Use of gene probes and adhesion tests to characterise Escherichia coli belonging to enteropathogenic serogroups isolated in the United Kingdom.

Nine hundred and twenty-five Escherichia coli isolates from cases of diarrhoea in the United Kingdom and belonging to enteropathogenic E. coli (EPEC) O serogroups were examined for virulence properties. The tests included adhesion to HEp-2 cells, the fluorescence actin staining (FAS) test (which correlates with the ability to cause attaching and effacing lesions) and DNA hybridisations with probes to detect sequences for eaeA (E. coli attaching and effacing factor), EAF (EPEC adherence factor), verocytotoxins VT1 and VT2, enteroaggregative E. coli and diffusely adherent E. coli. The O serogroups examined were 18, 26, 44, 55, 86, 111, 114, 119, 125, 126, 127, 128 and 142. Six hundred and sixty strains (71.4%) hybridised with at least one of the DNA probes. Over 80% of strains in O serogroups 26, 55, 119, 125, 127 and 142 and 41% of strains of serogroups 86, 111, 114, 126 and 128 hybridised with the eae probe and most showed localised attachment and were FAS-positive. However, <10% of these eae probe-positive strains hybridised with the EAF probe. Eighty-four of 232 strains in O serogroups 44, 86, 111, and 126 were enteroaggregative. VT genes were detected in 57 of 402 strains in O serogroups 26, 55, 111 and 128. Identification of EPEC by serogrouping was shown to be an effective method of identifying strains with pathogenic potential, although the organisms were diverse in their properties.

Catabolite repression of the adhesion of Vero cytotoxin-producing Escherichia coli of serogroups O157 and O111.

The virulence traits that mediate Vero cytotoxin-producing E. coli (VTEC) adherence are unclear. Many VTEC strains possess the eaeA gene which is involved in the attaching and effacing effects of enteropathogenic E. coli (EPEC). Most eae-positive VTEC adhered to HEp-2 cells in a localized manner; however some strains did not adhere. Thus we investigated the adhesion of poorly adherent strains, especially those of serogroups O111 and O157. To establish a model, the adherence to HEp-2, INT407 and Caco-2 cells of 12 O157 VTEC and six O111 VTEC isolated from cases of human infection were studied after growth of the bacteria under different conditions. For adhesion tests mannose is usually added during prior broth culture of the bacteria, and during the period of attachment, so that any adhesion due to mannose-sensitive type 1 pili is inhibited. Bacteria cultured in peptone water in the absence of mannose adhered to all three lines; there were localized clusters of bacteria on 1%-82% cells, whether mannose was present during the attachment period or not. Bacteria grown in the presence of D-mannose, or any other sugar that was metabolized, showed little adherence (range 0-9%). alpha-Methyl-glucoside also caused marked inhibition of adhesion. It was concluded that inhibition of adhesion was due to catabolite repression.

Detection and differentiation of the gene for toxin co-regulated pili (tcpA) in Vibrio cholerae non-O1 using the polymerase chain reaction.

The polymerase chain reaction has been used to differentiate the gene which encodes the toxin co-regulated pili (tcpA) of the El Tor and classical biotypes of Vibrio cholerae O1. The same PCR primers were applied to strains belonging to non-O1 serogroups that produced cholera toxin. The size of fragment amplified was either identical to the tcpA of biotype El Tor (471 bp) or to the tcpA of biotype classical (617 bp). All strains belonging to the novel epidemic serogroup O139 generated a 471-bp fragment identical to El Tor tcpA. The present study suggests that there may be an association between non-O1 serogroup and tcpA type.

Isolates of Escherichia coli O44:H18 of diverse origin are enteroaggregative.

One hundred thirteen strains of Escherichia coli O44:H18 isolated in several countries over 25 years were examined for adhesion to tissue culture cells and for hybridization with DNA probes. Fifty-nine strains were from sporadic cases of infection; 54 were from 12 outbreaks. Of the 113 strains, 85 showed aggregative adhesion to HEp-2 cells; 36 were from sporadic cases and 49 were from 9 outbreaks. All adhesive strains hybridized with the probe for enteroaggregative E. coli (EAggEC) and 1 nonadhesive strain was also positive. However, 80 of the 86 EAggEC probe-positive strains also hybridized with the probe for diffusely adherent E. coli (DAEC) derived from the daaC gene of strain F1845. The EAggEC and DAEC probes hybridized to different fragments of a large plasmid in the O44:H18 strains.

Hybridization of strains of Escherichia coli O157 with probes derived from the eaeA gene of enteropathogenic E. coli and the eaeA homolog from a Vero cytotoxin-producing strain of E. coli O157.

A total of 375 Escherichia coli O157 strains were tested by colony hybridization with the eae probe from the central portion of the eaeA gene of the classical enteropathogenic E. coli strain E2348/69. They were also tested with a probe, eaeO157, from the C-terminal end of the eae gene homolog from a Vero cytotoxin (VT)-producing strain of E. coli (VTEC) of serotype O157:H7. Both probes hybridized with all 246 O157:H7 or H- VTEC strains tested. The majority were from human infections, and the remainder were from cattle. A further 10 strains (H7 or H-) hybridized with both eae and eaeO157 sequences but not with VT probes. They resembled O157 VTEC and were probably naturally occurring derivatives that had lost VT genes. The remaining 119 strains of O157 were from human, animal, and food sources and belonged to 16 H types other than H7 or were H-. They were VT negative and differed in their properties from O157 VTEC: generally they fermented sorbitol in 1 day, produced beta-glucuronidase, and could not be phage typed by the scheme for O157 VTEC. The eae probe but not the eaeO157 sequence hybridized with 18 H8 or H39 strains, predominantly from human diarrhea. The remaining 101 VT-negative strains hybridized with neither probe. However, 16 strains of O157:H45 hybridized with a probe for diffusely adherent E. coli and attached to HEp-2 cells in a diffuse pattern. Serogroup O157 comprises strains with heterogeneous properties. The eaeO157 probe is a valuable addition to the VT probes used to differentiate O157 strains.

Prospective study of verocytotoxin-producing, enteroaggregative and diffusely adherent Escherichia coli in different diarrhoeal states.

One hundred and eighty-one stool specimens from patients with various types of diarrhoea (135 patients) or from non-diarrhoeal controls (23 acute medical patients, 23 inflammatory bowel disease in remission) were investigated using a colony-blot DNA hybridization assay for the presence of Verocytotoxin-producing (VTEC), enteroaggregative (EAggEC) and diffusely adherent (DAEC) Escherichia coli. Twelve patients had probe-positive EAggEC in the stool and 8 of these had diarrhoea, 6 following recent travel. Eight patients had DAEC, 7 of whom had travellers' diarrhoea. Six of 10 (60%) travellers with gastroenteritis, but without a recognized enteric pathogen, were positive for EAggEC (4) or DAEC (2). Five of 10 (50%) travellers with gastroenteritis related to a recognized enteric pathogen also had DAEC identified in their stool. Of the 23 acute medical control patients 11 had been abroad, 4 of these were immigrants and had EAggEC. VTEC were not found and, with one exception, immunoassays for antibodies to E. coli O 157 and O 2 lipopolysaccharides were negative.

Ability of human sera to neutralise the activity of Vero cytotoxins VT1, VT2 and variant forms of VT2.

Sera, from 17 patients with diarrhoea or haemolytic uraemic syndrome, and six healthy adults, were tested for neutralisation of Vero cytotoxins (VT). For all 17 patients there was evidence of infection with Escherichia coli O157. Sera from two controls but from none of the patients neutralised VT1, although two patients were infected by strains producing VT1 and VT2. Sera from all six controls and 14 patients neutralised VT2 derived from strains 933 and E32511, but not variant forms of VT2 derived from strains E32511, E57, B2F1 and H.1.8. This neutralising activity warrants further investigation, especially as many O157 VTEC carry both VT2 and VT2 variant genes.

The use of gene probes, immunoassays and tissue culture for the detection of toxin in Vibrio cholerae non-O1.

Vibrio cholerae non-O1 strains were screened for the presence of cholera enterotoxin (CT) genes by means of digoxigenin-labelled polynucleotide CTA and CTB probes. In-vitro production of CT was investigated by the Y1 mouse adrenal cell assay, enzyme-linked immunosorbent assay (ELISA) and a commercial, reversed passive latex agglutination (RPLA) kit. Only two (0.25%) of 790 strains tested gave positive results with the CTA and CTB probes. The production of other bacterial cytotoxin(s) made it impossible to use the characteristic cell-rounding effect on Y1 cells for the detection of CT. CT production by the probe-positive strains was confirmed by the immunoassays. Two hundred and fifty-two of the 788 probe-negative strains were tested by both cell assay and immunoassays. Of these, 90% produced cytotoxin(s) in the cell assay. In addition, 37% gave positive results in CT-ELISA, but negative results with LT-ELISA and VET-RPLA. These results indicate the presumed presence of a toxin in V. cholerae non-O1 that is able to bind GM1 and react with antisera to CT, but which is not identical to CT.

Adhesion to and invasion of human colon carcinoma Caco-2 cells by Aeromonas strains.

The enteropathogenicity of Aeromonas strains that showed mannose-resistant adhesion to INT407 cells was evaluated by infecting Caco-2 cells and observing them by light and electronmicroscopy. Five of six strains adhered in large numbers to Caco-2 cells in the presence of mannose and caused cytopathic effects. Two strains of Aeromonas spp. seemed to invade Caco-2 cells, as membrane-bound bacteria were seen within the cytoplasm of these cells; however, staining by acridine orange-crystal violet appeared to show intracellular fluorescent bacteria in three strains. Fimbriae did not appear to play an important role in adhesion because fimbrial structures were not seen by transmission electronmicroscopy. Adhesion of four strains was inhibited by the addition of L-fucose. The strains were negative in the fluorescence actin staining test, which in enteropathogenic Escherichia coli strains correlates with the ability to attach and efface intestinal microvilli. The DNA of the Aeromonas strains did not hybridise with the E. coli eae and ipaB probes, associated with attaching and effacing ability and invasion, respectively. These results give support to the enteropathogenicity of adhesive strains of Aeromonas spp., although the mechanisms of adhesion, and possibly invasion, remain to be elucidated.

Virulence properties of Escherichia coli strains belonging to serogroups O26, O55, O111 and O128 isolated in the United Kingdom in 1991 from patients with diarrhoea.

Some strains of Escherichia coli belonging to serogroups O26, O55, O111 or O128 produce Vero cytotoxin (VT). These serogroups are included in the range of enteropathogenic E. coli (EPEC) serogroups for which commercial antisera are available. In an attempt to obtain information on VT-producing strains other than those of serogroup O157, 122 strains belonging to these four serogroups and isolated in 1991 from patients with diarrhoea in the United Kingdom were tested for hybridization with VT probes. Only 18 of the 122 strains were VT-positive and these were O26 or O128. However 90 strains hybridized with the E. coli attaching and effacing (eae) probe (including 14 VT-positive strains) and 17 with the enteroaggregative E. coli (EAggEC) probe. For 78 eae-positive and 9 EAggEC-positive strains, tissue culture tests correlated with the probe results as the strains gave, respectively, either localized adhesion and a positive fluorescent-actin staining test or a characteristic aggregative attachment. A total of 111 of the 122 strains belonging to serogroups O26, O55, O111 or O128 possessed properties that may be associated with the ability to cause human diarrhoeal disease, and similar studies are needed on strains from the other classical EPEC serogroups.

A longitudinal study of Vero cytotoxin producing Escherichia coli in cattle calves in Sri Lanka.

Two cohorts of 10 and 16 calves were followed at weekly or fortnightly intervals from 4-28 and 1-9 weeks respectively to determine whether natural infection by Vero cytotoxin (VT) producing Escherichia coli (VTEC) occurred. Ninety-one of 171 (53%) faecal specimens were VTEC positive and 20-80% of animals at any given time excreted VTEC. Of 104 VTEC strains studied further, 6 different serogroups (O 22.H16; O 25.H5; O 49.H-; O 86.H26; O 88.H25; O 153.H12) and an untypable strain (O? .H21) were identified. All strains belonging to the same serotype had identical profiles of reactivity with DNA probes to toxins VT1 or 2, LTI or II and a probe (CVD419) derived from a plasmid carried by enterohaemorrhagic Escherichia coli O 157.H7. Four of these serotypes were found in the faecal flora of the calves, taken as a group, throughout the 4-month study period. Sixty percent of the strains hybridized with the probe for VT1, 4% with the probe for VT2, and 36% with both probes. Faecal VTEC were significantly associated with overt diarrhoeal illness in animals < 10 weeks of age, but no characteristic profile of markers (serotype or hybridization pattern) in E. coli isolates was associated with diarrhoea. A serological response to VT1 was detected in some animals, but faecal VT1 VTEC excretion persisted in spite of seroconversion. VT1 seroconversion was not associated with diarrhoea. A serological response to VT2 was not detected even in those animals excreting VT2 VTEC in the faeces.

Properties of Vero cytotoxin-producing Escherichia coli isolated from human and non-human sources.

Vero cytotoxin-producing Escherichia coli (VTEC) are an important cause of disease in man and animals. In addition to the production of VT these strains may possess other properties that are required for full virulence. Examples of some recent molecular studies are reviewed. Use of oligonucleotide probes and the polymerase chain reaction provide methods for the identification and typing of different VT genes. Several VTEC have the ability to cause attaching and effacing lesions of the epithelial microvilli. Hybridization experiments with the eae probe (E. coli attaching and effacing) showed homology with VTEC of human origin of eight different O serogroups. Properties of VTEC from human infections have been compared to strains isolated from animals and foods.

Strains of Escherichia coli O157:H8 from human diarrhoea belong to attaching and effacing class of E coli.

AIMS: To determine whether 17 Escherichia coli O157:H8 strains isolated from patients with diarrhoea in the United Kingdom were putative pathogens. METHODS: The strains had been isolated by the use of O157 antiserum, available for the detection of Vero cytotoxin (VT) producing strains of E coli O157 that are usually of flagellar (H) type 7, but may also be non-motile. The strains were examined for VT production, for their ability to adhere to HEp-2 cells, and for hybridisation with several DNA probes that recognise pathogenic properties of E coli. Their ability to ferment sorbitol and to produce beta-glucuronidase was also investigated, as these tests are used to discriminate VT positive O157 strains. RESULTS: The O157:H8 strains did not produce VT. All gave localised attachment to HEp-2 cells, associated with a positive fluorescence-actin staining test, and all hybridised with the E coli attaching and effacing (eae) probe. In addition to the difference in VT production, O157:H8 strains could be distinguished from VT positive O157 strains by their beta-glucuronidase activity, their failure to produce enterohaemolysin, and their lack of hybridisation with the CVD419 probe derived from a plasmid in an O157:H7 strain. CONCLUSIONS: The 0157:H8 strains had in vitro properties characteristic of the class of E coli that causes attaching and effacing lesions in epithelial intestinal cells. They may therefore be considered a putative cause of diarrhoea but their prevalence remains to be established. Several O157:H8 strains failed to ferment sorbitol in agar plates and therefore could be misidentified as VT positive O157 strains. Confirmatory tests for VT production are needed when O157 strains are isolated from faeces.

Properties of Vero cytotoxin-producing Escherichia coli of human origin of O serogroups other than O157.

The 48 Vero cytotoxin-producing Escherichia coli (VTEC) examined for properties associated with virulence were of human origin and represented 17 O serogroups other than O157 and O26. Only Vero cytotoxin production was common to all the strains. About 60% produced enterohemolysin and hybridized with the CVD419 probe derived from plasmid sequences of E. coli O157. Thirteen strains gave localized adherence (LA) to HEp-2 cells. All of these hybridized with the E. coli attaching and effacing (eae) gene probe and were positive in the fluorescence actin staining test, properties characteristic of strains that efface intestinal microvilli. A further 5 strains were eae probe-positive but did not give LA. None of the VTEC hybridized with a probe specific for the enteropathogenic E. coli adherence factor. Seven strains adhered to HEp-2 cells in a diffuse or aggregative pattern but did not hybridize with probes for these phenotypes. Non-O157 E. coli strains are diverse in their properties, although some may share virulence mechanisms with other diarrheogenic E. coli.

Virulence properties of Enterobacteriaceae isolated from the small intestine of children with diarrhea.

Enterobacteriaceae isolated from the duodena of Peruvian children with persistent diarrhea (PD) have been examined for virulence factors and compared with Enterobacteriaceae isolated from children with acute diarrhea, those convalescent from PD and diarrhea-free controls. Escherichia coli were isolated from 42 of 186 (23%) of the aspirates. All 11 children with PD in whom multiple E. coli colonies were examined were colonized by a single serotype. DNA probes identified enterotoxigenic E. coli in 2 of 89 (2.2%) PD aspirates and 2 of 38 (5.3%) acute diarrhea aspirates and enteroaggregative E. coli in one PD and one control aspirate. Strains positive with the enteropathogenic E. coli adherence factor probe were identified from 2 of 89 (2.2%) patients with PD and 1 of 34 (2.9%) controls. A subset of 12 E. coli strains failed to show adhesion to human duodenal enterocytes although 5 of 9 showed sparse but polar attachment to ileal cells from a child with short bowel syndrome and PD. Three of 10 Enterobacteriaceae (two E. coli, one Klebsiella species) caused diarrhea in the reversible ileal tie adult rabbit model. Colonization with virulent Enterobactericeae did not explain the majority of episodes of PD. Examination of these duodenal bacteria in the rabbit model revealed some that caused diarrhea but were not recognized pathogens.

A method of enhancing verocytotoxin production by Escherichia coli.

The number of verocytotoxin producing Escherichia coli (VTEC) present in the faeces during an infection may be very low, making their detection difficult. We report a method for enhancing toxin production by VTEC using mitomycin C as an inducing agent with the aim of improving the detection of VTEC. In pure culture, mitomycin C enhanced toxin production up to 100-fold. When applied to mixed faecal culture, toxin could be detected in mitomycin C treated samples when standard cultures were negative and when substantially fewer verocytotoxin-producing bacteria were present. Use of this method may aid in the detection of VTEC and is appropriate for use in the routine diagnostic laboratory.

Identification of enteropathogenic Escherichia coli isolated in Britain as enteroaggregative or as members of a subclass of attaching-and-effacing E. coli not hybridising with the EPEC adherence-factor probe.

Strains of Escherichia coli from sporadic cases of diarrhoea and belonging to serotypes O44:H18, O55:H7, O111ab:H21, O111ab:H25 or O126:H27 were examined for virulence properties. With the exception of O111ab:H25 these are considered to be classical enteropathogenic E. coli (EPEC) serotypes. The strains had been isolated in Britain from the faeces of children less than 3 years old. Of the serotypes examined, 7 of 13 O44:H18 strains, all of 10 O111ab:H21 strains and 13 of 21 O126:H27 strains belonged to the enteroaggregative class of E. coli (EAggEC) that attached to HEp-2 cells in the characteristic aggregative pattern and hybridised with the EAggEC probe. They also caused mannose-resistant haemagglutination of rat erythrocytes, a property which may be a useful marker for their identification. Strains of O44:H18 with similar properties were also isolated from three small outbreaks in Britain, one of which involved elderly patients. EAggEC have not been considered previously as aetiological agents of diarrhoea in developed countries and have rarely been reported as belonging to EPEC serotypes. All 15 O55:H7 strains and seven of eight O111ab:H25 strains were also considered to be potentially diarrhoeagenic as they gave localised attachment (LA) to HEp-2 cells that resulted in a positive fluorescence actin-staining test. This test is considered to correlate with the attaching-and-effacing virulence mechanisms of EPEC in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Surveys of human enterotoxigenic Escherichia coli from three different geographical areas for possible colonization factors.

Enterotoxigenic Escherichia coli (ETEC) from Burma, central Africa (Rwanda and Zaire) and Peru, were screened by enzyme-linked immunoassays for the colonization factor antigens (CFAs) and putative colonization factors (PCFs): CFA/I, CFA/II, which consists of three coli surface-associated (CS) antigens, CS1, CS2 and CS3, CFA/III, CFA/IV (CS4, CS5, CS6), CS7, PCFO9, PCFO159. H4, PCFO166, and CS17. The highest proportion of ETEC with identifiable colonization factors (71%) were found in the strains from Burma, which were mainly positive for CFA/I (38%), but strains producing CFA/II (4%), CFA/IV (11%), CS7 (10%), CS17 (4%), PCFO159, H4 (2%) and PCFO166 (2%) were also found. Sixty-nine percent of the ETEC from central Africa were positive for known colonization factors. While CFA/I positive strains were important (12%), a higher number of ETEC producing CFA/IV (33%) and CS17 (24%) were found. Fifty-two percent of the Peruvian strains produced identifiable colonization factors. The largest group of strains produced antigens of the CFA/IV complex (17%), while ETEC producing CFA/II (6%), CFA/III and CS6 (2%), CS7 (6%), PCFO9 (6%), PCFO166 (8%) and CS17 (7%) were also found. These surveys show that there is a considerable variation in the proportions and types of colonization factor found in different geographical areas. From 29 to 48% of the ETEC did not possess an identifiable colonization factor. These were particularly of the LT only producing type. These results have important implications for vaccine formulation.