Avirulent viable but non culturable cells of Listeria monocytogenes need the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

The aim of this study was to assess the efficiency of the embryonated egg model to recover Viable But Non Culturable (VBNC) cells of Listeria monocytogenes. L. monocytogenes cells were incubated in filtered sterilised distilled water. The VBNC state was obtained after a 25 to 47 days incubation period (concentration of culturable cells less than 1 cfu cfu/mL). Fifteen days after the VBNC state was reached, non culturability was checked in various media. One milliliter of each VBNC suspension that contained 10(4) metabolically active cells (i. e. Direct Viable Count + cells) was inoculated into the vitellus fluid of embryonated and non- embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs (18/32), but not in the non- embryonated eggs (1/32). The recovery rate was higher after culture of the vitellus fluid plus embryo (18/32) than after culture of the vitellus fluid alone (6/32). The results indicate that the embryo likely plays a prominent part in the recovery process. The virulence of recovered cells was assessed by the ability to form plaques in HT-29 cell monolayers and by the ability to colonise mouse spleens. Although the cells were classified as avirulent when in the VBNC state, the virulence was recovered after resuscitation.