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Potential of Escherichia coli O157:H7 to persist and form viable but non-culturable cells on a food-contact surface subjected to cycles of soiling and chemical treatment

Marouani-Gadri, Nesrine, Firmesse, Olivier, Chassaing, Danielle, Sandris-Nielsen, Dennis, Arneborg, Nils, Carpentier, Brigitte
International journal of food microbiology 2010 v.144 no.1 pp. 96-103
Escherichia coli O157:H7, viability, cell culture, food contact surfaces, simulation models, chemical treatment, pathogen survival, cleaning, disinfection, bacterial contamination, biofilm, Pseudomonas fluorescens, food sanitation
Our aim was to assess the potential of Escherichia coli O157:H7 to persist in a processing environment. We studied E. coli behaviour under conditions modelling those of meat plants to establish one initial bacterial load that allows persistence and another that does not. Polyurethane coupons (3.5cm²) were contaminated once with E. coli in meat exudate before being subjected daily to a cleaning product and a disinfectant, both at half the recommended in-use concentrations, and a further soiling with the exudate. This procedure aimed to model what occurs in harbourage sites. Because previous experiments showed that persistence could not be achieved at 15°C (temperature of slaughter halls), we incubated the coupons at 20°C. Viable cells were determined by ethidium monoazide-qPCR (EMA-qPCR). When the first chemical treatment (CT) was applied to 24-hour biofilms with 5.4logCFU/cm², cells were no longer detectable after the first week. However, on 66-hour biofilms with 6.7log CFU/cm², after initially decreasing, E. coli numbers reached 6.6logCFU/cm² and 8.3logviable cells/cm² on the 11th day. When E. coli was cultured with a Comamonas testosteroni previously shown to increase E. coli biofilm formation, and subjected to CT on alternate days, E. coli stabilized at 4.6logCFU/cm² before the CT, from the 5th day of the experiment. The killing and detachment effects of the CT decreased over time and PCR quantification detected a resumption of growth after 2days (CT on alternate days) or 3days (daily CT). Intracellular pH (pHi) of individual cells was determined during an experiment in which the CT was applied on alternate days. The proportion of cells with no proton gradient towards the environment (pHi≤5.4) increased after the CT as expected. But during the first week of the experiment only, a further increase in this proportion occurred 24h after the CT, suggesting that some of the surviving viable but non-culturable cells finally died. This study shows that conditions leading to E. coli O157:H7 persistence are not likely to arise when good refrigeration and hygiene practices are applied, and highlights the usefulness of EMA or PMA-qPCR as a complement to CFU determination in studying bacterial survival after cleaning and disinfection.