Detection,isolation, and molecular subtyping of Escherichia coli O157:H7 and Campylobacter jejuni associated with a large waterborne outbreak

The largest reported outbreak of waterborne Escherichia coli O157:H7 in the United States occurred in upstate New York following a county fair in August 1999. Culture methods were used to isolate E. coli O157:H7 from specimens from 128 of 775 patients with suspected infections. Campylobacter jejuni was also isolated from stools of 44 persons who developed diarrheal illness after attending this fair. There was one case of a confirmed coinfection with E. coli O157:H7 and C. jejuni. Molecular detection of stx(1) and stx(2) Shiga toxin genes, immunomagnetic separation (IMS), and selective culture enrichment were utilized to detect and isolate E. coli O157:H7 from an unchlorinated well and its distribution points, a dry well, and a nearby septic tank. PCR for stx(1) and stx(2) was shown to provide a useful screen for toxin-producing E. coli O157:H7, and IMS subculture improved recovery. Pulsed-field gel electrophoresis (PFGE) was used to compare patient and environmental E. coli O157:H7 isolates. Among patient isolates, 117 of 128 (91.5%) were type 1 or 1a (three or fewer bands different). Among the water distribution system isolates, 13 of 19 (68%) were type 1 or 1a. Additionally, PFGE of C. jejuni isolates revealed that 29 of 35 (83%) had indistinguishable PFGE patterns. The PFGE results implicated the water distribution system as the main source of the E. coli O157:H7 outbreak. This investigation demonstrates the potential for outbreaks involving more than one pathogen and the importance of analyzing isolates from multiple patients and environmental samples to develop a better understanding of bacterial transmission during an outbreak.     

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.     

Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing.

Two hundred thirty-three isolates of Escherichia coli O157:H7 were analyzed by both pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. All 26 isolates from persons whose illness was associated with a recent multistate outbreak of E. coli O157:H7 infections linked to the consumption of undercooked hamburgers and all 27 isolates from incriminated lots of hamburger meat had the same phage type and the same PFGE pattern. Twenty-five of 74 E. coli O157:H7 isolates from Washington State and 10 of 27 isolates from other states obtained during the 6 months before the outbreak had the same phage type as the outbreak strain, but only 1 isolate had the same PFGE pattern. PFGE thus appeared to be a more sensitive method than bacteriophage typing for distinguishing outbreak and non-outbreak-related strains. The PFGE patterns of seven preoutbreak sporadic isolates and five sporadic isolates from the outbreak period differed from that of the outbreak strain by a single band, making it difficult to identify these isolates as outbreak or non-outbreak related. Phage typing and PFGE with additional enzymes were helpful in resolving this problem. While not as sensitive as PFGE, phage typing was helpful in interpreting PFGE data and could have been used as a simple, rapid screen to eliminate the need for performing PFGE on unrelated isolates.     

Long-term shedding and clonal turnover of enterohemorrhagic Escherichia coli O157 in diarrheal diseases.

To investigate the length of time that Shiga-like toxin-producing Escherichia coli O157 is excreted after the onset of diarrhea, 456 serial stool specimens were obtained from 53 children. E. coli O157 cells were identified by the use of DNA probes followed by agglutination with a specific antiserum. Specimens were collected until three consecutive stool samples (collected within 9 days) were negative for E. coli O157. The median durations of shedding were 13 days (range, 2 to 62 days) in patients with diarrhea or hemorrhagic colitis and 21 days (range, 5 to 124 days) in patients that developed hemolytic uremic syndrome. In 36 (68%) of the patients, only the first culture was O157 positive, and the three cultures that followed were negative. In 7 (13%) of the patients, E. coli O157 cells were shed for more than 32 days after the onset of diarrhea; these long-term shedders were clinically asymptomatic by the end of this period. In 12 patients, one or two serial O157-negative cultures, obtained up to 8 days after a positive culture, were followed by another positive culture. Comparison of the first and last E. coli O157 isolates by pulsed-field gel electrophoresis revealed that in three of the seven long-term shedders, pulsed-field gel electrophoresis types varied. In two cases, a Shiga-like toxin gene was apparently lost during infection. The observation of long-term shedding accompanied by genotypic turnover has epidemiological and diagnostic implications.     

Impact of free verotoxin testing on epidemiology of diarrhea caused by verotoxin-producing Escherichia coli.

During a 10-week period in the summer of 1990, an epidemiologic investigation of the prevalence of verotoxin (VT)-producing Escherichia coli infection was conducted in Calgary, Alberta, Canada. Consecutive stool specimens (n = 3,577) were cultured for E. coli O157:H7, and fecal filtrates were tested for free VTs (FVTs). E. coli O157:H7 was recovered from 22 specimens (0.6%), but VT was detected in 74 specimens (2.1%). Sixty-nine stool specimens positive for FVTs or E. coli O157:H7 were probed for VT genes by colony blot hybridization; 22 of 38 VT gene probe-positive isolates were non-O157:H7 E. coli organisms. Fourteen of 22 strains could not be induced to produce VT in vitro, despite the presence of FVTs in the stool sample, positivity on colony blot hybridization, positive PCR probes with the primers described by Pollard et al. (D. R. Pollard, W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee, J. Clin. Microbiol. 28:540-545, 1990) or Gannon et al. (V. P. Gannon, R. K. King, J. Y. Kim, and E. J. Golsteyn-Thomas, Appl. Environ. Microbiol. 58:3809-3815, 1992) (but not those described by Karch and Meyer [H. Karch and T. Meyer, J. Clin. Microbiol. 27:2751-2757, 1989]), and positive Southern blot analysis of isolates in 10 of 14 strains. The patient survey questionnaire showed that E. coli O157:H7 infection was associated with bloody diarrhea of short duration, whereas infection with other serotypes or persistence of FVT only was associated with longer-duration nonbloody diarrheal illness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Performance of the ImmunoCard STAT! E. coli O157:H7 test for detection of Escherichia coli O157:H7 in stools

ImmunoCard STAT! E. coli O157:H7 (Meridian Diagnostics, Inc., Cincinnati, Ohio) is a novel rapid (10-min) test for the presence of Escherichia coli O157:H7 in stools. The test may be performed either directly on stool specimens or on an overnight broth culture of stool. In a multicenter prospective study, 14 of 14 specimens positive by culture for E. coli O157:H7 were positive by the ImmunoCard STAT! O157:H7 test, and there were no false positives from 263 culture-negative specimens. In a retrospective study, the test was positive in 339 (81%) of 417 stored culture-positive specimens and the specificity was 95% (98 of 103 specimens). No false positives were associated with alternate stool pathogens. The ImmunoCard STAT! O157:H7 test has high sensitivity and specificity.     

Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster.

Genomic DNAs prepared from 168 isolates of Escherichia coli O157:H7 were analyzed for restriction fragment length polymorphisms on Southern blots probed with bacteriophage lambda DNA. The isolates analyzed included strains from a recent large multistate outbreak of E. coli O157:H7 infection associated with consumption of poorly cooked beef in restaurants, a day-care center cluster, and temporally and geographically unrelated isolates. E. coli O157:H7 isolates recovered from the incriminated meat and from 61 (96.8%) of 63 patients from Washington and Nevada possessed identical lambda restriction fragment length patterns. The lambda restriction fragment length polymorphisms observed in 11 (91.7%) of 12 day-care center patients were identical, but they differed from that of the strain associated with the multistate outbreak. E. coli O157:H7 from 42 patients temporally or geographically unrelated to either cluster of infection possessed unique and different lambda restriction fragment length patterns, except for paired isolates from three separate clusters of infection. These data demonstrate that the hybridization of DNA digests of E. coli O157:H7 with radiolabelled bacteriophage lambda DNA can be a useful, stable, and discriminatory epidemiologic tool for analyzing the linkage between strains of E. coli O157:H7.     

Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli.

An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.     

Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7 in the bovine host

Escherichia coli O157:H7 causes bloody diarrhea and potentially fatal systemic sequelae in humans. Cattle are most frequently identified as the primary source of infection, and E. coli O157:H7 generally colonizes the gastrointestinal tracts of cattle without causing disease. In this study, persistence and tropism were assessed for four different E. coli O157:H7 strains. Experimentally infected calves shed the organism for at least 14 days prior to necropsy. For the majority of these animals, as well as for a naturally colonized animal obtained from a commercial beef farm, the highest numbers of E. coli O157:H7 were found in the feces, with negative or significantly lower levels detected in lumen contents taken from the gastrointestinal tract. Detailed examination demonstrated that in these individuals the majority of tissue-associated bacteria were adherent to mucosal epithelium within a defined region extending up to 5 cm proximally from the recto-anal junction. The tissue targeted by E. coli O157:H7 was characterized by a high density of lymphoid follicles. Microcolonies of the bacterium were readily detected on the epithelium of this region by immunofluorescence microscopy. As a consequence of this specific distribution, E. coli O157:H7 was present predominantly on the surface of the fecal stool. In contrast, other E. coli serotypes were present at consistent levels throughout the large intestine and were equally distributed in the stool. This is a novel tropism that may enhance dissemination both between animals and from animals to humans. The accessibility of this site may facilitate simple intervention strategies.     

Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H-

The plasmid-borne sfpA gene encodes the pilin subunit in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H-. We investigated the distribution of sfpA among 600 E. coli isolates comprising the complete E. coli standard reference (ECOR) and diarrheagenic E. coli (DEC) strain collections and clinical isolates associated with enteric disease. sfpA was detected in DEC3F SF EHEC O157:H- strain 493/89, each of 107 SF EHEC O157:H- clinical isolates, and 14 Shiga toxin-negative SF E. coli O157:H- strains which contained eae, which encodes gamma-intimin, and fliC, which encodes the H7 antigen. sfpA was absent from all other strains, including the ECOR strain collection, all non-SF EHEC O157:H7 strains, and all E. coli O55:H7 strains (E. coli O55:H7 is the postulated ancestor of Shiga toxin-producing E. coli [STEC] O157). These results suggest that there was a single acquisition of the sfpA gene in the nonmotile SF E. coli O157 branch, presumably after the eae-encoding pathogenicity island (the locus of enterocyte effacement) was acquired and motility was lost. We then applied the sfpA PCR in combination with rfbO157, stx, and eae PCRs to screen 636 stool samples from patients with diarrhea or hemolytic-uremic syndrome for SF STEC O157:H-. In 27 cases, the simultaneous presence of the sfpA, eae, and rfbO157 amplicons indicated the presence of SF E. coli O157:H- strains, and the result was subsequently confirmed by isolation. All but two of these strains possessed stx2. None of the other stool samples was positive by the sfpA PCR; 59 of these stool samples contained EHEC O157:H7. The sfpA gene can be recommended as a target for screening for SF E. coli O157:H-.     

Comparison of a direct fecal Shiga-like toxin assay and sorbitol-MacConkey agar culture for laboratory diagnosis of enterohemorrhagic Escherichia coli infection.

A direct fecal Shiga-like toxin assay (DSLTA) was used to prospectively screen 9,449 unselected stool samples, received at the British Columbia Provincial Health Laboratories and the Metropolitan Laboratories of Vancouver, for Shiga-like toxin I and Shiga-like toxin II. The results were compared with results of routine stool culture on sorbitol-MacConkey agar (SMAC) for Escherichia coli O157:H7. Of 80 specimens positive by either method, 59 (74%) and 74 (93%) were positive by SMAC and DSLTA, respectively; 53 (66%) were positive by both methods, 21 (26%) were positive by DSLTA only, and 6 (7%) were positive by SMAC only. On further screening, Shiga-like toxin-producing E. coli were detected in 8 (38%) of the 21 stools positive by DSLTA only, including serotypes O157:H7 (1 stool), O26:K60 (5 stools), O128:K67 (1 stool), and O103:H2 (1 stool). For the remaining 13 stools in which no SLTEC was found but DSLTA was positive, clinical information revealed that 11 of 12 patients had diarrheal illnesses, and 4 of these 11 had bloody diarrhea or hemolytic-uremic syndrome. Stools positive only by SMAC were collected earlier in the illness than stools positive by DSLTA, suggesting that free fecal toxin levels may be too low to detect at this time. Overall we found that DSLTA detected 19% more positive specimens than SMAC and that Shiga-like toxin-producing E. coli serotypes other than E. coli O157:H7 are causing disease in the province of British Columbia, Canada.     

Molecular epidemiology of a fast-food restaurant-associated outbreak of Escherichia coli O157:H7 in Washington State.

We studied the molecular epidemiology of the recent fast-food restaurant chain-associated Escherichia coli O157:H7 outbreak in Washington State. Genomic DNAs prepared from strains isolated from 433 patients were probed with radiolabelled Shiga-like toxin (SLT) I and SLT II genes and bacteriophage lambda DNA and were subsequently analyzed for their restriction fragment length polymorphism (RFLP) patterns. The SLT RFLP and lambda RFLP profiles of an E. coli O157:H7 strain isolated from the incriminated beef and prototype patient were compared with those of the patient isolates for determination of the concordance between patterns. Of the 377 patients with primary and secondary cases of infection epidemiologically linked to the outbreak, isolates from 367 (97.3%) of the patients displayed SLT RFLP and lambda RFLP profiles identical to those of the outbreak strains. Isolates from 10 of the 377 (2.6%) patients possessed SLT RFLP and lambda RFLP profiles different from those of the outbreak strains, and the patients from whom those isolates were obtained were subsequently characterized as having non-outbreak-related infections. The E. coli O157:H7 strains isolated from 31 of 44 (70.4%) patients who were epidemiologically excluded from the outbreak were linked to the outbreak by RFLP typing. Our results indicate that SLT RFLP and lambda RFLP analyses are stable and sensitive methods, and when they are used in conjunction with an epidemiological investigation they could result in an earlier recognition of outbreaks and their sources, hence prompting measures to prevent the continued transmission of E. coli O157:H7.     

Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle.

We examined 1,266 fecal specimens from healthy cattle during the investigations of two sporadic cases of hemolytic uremic syndrome associated with raw milk consumption and an outbreak of gastroenteritis and hemolytic uremic syndrome caused by Escherichia coli serotype O157:H7. We collected specimens from heifers, calves, and adult cows on 22 farms, in a stockyard, and in a packing house. We also collected 3 raw hamburger specimens from a restaurant and 23 raw milk samples from two farms. All specimens were examined for E. coli O157:H7 by using sorbitol-MacConkey agar, H immobilization, O157 agglutination, and tissue culture cytotoxicity. E. coli O157:H7 was isolated from 16 heifers or calves and 1 adult cow on 22 farms, 1 stockyard calf, 2 beef specimens, and 1 raw milk sample. Selected fecal specimens were also examined for the presence of other Shiga-like-toxin-producing E. coli (SLTEC) by testing polymyxin B extracts of colony sweeps and then testing individual colonies for toxin production. SLTEC other than O157 was isolated from 8 of 10 farms investigated and from the stockyard; 8% of adult cows and 19% of heifers and calves were positive for SLTEC. Several animals were positive for SLTEC by colony sweep only. This investigation demonstrates that dairy cattle are a reservoir of E. coli O157:H7 and other SLTEC.     

Profile of Escherichia coli O157:H7 pathogen responsible for hamburger-borne outbreak of hemorrhagic colitis and hemolytic uremic syndrome in Washington.

We analyzed Escherichia coli O157:H7 isolates from stool samples of five patients who had bloody diarrhea and were infected during a large food-borne outbreak of hemorrhagic colitis in Washington state. The isolates were assessed for Shiga-like toxin profile, adherence and plasmid traits, mouse virulence, capsule, and enterohemolysin production. The profiles of the five isolates were indistinguishable from each other and similar to that of E. coli O157:H7 strain EDL933, an organism responsible for a similar hamburger-associated food poisoning episode in 1982.     

The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections

BACKGROUND: Children with gastrointestinal infections caused by Escherichia coli O157:H7 are at risk for the hemolytic-uremic syndrome. Whether antibiotics alter this risk is unknown. METHODS: We conducted a prospective cohort study of 71 children younger than 10 years of age who had diarrhea caused by E. coli O157:H7 to assess whether antibiotic treatment in these children affects the risk of the hemolytic-uremic syndrome and to assess the influence of confounding factors on this outcome. Estimates of relative risks were adjusted for possible confounding effects with the use of logistic-regression analysis. RESULTS: Among the 71 children, 9 (13 percent) received antibiotics and the hemolytic-uremic syndrome developed in 10 (14 percent). Five of these 10 children had received antibiotics. Factors significantly associated with the hemolytic-uremic syndrome were a higher initial white-cell count (relative risk, 1.3; 95 percent confidence interval, 1.1 to 1.5), evaluation with stool culture soon after the onset of illness (relative risk, 0.3; 95 percent confidence interval, 0.2 to 0.8), and treatment with antibiotics (relative risk, 14.3; 95 percent confidence interval, 2.9 to 70.7). The clinical and laboratory characteristics of the 9 children who received antibiotics and the 62 who did not receive antibiotics were similar. In a multivariate analysis that was adjusted for the initial white-cell count and the day of illness on which stool was obtained for culture, antibiotic administration remained a risk factor for the development of the hemolytic uremic syndrome (relative risk, 17.3; 95 percent confidence interval, 2.2 to 137). CONCLUSIONS: Antibiotic treatment of children with E. coli O157:H7 infection increases the risk of the hemolytic-uremic syndrome.     

Screening for Escherichia coli O157:H7 in Illinois

Objective: To determine the incidence of infection with Escherichia coli O157:H7 in a tertiary referral center in Chicago, where a similar study had been performed in 1984, to evaluate cases of disease reported to the Illinois Department of Public Health (IDPH) in 1993, and to determine laboratory practices used to detect this infection throughout the state.
Methods: During a 6-month period in 1993, all stool specimens at Rush-Presbyterian-St Luke's Medical Center (RPSLMC) were tested for E. coli O157:H7. Reports of diagnosed E. coli O157:H7 cases investigated by IDPH were also reviewed. A survey of 73 hospitals in the Chicago area was performed to determine routine culturing practices, specifically, the selection of stool specimens for evaluation for this pathogen.
Results: In the RPSLMC survey, two cases were identified among 1985 samples (incidence 0.1%), similar to the 0.08% incidence detected in a similar study conducted at the same institution in 1984. Through passive surveillance, the IDPH received 44 reports of E. coli O157:H7 in 1993. The hospital survey revealed that, in the seven labs testing all stool specimens for E. coli O157:H7, an incidence of 16/8137 specimens (0.2%) was determined.
Conclusions: These data suggest that sporadic E. coli O157:H7 remains uncommon in Illinois and that the incidence may not have changed over a 9-year period. The low yield and substantial cost of culturing all stools suggest that only specimens from patients with bloody diarrhea should be evaluated routinely in areas of low endemicity.     

Direct detection of verotoxin-producing Escherichia coli in stool samples by PCR.

A method for the rapid detection of verotoxin-producing Escherichia coli in stool samples by PCR was evaluated. Verotoxin-1 and verotoxin-2 genes in DNA extracted directly from stool samples were amplified with oligonucleotide primers. Stools spiked with control organisms, E. coli C600 (H19B) (verotoxin-1) or E. coli C600 (933W) (verotoxin-2), demonstrated that verotoxin-1-containing organisms could be detected at 10(2) CFU per 0.1 g of stool and verotoxin-2-containing organisms could be detected at 10(7) CFU per 0.1 g of stool. Testing of stool samples from patients with diarrhea showed a high concordance between PCR positivity and the presence of verotoxin-producing E. coli, determined by isolation of serotype O157:H7 on sorbitol-MacConkey medium (34 of 35 stool samples) or by colony blots with gene probes (19 of 21 stool samples). Conversely, only 1 of 20 (5.0%) stool samples that were O157:H7 culture negative and colony blot negative and that contained free verotoxin only was positive by PCR. As well, only 4 of 145 (2.8%) stool samples that were negative for serotype O157:H7 or free verotoxin were PCR positive. PCR of DNA extracted directly from stool samples provides a rapid method for the detection of stool samples containing verotoxin-producing E. coli compared with colony blot testing.     

Variation in the persistence of Escherichia coli O157:H7 in experimentally inoculated 6-week-old conventional lambs

Six-week-old lambs were inoculated orally with 10(9) cfu of an antibiotic-resistance marked four-strain mixture of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 to investigate faecal excretion and intestinal colonisation. In the first experiment, three E. coli O157:H7 isolates were not detected in the faeces of any lambs beyond day 8 post inoculation (pi), or from any of the tissues derived from inoculated animals. One strain, 140065 Nal(r), was isolated from the caecum and colon of one lamb on day 9 pi, from the rectum of another on day 22 pi and persisted in the faeces for up to 28 days pi. All animals remained clinically normal throughout the study period and histological evidence of adhesion of E. coli O157:H7 to the intestinal mucosa was not found. In a separate experiment, four 6-week-old lambs were inoculated orally with 10(9) cfu of E. coli O157:H7 strain 140065 Nal(r) alone. Faecal samples were positive for this strain until the end of the experiment (day 19 pi). This strain was also recovered from the gastrointestinal tract of lambs on days 6, 18 and 19 pi, but was not isolated at day 17 pi. When sampled separately, rectum and terminal colon contents contained higher numbers of the inoculated strain than the intestinal tissue at these sites. Animals inoculated with O157:H7 strain 140065 Nal(r) alone produced soft faeces from day 5 pi onwards. Although attaching and effacing lesions were observed in the caecum, proximal colon and rectum in one animal on day 18 pi, the adherent bacteria did not stain with antiserum raised against the O157 antigen.     

A severe outbreak of Escherichia coli O157:H7--associated hemorrhagic colitis in a nursing home

In September 1985, an outbreak of Escherichia coli O157:H7 enteritis affected 55 of 169 residents and 18 of 137 staff members at a nursing home. The outbreak was characterized by two phases: a primary wave whose source was probably a contaminated sandwich meal and a secondary wave compatible with person-to-person transmission of infection. Among the elderly residents, the incubation period was 4 to 9 days (mean, 5.7 +/- 1.2). Older age and previous gastrectomy increased the risk of acquiring the infection (P = 0.01 and 0.03, respectively). Antibiotic therapy during exposure was associated with acquiring a secondary infection (P = 0.001). Hemolytic uremic syndrome developed in 12 affected residents (22 percent), 11 of whom died. Overall, 19 (35 percent) of the affected residents died, 17 (31 percent) from causes attributable to their infection. Antibiotic therapy after the onset of symptoms was associated with a higher case fatality rate in the more severe cases, possibly because patients with more severe disease tended to be treated with antibiotics. There were no complications or deaths among the affected members of the staff. Evidence of infection by verotoxin-producing E. coli O157:H7 was detected in 30 of 70 cases on the basis of isolation of this organism or demonstration of free verotoxin in stools. All isolates belonged to the same phage type. The high morbidity and mortality associated with this condition emphasize the need for proper food hygiene, rapid identification of outbreaks of disease, and prompt institution of infection-control techniques among the institutionalized elderly.     

Role of Escherichia coli O157:H7 virulence factors in colonization at the bovine terminal rectal mucosa

The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the approximately 92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, delta tir, and delta eae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.