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Assessment of Viability of Stressed Campylobacter jejuni Cells by Plate Count and Fluorescence Techniques

There is an increasing interest in development of rapid methods for microbial viability determination, which is crucial for detection and enumeration of spoilage and pathogenic microorganisms, evaluation of antimicrobial efficiency of food preservation treatments etc. Traditional plate counting underestimates the real viable portion of microbial population, especially when cells are exposed to environmental stresses resulting in sublethal injuries of the cells - these could remain still virulent but undetected by routine plating methods. Campylobacter jejuni is currently recognized as a leading foodborne pathogen in many countries. Its ability of transforming into a viable but nonculturable (VBNC) state, undergoing alteration in cell morphology, is well documented. The aim of this study was to follow up survival of C. jejuni cells from exponential or stationary growth phase after exposure to different environmental stresses. Starvation of cells in combination with heat or oxidative shock in laboratory model system was tested. Cell morphology and membrane integrity changes were evaluated using the LIVE/DEAD Baclight bacterial viability kit (BacLightTM) and subsequent fluorescent microscopy. In addition flow cytometric measurement after SYTO 9 and propidium iodide staining was folowed. The results of fluorescent measurements were comparativelly evaluated to CFU counts detected on selective and non-selective Campylobacter agar plates. It was confirmed that Campylobacter jejuni cells shift from actively growing spiral cells to noncultivable coccoid forms and remain viable during prolonged exposure to stress conditions. Comparatively, microbial survival was the best in 5h-starved cells from exponential growth phase. Important differences in viability during applied stresses were detected and will be presented. Flow cytometry analysis showed that new population of cells appeared in starved cells exposed to oxidative stress. The consequences of the exposure of Campylobacter cells to environmental stresses and subsequent changes in physiological state of the cells will be discussed. The impact of these changes on Campylobacter cells infectivity and virulence properties are further aims of our study.

Campylobacter; viability; VBNC; fluorescence techniques; microbial stress response

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