Stability and Infectivity of Cytolethal Distending Toxin Type V Gene-Carrying Bacteriophages in a Water Mesocosm and under Different Inactivation Conditions

Abstract

Two cytolethal distending toxin (Cdt) type V-encoding bacteriophages (⌽62 and ⌽125) were induced spontaneously from their wild-type Escherichia coli strains and from the lysogens generated in Shigella sonnei. The stability of Cdt phages was determined at various temperatures and pH values after 1 month of storage by means of infectivity tests using a plaque blot assay and analysis of phage genomes using real-time quantitative PCR (qPCR): both were highly stable. We assessed the inactivation of Cdt phages by thermal treatment, chlorination, UV radiation, and in a mesocosm in both summer and winter. The results for the two Cdt phages showed similar trends and were also similar to the phage SOM23 used for reference, but they showed a much higher persistence than Cdt-producing E. coli. Cdt phages showed maximal inactivation after 1 h at 70°C, 30 min of UV radiation, and 30 min of contact with a 10-ppm chlorine treatment. Inactivation in a mesocosm was higher in summer than in winter, probably because of solar radiation. The treatments reduced the number of infectious phages but did not have a significant effect on the Cdt phage particles detected by qPCR. Cdt phages were quantified by qPCR in 73% of river samples, and these results suggest that Cdt phages are a genetic vehicle and the natural reservoir for cdt in the environment. Cytolethal distending toxin (Cdt) is produced by different pathogenic microorganisms, including Escherichia coli, Shigella dysenteriae (29), and Campylobacter spp. (18), among others (15,22,33). Its cytolethal distending action consists of blocking the G 2 and M phases during mitosis, which distends the cells, since cell division ceases but growth continues.Cdt consists of three subunits (CdtA, CdtB, and CdtC) that are encoded by three adjacent genes. The catalytic subunit CdtB is homologous to DNase I and is the most conserved gene due to its essential role in cellular toxicity. The other subunits act as binding proteins that deliver CdtB into target cells, thus producing the cytotoxic effect (13). Five variants of the toxin have been reported in E. coli. In some of these variants, the cdt genes are flanked by lambda-like and bacteriophage P2 genes (Cdt-I and Cdt-IV) (32). Some variants are present in inducible lambdoid prophages, such as Cdt-I (5) and Cdt-V (2), or are encoded in pVir, a conjugative plasmid (Cdt-III) (30).Recent genomic sequencing of different E. coli strains indicates that a large part of the genome consists of bacteriophage genes and that they constitute an important mechanism for adaptation to new hosts through the horizontal transfer of virulence genes (7). Cdt genes in E. coli occur independently of the presence of other virulence traits (21), which strongly suggests that they are independently acquired by horizontal gene transfer, probably by means of bacteriophages. cdt can be transduced from a cdt-positive bacteria to a cdt-negative strain by means of bacteriophages (2,5). This is an important process because this gene exchange can lead to the emergence of virulen...

Persistence of Infectious Shiga Toxin-Encoding Bacteriophages after Disinfection Treatments

“…Our results are also consistent with reports on other E. coli phages after either thermal treatment (46,47), UV treatment (48,49), or chlorination (7,21,50). Chlorinating water with 10 ppm of chlorine would inactivate bacteria, which would be considered suitable for consumption according to current practices in many parts of the world.…”

“…In terms of UV treatment, we calculated estimated UV doses of 5.94, 29.7, 59.4, and 178.2 mJ/cm 2 at each time, and the approach allowed comparison between microorganisms, as shown previously (7,21). The Stx phages ⌽534 and ⌽557 were not particularly sensitive to UV treatment and showed reductions in PFU of 2.2 log 10 units after 30 min.…”

“…Radiation and oxidation agents have been reported to result in modifications to viral proteins (capsid protein backbone cleavage) and nucleic acids (40). Nevertheless, important variations can be observed depending on the virus assayed, and previous studies in the same mesocosm system with other phages showed similar reduction of infectious phages but almost no reduction when evaluating phage genomes by qPCR (21).…”

“…However, Stx phages may still be detected in chlorinated samples. Stx phages are not the only example of phages encoding virulence genes that persist under certain inactivation conditions more than their bacterial hosts (21). Moreover, some environmental conditions, such as sunlight, could even cause the induction of temperate bacteriophages (51).…”

Evolution of a Self-Inducible Cytolethal Distending Toxin Type V-Encoding Bacteriophage from Escherichia coli O157:H7 to Shigella sonnei

“…Recently, environmental strains harboring Cdt-V-encoding phages were described (14). Moreover, Cdt phages were detected in fecally polluted waters (14,16) and showed high persistence under different disinfection processes and under natural inactivation conditions (16). Therefore, these phages may be interesting candidates for the mobilization of cdt genes in the environment.…”