First-time isolation and characterization of a bacteriophage encoding the Shiga toxin 2c variant, which is globally spread in strains of Escherichia coli O157

Abstract

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.

FIG. 1.

Subtyping of stx genes in STEC O157 strains and in C600 transductants. HincII digest of stx-specific PCR products. Lanes: 1, C600 (H19) (stx₁); 2, E32511 (stx₂ and stx_2c); 3, CB2851 (stx_2c); 4, C600 (phage 2851) (stx_2c); 5, C600 phage H19 and phage 2851 (stx₁ and stx_2c); M, DNA Hyperladder II (range, 100 to 1,000 bp; Bioline GmbH, Luckenwalde, Germany).

FIG. 2.

Electron micrograph of CsCl-purified phage 2851 particles. Bar = 100 nm.

FIG. 3.

Restriction patterns and cross-hybridizations of DNAs of phages lambda (Stx negative), H19 (Stx1), 933W (Stx2), and 2851 (Stx2c). Shown are AccI digests of phage DNAs. Lanes 1, lambda; lanes 2, H19; lanes 3, 933W; lanes 4, CB2851. (A) Restriction pattern. (B) Southern hybridization using genomic DNA of lambda cut with AccI as a probe. (C) Southern hybridization using H19 DNA cut with AccI as a probe. (D) Southern hybridization using 933W DNA cut with AccI as a probe. (E) Southern hybridization using 2851 DNA cut with AccI as a probe.

FIG. 4.

Structures of the stx_2c genes and flanking regions of phage 2851 and prophage Nil2. The grey shaded area indicates the overlapping sequenced DNA (identity > 99%). The hatched boxes (no. 1 to 6) represent genetic elements of phage 2851 DNA (Table 1). The black boxes (no. 7 to 9) represent PCR products derived from phage 2851 DNA used for diagnostic PCR (Table 1). t_R, putative terminators involved in antitermination by Q.