Verocytotoxin Genes Research Articles

Verocytotoxin-producing Escherichia coli infection: The Hong Kong experience.

Infection by verocytotoxin-producing Escherichia coli (VTEC) is prevalent in many parts of the world but relatively uncommon in Asia, except Japan. A territory wide screening for VTEC (April to August 1996) in diarrhoeal stool samples sent to six hospital microbiology laboratories in Hong Kong revealed only four isolates of VTEC and one isolate of E. coli O157:NM in 1003 specimens (incidence 0.5%). Two isolates carrying the verocytotoxin (VT) genes belonged to the O157:H7 serotype while the other two were non-O157. One non-toxigenic E. coli O157:NM was also isolated. All isolates positive for VT genes by polymerase chain reaction (PCR) were also positive for the Vero toxin assayed by the Vero cell culture. The 97 kDa eaeA outer membrane protein gene and 60 MDa fimbrial plasmid pcVD419 were present only in the two O157:H7 isolates. All patients presented with uncomplicated watery diarrhoea; no one suffered from haemorrhagic colitis or the haemolytic uraemic syndrome. All patients recovered uneventfully without antibiotic treatment. Although VTEC infection is still uncommon in Hong Kong, continued surveillance is essential to prevent future outbreaks.

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.

Risk factors for infection with verocytotoxigenic Escherichia coli in cattle on Ontario dairy farms

Risk factors for prevalent infection with verocytotoxigenic Escherichia coli (VTEC) were studied in a random sample of 886 cows and 592 calves under 3 months of age on 80 randomly selected dairy farms in southern Ontario. Fecal-culture supernatants from each animal were screened for verocytotoxicity using a Vero cell assay (VCA) and for verocytotoxin (VT) genes by a polymerase chain reaction (PCR) procedure. Up to 20 E. coli isolates from positive samples were tested for VT production using VCA and PCR. VTEC isolates were serotyped. Farm managers were interviewed using a standardized questionnaire to obtain information on farm- and individual animal-level management practices and characteristics. There was a significant ( P<0.001) positive association between age of calves and their VTEC infection status, and calves were significantly more likely to be infected than cows. The proportion of calves infected on the farm was positively associated with both the use of regular pails for feeding calves (as opposed to nipple bottles or nipple pails) and bringing new animals into the herd in the previous year.

Enterobacteriaceae found in high numbers in fish, minced meat and pasteurised milk or cream and the presence of toxin encoding genes

Enterobacteriaceae were found in high numbers after storage at 7°C in 6% of consumers packs of pasteurised milk or cream, in 31% of retailed fish and in 100% of retail packs of minced meat. Seventy two fresh-water fishes, 40 packs of minced meat and 430 milk packs were sampled. One hundred and eighty four isolates were randomly picked from Tryptone glucose extract (TGE) agar (30°C for 3d) or Violet red bile glucose (VRBG) agar (37°C for 1d). In minced meat, Serratia liquefaciens, Hafnia alvei, Rahnella aquatilis were frequently encountered. On fish, the most frequently found species were R. aquatilis, and in milk, the dominating species were S. liquefaciens, H. alvei and R. aquatilis. One to three isolates of Citrobacter freundii were found in all three food categories. Using a polymerase chain reaction (PCR) technique, the gene of Escherichia coli heat-labile toxin ( l t) was indicated in one fish isolate of R. aquatilis whereas heat-stable toxin genes ( s t) were indicated in four H. alvei isolates, two originating from fish and two from minced meat. Positive PCR-reaction for vero cytotoxin genes were found in one H. alvei strain originating from fish ( vt1), in two S. liquefaciens strains from minced meat ( vt2), and in a C. freundii reference strain. One of the st-positive H. alvei strains from meat harboured the eaeA gene involved in the attaching phenotype of enteropathogenic E. coli.

Isolation and virulence factors of vero cyto toxinproducing Escherichia coli in human stool samples

OBJECTIVE: To evaluate the isolation rate of O157 and non-O157 verocytotoxin-producing Escherichia coli (VTEC) strains, to study the occurrence of additional virulence factors and to correlate these with clinical symptoms. METHODS: Over more than 5 years, 17 296 unduplicated fecal samples submitted for routine culture were screened for VTEC by a single PCR detecting VT1, VT2 and its variants. Verocytotoxin B subunit genotypes of the isolates obtained by testing individual colonies in positive samples were determined by a polymerase chain reaction---restriction fragment length polymorphism (PCR---RFLP) technique, the eaeA gene and the 60-MDa virulence plasmid by PCR, and the hemolytic phenotype by using CaCl2-washed blood agar. RESULTS: Verocytotoxin genes were found in 1.02% of the samples. Non-O157 VTEC strains were isolated in 0.66% and O157 in 0.17%. Overall, VTEC was less frequently isolated than Campylobacter and Salmonella but more frequently than Yersinia and Shigella. All cases except two siblings were epidemiologically unrelated. Cases of hemolytic uremic syndrome (HUS) were only observed in association with serogroup O157, which seems to be more pathogenic than the non-O157 strains. Among non-O157 VTEC strains, eaeA-positive strains are more frequently associated with clinical symptoms than are eaeA-negative strains. Other virulence factors correlate less closely with the presence of symptoms. CONCLUSIONS: VTEC is the third bacterial intestinal pathogen in our study population. All stool samples from patients with diarrhea should be screened for the most frequent serogroup, O157, or, if this is not possible, at least those from patients with bloody diarrhea. Non-O157 VTEC strains, especially if they are eaeA positive, are also associated with diarrhea, more often non-bloody. PCR or the new commercially available immunoassays could be used in selected cases, e.g. in patients suffering from HUS and in cases of outbreaks.

Vero cytotoxin-producing Escherichia coli, particularly serogroup O157, associated with human infections in England and Wales: 1992-4.

Investigations were performed by the Laboratory of Enteric Pathogens on Vero cytotoxin-producing Escherichia coli (VTEC) in England and Wales from 1992-4. Bacterial isolates, faeces and sera obtained from patients with diarrhoea, bloody diarrhoea and haemolytic uraemic syndrome were examined. Using serotyping, Vero cytotoxin gene probing and serodiagnostic tests for E. coli O157, evidence of infection was detected in 543, 434 and 491 individuals in 1992, 1993 and 1994 respectively; VTEC of serogroup O157 were isolated from 470, 385 and 411 cases. The O157 VTEC strains belonged to at least 19 different phage types (PT) although 84% belonged to PT2, PT49, PT8, PT1 or PT4. Antibodies to E. coli O157 lipopolysaccharide were detected in 13% of the cases. The average annual rate of infection with O157 VTEC was 0.83/100000 and 12% of the 1458 individuals with evidence of infection with VTEC or E. coli O157 developed haemolytic uraemic syndrome. There were at least 18 general outbreaks and many family outbreaks.

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.

Prevalence and clinical significance of vero-cytotoxin-producing Escherichia coli (VTEC) isolated from cattle in herds with and without calf diarrhoea.

To determine the clinical significance and prevalence of Vero cytotoxin (VT)-producing E. coli (VTEC) in Swiss cattle, faecal swabs and milk samples of 93 cattle from two farms with calf diarrhoea, and of 54 cattle from two similar farms without clinical problems, were screened for VTEC by PCR and colony-blot hybridization. On average, 21% of all cows were positive for VTEC by PCR, without differences between farms with and without diarrhoea problems. By contrast, VTEC were detected by PCR in 60% of animals younger than 2 years from farms with diarrhoea problems, whereas only 32% of comparable control animals from farms without clinical problems had VTEC. No VTEC were detected in the milk samples. A variety of toxin types, antimicrobial-susceptibility patterns and serotypes were found by colony-blot hybridization, among the VTEC in individual herds. However, there was a significant relationship between the VT1-only toxin type and the presence of antimicrobial resistance and clinical disease in calves. No association could be found between levels of VTEC excretion and the presence of diarrhoea. This is the first study to report systematic quantitative data on numbers of VTEC in faecal samples of cattle positive for VT genes by PCR.

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.

Examination of raw beef products for the presence of Vero cytotoxin producing Escherichia coli, particularly those of serogroup O157

Fifty-four of 310 (17%) samples of raw beef products contained Vero cytotoxin (VT)-producing Escherichia coli (VTEC) detected by DNA probes for the VT genes. VTEC strains examined in detail from a selection of the positive samples belonged to several O serogroups, some of which have been associated with human diarrhoea or haemolytic uraemic syndrome. Some of the strains possessed properties that may contribute to virulence in man. None of the food samples contained VT-producing E. coli O157 when tested by a combination of VT probe tests and colony immunoblotting with commercially available anti-O157 serum. Quantification of the immunoblotting technique indicated that O157 VTEC could be recovered from artificially-inoculated meat samples at a level of less than one organism per gram. Five of the food samples carried E. coli O157 strains that did not produce VT and differed in other properties from O157 VTEC.

Vero cytotoxin-producing Escherichia coli, particularly serogroup O 157, associated with human infections in the United Kingdom: 1989-91.

This survey reports the results of investigations performed by the Laboratory of Enteric Pathogens (LEP), to identify evidence of human infection with Vero cytotoxin-producing Escherichia coli (VTEC) in the UK during the period 1989-91. Bacterial isolates, faecal specimens and serum samples were received from patients suffering from diarrhoea, bloody diarrhoea and haemolytic uraemic syndrome. Using serotyping, Vero cytotoxin gene probing and an ELISA for serum antibodies to E. coli O 157, evidence of infection was detected in 232, 428 and 615 individuals in 1989, 1990 and 1991 respectively. Of these individuals, 15% were reported as having HUS. Vero cytotoxin-producing E. coli O 157 was the most frequently encountered serogroup, with isolations from a total of 1092 individuals over the 3-year period. The incidence of VTEC infection increased from 0.41/100,000 in 1989 to 1.07/100,000 in 1991. The area with the highest rate of infection in each year was Scotland, increasing from 1.37/100,000 in 1989 to 3.97/100,000 in 1991.

Detection of entero- and verocyto-toxin genes in Escherichia coli from diarrhoeal disease in animals using the polymerase chain reaction

Oligonucleotide primers were designed for the specific polymerase chain reaction (PCR) amplification of the enterotoxins STIa and LTI and of the verocytotoxins VT1 and VT2. All of 184 E. Coli isolates from cases of diarrhoea from pigs, cattle and sheep gave identical toxin profiles by PCR and gene probe. Differentiation between VT2 and VT2v was achieved using two oligonucleotide primers pairs in PCR and showed that all of 34 VT2 + porcine isolates, of which 23 were 0138:K1, harboured VT2v whereas 20 VT2 + bovine and ovine isolates harboured VT2. No isolate harboured both VT2 polymorphs. Simplified methods for sample preparation for PCR were examined and showed that PCR was not inhibited by direct addition of broth culture to the reaction mixture.

Detection of verotoxin-producing Escherichia coli using polymerase chain reaction from dairy cattle

The vero cytotoxin (VT) is responsible for hemorrhagic colitis and hemolytic uremic syndrome. Polymerase chain reaction (PCR) was used to detect VT-producing coliform bacteria from dairy cattle. It was found that 39 (33.3%) of the 117 fecal samples examined were recognized with VT genes in BGLB enrichment broth by the PCR method (named BGLB-PCR). Of the VT-positive samples, 31 samples (26.5%) were found to have VT-producing Escherichia coli. Frequencies of isolation in younger cattle (under 5 months) were 31.3-32.9%. On the other hand, the PCR method using the bacterial suspension of some colonies from DHL selective isolation medium (named DHL-PCR), was used for 105 samples. The DHL-PCR was validated according to the number of colonies tested for detecting VTEC. When using E. coli strains which have been stored after isolation by the conventional culture method, the VT-producing strains found were 7 (10.3%) of the 68 isolates tested. The 101 out of the 108 VTEC strains from cattle were classified into 14 O groups. 4 O serogroups (O26, O111, O145, O157) from 60% of VTEC positive cattle, were also the most common in humans with diarrhea. All E. coli O157:H7 isolates failed to ferment after 48 hrs and to hydrolyze 4-methyl-umbelliferyl-beta-D-glucuronide (MUG). These results suggests that cattle may play an important role in human VTEC infections. The BGL B-PCR technique is usefull in ecological studies for VT-producing pathogens.

Examination of retail chickens and sausages in Britain for vero cytotoxin-producing Escherichia coli

Samples from chickens and pork sausages were examined for the presence of Vero cytotoxin-producing Escherichia coli by using DNA probes for the Vero cytotoxin genes. Hybridization was detected in 25% of the 184 sausage samples, but none of the chickens was positive. No E. coli O157:H7 strains were isolated, and serotyping showed that the Vero cytotoxin-producing E. coli strains belonged to eight different O serogroups and that six strains had an unidentifiable O antigen.

Applications of DNA probes for Vero cytotoxin-producing Escherichia coli

Vero cytotoxin-producing Escherichia coli (VTEC) are now recognized as important aetiological agents in human disease. The symptoms of VTEC infection range from mild non-bloody diarrhoea to severe conditions such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Two types of Vero cytotoxin (VT), VT1 and VT2, have been identified. The genes controlling production of VT1 and VT2 are phageencoded in several E. coli strains and DNA probes have been developed from cloned genes derived from these VT phages. Recently, synthetic oligonucleotide probes for VTEC have also been prepared and evaluated. The VT probes have been labelled radioactively and also non-radioactively with digoxigenin and biotin. Present applications of VT probes include detection of VTEC in faecal samples from patients with diarrhoea, HC or HUS and also the examination of different foods for the presence of VTEC. The proportion of VTEC in the faecal flora or foods may be low, often less than 1%. The use of DNA probes allows several hundred colonies from a sample to be examined and by this technique VT genes were detected in 30–40% of faecal specimens from patients with HC or HUS. Use of methods such as the polymerase chain reaction for amplification of the target genes combined with DNA probes should result in an increased sensitivity for the detection of VTEC.

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.

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.

Vero cytotoxin production and presence of VT genes in Escherichia coli strains of animal origin.

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 O138, O139 and O141, were isolated from cases of oedema disease. In general, the porcine isolates produced toxin at a lower level than the bovine strains.

Vero cytotoxin production and presence of VT genes in strains ofEscherichia coliandShigella

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.

Vero cytotoxin production and presence of VT genes in strains of Escherichia coli and Shigella

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.