VT2 Genes Research Articles

Prevalence and characteristics of human and bovine verotoxigenic Escherichia coli strains isolated in Galicia (north-western Spain).

An epidemiological study was carried out to determine the incidence and the serotypes of verotoxigenic Escherichia coli (VTEC) that cause infections in Galicia (north-western Spain). Although, VTEC strains were isolated from 55 (14%) of the 387 calves sampled and the majority of bovine VTEC strains belonged to serotypes (026:H11 or H-, 091:H21, O103:H2, 0105:H18, O111:H-O113:H21, O126:H-, O128:H- and O157:H7 or H-) previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) in other countries, VTEC are not a common cause of human infections in Spain. Thus, VTEC (O26:H11 and O86:H10) were isolated from only 3 (0.6%) of the 482 children with diarrhoea investigated. We examined the 69 (3 humans and 66 bovines) VTEC strains that were initially isolated as E. coli producing a toxin cytotoxic to Vero and HeLa cells by polymerase chain reaction (PCR) using specific primers for VT1, VT2 and eae genes. PCR showed that 38 (55%) of VTEC strains carried VT1 genes. 18 (26%) possessed VT2 genes, and 10 (14%) carried both VT1 and VT2 genes. Three (one human and two bovine) strains which were formerly VTEC had lost the ability to produce verotoxins upon subculture and became negative for VT1 and VT2 by PCR. In total 35 (51%) of 69 VTEC strains, including the two human VT1+ strains of serotype O26:H11, were positive for eae sequences when tested by PCR. Presence of the eae gene was significantly more frequent (100%; 21/21) among VTEC strains with serotypes (O26:H11, O111:H-, O157:H-and O157:H7) considered as enterohaemorrhagic E. coli (EHEC) than among VTEC strains with non-EHEC serotypes (29%; 14/48) (p < 0.001). Results obtained in this study indicate that cattle may be an important source of VTEC involved in human disease. However, severe clinical syndromes caused by VTEC, such as HC and HUS, are uncommon in Spain, in comparison with North America and the UK. In any case, VTEC disease can appear on the scene very suddenly, as occurred in the UK and North America in the 1980s.

Pheno-genotyping of verotoxin 2 (VT2)-producing Escherichia coli causing haemorrhagic colitis and haemolytic uraemic syndrome by direct analysis of patients' stools.

The subtype of verotoxin 2 (VT2) found in 22 VT2-positive stool samples from severely diseased Italian and German children with haemorrhagic colitis or haemolytic uraemic syndrome, or both, and that produced by the corresponding VT-producing Escherichia coli (VTEC) strains isolated from the stools were studied by cytotoxicity seroneutralisation assays and by polymerase chain reaction (PCR) amplification of the VT2 B-subunit gene, followed by restriction fragment length polymorphism (RFLP) analysis. The free faecal toxin was serotyped as the classical VT2 in 21 stool samples, and as the VT2 variant VT2c in one. For all but one of the VTEC isolates, the toxin phenotype was consistent with the type of VT produced in vivo and found in the corresponding stool samples. Genotyping was in agreement with phenotyping for those strains harbouring a single type of VT2 gene. Three O157:H7 isolates carrying both VT2 and VT2c genes had the VT2 phenotype, instead of the expected VT2c phenotype. Direct PCR analysis of stools detected VT genes in only 11 of 20 VT-positive stool samples suggesting that the Vero cell cytotoxicity assay is more sensitive in diagnosing VTEC infection. Immunological and genetic subtyping of VT2 performed directly on stool samples from patients with haemolytic uraemic syndrome could be a useful complementary approach to understanding the role of the different types of VT in this syndrome.

Characterization of verocytotoxin-producingEscherichia coliO157 isolates from patients with haemolytic uraemic syndrome in Western Europe

Fifty verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O157 were characterized by phage typing, polymerase chain reaction (PCR) for VT genes and the E. coli attaching and effacing (eae) gene, and random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting. The collection represented isolates obtained from patients with diarrhoea-associated haemolytic-uraemic syndrome (D+ HUS) and their family contacts, isolated in the Netherlands, Belgium and Germany between 1989 and 1993. Based on isolates from separate families (n = 27) seven different phage types were identified, types 2 (44%) and 4 (33%) were predominant. Eighty-five percent of the strains contained only VT2 gene sequences and 15% both VT1 and VT2. All strains of the dominant phage types 2 and 4 carried the VT2 gene. Strains that belonged to the minor phage types 8, 14, 32 carried both VT1 and VT2 genes, with the exception of two isolates identified as phage types 49 and 54 which contained only VT2 genes. All O157 VTEC strains possessed the chromosomally-located eae gene, which indicates its usefulness as virulence marker. RAPD-PCR fingerprinting identified four distinct banding patterns, with one profile found among 79% of the strains. Based on the combined results of all typing methods used in this study, the collection of 50 O157 VTEC strains could be divided into nine distinct groups. Strains isolated from different persons within one family could not be distinguished by any of these methods. The data suggest that O157 VTEC strains are members of one clone that has become widely distributed.

Some characteristics of verotoxin-producing Escherichia coli strains isolated from sporadic diarrhea in Akita Prefecture

Six sporadic cases of VTEC infection have been confirmed in Akita Prefecture from June. 1991 to Nov. 1994. Six VTEC strains isolated in these cases were examined for their serotype, Vero toxin type, existence of eae gene and 60 MDA plasmid, and CVD probe reactivity. Of the 6 isolates, 5 were O157:H7. Two isolates of which possessed TV-1 and VT-2 genes and the rest of 3 possessed VT-1 and VT-2vh genes, VT-2 gene, respectively. One isolate was O26:NM possessing VT-1 gene. A primer pair, designated as EA-1 and EA-2, was designed for detecting eae gene in VTEC. Examination of the 6 VTEC isolates by PCR using EA-1 and EA-2 primers indicated that all of the isolates were eae positive. This was further confirmed by dot blot hybridization using a eae probe. All of the 6 isolates also harbored approximately 60 MDa plasmid, which hybridized with the CVD419 probe in southern blot analysis. These results supported the hypothesis that eae gene and the 60 MDa plasmid might play a role in the mechanism for VTEC to cause diseases including diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Diversity observed in plasmid profile and VT type of the 6 isolates suggested that they were originated from independent sources, which could not be identified.

ＰＣＲ法による食肉およびふん便中の下痢原性大腸菌の迅速検査法の検討

A direct polymerase chain reaction method to EC cultured broth (named EC-PCR) for screening detection of enteropathogenic E. coli (ETEC, VTEC and EIEC) in foods and feces is described.Eleven E. coli strains belonging to three groups (4ETEC: 06, 025, 0128, 0148; 4VTEC: 026, 0111, 0145, 0157; and 3EIEC: 028ac, 0124, 0164) were used for this study. Four pairs of oligonucleotide primers homologous to LT, ST, VT and EIEC genes were used in combination. The culture condition at 37-43°C for 16-20 h in EC broth was most suitable for the EC-PCR method, and no cross readings were observed with other bacteria, substances in meats or feces.After a 20-h enrichment step, it was possible to detect fewer than 10 to 102 bacteria per g of the aritificially inoculated meat. E. coli was easily detected because of low contamination with other bacteria which disturb the detection of E. coli. However, in feces, it ranged from 103 to 104 cfu per g. The large number of bacteria with feces are the main limiting factor of the EC-PCR detection assay. All E. coli strains examined were detected from all the enrichment cultures on both the EC-PCR and the culture methods. In two sporadic cases and two food poisoning cases, the enteropathogenicity of E. coli isolates from patients was rapidly judged by the EC-PCR method.These findings were consistent with those of the culture method. Thus, the findings suggest that the EC-PCR method is a suitable, sensitive and rapid method for detection of the potentially enteropathogenic E. coli.

Isolation of verotoxin-producing Escherichia coli from pediatric patients suffering from enteritis with bloody stool and intussusception

We tried to isolate verotoxin-producing Escherichia coli (VTEC) on sorbitol-MacConkey (SMAC) agar and in part by the polymerase chain reaction (PCR) method from sporadic enteritis patients with bloody stools and intussusception patients who came to three pediatric clinics in the Fukuoka area from October 1990 to September 1994. VTEC O157:H7 strains were isolated from 6 (10.5%) of 57 patients with bloody stools, Campylobacter spp. 15 (26.3%), Salmonella spp. 14 (24.6%) and Yersinia enterocolitica 2. We were not able to detect VT genes by PCR from 11 of 20 patients from whose stools no causative bacteria were isolated. Massive fresh bloody stools following frequent watery diarrhea were typical of the VTEC enteritis patients. Only 1 patient had fever and 2 had leukocytosis, but the C-reactive protein in all of them was below 1+. The VTEC strains were isolated during the summer season, 1 in June, 2 in July, and 3 in September. Since in the area O157:H7 appeared to be the most prevalent VTEC serotype, SMAC is very useful for screening VTEC in bloody stools. VTEC seems to be a rare pathogen of intussusception because the organisms were detected from none of the 30 patients.

Genes coding for Shiga-like toxins in bovine verotoxin-producing Escherichia coli (VTEC) strains belonging to different O:K:H serotypes

Forty-six verotoxin-producing Escherichia coli (VTEC) strains isolated from diarrhoeic and healthy calves in Spain were examined for DNA sequences homologous to genes for verotoxins (VT1 and VT2) and enterotoxins (LT-I, LT-II, STaH, STaP and STb). Hybridisation showed that 26 (57%) of VTEC strains carried VT1 genes, 13 (28%) possessed VT2 genes, and 7 (15%) carried both VT1 and VT2 genes. No VTEC strains hybridised with DNA probes for enterotoxins. A correlation was found between the serotype and type of VT produced. Thus, all strains of serotypes O26:K-:H11 (13 strains), O103:K-:H2 (3 strains) and O128:K?:H- (4 strains) hybridised with the VT1 probe only, whereas all strains of serotypes O4:K-:H4 (3 strains) and O113:K-:H21 (4 strains) were positive with the VT2 probe only. By contrast, O81:K?:H28 (2 strains) and O157:K-:H- (2 strains) strains hybridised with both VT1 and VT2 probes. One strain of serotype O157:K-:H7 was VT2 positive.

Biological characters of enterohemorrhagic Escherichia coli isolates from diarrhea patients in Saitama (1990-1992)

A total of 16 strains of Enterohemorrhagic Escherichia coli (EHEC) isolated from diarrea patients in Saitama from 1990 to 1992 were tested for their serotype, verotoxin production, biochemical characteristics, antibiotics sensitivity and plasmid profiles. By serotype analysis, 14 strains from two outbreaks and 12 sporadic cases were classified as type O157:H7, one as O111:H-(not motility) and one as O128:H2. Typing of verotoxin by gene analysis using Polymerase Chain Reaction (PCR) showed that 9 of O157:H7 strains including two cases from outbreaks and O128:H2 have VT1 and VT2 genes, other O157:H7 have the VT2 gene and O111:H-has only the VT1 gene. Biochemical characteristic analysis indicated two strains of O157:H7 type from outbreaks were biotype II and the rest of O157:H7 were biotype I. One of the O157:H7 strain from a sporadic case showed positive for urease production. According to sensitivity tests against antibiotics, out of the O157:H7 group, one strain was resistant against ABPC, one against SM and two strains resistant to SM-TC. For plasmid profiles, all strains had 94 Kb plasmids and several smaller sizes of plasmids. But 5 strains of O157:H7 had 94 Kb plasmid only.

Use of digoxigenin-labelled oligonucleotide DNA probes for VT2 and VT2 human variant genes to differentiate Vero cytotoxin-producing Escherichia coli strains of serogroup O157

Digoxigenin-labelled oligonucleotide DNA probes specific for B-subunit genes of Vero cytotoxin 2 (VT2) and a variant of VT2 (VT2vha) were used to differentiate 116 strains of Escherichia coli serogroup O157 belonging to phage types 1, 2, 4, 8, 14, and 49. Of these strains, 38% had sequences for both VT2 and VT2vha, 38% had sequences for VT2 only, and 24% had sequences for VT2vha only. Oligonucleotide probe hybridization subdivided strains of all of the phage types except phage type 1. The greatest variation in toxin gene pattern was observed with strains of phage type 14, for which there were six distinct patterns when the presence or absence of VT1 genes was also considered. Two strains from each phage type group were examined for bacteriophages encoding VT production. Two of the six VT2vha-producing strains carried phage from which DNA hybridized with the VT2vha-specific probe. Phages were not detected in the remaining four VT2vha strains, suggesting that genes may be chromosomally located or associated with a defective prophage. In contrast, seven of the eight VT2 strains carried phages from which DNA hybridized with the VT2-specific probe. Two strains of E32511 (O157:H-) were also investigated. One strain (E32511A) possessed gene sequences for both VT2 and VTvha and was shown to carry phage possessing gene sequences for VT2. With strain E32511B, however, phages were not detected and DNA hybridized only with the VT2vha probe. Analysis of total genomic DNA digested with restriction endonuclease EcoRI showed that polymorphisms were seen with VT2 strains and not with VT2vha strains.

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.

Biological characters of the clinically isolated strains of Escherichia coli O157:H7 in Canada

Biological characters of enterohemorrhagic Escherichia coli (EHEC) strains isolated from food poisoning cases in Canada, 1987-1988 were examined in comparison with that isolated in Saitama, 1990. The Saitama strain was found to belong to biotype I, and biotypes I and II were found in Canada. Most of the strains including the Saitama strain were found to have siderophilins suggesting their advantageous growth in vivo. An enteroinvasive strain with 140 megadalton (Md) plasmid and two colicinogenic strains with 4.8 Md plasmids were detected. All strains were found to have 60 Md plasmids. This plasmid might be specific for this serotype. A few strains were resistant to streptomycin (SM) and/or tetracycline (TC), and a strain transferred its SM-TC resistances by conjugation. Many strains including the Saitama strain were found to produce vero cytotoxin (VT) types 1 and 2 (VT1 and VT2). A few type 2 and untypable toxin producing strains were also detected. VT titers produced by untypable toxin producing strains were over 10 times lower than those by other strains. High annealing temperature for PCR amplification of VT1 gene and a variety of annealing temperatures of VT2 gene suggested that the nucleotide sequences for VT1 gene were well preserved, but that those for VT2 might have some mutations.

A healthy carrier of verotoxin-producing Escherichia coli, who was detected by periodic feces examinations

Verotoxin-producing Escherichia coli (VTEC) was rapidly detected by the PCR method in one of the 9 feces samples. They were collected from the people who had been cooking meals for patients on a periodic feces examination on November 25, 1992. It was confirmed by PCR that the isolate had both VT1 and VT2vh toxic genes and showed cytotoxicity on Verocells. In this case, the isolate was highly susceptible to common antibiotic agents, it was removed by the administration of tosfulaxacin. Some isolates which had the same properties as those of the strain described in "VITEK GNI card" and the antimicrobial susceptibility and VT toxin types, were detected in 2 of the 3 members of her family, they were healthy carriers and did not have any subjective symptoms such as diarrhea or abdominal pain. In order to detect Verotoxic genes, the sample of feces preincubated for 3 h in trypticase broth was subjected to PCR. We recommend that this method is much more useful because of rapid detection and identification of VTEC compared with the classical culture method.

Polymerase chain reaction for detection of verocytotoxigenic Escherichia coli isolated from animal and food sources

Animals and their by-products have been implicated as important sources of verocytotoxigenic Escherichia coli (VTEC) associated with disease in humans. VTEC comprise a wide range of serotypes and produce a variety of closely related verocytotoxins (VT). A pair of oligonucleotide primers, targeting conserved sequences found in VT1, VT2 and VTE genes, was used to develop a polymerase chain reaction (PCR) procedure to detect all types of VTEC. Supernatants of boiled broth cultures of VTEC (223 strains) isolated from ground beef, ground pork, raw milk, bovine faeces and porcine faeces; non-VTEC E. coli (72 strains); and other enteric and food bacteria (76 strains) were tested by PCR. The verocytotoxigenicity of these strains was verified by the Vero cell assay. All 223 VTEC isolates, comprising over 50 different serotypes, were detected by the PCR procedure. Shigella dysenteriae type 1 was the only other bacterium that was positive in this assay. As little as 1 pg of VTEC DNA and as few as 17 cfu of VTEC could be detected with this method. The results indicate that these primers detect VTEC over a wide range of serotypes. This method may be applicable as a screening procedure for the detection of VTEC in samples of foods and faeces.

Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype.

Non-radioactively labelled polynucleotide and oligonucleotide DNA probes, for selectively detecting Escherichia coli strains producing Vero cytotoxins VT1, VT2 and VT2 variant

Vero cytotoxin producing Escherichia coli (VTEC) were detected in faecal specimens and bacterial isolates, using non-radioactively labelled polynucleotide and oligonucleotide DNA probes specific for Vero cytotoxin (VT) genes. VT1 and VT2 structural gene sequences, previously cloned and used for radioactive probes, were labelled with digoxigenin or biotin. Oligonucleotide gene sequences coding for the A subunit of VT1, VT2 and VT2 variant were labelled with digoxigenin. The VT1 and VT2 probes were specific for detecting VT1 and VT2 gene sequences and gave very similar results to those obtained using the radioactive label 35S as a standard. The VT2 variant probe hybridized only with the strains of porcine origin. For the range of isolates tested, there was little significant difference in specificity and sensitivity between the digoxigenin-labelled polynucleotide and oligonucleotide probes. The biotin system gave rise to more non-specific effects, particularly with some non- E. coli strains, and was therefore less reliable. All of the digoxigenin-labelled probes gave satisfactory results after several times re-use, which is of importance when considering cost.

DNA probes for the detection of toxin genes in Escherichia coli isolated from diarrhoeal disease in cattle and pigs

DNA gene probes specific for genes coding for heatt labile toxin (LT), heat stable toxins (STpa, STpb) and Vero-cell toxins (VT1, VT2) were used to examine 1031 diarrhoeal disease isolates of E. coli (345 from cattle and 686 from pigs). Of the bovine strains, 60 hybridized with the STpa probe and most possessed the K99 (F5) or F41 adhesin. Five bovine strains possessed STpb genes and five either VT1 or VT2 genes. Of the porcine strains, 245 hybridized with one or more gene probes. Of 160 K88 (F4) positive strains, 133 possessed both LT and STpb genes, whilst 17 possessed LT or STpb or STpa alone or in combination. Ten K88 strains did not possess toxin genes. Isolates bearing the K99 (F5) adhesin possessed either STpa, STpb and VT2 genes alone or in combination; in one isolate only the LT gene was detected. Isolates belonging to serogroup 0138:K81 were more heterogeneous as to their toxin genes; of the 60 strains, fourteen carried only VT2 genes, thirty-two carried VT2, STpa and STpb genes, one carried LT, VT2, STpa and STpb genes, two carried STpb gene, four carried STpa and STpb genes, one carried LT and VT2 genes, two carried LT and STpa genes, whilst four carried none. Twenty-four percent of all toxigenic strains apparently did not possess adhesins.

Plasmid and chromosomal related toxin polymorphism of Escherichia coli serogroup O138; plasmid transfer and co-integration with pRP4

DNA-DNA hybridisation was used to examine 89 isolates of Escherichia coli serogroup O138 for toxin gene content and location. Sixty-six isolates encoded toxin genes; 29 were STpa STpb VT2, 24 were VT2, four were STpa STpb, three were STpb VT2, three were STpb VT2LT and one was STpa. None were K88-, K99- or F41-positive. The VT toxin gene was chromosomally located in all VT+ isolates tested whereas STpa, STpb and LT were plasmid borne. In co-transfer experiments with pRP4 as the mobilising plasmid, VT2 genes were not transferred to recipient E. coli K12 whereas STpa, STpb and LT genes were. Two VT2-positive isolates inhibited pRP4 transfer to K12 by a factor of 10(6) and rare transconjugants harboured a co-integrate plasmid comprising pRP4 (54kb) and a cryptic host plasmid (145 kb). In a K12 background, the co-integrate had transfer and incompatibility properties of pRP4 whereas in both progenitor E. coli O138 backgrounds the co-integrate was transfer inhibited and in one case resolved to give original plasmids.

Examination of strains belonging to enteropathogenic Escherichia coli serogroups for genes encoding EPEC adherence factor and Vero cytotoxins

Four hundred and forty-nine strains isolated from patients with diarrhoea and belonging to 13 enteropathogenic Escherichia coli (EPEC) O serogroups were tested with a DNA probe for the EPEC adherence factor (EAF). Positive results were obtained with only 36 strains; they belonged to 10 O serogroups and flagellar typing showed they were usually of the "classical" EPEC serotypes. Thirty-four of the 36 EAF-positive strains showed localised adhesion to HEp-2 cells. The two remaining strains, of serotypes O114:H2 and O127:H4, showed low level or no adhesion to HEp-2 cells. No colonies hybridising with the EAF probe were identified in cultures from 115 faecal specimens from healthy children. Sixteen of the 449 strains hybridised with one or both probes for the Vero cytotoxin genes VT1 and VT2; 15 of the 16 strains belonged to serogroups O26 and O128. None of the strains hybridised with both the EAF and VT gene probes. These studies show that the great majority of strains belonging to EPEC O serogroups do not possess the EPEC adherence factor or carry VT genes.

Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction

A set of four synthetic oligonucleotide probes derived from sequences of the VT1 (Shiga-like toxin I [SLT-I]) and VT2 (SLT-II) genes were used in a polymerase chain reaction (PCR) amplification procedure to detect these genes in some enteric pathogens. A total of 40 verotoxin-producing Escherichia coli strains and 43 isolates of other recognized enteric pathogens were studied. PCR amplification products identifying the VT1 and VT2 gene sequences were observed only in nucleic acid extracted from strains found to be VT positive in traditional tissue culture assays. Template nucleic acid extracted from other gram-negative bacteria was found to be negative with the exception of five isolates of Shigella dysenteriae type 1 in which good amplification with the VT1 probe was observed. The oligonucleotide probes clearly distinguished VT1 and VT2 strains of E. coli and did not give specific amplification with nucleic acid from VTe (a SLT-II variant)-producing E. coli. VT1 or VT2 genes or both were not detected in E. coli K-12 strain C600 or HB101 or in strains known to express other virulence factors, such as enterotoxins, adhesins, hemolysins, or unrelated cytotoxins. The sensitivity of the PCR procedure for detection of both VT1 and VT2 genes was determined to be 1 ng of total nucleic acid. Furthermore, the VT1 gene was easily detected when only 100 pg of nucleic acid was used as the template in the PCR procedure.

Heterogeneity of Escherichia Coli Phages Encoding Vero Cytotoxins: Comparison of Cloned Sequences Determining VT1 and VT2 and Development of Specific Gene Probes

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.