Coli O157 Strains Research Articles

Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.

Effects of mucin and its carbohydrate constituents on Escherichia coli O157 growth in batch culture fermentations with ruminal or fecal microbial inoculum1

In cattle, Escherichia coli O157 generally persists in the large intestine more often than in the rumen. In contrast to the rumen, the large intestine is lined by an epithelial membrane that secretes mucus. We hypothesize that substrates contained in intestinal mucus may constitute a source of energy that is preferentially used by E. coli O157. Therefore, our objective was to test the effects of mucin and its carbohydrate constituents on in vitro growth of E. coli O157 in ruminal or fecal microbial fermentations. Ruminal contents and feces were collected from a ruminally cannulated donor steer fed a corn grain-based finishing diet. Ruminal contents were strained through 2 layers of cheesecloth and incubated at 39 degrees C for 1 h; the floating hay mat was removed with a vacuum suction; and the remaining material was utilized as rumen microbial inoculum. Feces were suspended in physiologic saline to increase fluidity, blended, and strained through 2 layers of cheesecloth. The resulting fluid was utilized as fecal microbial inoculum. Fermentations (50 mL) were performed in serum bottles with a 2:1 mineral buffer to microbial inoculum ratio. Substrates (mucin, fucose, galactose, mannose, gluconic acid, galacturonic acid, glucuronic acid, galactosamine, and glucosamine) were added at 10 mg/mL. A mixture of 5 strains of nalidixic acid-resistant (Nal(R)) E. coli O157 strains was added to each fermentation and concentrations were determined after 0, 6, 12, and 24 h of incubation. In ruminal fermentations, fucose, mannose, glucuronic acid, galacturonic acid, glucosamine, galactosamine, and mucin had no effect on Nal(R) E. coli O157 concentration compared with the control (no substrate added) fermentation. At 24 h of fermentation, the mean concentration of Nal(R) E. coli O157 in fermentations with galactose was less than the control. However, including gluconic acid as substrate increased Nal(R) E. coli O157 concentration at 24 h. In fecal fermentations, mannose, galactose, gluconic acid, glucuronic acid, galacturonic acid, glucosamine, and mucin increased Nal(R) E. coli O157 growth compared with control at 24 h, whereas galactosamine and fucose did not. Gluconic acid was the most stimulatory substrate, increasing Nal(R) E. coli O157 by more than 1.0 log in ruminal fermentations and 2.0 log in fecal fermentations. In summary, availability of mucous constituents, particularly gluconic acid, may explain the greater prevalence of E. coli O157 in the large intestine compared with the rumen of the digestive tract.

Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Using colony blot hybridization with stx(2) and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx(2)-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx(2)-positive isolates fliC(H7), non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx(2)-positive strains by loss of stx(2) during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx(2)-positive and stx-negative isolates demonstrated that yecE was occupied in stx(2)-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx(2)-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx(2)-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx(2)-harboring phages. The ability to recycle stx(2), a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.

Detection of toxB , a Plasmid Virulence Gene of Escherichia coli O157, in Enterohemorrhagic and Enteropathogenic E. coli

The virulence plasmid of Escherichia coli O157 strain EDL933 carries a 10-kb putative virulence gene designated toxB. Little is known about the distribution of this gene among E. coli O157 strains or its presence in other enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) strains. We developed PCR and hybridization tools for the detection of the entire toxB sequence and investigated its presence in a collection of EHEC O157 strains and other EHEC and EPEC strains belonging to different serogroups and isolated from different sources. The EHEC O157 strains reacted with all of the PCR primers and probes used, thus indicating the presence of a complete toxB gene regardless of the human or bovine origin of the isolates. Similar positive reactions were observed for about 50% of the EHEC O26 strains tested and a few other EHEC and EPEC strains. However, the size of the DNA fragments hybridizing with the toxB probes differed from that of the positive fragments from EHEC O157, suggesting a polymorphism in the toxB genes present in the different E. coli serogroups. Moreover, several EHEC and EPEC strains belonging to different serogroups reacted with only some of the genetic tools used, suggesting either the existence of major variants of toxB or the presence of fragments of the gene. Southern blotting analysis showed that toxB sequences were located on large plasmids in EHEC and EPEC O26 as well.

Escherichia coliO157 outbreak associated with fresh unpasteurized goats' cheese

A family cluster of three cases of Escherichia coli O157 infection was identified in France. Two cases developed haemolytic-uraemic syndrome. The source was fresh unpasteurized goats' cheese, produced by an independent producer. Three E. coli O157 strains, isolated from one HUS case and faeces of one cow and one goat, were indistinguishable by toxin type and PFGE pattern.

First-Time Isolation and Characterization of a Bacteriophage Encoding the Shiga Toxin 2c Variant, Which Is Globally Spread in Strains of Escherichia coli O157

A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E. coli K-12 laboratory strains C600 and MG1655. Production of Stx2c was found in the wild-type E. coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin. Phage 2851 is the first reported viable bacteriophage which carries an stx(2c) gene. Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda. Sequence analysis of an 8.4-kb region flanking the stx(2c) gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages. Phage 2851 showed lysis of E. coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages. Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E. coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E. coli O157 strains. Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E. coli O157 strains producing Stx2c. The phage 2851 q and o genes were frequently detected in Stx2c-producing E. coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E. coli O157 that were isolated in different locations and time periods.

Prevalence and characteristics of Escherichia coli O157 from major food animals in Korea

Escherichia coli O157:H7/NM ( E. coli O157) is now recognized as an important cause of diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome worldwide. There have been several cases of human E. coli O157 infection in Korea since it was first isolated from a patient with hemolytic-uremic syndrome in 1998. Meat, other foods, and recreational and drinking water contaminated with animal feces are probably the major sources of the E. coli O157 infection. In this study, we investigated the prevalence of E. coli O157 in fecal and meat samples of cattle, pigs and chicken in Korea from April 2000 to July 2002. Eighty-six (3.03%) of 2843 samples were positive for E. coli O157. Most of the E. coli O157 strains were isolated from fecal samples of beef and dairy cattle from May to October of each year. Of 86 E. coli O157 isolates, 73 were serotype O157:H7 and 13 were serotype O157:NM. Polymerase chain reaction (PCR) analysis of E. coli O157 virulence markers revealed that all O157:H7/NM isolates were positive for EhlyA, eaeA and rfb O157, and 77 isolates were positive for stx1 and/or stx2. Cytotoxicity analysis revealed that many of the E. coli O157 isolates showed high cytotoxicity on Vero cells. Our data suggest that the majority of Korean E. coli O157 isolates from food animals can cause serious diseases in humans.

Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents.

The mechanisms by which bacteria resist killing by antibiotics and biocides are still poorly defined, although repeated exposure to sublethal concentrations of antibacterial agents undoubtedly contributes to their development. This study aimed both to investigate the potential of Salmonella enterica and Escherichia coli O157 for adaptive resistance to commonly used biocides and to determine any cross-resistance to antibiotics. Strains were repeatedly passaged in media containing increasing concentrations of a biocide or antibiotic until adaptive resistance was obtained. A wide panel of antimicrobial agents was then screened by using the adapted strain to determine cross-resistance, if any. Adaptive resistance was readily achieved for both S. enterica and E. coli O157. Cross-resistance in adaptively resistant S. enterica varied with the serotype; Salmonella enterica serovar Enteritidis expressed cross-resistance to chloramphenicol, whereas Salmonella enterica serovar Typhimurium expressed cross-resistance to chlorhexidine. Benzalkonium chloride-resistant Salmonella enterica serovar Virchow showed elevated resistance to chlorhexidine; however, chlorhexidine-resistant Salmonella serovar Virchow did not demonstrate reciprocal cross-resistance to benzalkonium chloride, suggesting specific rather than generic resistance mechanisms. E. coli O157 strains acquired high levels of resistance to triclosan after only two sublethal exposures and, when adapted, repeatedly demonstrated decreased susceptibilities to various antimicrobial agents, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. These observations raise concern over the indiscriminate and often inappropriate use of biocides, especially triclosan, in situations where they are unnecessary, whereby they may contribute to the development of microbial resistance mechanisms.

An outbreak of Escherichia coli O157 infection following exposure to a contaminated building.

Infection with Escherichia coli O157 causes an estimated 70 000 diarrheal illnesses per year in the United States and can result in hemolytic-uremic syndrome and death. Environmental contamination with E coli O157 may be a public health problem. To determine risk factors for E coli O157 infection during an outbreak investigation at a county fair and to evaluate environmental contamination as a possible cause of the outbreak. Case-control study of 23 patients (median age, 15 years) and 53 age-matched controls who had attended the Lorain County, Ohio, fair between August 20 and August 26, 2001. Case-patients had laboratory-confirmed E coli O157 infection, hemolytic-uremic syndrome, or bloody diarrhea within 7 days of attending the fair; controls attended the fair and did not have diarrhea. Risk factors for infection and isolates of E coli O157 from environmental specimens. Six (26%) case-patients were hospitalized and 2 (9%) developed hemolytic-uremic syndrome. Case-patients were more likely than controls to have visited building A (a multipurpose community facility on the fairgrounds; matched odds ratio [MOR], 21.4 [95% confidence interval [CI], 2.7-170.7]). Among visitors to building A, illness was independently associated with attending a dance in the building (MOR, 7.5; 95% CI, 1.4-41.2), handling sawdust from the floor (MOR, 4.6; 95% CI, 1.1-20.0), or eating and/or drinking in the building (MOR, 4.5; 95% CI, 1.2-16.6). Twenty-four (44%) of 54 specimens collected from building A 6 weeks after the fair grew Shiga toxin-producing E coli O157. Isolates from sawdust, the rafters, and other surfaces were identical by molecular fingerprinting to patient isolates. Sawdust specimens collected 42 weeks after the fair also grew the same E coli O157 strain. Absence of evidence implicating specific food or beverage sources and the recovery of E coli O157 from the rafters suggest that airborne dispersion of bacteria contributed to the contamination. Because E coli O157 can survive in the environment for more than 10 months, humans may be at risk of infection long after an environment is initially contaminated.

Distinctiveness of the genomic sequence of Shiga toxin 2-converting phage isolated from Escherichia coli O157:H7 Okayama strain as compared to other Shiga toxin 2-converting phages

Shiga toxin 2-converting phage was isolated from Escherichia coli O157:H7 associated with an outbreak that occurred in Okayama, Japan in 1996 (M. Watarai, T. Sato, M. Kobayashi, T. Shimizu, S. Yamasaki, T. Tobe, C. Sasakawa and Y. Takeda, Infect. Immun. 61 (1998) 3210–3204). In this study, we analyzed the complete nucleotide sequence of Shiga toxin 2-converting phage, designated Stx2φ-I, and compared it with three recently reported Stx2-phage genomes. Stx2φ-I consisted of 61,765 bp, which included 166 open reading frames. When compared to 933W, VT2-Sakai and VT2-Sa phages, six characteristic regions (regions I–VI) were found in the Stx2 phage genomes although overall homology was more than 95% between these phages. Stx2φ-I exhibited remarkable differences in these regions as compared with VT-2 Sakai and VT2-Sa genes but not with 933W phage. Characteristic repeat sequences were found in regions I–IV where the genes responsible for the construction of head and tail are located. Regions V and VI, which are the most distinct portion in the entire phage genome were located in the upstream and downstream regions of the Stx2 operons that are responsible for the immunity and replication, and host lysis. These data indicated that Stx2φ-I is less homologous to VT2-Sakai and VT2-Sa phages, despite these three phages being found in the strains isolated at the almost same time in the same geographic region but closely related to 933W phage which was found in the E. coli O157 strain 933W isolated 14 years ago in a different geographic area.

DNA fingerprinting of Shiga-toxin producing Escherichia coli O157 based on Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA)

BackgroundThe ability to react early to possible outbreaks of Escherichia coli O157:H7 and to trace possible sources relies on the availability of highly discriminatory and reliable techniques. The development of methods that are fast and has the potential for complete automation is needed for this important pathogen.MethodsIn all 73 isolates of shiga-toxin producing E. coli O157 (STEC) were used in this study. The two available fully sequenced STEC genomes were scanned for tandem repeated stretches of DNA, which were evaluated as polymorphic markers for isolate identification.ResultsThe 73 E. coli isolates displayed 47 distinct patterns and the MLVA assay was capable of high discrimination between the E. coli O157 strains. The assay was fast and all the steps can be automated.ConclusionThe findings demonstrate a novel high discriminatory molecular typing method for the important pathogen E. coli O157 that is fast, robust and offers many advantages compared to current methods.

Polymerase Chain Reaction Assay for Detection of Escherichia coli O157:H7 and Escherichia coli O157:H−

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non–E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H− and E. coli O157:H7. Since the DNA sequence from base 15 to base 1008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H− and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 × 100 to 3.5 × 102 CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42°C for 18 h and for the conventional cultural method.

Differences in Levels of Secreted Locus of Enterocyte Effacement Proteins between Human Disease-Associated and Bovine Escherichia coli O157

Ongoing extensive epidemiological studies of verotoxin-carrying Escherichia coli O157 (stx(+) eae(+)) have shown this bacterial pathogen to be common in cattle herds in the United States and the United Kingdom. However, the incidence of disease in humans due to this pathogen is still very low. This study set out to investigate if there is a difference between strains isolated from human disease cases and those isolated from asymptomatic cattle which would account for the low disease incidence of such a ubiquitous organism. The work presented here has compared human disease strains from both sporadic and outbreak cases with a cross-section, as defined by pulsed-field gel electrophoresis, of E. coli O157 strains from cattle. Human (n = 22) and bovine (n = 31) strains were genotyped for carriage of the genes for Shiga-like toxin types 1, 2, and 2c; E. coli secreted protein genes espA, espB, and espP; the enterohemolysin gene; eae (intimin); ast (enteroaggregative E. coli stable toxin [EAST]); and genes for common E. coli adhesins. Strains were also phenotyped for hemolysin, EspP, Tir, and EspD expression as well as production of actin and cytoskeletal rearrangement associated with attaching and effacing (A/E) lesions on HeLa cells. The genotyping confirmed that there was little difference between the two groups, including carriage of stx(2) and stx(2c), which was similar in both sets. ast alleles were confirmed to all contain mutations that would prevent EAST expression. espP mutations were found only in cattle strains (5 of 30). Clear differences were observed in the expression of locus of enterocyte effacement (LEE)-encoded factors between strains and in different media. EspD, as an indicator of LEE4 (espA, -B, and -D) expression, and Tir levels in supernatants were measured. Virtually all strains from both sources could produce EspD in Luria-Bertani broth, although at very different levels. Standard trichloroacetic acid precipitation of secreted proteins from tissue culture medium produced detectable levels of EspD from the majority of strains of human origin (15 of 20) compared with only a few (4 of 20) bovine strains (P < 0.001), which is indicative of much higher levels of protein secretion from the human strains. Addition of bovine serum albumin carrier protein before precipitation and enhanced detection techniques confirmed that EspD could be detected after growth in tissue culture medium for all strains, but levels from strains of human origin were on average 90-fold higher than those from strains of bovine origin. In general, levels of secretion also correlated with ability to form A/E lesions on HeLa cells, with only the high-level protein secretors in tissue culture medium exhibiting a localized adherence phenotype. This research shows significant differences between human- and bovine-derived E. coli O157 (stx(+) eae(+)) strains and their production of certain LEE-encoded virulence factors. These data support the recent finding of Kim et al. (J. Kim, J. Nietfeldt, and A. K. Benson, Proc. Natl. Acad. Sci. USA 96:13288-13293, 1999) proposing different E. coli O157 lineages in cattle and humans and extend the differential to the regulation of virulence factors. Potentially only a subset of E. coli O157 isolates (stx(+) eae(+)) in cattle may be capable of causing severe disease in humans.

Cattle water troughs as reservoirs of Escherichia coli O157.

Environmental survival of Escherichia coli O157 may play an important role in the persistence and dissemination of this organism on farms. The survival of culturable and infectious E. coli O157 was studied using microcosms simulating cattle water troughs. Culturable E. coli O157 survived for at least 245 days in the microcosm sediments. Furthermore, E. coli O157 strains surviving more than 6 months in contaminated microcosms were infectious to a group of 10-week-old calves. Fecal excretion of E. coli O157 by these calves persisted for 87 days after challenge. Water trough sediments contaminated with feces from cattle excreting E. coli O157 may serve as a long-term reservoir of this organism on farms and a source of infection for cattle.

ラピブロットＥｓｃｈｅｒｉｃｉａ ｃｏｌｉ Ｏ１５７（栄研）の評価

Rapiblot E. coli O157 is based on the use of anti-E. coli O157 antibody-coated Polymacron, as a high-surface-area immunoadsorbent for rapid capture and subsequent immunoenzyme detection of E. coli O157 antigens extracted from test samples by heating at 100°C for 10 minutes.Atotal of 24 non-E. coli O157 strains and various gram-negative and gram-positive bacteria tested gave negative results.The detection limit of Rapiblot E. coli O157 was 105cfu/ml in assays of E. coli O157 strains in broth suspension prepared by the spread plate method.This highly specific and sensitive selective antibody procedure was 98% effective when tested against IMS using ground beef and radish sprouts.The results obtained by Rapiblot E. coli O157 were the same or better than those of other commercial kits to detect E. coli O157.Rapiblot E. coli O157 was therefore considered effective for the detection of E. coliO157 in food.

Isolation and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep.

In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup 0157. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of E. coli O157 strains that are potentially pathogenic for humans.

Structure and function of plasmid pColD157 of enterohemorrhagic Escherichia coli O157 and its distribution among strains from patients with diarrhea and hemolytic-uremic syndrome.

In this study, pColD157, a 6.7-kb colicinogenic plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL40cu, was characterized by restriction mapping and determination of its complete nucleotide sequence. The sequence consists of 6,675 bp and shows a high degree of similarity to the nucleotide sequence of colicinogenic plasmids pColD-CA23 and pColK. Seven potential genes were located on pColD157, three of which were closely related (>97.9%) to the colicin D structural gene and the corresponding immunity and lysis genes of plasmid pColD-CA23, and these were therefore designated cda, cdi, and cdl, respectively, using the reference extension -CL40 for differentiation. The adjacent 3' region is related to the origin of replication of pColD-CA23. In contrast, the remaining part of the plasmid harbors a cluster of genes, closely related to the mobilization genes of pColK, which is followed by a 0.3-kb stretch homologous to the pColK resolution function. These determinants were designated mbdA, mbdB, mbdC, and mbdD and cdr, respectively. Southern blot analysis was performed with a probe specific for the cda gene of pColD157 and two groups of EHEC O157:H7 isolates from patients with diarrhea or hemolytic-uremic syndrome resident in Germany. Whereas 16 of 46 E. coli O157 strains isolated between 1987 and 1991 harbored plasmid pColD157, only 1 of 50 strains isolated during 1996 carried this plasmid. In addition, all strains harboring plasmid pColD157 were shown to have colicinogenic activity.

Detection and characterization of thefimAgene ofEscherichia coliO157:H7

An expected 850-bp DNA fragment containingfimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains ofEscherichia coliusing the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13HinPI and fourSau96I restriction profiles among these 39E. colistrains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared byE. coliO157:H7, O157:H- and a few O55 serotype strains. DNA sequence analysis of thefimAregion demonstrated thatE. coliO157:H7 strain 933 and O157:H−strain E32511 contained identical DNA sequences that were distinct from otherE. colistrains, especially a 16-bp sequence 5′ tofimAthat was conspicuously absent only inE. coliO157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from allE. coliO157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detectE. colistrains of the O157:H7 serotype.