Vet. Res. 36 (2005) 589-599
DOI: 10.1051/vetres:2005018

Avirulence of Viable But Non-Culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

Jean Michel Cappelier^a, Valérie Besnard^a, Sylvie Roche^b, Nathalie Garrec^a, Etienne Zundel^b, Philippe Velge^b and Michel Federighi<sup>a

a</sup>  Unité Mixte de Recherche SECALIM INRA-ENVN, École Nationale Vétérinaire de Nantes, BP 40706, Route de Gachet, 44307 Nantes, France
^b  Institut National de la Recherche Agronomique, Laboratoire de Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France

(Received 14 May 2004; accepted 6 December 2004)

Abstract - The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 °C and Scott A strain at 4 °C. No culturable bacteria were detected in the VBNC state, although 10⁴ active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state.

Key words: VBNC / virulence / Listeria monocytogenes / plaque forming assay

Corresponding author: Jean Michel Cappelier cappelier@vet-nantes.fr

© INRA, EDP Sciences 2005