ISBN:  Unavailable

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Page count: 92

ABSTRACT: Over 30 bacterial species, including Vibrio parahaemolyticus, have been shown to enter the viable but nonculturable (VBNC) state where cells maintain viability but are no longer able to be cultured on routine microbiological media. Kanagawa phenomenon (hemolysin) positive (KP) strains of V. parahaemolyticm are isolated primarily from patients, whereas>99% of strains isolated from the environment are KP−. Thus, we hypothesized that the KP strains may enter into the VBNC state faster than KP− strains, accounting for the lack of KP+ strains isolated from marine waters. In this study, KP+ and KP− strains of V. parahaemolyticus were induced to enter into the VBNC state following nutrient downshift and incubation at 5̊C in artificial seawater (ASW), phosphate buffered saline, or 20% ASW. Also included in these studies were modified strains of V parahaemolyticus, one of which lacked the chromosomal genes to produce hemolysin while the other mutant had the hemolysin genes restored on a plasmid. No differences were noted in the times that it took the KP+ strains versus the KP− strains to enter the VBNC state. Moreover, VBNC cells could be restored to a culturable state on a routine medium following overnight temperature upshift. Using in situ hybridization, the ribosomal RNA content of a KP+ and a KP− strain of V parahaemolyticus was measured using fluorescent probes specific for the 16S and 23S rRNA subunits during entry into and exit from the VBNC state as well as during starvation. Results indicate that a significant loss of ribosomal content occurs as cells enter into the VBNC state, although this level was adequate to allow resuscitation.