Stability and survival of VBNC cells - conceptual and practical implications

Cold-stressed populations of the estuarine bacterium Vibrio vulnificus constitute model systems for the “viable but nonculturable” (VBNC) state, and are comprised of nonrecoverable, injured, and fully culturable cells. The nonculturable cells display increased sonication resistance, but also substantial degradation of nucleic acids, raising the possibility that these “VBNC” cells are, in spite of their stability, in fact sublethally or lethally injured. As the standard criteria for viability of VBNC cells (cellular activity or uptake of substrate) are neither necessary nor sufficient criteria, recovery of these cells needs to be shown to prove their viability. Recoverable VBNC cells, however, are not truly nonculturable. In order to resolve this contradiction in terms, it is here proposed to apply the term “viable” only to recoverable cells, and the term “active but nonculturable” (ABNC) for nonrecoverable (non-viable) cells such as lethally injured and senescent cells. Cells which recover only under specific “resuscitation” conditions are to be considered either sublethally injured or, if cellular activity is initially low, dormant. It is suggested that this classification allows the replacement of the “VBNC” category by a more consistent terminology. The term “viable but nonculturable” (VBNC) has been coined to describe a state from which bacterial cells can not be recovered, but in which they maintain certain features of viable cells, such as cellular integrity and activity. It appears to be a common observation that bacteria enter such a “VBNC” state under environmental or laboratory conditions [19, 24, 29]. This “non-recoverable” state has often been interpreted to be a consequence of dormancy. It has to be pointed out, however, that dormancy is characterised by the absence of significant cellular activity, and that dormancy is by definition a reversible phenomenon dormant cells are hence typically inactive and culturable [15]. Is it possible that “VBNC” cells dwell in a genetically determined “refractory” state other than dormancy in which cell division is blocked? Can some of the observations be due to mutations or infections with bacteriophages? Or are “VBNC” cells merely injured or debilitated by the exposure to stressful conditions and, if so, is this injury sublethal or lethal [19]? These questions have tremendous significance for medical microbiology, epidemiology and general microbial ecology. It has been proposed that the discrepancy between plate counts and total counts in natural environments (“Great Plate Count Anomaly”) may be partly explained by bacteria in a “VBNC state”, and that this state may constitute an adaptive strategy of non-sporeforming bacteria allowing survival under adverse conditions [29]. If this were the case, VBNC cells of pathogenic bacteria would provide a potentially To Grow or Not to Grow: Nonculturability Revisited huge and dangerous reservoir for infection which evades detection by most standard methods [8, 24, 26, 28, 30, 34, 36]. What approaches and methods can be employed to shed light on this elusive issue? The methods normally used for the examination of “VBNC” cells are based on cellular activity or uptake of substrate, which are, however, neither necessary nor sufficient criteria for viability. Dormant cells, for example, may not display any detectable activity or uptake. Cells, which have acquired lethal DNA damage, on the other hand, may still display activity. Necessary viability criteria include cellular integrity, intact membrane barrier, presence of DNA and RNA, and potential for protein synthesis. But these are not sufficient criteria, as even lethally injured (non-viable) cells can still display remnants of such activity and integrity. For example, cells can stay morphologically intact even after autoclaving. The ability of a cell to recover and grow (as manifested by “culturability”) is so far the only sufficient criterion for viability, but it may not be considered a necessary criterion. Some standard media and conditions, for example, may be unsuitable for a cell in a certain physiological state to initiate growth or to form visible colonies. So far it has been impossible to devise reliable criteria for viability which are both necessary and sufficient. While use of necessary criteria as the basis of viability assays can create false-positive results, the use of sufficient criteria has the potential of leading to falsenegative results. The choice of approach obviously depends on whether it is intended to estimate the maximum possible risk (thus including all “possibly viable” cells) or whether viability in the strict sense is determined (including only cells that have been proven to be viable). It is here suggested to use the term “viable” only for cells that have been unequivocally shown to be fully viable which, because of above considerations, means that they have to be recoverable. Thus, ultimately, for the practical purpose of detecting viable cells, culturability (defined as the ability to recover under at least one of any conditions, including resuscitation treatments) is proposed to have the status of a necessary criterion for viability. Several groups of investigators have attempted to resolve the issue of whether VBNC cells are truly viable and whether they can be resuscitated [3-5, 7, 9, 10, 12, 13, 16-18, 21, 22, 24, 31, 33, 35]. One of the model organisms for the study of “VBNC phenomena” is the estuarine human pathogen Vibrio vulnificus [25]. Incubation of V. vulnificus under conditions mimicking cold estuarine environments leads to the formation of heterogeneous cell populations comprised of recoverable (“culturable”) cells, and non-recoverable (“nonculturable”) cells [25, 31, 33]. In this experimental system, the part of the population that remains culturable decreases during cold incubation, and contains a fraction of injured cells; the nonculturable cells can not grow in either liquid or solid media, and thus have to be considered “non-recoverable” in the absolute sense [31, 33]. The non-recoverable presumed VBNC cells, however, display improved sonication resistance as compared to growing cells, indicating increased cellular stability [31]. In fact, after several weeks of cold incubation, stability of non-recoverable cells is equal to that featured by (culturable) starved cells which are characterised by high autolysis-resistance. Protein synthesis is undetectable in “aged” nonculturable cells, and the nucleic acids of the majority of these cells are highly degraded, which might explain the loss of culturability