The Viable but Nonculturable State in Bacteria

Oliver James D. The Microbiological Society of Korea 2005 The journal of microbiology Vol.43 No.1

It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the 'viable but nonculturable' (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

The Ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina Estuaries

Karen Dyer Blackwell,James D. Oliver 한국미생물학회 2008 The journal of microbiology Vol.46 No.2

While numerous studies have characterized the distribution and/or ecology of various pathogenic Vibrio spp., here we have simultaneously examined several estuarine sites for Vibrio vulnificus, V. cholerae, and V. parahaemolyticus. For a one year period, waters and sediment were monitored for the presence of these three pathogens at six different sites on the east coast of North Carolina in the United States. All three pathogens, identified using colony hybridization and PCR methods, occurred in these estuarine environments, although V. cholerae occurred only infrequently and at very low levels. Seventeen chemical, physical, and biological parameters were investigated, including salinity, water temperature, turbidity, dissolved oxygen, levels of various inorganic nutrients and dissolved organic carbon, as well as total vibrios, total coliforms, and E. coli. We found each of the Vibrio spp. in water and sediment to correlate to several of these environmental measurements, with water temperature and total Vibrio levels correlating highly (P<0.0001) with occurrence of the three pathogens. Thus, these two parameters may represent simple assays for characterizing the potential public health hazard of estuarine waters.

Changes in Membrane Fatty Acid Composition during Entry of Vibrio vulnificus into the Viable But Nonculturable State

Ashley P. Day,James D. Oliver 한국미생물학회 2004 The journal of microbiology Vol.42 No.2

Vibrio vulnificus, a Gram-negative bacterium found in estuarine waters, is responsible for over 95%of all seafood-related deaths in the United States. As a result of a temperature downshift to 5oC, this organism enters the viable but nonculturable (VBNC) state. Changes in the membrane fatty acid (FA) composition of V. vulnificus may be a contributing factor to the ability of this organism to enter into and survive in the VBNC state. This hypothesis was tested by incubating the organism at 5oC in artificial sea water and analyzing the cells’ FAs during the initial hours of temperature and nutrient downshift. Prior to downshift, the predominant FAs were 16:0, 16:1 and 18:0. During the first four hours of downshift, statistically significant changes occurred in 15:0, 16:1, 16:0, 17:0, and 18:0. These results indicate that changes in FA composition occur prior to entry of V. vulnificus into the VBNC state, suggesting that the ability to maintain membrane fluidity may be a factor in this physiological response. Cells in which fatty acid synthesis was inhibited did not survive, indicating that active fatty acid metabolism is essential for entry of cells into the VBNC state.

Changes in Membrane Fatty Acid Composition during Entry of Vibrio vulnificus into the Viable But Nonculturable State

Day, Ashley P.,Oliver, James D. The Microbiological Society of Korea 2004 The journal of microbiology Vol.42 No.2

Vibrio vulnificus, a Gram-negative bacterium found in estuarine waters, is responsible for over 95% of all seafood-related deaths in the United States. As a result of a temperature downshift to 5$^{\circ}C$, this organism enters the viable but nonculturable (VBNC) state. Changes in the membrane fatty acid (FA) composition of V. vulnificus may be a contributing factor to the ability of this organism to enter into and survive in the VBNC state. This hypothesis was tested by incubating the organism at 5$^{\circ}C$ in arti-ficial sea water and analyzing the cells' FAs during the initial hours of temperature and nutrient down-shift. Prior to downshift, the predominant FAs were 16:0, 16:1 and 18:0. During the first four hours of downshift, statistically significant changes occurred in 15:0, 16:1, 16:0, 17:0, and 18:0. These results indicate that changes in FA composition occur prior to entry of V. vulnificus into the VBNC state, suggesting that the ability to maintain membrane fluidity may be a factor in this physiological response. Cells in which fatty acid synthesis was inhibited did not survive, indicating that active fatty acid metab-olism is essential for entry of cells into the VBNC state.

The Viable But Nonculturable State of Kanagawa Positive and Negative Strains of Vibrio parahaemolyticus

Bates, Tonya C.,Oliver, James D. The Microbiological Society of Korea 2004 The journal of microbiology Vol.42 No.2

Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gas-troenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP$\^$+/) strains of V. parahaemolyticus are isolated from patients, whereas >99% of strains isolated from the environment do not produce this hemolysin (i.e. are KP$\^$-/). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but noncultur-able (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media. We speculated that KP$\^$+/ and KP$\^$-/ strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of cul-turable KP$\^$+/ strains isolated from estuarine waters. The response of eleven KP$\^$+/ and KP$\^$-/ strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP$\^$+/ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.

The Viable But Nonculturable State of Kanagawa Positive and Negative Strains of Vibrio parahaemolyticus

Tonya C. Bates,James D. Oliver 한국미생물학회 2004 The journal of microbiology Vol.42 No.2

Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gastroenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP+) strains of V. parahaemolyticus are isolated from patients, whereas >99% of strains isolated from the environment do not produce this hemolysin (i.e. are KP-). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but nonculturable (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media. We speculated that KP+ and KP- strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of culturable KP+ strains isolated from estuarine waters. The response of eleven KP+ and KP- strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP+ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.