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

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 10 4 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. VBNC / virulence / Listeria monocytogenes / plaque forming assay

Avirulent Viable But Non Culturable cells ofListeria monocytogenesneed the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

“…These strains were chosen because of their origin (clinical and food isolates) and because they were previously used in VBNC studies in which optimal conditions to induce the VBNC state were described [3,5,12]. Two subcultures were performed under similar conditions and then the cells were placed in filtered sterilised distilled water (microcosm water) in order to induce the VBNC state.…”

“…I), non culturability was [12]. Then, 1 mL from each of the microcosm water samples was inoculated into embryonated eggs and into non embryonated eggs.…”

“…The virulence tests were described in Cappelier et al [12]. Briefly, the Listeria monocytogenes strains (culturable, VBNC and recovered cells) were tested with HT-29 cell monolayers in a plaqueforming assay [48,49] and the strains were also tested for their potential to colonise mouse spleens (Swiss female OF1) after intravenous inoculation [1,8] according to the methods described by Roche et al [42].…”

“…Briefly, the Listeria monocytogenes strains (culturable, VBNC and recovered cells) were tested with HT-29 cell monolayers in a plaqueforming assay [48,49] and the strains were also tested for their potential to colonise mouse spleens (Swiss female OF1) after intravenous inoculation [1,8] according to the methods described by Roche et al [42]. The level of virulence determined by in vitro (PFA assay) and in vitro (inoculation in mice) techniques, for each strain used, in the culturable state and in the VBNC state were previously described in Cappelier et al [12] (Tab. II).…”

“…The existence of the VBNC state in a foodborne pathogen like Listeria monocytogenes, which exhibits a high lethality rate, needs to be taken into account. For these reasons, the levels of virulence of Listeria monocytogenes VBNC cells were assessed by in vitro and in vivo tests in Cappelier et al [12]. In addition, the ability of such VBNC cells to recover a culturable state must be investigated.…”