Nonculturability: adaptation or debilitation?

Authors


*Corresponding author. Tel.: +61 (2) 9385 2102; Fax: +61 (2) 9385 1591; E-mail: S.Kjelleberg@UNSW.edu.au

Abstract

Investigation of bacterial survival in natural environments has indicated that some organisms lose culturability on appropriate media under certain conditions and yet still exhibit signs of metabolic activity and thus viability. This reproducible loss of culturability by many bacterial species led to the description of bacterial cells in this state as ‘viable but nonculturable’ (VBNC). It is suggested that the VBNC state is part of the life cycle of non-differentiating bacteria induced by environmental stress. The purpose of this review is to summarize some of the reports which support and refute the hypothesis that the VBNC response is a programmed response. Methods currently used in the determination of viability will be discussed with respect to their advantages and disadvantages. Reports which indicate resuscitation in vitro and in vivo, as well as those that show retention of infectivity and pathogenicity in the case of pathogenic organisms are presented as well.

1Introduction

Bacterial populations in the environment are frequently exposed to stresses due to limitations and changes in nutrient availability, temperature, salinity, solar illumination, and oxygen saturation. Thus, the persistence of bacteria in the environment is to a great part determined by their ability to endure these stresses. While it is generally accepted that non-differentiating bacteria are capable of developing starvation and stress resistance through a series of differentiation-like genetic programs [1], the existence and nature of a viable but nonculturable (VBNC) state has been the topic of intense debate for over 15 years. The formation of VBNC cells has been proposed by some as a survival strategy [2] and as such, is an active process involving the induction of global control networks leading to sequentially regulated differentiation responses. These cells should therefore also have the ability to reverse this program and ‘resuscitate’ when conditions become favorable for growth. Conversely, others argue that the VBNC state may be a moribund condition in which cells become progressively debilitated until cell death finally occurs. These cells may maintain signs of metabolic activity or respiration for some time but are not able to ‘resuscitate’.

Only those bacteria which have been shown to enter the VBNC state in response to natural environmental stresses will be addressed in this review. Sublethal injury which occurs as a result of exposure to treatments such as freeze/thaw, antibiotics, chlorine or other chemicals are not considered germane to the VBNC response and thus are not within the scope of this review. Injury-induced changes in cell populations may appear to be similar to the VBNC state, but instead these cells are culturable under the proper conditions and do not need to be resuscitated to become culturable. In particular, emphasis will be placed on the marine bacterium, Vibrio vulnificus, which is one of the paradigm organisms for the study of the VBNC response. This marine organism is of particular interest due to its ecology and its ability to produce lethal infections in persons who consume raw shellfish.

2The viable but nonculturable state

It is now generally accepted that a number of culturable nondifferentiating bacteria, upon encounter with certain environmental stresses, are not recoverable by normal culture techniques. It has been proposed that viability may be maintained in the absence of culturability, and that this VBNC response is analogous to the stress responses of the differentiating bacteria (e.g. spore formation). Thus the VBNC response may be a genetically programmed physiological response of some bacteria which enhances survival during environmental stress. To complete this analogy, the VBNC cells must be able to exit this dormant survival state and return to an actively metabolizing state when conditions become favorable. While it is now generally accepted that certain conditions can induce the VBNC response in bacteria, reports of resuscitation have been met with skepticism and it is uncertain whether the VBNC state represents a life cycle process or end-of-life process. Therefore, the VBNC debate centers around two related points. Firstly, is the VBNC response a genetically determined developmental cycle principally analogous to the starvation response of other differentiating bacteria? Secondly, are VBNC cells capable of resuscitation, or is the increase in cell numbers reported in a large number of publications on this issue merely the result of growth of a few viable cells? This review addresses the first point by looking at reproducible, predictable changes in metabolic activity and cell features such as membrane alterations, RNA and DNA content, and maintenance of virulence. With regard to point two, reports of both in vivo and in vitro resuscitation are discussed.

3VBNC formation

There have been numerous reports of bacterial species which enter the VBNC state (for a review see [2]) as well as some reports of resuscitation (Table 1). Conditions shown to induce nonculturability differ according to the organism and include such diverse factors as starvation, salinity, visible light and temperature ([2] and references therein).

Table 1.  Studies investigating the viable but nonculturable response
BacteriumResuscitation method
Aeromonas (Vibrio) salmonicidaNutrient addition
Agrobacterium tumefaciens 
Alcaligenes eutrophus 
Campylobacter jejuniAnimal passage
Enterobacter aerogenes 
E. cloacae 
Enterococcus faecalis 
Escherichia coliRabbit ileal loop passage
Helicobacter pylori 
Klebsiella pneumoniae 
Legionella pneumophilaInjection into embryonated eggs
Micrococcus flavus 
M. luteusResuspension in fresh medium with lactate and supernatant from stationary phase cells
Pasteurella piscicidaAddition of nutrient
Pseudomonas aeruginosa 
P. fluorescensTransfer of N-starved cells to fresh medium without glucose
P. putida 
P. syringae 
Rhizobium meliloti 
Salmonella enteritidisNutrient addition
S. typhimurium 
Shigella dysenteriae 
S. flexneri 
S. sonnei 
Vibrio anguillarum 
V. campbelli 
V. choleraeRabbit ileal loop passage, heat shock, human intestine
V. fischeri 
V. harveyi 
V. mimicus 
V. natriegens 
V. parahaemolyticus 
V. proteolyticus 
V. vulnificusTemperature upshift, injection into mice, environmental chamber
Yersinia ruckeriAddition of nutrient

Low temperature is the only factor required for inducing the VBNC state in V. vulnificus as demonstrated in microcosms containing artificial seawater as well as complex nutrient media [2]. When incubated at low temperature, V. vulnificus enters the VBNC state [3], while at room temperature the organism exhibits a classic starvation response [4].

The kinetics of VBNC formation are affected by the nutritional state of the population. Cells entering into stationary phase or starvation conditions prior to low temperature incubation delay VBNC formation [3, 5]. Indeed, incubating V. vulnificus under starvation conditions for as little as 1 h prior to incubation at 4°C significantly decreases the rate at which cells become nonculturable [3, 4].

We have shown that carbon (or multiple-nutrient) and phosphorus starvation are conditions that induce maintenance of culturability during cold exposure [4]. Comparison of the proteins induced during carbon and phosphorus starvation in V. vulnificus led us to the conclusion that carbon and phosphorus starvation may elicit maintenance of culturability via different proteins and most likely by employing different multigenic pathways.

Carbon starvation has previously been reported to induce a temporal progression of protein subsets [1]. Additionally, induction of these starvation proteins has been shown to be ‘cross-protective’ against other stresses [6] such as temperature, oxidative and osmotic stresses, UV, near UV, cadmium chloride and solvent exposure. We have also observed that addition of inhibitors which prevent the development of the starvation program in V. vulnificus also prevents maintenance of culturability during cold incubation (unpublished results). Based on the observations that starvation prior to cold exposure allows for maintenance of culturability and that inhibition of the starvation response prevents this maintenance of culturability, it is our hypothesis that these starvation-induced proteins may provide a protective effect against various stresses, including that of low temperature.

4Methods of assessing the VBNC state

One point of discussion related to the VBNC debate is the determination of viability. A number of methods have been proposed to assess the viability of nonculturable cells, all of which have advantages and disadvantages. Hence, there is no one method that has been agreed upon as suitable in all cases. These methods assess viability by one of two criteria, demonstration of metabolic activity or maintenance of cellular structures.

Methods which have been used as indications of cellular metabolic activity include the use of microautoradiography [7] as well as inducible enzyme activity [8] as an indicator of de novo protein synthesis, the direct viable count method (DVC) [9] which is based on the enlargement of cells upon addition of nutrient, and the reduction of tetrazolium salts as an indication of an active electron transport chain [10]. There are, however, disadvantages associated with the use of these methods. DVC and tetrazolium salt reduction assays require nutrient addition [10, 11] and are thus dependent upon the ability of the organism to respond to the nutrient supplied. Measurements of respiration also have their drawbacks. A range of factors has been shown to affect formazan deposit formation from tetrazolium salt reduction [11], and a recent report showed that the tetrazolium salt, 5-cyano-2,3-ditolyl tetrazolium chloride, inhibits bacterial metabolism [12].

Cell viability assays have also been developed based on the staining of cells with fluorochromes. These methods assess viability by the maintenance of stable cellular structures. Acridine orange direct counts [13] and 4′,6-diamidino-2-phenylindole staining [14] have been used as an indication of the maintenance of intact nucleic acids. Rhodamine 123 has been used extensively as an indicator of membrane potential and with the development of flow cytometry, there has been a surge of methods for characterization of the physiological status of the cells [15]. Permeability of these dyes may be a problem in some organisms. While many of these methods have limitations, it is apparent that VBNC cells maintain certain characteristics of viable cells, such as the potential for metabolic activity and respiration as well as cellular integrity.

5Stability of nonculturable cells

Cells which enter the VBNC state undergo some definite, predictable changes which allow them to persist in the environment for extended periods of time. These changes involve stabilization of the cell wall and membrane, thereby increasing the stability of the cell. Cells which enter the VBNC state appear to maintain gross membrane integrity although changes in membrane composition have been reported. For example, there are reports of cells maintaining a normal cytoplasmic membrane with a decrease of up to 60% of the major fatty acid species compared with culturable cells [16, 17] with the concomitant appearance of new long-chain fatty acids.

Likewise, it was reported that changes in phospholipid composition occurred in Micrococcus luteus[18]. Total amounts of lipid and protein decreased rapidly [19] with the cytoplasm becoming more dense as cells became VBNC. These cells maintained morphological integrity, including an intact nucleoid and structured cytoplasm, with a thickening of the cell wall while exhibiting a loss of permeability barrier by the cytoplasmic membrane. The authors propose this loss of permeability barrier to be one of the reasons for loss of culturability.

The outer and cell membrane of VBNC cells of Vibrio cholerae were found to be intact with a thickening of the peptidoglycan layer observed [20]. Likewise, electron micrographs of VBNC Campylobacter jejuni[21] revealed intact but asymmetric membrane structures. Studies with Aeromonas salmonicida[22] revealed a decrease in protein content and DNA with no loss of lipopolysaccharide occurring.

Weichart and Kjelleberg [23] have examined the stability of nonculturable cells of V. vulnificus. They report that as cells become nonculturable, there is an increase in the resistance to sonication. Cells which have been VBNC for several weeks display sonication resistance which resembles the resistance of starved cells. In addition, these cells resist lysis upon return to room temperature.

Thus, it is apparent that these changes in the cell wall and membranes allow for long-term stability and persistence. These alterations of the membrane may be analogous to the development of resistant cells during starvation, which is believed to occur partly as a result of changes in cell wall cross-linking. Whatever the mechanism, it is evident that these changes are found in several organisms and serve to allow for cellular stability.

6Nucleic acids of nonculturable cells

A number of studies have focused on the nucleic acids of VBNC cells. PCR has been used successfully in some cases for the detection of VBNC cells [24, 25]. However, others have shown that increased amounts of DNA of VBNC cells are required for amplification to occur [26]. It is possible that there is a condensation of DNA upon entry into the VBNC state. This possibility, along with the concurrent thickening of the cell wall, may make PCR less efficient in VBNC cells compared to culturable cells.

In some cases, VBNC cells have been reported to maintain normal amounts of DNA while in other cases DNA amounts decrease. Most studies, however, report decreases of RNA content in VBNC cells. Cells of M. luteus[19] maintain a constant DNA content and exhibit a 50% decrease in RNA content when becoming VBNC. VBNC cells of V. vulnificus have been shown to possess reduced ribosomal and nucleic acid material [2]. It has recently been reported for V. vulnificus[27] that prolonged cold exposure of VBNC cells leads to a gradual degradation of DNA and RNA in an increasing fraction of the population. In a similar study, Yamamoto et al. [25] found that a majority of VBNC cells of Legionella pneumophila contained degraded nucleic acids. This indicates that a small subpopulation maintains intact nucleic acids, and may retain viability and thus the potential to resuscitate, while the majority of the population undergoes a gradual loss of RNA and then DNA.

Based on these observations, it is our hypothesis that there are two phases of the formation of VBNC cells: (i) transition to the VBNC state with concomitant loss of culturability while cellular integrity and intact nucleic acids are maintained, and (ii) gradual loss of cellular integrity and degradation of RNA and DNA ultimately leading to loss of viability.

7Do VBNC cells of pathogenic organisms retain virulence?

The maintenance of potential pathogenicity by VBNC cells is supported by numerous reports of in vivo resuscitation. Experiments with animal models have shown that nonculturable cells may be resuscitated by animal passage and may, in some cases, retain virulence. VBNC V. cholerae and enteropathogenic Escherichia coli were shown to regain culturability after animal passage [28]. Similarly, VBNC cells of E. coli were resuscitated by introduction into ligated rabbit ileal loops, after which culturable cells were recovered [29]. Human volunteers developed clinical symptoms of cholera after ingestion of VBNC V. cholerae, with the pathogen subsequently being isolated in the culturable form from stools of the volunteers [30]. Culturable cells were isolated from feces of volunteers who ingested cells which had been VBNC for 23 days, but not from those ingesting cells of V. cholerae that had been VBNC for 4 weeks indicating that maintenance of infectivity of VBNC cells may be confined to ‘young cells’ while those that have been in the VBNC state for longer may lose infectivity. This loss of infectivity of VBNC cells that occurs with time is further evidence for the proposed stages of the VBNC state. Those cells in the first stage maintain the potential for resuscitation and pathogenicity, while cells in the later stage are in the process of gradual loss of cellular integrity and are thus not capable of resuscitation or colonization.

In another study, suspensions of four different C. jejuni strains which were shown to be nonculturable were fed to suckling mice [31]. Colonization of mice was established by two of the four strains. Similarly, VBNC C. jejuni were resuscitated upon passage through rat gut [32] and were shown to colonize 1-week-old chicks [33]. In contrast, others have demonstrated [34] a lack of colonization of the intestines of 1-day-old chicks when VBNC cells of C. jejuni were administered orally. Likewise, no resuscitation was observed in simulated stomach, ileal, or colon environments [35].

The injection of chick embryos with VBNC L. pneumophila resulted in lethal infection [36] indicating maintenance of virulence of the nonculturable cells. VBNC cells of Shigella dysenteriae were found to remain cytopathic to cultured HeLa cells [7], to maintain biologically active Shiga toxin and to adhere to intestinal epithelial cells [24]. Likewise, V. vulnificus cells which had been VBNC for >8 months were shown to be cytopathic to murine macrophage cultures and resuscitation of cells was demonstrated [2]. Furthermore, maintenance of virulence for VBNC cells of V. vulnificus was demonstrated by Oliver and Bockian [37] when infection of mice with <0.04 CFU resulted in death. It was reported, however, that virulence decreases significantly as cells enter the VBNC state and continues to decrease with time after cells become nonculturable, similar to V. cholerae.

Collectively, the studies discussed above indicate that cells which were initially VBNC retain the capacity to cause disease and are therefore still active. However, at this point it is not obvious if VBNC cells are sufficient to cause disease or if they must first resuscitate and can then cause disease. Furthermore, it appears that even though they are initially VBNC, cells can be activated upon passage through a host. This suggests that some factor is supplied by the host to induce resuscitation and completion of the VBNC cycle. It is unclear what factors are required for resuscitation; it could be an environmental factor or a signal from other actively growing bacterial cells. Unequivocal determination of these factors may come from attempts to resuscitate cells in vitro.

8Reports of in vitro resuscitation

A principal controversy relates to whether reports of in vitro resuscitation depict true resuscitation of viable cells or growth of a few viable cells which escaped detection. There have been numerous reports of resuscitation induced by mechanisms such as nutrient addition [38], transfer to fresh medium [39], temperature upshift [40], and heat shock [41]. In most of these reports, resuscitation could only be accomplished with those cultures which had been VBNC for a short period of time. There have also been a number of reports which refute these data and indicate that resuscitation was nonetheless due to regrowth of a few viable cells remaining in the cultures [5, 17].

More recently, there have been publications on resuscitation in conditions where regrowth of viable cells is less likely to have occurred. V. vulnificus was shown to resuscitate when placed in situ in warm marine water [42]. Roth et al. [43] demonstrated a rapid loss of culturability when cells of E. coli were exposed to 0.8 M NaCl. These cells accumulated ATP intracellularly and were resuscitated by the addition of the osmoprotectant betaine to the culture. After the addition of betaine, the number of colony forming units returned to the control level within 2 h. This resuscitation was seen even when chloramphenicol was added to the culture, precluding the possibility of growth of a few cells.

Heat shock treatment induced resuscitation of VBNC cells of V. cholerae[44]. These cells had been nonculturable for up to 86 days and became culturable on solid media after exposure to 45°C for 1 min. Growth of culturable cells was not detected without prior heat shock.

A detailed description of resuscitation has been recently reported for M. luteus. While these cells have been reported to be ‘dormant’ as opposed to VBNC [15] and thus show no evidence of metabolic activity when assessed by the methods used to indicate activity in VBNC cells, the results from this study may nonetheless be applicable to resuscitation of VBNC cells. It was reported that in a microcosm exhibiting a culturability of <0.001%, 70% of the cells could be lysed upon inoculation into fresh lactate minimal medium containing penicillin, showing that a significant portion of the cells had the capacity to engage in cell wall synthesis. In a recent publication [45], it was shown that this population is heterogeneous and consists of subpopulations of viable, nonculturable and dead cells. The resuscitation capacity of these populations was 1000–100 000-fold greater when samples were diluted into liquid media containing supernatants taken from stationary phase of batch cultures, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells [46]. It was demonstrated that the presence of viable cells in a population before resuscitation is a requirement for the recovery of dormant cells [47].

This requirement for the presence of viable cells in resuscitation cultures may help to explain the inconsistency of resuscitation experiments. This requirement may be due to either the presence of the cells themselves or the secretion of some signal which ‘awakens’ the VBNC population. Recently, such a ‘resuscitation factor’ has been purified and characterized [48]. These authors report the resuscitation of dormant cells of M. luteus was increased 100-fold when the purified factor was added to cultures of dormant cells at ng ml−1 concentrations. Clearly, the possibility of signal molecules involved in the entry into and exit from the VBNC state has far-reaching ramifications.

9Conclusions

It is well known that in nature, survival over extended periods of nutrient deprivation and exposure to other environmental stresses is the rule rather than the exception. It seems reasonable, therefore, that cells have developed strategies of survival under these conditions and that several different stress-induced responses exist. Regardless of the inducing stimulus for VBNC formation, consistent physiological changes occur including size reduction, decrease in RNA content and in some instances DNA content, condensation of the cytoplasm, inability to multiply, reduction of metabolic activity, increased resistance to certain environmental stresses, and persistence in the environment. It has been demonstrated that dead cells when released into soil microcosms disappear very rapidly, while VBNC cells persist for long periods of time [49].

The most likely explanation for the accumulated data remains that cells in the environment are induced, by some factor, to enter this state of reduced metabolism described as the VBNC state. The inducing factor is some environmental stress, but cells undergoing the transition may produce a signal which other cells can detect and respond to as well. It has been shown that supernatants from nonculturable or starving cells [5, 18] can induce nonculturability in growing cells. In addition, just as quorum-sensing bacteria produce extracellular signals, growing cells may produce a factor or signal needed for resuscitation to occur [46, 47] as has been reported for M. luteus. This may explain the discrepancies seen in numerous resuscitation attempts. More than likely, the factors needed for resuscitation remain elusive in most cases.

In several cases, VBNC cells have been shown to maintain some potential for metabolic activity as well as possible infectivity. It has been demonstrated that V. cholerae 01 can be detected in the natural environment through the use of fluorescent antibodies in large numbers of samples from which the organism could not be cultured. Enterotoxigenic E. coli was shown to produce enterotoxin while in the VBNC state [50]. The fact that these pathogens can persist in the environment and produce toxin while not culturable is a true public health concern. Thus, study of the VBNC state is not merely one of pure curiosity but rather one that involves genetics, physiology and public health concerns.

Numerous studies have indicated the heterogeneous nature of most cultures of VBNC cells. Recent work with M. luteus[48] indicates that a culture starved for extended periods of time consists of viable, nonculturable and dead cells. It has been shown for both V. vulnificus[27] and L. pneumophila[25] that the VBNC cultures contain some cells with intact DNA and RNA (nonculturable) while a significant proportion of the population consists of cells which maintain DNA and RNA that has undergone degradation (nonviable cells). A similar trend was seen for induced enzyme activity with a subpopulation remaining responsive while the activity was lost in other cells [8]. Similarly, these same trends were observed regarding toxicity and long- versus short-term VBNC cells.

The physiological and molecular basis for entry into and exit from the VBNC state remains obscure. We propose that there are subpopulations within VBNC cultures and that these subpopulations are a reflection of the stages of VBNC formation. In the initial stage, cells lose culturability while maintaining intact membranes and RNA and DNA. These cells thus maintain the potential for resuscitation (viability). In the later stages of the VBNC state cells gradually experience degradation of nucleic acids and thus lose the potential for resuscitation (nonviable). Definitive proof that the VBNC state represents a true programmed physiological response will depend on the identification and characterization of the genetic determinants that regulate this response, which will allow for an understanding of the biology of this phenomenon.

Acknowledgements

Work in the authors’ laboratory is supported by grants from the Australian Research Council and the Centre for Marine Biofouling and Bio-Innovation.

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