Bacteriophage-encoding cytolethal distending toxin type V gene induced from nonclinical Escherichia coli isolates

Abstract

Cytolethal distending toxin (Cdt) is produced by a variety of pathogenic bacteria, including pathogenic serotypes of Shiga toxin-producing Escherichia coli (STEC). The Cdt family comprises five variants (Cdt-I to Cdt-V) encoded by three genes located within the chromosome or plasmids or, in the case of Cdt-I, within bacteriophages. In this study, we evaluated the occurrence of the cdt gene in a collection of 140 environmental STEC isolates. cdt was detected in 12.1% of strains, of which five strains carried inducible bacteriophages containing the Cdt-V variant. Two Cdt-V phages of the Siphoviridae morphology lysogenized Shigella sonnei, generating two lysogens: a single Cdt phage lysogen and a double lysogen, containing a Cdt phage and an Stx phage, both from the wild-type strain. The rates of induction of Cdt phages were evaluated by quantitative PCR, and spontaneous induction of Cdt-V phage was observed, whereas induction of Stx phage in the double lysogen was mitomycin C dependent. The Cdt distending effect was observed in HeLa cells inoculated with the supernatant of the Cdt-V phage lysogen. A ClaI fragment containing the cdt-V gene of one phage was cloned, and sequencing confirmed the presence of Cdt-V, as well as a fragment downstream from the cdt homolog to gpA, encoding a replication protein of bacteriophage P2. Evaluation of Cdt-V phages in nonclinical water samples showed densities of 10(2) to 10(9) gene copies in 100 ml, suggesting the high prevalence of Cdt phages in nonclinical environments.

Fig. 1.

Colony hybridization showing the cdt-positive strains (47, 62, 78, 115, and 125) and the Cdt lysogens S(Φ62) and S(Φ125) obtained using S. sonnei as the host strain (A) and plaque blot hybridization with the cdtB-DIG probe of phages spotted onto the S. sonnei host strain (B).

Fig. 2.

Micrographs of Cdt phages and Stx phages induced from lysogens. (A) Siphoviridae morphological type of bacteriophages induced from lysogens S(Φ62) and S(Φ125). (B) Podoviridae morphological type of bacteriophages induced from lysogen S(Φ62). Bar, 100 nm.

Fig. 3.

Densities of Cdt phages (log₁₀ GC) and Stx phages (log₁₀ GC) with or without mitomycin C induction from wild-type strains 62 and 125 and lysogens S(Φ62) and S(Φ125), as evaluated by real-time qPCR.

Fig. 4.

Southern blot analysis for detection of cdt fragment in an XmnI-restricted bacterial DNA from S. sonnei 866, lysogens S(Φ62) and S(Φ125) (A); in ClaI- and EcoRI-restricted phage DNA obtained from lysogen S(Φ125) (B); and genetic organization of the 3,211-bp ClaI fragment of phage Φ125 (C).

Fig. 5.

Distending effect of sterile culture filtrates from lysogen S(Φ125) on HeLa cells after 96 h of incubation. (A and C) Lysogen S(Φ125) with and without mitomycin C induction, respectively. (B) S. sonnei strain 866. (D) HeLa cells incubated for 96 h. Bar, 200 μm.

Fig. 6.

The left chart shows the densities of cdt genes (log₁₀ GC/100 ml) in phage DNA isolated from 16 urban sewage samples and 15 wastewater samples from cattle. The left table summarizes the percentages of positive samples, geometric means, and maximum and minimum values of cdt-V (GC/100 ml) in phage DNA isolated from the human and cattle samples. The right chart and table show the densities of cdt genes (log₁₀ GC/100 ml) in bacterial DNA isolated from 18 urban sewage samples.