Evidence of Viable But Non-Culturable state in Listeria monocytogenes by direct viable count and CTC-DAPI double staining

Abstract

Viable but Non-culturable (VBNC) state in bacteria was detected originally in environmental microbiology studies. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and moreover the cells remain potentially pathogenic under favourable conditions. But the VBNC state has not been yet described in Listeria monocytogenes. Production of VBNC L. monocytogenes cells was studied using four strains resuspended in microcosm water. Various strains of L. monocytogenes were resuspended in filtered sterilized water adjusted to pH 6·0, incubated at 20°C and 4°C with or without NaCl supplementation (0%, 7%), with gentle shaking at 100 rpm. The culturability of starved cells suspensions was determined by spread plate count (PCA). The cells activity was measured by a Direct Viable Count technique and by CTC-DAPI double staining. Two strains of four (CNL 895807, Scott A) exhibited a VBNC state. They lost their culturability and maintained a cellular activity, even after 10 weeks of starvation in microcosm water.