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.     

The use of serodiagnosis in the retrospective investigation of a nursery outbreak associated with Escherichia coli O157:H7.

AIMS: To use serology to investigate an outbreak of verocytotoxin (VT) producing Escherichia coli O157 in a hospital nursery, following the detection of faecal E coli O157 (phage type 49) producing VT type 2. METHODS: ELISA and immunoblotting techniques, based on lipopolysaccharide (LPS) purified from E coli O157; diagnostic bacteriology; serotyping and phage typing; DNA probes for VT. RESULTS: 29 of 126 sera contained antibodies to the LPS of E coli O157: 10 were from children, three were from staff, and 11 were from hospital kitchen staff. Five parents of children attending the nursery were antibody positive. Sixty four sera from other hospital staff and controls did not contain antibodies to the LPS of E coli O157. CONCLUSIONS: Serology detected evidence of infection with E coli O157 in 23% of sera examined. By bacteriology alone, only a single case of infection with E coli O157 would have been detected. Serology is valuable in providing evidence of infection with E coli O157.     

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.     

A novel cryohemagglutinin associated with adherence of enteroaggregative Escherichia coli.

Strain O42 (serotype O44:H18) of enteroaggregative Escherichia coli (EAggEC) has been shown to be pathogenic in volunteer experiments. This strain exhibited plasmid (pO42)-encoded D-mannose-resistant hemagglutinating activity (MRHA) that was detected only at low temperatures (e.g., 0 degrees C) and only with human erythrocytes. The production of this cryogenic MRHA (cryo-MRHA) was observed when the bacteria were grown in liquid media and was strictly regulated by bacterial growth temperatures. Transposon-insertion mutagenesis revealed that this MRHA is associated with (i) bacterial clump formation in liquid cultures, (ii) bacterial adherence to HEp-2 cells as well as (Formalin-fixed) human colonic mucosa, and (iii) production of a 16-kDa outer membrane protein. The PCR designed on the basis of the determined cryo-MRHA-associated DNA sequence sharply distinguished strain O42 from eight other EAggEC strains whose MRHAs were detected at both cold and room temperatures to the same (or similar) extent. Strain O42 possessed a surface layer that may enhance the pO42-mediated adherence. The data suggest that a plasmid-encoded cryo-MRHA is a candidate for a major adhesin of EAggEC strain O42.     

Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype.

Two outbreaks of hemorrhagic colitis, a newly recognized syndrome characterized by bloody diarrhea, severe abdominal pain, and little or no fever, occurred in 1982. No previously recognized pathogens were recovered from stool specimens from persons in either outbreak. However, a rare E. coli serotype, O157:H7, was isolated from 9 of 20 cases and from no controls. It was also recovered from a meat patty from the implicated lot eaten by persons in one outbreak. No recovery of this organism was made from stools collected 7 or more days after onset of illness; whereas 9 of 12 culture-positive stools had been collected within 4 days of onset of illness. The isolate was not invasive or toxigenic by standard tests, and all strains has a unique biotype. Plasmid profile analysis indicates that all outbreak-associated E. coli O157:H7 isolates are closely related. These results suggest that E. coli O157:H7 was the causative agent of illness in the two outbreaks.     

Comparative virulence characterization of the Shiga toxin phage-cured Escherichia coli O104:H4 and enteroaggregative Escherichia coli

Escherichia coli O104:H4 (E. coli O104:H4), which caused in 2011 a massive foodborne outbreak in Germany, is characterized by an unusual combination of virulence traits. E. coli O104:H4 contains a prophage-encoded Shiga toxin (Stx) gene, which is the cardinal virulence factor of enterohemorrhagic E. coli (EHEC). However, the outbreak strain shares highest DNA sequence similarity with enteroaggregative E. coli (EAEC) and displays the EAEC-characteristic tight adherence to epithelial cells. The virulence potential of the underlying EAEC background has not been investigated and it is therefore not clear whether E. coli O104:H4 displays distinct virulence characteristics in comparison to prototypical EAEC. In this study, we performed a detailed comparative phenotypic characterization of the Stx phage-cured E. coli O104:H4 strain C227-11φcu, the closely related EAEC strain 55989 and two other well-characterized EAEC strains 042 and 17-2 with focus on virulence traits. C227-11φcu displayed superior aggregative adherence phenotype to cultured HCT-8 epithelial cells, adhering with 3–6 times more bacteria per epithelial cells than the tested EAEC strains. Otherwise, C227-11φcu showed similar virulence characteristics to its closest relative 55989, i.e. strong acid resistance, good biofilm formation and cytotoxic culture supernatants. Furthermore, C227-11φcu was characterized by significantly weaker motility and pro-inflammatory properties than 55989 and 042, nevertheless stronger than 17-2. Taken together, C227-11φcu displayed mostly robust, but not outstanding virulence characteristics in comparison to the tested EAEC. Therefore, it appears likely that the combination of Stx production and EAEC characteristics in general, rather than an exceptionally potent EAEC background resulted in the unusual virulence of the E. coli O104:H4. Thus, the emergence of such hypervirulent strains in the future might be more likely than previously anticipated.     

Characterization of genes associated with internalization of enteroaggregative Escherichia coli

On animal models enteroaggregative Escherichia coli (EAEC) can cause mild, but significant mucosal damage, suggesting the invasive capability of these strains. In the study we investigated the ability of typical, aggR-positive and atypical, aggR-negative EAEC isolates to enter intestinal epithelial Int407 cells in relation to the distribution of genes encoding the putative invasins described among pathogenic E. coli categories. The results demonstrated that regardless of origin and affiliation to typical and atypical EAEC, most isolates examined were internalized by the epithelial cells to different extent. Although as many as 50 (84.3%) EAEC demonstrated a variety of combinations of the aggB, afaD, ipaH and tia genes determined, there was no correlation between the invasion efficiency of these strains and the presence of any particular gene involved in invasion. Most of EAEC examined belonged to phylogenetic group B2 and D.     

Infection of gnotobiotic pigs with an Escherichia coli O157:H7 strain associated with an outbreak of hemorrhagic colitis.

Gnotobiotic pigs inoculated with an Escherichia coli O157:H7 strain isolated from a human with hemorrhagic colitis developed anorexia, lethargy, and watery diarrhea. Bacteria diffusely colonized the cecum and colon surfaces and the crypt epithelium. At bacterial attachment sites, microvilli were effaced, and epithelial cells were irregularly shaped, rounded, or detached. Submucosa, lamina propria, and mesentery were markedly edematous and contained many inflammatory cells.     

O148 Shiga toxin-producing Escherichia coli outbreak: microbiological investigation as a useful complement to epidemiological investigation

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O148 infection occurred among wedding attendees in France in June 2002. A retrospective cohort study was performed and ten cases were identified, including two adults with haemolytic uraemic syndrome (HUS). The analytical study revealed that > 80% of affected individuals had eaten lightly roasted mutton and poultry paté, but only the consumption of paté tended to be associated with illness (relative risk 3.4; 95% CI 0.8-14.4). Left-overs (cooked mutton and raw offal) and processed foods (paté) from the same batches as served at the party were sampled. Human, food and environmental samples were examined for the Shiga toxin (stx) gene and virulence traits by PCR. Stx-positive samples were cultured for STEC. HUS cases were tested for serum antibodies against 26 major STEC serogroups. An STEC O26 strain (stx1, eae, ehxA) was isolated from one case with diarrhoea, and an STEC O148 strain (stx2c) from one case of HUS. Serum antibodies against O26 were not detected in either of these patients; antibodies against O148 were not tested. Three STEC strains were isolated from the mutton and the offal (stx2c, O148), and two from the paté (stx2c, O-X and O-Y). The isolates from the mutton were indistinguishable from the human stx2c isolate, whereas the paté isolates differed. Although four different STEC strains were identified in patients and foods, the results of molecular subtyping, in conjunction with analysis of food consumption patterns, strongly suggested that this outbreak was caused by mutton contaminated with STEC O148.     

Susceptibility patterns of enteroaggregative Escherichia coli associated with traveller's diarrhoea: emergence of quinolone resistance.

Enteroaggregative Escherichia coli (EAggEC) isolates were identified as a cause of traveller's diarrhoea in 50 (9%) of 517 patients and their antimicrobial susceptibility was determined. Molecular epidemiological characterisation and investigation of the mechanisms of acquisition of quinolone resistance among nalidixic acid-resistant EAggEC strains was performed. Seventeen (34%) of 50 patients needed antimicrobial therapy, because of persistence of symptoms in nine cases and the severity of symptoms in eight cases. Ampicillin and tetracycline resistance was high, whereas chloramphenicol and co-trimoxazole showed moderate activity and amoxicillin plus clavulanic acid, nalidixic acid and ciprofloxacin showed very good activity. Resistance to nalidixic acid was demonstrated in three isolates, two from patients who had travelled to India. In all three strains the resistance was linked to mutations in the gyrA gene alone or in both gyrA and parC genes. Although ciprofloxacin shows excellent in-vitro activity and could be useful in the treatment of traveller's diarrhoea in patients travelling abroad, it may not be useful in patients who have journeyed to India or to Mexico.     

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.     

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.     

Responses of cattle to gastrointestinal colonization by Escherichia coli O157:H7

Recent research has established that the terminal rectum is the predominant colonization site of enterohemorrhagic Escherichia coli O157:H7 in cattle. The main aim of the present work was to investigate pathological changes and associated immune responses at this site in animals colonized with E. coli O157:H7. Tissue and gastrointestinal samples from a total of 22 weaned Holstein-cross calves challenged with E. coli O157:H7 were analyzed for bacterial colonization and pathology. Five unexposed age-matched calves were used as comparative negative controls. E. coli O157:H7 bacteria induced histopathological alterations of the rectal mucosa with enterocyte remodeling. This was often associated with removal of the colonized epithelial layer. Immunogold labeling and transmission electron microscopy (TEM) showed E. coli O157 bacteria on pedestals, as part of attaching and effacing lesions. These pathological changes induced a local infiltration of neutrophils that was quantified as larger in infected animals. Rectal mucosal immunoglobulin A responses were detected against the E. coli O157:H7 antigen. This work presents evidence that E. coli O157:H7 is not a commensal bacteria in the bovine host and that the mucosal damage produced by E. coli O157:H7 colonization of the terminal rectum induces a quantifiable innate immune response and production of specific mucosal antibodies.     

Ruminant gastrointestinal cell proliferation and clearance of Escherichia coli O157:H7

Human infections with Escherichia coli O157:H7 cause hemorrhagic colitis that can progress to a life-threatening sequelae. The most common mode of disease transmission is ingestion of contaminated bovine food products, and it is well established that E. coli O157:H7 is a transient member of the bovine microbiota. However, the conditions that induce acquisition and subsequent clearance of this bacterium from the ruminant gastrointestinal tract (GIT) are not understood. Evidence that the rates of epithelial cell proliferation in the lower GIT of cattle are associated with the duration animals remained E. coli O157:H7 culture positive is presented. Cattle with slower rates of intestinal cell proliferation in the cecum and the distal colon were culture positive significantly longer than cohort cattle with faster cell proliferation rates. Cell death rates (apoptotic indices) between the short- and long-term culture-positive animals were not different. Typical grain-based finishing diets and forage-based growing diets did not effect GIT cell proliferation or the duration animals remained E. coli O157:H7 culture positive. To identify a dietary intervention that would effect GIT cell proliferation, we used sheep as a model ruminant. A fasting-refeeding regime that increased the rate of GIT cell proliferation was developed. The fasting-refeeding protocol was used in cattle to test the hypothesis that feeding interventions that increase the rate of GIT cell proliferation induce the clearance of E. coli O157:H7 from the bovine GIT.     

Pet toxin from enteroaggregative Escherichia coli produces cellular damage associated with fodrin disruption

Pet toxin is a serine protease from enteroaggregative Escherichia coli which has been described as causing enterotoxic and cytotoxic effects. In this paper we show that Pet produces spectrin and fodrin (nonerythroid spectrin) disruption. Using purified erythrocyte membranes treated with Pet toxin, we observed degradation of alpha- and beta-spectrin chains; this effect was dose and time dependent, and a 120-kDa protein fraction was observed as a breakdown product. Spectrin degradation and production of the 120-kDa subproduct were confirmed using specific antibodies against the alpha- and beta-spectrin chains. The same degradation effect was observed in alpha-fodrin from epithelial HEp-2 cells, both in purified cell membranes and in cultured cells which had been held in suspension for 36 h; these effects were confirmed using antifodrin rabbit antibodies. The spectrin and fodrin degradation caused by Pet is related to the Pet serine protease motif. Fluorescence and light microscopy of HEp-2 Pet-treated cells showed morphological alterations, which were associated with irregular distribution of fodrin in situ. Spectrin and fodrin degradation by Pet toxin were inhibited by anti-Pet antibodies and by phenylmethylsulfonyl fluoride. A site-directed Pet mutant, which had been shown to abolish the enterotoxic and cytotoxic effects of Pet, was unable to degrade spectrin in erythrocyte membranes or purified spectrin or fodrin in epithelial cell assays. This is a new system of cellular damage identified in bacterial toxins which includes the internalization of the protease, induction of some unknown intermediate signaling steps, and finally the fodrin degradation to destroy the cell.