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

Abstract

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.Vibrio vulnificus is a gram-negative bacterium found in estuarine and coastal waters, including the East, Gulf, and Pacific Coasts of the United States and coastal waters throughout the world (15,26,27,35,46), at high numbers in bivalve mollusks. This bacterium is capable of causing rapidly fatal septicemia and wound infections subsequent to the ingestion of raw or undercooked seafood or following the exposure of wounds to water containing this pathogen (27). The ingestion of raw shellfish, especially oysters, is the most common means of developing primary septicemia, which affects individuals who either are immunocompromised or display underlying hepatic disorders with increased serum iron levels almost exclusively (32). In fact, it has been established experimentally that elevated serum iron levels highly correlate with the growth of V. vulnificus in human serum (25,44,52). V. vulnificus is of extreme significance to the seafood industry, as it causes more seafood-borne deaths than any other organism in the United States and also has the highest case fatality rate of any food-borne infection reported in this country (23). Indeed, 30 to 40 cases resulting in death occur each year in the United States. Interestingly, approximately 90% of all who suffer from V. vulnificus-induced primary septicemia are males, due to the apparent protection from the endotoxin by estrogen in females (24).The environment for V. vulnificus, as for all estuarine bacteria, consists of constantly f...

The Biology of Vibrio vulnificus

“…VBNC bacteria are organisms that fail to grow and develop colonies on the media they are normally cultured on, but their metabolic activity capabilities indicate that they are still alive (98). Several reviews of this dormancy state have been published (99,100,101,102,103,104,105,106,107,108), and the list of bacteria known to enter this state is constantly growing. We have also employed diffusion chambers to demonstrate that this phenomenon occurs not only in the lab, but also in situ in estuarine waters (105).…”

State transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow cytometry

“…VBNC cells lose their viability very quickly as assessed by plate count on agar, while they are able to maintain some metabolic activity or cellular integrity as evaluated using other methods (16,17). Up to now, few studies on viability and activity changes in VBNC V. parahaemolyticus cells have been carried out using alternative methods to standard techniques.…”

“…Moreover, since some authors have demonstrated the ability of V. parahaemolyticus to ''resuscitate'' from the VBNC state (17,32,33), FCM analyses was used to evaluate physiological and morphological features of the V. parahaemolyticus vegetative form after resuscitation.…”

Biological changes of Enterococcus faecalis in the viable but nonculturable state

“…In this state, bacteria lose their ability to form colonies on culture media (Lleò et al, 1998), but remain alive and capable of producing antigen components (Smith and Oliver, 2006) and virulence factors (Pruzzo et al, 2002). When permissive conditions are restored, VBNC cells may resume cell division (Lleò et al, 2001) and participate in disease transmission and persistence (Xu et al, 1982).…”

“…When bacteria enter the VBNC state, they lose their ability to form colonies on culture media (Lleò et al, 1998), but continue to express antigen components (Smith and Oliver, 2006) and virulence factors (Pruzzo et al, 2002). VBNC bacteria may be able to resume cell division and promote disease transmission and persistence when permissive conditions are restored (Lleò et al, 2001).…”