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.     

Culture confirmation of Escherichia coli serotype O157:H7 by direct immunofluorescence.

An evaluation of a fluorescein-labeled, polyclonal, affinity-purified goat antibody to Escherichia coli serotype O157:H7 (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) was conducted to determine the efficacy of this research reagent for the rapid direct immunofluorescence identification of E. coli O157:H7 isolated from fecal specimens cultured on sorbitol-MacConkey (SMAC) agar. The E. coli O157:H7 fluorescent-antibody conjugate proved to be 100% sensitive and specific for the rapid identification of non-sorbitol-fermenting E. coli O157:H7 from SMAC agar inoculated with fecal specimens. The addition of SMAC agar to the battery of primary isolation media routinely used for fecal specimens resulted in the identification of 16 patients with E. coli O157:H7 disease from a total of 799 fecal specimens cultured during 1988.     

Shiga toxin-producing Escherichia coli in children: diagnosis and clinical manifestations of O157:H7 and non-O157:H7 infection

Shiga toxin-producing Escherichia coli (STEC), a cause of food-borne colitis and hemolytic-uremic syndrome in children, can be serotype O157:H7 (O157) or other serotypes (non-O157). E. coli O157 can be detected by culture with sorbitol-MacConkey agar (SMAC), but non-O157 STEC cannot be detected with this medium. Both O157 and non-O157 STEC can be detected by immunoassay for Shiga toxins 1 and 2. The objectives of this study were first to compare the diagnostic utility of SMAC to that of the Premier EHEC enzyme immunoassay (Meridian Diagnostics) for detection of STEC in children and second to compare the clinical and laboratory characteristics of children with serotype O157:H7 STEC and non-O157:H7 STEC infections. Stool samples submitted for testing for STEC between April 2004 and September 2009 were tested by both SMAC culture and the Premier EHEC assay at Children's Hospital Boston. Samples positive by either test were sent for confirmatory testing and serotyping at the Hinton State Laboratory Institute (HSLI). Chart review was performed on children with confirmed STEC infection. Of 5,110 children tested for STEC, 50 (0.9%) had STEC infection confirmed by culture; 33 were O157:H7 and 17 were non-O157:H7. The Premier EHEC assay and SMAC culture detected 96.0% and 58.0% of culture-confirmed STEC isolates (any serotype), respectively, and 93.9% and 87.9% of STEC O157:H7 isolates, respectively. There were no significant differences in disease severity or laboratory manifestations of STEC infection between children with O157:H7 and those with non-O157 STEC. The Premier EHEC assay was significantly more sensitive than SMAC culture for diagnosis of STEC, and O157:H7 and non-O157:H7 STEC caused infections of similar severity in children.     

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.     

Comparison of Escherichia coli O157:H7 antigen detection in stool and broth cultures to that in sorbitol-MacConkey agar stool cultures

We evaluated the Meridian IC-STAT direct fecal and broth culture antigen detection methods with samples from children infected with Escherichia coli O157:H7 and correlated the antigen detection results with the culture results. Stools of 16 children who had recently had stool cultures positive for this pathogen (population A) and 102 children with diarrhea of unknown cause (population B) were tested with the IC-STAT device (direct testing). Fecal broth cultures were also tested with this device (broth testing). The results were correlated to a standard of the combined yield from direct culture of stools on sorbitol-MacConkey (SMAC) agar and culture of broth on SMAC agar. Eleven (69%) of the population A stool specimens yielded E. coli O157:H7 when plated directly on SMAC agar. Two more specimens yielded this pathogen when the broth culture was similarly plated. Of these 13 stool specimens, 8 and 13 were positive by direct and broth testing (respective sensitivities, 62 and 100%). Compared to the sensitivity of a simultaneously performed SMAC agar culture, the sensitivity of direct testing was 73%. Three (3%) of the population B stool specimens contained E. coli O157:H7 on SMAC agar culture; one and three of these stool specimens were positive by direct and broth testing, respectively. The direct and broth IC-STAT tests were 100% specific with samples from children from population B. Direct IC-STAT testing of stools is rapid, easily performed, and specific but is insufficiently sensitive to exclude the possibility of infection with E. coli O157:H7. Performing the IC-STAT test with a broth culture increases its sensitivity. However, attempts to recover E. coli O157:H7 by culture should not be abandoned but, rather, should be increased when the IC-STAT test result is positive.     

Comparison of sorbitol MacConkey agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) and specifically serotype O157:H7 are a significant cause of hemorrhagic gastrointestinal disease and the hemolytic uremic syndrome. Methods currently used in clinical microbiology labs, such as sorbitol-MacConkey (SMAC) agar, reliably detect only O157:H7. We have evaluated a two-step method that has the potential to identify and isolate all EHEC serotypes, including serotype O157:H7. This method utilizes a chromogenic selective-differential medium for the isolation of E. coli together with an enzyme-linked immunosorbent assay (ELISA) that detects the Shiga-like toxins Stx1 and Stx2. Both are commercially available and usable in a wide range of clinical microbiology laboratories. Compared to a Vero cell cytotoxic assay, SMAC had sensitivities of 23.5% for the identification of all EHEC serotypes and of 50.0% for the identification of O157:H7 alone. The two-step method had sensitivities of 76.5 and 100%, respectively. The ELISA alone had a sensitivity of 82.4% in the detection of Stx1 and Stx2. The specificity was 100% in all cases. Overall, 14 EHEC isolates were obtained: 8 (58%) O157:H7, 2 (14%) O26, 2 (14%) O111:NM, 1 (7%) O103:H2, and 1 (7%) O121:H19. All but one were isolated during the months of May to September. The two-step method was found to be considerably more expensive than SMAC for both positive and negative samples.     

Immunological characterization of Escherichia coli O157:H7 intimin gamma1.

Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a+ expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a+. Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin types alpha (O serogroups 86, 127, and 142), beta1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), gamma 1 (O serogroups 55, 145, and 157), gamma 2 (O serogroups 111 and 103), and epsilon (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intgamma1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin gamma 1-producing E. coli strains such as E. coli O157:H7.     

Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus.

Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood. We performed TnphoA mutagenesis on E. coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression. Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U. H. Stroeher, L. E. Karageorgos, R. Morona, and P. A. Manning, Proc. Natl. Acad. Sci. USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase. This open reading frame was designated rfbE(EcO157:H7). The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E. coli. rfbE(EcO157:H7) is conserved in nontoxigenic E. coli O157 strains expressing a variety of other flagellar antigens but is not found in E. coli O55:H7 strains, which are more closely related to E. coli O157:H7. Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E. coli O157:H7 parent, but they did not differ in other phenotypes. Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12. We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E. coli rfb genes occurred independently in E. coli O157:H7 and unrelated O157 strains, and that the O side chain of E. coli O157:H7 lipopolysaccharide interferes with the adherence of E. coli O157:H7 to epithelial cells.     

Bovine immune response to shiga-toxigenic Escherichia coli O157:H7

Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+ O157. Following the challenge, levels of fecal shedding of Stx2+ O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx- O157, but not those inoculated with Stx2+ O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+ O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.     

Molecular characterization of the gene encoding H antigen in Escherichia coli and development of a PCR-restriction fragment length polymorphism test for identification of E. coli O157:H7 and O157:NM.

Recent outbreaks of disease caused by Escherichia coli O157:H7 have focused much attention on this newly emerged pathogen. Identification of the H7 flagellar antigen is critical for the confirmation of E. coli O157:H7; however, clinical isolates are frequently nonmotile and do not produce detectable H antigen. To further characterize nonmotile isolates (designated NM), we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) test to identify and characterize the gene encoding the H antigen (fliC) in E. coli. The entire coding sequence of fliC was amplified by PCR, the amplicon was restricted with RsaI, and the restriction fragment pattern was examined after gel electrophoresis. Two hundred eighty E. coli isolates representing serotypes O157:H7 and O157:NM, flagellar antigen H7 groups associated with other O serogroups, and all other flagellar antigen groups were analyzed. A single restriction pattern (pattern A) was identified for O157:H7 isolates, O157:NM isolates that produced Shiga toxin (formerly Shiga-like toxin or verotoxin), and 16 of 18 O55:H7 isolates. Flagellar antigen group H7 isolates of non-O157 serotypes had one of three banding patterns distinct from pattern A. A wide variety of patterns were found among isolates of the other 52 flagellar antigen groups; however, none was identical to the O157:H7 pattern. Thirteen of 15 nonmotile strains that did not produce the A pattern had patterns that matched those of other known H groups. The PCR-RFLP in conjunction with O serogroup determination will be useful in identifying E. coli O157:H7 and related strains that do not express immunoreactive H antigen and could be expanded to include other clinically important E. coli strains.     

Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.     

Rectoanal mucosal swab culture is more sensitive than fecal culture and distinguishes Escherichia coli O157:H7-colonized cattle and those transiently shedding the same organism

Enrichment and direct (nonenrichment) rectoanal mucosal swab (RAMS) culture techniques were developed and compared to traditional fecal culture for the detection of Escherichia coli O157:H7 in experimentally infected and naturally infected cattle. Holstein steers (n = 16) orally dosed with E. coli O157:H7 were sampled after bacterial colonization starting 15 days postinoculation. Enrichment RAMS cultures (70.31% positive) were more sensitive than enrichment fecal cultures with 10 g of feces (46.88% positive) at detecting E. coli O157:H7 (P < 0.01). Holstein bull calves (n = 15) were experimentally exposed to E. coli O157:H7 by penning them with E. coli O157:H7-positive calves. Prior to bacterial colonization (1 to 14 days postexposure), enriched fecal cultures were more sensitive at detecting E. coli O157:H7 than enriched RAMS cultures (P < 0.01). However, after colonization (40 or more days postexposure), the opposite was true and RAMS culture was more sensitive than fecal culture (P < 0.05). Among naturally infected heifers, enriched RAMS or fecal cultures were equally sensitive (P = 0.5), but direct RAMS cultures were more sensitive than either direct or enriched fecal cultures at detecting E. coli O157:H7 (P < 0.01), with 25 of 144, 4 of 144, and 10 of 108 samples, respectively, being culture positive. For both experimentally and naturally infected cattle, RAMS culture predicted the duration of infection. Cattle transiently shedding E. coli O157:H7 for <1 week were positive by fecal culture only and not by RAMS culture, whereas colonized animals (which were culture positive for an average of 26 days) were positive early on by RAMS culture. RAMS culture more directly measured the relationship between cattle and E. coli O157:H7 infection than fecal culture.     

Detection of Escherichia coli O157:H7 using chicken immunoglobulin Y

A sandwich ELISA technique was examined to detect Escherichia coli O157:H7 using chicken anti-E. coli O157:H7 IgY as the capture-antibody and an anti-E. coli O157 mouse mAb conjugated with biotin as the detection antibody. The anti-E. coli O157:H7 IgY was harvested from eggs laid by hens (23 weeks of age, Single Comb White Leghorn) immunized with formalin-killed E. coli O157:H7. The IgY was purified by water dilution methods and gel chromatography on Sephacryl S-300 followed by ammonium sulfate precipitation. The sensitivity (CFU/ml) of sandwich ELISA for the E. coli O157:H7 was repeatedly examined with 10 replicates of each sample and a standard curve was plotted. The sandwich ELISA can detect as low as 40CFU/ml of E. coli O157:H7. The data suggest that chicken IgY-based sandwich ELISA provides a reliable, inexpensive and sensitive assay for the detection of the food-borne pathogen E. coli O157:H7.     

Escherichia coli O157:H7 and O157:H− Strains That Do Not Produce Shiga Toxin: Phenotypic and Genetic Characterization of Isolates Associated with Diarrhea and Hemolytic-Uremic Syndrome

We have isolated one sorbitol-nonfermenting (SNF) Escherichia coli O157:H7 isolate and five sorbitol-fermenting (SF) E. coli O157:H(-) isolates that do not contain Shiga toxin (Stx) genes (stx). Isolates originated from patients with diarrhea (n = 4) and hemolytic-uremic syndrome (HUS) (n = 2). All isolates harbored a chromosomal eae gene encoding gamma-intimin as well as the plasmid genes E-hly and etp. The E. coli O157:H7 isolate was katP and espP positive. Respective sera obtained from the patient with HUS contained antibodies to the O157 lipopolysaccharide antigen. The stx-negative E. coli O157:H7 isolate is genetically related to stx-positive SNF E. coli O157:H7. All stx-negative SF E. coli O157:H(-) isolates belong to the same genetic cluster and are closely related to stx-positive SF E. coli O157:H(-) isolates. Our data indicate that stx-negative E. coli O157:H7/H(-) variants may occur at a low frequency and cannot be recognized by diagnostic methods that target Stx.     

Selection of an Escherichia coli O157:H7 bacteriophage for persistence in the circulatory system of mice infected experimentally

A bacteriophage lytic for Escherichia coli O157:H7 was isolated from bovine manure. Following in-vivo selection, the phage acquired the capacity to persist in the circulatory system of mice for at least 38 days. When mice were infected experimentally with E. coli O157:H7 (10(7) CFU/mouse), simultaneous injection of the mice with phage (10(8) PFU/mouse) cleared E. coli O157:H7 from the mice within 48 h.     

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.     

Clonal structure and pathogenicity of Shiga-like toxin-producing, sorbitol-fermenting Escherichia coli O157:H-.

We compared a collection of sorbitol-fermenting (SF) Escherichia coli O157:H- strains with SF E. coli O157:H45 and non-SF E. coli O157:H7 and E. coli O157:H- strains by pulsed-field gel electrophoresis. The SF E. coli O157:H- strains had identical or closely related XbaI patterns that differed markedly from those for the other E. coli O157 strains. Plasmid content and the presence of Shiga-like toxin-converting phages were determined for the SF E. coli O157:H- strains, indicating that these strains harbor a single 90-kb plasmid. They are lysogenized by toxin-converting phages and harbor the eae gene. Nonmotile E. coli O157 strains were observed to adhere more efficiently to HEp-2 cells than the motile strains. From their phenotypic and genotypic features, the SF E. coli O157:H- strains may well represent a new clone with non-SF E. coli O157:H7 pathogenic characteristics.     

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.     

Identification and distribution of the enterohemorrhagic Escherichia coli factor for adherence (efa1) gene in sorbitol-fermenting Escherichia coli O157: H-

Sorbitol-fermenting (SF) Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H- strains are emerging as causes of hemorrhagic colitis and the hemolytic-uremic syndrome in Europe. Using subtractive hybridization between SF STEC O157:H- strain 493/89 and STEC O157:H7 strain EDL933, three different fragments, of approximately 700 bp in length, were identified. Each demonstrated > 99% homology to genes encoding the enterohemorrhagic E. coli factor for adherence (efa1) and lymphostatin (lifA). Therefore, a cosmid library was constructed from SF STEC O157:H- strain 493/89, and one clone containing these fragments was sequenced. This sequencing demonstrated a 9669-bp open reading frame (ORF) that had 99.9% sequence homology to efa1 of STEC O111:H- strain E45035 and to lifA of an enteropathogenic E. coli O127:H6 strain E2348/69. In STEC O157:H7 strain EDL933, only small (ca. 3 kb) initial and terminal fragments of this ORF are present. PCR analysis with primers complementary to the efa1/lifA sequence of strain 493/89 indicated that the complete sequence is present in each of 10 SF STEC O157:H- isolates but in none of 10 STEC O157:H7 strains investigated. The presence of the complete efa1/lifA also in both tested E. coli O55:H7 strains supports the hypothesis that SF STEC O157:H- are phylogenetically closer to the proposed E. coli O55:H7 ancestor than STEC O157:H7. Our data demonstrate the presence of a potential virulence gene in SF STEC O157:H- that is only rudimentarily present in STEC O157:H7.