Resuscitation from the VBNC State

For the VBNC response to represent a true survival response it must be possible for the cells to exit this dormant state and return to a fully active and culturable state. Such a reversal in physiology is termed resuscitation, and often is triggered simply by the removal of the stress which initially induced the VBNC response. In V. vulnificus, for example, exit from the low temperature-induced VBNC state is triggered by a temperature upshift (e.g., from 5°C to room temperature). After such a shift, culturable cells rapidly (typically within 8 hours) begin to appear, and population levels approximately equaling the original levels are generally observed within 12-24 hours (Fig. 1.4). During this time, the small coccoid cells which result as the cells enter the VBNC state are replaced by rods typical in size for V. vulnificus.13

Table 1.1. Bacteria described to enter the VBNC state

Aeromonas salmonicida

Agrobacterium tumefaciens

Campylobacter jejuni

Enterobacter aerogenes

Enterococcus faecalis

Escherichia coli (including EHEC strains)

Helicobacter pylori

Klebsiella pneumoniae

Lactobacillus plantarum

Legionella pneumophila

Micrococcus luteus

M. varians

Pasteurella piscida

Pseudomonas aeruginosa

P. fluorescens

P. putida

P. syringae

Salmonella enteritidis S. typhimurium Shigella dysenteriae S. flexneri S. sonnei Vibrio anguillarum V. campbellii V. cholerae V. fischeri V. harveyi V. mimicus V. natriegens V. parahaemolyticus V. proteolytica

V. vulnificus (biotypes 1 and 2)

Fig. 1.4. Changes in culturable cell (plate) counts and cell morphology during temperature downshift to 5°C and subsequent resuscitation of the nonculturable cells by incubation at ca. 22°C. Reprinted with permission from: Nilsson L et al. J Bacteriol 1991; 173:5054-5059.

While entry into a VBNC state has been described by many researchers and for many different bacterial species, demonstrating resuscitation for these cells has not always been a simple matter. Indeed, while some bacteria like V vulnificus can be resuscitated by a simple reversal of the inducing stress, in others it has been quite difficult to show. We now realize that resuscitation may be an extremely complex event, one which may be quite difficult to demonstrate in the lab. In the case of L. pneumophila, simple addition of nutrients to the cells (which enter the VBNC state in response to nutrient deprivation) does not reverse the dormancy. It was found, however, that the addition of certain amoebae, which are natural hosts to this bacterium in the aquatic environment, does allow resuscitation of this causative agent of Legionnaire's disease.14

Other problems also exist in demonstrating resuscitation from the VBNC state. It has been difficult to overcome the argument that what was being observed in the name of "resuscitation" was, in fact, regrowth of a few culturable cells which had escaped detection during plating of the population under study. However, we have recently presented very strong evidence that, at least in the case of V. vulnificus, true resuscitation does occur.15 Our studies employed extensively diluted populations of VBNC cells in which it was statistically impossible that any culturable cells were present. Resuscitation of these populations occurred at such a rapid rate that, if it were due to regrowth of culturable cells, they would have to have had a doubling time of approximately 6 minutes. This is clearly an impossible generation time for cells incubated at a suboptimal temperature without nutrients or aeration (Fig. 1.5). We also observed that the presence of nutrients appears to inhibit (but not kill) VBNC cells, and this may be the reason such cells are "nonculturable" when plated onto the high organic nutrient media routinely employed for bacterial culture.

Fig. 1.5. Time required for resuscitation of VBNC V. vulnificus cells. Cells from a VBNC microcosm (< 3.3 x 101 cfu/ml) were shifted to room temperature and aliquots removed at hourly intervals and plated onto HI agar. Reprinted with permission from: Whitesides MD, Oliver JD. Appl Environ Microbiol 1997; 63:1002-1005.

Fig. 1.5. Time required for resuscitation of VBNC V. vulnificus cells. Cells from a VBNC microcosm (< 3.3 x 101 cfu/ml) were shifted to room temperature and aliquots removed at hourly intervals and plated onto HI agar. Reprinted with permission from: Whitesides MD, Oliver JD. Appl Environ Microbiol 1997; 63:1002-1005.

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