Evolution of enterohemorrhagic Escherichia coli hemolysin plasmids and the locus for enterocyte effacement in shiga toxin-producing E. coli

This study assessed the diversity of the enterohemorrhagic Escherichia coli (EHEC) hemolysin gene (ehxA) in a variety of Shiga toxin-producing E. coli (STEC) serotypes and the relationship between ehxA types and virulence markers on the locus for enterocyte effacement (LEE). Restriction fragment length polymorphism of the ehxA gene and flanking sequences and of the E. coli attaching and effacing (eae) gene was determined for 79 EHEC hemolysin-positive STEC isolates of 37 serotypes. Two main groups of EHEC hemolysin sequences and associated plasmids, which corresponded to the eae-positive and the eae-negative isolates, were delineated. Comparisons of the ehxA gene sequences of representative isolates of each group showed that this gene and the rest of the EHEC hemolysin operon are highly conserved. Digestion of an ehxA PCR product with the restriction endonuclease TaqI showed a unique restriction pattern for eae-negative isolates and another one for isolates of serotypes O157:H7 and O157:NM. A conserved fragment of 5.6 kb with four potential open reading frames was identified on the EHEC hemolysin plasmid of eae-positive STEC. Phylogenetic analysis of a subset of 27 STEC isolates, one enteropathogenic E. coli isolate, and a K-12 reference isolate showed that eae-positive STEC isolates all belong to a single evolutionary lineage and that the EHEC hemolysin plasmid and the ehxA gene evolved within this lineage without recent horizontal transfer. However, the eae gene and the LEE appear to have been transferred horizontally within this STEC lineage on several occasions. The reasons for the lack of transfer or maintenance of the LEE in other STEC lineages are not clear and require further study.     

Molecular microbiological investigation of an outbreak of hemolytic-uremic syndrome caused by dry fermented sausage contaminated with Shiga-like toxin-producing Escherichia coli

Shiga-like toxin-producing Escherichia coli (SLTEC) strains are a diverse group of organisms which are known to cause diarrhea and hemorrhagic colitis in humans. This can lead to potentially fatal systemic sequelae, such as hemolytic-uremic syndrome (HUS). Strains belonging to more than 100 different O:H serotypes have been associated with severe SLTEC disease in humans, of which only O157 strains (which are uncommon in Australia) have a distinguishable cultural characteristic (sorbitol negative). During an outbreak of HUS in Adelaide, South Australia, a sensitive PCR assay specific for Shiga-like toxin genes (slt) was used to test cultures of feces and suspected foods. This enabled rapid confirmation of infection and identified a locally produced dry fermented sausage (mettwurst) as the source of infection. Cultures of feces from 19 of 21 HUS patients and 7 of 8 mettwurst samples collected from their homes were PCR positive for slt-I and slt-II genes. SLTEC isolates belonging to serotype O111:H- was subsequently isolated from 16 patients and 4 mettwurst samples. Subsequent restriction fragment length polymorphism analysis of chromosomal DNA from these isolates with slt-specific probes indicated that at least three different O111:H- genotypes were associated with the outbreak. Pulsed-field gel electrophoresis of genomic DNA restricted with XbaI showed that two of these restriction fragment length polymorphism types were closely related, but the third was quite distinct. However, SLTEC strains of other serotypes, including O157:H-, were also isolated from some of the HUS patients.     

Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans

Associations between known or putative virulence factors of Shiga toxin-producing Escherichia coli and disease in humans were investigated. Univariate analysis and multivariate logistic regression analysis of a set of 237 isolates from 118 serotypes showed significant associations between the presence of genes for intimin (eae) and Shiga toxin 2 (stx2) and isolates from serotypes reported in humans. Similar associations were found with isolates from serotypes reported in hemorrhagic colitis and hemolytic-uremic syndrome. The enterohemorrhagic E. coli (EHEC) hemolysin gene was significantly associated with isolates from serotypes found in severe diseases in univariate analysis but not in multivariate logistic regression models. A strong association between the intimin and EHEC-hemolysin genes may explain the lack of statistical significance of EHEC hemolysin in these multivariate models, but a true lack of biological significance of the hemolysin in humans or in disease cannot be excluded. This result warrants further investigations of this topic. Multivariate analysis revealed an interaction between the eae and stx2 genes, thus supporting the hypothesis of the synergism between the adhesin intimin and Shiga toxin 2. A strong statistical association was observed between the stx2 gene and severity of disease for a set of 112 human isolates from eight major serotypes. A comparison of 77 isolates of bovine origin and 91 human isolates belonging to six major serotypes showed significant associations of the genes for Shiga toxin 1 and EspP protease with bovine isolates and an increased adherence on HEp-2 cell cultures for human isolates, particularly from diarrheic patients and healthy persons.     

Clonal diversity of Chilean isolates of enterohemorrhagic Escherichia coli from patients with hemolytic-uremic syndrome, asymptomatic subjects, animal reservoirs, and food products

To determine clonal relationship among Chilean enterohemorrhagic Escherichia coli (EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of E. coli O157, 5 of E. coli O111, and 5 of E. coli O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44 E. coli O157 isolates analyzed, of which the genetic profile stx1+ stx2+ hly+ eae+ was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the stx1 and stx2 toxin-encoding genes and were eaeA positive. Only 9 of 13 isolates from human controls were stx1+ stx2+, and 8 carried the eaeA gene. Comparison of profiles obtained by PFGE of XbaI-digested genomic DNA showed a great diversity among the E. coli O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype stx1+ stx2+ eae+ and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile.     

eaeA genes in Escherichia coli derived from Japanese patients with sporadic diarrhea

To investigate the prevalence of attaching and effacing Escherichia coli, we examined 364 strains isolated from the feces of 9,684 patients with diarrhea at the Anjo Kosei Hospital in Japan for the presence of eaeA. Twenty-nine (8%) of the strains were eaeA positive. Of enteropathogenic E. coli (EPEC), 11 of the 87 (13%) strains were for the positive eaeA gene. The serotypes and the numbers of eaeA-positive strains among the strains tested were as follows: O26:H-(2/3), O55:H7 (4/4), O55:H-(2/ 2) and O128:H2(3/3). Two enterohemorrhagic E. coli (EHEC) strains (Verotoxin positive O157:H7) were also eaeA positive. Among 260 non-EPEC strains that were not categorized as diarrheagenic E. coli, 16 (6%) were eaeA positive. Those serotypes were as follows: O15:H2, O20:H6, O28:H28, O63:H6. O153:H7, O28:H6, O153:H19 and O157:H45. EPEC strains including O18:H7 and six other serotypes, enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC) were all eaeA negative.     

Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli

An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.     

Genotype analysis of enterohemorrhagic Escherichia coli O157:H7 strains isolated in Iwate Prefecture from 1996 to February 1997

Genotypes of 38 isolates of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolated from 11 sporadic cases and one outbreak in Iwate Prefecture from 1996 to February 1997 were studied by pulsed-field gel electrophoresis (PFGE), in comparison with a strain of EHEC O157:H7 isolated in 1992 in Ohazama-Cho, Iwate Prefecture, and two isolates of EHEC O157:NM. In order to substantiate the genotypes classified by PFGE, Southern blotting was performed to investigate integration sites of the Shiga toxin genes (stx) in the XbaI-digested genome DNA fragments. The stx1 gene existed on an approximately 130 kb fragment in all isolates except two ones. On the other hand, the stx2 gene was observed on 11 DNA fragments in different length from 600 kb to 155 kb, indicating that the stx2 gene integrates into more heterogeneous sites of genome DNA than stx1 does. From these analyses, EHEC O157:H7 isolates examined were classified into 7 genotypes. Since half of the isolates were the same genotype as that of the isolate in 1992, it is suggested that this type of EHEC O157:H7 strain is expanding from Ohazama-Cho and Morioka City in Iwate Prefecture.     

Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes

A polymerase chain reaction (PCR) technique for the identification of Shiga-like toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.     

EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V

In this study, we identified and characterized a novel secreted protein, the extracellular serine protease EspP, which is encoded by the large plasmid of enterohaemorrhagic Escherichia coli (EHEC) O157:H7. The corresponding espP gene consists of a 3900 bp open reading frame that is able to encode a 1300-amino-acid protein. EspP is synthesized as a large precursor which is then processed at the N- and C-termini during secretion. It can be grouped into the autotransporter protein family. The deduced amino acid sequence of EspP showed homology to several secreted or surface-exposed proteins of pathogenic bacteria, in particular EspC of enteropathogenic E. coli and IgA1 proteases from Neisseria spp. and Haemophilus influenzae. Hybridization experiments and immunoblot analysis of clinical EHEC isolates showed that EspP is widespread among EHEC of the serogroup O157 and that it also exists in serogroup 026. A specific immune response against EspP was detected in sera from patients suffering from EHEC infections. Functional analysis showed that EspP is a protease capable of cleaving pepsin A and human coagulation factor V. Degradation of factor V could contribute to the mucosal haemorrhage observed in patients with haemorrhagic colitis.     

Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.     

Diversity of DNA sequences among enterohemorrhagic Escherichia coli O157:H7 detected by PCR-based DNA fingerprinting

A random amplified polymorphic DNA(RAPD) fingerprinting method has been developed to differentiate Enterohemorrhagic Escherichia coli(EHEC) O157:H7 isolates. This method uses an oligonucleotide of arbitrarily chosen sequence to prime DNA synthesis from pairs of sites to which it is matched or practically matched and results in strain-specific arrays of DNA products. By the RAPD analysis using A07(5'-TGCCTCGCACCA-3'), EHEC strains tested in this study were found to be divided into 5 groups. The RAPD arrays among 5EHEC clinical isolates from a single outbreak were identical from each other, and different from other origin. The data indicate that the RAPD method is feasible for investigating the source of an outbreak associated with EHEC O157:H7 infection.     

Verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC in 1-30-days-old diarrhoeic dairy calves

Faecal samples from 221, 1-30-days-old, diarrhoeic dairy calves were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC. Calves were grouped according to their age (1-7, 8-14, 15-21 and 22-30 days) and analyses of prevalences were done by Mantel-Haenzsel chi 2-test for trend. VTEC and eae-positive non-VTEC were detected in 20 (9.0%) and 18 (8.1%) of the diarrhoeic calves, respectively. A significant age-associated increase in the prevalence of VTEC (p = 0.0001), but not in the prevalence of eae-positive non-VTEC (p = 0.381), was found. Significant differences in VTEC prevalence were found between the age-group 22-30 days and in all other age-groups. 43 (5.0%) of the 861 E. coli isolates from the 221 diarrhoeic calves were VTEC, and 30 (69.8%) of these strains produced VT1 only. More than one-half of the VTEC strains (55.8%) were positive for the eae gene and all these eae-positive VTEC strains produced VT1 only. A high percentage (76.7%) of VTEC strains belonged to E. coli serogroups (O4, O26, O39, O91, O113, O128 and O145) associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. 51 (5.9%) of the E. coli strains studied were eae-positive non-VTEC and the serogroups most prevalent among these strains were O4, O14, O26 and O123. Only four of the eae-positive strains were also espB-positive by hybridization with a probe from a human EPEC isolate and none of these strains produced VT.     

Identification of new verocytotoxin type 2 variant B-subunit genes in human and animal Escherichia coli isolates

The sequence of a verocytotoxin 2 (VT2) variant gene that was untypeable by the B subunit PCR and restriction fragment length polymorphism analysis (PCR-RFLP) method described by Tyler et al. (S. D. Tyler, W. M. Johnson, H. Lior, G. Wang, and K. R. Rozee, J. Clin. Microbiol. 29:1339-1343, 1991) was determined and compared with published sequences. It was highly homologous to two recently reported VT2 variant sequences. The PCR-RFLP method described by Tyler et al. was extended to include these new sequences. New VT2 variants were identified in 65 of 359 VT-producing Escherichia coli (VTEC) with newly designed primers (VT2-cm and VT2-f) and were characterized as well by restriction analysis of the amplification products obtained with another VT2-specific primer pair (VT2-e and VT2-f). The VT genes harbored by 64 of these isolates proved to be untypeable by Tyler's PCR-RFLP method because no amplification was obtained with the primers used with this method (VT2-c and VT2-d). The last isolate harbored the new variant gene in addition to VT2vh-a. None of the isolates harboring these new toxin genes belonged to serogroups O157, O26, O103, O111, and O145. All 65 isolates were negative for the eaeA gene and were significantly less frequently enterohemolytic or positive for the enterohemorrhagic E. coli (EHEC) virulence plasmid than non-O157 VTEC isolates harboring other VT2 genes. They were also less frequently isolated from patients with EHEC-associated symptoms. The extended PCR-RFLP typing method is a useful tool to identify less-virulent VTEC isolates and for VT genotyping in epidemiological studies with non-O157 strains.     

Partial inhibition of amplifications by primers of EHEC genes

Diagnostic value of multiplex polymerase chain reaction (PCR) was examined by using three primer pairs, specific for the common conserved region of stx1 and stx2, eae and an enterohaemolysin A gene (ehxA). The sensitivity in respect of each amplicon decreased with three exponents comparing to the individual PCR reactions. These PCR reactions were partially inhibited by the presence of certain additional primers. This inhibitory effect was template-concentration dependent, and was partially balanced by usage of increased amount of dNTP. Taq DNA polymerase in a range of 0.3-1.25 U/reaction did not influence the inhibition. The same inhibition was detected if the annealing temperature was changed from 48 degrees C to 57 degrees C. Pairs of EHEC primers inhibited a Salmonella enteritidis virulence-plasmid specific gene amplification, as well. Theoretical inhibiting effects were predicted by Primer Premier software but our observations can be sufficiently explained neither by the competitions between the specific and aspecific amplifications nor by the inhibition caused by dimerization of primers.     

Isolation and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep

In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup 0157. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of E. coli O157 strains that are potentially pathogenic for humans.     

Construction and characterization of avian Escherichia coli cya crp mutants

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.     

Enteropathogenicity markers in Escherichia coli isolated from infants with acute diarrhoea and healthy controls in Rio de Janeiro, Brazil

Faeces from urban children < 2 years old with acute diarrhoeal illness and from non-diarrhoeal infants (controls) were examined for Escherichia coli and other enteropathogens. A total of 990 E. coli isolates from 100 patients and 50 controls was tested for enteropathogenic E. coli (EPEC) serotype (O:H), adherence to HEp-2 cells after incubation for 3 and 6 h, fluorescent actin staining (FAS), DNA hybridisation with EAF, eaeA, STh, STp and EAggEC probes and production of heat-labile enterotoxin (LT) and verocytotoxin (VT) with Y1 and Vero cells. EPEC were the most prevalent enteropathogens in patients (32.7%; and 14% in controls). Enteroinvasive E. coli (EIEC) and Vero cytotoxin-producing E. coli (VTEC) were not detected. The rate of isolation of enterotoxigenic E. coli (ETEC) was identical in both groups. Among the EPEC isolates the prevalent serotypes were O111:H2, O55:NM and O119:H6. Localised adherence (LA) was found significantly more frequently in isolates from patients (19.6%) than controls (2.1%). All LA-positive EPEC isolates were FAS+ and eaeA+, but only 75.2% of them hybridised with the EAF probe. Diffusely adhering E. coli (DAEC) and enteroaggregative E. coli (EAggEC) were found with equal frequency in patients and controls. Twenty-seven E. coli isolates were negative for EAF but positive for eaeA and FAS and produced LA in 6-h adherence tests. These EAF-/eaeA+ strains were the only putative enteropathogen identified in seven patients and were not found in controls. The ability of these strains to elicit ultrastructural cell alterations and cell-signalling events was evaluated in Caco-2 cells (human colon carcinoma cell line) by the gentamicin invasion assay and by transmission electron microscopy. The numbers of intracellular bacteria in cell invasion tests varied from 0.4% to 1.6% of the cell-associated bacteria after a 6-h incubation period. Tyrosine phosphorylation of host cell proteins was assessed in HEp-2 cells by immunofluorescence microscopy and all strains gave positive results. EAF-/eaeA+ E. coli strains express most of the virulence properties found among true EPEC strains and can be a relevant cause of infant diarrhoea in developing countries.     

Detection of Escherichia coli O157:H7 in the beef marketed in Malaysia

Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources.     

Generation of Escherichia coli intimin derivatives with differing biological activities using site-directed mutagenesis of the intimin C-terminus domain

Intimins, encoded by eae genes, are outer membrane proteins involved in attaching-effacing (A/E) lesion formation and host cell invasion by pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium. A series of intimins, harbouring specific mutations close to the C-terminus, were constructed using pCVD438, which encodes the eae gene from EPEC strain E2348/69. These mutant plasmids were introduced into EPEC strain CVD206 and C. rodentium strain DBS255, which both contain deletion mutations in their eae genes. CVD206, CVD206(pCVD438) and CVD206(pCVD438) derivatives were assessed for their ability to promote A/E lesion formation or invasion of HEp-2 cells and to induce A/E lesions on fresh human intestinal in vitro organ cultures (IVOC). The pathogenicity of C. rodentium DBS255 harbouring these plasmid derivatives was also studied in mice. Here, we report that intimin-mediated A/E lesion formation can be segregated from intimin-mediated HEp-2 cell invasion. Moreover, adherence to IVOC, EPEC-induced microvillus elongation and colonization of the murine intestine by C. rodentium were also modulated by the modified intimins.     

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.