We determined the nucleotide sequence of the Shiga-like toxin-1 (SLT-1) genes carried by the toxinconverting bacteriophage H-19B. Two open reading frames were identified; these were separated by 12 base pairs and encoded proteins of 315 (A subunit) and 89 (B subunit) amino acids. The predicted protein subunits had N-terminal hydrophobic signal sequences of 22 and 20 amino acids, respectively. The predicted amino acid sequence of the B subunit was identical to that of the B subunit of Shiga toxin. The A chain of ricin was found to be significantly related to the predicted Al fragment of the SLT-1 A subunit. SI nuclease protection experiments showed that the two cistrons formed a single transcriptional unit, with the A subunit being proximal to the promoter. A probable promoter was identified by primer extension, and transcription was found to increase dramatically under conditions of iron starvation. A 21-base-pair sequence with dyad symmetry was found in the region of the SLT-1 -10 sequence, which was found to be 68% homologous to a region of dyad symmetry found in the -35 region of the promoter of the iucA gene op plasmid ColV-K30, which specifies the 74,000-dalton ferric-aerobactin receptor protein. Shigella dysenteriae 1 produces a toxin which is cytotoxic to eucaryotic cell lines (8). Binding to the glycolipid globotriaosylceramide (Gb3) membrane receptor is mediated by a pentamer of 7-kilodalton (kDa) B subunits, while the 31-kDa A subunit, after proteolytic nicking and reduction, inhibits protein synthesis by catalytic inactivation of the 60S ribosomal subunit (8,20,34,35). O'Brien and LaVeck (30,31) have shown that some Escherichia coli strains produce large amounts of a cytotoxin which appeared very similar to Shiga toxin with respect to its subunit structure and mechanism of action. It was completely neutralized by antiserum raised against Shiga toxin and was named Shiga-like toxin 1 (SLT-1) (28, 42). Recently, Strockbine et al. (42) have characterized a second cytotoxin, also produced by E. coli, which is related to SLT-1 at the DNA sequence level but is not neutralized by antiserum raised against Shiga toxin or SLT-

No abstract

Some strains of Escherichia coli produce a protein which is cytotoxic for Vero cell and HeLa cell monolayers. This toxin is very similar to the toxin of Shigella dysenteriae 1 and has been named verotoxin or E. coli Shiga-like toxin. It has been shown that toxin conversion is due to a group of bacteriophages, one of which has been designated H-19B. In this study we report hybridization experiments showing that part of the H-19B genome is homologous to phage lambda. We have cloned a 1.7-kilobase BalI-BglII fragment from the genome of H-19B into pUC18. The recombinant plasmid confers the ability to produce high levels of Shiga-like toxin on transformed E. coli cells. We demonstrate using an in vitro transcription/translation system that the cloned fragment specifies the two verotoxin subunit peptides which have masses of 31 and 5.5 kilodaltons. The identity of peptides was confirmed by immunoprecipitation with verotoxin antiserum and protein A-Sepharose beads.

Agar dilution antimicrobial susceptibility testing of Camphylobacter jejuni showed that erythromycin, clindamycin, nitrofurantoin, and gentamicin were the most active compounds, inhibiting 90% of the isolates at a concentration of 1 ,tg/ ml or less. The frequency of high-level erythromycin resistance was 1%. Erythromycin-resistant isolates showed cross-resistance to clindamycin. All strains were inhibited by chloramphenicol at -8,ig/ml. About 20% of the isolates were resistant to tetracycline at 4 ,ug/ml. All strains were highly resistant to novobiocin, bacitracin, vancomycin, and trimethoprim and resistant to rifampin. The minrimal inhibitory concentrations (MICs) of metronidazole ranged from _0.5 to 128 ,g/ ml. The susceptibility of strains to sulfamethoxazole and polymyxin B sulfate was markedly influenced by inoculum size. The MICs of polymyxin B sulfate were significantly higher at 42 than 36°C. All strains were inhibited by nalidixic acid at 32 ,Ag/ml. In the penicillin group, ampicillin was the most active compound, inhibiting only about three-quarters of the strains at 8 ,ug/ml. The cephalosporins as a group showed only moderate to poor activity, the most active cephalosporin being cefotaxime, which inhibited about 90% of the strains at 8 ,ug/ml. The use of antibiotics in selective media is discussed.

We and others have noted that there are serological differences between verotoxin 2 (VT2) (also known as Shiga-like toxin II) produced by Escherichia coli C600(933W) and the VT2 variant (VT2v) produced by strain E32511. Recent reports have described nucleotide sequence differences between the VT2v B subunit cistron of E32511 and B2F1 and that of VT2. We have confirmed the sequence differences and have used them to design oligonucleotide probes which differentiate the B subunit cistron of VT2v from that of VT2. Isolates of VT-producing E. coli obtained from human as well as food and veterinary sources were classified according to the toxin phenotype by using a toxin neutralization assay with VT2-specific monoclonal antibody and VT2v-specific polyclonal antisera. Using the oligonucleotide probes in colony hybridization, we detected 35 of 35 VT2 producers and 16 of 16 VT2v producers. One VT2 producer was falsely identified as containing the VT2v gene. The E32511 strain in our collection hybridized only with the VT2-specific probe. Southern hybridization of radiolabeled oligonucleotide probes showed that strains carried zero to one copy of the VT2 gene and zero to two copies of the VT2v gene. We conclude that colony hybridization with the VT2-and VT2v-specific probes is highly predictive of the toxin phenotypes in the clinical isolates described in this study.

Agar dilution antimicrobial susceptibility testing showed that Campylobacter jejuni was significantly more resistant than Campylobacter fetus subsp. fetus (intestinalis) to cephalosporin C, cephaloridine, cephalothin, cefazolin, and cefamandole. No species differences in susceptibility were noted with cephalexin, cefotaxime, and cefoxitin. Rapid species differentiation on the basis of an antibiogram could be achieved with the disk diffusion method. C jejuni failed to produce a zone of inhibition around a 30-microgram cephalothin disk but produced a significant zone around a 30-microgram nalidixic acid disk. C. fetus subsp. fetus (intestinalis) produced exactly the reverse pattern.

This study assessed the diversity of the enterohemorrhagicEscherichia coli (EHEC) hemolysin gene (ehxA) in a variety of Shiga toxin-producing E. coli (STEC) serotypes and the relationship between ehxA types and virulence markers on the locus for enterocyte effacement (LEE). Restriction fragment length polymorphism of the ehxA gene and flanking sequences and of the E. coli attaching and effacing (eae) gene was determined for 79 EHEC hemolysin-positive STEC isolates of 37 serotypes. Two main groups of EHEC hemolysin sequences and associated plasmids, which corresponded to the eae-positive and the eae-negative isolates, were delineated. Comparisons of the ehxA gene sequences of representative isolates of each group showed that this gene and the rest of the EHEC hemolysin operon are highly conserved. Digestion of anehxA PCR product with the restriction endonucleaseTaqI showed a unique restriction pattern foreae-negative isolates and another one for isolates of serotypes O157:H7 and O157:NM. A conserved fragment of 5.6 kb with four potential open reading frames was identified on the EHEC hemolysin plasmid of eae-positive STEC. Phylogenetic analysis of a subset of 27 STEC isolates, one enteropathogenic E. coliisolate, and a K-12 reference isolate showed thateae-positive STEC isolates all belong to a single evolutionary lineage and that the EHEC hemolysin plasmid and theehxA gene evolved within this lineage without recent horizontal transfer. However, the eae gene and the LEE appear to have been transferred horizontally within this STEC lineage on several occasions. The reasons for the lack of transfer or maintenance of the LEE in other STEC lineages are not clear and require further study.

Escherickhia coli H.I.8, an 0128 infant diarrhea isolate, produces low titers of a unique Shiga-like toxin (SLT), called SLT-IIva, which is a variant of SLT-II. We investigated induction of toxin synthesis and the putative association of a bacteriophage with toxin synthesis. Induction of broth cultures of strain H.I.8 with mitomycin yielded a 3,000-fold increase in SLT-IIva, production of a colicin, and appearance of a bacteriophage. Southern hybridization demonstrated that the genes for SLT-IIva were not carried by the bacteriophage.