Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes

A viable but non-culturable (VBNC) bacterial state was originally detected in studies in environmental microbiology. In particular, this state has bee n demonstrated for a number of human pathogens (Escherichia coli, Salmonell a enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter je juni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and the cells rem ain potentially pathogenic under favourable conditions. But, as far as we k now, the VBNC state has not been yet described in Listeria monocytogenes. I n most studies, this has been assessed by the Kogure procedure based on cel lular elongation in the presence of DNA gyrase inhibitors. The antibiotic u sed was nalidixic acid in order to prevent DNA replication, only efficient in Gram-negative bacteria studies. In this study, we describe a new DVC pro cedure to detect and count viable of L. monocytogenes suspended in filtered , sterilized distilled water. We used different concentrations of ciproflox acin, efficient both in Gram-negative and Gram-positive bacteria. Bacteria cells were removed and resuspended in BHI broth, with yeast extract and cip rofloxacin. The mixture was incubated at different incubation times at 37 d egrees C. After different incubation times, cells were filtered through an isopore polycarbonate black membrane filter and covered with a DAPI solutio n or orange acridine. The filters were prepared and examined by epifluoresc ence microscopy. Elongated cells were counted as viable cells, whereas norm al size was regarded as nonactive ones. This method allows determination of ciprofloxacin concentration and incubation time optimal to detect maximum viable cells percentage in L. monocytogenes.