Antimicrobial resistance of Shiga toxin (verotoxin)-producing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in Spain

A total of 722 Shiga toxin-producing Escherichia coli (STEC) isolates recovered from humans, cattle, ovines and food during the period from 1992 to 1999 in Spain were examined to determine antimicrobial resistance profiles and their association with serotypes, phage types and virulence genes. Fifty-eight (41%) out of 141 STEC O157:H7 strains and 240 (41%) out of 581 non-O157 STEC strains showed resistance to at least one of the 26 antimicrobial agents tested. STEC O157:H7 showed a higher percentage of resistant strains recovered from bovine (53%) and beef meat (57%) than from human (23%) and ovine (20%) sources, whereas the highest prevalence of antimicrobial resistance in non-O157 STEC was found among isolates recovered from beef meat (55%) and human patients (47%). Sulfisoxazole (36%) had the most common antimicrobial resistance, followed by tetracycline (32%), streptomycin (29%), ampicillin (10%), trimethoprim (8%), cotrimoxazole (8%), chloramphenicol (7%), kanamycin (7%), piperacillin (6%), and neomycin (5%). The multiple resistance pattern most often observed was that of streptomycin, sulfisoxazole, and tetracycline. Ten (7%) STEC O157:H7 and 71 (12%) non-O157 strains were resistant to five or more antimicrobial agents. Most strains showing resistance to five or more antimicrobial agents belonged to serotypes O4:H4 (4 strains), O8:H21 (3 strains), O20:H19 (6 strains), O26:H11 (8 strains eae-beta1), O111:H- (3 strains eae-gamma2), O118:H- (2 strains eae-beta1), O118:H16 (5 strains eae-beta1), O128:H- (2 strains), O145:H8 or O145:H- (2 strains eae-gamma1), O157:H7 (10 strains eae-gamma1), O171:H25 (3 strains), O177:H11 (5 strains eae-beta1), ONT:H- (3 strains/1 eae-beta1) and ONT:H21 (2 strains). Interestingly, most of these serotypes, i.e., those indicated in bold) were found among human STEC strains isolated from patients with hemolytic uremic-syndrome (HUS) reported in previous studies. We also detected, among non-O157 strains, an association between a higher level of multiple resistance to antibiotics and the presence of the virulence genes eae and stx(1). Moreover, STEC O157:H7, showed an association between certain phage types, PT21/28 (90%), PT23 (75%), PT34 (75%), and PT2 (54%), with a higher number of resistant strains. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in animals is highly recommended.     

Clonal structure of Shiga toxin (Stx)-producing and beta-D-glucuronidase-positive Escherichia coli O157:H7 strains isolated from outbreaks and sporadic cases in Hokkaido, Japan

A total of 22 clonal phenotypic variants of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 was isolated from six different locations in Hokkaido, Japan. These isolates were negative for sorbitol fermentation but positive for beta-D-glucuronidase (GUD+). They carried eaeA, EHEC-hlyA, pas and etpD genes like typical E. coli O157:H7 and, in addition, st1 and stx2 genes. However, they were shown to lack katP and espP genes that are present in typical STEC O157:H7. All these atypical GUD+ STEC O157:H7 isolates had very similar antimicrobial susceptibilities. Pulsed-field gel electrophoresis analysis with XbaI, SfiI, SwaI, SpeI and NotI indicated that they were identical or closely related to one another. From their phenotypic and genotypic features, these GUD+ STEC O157:H7 isolates may represent a distinct clone among STEC O157.     

Shiga toxin-producing Escherichia coli in children: diagnosis and clinical manifestations of O157:H7 and non-O157:H7 infection

Shiga toxin-producing Escherichia coli (STEC), a cause of food-borne colitis and hemolytic-uremic syndrome in children, can be serotype O157:H7 (O157) or other serotypes (non-O157). E. coli O157 can be detected by culture with sorbitol-MacConkey agar (SMAC), but non-O157 STEC cannot be detected with this medium. Both O157 and non-O157 STEC can be detected by immunoassay for Shiga toxins 1 and 2. The objectives of this study were first to compare the diagnostic utility of SMAC to that of the Premier EHEC enzyme immunoassay (Meridian Diagnostics) for detection of STEC in children and second to compare the clinical and laboratory characteristics of children with serotype O157:H7 STEC and non-O157:H7 STEC infections. Stool samples submitted for testing for STEC between April 2004 and September 2009 were tested by both SMAC culture and the Premier EHEC assay at Children's Hospital Boston. Samples positive by either test were sent for confirmatory testing and serotyping at the Hinton State Laboratory Institute (HSLI). Chart review was performed on children with confirmed STEC infection. Of 5,110 children tested for STEC, 50 (0.9%) had STEC infection confirmed by culture; 33 were O157:H7 and 17 were non-O157:H7. The Premier EHEC assay and SMAC culture detected 96.0% and 58.0% of culture-confirmed STEC isolates (any serotype), respectively, and 93.9% and 87.9% of STEC O157:H7 isolates, respectively. There were no significant differences in disease severity or laboratory manifestations of STEC infection between children with O157:H7 and those with non-O157 STEC. The Premier EHEC assay was significantly more sensitive than SMAC culture for diagnosis of STEC, and O157:H7 and non-O157:H7 STEC caused infections of similar severity in children.     

Molecular Profiling of Escherichia coli O157:H7 and Non-O157 Strains Isolated from Humans and Cattle in Alberta,Canada

Virulence markers in Shiga toxin-producing Escherichia coli (STEC) and their association with diseases remain largely unknown. This study determines the importance of 44 genetic markers for STEC (O157 and non-O157) from human clinical cases and their correlation to disease outcome. STEC isolated from a cattle surveillance program were also included. The virulence genes tested were present in almost all O157:H7 isolates but highly variable in non-O157 STEC isolates. Patient age was a significant determinant of clinical outcome.     

Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.     

Analysis of antimicrobial resistance genes detected in multidrug-resistant Salmonella enterica serovar Typhimurium isolated from food animals

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is the most prevalent penta-resistant serovar isolated from animals by the U.S. National Antimicrobial Resistance Monitoring System. Penta-resistant isolates are often resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. To investigate MDR in Salmonella Typhimurium (including variant 5-), one isolate each from cattle, poultry, and swine with at least the ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline phenotype were selected for each year from 1997 to 2007 (n = 33) for microarray analysis of antimicrobial resistance, incompatibility IncA/C, and HI1 plasmid genes. Cluster analysis based on these data separated 31 of the isolates into two groups A and B (15 and 16 isolates, respectively). Isolates in group A were phage type DT104 or U302 and were mostly swine isolates (7/15). Genes detected included intI1, bla(PSE-1), floR, aadA, sulI, tet(G), and tetR, which are often found in Salmonella Genomic Island I. Isolates in group B had numerous IncA/C plasmid genes detected and were mostly cattle isolates (9/16). Genes detected included bla(CMY-2), floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR, which are often found on MDR-AmpC IncA/C plasmids. The IncA/C replicon was also detected in all group B isolates. The two remaining isolates did not cluster with any others and both had many HI1 plasmid genes detected. Linkage disequilibrium analysis detected significant associations between plasmid replicon type, phage type, and animal source. These data suggest that MDR in Salmonella Typhimurium is associated with DT104/Salmonella Genomic Island I or IncA/C MDR-AmpC encoding plasmids and these genetic elements have persisted throughout the study period.     

Verocytotoxin-producing Escherichia coli (VTEC) O157 and other VTEC from human infections in England and Wales: 1995-1998

A total of 3429 isolations of verocytotoxin-producing Escherichia coli O157 (VTEC O157) was confirmed from human sources in England and Wales during the period 1995-1998. The largest annual total was 1087 in 1997. Most infections occurred in the third quarter of each year. The overall rate of infection ranged from 1.28 to 2.10/100,000 population and showed regional variation. The highest incidence was in children aged 1-4 years. Annually, between 5% and 11% of strains were from patients who had travelled abroad. There were 67 general outbreaks of infection represented by 407 (11.9%) VTEC O157 isolates. Outbreaks involved transmission by contaminated food or water, person-to-person spread and direct or indirect animal contact, and five were associated with foreign travel. The majority (76%) of strains carried verocytotoxin (VT) 2 genes and 23.3% were VT1+VT2. Most strains had the flagellar antigen H7, but c. 14% were non-motile. Approximately 20% of isolates were resistant to antimicrobial agents, predominantly streptomycin, sulphonamides and tetracycline. In addition to VTEC O157, strains of serogroup O157 that did not possess VT genes were identified. These were either derivatives of VTEC O157 that had lost VT genes or were strains with H antigens other than H7 that have never been associated with VT production. Strains of VTEC other than O157 were characterised. Most were associated with diarrhoea, bloody diarrhoea or haemolytic uraemic syndrome and had virulence markers in addition to VT.     

Shiga toxin-producing Escherichia coli in Central Greece: prevalence and virulence genes of O157:H7 and non-O157 in animal feces, vegetables, and humans

In Greece, Shiga toxin-producing Escherichia coli (STEC) have only been sporadically reported. The objective of this study was to estimate the prevalence of STEC and Escherichia coli O157:H7 in farm animals, vegetables, and humans in Greece. A total number of 1,010 fecal samples were collected from farm animals (sheep, goats, cattle, chickens, pigs), 667 diarrheal samples from humans, and 60 from vegetables, which were cultured in specific media for STEC isolates. Enzyme-linked immunosorbent assay (ELISA) was used to detect toxin-producing colonies, which, subsequently, were subjected to a multiplex polymerase chain reaction (PCR) for stx1, stx2, eae, rfbE _O157, and fliC _h7 genes. Eighty isolates (7.9 %) from animal samples were found to produce Shiga toxin by ELISA, while by PCR, O157 STEC isolates were detected from 8 (0.8 %) samples and non-O157 STEC isolates from 43 (4.2 %) samples. STEC isolates were recovered mainly from sheep and goats, rarely from cattle, and not from pigs and chickens, suggesting that small ruminants constitute a potential risk for human infections. However, only three human specimens (0.4 %) were positive for the detection of Shiga toxins and all were PCR-negative. Similarly, all 60 vegetable samples were negative for toxin production and for toxin genes, but three samples (two roman rockets and one spinach) were positive by PCR for rfbE _O157 and fliC _h7 genes. These findings indicate that sheep, goats, cattle, and leafy vegetables can be a reservoir of STEC and Escherichia coli O157:H7 isolates in Greece, which are still rarely detected among humans.     

Probiotic bifidobacteria protect mice from lethal infection with Shiga toxin-producing Escherichia coli O157:H7

The anti-infectious activity of probiotic Bifidobacteria against Shiga toxin-producing Escherichia coli (STEC) O157:H7 was examined in a fatal mouse STEC infection model. Stable colonization of the murine intestines was achieved by the oral administration of Bifidobacterium breve strain Yakult (naturally resistant to streptomycin sulfate) as long as the mice were treated with streptomycin in their drinking water (5 mg/ml). The pathogenicity of STEC infection, characterized by marked body weight loss and subsequent death, observed in the infected controls was dramatically inhibited in the B. breve-colonized group. Moreover, Stx production by STEC cells in the intestine was almost completely inhibited in the B. breve-colonized group. A comparison of anti-STEC activity among several Bifidobacterium strains with natural resistance to streptomycin revealed that strains such as Bifidobacterium bifidum ATCC 15696 and Bifidobacterium catenulatum ATCC 27539(T) did not confer an anti-infectious activity, despite achieving high population levels similar to those of effective strains, such as B. breve strain Yakult and Bifidobacterium pseudocatenulatum DSM 20439. The effective strains produced a high concentration of acetic acid (56 mM) and lowered the pH of the intestine (to pH 6.75) compared to the infected control group (acetic acid concentration, 28 mM; pH, 7.15); these effects were thought to be related to the anti-infectious activity of these strains because the combination of a high concentration of acetic acid and a low pH was found to inhibit Stx production during STEC growth in vitro.     

Prevalence of EHEC O157:H7 in patients with diarrhoea in Lagos, Nigeria

The prevalence of sorbitol-nonfermenting Escherichia coli O157:H7 (EHEC) was assessed in 100 patients with diarrhoea by stool culture on sorbitol MacConkey agar. The cytotoxicity of the EHEC strains was analysed by Vero cell assay and the antimicrobial susceptibility pattern of the isolates was determined. Detection rate of EHEC O157:H7 was 6%. Five of the six patients were males. Three of the isolates were from children and one was from a teenager. All strains induced cytotoxic effects in the Vero cell assay. All isolates were susceptible to most of the antimicrobials tested. The results showed that diarrhoea caused by EHEC O157:H7, a potentially life-threatening pathogen, has remained common particularly among the child population of Lagos during the past 10 years (5). There must therefore be adequate meat and food inspection to improve the general hygiene of local fast food restaurants, so-called 'bukkas', which are regarded as likely sources of infection.     

Detection and characterization of class 1 integrons in enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) strains isolated from humans, cattle, and food and belonging to serogroups O26 (7 strains), O111 (19 strains), and O157 (70 strains) were examined for susceptibility to 11 antimicrobial drugs. Fifty-nine strains showing resistance to at least one of the drugs were examined by PCR for the presence of class 1 integrons, which were identified in 17 strains. Integrons were found more frequently in strains belonging to serogroups O111 and O26 than in the O157 isolates. DNA sequence analysis demonstrated that most of the integrons contained the aadA1 gene cassette conferring resistance to streptomycin/ spectinomycin, alone or associated with the drfA1 gene cassette conferring resistance to trimethoprim. One integron, identified in a O157:H7 strain, carried the aadA2 and dfrA12 gene cassettes, conferring resistance to streptomycin/spectinomycin and trimethoprim, and the open reading frame F (OrfF) encoding unknown functions. Most of the integrons were carried by Tn21 derivative transposons and were transferable by conjugation to an E. coli K-12 strain. In conclusion, integrons and antibiotic resistance genes can be frequently found in EHEC strains, particularly E. coli O111 and E. coli O26, and their presence could complicate therapeutic trials.     

Variation in clonality and antibiotic-resistance genes among multiresistant Salmonella enterica serotype typhimurium phage-type U302 (MR U302) from humans, animals, and foods

Since 1990 multiresistant (MR) Salmonella enterica serotype Typhimurium definitive phage-type (DT) 104 (MR DT104) and closely related phage types have emerged as a worldwide health problem in humans and food animals. In this study the presence of the blaCARB-2 (ampicillin), cmlA (chloramphenicol), aadA2 (streptomycin/spectinomycin), sul1 (sulphonamide), and tetG (tetracycline) resistance genes in isolates of one such phage type, U302, have been determined. In addition blaTEM primers have been used for the detection of TEM-type beta-lactamases. Isolates have also been characterized by plasmid profile and pulsed field gel electrophoresis (PFGE). Thirty-three of 39 isolates were positive for blaCARB-2, cmlA, aadA2, sul1 and tetG, four for blaTEM, aadA2 and sul1, one for aadA2 and sul1, and one for blaTEM only. blaTEM-mediated ampicillin resistance was transferred to Escherichia coli K12 from three isolates along with other resistance markers, including resistance to chloramphenicol, streptomycin, spectinomycin, sulphonamides, and tetracyclines. Strains carried up to 6 plasmids and 34 plasmid profiles were identified. Although the majority of strains (33/39) produced a PFGE profile identical to that predominant in MR DT104, six different patterns were generated demonstrating the presence of various clones within MR U302. The results show that the majority of the MR U302 strains studied possessed the same antibiotic resistance genes as MR DT104. However, isolates with distinctive PFGE patterns can have different mechanisms of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines. Such resistance genes may be borne on transmissible plasmids.     

Identification and distribution of the enterohemorrhagic Escherichia coli factor for adherence (efa1) gene in sorbitol-fermenting Escherichia coli O157: H-

Sorbitol-fermenting (SF) Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H- strains are emerging as causes of hemorrhagic colitis and the hemolytic-uremic syndrome in Europe. Using subtractive hybridization between SF STEC O157:H- strain 493/89 and STEC O157:H7 strain EDL933, three different fragments, of approximately 700 bp in length, were identified. Each demonstrated > 99% homology to genes encoding the enterohemorrhagic E. coli factor for adherence (efa1) and lymphostatin (lifA). Therefore, a cosmid library was constructed from SF STEC O157:H- strain 493/89, and one clone containing these fragments was sequenced. This sequencing demonstrated a 9669-bp open reading frame (ORF) that had 99.9% sequence homology to efa1 of STEC O111:H- strain E45035 and to lifA of an enteropathogenic E. coli O127:H6 strain E2348/69. In STEC O157:H7 strain EDL933, only small (ca. 3 kb) initial and terminal fragments of this ORF are present. PCR analysis with primers complementary to the efa1/lifA sequence of strain 493/89 indicated that the complete sequence is present in each of 10 SF STEC O157:H- isolates but in none of 10 STEC O157:H7 strains investigated. The presence of the complete efa1/lifA also in both tested E. coli O55:H7 strains supports the hypothesis that SF STEC O157:H- are phylogenetically closer to the proposed E. coli O55:H7 ancestor than STEC O157:H7. Our data demonstrate the presence of a potential virulence gene in SF STEC O157:H- that is only rudimentarily present in STEC O157:H7.     

Chronological study of antibiotic resistances and their relevant genes in Korean avian pathogenic Escherichia coli isolates

Antibiograms and relevant genotypes of Korean avian pathogenic Escherichia coli (APEC) isolates (n = 101) recovered between 1985 and 2005 were assessed via disc diffusion test, PCR, restriction enzyme analysis, and sequencing. These isolates were highly resistant to tetracycline (84.2%), streptomycin (84.2%), enrofloxacin (71.3%), and ampicillin (67.3%), and most of the tetracycline, streptomycin, enrofloxacin, and ampicillin resistances were associated with tetA and/or tetB, aadA and/or strA-strB, mutations in gyrA and/or parC, and TEM, respectively. Class 1 integrons were detected in 40 isolates (39.6%), and a variety of gene cassettes conferring streptomycin (aadA), gentamicin (aadB), and trimethoprim (dfr) resistances were identified: aadA1a (27.5%), dfrV-orfD (2.5%), aadB-aadA1a (2.5%), dfrI-aadA1a (47.5%), dfrXVII-aadA5 (12.5%), and dfrXII-orfF-aadA2 (7.5%). In addition, several types of common promoters (P(ant)) of the gene cassettes (hybrid P1, weak P1, or weak P1 plus P2) and single-nucleotide polymorphisms in aadA1a were identified. The results of a chronological analysis demonstrated significant and continuous increases in the frequencies of resistances to several antibiotics (tetracycline, streptomycin, enrofloxacin, ampicillin, and trimethoprim-sulfamethoxazole) and of the relevant resistance genes (tetA, strA-strB, and TEM), mutations in gyrA and parC, and multidrug-resistant APEC strains during the period 2000 to 2005.     

Heterogeneity among virulence and antimicrobial resistance gene profiles of extraintestinal Escherichia coli isolates of animal and human origin

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates collected from different infected animals and from human patients with extraintestinal infections in 2001 were characterized for their phenotypic and genotypic antimicrobial resistance profiles, genotypes, and key virulence factors. Among the 10 antimicrobial agents tested, resistance to ampicillin, tetracycline, and sulfonamides was most frequent. Multiresistant strains were found in both the animal and the human groups of isolates. Resistance gene distribution was assessed by colony hybridization. Similar antibiotic resistance patterns could be observed in the animal and the human isolates. Although some resistance genes, such as bla(TEM), sulI, and sulII, were equally represented in the animal and human ExPEC isolates, differences in the distributions of tetracycline [tet(D)], chloramphenicol (catI, catIII, and floR), and trimethoprim (dhfrI, dhfrV, dhfrVII, and dhfrXIII) resistance genes were observed between the animal and the human isolates. Approximately one-third of the ExPEC isolates possessed a class 1 integron. The four major different variable regions of the class 1 integron contained aminoglycoside (aadA1, aadA2, aadA5, and aadA6) and/or trimethoprim (dhfrIb, dhfrXII, and dhfrXVII) resistance genes. The ExPEC strains belonged to different phylogenetic groups, depending on their host origin. Strains isolated from animal tissues belonged to either a commensal group (group A or B1) or a virulent group (group B2 or D), while the majority of the human isolates belonged to a virulent group (group B2 or D). Although the limited number of isolates evaluated in the present study prevents firm epidemiological conclusions from being made, on a more global scale, these data demonstrate that extraintestinal isolates of E. coli can possess relatively distinct intra- and intergroup resistance gene profiles, with animal isolates presenting a more heterogeneous group than human isolates.     

Production of attaching-effacing lesions in ligated large intestine loops of 6-month-old sheep by Escherichia coli O157:1H7

Shiga-toxigenic Escherichia coli O157:H7 (STEC O157:H7) is associated with potentially fatal human disease, and a persistent reservoir of the organism is present in some farm animal species, especially cattle and sheep. The mechanisms of persistent colonisation of the ruminant intestine by STEC O157:H7 are poorly understood but may be associated with intimate adherence to eukaryotic cells. Intimate adherence, as evidenced by induction of attaching-effacing (AE) lesions by STEC O157, has been observed in 6-day-old conventional lambs after deliberate oral infection but not in older animals. Thus, the present study used a ligated intestinal loop technique to investigate whether STEC O157:H7 and other attaching-effacing E. coli may adhere intimately to the sheep large intestinal mucosa. To do this, four STEC O157:H7 strains, one STEC O26:K60:H11 and one Shiga toxin-negative E. coli O157:H7 strain, suspended in either phosphate-buffered saline or Dulbecco's modified Eagle's medium, were inoculated into ligated spiral colon loops of each of two lambs. The loops were removed 6 h after inoculation, fixed and examined by light and electron microscopy. AE lesions on the intestinal mucosa were produced by all the inoculated strains. However, the lesions were sparse and small, typically comprising bacterial cells intimately adhered to a single enterocyte, or a few adjacent enterocytes. There was little correlation between the extent of intimate adherence in this model and the bacterial cell density, pre-inoculation growth conditions of the bacteria or the strain tested.     

Characterization of antimicrobial resistance among Escherichia coli O111 isolates of animal and human origin

Fifty isolates of Escherichia coli serogroup O111 recovered from humans and various animal species over a 24-year period (1976-1999) were examined for typical virulence-associated factors and susceptibilities to antimicrobials of human and veterinary significance. Nine H (flagellar) types were identified including nonmotile (n = 24), 32 (n = 12), negative (n = 5), and 56 (n = 3). Thirty-five (70%) isolates possessed at least one Shiga-toxin-producing E. coli (STEC)-associated virulence determinants (eae, stxl, stx2, hlyA) via PCR analysis. Of these 35 isolates, 20 possessed eae, stxl, and hlyA genes, whereas three isolates possessed eae, stxl, stx2, and hylA genes. Multiple antibiotic resistance was observed in 70% of the 50 E. coli O111 isolates. The majority of isolates displayed resistance to streptomycin, sulfamethoxazole, tetracycline, and kanamycin. Bacterial resistance to ampicillin, gentamicin, chloramphenicol, trimethoprim and apramycin was also observed. Integrons were identified in 23 (46%) of the E. coli isolates assayed, with a 1-kb amplicon being most frequently observed. DNA sequencing of these integrons revealed the presence of the aadA gene, encoding resistance to streptomycin. Two integrons of 1.5 and 2 kb contained the aadA2 and either dfrI or dfrXII genes, encoding resistance to streptomycin and trimethoprim, respectively. Integrons were also identified from isolates dating back to 1982. Isolates were further genetically characterized via ribotyping, which identified 15 distinct ribogroups, with 62% of isolates clustering into four major ribogroups. Certain riboprint patterns from different animal species, including humans, were observed in isolates spanning the 24-year collection period, suggesting the dissemination of specialized pathogenic O111 clones.     

Distribution of virulence factors,antimicrobial resistance genes and phylogenetic relatedness among Shiga toxin-producing Escherichia coli serogroup O91 from human infections

Shiga toxin-producing Escherichia coli (STEC) belonging to the serogroup O91 are among the most common non-O157 STEC serogroups associated with human illness in Europe. This study aimed to analyse the virulence factors, antimicrobial resistance genes and phylogenetic relatedness among 48 clinical STEC O91 isolates collected during 2003–2019 in Switzerland. The isolates were subjected to whole genome sequencing using short-read sequencing technologies and a subset of isolates additionally to long-read sequencing. They belonged to O91:H10 (n = 6), O91:H14 (n = 40), and O91:H21 (n = 2). Multilocus sequence typing showed that the O91:H10 isolates all belonged to sequence type (ST)641, while the O91:H14 isolates were assigned to ST33, ST9700, or were non-typeable. Both O91:H21 isolates belonged to ST442. Shiga toxin gene stx1a was the most common Shiga toxin gene subtype among the isolates, followed by stx2b, stx2d and stx2a. All isolates were LEE-negative and carried one or two copies of the IrgA adhesin gene iha. In a subset of long-read sequenced isolates, modules of the Locus of Adhesion and Autoaggregation pathogenicity island (LAA-PAI) carrying iha and other genes such as hes, lesP or agn43 were identified. A large proportion of STEC O91:H14 carried the subtilase cytotoxin gene subA, colicin genes (cba, cea, cib and cma) or microcin genes (mcmA, mchB, mchC and mchF). STEC O91:H14 were further distinguished from STEC O91:H10/H21 by one or more virulence factors found in extraintestinal pathogenic E. coli (ExPEC), including hlyF, iucC/iutA, kpsE and traT. The hlyF gene was identified on a novel mosaic plasmid that was unrelated to hlyF+ plasmids described previously in STEC. Core genome phylogenetic analysis revealed that STEC O91:H10 and STEC O91:H21 were clonally conserved, whereas STEC O91:H14 were clonally diverse. Among three STEC O91:H14 isolates, a number of resistance genes were identified, including genes that mediate resistance to aminoglycosides (aadA, aadA2, aadA9, aadA23, aph(3'')-Ib and aph(6)-Id), chloramphenicol (cmlA), sulphonamides (sul2 and sul3), and trimethoprim (drfA12). Our data contribute to understanding the genetic diversity and differing levels of virulence potential within the STEC O91 serogroup.     

Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was isolated for the first time from Musca domestica L. A total of 310 fly samples was collected from 4 different farms in Obihiro-City, Hokkaido, in the summer and autumn of 1997;5 samples carried E. coli serotype O157:H7. Using ELISA and Vero cell cytotoxicity assay, 3 isolates from 1 cattle farm produced both active Shiga-toxin type 1 (Stx1) and 2 (Stx2). These isolates also carried hemolysin and eaeA genes and harbored the 90-kb virulence plasmid of EHEC O157:H7. Based on plasmid profiles, antibiotic patterns, polymerase chain reaction (PCR)-based DNA finger printing analysis using random amplified polymorphic DNA, pulsed field gel electrophoresis analysis, and DNA sequences of stx1 and stx2, all 3 isolates from fly samples were identical. These results indicate that the house fly is capable of carrying the toxigenic EHEC O157:H7 involved in human disease.