacterial genes is specific to EHEC 1. Split decomposition analysis of gene content revealed that O111:H8 was more genetically uniform and distinct from other EHEC 2 strains, with respect to the Sakai O157:H7 gene distribution. Serotype O26:H11 was the most heterogeneous EHEC 2 subpopulation, comprised of strains with the highest as well as the lowest levels of Sakai gene content conservation. Of the 979 parsimoniously informative genes, 15% were found to be compatible and their distribution in EHEC 2 clustered O111:H8 and O118:H16 strains by serotype. CGH data suggested divergence of the LEE island from the LEE1 to the LEE4 operon, and also between animal and human isolates irrespective of serotype. No correlation was found between gene contents and geographic locations of EHEC 2 strains.The gene content variation of phage-related genes in EHEC 2 strains supports the hypothesis that extensive modular shuffling of mobile DNA elements has occurred among EHEC strains. These results suggest that EHEC 2 is a multiform pathogenic clonal complex, characterized by substantial intra-serotype genetic variation. The heterogeneous distribution of mobile elements has impacted the diversification of O26:H11 more than other EHEC 2 serotypes.
Project description:Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. The increasing incidence of non-O157 STEC has posed a great risk to public health. Besides the Shiga toxin (Stx), the adherence factor, intimin, coded by eae gene plays a critical role in STEC pathogenesis. In this study, we investigated the prevalence and polymorphisms of eae gene in non-O157 STEC strains isolated from different sources in China. Among 735 non-O157 STEC strains, eae was present in 70 (9.5%) strains. Eighteen different eae genotypes were identified in 62 eae-positive STEC strains with the nucleotide identities ranging from 86.01% to 99.97%. Among which, seven genotypes were newly identified in this study. The eighteen eae genotypes can be categorized into five eae subtypes, namely ?1, ?1, ?1, ?3 and ?. Associations between eae subtypes/genotypes and serotypes as well as origins of strains were observed in this study. Strains belonging to serotypes O26:H11, O103:H2, O111:H8 are associated with particular eae subtypes, i.e., ?1, ?1, ?, respectively. Most strains from diarrheal patients (7/9, 77.8%) carried eae-?1 subtype, while most isolates from cattle (23/26, 88.5%) carried eae-?3 subtype. This study demonstrated a genetic diversity of eae gene in non-O157 STEC strains from different sources in China.