The viable but nonculturable phenotype: a crossroads in the life-cycle ...

Rev Environ Sci Biotechnol (2009) 8:245–255 DOI 10.1007/s11157-009-9159-x

REVIEW PAPER

The viable but nonculturable phenotype: a crossroads in the life-cycle of non-differentiating bacteria? Isabel Barcina Æ Ine´s Arana

Published online: 26 April 2009 Ó Springer Science+Business Media B.V. 2009

Abstract In nature, prokaryotes must face alternating periods of prosperity and adversity. Differentiating bacteria confront situations of adversity by developing resistant structures. When there is a plentiful period, they adopt a vegetative state and when the period is adverse, a resistant structure, thereby completing a cycle. Non-differentiating bacteria do not develop such morphological distinct resistant structures. It has been proposed that many of these bacteria withstand periods of adversity by adopting the viable but nonculturable phenotype (VBNC). Bacteria of this phenotype conserve detectable metabolic function but become unculturable. Is it possible that the VBNC phenotype can revert to a culturable state, and vice versa, thus establishing a life-cycle? This review presents and evaluates different hypotheses regarding this question. Moreover, it attempts to analyse and proffer answers to other questions related to this phenotype. Is this a successful phenotype which prolongs survival? Is this a strategy for the survival of individual cells, or is it a strategy for the survival of a population? Finally, is it possible that this phenotype is, in fact, an example of altruistic death?

I. Barcina (&) I. Arana Department of Immunology, Microbiology and Parasitology, Faculty of Science and Technology, University of the Basque Country, 644 PO, 48080 Bilbao, Spain e-mail: [email protected]

Keywords Non-differentiating bacteria Survival strategy Viable but nonculturable

1 Introduction Prokaryotes are ubiquitous, although specific abiotic and biotic conditions are required for each species’ optimum development. However, the environmental conditions in which prokaryotes live generally suffer great seasonal and circadian changes, such as temperature variations, changes in levels of solar radiation and in the availability of nutrients, the latter alternating between situations of feast and famine. These changes are particularly pronounced in aquatic environments. What is more, great masses of prokaryotes are transported from their natural habitats to other habitats, thereby losing their status of autochthonous bacteria, on occasions the dominant species, and becoming allochthonous bacteria, often in a minority. In this context, one of the cases which attract most interest, due to its evident repercussions for public health, is that of the number of intestinal bacteria and bacteria of other types contained in the wastewater which enters our waterways. It is easier to triumph, grow and colonise in times of prosperity than it is to survive during periods of adversity. The perdurability of species largely depends upon their ability to survive in adverse conditions (Colwell 2000; Morita 1980). When environmental

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conditions become adverse, bacteria must be able to withstand stress and to adopt strategies which permit them to survive until suitable conditions for growth and development are re-established. The expression of an advantageous phenotype ensures continuance and even proliferation of prokaryotes in a restrictive or unfavourable environment. Some bacterial genera confront adverse conditions by developing resistant structures, for example: endospores, myxospores, cysts, chlamidospores and achinetes. These structures are formed by vegetative cells undergo transformation processes. Perhaps the best known case is that of the formation of the endospore which is particularly interesting not only because of the complexity of the transformation process itself, but also because it is an example of an authentic process of differentiation (Eichenberger 2007; Schaechter et al. 2006). When environmental conditions become suitable once more, these resistant forms once again transform themselves into vegetative cells, and in this way complete a cycle of life. The high growth rate of prokaryotes favours the re-establishment of large populations and thus assures the perdurability of the species. Nevertheless, very few bacteria are able to develop resistant structures, but the success that prokaryotes have had in colonising the planet, and the abundance of both autochthonous and allochthonous bacteria in ecosystems, leads to the following question: how do prokaryotes which do not develop resistant structures withstand adverse conditions?

2 The viable but nonculturable state In the 1980s, the application of methods which showed cellular activity combined with the use of the epifluorescence microscope permitted it to be demonstrated that, in adverse conditions, some bacteria were able to maintain detectable metabolic functions although they were nonculturable by the methods available. Xu et al. (1982) defined this phenomenon as the viable but nonculturable (VBNC) state. Both the influence of environmental factors in the dynamics of the formation of this phenotype, as well as the morphological characteristics and the physiological properties of cells in the VBNC state, including gene expression and the modulation of novel proteins, have been widely studied (Arana and Barcina 2008; Asakura et al. 2007,

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Rev Environ Sci Biotechnol (2009) 8:245–255

2008; Barcina et al. 1997; Coutard et al. 2007; Cuny et al. 2005; Day and Oliver 2004; Lleo´ et al. 2000; Muela et al. 1999, 2008; Smith and Oliver 2006; Srinivasan et al. 1998; Wong et al. 2004a). The number of species known to enter the VBNC state constantly increases (for a review see Oliver 2005). The list includes Gram positive and Gram negative bacteria, non-pathogens and a large number of human pathogens (i.e. Campylobacter spp., Klebsiella spp., Legionella, Lactobacillus plantarum, Lactococcus lactis, Micrococcus spp., Mycobacterium tuberculosis, Pseudomonas spp., Salmonella spp., Vibrio spp., etc.), as well as the universal indicators of faecal contamination, Escherichia coli and Enterococcus faecalis. This raises serious questions about, for example, the interpretation of routine water or food testing and testing the effects of antibiotics or the sterility of medicinal drugs, all cases in which this VBNC state appears to mask the actual number of viable cells, constituting a factor which should therefore be taken into account during microbial inspection. Consequently, this is not merely a question of shedding light upon a scientific enigma, but, due to the importance of bacterial populations in the VBNC state and the influence which they have on the lives of human beings, it is absolutely necessary to establish the biological significance of this phenomenon. When this objective is achieved, it will permit the clarification of certain questions which at present remain unanswered. Firstly, does the VBNC phenotype form part of the life-cycle of non-differentiating bacteria? Can the existence of this phenotype be considered as a survival strategy within a population, leading to the maintenance of some species? Moreover, is it possible that different strategies of perdurability derive from the VBNC phenotype? The aim of this review is not only to give an overall view of the VBNC phenotype, but also to look into the underlying molecular mechanisms which are involved in entering and leaving this state, and their biological significance.

3 The role of the VBNC phenotype. A phenotype for situations of adversity? Since this state was described, the interpretation of its significance has provoked a great deal of controversy within the scientific community. Firstly, some critics were of the opinion that cells in the VBNC state were,


in fact, dead cells. Those who defended this thesis maintained that the techniques used to detect cells in this state only revealed the existence of residual biochemical activity outside the regulatory cellular pathways (Kell et al. 1998; Villarino et al. 2000). Furthermore, it was argued that the expression viable but nonculturable was an oxymoron given that for decades a lack of culturability had been a synonym of non-viability (Barer and Harwood 1999; Mukamolova et al. 2003). It is true that the terminology which surrounds the VBNC state is confusing and has been criticised by a great many researchers. Barer et al. (2000) proposed that these cells lacking culturability may be unambiguously considered as active but nonculturable (ABNC) cells. Probably, both this proposed term and others provide a more exact description but none have been totally accepted or globally adopted by the scientific community (Barer et al. 2000; Kell et al. 1998; Mukamolova et al. 2003). Given the controversy generated by the term VBNC, a consensus of opinion in respect of this would be an advantage.

3.1 A step in the life-cycle of some non-differentiating bacteria Nowadays, the most widespread opinion is that the VBNC phenotype is made up of live cells which have physiological activity, but which are infertile while in the VBNC state. Using molecular techniques, gene transcription and the synthesis and/or modulation of proteins in cells in the VBNC state have been demonstrated (Asakura et al. 2007, 2008; Coutard et al. 2005; Fischer-Le Saux et al. 2002; Gunasekera et al. 2002; Heim et al. 2002; Karunasagar and Karunasagar 2005; Lleo´ et al. 2000; Smith and Oliver 2006). These facts ratify unequivocally that populations in the VBNC state are formed by live cells. Based on this premise, many studies have been aimed at establishing whether these cells are able to recover their capacity to generate new cells (Gupte et al. 2003; Lleo´ et al. 2001; Magarinõs et al. 1997; Ohtomo and Saito 2001; Whitesides and Oliver 1997; Wong et al. 2004b). If it is possible to pass from the VBNC state to the culturable state, then perhaps the VBNC phenotype could be considered as part of the life-cycle of non-differentiating bacteria. The cycle begins when environmental

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conditions change from those suitable for growth to those which are adverse for it. Culturable cells then enter a viable but nonculturable state, this being the natural phenotype for situations which are inadequate for growth, and later return to the culturable phenotype, once the environmental stress has disappeared. Unfortunately, demonstrating that VBNC cells are able to recover their culturability is not an easy task (Arana et al. 2007; Arana and Barcina 2008). Apart from the complexity of the process itself, there are methodological problems which still remain unresolved. Research is carried out on heterogeneous populations comprised of both culturable and nonculturable cells, which means that in the majority of cases it is practically impossible to discern if an increase in culturable cells within a given population is due to what is termed as the resuscitation of the VBNC cells or, on the other hand, to the growth of culturable cells which are hidden within the general population (Bogosian et al. 2000; Jiang and Chai 1996; Kell et al. 1998). Different methodological strategies have been proposed orientated towards obtaining homogenous populations exclusively comprised of VBNC cells. Most of these strategies are based on methods using dilution (Oliver 2005; Wai et al. 2000), which are frequently combined with the use of inhibitors of cellular replication aimed at inhibiting growth in the residual culturable population (Basaglia et al. 2007; Ohtomo and Saito 2001; Wong et al. 2004b). Another approach which has been used is that of the physico-chemical separation of culturable and nonculturable cells using density-gradient centrifugation (Arana et al. 2008; Cuny et al. 2005; Desnues et al. 2003). With the objective of stimulating resuscitation, the use of diluted culture mediums has been recommended (Dukan et al. 1997; Whitesides and Oliver 1997), thereby avoiding an increase in cellular stress derived from the oxidative metabolism (Wong et al. 2004b). The addition of quenchers of free-radicals to the culture mediums (Mizunoe et al. 2000; Wai et al. 2000), the use of semi-solid mediums to encourage diffusion of the nutrients (Wai et al. 2000), and/or the stimulation of resuscitation by using different biological molecules (Kaprelyants et al. 1994; Mukamolova et al. 1999) have also been proposed. In the case of pathogenic bacteria, resuscitation has been

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approached by the inoculation of natural hosts with populations of VBNC cells (Baffone et al. 2006; Dhiaf and Bakhrouf 2004). Arana et al. (2007) report that in addition to the methodological problems explained above, there have also been several other factors which have contributed to the disparity among the published results and the varying opinions of the scientific community. Among these factors, the following should be highlighted: the lack of stringent controls in many studies, the time spent by the cells in the VBNC state, the differences among the experimental designs used, the use of cell populations subjected to very different adverse conditions which provoke a loss of culturability due to different lesions, and obviously, the complexity of the biological process itself. It is within this scenario that some authors have demonstrated that true resuscitation does occur, at least in some bacteria. The removal of environmental stress by temperature up-shift from 5 to 20°C results in the resuscitation of Vibrio vulnificus (Whitesides and Oliver 1997) and Helicobacter (Kurokawa et al. 1999). The same strategy, the removal of environmental stress, led to the resuscitation of Salmonella (Gupte et al. 2003), Enterococcus (Lleo´ et al. 2001), Pasteurella pisticida (Magarinõs et al. 1997), diverse species of Vibrio (Nilsson et al. 1991; Wong et al. 2004b), E. coli (Ohtomo and Saito 2001), Aeromonas (Maalej et al. 2004; Wai et al. 2000) and Micrococcus (Mukamolova et al. 1998), etc. Authors defend, at least in the case these bacteria, that the VBNC state forms part of their life-cycle and therefore, constitutes a survival strategy in the face of adversity. Nevertheless, other studies have reported that nonculturable bacteria cannot be resuscitated (Arana et al. 2007; Kolling and Matthews 2001; Ziprin et al. 2003). Irrespective of the contradictions concerning the capacity for resuscitation of some bacteria, the actual number of species in which resuscitation has been clearly demonstrated is very low in comparison with the number of bacterial species which are known to adopt the VBNC phenotype. Furthermore, if we take into account the fact that cells may remain in this state for long periods of time (Amel et al. 2008; Flint 1987; Srinivasan et al. 1998), it is of fundamental importance to consider the role which populations of bacteria in the VBNC state play in nature.

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3.2 A state whose role is based on its activity and the numerical importance of its populations There is an underlying idea that the VBNC phenotype is only of interest if cells in this state are able to recover culturability, that is to say, if the cells undergo a process of resuscitation (Keep et al. 2006; Oliver 2005). Clearly, there are obvious and serious consequences if pathogenic bacteria or other types of bacteria are able to regain the capacity to colonise and invade, but we must not limit ourselves to only considering this option. Bacterial populations in the VBNC state have importance in their own right, independently of whether or not they have the capacity to resuscitate. These VBNC populations maintain activity and therefore, participate in the functioning of ecosystems, in the carbon cycle and in energy production. Moreover, pathogenic bacteria in this state are still able to produce toxins, thus having a negative effect on their hosts (Huq et al. 2000; Karunasagar and Karunasagar 2005). We must not forget that although bacteria in this state drastically reduce their activity, their density may be such that the global result of this activity can be of quantitative importance.

3.3 A phenotype which participates in the maintenance of species? A survival strategy of populations? We have set out arguments which show that the VBNC phenotype could form part of the life-cycle of certain non-differentiating bacteria and that it could contribute to the functioning of ecosystems. Nevertheless, every day there are more authors who consider this cellular state as a preamble to cell death (Aertsen and Michiels 2004; Bogosian and Bourneuf 2001; Nystro¨m 2001, 2003). In this context, Joux and Lebaron (2000) report that adverse conditions in a marine environment cause a progressive deterioration in Salmonella typhimurium, a progressive decay of measurable physiological capacities which lead to cell death. Desnues et al. (2003) associate the process with increased and irreversible oxidative damage in nonculturable E. coli cells which affects various compartments of the cells. In both studies, the process


culminates with a population which is largely made up of cells with no apparent activity, cells which are non-viable (perhaps, dead cells?). A similar chain of events culminating in the degeneration of the nucleoid is also described by Muela et al. (1999) for E. coli populations maintained in river water. A global analysis of the dynamics of cellular subpopulations throughout the survival process shows that the VBNC phenotype by no means constitutes the largest subpopulation and, therefore, does not appear to be a successful phenotype, but rather a transitory one (Arana et al. 2007). The aforesaid leads us to ask two questions. What is the role of this phenotype in the survival process? Is it possible that the dynamics of the VBNC subpopulation are aimed at maintaining the remaining culturable population? A review of the published studies of the last 20 years on the survival of bacteria, principally Gram negatives in hostile environments, confirms the maintenance of a residual population (102–104 cells ml-1) which continues to be culturable despite the deterioration of the majority of the population (Arana et al. 2001; Flint 1987; Muela et al. 1999; Ritchie et al. 2003). This fact is attributed to the intrinsic heterogeneity of bacterial populations and may be an extra mechanism to assure perdurability of the species. Barer and Harwood (1999) propose that in a microcosm containing a single species of bacterium, survival may be viewed as a population phenomenon, the aim of which is the perdurability of the species represented as the overall population. Aertsen and Michiels (2004) report that following cell division; the distribution of mRNA and proteins among the daughter cells is not symmetric, which means that there is proteomic heterogeneity among the cells which form a population. These differences may result in different morphological and physiological phenotypes, especially if this unequal protein share involves minority proteins. Bacteria can protect themselves from various types of stress at the cost of suspending their growth. According to Kussell et al. (2005), in nutrient-rich environments, in the absence of such stress, bacteria face a crucial choice between two strategies: proliferate and risk death if stress conditions are encountered or suppress growth and be protected under such conditions. Persistence represents a risk-reducing solution to this dilemma, a kind of insurance policy

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wherein the majority of the population proliferates quickly under growth conditions, but a small fraction significantly suppresses growth. These slow growing cells can save the population from extinction during times of stress. The actual investment in such an insurance policy, namely the number of persister cells, is known to be genetically determined and could therefore be subject to evolutionary adaptation (Kussell et al. 2005; Shah et al. 2006). Several authors (Aertsen and Michiels 2004; Arana et al. 2004; Cuny et al. 2005) have proposed that in the case of serious damage by stressful factors, some cells may excrete organic molecules which then provide for other members of the population thereby assuring species survival until better times come. Arana et al. (2004) simultaneously studied the changes in an E. coli population in adverse conditions and the chemical variations in the surrounding medium. They detected that throughout the survival process, which included entrance into the VBNC state, E. coli released into its surroundings organic molecules (aminoacids, proteins and carbohydrates). The variations in the concentrations of aminoacids and carbohydrates in the medium throughout the survival process indicated the coexistence of a double process of excretion and uptake. We propose that the VBNC phenotype may represent an intermediate stage in an altruistic death process which forms part of a survival strategy of species. As part of the response to environmental stress, a percentage of the population releases organic molecules into the surrounding medium. The excretion of these molecules may, among other things, cause a loss of culturability and the consequent appearance of a VBNC subpopulation. At the same time, the organic material excreted may be used by other members of the population to repair cell damage and alleviate stress. In this way, a culturable subpopulation can be maintained which will favour growth and expansion of the species once a suitable change in environmental conditions has taken place. The coexistence of altruistics or survivors in bacterial populations is a successful survival strategy, and an illustration of multicellular behaviour in bacteria. Most available data on this subject comes from studies carried out in batch cultures. To extrapolate the proposed hypotheses to the behaviour of bacteria in natural conditions is a task which undoubtedly involves risks and must therefore be undertaken with

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great care. It is unlikely that material excreted by free E. coli cells in a river can be taken by other members of the same species. If this is the case, then the organic molecules will form part of the dissolved organic carbon pool which can be assimilated by the ecosystem’s microbial population. However, if we concentrate on more specific environments such as biofilms or aggregates, omnipresent in any type of habitat, then the environmental conditions are more similar to those in the aforementioned studies and thus, the hypotheses proposed are able to be extrapolated. A biofilm is a complex association of microorganisms, made up of just one or several species, and which adheres to a surface. An aggregate also consists of an association of microorganisms, but this does not adhere to a surface. In oligotrophic aquatic mediums there is a widespread alternation between cells in a planctonic state and a bentonic state. The bacteria inside a biofilm or those of an aggregate form a coordinated, functional community. Costerton et al. (1995) and Guerrero and Berlanga (2001) propose that bacteria inside a biofilm are protected from sudden changes in their environmental conditions by maintaining a primitive homeostasis and they emphasise the positive selection of biofilms in present day ecosystems. Today, there is an absolute consensus of opinion which considers aggregation to be a strategy to face the stress imposed by adverse conditions (Grossart et al. 2003; Shoji et al. 2008). In a natural environment, biofilms can also provide a safe haven for pathogenic bacteria, protecting them from a variety of physicochemical stresses, including UV light, oxidative stress, and biocidal agents. Additionally, since bacteria in natural environments are subject to predation by protozoa, bacteriovorous microorganisms like Bdellovibrio spp., and bacteriophages, it is probable that biofilms provide a mechanism for the persistence of bacterial pathogens in the environment. September et al. (2007) reports the presence of biofilms containing E. coli, Aeromonas, and Pseudomonas spp. in drinking water, which signifies a potential risk to human health if pathogenic forms are present. Furthermore, it has been suggested that biofilms play a significant role in the transmission and persistence of human diseases (Huq et al. 2008). Both biofilms and aggregates form a set of microhabitats which are interrelated. In the superficial layers which are in contact with the surrounding

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medium, the oxygen tension and the availability of nutrients may be, depending on the ecosystem involved, optimum for bacterial growth. In the interior, the situation is less favourable given that both the level of oxygen and that of nutrients is diminished, either because of consumption or due to problems of diffusion. In these internal layers, in which conditions which are unsuitable for growth predominate, cellular interaction, without the hindrance of physical distance, becomes especially relevant. In the interior of an aggregate or a biofilm, the organic material excreted by some cells can be used by other cells of the same species or by cells of different species to repair lesions and to survive, thereby assuring the survival of the species.

4 Molecular approach Bogosian and Bourneuf (2001) report that cells exposed to hostile environments become nonculturable due to cellular deterioration, which can be a stochastic phenomenon, whereas Aertsen and Michiels (2004) and Nystro¨m (2003) report that cells exposed to hostile environments become nonculturable due to cellular deterioration which is genetically programmed. Morphological and physiological changes in cells during the transition from culturable to the VBNC state have been exhaustively studied (Arana and Barcina 2008; Barcina et al. 1997; Cuny et al. 2005; Oliver 2005). Comparatively, the knowledge about the underlying molecular mechanisms is limited. Aertsen and Michiels (2004) report that although the VBNC phenomenon is thought of as a protective response, comparable with sporulation (Bacillus) or dormancy (Micrococcus luteus), so far no mutations or genes have been found as evidence for an underlying pathway triggered in response to stress. However, the expression (transcription) of genes in VBNC populations has been demonstrated (Coutard et al. 2005; Desnues et al. 2003; Fischer-Le Saux et al. 2002; Gunasekera et al. 2002; Lleo´ et al. 2000; Smith and Oliver 2006). Obviously, merely proving the existence of gene activity in this state does not shed light upon the molecular mechanism which regulates the transition of cells from the culturable to the VBNC state. Proteomics enables one to determine the tools that organisms use to survive and proliferate in a given


environment (Nystro¨m 2001). This molecular approach has revealed that new proteins are synthesised when the environmental conditions become inadequate for growth. Proteins are known to be expressed in response to thermal stress, oxidative stress, starvation and pH changes etc. Some of these protein species are specific, while others provide cross protection against various types of stress (Blankenhorn et al. 1999; Etchegaray and Inouye 1999). The studies carried out by Hengge-Aronis (1993, 2002) and other researchers (Ferenci 2001; Klauck et al. 2007) have helped to discover the genetic and metabolic changes which take place in the transition from active growth to stasis. It is probable that a similar approach is a useful tool for the analysis of the changes in the proteome which accompany the transition from the culturable state to the VBNC state. Therefore, once the changes in protein expression and modulation are known, it may be possible to establish which genes are involved in this process. Given the large number of proteins contained in a microorganism, the comparative analysis of the total proteome may not only be extremely complex, but may also lead to error, especially if the changes involve minority proteins which could play a role in the process. The studies carried out in this field demonstrate that fractioning and simplifying the samples can greatly improve the chances of identification and assignment of function to lower-abundance proteins (Wang et al. 2006). On the other hand, the use of bacteria whose entire genome sequence is known, plus the development of the 2-DE protein database, provides an opportunity for the investigation of gene expression responding to environmental changes to establish the key proteins which control bacterial growth and death. Recently, the protein expression patterns between culturable and VBNC populations have been compared. Thus, for E. coli (Asakura et al. 2007, 2008; Muela et al. 2008) or Ent. faecalis (Heim et al. 2002) differences between proteomes indicated a distinct physiological state. Kong et al. (2004) demonstrate that low temperature incubation of V. vulnificus turn out to loss of catalase activity, making the cells hydrogen peroxide sensitive, and paralleling the entry into the VBNC state. Moreover, Asakura et al. (2007) report that hydrogen peroxide induces populations of E. coli O157:H7 in the stationary phase to pass to the VBNC

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state. In this case, at the time when the VBNC state is adopted, there is a simultaneous and significant decrease in the expression of hydrophobic proteins. Among other changes, those which particularly stand out are the over-expression of the OmpW protein in the VBNC state, together with the prevalence of the EF-TU protein that was identified as the most consistent protein, even after 48 h of exposure to hydrogen peroxide in the total absence of a culturable population. The behaviour of the EF-TU protein coincides totally with the results found in E. coli by Muela et al. (2008). Heim et al. (2002) only detect a protein spot (approximate molecular mass of 30 kDa at pI 6) exclusive to VBNC cells, and Muela et al. (2008) do not detect any outer membrane proteins which are exclusive to the VBNC state in E. coli. However, despite the fact that the conclusions of these two studies do not coincide, in both studies significant changes in the expression of proteins were seen during the culturable-VBNC transition. Heim et al. (2002) maintain that the VBNC state constitutes a physiologically distinct state within the life-cycle of Ent. faecalis, which is activated in response to multiple environmental stresses, whereas Muela et al. (2008) report that the most important changes in gene expression take place before entrance into the VBNC state, while the cells are confronting the stress and still remain culturable. The latter authors’ interpretation of entrance into the VBNC state is that it may be considered as an absence of or a failure in the response to stress which leads to the loss of culturability. When evaluating the apparent disparity between the conclusions of both studies, we must not forget the substantial difference in the behaviour of these two bacteria. Lleo´ et al. (2001, 2003) show that, after entering the VBNC state, Ent. faecalis are susceptible to resuscitation and they concluded that the VBNC state forms part of the life-cycle of this species. However, Arana et al. (2007) and Bogosian and Bourneuf (2001) report that E. coli cells are not able to resuscitate from this state. Similar studies (Orrunõ 2009) in which the changes of the subproteome of the outer membrane of Pseudomonas fluorescens CHA0 under different conditions of stress are analysed, found that while temperature provoked significant changes, starvation was responsible for the expression of only two outer membrane proteins.

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Although proteomic is considered to be a very useful tool with which to know the molecular mechanism which underlies the cultivable/VBNC/ cultivable transitions, the studies which have so far been carried out are by no means conclusive. It seems clear that the VBNC phenotype, apart from representing a bacterial response to stress, is not a definitive state but a phenotype of transition, a crossroads in the life-cycle of non-differentiating bacteria. The most likely options for a cell after adopting the VBNC phenotype appear to be either resuscitation or participation in a process of altruistic death. We have the necessary tools with which to study this phenomenon and they are easily available, but nevertheless, our knowledge is still insufficient. We have before us an exciting field of study which is not only of great biological interest, but which also has important repercussions for society. Acknowledgments We thank to Adele Hopley for assistance with the translation to English.

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