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 (Φ62 and Φ125) 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.

Fig 1

Stability of Cdt phages and SOM23 at various temperatures (4, 22, and 37°C) (A) and different pHs (3, 7, and 9 after 1 month of storage at 4°C) (B). The results of infectivity presented (PFU/ml) for Φ62 and Φ125 correspond to plaque blot hybridization results. Infectivity of phage SOM23 was evaluated using plaque assays, which was included as a control. The charts show the results of one representative experiment. The tables present the averages (with the standard deviations [SD] indicated in parentheses) of the slopes of the logarithmic regression lines obtained in three replicas of each experiment for infectious phages and qPCR.

Fig 2

Persistence of Cdt phages Φ62 and Φ125, SOM23, and Cdt-STEC under thermal treatment at 60 and 70°C (A) to UV treatment (B) to 10 ppm of chlorine (C) through time. The charts on the left show logarithmic values of PFU, CFU or GC/ml. The tables on the right present the averages (SD) of the slopes of the logarithmic regression lines obtained in three replicas of each experiment. Dotted lines indicate values below the limit of detection.

Fig 3

Persistence of Cdt phages (qPCR and infectivity), SOM23 (plaque assay) and Cdt-STEC (colony counts) to inactivation processes in a mesocosm in summer and winter. Each chart shows the results of one representative experiment in each season. The table presents the averages (SD) of the slopes of the equation of the logarithmic regression lines obtained with three replicas of the experiment. The dotted line indicates values below the limit of detection.