The fate of verocytotoxigenic Escherichia coli C600φ3538(Δvtx2 ::cat) and its vtx2 prophage during grass silage preparation.

AIMS: Silage is grass, preserved by fermentation and used as winter feed for cattle. The impact of a range of current grass silage preparation practices on the survival of Escherichia coli C600φ3538(Δvtx2 ::cat) and on the induction, release and infectivity of free phage were investigated. METHODS AND RESULTS: Wilted and fresh grass samples, from plots with and without slurry application, were ensiled with or without formic acid. Each treatment combination was inoculated with approximately 6 log10 CFU per g E. coli C600φ3538(Δvtx2 ::cat) (donor strain) and E. coli C600::kanamycinR (recipient strain) in test-tube model silos and incubated in the dark at 15°C. The physico-chemical (pH, ammonia, ethanol, lactic acid and volatile fatty acids) and microbiological (total viable counts, TVC, total Enterobacteriaceae counts, TEC, E. coli counts, ECC and lactic acid bacteria, LAB) properties of each fermentation were monitored throughout the experiment as were the concentrations of E. coli C600φ3538(Δvtx2 ::cat), E. coli C600::kanamycinR , free phage and transductants, using culture and PCR-based methods. Over the course of the experiment the pH of the grass samples typically decreased by 2 pH units. TVC, TEC and ECC decreased by up to 2·3, 6·4 and 6·2 log10 CFU per g, respectively, while the LAB counts remained relatively stable at 5·2-7·1 log10 CFU per g. Both donor and recipient strains decreased by approximately 5 log10 CFU per g. Free phages were detected in all treatments and transductants were detected and confirmed by PCR in the silo containing wilted grass, pretreated with slurry and ensiled without formic acid. CONCLUSIONS: Verocytotoxigenic E. coli may survive the ensiling process and the conditions encountered are sufficient to induce vtx2 bacteriophage leading to low levels of phage-mediated vtx2 gene transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: These studies suggest that the ensiling of grass may create an environment which facilitates the emergence of new verocytotoxigenic E. coli.

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

AIMS: Cattle are the main reservoir of verocytotoxigenic Escherichia coli (VTEC), food-borne pathogens that express verocytotoxins (vtx) encoded by temperate bacteriophage. Bovine faeces and unturned manure heaps can support the survival of VTEC and may propagate and transmit VTEC. This study investigated the survival of a vtx2 bacteriophage, φ24B ::Kan, in bovine faeces and slurry. The survival of an anti-Escherichia coli O157:H7 lytic bacteriophage, e11/2, was examined in the same matrices, as a possible bio-control option for VTEC. METHODS AND RESULTS: Samples were inoculated with φ24B ::Kan and/or e11/2 bacteriophage at a concentration of 7-8 log10  PFU g(-1)  (faeces) or ml(-1) (slurry), stored at 4 and 14°C and examined every 2 days for 36 days. The ability of φ24B ::Kan to transduce E. coli cells was examined. Moreover, E. coli concentrations in the faeces and slurry were monitored throughout the experiment as were the pH and aw (faeces only). Both bacteriophages survived well in faeces and slurry. In addition, φ24B ::Kan was able to form lysogens. CONCLUSIONS: φ24B ::Kan and e11/2 phage can survive and remain infective in bovine faeces and slurry for at least 30 days under representative Irish temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: Bovine faeces and slurry may act as a reservoir for vtx bacteriophages. The survival of the anti-O157 phage suggests it may be a suitable bio-control option in these matrices.

The Survival of a Temperate vtx Bacteriophage and an Anti-Verocytotoxigenic Escherichia coli O157 Lytic Phage in Water and Soil Samples.

Verocytotoxigenic (vtx) Escherichia coli (VTEC) are zoonotic foodborne pathogens with the vtx operon encoded by lambdoid bacteriophage (phage). Despite much research on the host bacteria, similar data on the persistence of verocytotoxin converting phage and the ecological niches where transduction occurs are lacking and novel VTEC of important public health significance, have and continue to emerge. This study investigated the survival of a temperate vtx bacteriophage (24B ::kanamycinR ) in water (raw farm, pasteurized farm, laboratory tap and autoclaved purified water) and soil (sandy loam and loam soil). It also examined the persistence of an anti-VTEC lytic phage (e11/2) in the same matrices as this may be one option for controlling the emergence of novel VTEC, especially in farm ecological niches where other control options, such as chemical, heat or high pressure treatments, are not feasible. Samples inoculated with 24B ::kanamycinR and e11/2 bacteriophage (8 log10 pfu/ml or pfu/g) separately were incubated at 4°C and 14°C, representative Irish Winter and Summer temperatures, respectively, and tested every 2 days for 40 days. The transduction of 24B ::kanamycinR was also continuously assessed. Both phages survived with reductions observed, regardless of matrix or storage temperature. Moreover, 24B ::kanamycinR was able to transduce its host E. coli strain. It was therefore concluded that aquatic and soil environments on farms may serve as a vtx phage reservoir and transduction point but anti-VTEC phage is a possible biocontrol option.