Characterization of Escherichia coli serotype O157:H7.

A total of 174 strains of Escherichia coli serotype O157:H7 representing human isolates obtained from outbreaks and sporadic cases of hemorrhagic colitis, hemolytic-uremic syndrome, and nonbloody diarrheal illnesses as well as from asymptomatic carriers across Canada and the United States were examined. E. coli serotype O157:H7 possessed distinct biochemical markers, a 100% negative reaction for beta-glucuronidase and sorbitol, and a 100% positive reaction for raffinose and dulcitol; all strains otherwise were biochemically typical of E. coli. The vast majority (97%) of the strains were susceptible to commonly used antimicrobial agents. All strains produced readily detectable levels of Verotoxin; however, with polymyxin extraction, nearly 50% of the strains showed up to a 10-fold increase in the toxin level. None were found to mediate hemagglutination of human group A erythrocytes with or without D-mannose. The majority (approximately 70%) of the strains showed localized and diffuse adherence to HEp-2 cells and Henle 407 cells, and the adherence patterns were not very different from those observed among other E. coli strains. Twenty phage types were recognized, with phage types 1 and 2 accounting for 65% of the test strains. Plasmid analysis indicated three basic plasmid profiles: profile I was characterized by 68.7- and 4.2-megadalton (MDa) plasmids (62% of strains), profile II was characterized by 66.2- and 1.8-MDa plasmids (20% of strains), and profile III was characterized by a 62.5-MDa plasmid (18% of strains). A small number (19%) of the strains carried at least one additional plasmid over the basic complements, and these could be considered to constitute a miscellaneous category. None of the above-described characteristics of E. coli serotype O157:H7 could be directly correlated with one another, with the nature of infection, or with the geographical distribution of strains.     

Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya.

A total of 26% of the pneumococci isolated from an outpatient clinic in Nairobi, Kenya, during 1991 to 1992 had intermediate levels of penicillin resistance. Gene fingerprinting and DNA sequencing were used to distinguish the penicillin-binding protein (PBP) 1A, 2B, and 2X genes in 23 resistant isolates. Isolates were grouped into those that had identical forms of each of the three PBP genes (fingerprint groups) and those that had identical rRNA gene restriction patterns (ribotypes). Both methods divided the isolates into 11 groups. In a few cases, horizontal gene transfer appeared to have distributed an identical altered PBP gene into different pneumococcal lineages. Eight isolates were indistinguishable by ribotyping or multilocus enzyme electrophoresis and contained identical PBP 1A genes. Although these isolates were therefore members of the same clone, they were divided into two fingerprint groups which contained different PBP 2X and 2B genes. Presumably, members of this clone have acquired different altered PBP 2X and 2B genes on two separate occasions. One of these fingerprint groups contained isolates of serotype 14, whereas the other contained isolates of both serotypes 14 and 7. The identification of isolates in the latter group that are identical by all criteria, except serotype, implies the occurrence of a change in serotype. The predominant serotypes of the penicillin-resistant pneumococci from Nairobi were serotypes 14 and 19. In both cases, isolates of the same serotype which required the same MIC of penicillin were not members of a single clone, indicating that identity of serotype and MIC are not sufficient criteria for defining clones of resistant pneumococci even when the bacteria are isolated from a single clinic.     

Characterization of flagella purified from enterohemorrhagic, vero-cytotoxin-producing Escherichia coli serotype O157:H7.

Escherichia coli of the serotype O157:H7 has recently been isolated in human fecal specimens in association with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome. The aim of this study was to characterize the flagellin protein subunit constituents of flagellar filaments from E. coli O157:H7 strain CL-56. Flagellin isolated from a reference Salmonella enteritidis strain was used for comparison. Flagella were dissociated by incubation of bacteria under acidic conditions, centrifugation, and differential ammonium sulfate precipitation. Reconstituted flagellar filaments were demonstrated by three complementary methods: transmission electron microscopy, antigenic reactivity with H7 antiserum by a dot blot immunoassay, and immunogold localization of antiserum raised to the purified antigen to intact flagella on whole E. coli O157:H7. On sodium dodecyl sulfate-polyacrylamide gels flagellin proteins from E. coli O157:H7 demonstrated an apparent Mr of 66,000. The isoelectric point of E. coli O157:H7 flagellin was 5.42. By immunoblotting, H7 flagellin proteins were shown to be immunogenic. They induced a systemic immune response both in rabbits challenged with whole bacteria and in a human previously infected with E. coli O157:H7.     

Invasive strains of Escherichia coli belonging to serotype O121:NM.

Ten strains of Escherichia coli isolated from 10 travellers with sporadic diarrhea who were returning to Tokyo, Japan, from abroad were found to be of serotype O121:NM and were positive in the Serény test for invasiveness; this suggests that this serotype can cause a shigellosis-like illness in humans. The E. coli O121:NM strains were positive in the cell invasion test in HeLa cells. Analysis of the plasmid content of these strains showed that they contained a high-molecular-mass plasmid of 120 to 140 MDa which has been associated with invasiveness and were positive in an enzyme-linked immunosorbent assay for detection of the virulence plasmid-encoded proteins of Shigella spp. and enteroinvasive E. coli.     

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.     

Clonal analysis of Escherichia coli serotype O6 strains from urinary tract infections.

A total of 36 Escherichia coli urinary tract isolates (UTI) of serotype O6, with different combinations of capsule (K) and flagellin (H) antigens, were analysed according to the outer membrane pattern (OMP), serum resistance properties, mannose-resistant hemagglutination using various types of erythrocytes, and also for the genetic presence and the expression of P-fimbriae, S fimbriae/F1C fimbriae, Type 1 fimbriae, aerobactin and hemolysin. Twenty selected strains were further analysed by pulsed field gel electrophoresis (PFGE), elaborating genomic profiles by XbaI cleavage and subsequent Southern hybridization to virulence-associated DNA probes. It could be shown that O6 UTI isolates represent a highly heterogeneous group of strains according to the occurrence and combination of these traits. Relatedness on the genetic and the phenotypic level was found for some of the strains exhibiting the same O:K:H:F serotype. DNA long-range mapping further indicated some interesting features, according to the copy number and the genomic linkage of virulence genes.     

Organization of aerobactin, hemolysin, and antibacterial resistance genes in lactose-negative Escherichia coli strains of serotype O4 isolated from children with diarrhea.

Epidemiologically related, non-lactose-fermenting (NLF) Escherichia coli strains of serotype O4 have been isolated at a high frequency from children with diarrhea in Somalia (M. Nicoletti, F. Superti, C. Conti, A. Calconi, and C. Zagaglia, J. Clin. Microbiol. 26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF O4 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FI (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160- to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40- to 140-kb IncFII plasmid sharing homology with the RepFIIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and closely resembles that of the reference hemolytic plasmid pHly152; and (v) Hly- mutants obtained by transposon insertion mutagenesis are not cytotoxic to HeLa cell monolayers, indicating that hemolysin is responsible for the high cytotoxic activity we have previously reported for these strains. The structural organization of the plasmid-encoded aer operon, together with the finding that those plasmids also carry antibiotic resistance genes, indicates that the IncFI plasmid of the NLF O4 E. coli strains studied more closely resembles aer-encoding virulence IncFI Salmonella R plasmids than E. coli ColV plasmids. The data presented here cannot rule out whether the strains examined are potentially intestinal or extraintestinal pathogens. Nevertheless, the genetic organization of the virulence genes, together with the epidemiological behavior and the wide spectrum of antibiotic resistance of the NLF O4 E. coli strains, indicates that these strains are structured as typical E. coli pathogenic isolates of human origin.     

Molecular characterisation of verocytotoxin-producing Escherichia coli of serogroup O111 from different countries.

A collection of epidemiologically unrelated verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O111 isolated from human patients and cattle with diarrhoeal disease in five different countries were characterised by determination of their VT genotypes, the presence of other virulence factors such as the intimin-coding eae gene and the enterohaemorrhagic E. coli (EHEC) plasmid, and their antibiotic susceptibility patterns. The genetic relatedness among isolates was evaluated by genomic DNA fingerprinting techniques such as restriction fragment length polymorphism analysis of ribosomal RNA genes (ribotyping) and pulsed-field gel electrophoresis. The results indicated that the VTEC O111 examined belong to two distinct clonal lineages. The first group was constituted mainly of non-motile, eae-positive, EHEC plasmid-positive isolates from both man and cattle. The second lineage was represented by an O111:H2 epidemic strain, isolated during an outbreak of haemolytic uraemic syndrome in France and exhibiting an unusual combination of virulence factors: VT production and aggregative adhesion to HEp-2 cells associated with an enteroaggregative E. coli (EAEC) plasmid.     

Enteropathogenic Escherichia coli diarrhea in hospitalized children in Bangladesh.

The role of enteropathogenic Escherichia coli (EPEC) was evaluated in a group of children with endemic diarrhea admitted to Dhaka Shishu Hospital in Dacca, Bangladesh. EPEC was detected in fecal samples of 23% of 104 cases and 8% of 74 concurrent control children. The most commonly isolated EPEC strains were serogroups O20a, O20c:K61; O20a, O20b:K84; O26:K60; and O18a, O18c:K77. Except for O26:K60, these groups had not been reported from Bangladesh. On testing for enterotoxin production, only two strains (serogroups O26:K60, O18a, and O18c:K77) were enterotoxigenic. None was enteroinvasive as tested in the guinea pig conjunctivitis model. Our study supports the concept that EPEC may be an important cause of endemic diarrhea in Bangladesh.     

Inhibition of Escherichia coli serotype O157:H7 by bromthymol blue.

Bromthymol blue, at a concentration of 0.1% in tryptose-glucose broth, inhibited growth of 98.4% of Escherichia coli serotype O157:H7 isolates but only 0.8% of E. coli non-O157:H7 isolates after an overnight incubation at 44.5 degrees C, but not 35 degrees C. The inhibition was dependent on temperature, density of inoculum, bromthymol blue concentration, time of incubation, and composition of the medium. Compared with serologic typing, the inhibition had sensitivity, specificity, predictive values of the positive and negative tests, and overall agreement between the two tests of 98.4, 99.2, 98.4, 99.2, and 98.9%, respectively. The inhibition could be useful as a presumptive test to identify E. coli isolates of serotype O157:H7, especially in laboratories that do not have serotyping capabilities.     

Latex agglutination test for detection of Escherichia coli serotype O157.

The value of a latex agglutination test (Escherichia coli O157 latex test; Oxoid Ltd.) for rapid presumptive detection of E. coli serotype O157:H7 was determined by laboratory trials and during an outbreak of hemorrhagic colitis. The latex test was found to be a simple, highly efficient and reliable test in detecting E. coli O157:H7 with 100% sensitivity and specificity. It was also found that sorbitol-MacConkey agar cultures were not as useful for food samples as they were for fecal specimens in screening for E. coli O157:H7, but the use of the latex screen was particularly efficient in this setting.     

Biotype, serotype, and pathogenicity of attaching and effacing enteropathogenic Escherichia coli strains isolated from diarrheic commercial rabbits.

A total of 568 strains of Escherichia coli isolated from healthy and diarrheic rabbits were separated into 11 different biotypes according to the fermentation patterns of four carbohydrates. Strains belonging to biotypes 1 to 3, 6, and 8 induced lesions characteristic for attaching and effacing E. coli (AEEC). They attached to the intestinal epithelium of the terminal small intestine and the large intestine of 5-week-old rabbits after experimental infection and caused effacement of the microvillous brush border. However, pathogenicity for weaned rabbits, as judged by diarrhea score, anorexia, and reduced weight gain, varied according to the biotypes of the strains. Strains belonging to biotypes 1 and 6 produced only discrete clinical signs, strains belonging to biotypes 2 and 3+ (motile) induced diarrhea and growth depression, whereas strains belonging to biotypes 3- (immotile) and 8 caused severe clinical signs and high mortality. This confirms evidence from the field. Biotypes 3- and 8, accounting for 35.5 and 7.1% of AEEC strains in weaned diarrheic rabbits, respectively, were not detected in weaned healthy rabbits, while biotype 2 was the predominant strain in weaned healthy rabbits (62.3%). Finally, serotyping showed a close relationship between biotype and serotype of the AEEC examined. Most strains of biotypes 1+ and 2+ tested were O109:K-:H2 and O132:K-:H2, respectively, whereas all strains tested of biotype 3- were O15:K-:H- and those of biotype 8 were O103:K-:H2. These data indicate that specific clones of AEEC might be involved in juvenile rabbit enteritis. It was concluded that determination of biotypes allows the screening of highly pathogenic AEEC in weaned rabbits (biotypes 3- and 8).     

Catalase-negative Escherichia coli isolated from blood.

A catalase-negative variant of Escherichia coli was isolated from the blood of a patient with acute leukemia who had been treated with various antibiotics and gentamicin. This small-colony variant grew almost as actively under anaerobic conditions as its large-colony revertant or E. coli NIHJ JC-2. The variant was resistant to gentamicin, in contrast with the revertant. Streptomycin and hemin stimulated growth of the variant slightly. With repeated subculturing the variant tended to increase slightly in colony size with coincident recovery of weak catalase production. The possibility that such a variant may have been induced by gentamicin was indicated.     

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.     

O serogroups, biotypes, and eae genes in Escherichia coli strains isolated from diarrheic and healthy rabbits.

A total of 305 Escherichia coli strains isolated from diarrheic and healthy rabbits in 10 industrial fattening farms from different areas of Spain were serotyped, biotyped, and tested for the presence of the eae gene and toxin production. The characteristics found in strains isolated from healthy rabbits were generally different from those observed in E. coli strains associated with disease. Thus, strains with the eae gene (74% versus 22%); strains belonging to serogroups O26, O49, O92, O103, and O128 (64% versus 12%); rhamnose-negative strains (51% versus 5%); and rhamnose-negative O103 strains with eae genes present (41% versus 1%) were significantly (P < 0.001 in all cases) more frequently detected in isolates from diarrheic animals than in those from healthy rabbits. Whereas a total of 35 serogroups and 17 biotypes were distinguished, the majority of the strains obtained from diarrheic rabbits belonged to only four serobiotypes, which in order of frequency were O103:B14 (72 strains), O103:B6 (16 strains), O26:B13 (12 strains), and O128:B30 (12 strains). These four serobiotypes accounted for 48% (112 of 231) and 5% (4 of 74) of the E. coli strains isolated from diarrheic and healthy rabbits, respectively. Only six strains were toxigenic (three CNF1+, two CNF2+, and one VT1+). We conclude that enteropathogenic E. coli strains that possess the eae gene are a common cause of diarrhea in Spanish rabbit farms and that the rhamnose-negative highly pathogenic strains of serotype O103:K-:H2 and biotype B14 are especially predominant. Detection of the eae gene is a useful method for the identification of enteropathogenic E. coli strains from rabbits. However, a combination of serogrouping and biotyping may be sufficient to accurately identify the highly pathogenic strains for rabbits.     

Characterization of Enterohemorrhagic Escherichia coli O111 and O157 Strains Isolated from Outbreak Patients in Japan

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx₂ and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx₂ isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx₁, stx₂, and stx₁ stx₂), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx₂ isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx₂ to an stx-negative strain may have occurred during infection.     

Heat-labile enterotoxin produced by Escherichia coli serogroup O149 isolated from diarrheic calves.

Nine strains of Escherichia coli serogroup O149 isolated from diarrheic calves were found to produce a heat-labile enterotoxin. The heat-labile enterotoxin activity was destroyed by heat (85 degrees C/30 min) and was neutralized by rabbit anti-heat-labile enterotoxin. One of the strains also produced a heat-stable enterotoxin.