Vero cytotoxin-producing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes

Faecal specimens from 66 children with haemolytic uraemic syndrome in the United Kingdom were examined for strains of Escherichia coli producing Vero cytotoxin (VT). Initially, conventional bacteriological methods were used to identify colonies of E. coli which were then tested for VT production. Subsequently, specific DNA probes for VT1 and VT2 were used in hybridisation tests to detect VT-producing E. coli (VTEC). VTEC strains were isolated from 19 cases and in 15 they belonged to serogroup O157. Fourteen of these O157 strains possessed the flagellar antigen H7 and one was non-motile. The VTEC strains from the remaining four cases belonged to serotypes O26:H11, O104:H2, O153:H25, and O163:H19 together with a rough VT+ strain with flagellar antigen H51. The O157 strains hybridised with either the VT2 probe or both VT1 and VT2 probes. The other VTEC strains hybridised with either the VT1 or VT2 probe. Confirmation of the production of VT1 and VT2 in vivo was obtained by the neutralisation of faecal VT with specific antisera raised against these two cytotoxins.     

Direct detection of verotoxin genes in stool samples by polymerase chain reaction in hemolytic uremic syndrome patients in France

Objective: To reassess the occurrence of verocytotoxin-producing Escherichia coli (VTEC) in French hemolytic uremic syndrome (HUS) patients.
Method: From March 1991 to January 1995, direct detection of verotoxin genes (VT) by the polymerase chain reaction (PCR) was performed on stool samples from 169 patients suffering from HUS.
Results: Fifty-one were PCR positive (30.1%); one was positive for the VT1 gene and the others for the VT2 gene. VTEC was isolated from only 32 of the 51 PCR-positive samples. E. coli O157:H7 was isolated from five patients. E. coli O111 was isolated from seven patients during an outbreak of HUS. Among the other VT2 E. coli strains, only four were serotypable. Of the 51 PCR-positive stools, 19 were culture negative for VTEC.
Conclusions: This study provides evidence that in France E. coli O157 and other VTEC serotypes are involved in HUS.     

Laboratory investigation of outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7.

A severe outbreak of hemorrhagic colitis occurred in London, Ontario, during the month of September 1985. A total of 55 residents and 18 employees of a nursing home developed diarrhea, and 17 residents (age range, 78 to 99 years) died. Specimens from 38 patients, 37 employees and contacts, and 10 autopsies were investigated for all enteric pathogens. Specimens were also planted on MacConkey-sorbitol agar. Fecal extracts were tested on Vero cells for cytotoxin (FVT). Escherichia coli isolates were serotyped and tested for verotoxin and beta-glucuronidase production. Of the 38 symptomatic patients, 26 were positive for FVT, verotoxin-producing E. coli (VTEC), or both. Of the 105 specimens that were examined from these 38 patients, FVT and VTEC were both positive in 30 specimens, FVT only was positive in 13 specimens, and VTEC only was positive in 4 specimens. None of the 27 specimens from 10 autopsies was positive for FVT or VTEC. No other enteric pathogen was found in any of the cases. All asymptomatic individuals were negative for both FVT and VTEC. Of 19 VTEC strains that were isolated, 18 belonged to serotype O157:H7. These 18 strains and 2 more strains that were obtained from sporadic cases that had occurred within the 2 previous months were found to give similar biochemical reactions in a 36-test identification system. All isolates of serotype O157:H7 were beta-glucuronidase negative and susceptible to the antimicrobial agents that are used to treat E. coli infections. Testing for FVT and VTEC was found to be the most sensitive and specific technique for the laboratory diagnosis of this disease. Negative sorbitol, positive raffinose, and negative beta-glucuronidase tests appeared to be consistent markers for aiding in the detection of E. coli O157:H7.     

Hemolytic uremic syndrome in Belgium: incidence and association with verocytotoxin-producing Escherichia coli infection

Objective: To evaluate the incidence of hemolytic uremic syndrome (HUS) in Belgium and to determine the role of verocytotoxin-producing Escherichia coli O 157:H7 and other serotypes (non-O 157 VTEC).
Methods: Twenty-two centers, including the seven university hospitals, registered prospectively all cases of HUS; they collected clinical samples for isolation of VTEC strains and serum for detection of specific O-lipopolysaccharide antibodies.
Results: Forty-seven cases of HUS (including five incomplete cases) were recorded. Three cases were seen in nonresidents. The incidence of complete HUS in Belgian residents was 4.3 cases/100 000 in children <5 years old, 1.8 cases/100 000 when all children <15 years were considered, and 0.42/100 000 when patients of all ages were taken into account. By combining bacteriologic and serologic results, evidence of VTEC infection was obtained in 64% of the patients, mainly but not exclusively in children with prodromal diarrhea. The 13 VTEC isolates belonged to serotypes O157:H7 (nine isolates), O26:H11, O121:H-, O145:H- and O172:H- (one each) and all produced VT2 (+VT2vh-a in three O157 strains) and were positive for the eaeA gene.
Conclusions: The incidence rate found in this study and the high mortality and morbidity linked with this syndrome warrant further registration of pediatric and post-diarrheic adult HUS cases and also examination of stools for both O157 and non-O157 VTEC strains. For effective prevention of this disease, further study of the serotypes and accessory virulence factors associated with HUS is needed.     

Serodiagnosis by passive hemagglutination test and verotoxin enzyme-linked immunosorbent assay of toxin-producing Escherichia coli infections in patients with hemolytic-uremic syndrome.

Eight cases of hemolytic-uremic syndrome in which no pathogens were isolated were diagnosed serologically by a passive hemagglutination assay and a verotoxin (VT; Shiga-like toxin) enzyme-linked immunosorbent assay (ELISA). The passive hemagglutination assay employed formalinized sheep erythrocytes sensitized with soluble native antigen or heat-treated antigen (lipopolysaccharide [LPS]) from Escherichia coli O26, O111, O128, and O157 or flagellar antigen of nine different H serogroups of E. coli: H2, H7, H8, H10, H11, H12, H18, H19, and H25. All patients had antibodies against the native antigen and/or the LPS of E. coli O157, but positive agglutination with H7 was observed only in one patient. In the VT-ELISA with plates coated with purified VT 1 or VT 2, antibody against VT 2 was observed in the sera of five of six patients examined, but none of the patients possessed VT 1 antibody. These results indicate that the causative pathogen in these eight hemolytic-uremic syndrome cases is likely to be VT-producing E. coli O157. The passive hemagglutination assay described here is a very sensitive, simple, and rapid method. This assay is highly recommended for the serological diagnosis of VT-producing E. coli infections, particularly in patients infected by serogroup O157 strains. Furthermore, the VT-ELISA is useful in studying the role of VT in hemolytic-uremic syndrome.     

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.     

Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle.

Verotoxin-producing Escherichia coli isolates from feces of healthy cattle were identified by DNA hybridization with verotoxin 1- and verotoxin 2-specific gene probes. Among 259 animals investigated, 28 (10.8%) were found to carry verotoxin-producing E. coli strains. Characterization of the verotoxin-producing isolates revealed a heterogeneous population in terms of serotype and toxin type. Nearly 40% of the strains belonged to serogroups known to be pathogenic for humans, i.e., O22, O39, O82, O91, O113, O116, O126, and O136. Two isolates from different bulls were identified as serotype O157:H7. Results obtained in this study indicate that cattle may be an important source of verotoxigenic E. coli involved in human disease.     

Verotoxin-producing Escherichia coli (VTEC) and Escherichia coli O157:H7 in medicine and food industry]

Escherichia coli O157:H7 is now recognised as an important human pathogen. Illness caused by E. coli O157:H7 infection can range from self limited, watery diarrhea to life-threatening manifestations such as hemorrhagic colitis, hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. The mode of transmission is primarily through food (e.g. undercooked minced beef products, especially beef burgers, raw cows' milk and cheese, contaminated pasteurised milk and untreated water.). Studies to date indicate that cattle is an important reservoir of the organism. Public health measures to control VTEC infection are broadly similar to the measures needed to control other gastro-intestinal infections. Because of the potential low infection dose, laboratory diagnosis of O157 VTEC in food samples has developed over recent years with the use of liquid enrichment and the development of methods such as immunomagnetic separation. VTEC of other serogroups than O157 have no reliable biochemical, serological or morphological characteristics (other than VT production itself) to distinguish them from commensal E. coli. Thus to detect VTEC other than O157 and phenotypic variants of E. coli O157 in food, we have to use methods for detection of verocytotoxin production and VT genes.     

Production and characterization of a monoclonal antibody specific for enterohemorrhagic Escherichia coli of serotypes O157:H7 and O26:H11

A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli O157:H7 and O26:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli O157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli O157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype O157:H7 (including strains of serotype O157 but not H7). The E. coli strains (all of serotype O26:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli O157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes O157:H7 and O26:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes O157:H7 and O26:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens.     

Hemolytic-uremic syndrome--microbiological study

In autumn 1988 in one locality of the North Bohemia region young children developed severe diarrhoea and the children were sent because of their serious clinical condition with the diagnosis of haemolytic-uraemic syndrome (HUS) to the Prague-Motol hospital. The probable aetiological agent which was detected was Escherichia coli producing verotoxin (VTEC)--026:H11, 026:H?, O157:H7, 05:H- and 01:H?. Biotyping and serotyping of the strains was made, the verotoxin level was assessed and their sensitivity to antibiotics was tested. Concurrently contacts were examined and suspect colonies of Escherichia coli were subjected to bio- and serotyping, however, none of the serovars found in the sick children was detected.     

Relationship between enterotoxin production and serotype in enterotoxigenic Escherichia coli.

We examined the relationship between serotype and enterotoxin production in 109 enterotoxigenic Escherichia coli strains isolated from 109 patients with severe cholera-like diarrhea in Dacca, Bangladesh. Of 69 strains producing both heat-labile and heat-stable toxins, 59 (86%) belonged to the one of four O serogroups, and 56 (81%) of these strains belonged to one of six O:K:H serotypes. In contrast, 34 strains producing only heat-stable toxin were distributed among 15 O serogroups, and six strains producing only heat-labile toxin were distributed among six O serogroups. Twelve strains producing heat-labile and heat-stable toxins and five strains producing heat-stable toxin were found which had the same serotype (O78:K-:H12) and biotype. It appears that at least in one geographic setting E. coli strains producing both heat-labile and heat-stable toxins are more restricted in their O groups and O:K:H serotypes than E. coli that produce only heat-stable toxin and that certain serobiotypes may commonly include strains which produce both toxin types.     

Verocytotoxin-producing Escherichia coli (VTEC) O157 and other VTEC from human infections in England and Wales: 1995-1998

A total of 3429 isolations of verocytotoxin-producing Escherichia coli O157 (VTEC O157) was confirmed from human sources in England and Wales during the period 1995-1998. The largest annual total was 1087 in 1997. Most infections occurred in the third quarter of each year. The overall rate of infection ranged from 1.28 to 2.10/100,000 population and showed regional variation. The highest incidence was in children aged 1-4 years. Annually, between 5% and 11% of strains were from patients who had travelled abroad. There were 67 general outbreaks of infection represented by 407 (11.9%) VTEC O157 isolates. Outbreaks involved transmission by contaminated food or water, person-to-person spread and direct or indirect animal contact, and five were associated with foreign travel. The majority (76%) of strains carried verocytotoxin (VT) 2 genes and 23.3% were VT1+VT2. Most strains had the flagellar antigen H7, but c. 14% were non-motile. Approximately 20% of isolates were resistant to antimicrobial agents, predominantly streptomycin, sulphonamides and tetracycline. In addition to VTEC O157, strains of serogroup O157 that did not possess VT genes were identified. These were either derivatives of VTEC O157 that had lost VT genes or were strains with H antigens other than H7 that have never been associated with VT production. Strains of VTEC other than O157 were characterised. Most were associated with diarrhoea, bloody diarrhoea or haemolytic uraemic syndrome and had virulence markers in addition to VT.     

Isolation of verotoxin-producing Escherichia coli O-rough:K1:H7 from two patients with traveler's diarrhea.

Two Escherichia coli O-rough:K1:H7 strains producing verotoxin 1 that were isolated from stool samples of two travelers with diarrhea who consulted our clinic after trips to the Indian Subcontinent and Central America were characterized. Both strains were sorbitol negative, the same phenotype presented by E. coli O157:H7, but in contrast they were beta-glucuronidase positive. Low-frequency restriction analysis of chromosomal DNA and pulsed-field gel electrophoresis and repetitive extragenic palindrome-PCR showed that both strains were epidemiologically related. The illness was self-limited in both cases but involved long-duration, watery diarrhea (10 to 50 days) accompanied by abdominal cramps and flatulence. This serotype should be taken into account as a possible cause of traveler's diarrhea.     

Unusual verotoxin-producing Escherichia coli associated with hemorrhagic colitis.

All strains of Escherichia coli isolated from cases of hemorrhagic colitis and sent to the Centers for Disease Control, Atlanta, Ga., over a 3-year period were assayed for toxicity in Vero cell cultures. Strains that produced moderate or high levels of verotoxin were characterized by serotype, biotype, antimicrobial resistance, plasmid profile, and adherence to HeLa cells. Over 200 isolates were typical O157:H7 strains. Six isolates were atypical O157:H7 strains; two were resistant to antimicrobial agents; one was indole negative, two were citrate positive, and one was urea positive. Six isolates were nonmotile O157 strains. All of these isolates were similar to typical O157:H7 strains by plasmid profile and negative or slow sorbitol fermentation. Eleven other verotoxigenic isolates did not possess the O157 antigen, had a variety of plasmid profiles, and were sorbitol positive. Two of the eleven were enteropathogenic serotypes (O111:NM and O26:H11), yet none were adherent to HeLa cells. We conclude that verotoxigenic E. coli associated with hemorrhagic colitis includes atypical O157 strains and other serotypes. Hence, investigators should use current screening methods with caution.     

Experimental infection of infant rabbits with verotoxin-producing Escherichia coli.

To study the pathogenesis of diarrheal disease due to verotoxin (VT)-producing Escherichia coli, 3-day-old rabbits were inoculated intragastrically with live E. coli O157:H7 (high VT producer), E. coli O113:K75:H21 (low VT producer), or O157:H45 (VT negative) and were examined for clinical symptoms, bacterial colonization, presence of detectable free VT in the intestines, and histological changes. Diarrhea developed consistently with 10(8) bacteria of E. coli O157:H7 but was observed only infrequently with even a higher dose of E. coli O113:K75:H21. VT-negative strains failed to cause diarrhea under the same experimental conditions. E. coli O157:H7 was recovered from the colon of infected animals in a significantly higher concentration than from the small intestine, and the clinical symptoms correlated with the presence of detectable free VT in the colon. Histological changes were seen mainly in the mid- and distal colon; these changes were characterized by a vast increase in apoptosis in the surface epithelium, increased mitotic activity in the crypts, mucin depletion, and a mild to moderate infiltration of neutrophils in the lamina propria and epithelium. Multiple foci of attached bacteria were seen on the surface epithelium of the gut-associated lymphoid tissue, cecum, and colon. Bacteria were never seen in epithelial cells or the lamina propria. These mucosal abnormalities as well as clinical symptoms were reproduced in infant rabbits by the intragastric administration of VT alone. These results are consistent with the hypothesis that VT plays a major role in the pathogenesis of diarrhea caused by E. coli O157:H7 and other VT-producing E. coli.     

Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli.

Sixty-four verotoxin-producing (VT+) Escherichia coli strains were analyzed for VT1- and VT2-specific DNA sequences and for production of hemolysin. Strains of human origin were of the following serotypes: O157:H7 or H-, O111:H8 or H-, O26:H11, O114:H4, and rough:H7. Strains of serotypes O157:H7, O113:H21, O116:H21, and rough:H- were from cattle, while those of serotype O139:K12:H1 were from pigs. All 64 isolates carried either VT1 or VT2 or both genes. Sixty of the strains (93.8%) were hemolytic (Hly+). The three O139:K12:H1 strains examined produced alpha-hemolysin, as shown by their reaction with the alpha-hemolysin-specific monoclonal antibody h2A and by DNA hybridization with an alpha-hly gene probe. The remaining 57 Hly+ strains (95%) produced a different type of hemolysin (enterohemolysin), which is genetically and serologically unrelated to alpha-hemolysin. The two types of hemolysin are further distinguished by the appearance of the lysis zone on blood agar and by the time interval for the detection of hemolysis. In contrast to alpha-hemolysin, enterohemolysin can be detected only on blood plates containing washed erythrocytes. The frequent association of enterohemolysin with verotoxin production (89%) makes it useful as an epidemiological marker for rapid and simple detection of potential VT+ E. coli.     

An improved selective medium for the isolation of Escherichia coli O157

Sorbitol-MacConkey medium has become widely used for the isolation of verotoxigenic (VT+) Escherichia coli O157. However, many organisms other than VT+ E. coli O157, especially other serogroups of E. coli and Proteus spp., may not ferment sorbitol, and thus may be confused initially with VT+ E. coli O157. Rhamnose is not fermented by VT+ E. coli O157, but is by most sorbitol non-fermenting E. coli of other serogroups. Cefixime is a cephalosporin antibiotic that is more active against Proteus spp. than against E. coli. Inclusion of rhamnose and cefixime in sorbitol-MacConkey agar improves its selectivity for the isolation of VT+ E. coli O157.     

Antibodies to Escherichia coli O157 in patients with haemorrhagic colitis and haemolytic uraemic syndrome.

Twenty four sera from patients with haemolytic uraemic syndrome or haemorrhagic colitis and healthy controls were examined for antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157. Faecal specimens from these patients were also examined for Vero cytotoxin producing E coli (VTEC) by DNA probes, and for faecal Vero cytotoxin. Eight patients with faecal E coli O157:H7 gave a strong antibody response to O157 LPS, shown by immunoblotting and an enzyme linked immunosorbent assay. Six symptomatic patients without evidence of faecal VTEC also gave a strong antibody response to O157 LPS. Sera from the remaining five patients and five healthy controls did not contain antibodies to E coli O157. The results suggest that the testing of sera from patients with haemorrhagic colitis or haemolytic uraemic syndrome by ELISA or immunoblot would prove valuable in addition to the established procedures for detecting VTEC, using DNA probes and testing for faecal Vero cytotoxin.     

Haemorrhagic colitis: detection of verotoxin producing Escherichia coli O157 in a clinical microbiology laboratory.

Faeces (n = 1319) were examined over three months for the presence of non-sorbitol fermenting, verotoxin producing Escherichia coli (serotype O157). Seven isolates were found, four from patients with bloodstained diarrhoea and three from patients with no evidence of blood in the faeces. Screening of all faecal samples with specific O157 antiserum for non-sorbitol fermenting organisms and agglutination was an important adjunct to clinical and microscopic findings and helped detect cases of verotoxin producing E coli which might otherwise have been missed.     

Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis.

Escherichia coli serotype O157:H7 is a recently recognized human pathogen associated with hemorrhagic colitis. Unlike most E. coli strains, E. coli O157:H7 does not ferment sorbitol. Therefore, the efficacy of MacConkey agar containing sorbitol (SMAC medium) instead of lactose as a differential medium for the detection of E. coli O157:H7 in stool cultures was determined in comparison with MacConkey agar. The relative frequency of non-sorbitol-fermenting (NSF) organisms other than E. coli O157:H7 in feces was low at 10 to 20% (95% confidence limits), and NSF organisms also occurred mostly in small numbers. In a field trial involving over 1,000 diarrheal stools, E. coli O157:H7 was isolated from 18 stools, all of which were from patients with bloody diarrhea. In every instance, the growth of E. coli O157:H7 on SMAC medium was heavy and occurred in almost pure culture as colorless NSF colonies in contrast to fecal flora, which are mostly sorbitol fermenting and hence appear pink on this medium, whereas on MacConkey agar cultures, the growth of E. coli O157:H7 was indistinguishable from fecal flora. SMAC medium permitted ready recognition of E. coli O157:H7 in stool cultures. Detection of E. coli O157:H7 on SMAC medium had a sensitivity of 100%, a specificity of 85%, and an accuracy of 86%. SMAC medium stool culture is a simple, inexpensive, rapid, and reliable means of detecting E. coli O157:H7, and we recommend routine use of SMAC medium especially for culturing bloody stools.