Incidence of enteritis of Enterobacteriaceae isolates possessing human colonization factor antigen. New human colonization factors

Of 462 Enterobacteriaceae strains including 435 Escherichia coli isolated from 250 patients, 298 haemagglutinating (HA) cultures were classified into 36 different HA groups. Sixteen of them belonged to Evan's I or II groups, although none possessed CF I or CF II antigen detectable by slide agglutination. Seventy-seven strains showed 4+ mannose resistant (MR) HA with human (53), bovine (2), chicken (6), guinea pig (7) or human and guinea pig (9) erythrocytes. These strains were significantly more frequent in patients under one year of age. Eighty-eight percent of the typable strains belonged to E. coli serogroups O1, O2, O4, O6, O18. HA positivity and fimbrial structures were correlated in 2 isolates (15/1, O18a, c:-K77: H-; 12/2/1 O1: K1: H .). Fimbriae of the two strains exhibited adhesive properties. Their fimbrial antigens differed serologically from each other and from those of the reference strains H 10407 and PB 176. Forty-nine of 4+ human MRHA strains showed variable reactions in the two sera for the new fimbrial antigens.     

Evaluation of colicins for inhibitory activity against diarrheagenic Escherichia coli strains, including serotype O157:H7.

Twenty-four Escherichia coli strains producing standard colicins were evaluated for inhibitory activity against 27 diarrheagenic E. coli strains of serotypes O15:H-, O26:(H11, H-), and O111:(H8, H11, H-), including O157:H7, representing diarrheagenic E. coli clones, 3, 4, 8, 9, and 10. Overlay techniques were used to assess inhibition on Luria agar and Luria agar supplemented with 0.25 micrograms of mitomycin C per ml to induce colicin production. As a group, the A colicins (Col) E1 to E8, K, and N inhibited 23 to 25 (85.2 to 92.6%) of the 27 diarrheagenic strains on mitomycin C-containing agar, whereas the most active group B colicins, Col D and Ia, inhibited 9 and 12 (33.3 and 44.4%), of the diarrheagenic strains, respectively. Col G and H and Mcc B17 inhibited 22 to 27 (81.5 to 100%) of the diarrheagenic strains on Luria agar but were suppressed on mitomycin C-containing agar medium. All O157:H7 strains evaluated were sensitive to Col E1 to E8, K, and N on mitomycin C-containing agar and to Col G and H and Mcc B17 on Luria agar. Sensitivity to colicins of the selected set of diarrheagenic strains was in the order diarrheagenic E. coli clone 9 > 4 > 3 > 10 > 8 and was not restricted to strains of a single clone or serotype. Strain 8C from clone 8 was resistant to most test colicins. There is potential for using colicins in foods and agriculture to inhibit sensitive diarrheagenic E. coli strains, including serotype O157:H7.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Verotoxin-producing Escherichia coli in Spain: prevalence,serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants,raw beef products,and humans

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.     

Comparison of pulsed-field gel electrophoresis patterns of antimicrobial-resistant Escherichia coli and enterococci isolates from the feces of livestock and livestock farmers in Japan

Seven hundred thirty-nine animal strains and 662 livestock-farmer strains, consisting of Escherichia coli and enterococci, were examined for their pulsed-field gel electrophoresis (PFGE) and antimicrobial-resistance patterns. Two hundred fifty-eight and 203 PFGE patterns were found among 739 animal strains isolated from animals comprising broilers, pigs and cattle, and 662 human strains isolated from livestock farmers, respectively, from 27 farms in Japan. These results demonstrated that the PFGE patterns found among E. coli and enterococci strains from animals and livestock-farmers were heterogeneous and considerably diverse. The strains having both the identical PFGE pattern and the same drug-resistance pattern were defined as a single clone in this study. Seven types of E. coli and enterococci clones were shared among animals within the same farms and between the different farms housing the same animal species. The 25 strains (3.4%) of 739 E. coli and enterococci animal strains belonged to these seven types of clones. Only three types of E. coli clones were shared among animals between the different farms housing different animal species, but no identical E. faecalis or E. faecium clones were found between different animal species farms. The 15 strains (2.0%) of 739 E. coli and enterococci animal strains belonged to these three types of clones. Additionally, the 11 strains (1.5%) of 739 E. coli and enterococci strains isolated from animals were identical clones to strains isolated from livestock farmers of the same farm. These results suggest that the transmission of animal clones to livestock farmers or vice versa is less common.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Attaching and effacing lesions in vivo and adhesion to tissue culture cells of Vero-cytotoxin-producing Escherichia coli belonging to serogroups O5 and O103

Certain isolates of Escherichia coli from humans and animals with enteric disease attach to enterocytes and cause 'attaching and effacing' (AE) lesions. E. coli strain S22-1, serotype O103:H2, isolated from a child with diarrhoea, contained two plasmids; one of these (pDEP12) hybridized with the CVD419 DNA probe derived from a plasmid found in E. coli O157:H7 and associated with expression of fimbriae and ability to adhere to Intestine 407 cells. Strain S102-9, serotype O5:H-, isolated from a calf with dysentery, contained six plasmids, one of which also hybridized with the CVD419 probe. Loss of pDEP12 coincided with reduced adhesion to HEp-2 or Intestine 407 cells cultured in vitro; reintroduction of this plasmid restored adhesiveness. Loss of the plasmid in strain S102-9 that hybridized with the CVD419 probe did not cause a decrease in adhesion. Accumulations of actin were seen in vitro in the fluorescence actin staining (FAS) test of strains S22-1, S102-9 and their derivatives, irrespective of the plasmid content of these strains or the prevalence of attached bacteria. Strain S22-1 and its plasmidless derivative caused AE lesions of equal severity in experimentally infected gnotobiotic piglets; piglets inoculated with an isolate from a healthy human or pig did not develop these lesions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transduction of porcine enteropathogenic Escherichia coli with a derivative of a shiga toxin 2-encoding bacteriophage in a porcine ligated ileal loop system

In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages Phi3538 (Deltastx(2)::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) with Phi3538 (Deltastx(2)::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions.     

A comparison of Shiga-toxin 2 bacteriophage from classical enterohemorrhagic Escherichia coli serotypes and the German E. coli O104:H4 outbreak strain

Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors.     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

Properties of E. coli of serological group 06:K13(L) isolated in acute intestinal diseases]

A study of 88 E. coli strains belonging to serological group 06: K13 (L) isolated from 50 children and 5 adults suffering from acute intestinal disturbances permitted to refer them to the serological types 06: K13 (L):H1 (82 strains) and 06: K13 (L): H--(6 strains), and to 5 biochemical types. Repeated isolation of E. coli belonging to serlogical group 06: K13 (L) in patients with acute intestinal diseases, and their absence in healthy children and adults indicated these microbes to be a possible etiological factor of the disease and to refer to the group group of enteropathogenic ones. Wide introduction of agglutinating sera 06: K13(L) into the work of practical laboratories is of expedience in connection with this.     

Vero cell toxins in Escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs

Sixty-eight of 519 strains of Escherichia coli and six of 10 strains of Pseudomonas aeruginosa produced toxins acting on Vero cells (VT+); all of 63 Salmonella, Shigella, Klebsiella, Enterobacter and Proteus strains were VT-. Most of the VT+ E. coli strains were from weaned pigs suffering from oedema disease and/or diarrhoea and belonged to serogroups O141:K85,88, O141:K85, O138:K81, and O139:K82; six VT+ E. coli strains were from diarrhoeic human babies, four of serogroup O26 and two of serogroup O128. The VT genes in two of the O26 strains and in the O128 strains were located in the genome of the phages with which they were lysogenized. One O141:K85,88 pig E. coli strain transferred its VT genes, probably by conjugation, to E. coli K12. The VTs of the human E. coli strains, the pig E. coli strains and the P. aeruginosa strains were antigenically different from each other; unlike the others, the P. aeruginosa VT was heat-resistant. Cell-free preparations of cultures of E. coli K12 to which the VT genes of the four human E. coli strains had been transferred caused fluid accumulation in ligated segments of rabbit intestine. Inoculated intravenously, they were lethal for mice and rabbits; similar preparations of E. coli K12 to which the VT genes of the pig E. coli strain had been transferred produced a disease in pigs that clinically and pathologically resembled oedema disease.     

The effect of volatile fatty acids in acidified fermented piggery effluent on shiga-toxigenic and non-toxic resident strains of Escherichia coli

AIMS: To examine the effects of acidified acidogenically fermented piggery effluent containing Volatile Fatty Acids (VFA) on shiga-toxigenic and resident strains of Escherichia coli (E. coli) as part of the development of a waste treatment process. METHODS AND RESULTS: Four shiga-toxigenic E. coli strains (O157:H7, 091.H-, 0111.H-, and 0123.H-) and four non-toxic resident enzootic strains were all killed by 3 h treatment with fermented piggery effluent liquor (153 mmol l(-1) total VFA) at pH 4.3. The shiga-toxigenic strains showed greater sensitivity after 1 h of treatment. The fermented liquor at pH 6.8 was not inhibitory. CONCLUSIONS: The shiga-toxigenic strains were no more resistant to the toxic effects of VFA than the non-toxic strains tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga-toxigenic strains and resident enzootic non-toxigenic strains are equally susceptible to inactivation by this waste treatment process and by acidified VFA in general.     

Occurrence of type-1C fimbriae on Escherichia coli strains isolated from human extraintestinal infections

Two monoclonal antibodies specific for type-1C fimbriae of Escherichia coli were produced. In enzyme-linked immunosorbent assay and immunoblotting the antibodies, which were of the IgG1 isotype, reacted with type-1C, but not with P or type-1 fimbriae of E. coli strain KS71. Immunoblotting and immunoprecipitation of crude fimbrial extracts from 25 strains invariably gave an apparent molecular weight of 17 000 for the type-1C fimbrillin. A total of 313 E. coli strains, isolated from patients with extraintestinal infection or from faeces of healthy children, were screened for the presence of type-1C fimbriae using both the monoclonal and polyclonal antibodies. Of these, 45 (14%) strains had type-1C fimbriae, with the highest frequency (27%) on strains isolated from patients with pyelonephritis. No faecal strain had type-1C fimbriae, and the frequency on the other diagnostic groups ranged from 11 to 15%. Thus, no direct correlation between type-1C fimbriae and bacterial virulence in human extraintestinal infections was found. Type-1C fimbriae were detected on only a few E. coli serotypes, notably on all O6:K2:H1 and O22:K13:H1 strains tested.     

Phylogenetic relationships among clonal groups of extraintestinal pathogenic Escherichia coli as assessed by multi-locus sequence analysis

The evolutionary origins of extraintestinal pathogenic Escherichia coli (ExPEC) remain uncertain despite these organisms' relevance to human disease. A valid understanding of ExPEC phylogeny is needed as a framework against which the observed distribution of virulence factors and clinical associations can be analyzed. Accordingly, phylogenetic relationships were defined by multi-locus sequence analysis among 44 representatives of selected ExPEC clonal groups and the E. coli Reference (ECOR) collection. Recombination, which significantly obscured the phylogenetic signal for several strains, was dealt with by excluding strains or specific sequences. Conflicting overall phylogenies, and internal phylogenies for virulence-associated phylogenetic group B2, were inferred depending on the specific dataset (i.e., how extensively purged of recombination), outgroup (Salmonella enterica and/or Escherichia fergusonii), and analysis method (neighbor joining, maximum parsimony, maximum likelihood, or Bayesian likelihood). Nonetheless, the major E. coli phylogenetic groups A, B1, and B2 were consistently well resolved, as was a major sub-component of group D and an ECOR 37-O157:H7 clade. Moreover, nine important ExPEC clonal groups within groups B2 and D, characterized by serotypes O6:K2:H1, O18:K1:H7, O6:H31, and O4:K+:H+ (from group B2), and O1:K1:H-, O7:K1:H-, O157:K+:H (non-7), O15:K52:H1, and O11/17/77:K52:H18 ("clonal group A") (from group D), were consistently well resolved, regardless of clinical background (cystitis, pyelonephritis, neonatal meningitis, sepsis, or fecal), host group, geographical origin, and virulence profile. Among the group B2-derived clonal groups the O6:K2:H1 clade appeared basal. Within group D, "clonal group A" and the O15:K52:H1 clonal group were consistently placed with ECOR 47 and ECOR 44, respectively, as nearest neighbors. These findings clarify phylogenetic relationships among key ExPEC clonal groups but also emphasize that recombination appears to obscure the oldest evolutionary relationships, despite extensive targeted sequencing and use of a wide range of analysis techniques.     

Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations,excisions, truncations,and evolutionary implications

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.     

Screening of Environmental DNA Libraries for the Presence of Genes Conferring Na+(Li+)/H+ Antiporter Activity on Escherichia coli: Characterization of the Recovered Genes and the Corresponding Gene Products

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na(+)(Li(+))/H(+) antiporter activity on the antiporter-deficient Escherichia coli strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive E. coli clones were obtained during the initial screening of 1,480,000 recombinant E. coli strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li(+)-resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na(+)/H(+) antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of E. coli K-12 containing nhaA, nhaR, and gef. This region is flanked by highly conserved insertion elements. The sequence identity with E. coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E. coli. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E. coli and enabled the antiporter-deficient E. coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na(+)/H(+) antiporter activity in everted membrane vesicles that were derived from the E. coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The K(m) values of both antiporters for Na(+) were approximately 10-fold higher than the values for Li(+).     

Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.     

Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.