In Situ and In Vitro Gene Expression by Vibrio vulnificus during Entry into, Persistence within, and Resuscitation from the Viable but Nonculturable State

Isolation of Vibrio vulnificus during winter months is difficult due to the entrance of these cells into the viable but nonculturable (VBNC) state. While several studies have investigated in vitro gene expression upon entrance into and persistence within the VBNC state, to our knowledge, no in situ studies have been reported. We incubated clinical and environmental isolates of V. vulnificus in estuarine waters during winter months to monitor the expression of several genes during the VBNC state and compared these to results from in vitro studies. katG (periplasmic catalase) was down-regulated during the VBNC state in vitro and in situ compared to the constitutively expressed gene tufA. Our results indicate that the loss of catalase activity we previously reported is a direct result of katG repression, which likely accounts for the VBNC response of this pathogen. While expression of vvhA (hemolysin) was detectable in environmental strains during in situ incubation, it ceased in all cases by ca. 1 h. These results suggest that the natural role of hemolysin in V. vulnificus may be in osmoprotection and/or the cold shock response. Differences in expression of the capsular genes wza and wzb were observed in the two recently reported genotypes of this species. Expression of rpoS, encoding the stress sigma factor RpoS, was continuous upon entry into the VBNC state during both in situ and in vitro studies. We found the half-life of mRNA to be less than 60 minutes, confirming that mRNA detection in these VBNC cells is a result of de novo RNA synthesis.     

In situ gene expression by Vibrio vulnificus

Strains of Vibrio vulnificus incubated in situ in natural estuarine waters during warm months continued to express katG (periplasmic catalase), rpoS (stress sigma factor), tufA (elongation factor), wza, and wzb (capsule synthesis). vvhA (hemolysin) was differentially expressed between environmental and clinical isolates. These results paralleled our in vitro findings.     

Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment.

Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months. Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, < 15 degrees C). In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca. 21 degrees C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state. These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not. This study indicates that the inability to isolate V. vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift.     

In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus.

Vibrio vulnificus is an estuarine bacterium responsible for 95% of all seafood-related deaths in the United States. The bacterium occurs naturally in molluscan shellfish, and ingestion of raw oysters is typically the source of human infection. V. vulnificus is also known to enter a viable but nonculturable (VBNC) state, wherein the cells are no longer culturable on routine plating media but can be shown to remain viable. Whether or not this human pathogen remains virulent when entering the VBNC state has not been definitively demonstrated. In this study, the VBNC state was induced through a temperature downshift to 5 degrees C, with cells becoming nonculturable (< 0.1 CFU/ml) within 7 days. As they became nonculturable, virulence was determined by employing an iron overload mouse model. At the point of nonculturability (7 days), injections of the diluted microcosm population resulted in death when < 0.04 CFU was inoculated, although > 10(5) cells in the VBNC state were present in the inoculum. Culturable cells of V. vulnificus, with identification confirmed through PCR, were recovered from the blood and peritoneal cavities of mice which had died from injections of cells present in the VBNC state for at least 3 days. Thus, our data suggest that cells of V. vulnificus remain virulent, at least for some time, when present in the VBNC state and are capable of causing fatal infections following in vivo resuscitation. Our studies also indicate, however, that virulence decreases significantly as cells enter the VBNC state, which may account, at least to some extent, for the decrease in infections caused by this bacterium during winter months.     

Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR

The abundance of Vibrio vulnificus in coastal environments has been linked to water temperature, while its relationship to salinity is less clear. We have developed a culture-independent, most-probable-number quantitative PCR approach to examine V. vulnificus population dynamics in Barnegat Bay, N.J. Based on the combined analysis of our results from Barnegat Bay and from the literature, the present data show that (i) V. vulnificus population dynamics are strongly correlated to water temperature and (ii) although the general trend is for V. vulnificus abundance to be inversely correlated with salinity, this relationship depends on salinity levels. Irrespective of temperature, high abundances of V. vulnificus are observed at 5 to 10 ppt, which thus appears to be the optimal salinity regime for their survival. At 20 to 25 ppt, V. vulnificus abundances show a positive correlation to salinity. Unsuccessful attempts to resuscitate V. vulnificus, combined with our inability to detect cells during the winter despite an assay adapted to detect viable but nonculturable (VBNC) cells, suggest that the decline and eventual disappearance of V. vulnificus from the water column during the winter months is due primarily to a significant reduction in population size and is not only the consequence of cells entering the VBNC state. These findings are in line with the hypothesis that the sediment serves as a refuge for a subpopulation of V. vulnificus over the winter and weather-driven mixing events during the spring initiate a summer bloom in the water column.     

Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State

Like many other gram-negative bacteria, the human pathogen Vibrio vulnificus is induced into a viable but nonculturable (VBNC) state by incubation at low temperatures. The ability of any bacterium to resuscitate from this dormant state would appear to be essential if the VBNC state is truly a survival strategy. The question as to whether the culturable cells which appear following removal of the inducing stress are a result of true resuscitation or of regrowth of a few residual culturable cells has long been debated. V. vulnificus was examined for its ability to resuscitate from this state following a temperature upshift. Several lines of investigation, including dilution studies, determination of the time necessary for appearance of a culturable population, and the effects of nutrient on recovery, all indicated that, at least for V. vulnificus, true resuscitation does occur. Our studies further suggest that nutrient is in some way inhibitory to the resuscitation of cells in the VBNC state and that studies which add nutrient in an attempt to detect resuscitation are able to detect only residual culturable cells which might be present and which were not inhibited by the added nutrient.     

Changes in membrane fatty acid composition during entry of Vibrio vulnificus into the viable but nonculturable state

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 degrees 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 degrees C in artificial 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 metabolism is essential for entry of cells into the VBNC state.     

The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus

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.     

Induction,resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water

Vibrio vulnificus, an important food-borne pathogen, is known to enter viable but nonculturable (VBNC) state under low temperature and low nutrition stress conditions. Present study examined the time required for induction of VBNC state and temperature which induces resuscitation of V. vulnificus YJ016. The change in cell morphology and gene expression during VBNC state and in resuscitated cells was also examined. V. vulnificus incubated in artificial sea water at 4 °C entered VBNC state after considerably extended time (70 days). An increase in temperature by 6 °C from the VBNC induction temperature (4 °C) resulted in resuscitation of VBNC cells; however, maximum resuscitation was observed when VBNC cells were held at 23 °C for 24 h. VBNC cells changed their morphology from comma shape to coccoid shape. Two rounds of induction of VBNC and resuscitation were possible with V. vulnificus cells; however, there was progressive reduction in number of resuscitated cells and after 190 days cells failed to resuscitate. Significant up-regulation of genes related to membrane proteins [porinH (10.4-fold), ompU (2.9-fold)], regulatory proteins [envZ (5.6-fold), toxR (4.5-fold), toxS (4.8-fold)], oxidative stress related protein katG (2.3-fold), cell division/maintenance proteins [ftsZ (4.3), mreB (6.5-fold)] and resuscitating promoter factor yeaZ (fourfold) was observed during resuscitation with respect to VBNC state indicating that these genes play a role during resuscitation. Gene expression data presented here would enhance our understanding of resuscitation of V. vulnificus from VBNC state. The results also highlight the importance of maintenance of low temperature during storage of seafood.     

Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

Entry of Vibrio cincinnatiensis into viable but nonculturable state and its resuscitation

Aims:  The aim was to characterize the viable but nonculturable (VBNC) state of Vibrio cincinnatiensis and its resuscitation.
Methods and Results:  Vibrio cincinnatiensis VIB287 was cultured in sterilized seawater microcosms at 4°C. Plate counts, direct viable counts and total counts were used. A large population of the V. cincinnatiensis became nonculturable after approx. 50 day at 4°C. Electron microscopy revealed that the VBNC cells changed from rod to coccoid and decreased in size. Resuscitation of VBNC cells was achieved by temperature upshift in nutrition of yeast extract and peptone by addition of catalase or compound vitamin B. The VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately. No death was observed in the group inoculated with the VBNC cells.
Conclusions:  Vibrio cincinnatiensis VIB287 could enter VBNC state in adverse environments. Resuscitation of VBNC cells occurred by addition of compound vitamin B or catalase to VBNC cells containing nutrient. The resuscitative cells might retain their pathogenicity.
Significance and Impact of the Study:  The study confirmed that V. cincinnatiensis could enter into VBNC state in seawater at low temperature and resuscitated. The resuscitative cells retained their pathogenicity, which may be important in future studies of ecology of V. cincinnatiensis .     

Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus

The viable but nonculturable (VBNC) suppression mutant formed platable cells at low temperature stress after inoculation in artificial seawater (ASW). Suppression subtractive hybridization was used to identify differentially expressed genes among cDNAs of the VBNC suppression mutant and the wild-type Vibrio vulnificus strain. Glutathione S-transferase was identified as a responsive gene of the VBNC suppression mutant in our assay, and was highly expressed from the VBNC suppression mutant at low temperature stress. Culturability tests revealed that the wild-type cells were sensitive to oxidative stress in the hydrogen peroxide (H(2)O(2)) and to 1-chloro-2,4-dinitrobenzene (CDNB) compared with the VBNC suppression mutant cells. Adding glutathione showed that many wild-type V. vulnificus cells maintained culturability in cold ASW. These results suggest that non-nutritional growth inhibitors, such as peroxide that accumulates at low temperatures, influence VBNC in V. vulnificus cells.     

Some properties of Vibrio vulnificus hemolysin

Some properties of hemolysin produced by Vibrio vulnificus were investigated. The hemolysin was heat labile, and the hemolytic activity was inhibited by adding cholesterol or divalent cations. Cholesterol inhibited the temperature-independent hemolysin-binding step, suggesting that cholesterol made up the binding site of the cell membrane, whereas the divalent cations inhibited the temperature-dependent membrane-degradation step. However, the V. vulnificus hemolysin was stable to oxygen and sulfhydryl reagents and was not inactivated by antiserum against streptolysin O, suggesting that the V. vulnificus hemolysin differs from oxygen-labile hemolysins which bind to cholesterol. The V. vulnificus hemolysin seems to be one of the exceptional cholesterol-binding hemolysins.     

Ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina

While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of V. vulnificus in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of Vibrio spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured. V. vulnificus isolates, along with estuarine bacteria, Vibrio spp., Escherichia coli organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including V. vulnificus. Isolates were confirmed as V. vulnificus by using hemolysin gene PCR or colony hybridization. V. vulnificus was isolated only when water temperatures were between 15 and 27 degrees C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of V. vulnificus in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both V. vulnificus (47%) and Vibrio spp. (48%).