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 (⌽534 and ⌽557) 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-log 10 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-log 10 reductions, respectively) and were highly inactivated (3 log 10 ) 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. Shiga toxin-producing Escherichia coli (STEC) strains are pathogenic and cause a wide range of diseases, with symptoms varying from noncomplicated diarrhea to the life-threatening hemolytic-uremic syndrome (HUS) (1, 2). STEC produces two immunologically distinct toxins known as Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), and both toxins present diverse variants (1).The genes encoding Stx in E. coli are located in the genomes of inducible temperate bacteriophages (Stx phages) (3). Induction of the lytic cycle of Stx phages causes an increase in production of Shiga toxin, which is the virulence factor responsible for severe complications associated with the infection, such as HUS (2, 4). In addition to the increase in Stx expression, the lysis of the cell caused by Stx phages allows the release of Stx outside the cell and also the dissemination of Stx phages. Free Stx phages spread within the gut and are excreted with the feces (5). In terms of occurrence in the environment, Stx phages have been found in water bodies containing fecal contamination of human or animal origin (6-12). Infectious Stx phages have also been detected in food samples, and despite the abundance of Stx phages in these samples, they showed levels of bacterial indicators (aerobic colony counts and E. coli) that make them acceptable for consumption according to European regulations (13).The widespread distribution of Stx phages in different environments indicates that they must be able to persist under diverse conditions. Previous studies suggested the persistence of environmental Stx phages (7). Newly developed molecular methods for the quantification of Stx phages (14), the development of new approaches for the optimal detection of plaques formed by infectious Stx phages (15), and optimized protocols for th...

An Environmental Shiga Toxin-Producing Escherichia coli O145 Clonal Population Exhibits High-Level Phenotypic Variation That Includes Virulence Traits

“…Infective Shiga toxin-encoding phages have previously been detected in various ecosystems and in fecal samples from animals and humans (54)(55)(56)(57)(58)(59), and transfer of stx genes has been demonstrated in water and food samples (60). These phages persisted better in nonhost environments than in their bacterial hosts and retained their infectious activities in harsh environments (61,62), implying the potential of stx acquisition by phage transduction in natural environments (63,64).…”

Contribution of cropland to the spread of Shiga toxin phages and the emergence of new Shiga toxin-producing strains

“…Free Stx phages are more persistent than bacteria 26 and are involved in the horizontal transduction of stx 27 , 28 . Their persistence and likelihood of transduction increase the risk of new STEC strains emerging in the environment and in food, particularly when food is consumed raw or only partially cooked.…”

Survival studies of a temperate and lytic bacteriophage in bovine faeces and slurry

“…Vtx bacteriophages replicate via the lysogenic cycle, while lytic bacteriophages such as e11/2 replicate via the lytic cycle (Rivas et al 2010). The transfer of vtx genes mediated by bacteriophages occurs via a process called transduction (Muniesa et al 1999;Allue-Guardia et al 2014).…”