Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance genes. 512 E. coli strains sourced from humans (n = 202), animals (n = 304) and the environment (n = 6) were screened for the presence of the intI1 gene. In 31/79 integron positive E. coli strains, the gene cassette regions could not be PCR amplified using standard primers. DNA sequence analysis of 6 serologically diverse strains revealed atypical integrons harboured the dfrA5 cassette gene and only 24 bp of the integron 3′-conserved segment (CS) remained, due to the insertion of IS26. PCR targeting intI1 and IS26 followed by restriction fragment length polymorphism (RFLP) analysis identified the integron-dfrA5-IS26 element in 27 E. coli strains of bovine origin and 4 strains of human origin. Southern hybridization and transformation studies revealed the integron-dfrA5-IS26 gene arrangement was either chromosomally located or plasmid borne. Plasmid location in 4/9 E. coli strains and PCR linkage of Tn21 transposition genes with the intI1 gene in 20/31 strains, suggests this element is readily disseminated by horizontal transfer.

2570Two hundred verocytotoxigenic and 216 non-verocytotoxigenic Escherichia coli (VTEC and non-VTEC), isolated from a variety of sources were tested for their resistances to 11 antimicrobial agents. The strains included isolates from domestic food animals and both symptomatic and asymptomatic infections in man. A much higher level of resistance was found among the non-VTEC than among the VTEC, regardless of source. The resistant VTEC isolated from animals were predominantly from specimens associated with sick animals. Antibiotic resistance was detected in only four of the 59 (6·8 %) VTEC of human origin, whereas more of the human non-VTEC possessed antibiotic resistance determinants. It was particularly noteworthy that 24/87 (28 %) strains isolated from healthy babies, who had neither contact with antibiotics nor had gastrointestinal symptoms for at least 2 weeks prior to the specimen being taken, were resistant to one or more of the antibiotics tested. INTRODUCTIONExtensive studies over the years, both by ourselves and others around the world, have shown that verocytotoxigenic Escherichia coli (VTEC) are commonly isolated from the faeces of ruminants such as cattle and sheep (Beutin et al., 1993;Djordjevic et al., 2001;Dorn et al., 1989;Fagan et al., 1999;Hornitzky et al., 2001;Kudva et al., 1997; Richter et al., 1997;Wells et al., 1991). In addition, there has been increasing concern of the possible development of resistance to antimicrobial agents in the Enterobacteriaceae, especially E. coli, as a result of the use of such agents in animal feed (Willis, 2000). With the recent emergence of strains of Salmonella Typhimurium DT104, resistant to four or more antimicrobial agents (Threlfall et al., 1997), the problem of such resistant organisms evolving and being transmitted through the food chain from cattle and sheep to man is becoming increasingly important throughout the world (Glynn et al., 1998;Levy & Cruz, 1999). In his review, Willis (2000) concludes that 'there is now good evidence that the use of antibiotics in agriculture is contributing to the problem of antibiotic resistance amongst pathogenic bacteria.'In her book, Garrett (1995) discusses the problems associated with the acquisition of antibiotic resistance factors by the intestinal organisms of domestic animals, as a result of the use of antibiotics in animal husbandry. She then discusses the emergence of VTEC, especially E. coli O157 : H7, and suggests that these may well also have emerged due to such antibiotic use. It seemed that if this hypothesis were true then VTEC isolated from cattle and sheep would be resistant to antibiotics. It was specifically to address this question that this study was initiated, especially as there are few studies in which the antibiotic sensitivities of VTEC have been determined. Most of these deal with human VTEC. Zhao et al. (2001) demonstrated that 39/50 (78 %) VTEC, mainly O157 : H7, but also including a range of serotypes, exhibited resistance to two or more antimicrobial classes. Most of these were isolate...

This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.

Two typical coliforms from an intestinal biopsy from an adult case of bloody diarrhoea carried genes encoding intimin-â, stx 1 and ehxA, and produced verocytotoxin 1 and enterohaemolysin in culture. Both were biochemically typical Escherichia coli O5 : H À , apart from producing urease. Such O5isolates represent a human pathogenic E. coli lineage. Case reportA 55-year-old man 3 months post autologous stem cell transplant for multiple myeloma presented to hospital with abrupt onset of bloody diarrhoea and fever (38 8C). He was commenced empirically on intravenous ciprofloxacin and metronidazole. General stool culture for commonly encountered enteric pathogens failed to yield a possible causative agent. Clostridium difficile toxin was not detected. A colonoscopy on the day of admission revealed multiple mucosal areas showing a granular erythematous appearance with changes predominating on the left side of the colon. A biopsy of the macroscopically abnormal area revealed numerous bacilli adhering and effacing the surface enterocytes with scant acute inflammatory cells, minor degenerative epithelial changes and focal epithelial sloughing (Fig. 1). The appearances were diagnosed as a bacterial colitis and the possibility of an attaching and effacing Escherichia coli (AEEC). Once the diagnosis was known, the antibiotics were ceased as ciprofloxacin has potential adverse effects. No further antibiotics were given and the patient improved over the next 3 days, the diarrhoea ceased and he went home.Regarding potential risk factors for exposure to Shiga toxinproducing E. coli (STEC), there was no recent travel history or contact with a farming environment. He worked in a food bar, where he sometimes ate cold peanuts and salami, and he had no knowledge of any reports of illness in any of the people who visited the shop around the time that his diarrhoea commenced. The studyThe biopsy material sent for microbiology was cultured for the presence of AEEC by smearing across the plates on MacConkey (MAC), sorbitol MacConkey (SMAC), and washed and unwashed sheep blood agars (WSBA and SBA). The majority of the colonies were coliform and of the same morphology, as well as a few staphylococci. The colonies all fermented lactose on MAC, sorbitol on SMAC, and were haemolytic on WSBA but not on SBA, suggesting a profuse growth of enterohaemolytic E. coli (Bettelheim, 1995) of similar phenotype. They were tested by the adaptation of the Denka-Seiken test ) and found to be verocytotoxigenic. Following standard previously described methods (Bettelheim et al., 2000) on two colonies, it was found that they were typical E. coli apart from being anaerogenic and urease producers. They were non-motile, and O serotyping showed them to belong to serogroup O5. Supernatants prepared from overnight growth in trypticase soy broth, when tested on Vero cells, gave the typical cytopathic effects of verocytotoxins at dilutions of up toAbbreviations: AEEC, attaching and effacing Escherichia coli; HUS, haemolytic uraemic syndrome; STEC, Shiga toxin-produc...

22"! 23"The lipopolysaccharide (O) and flagellar (H) surface antigens of Escherichia coli are targets 24"for serotyping that have traditionally been used to identify pathogenic lineages of E. coli. As 25"serotyping has several limitations, public health reference laboratories are increasingly 26"moving towards whole genome sequencing (WGS) for the rapid characterisation of bacterial 27"isolates. Here we present a method to rapidly and accurately serotype E. coli isolates from 28"raw, short read sequence data, leveraging the known genetic basis for the biosynthesis of O- 29"and H-antigens. Our approach bypasses the need for de novo genome assembly by directly 30"screening WGS reads against a curated database of alleles linked to known E. coli O-groups 31"and H-types (the EcOH database) using the software package SRST2. We validated our 32"approach by comparing in silico results with those obtained via serological phenotyping of 33"197 enteropathogenic (EPEC) isolates. We also demonstrated the utility of our method to 34"characterise enterotoxigenic E. coli (ETEC) and the uropathogenic E. coli (UPEC) epidemic 35"clone ST131, and for in silico serotyping of foodborne outbreak-related isolates in the public 36"GenomeTrakr database.

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.