Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding bacteriophage phi3538 isolated from Escherichia coli O157:H7

Abstract

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx(2) gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage phi3538(Deltastx(2)::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with phi3538(Deltastx(2)::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for phi3538(Deltastx(2)::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of phi3538(Deltastx(2)::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that phi3538(Deltastx(2)::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.

FIG. 1

Construction of a vector for gene replacement mutagenesis of the stx₂ gene. (A) PCR product of the entire stx₂ gene obtained by amplification with primers HSB1 and HSB3. (B) Ligation of the PCR product in vector pUC18. (C) Replacement of an internal EcoRV-HincII fragment of the stx₂ gene with an BsaAI-HincII fragment of pACYC184 covering the cat gene. (D) Ligation of the Δstx₂::cat construct in suicide vector pGP704. Restriction sites are depicted. The numbering in panel A corresponds to the beginning of the PCR product. Hatched bars depict the stx₂A and stx₂B subunit genes. The fragment derived from pACYC184 is noted by a dark box, and the cat gene is noted by a light box.

FIG. 2

Plaque hybridization. Phage lysates prepared from the lysogens E. coli O157:H⁻ strain 1193/89(Δstx₂::cat) (A) and E. coli O157:H7 strain 3574/92(Δstx₂::cat) (B) after mitomycin induction were mixed with E. coli C600 indicator bacteria in soft agar and poured onto LB agar. Plaques were transferred to nylon membranes and hybridized with a digoxigenin-labeled cat gene probe. Plasmid pACYC184 DNA was used as a positive control and is seen in the upper part of the membranes (large dots). stx₁B and stx₂B PCR products, respectively, were used as negative controls on the membranes.

FIG. 3

Agarose gel electrophoresis (A) and Southern blot hybridization (B) of phage DNA restricted with EcoRI. DNA of the original phage φ3538(Δstx₂::cat) (lanes 1) was compared with DNA from phage progeny that has been isolated from the lysogens 1193/89(Δstx₂::cat) (lanes 2), 3574/92(Δstx₂::cat) (lanes 3), 12860(Δstx₂::cat) (lanes 4), 7513/91(Δstx₂::cat) (lanes 5), and 1249/87(Δstx₂::cat) (lanes 6) and processed in E. coli laboratory strain DH5α as described in the text. M, molecular weight standard (1-kb DNA ladder; Gibco BRL, Eggenstein, Germany).