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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.

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.

SUMMARYEnterotoxigenicEscherichia 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.

Summary: Certain isolates of Escherichia coli from humans and animals with enteric disease attach to enterocytes and cause ‘attaching and effacing’ (AE) lesions. E. coli strain S22-1, serotype O103:H2, isolated from a child with diarrhoea, contained two plasmids; one of these (pDEP12) hybridized with the CVD419 DNA probe derived from a plasmid found in E. coli 0157:H7 and associated with expression of fimbriae and ability to adhere to Intestine 407 cells. Strain S102-9, serotype O5:H-, isolated from a calf with dysentery, contained six plasmids, one of which also hybridized with the CVD419 probe. Loss of pDEP12 coincided with reduced adhesion to HEp-2 or Intestine 407 cells cultured in vitro; reintroduction of this plasmid restored adhesiveness. Loss of the plasmid in strain S102-9 that hybridized with the CVD419 probe did not cause a decrease in adhesion. Accumulations of actin were seen in vitro in the fluorescence actin staining (FAS) test of strains S22-1, S102-9 and their derivatives, irrespective of the plasmid content of these strains or the prevalence of attached bacteria. Strain S22-1 and its plasmidless derivative caused AE lesions of equal severity in experimentally infected gnotobiotic piglets; piglets inoculated with an isolate from a healthy human or pig did not develop these lesions. These results indicate that the CVD419 probe is not specific for genes conferring the ability to adhere to HEp-2 or Intestine 407 cells by these E. coli and that the adhesins detected in vitro, or plasmid-encoded properties, are not required for strain S22-1 to cause AE lesions in gnotobiotic pigs or to cause the accumulation of actin in cells in vitro.

One hundred and twenty Vero cytotoxin-producing (VT + ) strains of Escherichia coli 0157 (of H type 7 or non-motile) isolated in the UK, failed to ferment sorbitol after 24 h or to hydrolyse 4-methylumbelliferyl-beta-D-glucuronide (MUG).This combination of properties was not found in 167 of 169 other E. coli strains, including VT + strains of other serogroups and VT − strains of serogroup 0157. As the two exceptions were rare VT − strains of serotype 0157:H7, we conclude that, although biochemical tests can aid the isolation of VT + 0157 strains, confirmation of VT production is necessary

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.

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 0157 VTEC carry both VT2 and VT2 variant genes.

Summary 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 ofVTEC from human infections have been compared to strains isolated from animals and foods.

Shiga toxin produced by strains of Shigella dysenteriae type 1 and Vero cytotoxins (Shiga-like toxins) produced by strains of Escherichia coli belong to a family of related toxins. Two major forms of Vero cytotoxin (VT) are recognised. Polyclonal antiserum to Shiga toxin neutralises the activity of VT1 (SLTI) but not VT2 (SLTII). Strains of E. coli produce VT1 only, VT2 only or both toxins. Some strains of animal origin, but none as yet of human origin, also produce heat labile enterotoxin or heat stable enterotoxin. By definition, VT is detected by its cytotoxic effect on a monolayer of Vero cells grown in tissue culture, with serum neutralisation tests to determine which toxins are present1. For routine testing, bacteria are grown in a medium such as trypticase soy broth and the toxin present in filtered culture supernatants is assayed. Cell bound toxin can be released by polymixin treatment or sonication to obtain maximum yields. Production of VT1, but not VT2, is increased in an iron-depleted medium2. HeLa cells have also been used to test for VT, but this cell line can no longer be recommended as there are variant forms of VT2, termed VT2v, that have litle or no effect on HeLa cells although they are cytotoxic for Vero cells. The first variant toxin to be identified, produced by strains causing porcine (o)edema disease, has been termed VT2vp3 or VT2e4. VT2vh5 and VT2va6 are variant toxins from strains of human origin. All the VT2 forms are neutralised at least to some extent by polyclonal antisera raised against one of the other VT2 forms.

Thirty-seven strains of Escherichia coli O26:H11 from infants and calves with diarrhea were examined for properties associated with enteropathogenic (EPEC) or enterohemorrhagic E. coli (EHEC). Strains were heterogeneous with respect to Vero cytotoxin (VT) production and hybridization with the EHEC plasmid-specific (CVD419) probe; 26 strains produced VT1; 1 produced VT2. Twenty-four of 27 VT+ strains and 5 of 10 VT- strains hybridized with the CVD419 probe and produced enterohemolysin; these properties are characteristic of EHEC. The strains did not hybridize with the EPEC adherence factor probe, a property characteristic of some EPEC. Nevertheless, 36 strains adhered to HEp-2 cells in a localized manner and were positive by the fluorescence actin staining (FAS) test that is considered to correlate with the ability to cause attaching and effacing lesions in vivo. EPEC and EHEC cause these lesions. Although the FAS test appeared to be the most general pathogenicity test for the O26:H11 strains, it could not be used to assign strains specifically to EPEC or EHEC groups.

Summary: Sequences encoding the production of a cytotoxin (VT) active on Vero cells were cloned in Escherichia coli K12 from a VT-determining phage that originated in E. coli strain H19 of serotype O26. H11. Subcloning resulted in the identification of a 2·5 kb fragment that still coded for VT production. Mutagenesis with transposon Tn1000 was used to map VT sequences and a 0·75 kb probe was developed. In colony hybridization tests with strains isolated from patients with haemolytic uraemic syndrome or diarrhoea, this probe derived from the H19 VT genes detected only some of the VT+ strains belonging to serogroup O157. A VT+ strain, E32511, serotype O157. H-, which was negative in colony hybridization was the source of another VT-determining phage from which VT sequences were cloned. Southern hybridization of the VT genes from E32511 with the H19 probe was negative under stringent conditions but there was weak homology under conditions of low stringency. These results indicate that there are differences in the VT genes of pathogenic E. coli.

Vero cytotoxin (VT) producing strains of Escherichia coli (VTEC), including isolates from cases of haemolytic uraemic syndrome and infantile diarrhoea, were used to determine the effect of iron availability on the production of intra- and extracellular VT, with particular interest in elevating toxin production by low-level toxin producing VTEC. Culturing bacteria under iron restriction resulted in growth retardation and a decrease in the production of VT. For the routine detection of both high- and low-level VT-producing E. coli, there was no advantage to be gained by growing bacteria under iron restriction or using disrupted bacterial cell preparations; on the contrary, testing culture supernatants from bacteria grown in iron-replete media for approximately 14 h proved to be the most sensitive and accurate method for detecting VT and the resultant identification of VTEC.

Strains of enteropathogenic Escherichia coli (EPEC), enteroinvasive E. coli (EIEC) and Shigella were examined for Vero cytotoxin (VT) production and tested for the presence of VT genes in DNA hybridisation experiments with specific probes for VT1 and VT2. Twenty seven of 104 EPEC strains produced VT and in 26 of the 27 strains VT genes were detected. The VT+ EPEC strains belonged to serogroups O26, O55 and O128. Strains belonging to 8 other EPEC serogroups including strains isolated from 13 outbreaks, were all negative using both techniques, as were the 5 EIEC strains. It is concluded that VT1 and VT2 are not virulence factors in most EPEC infections. VT1 was demonstrated in all Sh. dysenteriae type 1 strains but neither VT1 nor VT2 were detected in strains of Sh. flexneri, Sh. boydii or Sh. sonnei.

Summary: Two plasmids transferred from enterotoxigenic Escherichia coli (ETEC) of serotype 06. H16 and biotypes A and C coded for mannose-resistant haemagglutination (MRHA) and production of heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Both plasmids were non-autotransferring being mobilized most efficiently by the R plasmid R100-1. They were similar in their genetic properties being incompatible with each other and plasmids of the Inc group FI. The wild-type strains produced the colonization factor antigen II (CFA/II) which was made up of different coli surface antigens (CS). The biotype A strains produced CS1 and CS3 while the biotype C strains produced CS2 and CS3. These three antigens have the ability to cause MRHA. When plasmids coding for MRHA were transferred to K12 strains, the degree of haemagglutination was markedly reduced and only CS3 was produced. When both plasmids were transferred back into biotype A strains, good MRHA was restored and the strains produced CS1 and CS3. In a biotype C strain CS2 and CS3 were formed. The production of the antigens was compared by enzyme-linked immunosorbent assay (ELISA). The strains were also examined by electron microscopy where it was found that CS1 and CS2 were fimbrial antigens while CS3 was not.

SUMMARY: Sixty strains of Escherichia coli belonging to enteropathogenic serogroups (EPEC) were examined for the ability to adhere to HEp-2 cells, the possession of the genes encoding EPEC adherence factor (EAF) and the ability to express an outer-membrane protein (OMP) of 94 kDa thought to be involved in bacterial adhesion to eukaryotic cells. An absolute correlation was found between HEp-2 adhesion and the possession of the genes encoding EAF. An OMP of 94 kDa was observed in the SDS-PAGE profile of most adhesive strains. In some strains this protein was prone to proteolytic degradation. An antiserum raised to a HEp-2 adhesive strain of EPEC did not react with the 94 kDa OMP of all EPEC which were EAF-positive and HEp-2 adhesive, indicating some interstrain antigenic variation of this protein. Although this 94 kDa protein was surface-exposed, specific antibodies binding to the 94 kDa protein in situ in the outer membrane did not interfere with adhesion of EPEC to HEp-2 cells. Therefore, these studies question the value of this protein as a potential vaccine component.

Escherichia coli strains isolated from outbreaks of diarrheal disease were tested for the presence of adhesive factors. Fifty-one of these strains belonged to traditional infantile entero-pathogenic serotypes (EPEC) and 17 belonged to other serotypes. None of these strains were enterotoxigenic and none possessed colonization factors CFA/I or CFA/II, which have been described among strains of enterotoxigenicE. coli (ETEC). EnterotoxigenicE. coli strains from patients with diarrhea and strains which were neither EPEC nor ETEC from subjects without diarrhea were also examined. By means of a tissue culture technique using HEp-2 cells, a new adhesive factor was found to occur with greater frequency in EPEC strains. The adhesive factor was found less frequently in the other groups ofE. coli studied. It was distinct from type 1 pili and was not inhibited by the presence ofD-mannose.

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.

A total of 134 enterotoxigenicEscherichia coli (ETEC) of serogroups O25, O27, O148, and O159 were tested in the enzyme-linked immunosorbent assays for the colonization factor antigens I (CFA/I), CFA/II (coli surface antigens CS1, 2 and 3) and putative colonization factor (PCF) 8775 (CS4, 5 and 6). CS6 was detected without CS4 or CS5 in 94% of the strains of serogroup O25, 86% of strains of serogroup O27, 87% of strains of serogroup O148, and 29% of strains of serogroup, O159. The frequency with which CS6 occurs in ETEC of common serotypes without the antigens CS4 or CS5 suggests that it might be a colonization factor.

Six strains of Escherichia coli producing Vero cytotoxin (VTEC) and six strains of Shigella dysenteriae 1 were examined for the production of extra- and intracellular Verocytotoxin (VT) and Shiga toxin respectively, in relation to the growth medium and availabilityof iron. VTEC secreted less extracellular VT1 or VT2 when grown in trypticase soy broth(TSB) containing the iron chelator desferal, as compared to bacteria cultured in iron repleteTSB. Growth in TSB containing desferal resulted in increased production of intracellulartoxin by VT1 producing strains of E. coli ; but had little effect on production of intracellulartoxin by VT2 producing strains. Both extra- and intracellular levels of Shiga toxin wereincreased by growth in TSB containing desferal. Combining intra- and extracellular toxin titres, strains of E. coli producing VT1 gavehighest titres following growth in TSB where iron was bound to the chelating agent desferal,while strains of S. dysenteriae 1 produced highest levels of toxin when grown in syncase-glucosebroth made iron depleted using Chelex-100. Strains of S. dysenteriae 1 did not growin syncase-glucose broth containing desferal.

SUMMARYThe ability of certainEscherichia colistrains to produce enterotoxin is determined by transmissible plasmids. It is therefore possible that anyE. colistrain might be able to acquire such a plasmid and that the correlation between enterotoxigenicity and serotype might be random. However, recent studies show that the enterotoxigenic strains so far described belong to a restricted range of serotypes. Enterotoxigenic strains ofE. coliO6.H16 andE. coliO148.H28 have been associated with outbreaks of diarrhoea in several countries, therefore strains ofE. colibelonging to these serotypes were selected for further study.Twenty-three strains ofE. coliO6.H16 and 14 strains ofE. coliO148.H28 were examined; 20 strains ofE. coliO6.H16 and all 14 strains ofE. coli0148.H28 were enterotoxigenic but strains ofE. coliO6 with flagellar antigens other than H16 and strains ofE. coliO148 with flagellar antigens other than H28 were not enterotoxigenic. The examination of single colony subcultures derived from theE. coliO6.H16 strains showed that in some strains loss of enterotoxigenicity had occurred in a proportion of colonies.

Summary: Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than 0 6 and produced colonization factor antigen 11, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes. A non-autotransfer-ring plasmid, NTP165, from a strain of E. coli 0168.H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens. The CS antigens expressed after acquisition of plasmid NTPl65 depended on the recipient strain : a biotype A strain of serotype 0 6. H 16 expressed CS1 and CS3; a biotype C strain of serotype 0 6. H 16 expressed CS2 and CS3; strain K 12 and strain El9446 of serotype 0139. H28 expressed only CS3. An exceptional wild-type strain, E24377, of serotype 0139. H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lostthe plasmid coding for CS antigens produced both CSl and CS3 after the introduction of NTP165.

Twenty-three of 89 enterotoxigenic strains ofEscherichia coliwere resistant to one or more antimicrobial agents. Eleven strains transferred resistance directly and five transferred resistance after mobilization. In three cases a resistant recipient was enterotoxigenic. One of these strains contained a conjugative plasmid carrying genes for both drug resistance and enterotoxin production. In the two other strains genes for drug resistance and enterotoxin production were carried on separate co-transferable plasmids.

EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined. The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively. All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns. Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive. An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2. Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells. The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP). When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed. However, when EAF plasmids were transferred into E. coli K12 or non-EPEC E. coli the host strains either did not adhere or adhered poorly to the HEp-2 cells. These transconjugants did not express a 94 kDa OMP.

Summary: Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.

SUMMARY: Vero cytotoxin (VT) production has been studied in 34 Escherichia coli strains isolated from animals with enteric diseases. The strains were tested by DNA hybridization with probes for VT1 and VT2 sequences and also in toxin neutralization experiments with specific antisera. Twenty bovine strains, belonging to nine different O serogroups, produced VT1 or VT2 but not both toxins. In contrast, all 14 porcine strains of four different O serogroups produced VT2 only. Six of these porcine strains, belonging to serogroups 0138, 0139 and 0141, were isolated from cases of oedema disease. In general, the porcine isolates produced toxin at a lower level than the bovine strains.

SUMMARY: Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype Ol57: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The 0157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two 026 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the 026 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.