Persistence of infectious Shiga toxin-encoding bacteriophages after disinfection treatments

Abstract

In Shiga toxin-producing Escherichia coli (STEC), induction of Shiga toxin-encoding bacteriophages (Stx phages) causes the release of free phages that can later be found in the environment. The ability of Stx phages to survive different inactivation conditions determines their prevalence in the environment, the risk of stx transduction, and the generation of new STEC strains. We evaluated the infectivity and genomes of two Stx phages (Φ534 and Φ557) under different conditions. Infectious Stx phages were stable at 4, 22, and 37°C and at pH 7 and 9 after 1 month of storage but were completely inactivated at pH 3. Infective Stx phages decreased moderately when treated with UV (2.2-log10 reduction for an estimated UV dose of 178.2 mJ/cm(2)) or after treatment at 60 and 68°C for 60 min (2.2- and 2.5-log10 reductions, respectively) and were highly inactivated (3 log10) by 10 ppm of chlorine in 1 min. Assays in a mesocosm showed lower inactivation of all microorganisms in winter than in summer. The number of Stx phage genomes did not decrease significantly in most cases, and STEC inactivation was higher than phage inactivation under all conditions. Moreover, Stx phages retained the ability to lysogenize E. coli after some of the treatments.

FIG 1

(A) Micrographs of Stx phages Φ534 and Φ557. Bar, 100 nm. (B) Positive signals of plaques of lysis generated by Stx phages on E. coli strain WG5 after plaque blot hybridization with a DIG-labeled stx₂-A probe.

FIG 2

Stability of Stx phages after 1 month of storage at various temperatures (4°C, 22°C, and 37°C) and at different pHs (3, 7, and 9) after 1 month of storage at 4°C. Values for Stx phages are presented as the average of values obtained for phage Φ534 and phage Φ557. Infectious phages (inf) were evaluated by plaque blot and values expressed as PFU/ml. Phage genomes were evaluated by qPCR, and the results are expressed as GC/ml. Values for Stx phages are the averages of phage Φ534 and phage Φ557 in three independent experiments. The x axis is not presented at scale.

FIG 3

Logarithmic reduction calculated for Stx phages (infectivity and genomes), SC12 (infectivity), and STEC (colony counts) in an outdoor mesocosm in summer and winter. Values for Stx phages are presented as the average of values obtained for phage Φ534 and phage Φ557. All values are the average of three independent experiments conducted during the winter or summer season.