Abstract

Many bacteria, including a variety of important human pathogens, are known to respond to various environmental stresses by entry into a novel physiological state, where the cells remain viable, but are no longer culturable on standard laboratory media. On resuscitation from this ‘viable but nonculturable’ (VBNC) state, the cells regain culturability and the renewed ability to cause infection. It is likely that the VBNC state is a survival strategy, although several interesting alternative explanations have been suggested. This review describes the VBNC state, the various chemical and physical factors known to induce cells into this state, the cellular traits and gene expression exhibited by VBNC cells, their antibiotic resistance, retention of virulence and ability to attach and persist in the environment, and factors that have been found to allow resuscitation of VBNC cells. Along with simple reversal of the inducing stresses, a variety of interesting chemical and biological factors have been shown to allow resuscitation, including extracellular resuscitation-promoting proteins, a novel quorum-sensing system (AI-3) and interactions with amoeba. Finally, the central role of catalase in the VBNC response of some bacteria, including its genetic regulation, is described.

Introduction

Bacteria in the viable but nonculturable (VBNC) state fail to grow on the routine bacteriological media on which they would normally grow and develop into colonies, but are still alive (Oliver, 2000). Despite their typically low levels of metabolic activity, they are again culturable upon resuscitation. Since the pioneering study by Xu et al. (1982) over 25 years ago, a large body of literature has evolved from researchers worldwide documenting the existence of a VBNC state in a wide variety of bacteria. Most investigators believe it to be a survival strategy in response to harsh environmental conditions, and it is now clear that the VBNC state constitutes an important reservoir of pathogens in the environment (Lleò et al., 2007a). The medical implications of this fact are numerous (Sardessai, 2005). For example, it appears that the ‘latent’ or the ‘dormant’ phase of Mycobacterium tuberculosis infections represents the VBNC state in this pathogen (Shleeva et al., 2004; Young et al., 2009), and that the recurrence of tuberculosis years after a person was thought to be tuberculosis free is due to resuscitation of this pathogen from the VBNC state (Pai et al., 2000). The list of pathogenic bacteria that have adopted this lifestyle as a means of survival includes not only those that infect humans but also those that infect such diverse animals as fish (Magariños et al., 1994; Biosca et al., 1996; Rahman et al., 2001), corals (Banin et al., 2000; Israely et al., 2001) and sea urchins (Masuda et al., 2004). Many plant pathogenic bacteria entering this state have also been described (e.g. Grey & Steck, 2001; Imazaki & Nakaho, 2008; del Campo et al., 2009; Ordax et al., 2009). Indeed, the list of pathogens is ever increasing, and includes Campylobacter spp., Escherichia coli (including EHEC strains), Francisella tularensis, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, M. tuberculosis, Pseudomonas aeruginosa, several Salmonella and Shigella spp. and numerous pathogenic Vibrio spp. A list (likely incomplete) of these is provided in Table 1; a more complete list, including nonpathogens, can be found in Oliver (2005a).

Table 1

Pathogens known to enter the VBNC state

Aeromonas hydrophila Helicobacter pylori Serratia marcescens 
A. salmonicida Klebsiella aerogenes Shigella dysenteriae 
Agrobacterium tumefaciens K. pneumoniae S. flexneri 
Burkholderia cepacia K. planticola S. sonnei 
B. pseudomallei Legionella pneumophila Streptococcus faecalis 
Campylobacter coli Listeria monocytogenes Vibrio alginolyticus 
C. jejuni Mycobacterium tuberculosis V. anguillarum 
C. lari M. smegmatis V. campbellii 
Cytophaga allerginae Pasteurella piscicida V. cholerae 
Enterobacter aerogenes Pseudomonas aeruginosa V. harveyi 
E. cloacae P. syringae V. mimicus 
Enterococcus faecalis Ralstonia solanacearum V. parahaemolyticus 
E. hirae Rhizobium leguminosarum V. shiloi 
E. faecium R. meliloti V. vulnificus (types 1 & 2) 
Erwinia amylovora Salmonella enterica Xanthomonas campestris 
Escherichia coli (including EHEC) S. typhi X. axonopodis pv. citri 
Francisella tularensis S. typhimurium  
Aeromonas hydrophila Helicobacter pylori Serratia marcescens 
A. salmonicida Klebsiella aerogenes Shigella dysenteriae 
Agrobacterium tumefaciens K. pneumoniae S. flexneri 
Burkholderia cepacia K. planticola S. sonnei 
B. pseudomallei Legionella pneumophila Streptococcus faecalis 
Campylobacter coli Listeria monocytogenes Vibrio alginolyticus 
C. jejuni Mycobacterium tuberculosis V. anguillarum 
C. lari M. smegmatis V. campbellii 
Cytophaga allerginae Pasteurella piscicida V. cholerae 
Enterobacter aerogenes Pseudomonas aeruginosa V. harveyi 
E. cloacae P. syringae V. mimicus 
Enterococcus faecalis Ralstonia solanacearum V. parahaemolyticus 
E. hirae Rhizobium leguminosarum V. shiloi 
E. faecium R. meliloti V. vulnificus (types 1 & 2) 
Erwinia amylovora Salmonella enterica Xanthomonas campestris 
Escherichia coli (including EHEC) S. typhi X. axonopodis pv. citri 
Francisella tularensis S. typhimurium  

The existence of a VBNC state has been debated for many years (Bogosian & Bourneuf, 2001; Nyström, 2001; Oliver, 2005a, b; Barcina & Arana, 2009). At least some of the disagreement revolved around the phrase ‘viable but nonculturable’, which is most commonly used to describe this phenomenon (Barer & Harwood, 1997; Kell et al., 1998; Colwell & Grimes, 2000). However, the debate over whether a VBNC state truly exists has largely been put to rest, largely as a result of numerous molecular studies reported in recent years (discussed below). Several reviews on the VBNC state have also appeared in recent years, mostly in its support (McDougald et al., 1998; Colwell & Grimes, 2000; Edwards, 2000; Yamamoto, 2000; Rowan, 2004; Oliver, 2005a, b, 2006; Sardessai, 2005; Barcina & Arana, 2009) but a few opposed (Nyström, 2005; Sinton, 2006). An extensive review of the potential public health hazards specifically of food-borne pathogens has also been presented (Oliver, 2005a). The reader is referred to these reviews for discussions on various aspects of the VBNC state in general; more recent findings on the VBNC state in human pathogens are presented here.

Inducers of the VBNC state

A list of factors, both chemical and environmental, which have been reported to induce the VBNC state, are varied and numerous. It includes nutrient starvation (e.g. Cook & Bolster, 2007), incubation outside the normal temperature range of growth (e.g. Besnard et al., 2002; Maalej et al., 2004; Wong & Wang, 2004), elevated or lowered osmotic concentrations (e.g. Asakura et al., 2008; Wong & Liu, 2008), oxygen concentrations (Kana et al., 2008), commonly used food preservatives (Cunningham et al., 2009; Quirós et al., 2009), heavy metals (Ghezzi & Steck, 1999) and even exposure to white light (Gourmelon et al., 1994). A common response to such stresses by bacterial cells is their ultimate inability to develop into colonies on routine culture media (Fig. 1), even though the cells may remain viable for long periods of time.

Figure 1

Entry of Vibrio vulnificus into the VBNC state on incubation at 5°C. The total cell counts (□), culturable counts (○) and viable counts (•) are shown (from Oliver, 2005b).

Figure 1

Entry of Vibrio vulnificus into the VBNC state on incubation at 5°C. The total cell counts (□), culturable counts (○) and viable counts (•) are shown (from Oliver, 2005b).

Detection of VBNC cells

As cells in the VBNC state are no longer culturable, alternate nonculture methods must be used to demonstrate that cells in this state are alive. Commonly used are reagents (e.g. the BacLight® Live/Dead assay) designed to demonstrate, through direct microscopic examination, the presence of an intact cytoplasmic membrane. An increasingly popular molecular method is reverse transcriptase (RT)-PCR, which detects gene expression (see Gene expression by VBNC cells). Because the half-life of bacterial mRNA is typically only 3–5 min (Conway & Schoolnik, 2003), continued gene expression by nonculturable cells is considered an excellent indicator of bacterial cell viability. Other less commonly used methods are described in Oliver (2005b).

Cellular traits

Cells entering the VBNC state typically exhibit significant dwarfing (e.g. cells of Vibrio vulnificus are c. 2 μm long in the log phase, but 0.6 μm in diameter in the VBNC state). Such cells generally undergo major decreases in macromolecular synthesis and rates of respiration, but plasmids are retained and ATP levels and membrane potential remain high. Continued amino acid uptake and incorporation have been reported (Rahman et al., 1994), and a study of several probiotic Bifidobacterium spp. by Lahtinen et al. (2008) found all to retain high levels of rRNA, despite losing all culturability in fermented food products. Membrane fatty acids also change (Day & Oliver, 2004), as would be expected for cells undergoing environmental stress. Using proteomics, Muela et al. (2008) recently studied modifications in the outer membrane of E. coli cells as they entered the VBNC state. Cells were induced into this state by starvation, by incubation in seawater and/or by exposure to visible light. They observed a drastic rearrangement of the outer membrane subproteome with the appearance of over 100 new spots. Ten of these remained in the VBNC cells, although none were found to be exclusive to this state. In contrast, a proteome study on Enterococcus faecalis clearly demonstrated that the total cell protein profiles of cells entering the VBNC state are markedly different from those of exponentially growing or starved cells (Heim et al., 2002). Similarly, protein profiling of Vibrio parahaemolyticus revealed a number of upregulated proteins that remained at high levels for several weeks into the VBNC state (Lai et al., 2009).

Significant, and what might be characteristic biochemical changes in the cell walls of VBNC cells, have been well studied by Signoretto and colleagues. For example, Signoretto et al. (2002), in studying the cell wall peptidoglycan of E. coli entering the VBNC state, reported a threefold increase in unusual DAP–DAP cross-linking, an increase in mucopeptides bearing a covalently bound lipoprotein, and a shortening of the average length of glycan strands in comparison with exponentially growing cells. VBNC cells were also found to have an autolytic capability far higher than that of exponentially growing cells. Similar findings were reported by Signoretto et al. (2000) for E. faecalis. del Mar Lleo et al. (2007) examined the effects of several antibiotics acting on peptidoglycan or protein synthesis in E. faecalis and found several β-lactams to block resuscitation of VBNC cells. Surprisingly, vancomycin, even when used at 100 times its minimum inhibitory concentration (MIC), was totally ineffective in this regard. The authors suggested that this insensitivity is due to the lack of synthesis of d-ala-d-ala, the specific target of this antibiotic, by the metabolically relatively inactive (VBNC) cells. In that peptidoglycan rearrangements have been observed in both gram-positive and -negative cells as they enter the VBNC state (Costa et al., 1999; Signoretto et al., 2002), such events may be hallmarks of the VBNC state.

Virulence of cells in the VBNC state

While not all investigators have reported pathogens to be capable of initiating infection when in the VBNC state, many have done so. In some cases, the same investigators have reported an organism to be avirulent while in the VBNC state, for example Cappelier et al. (2005) studying L. monocytogenes, and have subsequently reported that certain conditions (e.g. incubation of an embryo in egg yolk) are required to observe virulence (Cappelier et al., 2007). In fact, it seems most likely that pathogens are not generally able to initiate disease when present in the VBNC state, but that virulence is retained and infection can be initiated following their resuscitation to the actively metabolizing state. Sun et al. (2008), for example, found that VBNC cells of Vibrio harveyi ceased to express the hemolysin gene and did not cause death when inoculated into zebra fish. However, resuscitated cells were lethal, indicating that VBNC V. harveyi cells retained pathogenic potential. Similarly, Oliver & Bockian (1995) reported V. vulnificus to lose virulence for mice in proportion to the length of time that the cells were in the VBNC state. The cells retained virulence, however, and even when fully nonculturable, were able to cause fatal infections, with resuscitation occurring within the mouse. Continued virulence for a variety of pathogenic vibrios has also been demonstrated by Baffone et al. (2003).

Antibiotic resistance of VBNC cells

One of the interesting and significant consequences of entry of pathogens into the VBNC state includes its effects on antibiotic resistance when pathogens are in this state (often in biofilms). As noted by del Mar Lleo et al. (2007), ‘… antibiotics which are highly active on growing cells do not necessarily act on VBNC (i.e. quiescent) cells’. It seems likely that, because VBNC cells demonstrate such low metabolic activity, they effectively become resistant to antibiotics, and yet are able to resuscitate and reinitiate infections. For example, Ehrlich et al. (2002) reported that antibiotic-resistant VBNC cells of Haemophilus influenzae present in biofilms are able to initiate chronic ear infections. In some reports, antibiotic resistance is exceptional. Vancomycin was reported to be effective against VBNC cells of E. faecalis only when at 500 times the MIC (Lleò et al., 2007a), and Anuchin et al. (2009) observed drastic increases in resistance to hydromycin and doxycyclin in dormant cells of Mycobacterium smegmatis compared with 48-h cultures. Another example is H. pylori, which produces gastric and duodenal ulcers, which are among the most widespread and common syndromes in the world (Kusters et al., 2006), and was shown by Adams et al. (2003) to rapidly enter the VBNC state when present in natural waters. Bates et al. (2003) subsequently reported that VBNC H. pylori cells are antibiotic resistant, which likely accounts for the frequent reinfections suffered by persons who undergo remission despite antibiotic treatment. Similarly, studies by Rivers & Steck (2001), following the work of previous investigators (Dominque et al., 1995; Mulvey et al., 2001), provided evidence suggesting that uropathogenic E. coli cells, typically not detected by standard methods, were not eliminated by antibiotic treatment. The recurrent urinary tract infections suffered by many individuals are thus likely a result of cells in this temporarily dormant state, which are able to resist antibiotic treatment and resuscitate back to the metabolically active state. Subsequent studies by Anderson et al. (2004) documented that VBNC cells were present not only in mouse but also in human urine specimens.

As a possible alternate strategy for becoming antibiotic resistant, Mason et al. (1995) reported a study in which over 90% of culturable E. coli cells were treated with 10 or 100 times the MIC of ciprofloxacin, and yet retained membrane potential and protein synthesis. The authors suggested that the results demonstrated a ‘quinolone-induced VBNC state’ in which the cells might be capable of continued pathogenesis.

Attachment and persistence of VBNC cells in the environment

Whether or not VBNC cells remain capable of attaching to surfaces appears to be species or strain dependent. Duffy & Dykes (2009) reported continued attachment by Campylobacter jejuni, and Cappelier et al. (1999b) observed this species to adhere to HeLa cells after resuscitation. In contrast, Lleò et al. (2007b) reported that while VBNC cells of several medically important Enterococcus spp. do not form biofilms on medical device materials, they continue to synthesize exopolysaccharides for a limited time, supporting their contention that such cells retain a public health risk. Signoretto et al. (2004) did report, however, that adhesion of nonculturable E. faecalis cells to plankton is an important mechanism for its persistence in aquatic environments, and Islam et al. (1994) found Vibrio cholerae O1 strains associated with freshwater cyanobacteria in Bangladesh. Rahman et al. (2001) concluded that VBNC cells of Aeromonas hydrophila can persist in aquatic environments for prolonged periods of time, although they gradually lose virulence to fish. Indeed, although data have not been reported on many species, it appears that cells may remain in the VBNC state for long periods. We found that Pseudomonas fluorescens cells can remain in this state in soil for over a year (Bunker et al., 2004), and Amel et al. (2008) showed that Vibrio fluvialis could be resuscitated 6 years after becoming VBNC in marine sediment. Unfortunately, how resistant VBNC cells are to environmental stresses has not been well studied. Wong & Wang (2004), studying V. parahaemolyticus, reported that VBNC cells of this human pathogen were ‘highly resistant to thermal (42 °C, 27 °C), low-salinity (0% NaCl), or acid (pH 4.0) inactivation’. Weichart & Kjelleberg (1996) reported that VBNC V. vulnificus cells exhibited an initial sonication sensitivity similar to that of growing cells, but that resistance increased with increased cold incubation, with final resistance equaling that of starved cells. Rowe et al. (1998) reported that VBNC cells of C. jejuni were significantly more sensitive to a variety of the quaternary ammonium compounds commonly used in food-processing operations compared with culturable cells, but that the VBNC cells were more resistant to chlorine than were culturable cells. Finally, Anuchin et al. (2009) reported dormant forms of M. smegmatis to show elevated resistance to heat (up to 80 °C).

Gene expression by VBNC cells

Some of the most exciting studies in the desire to understand the biology of cells in the VBNC state have been elucidated by investigations into various molecular aspects of these cells. This is exemplified by the use of RT-PCR to demonstrate continued gene expression in VBNC cells (e.g. Lleò et al., 2000, 2001). In addition to providing an essential insight into factors regulating the VBNC state, such studies have offered definitive proof that such cells remain metabolically active and are not dead. A few examples of such RT-PCR studies are described here. Yaron & Matthews (2002) found that a variety of genes, including mobA, rfbE, stx1 and those for 16S rRNA synthesis, were expressed in nonculturable E. coli O157:H7 cells. Similarly, Barrett (1998) and Saux et al. (2002) reported continued production of message for several genes after cells of V. vulnificus were in the VBNC state for as long as 4.5 months. Pai et al. (2000) found continued expression of antigen 85B in M. tuberculosis, and Gunasekera et al. (2002) reported the expression of gfp in VBNC cells of E. coli and Pseudomonas putida following pasteurization. Such findings are strong indications of viability, but whether or not the genes reported to be active are essential to the VBNC process is not known.

In a highly significant study, Vora et al. (2005) combined RT-PCR and a 90-plex PCR amplification scheme with microarray hybridization to analyze VBNC cells of V. cholerae O1, V. parahaemolyticus O3:K6 and V. vulnificus that had been incubated in artificial seawater (ASW) at 4 °C to induce the VBNC state. They reported that the ‘… unambiguous detection of mRNA species in each of the three nonculturable ASW cultures confirmed that (1) these bacteria were indeed VBNC, (2) bacteria in the VBNC state could be detected in this manner and (3) although VBNC, these strains continued to express known toxin (ctxAB, rtxA, hlyA, tl, tdh and vvhA) and virulence (tcpA and TTSS) genes, thus retaining their pathogenic potential’. Similarly, Pommepuy et al. (1996) showed retention of enteropathogenicity by VBNC E. coli cells through their continued production of enterotoxin. Thus, even when not actively multiplying, if a pathogen in the VBNC state is producing toxin, a potential public health concern exists. Virulence of pathogens in the VBNC state is reviewed in considerable detail in Oliver (2005a).

Using the membrane diffusion chambers pioneered by McFeters & Stuart (1972), we have been able to study in situ gene expression by various pathogens incubated in their natural aquatic environments. For example, we reported H. pylori cells to become nonculturable in a freshwater stream (Adams et al., 2003), and subsequently that they maintained the expression of murG, a glycosyltransferase (B. Adams & J.D. Oliver, unpublished data). This enzyme has been shown by Signoretto et al. (2002) to be required in the late stages of peptidoglycan assembly in E. faecalis cells entering the VBNC state, and may explain continued production of this enzyme in nonculturable cells. We also observed peptidoglycan synthesis and expression of the virulence factors CagA, VacA and UreA by H. pylori for at least 32 h while in this state (B. Adams & J.D. Oliver, unpublished data). These studies are in agreement with Nilsson et al. (2002), who showed the expression of these same virulence factors in vitro using nonculturable H. pylori cells. Such data provide further evidence that not only does this pathogen enter into and persist in the VBNC state in the environment, but that it likely remains infectious.

In subsequent in situ studies on the human pathogen, V. vulnificus (Jones & Oliver, 2009), we found continued expression of the various genes examined for as long as 4.5 days when the cells were in warm coastal waters (Smith & Oliver, 2006a; Jones et al., 2008). In contrast, when cells in three separate studies were incubated in situ in coastal waters with temperatures from 8 to 11 °C, several genes, including the hemolysin/cytolysin gene (vvhA) and two capsule-related genes (wza and wzb), ceased to be expressed in some strains as the cells entered the VBNC state (Smith & Oliver, 2006b). Regardless of whether the cells remained culturable or entered the VBNC state, continued expression of the major stress σ factor, RpoS, was observed (for as long as 14 days). This is consistent with the findings of Boaretti et al. (2003), who reported RpoS to be involved in the persistence of E. coli in the VBNC state.

Especially interesting in our in situ studies was our finding on expression of the katG gene (regulator of catalase production) and its role in resuscitation (see Why do cells enter the VBNC State?).

Resuscitation of cells from the VBNC state

The VBNC state can only be a significant means of survival if the cells are able to increase metabolic activity and again become culturable. Proving that true resuscitation of cells from the VBNC state occurs (as opposed to simple regrowth of a few undetected and culturable cells present in the VBNC population) has been problematic and a source of much of the disagreement concerning the validity of a VBNC state among bacteria (Barcina & Arana, 2009; Sachidanandham & Gin, 2009). While numerous investigations have reported on this aspect of the VBNC state, resuscitation has been most extensively studied in V. vulnificus (first reviewed by Oliver, 1995). The most common VBNC-inducing factor for many vibrios (as well as many other genera) is a simple temperature downshift. For example, V. vulnificus enters this state in response to temperatures of c. 10 °C. Numerous studies have found that a simple reversal of this stress (e.g. a temperature upshift) is sufficient to allow their resuscitation from the VBNC state (Gupte et al., 2003; Wong et al., 2004; Du et al., 2007a, b). Indeed, in V. vulnificus, this has been demonstrated in vivo in clams (Birbari et al., 2000) and in mice (Oliver & Bockian, 1995), in situ in estuarine waters (Oliver et al., 1995), as well as in vitro (Nilsson et al., 1991; Wolf & Oliver, 1992). Similarly, Bates & Oliver (2004) found that temperature down- and upshifts controlled the VBNC state in V. parahaemolyticus, with neither the presence nor the absence of the Kanagawa hemolysin genes (tdh1 and tdh2) having any effect on entry into or resuscitation from this state.

That a temperature upshift resulted in true resuscitation, as opposed to regrowth of undetected culturable cells, was shown conclusively by Whitesides & Oliver (1997). Recently, Abe et al. (2007) described a mutant of V. vulnificus that remained culturable at a low temperature. Suppression subtractive hybridization studies revealed that glutathione S-transferase was the responsive gene, which was highly expressed in the mutant at a low temperature.

Interestingly, several higher organisms may be biological mediators of resuscitation from the VBNC state. For example, L. pneumophila has been reported to enter the VBNC state following both starvation and hypochlorite treatment, but to resuscitate in the protozoans Acanthamoeba polyphaga (Garcia et al., 2007) and Acanthamoeba castellanii (Steinert et al., 1997). In another study, A. castellanii was found to induce the VBNC state in A. hydrophila (Rahman et al., 2008). In a different system, Sussman et al. (2003) reported the association of VBNC cells of the coral pathogen, Vibrio shiloi, with a marine fireworm. Other conditions found to allow resuscitation of pathogens include inoculation into yolk sacs of embryonated eggs (Cappelier et al., 1999b, 2007), into mice (Cappelier et al., 1999a) and into human volunteers (Colwell et al., 1996). Thus, a wide variety of bacterial–host associations may have special value in influencing the survival of bacteria, including those in the VBNC state.

Another exciting development in the reactivation of dormant cells is the role of a group of extracellular bacterial proteins, known as ‘resuscitation-promoting factors’ (Rpfs), which have been shown to induce resuscitation in M. tuberculosis and M. smegmatis (Mukamolova et al., 1998a, 2002; Shleeva et al., 2004). Studies by Mukamolova et al. (2006) indicate that at least some Rpfs are peptidoglycan hydrolases, involved in cell wall digestion and thus cell division (Hett et al., 2007). Thus, again, peptidoglycan rearrangement (c.f. Signoretto et al., 2000, 2002) appears to be prominent in the VBNC story. Rpf-like compounds have been identified in several other genera (Mukamolova et al., 1998b; Hett et al., 2007), and although not yet widely studied as an aspect of resuscitation, such activity may be common in the resuscitation of other genera. Most recently, Kana et al. (2008) showed that the Rpfs of M. tuberculosis are required not only for resuscitation but also for virulence. Interestingly, these authors also reported that the Rpfs were not required for in vitro growth.

Another ‘class’ of resuscitation factors was reported by Reissbrodt et al. (2002). This was described as a heat-stable ‘autoinducer of growth’, which was secreted by a variety of gram-positive and (primarily) gram-negative bacterial species when incubated in media containing the human catecholamine hormone, norepinephrine (Freestone et al., 1999). Norepinephrine is produced in large amounts in humans following severe tissue injury, and is thus considered to be a stress-related hormone. The bacterial growth stimulation observed in the presence of this hormone appeared to be due to non-nutritional factors (Lyte et al., 1996). These showed a high degree of cross-species activity, and appeared to be a family of signaling molecules (Freestone et al., 1999). Subsequently, Sperandio et al. (2003) identified the factors to represent a novel quorum-sensing system, which they termed AI-3. Because both epinephrine and norepinephrine could substitute for AI-3 in activating enterohemorrhagic E. coli virulence gene expression, and the effects of AI-3 and epinephrine/norepinephrine could be blocked by adrenergic receptor antagonists, they suggested that these compounds have a similar structure (Sperandio et al., 2003). Reissbrodt et al. (2002) found that the autoinducers present in the spent media of various bacteria resulted in resuscitation from the VBNC state of several strains of Salmonella enterica serovar Typhimurium and of two E. coli O157:H7 strains. The findings of Reissbrodt et al. (2002) and of Sperandio et al. (2003) would appear to have major implications for the resuscitation of enteropathogens from the VBNC state, especially those occurring in the human intestinal tract, at a time (e.g. tissue damage) when the host may be under significant physiological stress.

Why do cells enter the VBNC state?

It has been recognized for many years that H2O2 might play a significant role in inducing the VBNC state in a variety of bacteria, including E. coli (Mizunoe et al., 1999). We produced a catalase (katG)-negative mutant of V. vulnificus through inactivation of the katG regulator oxyR, which was thus incapable of degrading potentially fatal H2O2 (Kong et al., 2004). Whereas V. vulnificus enters the nonculturable state when exposed to temperatures <13 °C, the mutant cells were nonculturable on solid media even at room temperature. This observation suggested that one aspect of the VBNC state in this pathogen likely involves H2O2, either following its production by the cells when plated onto solid media, or its natural presence in solid media, coupled with an inability of the cells to detoxify this lethal metabolite. However, if cells entering into the VBNC state were plated onto conventional laboratory media (e.g. heart infusion agar) supplemented with peroxide-neutralizing agents (e.g. catalase), considerably enhanced culturability was seen (Fig. 2). We subsequently found that low temperature (the VBNC-inducer in this bacterium) prevents both catalase activity and its de novo synthesis (Fig. 3), rendering the cells highly sensitive (and nonculturable) to the peroxide present in culture media. Thus, low-temperature incubation resulted in cells that, due to this cold shock response, are nonculturable and yet remain viable.

Figure 2

Effect of anti-ROS agents on the culturability of Vibrio vulnificus following low-temperature incubation. Cells were incubated in ASW at 5°C for periods up to 36 days, with plating onto heart infusion (HI) agar (-○-) or HI agar supplemented with catalase (-•-) or pyruvate (-▪-). Whereas cells entered the VBNC state by 12 days, as indicated by culture on unsupplemented HI agar, continued culturability was observed under the same conditions when either of the H2O2-degrading agents was present in the medium. Reproduced from Kong et al. (2004).

Figure 2

Effect of anti-ROS agents on the culturability of Vibrio vulnificus following low-temperature incubation. Cells were incubated in ASW at 5°C for periods up to 36 days, with plating onto heart infusion (HI) agar (-○-) or HI agar supplemented with catalase (-•-) or pyruvate (-▪-). Whereas cells entered the VBNC state by 12 days, as indicated by culture on unsupplemented HI agar, continued culturability was observed under the same conditions when either of the H2O2-degrading agents was present in the medium. Reproduced from Kong et al. (2004).

Figure 3

Catalase activity and culturability of Vibrio vulnificus incubated at 5°C. Cells were incubated in ASW at 5°C, and examined for culturability on heart infusion agar (-○-) and for catalase activity (-•-) over time. A concomitant loss of catalase activity was observed as culturability decreased. Reproduced from Kong et al. (2004).

Figure 3

Catalase activity and culturability of Vibrio vulnificus incubated at 5°C. Cells were incubated in ASW at 5°C, and examined for culturability on heart infusion agar (-○-) and for catalase activity (-•-) over time. A concomitant loss of catalase activity was observed as culturability decreased. Reproduced from Kong et al. (2004).

Using the membrane diffusion chambers described above, we extended these studies by examining the effects of low temperature on culturability and the expression of the catalase gene (katG) in V. vulnificus when the cells were incubated in natural estuarine waters. Under these in situ conditions, we observed that katG underwent a demonstrable decrease in expression within as little as 30 min at 11 °C (Fig. 4). During these cold-temperature studies, the cells of all three V. vulnificus strains examined entered the VBNC state (<0.1 CFU mL−1) within 14 days, while the total and viable (BacLight® Live/Dead) direct microscopic counts remained elevated. Following a temperature upshift to c. 22 °C for 24 h, all three strains resuscitated to levels above 104 CFU mL−1. Resuscitation also resulted in renewed and full expression of katG. That peroxide is critical to the VBNC response in V. vulnificus has also been documented recently by Abe et al. (2007). This appears, to date, to be the only bacterium for which a molecular basis of the VBNC state has been described.

Figure 4

Detection of katG mRNA in three Vibrio vulnificus strains during in situ incubation in estuarine waters during cold and warm months. (a) Incubation for up to 14 days (time of study indicated in hours or days) at a water temperature of 11°C, and following resuscitation (‘R’; 24-h room temperature upshift of the 14-day VBNC cells). (b) In situ expression of tufA in V. vulnificus under the same conditions (11°C). (c) Incubation for up to 24 h in estuarine water at 21°C. Reproduced from Smith & Oliver (2006a, b). Copyright © American Society for Microbiology.

Figure 4

Detection of katG mRNA in three Vibrio vulnificus strains during in situ incubation in estuarine waters during cold and warm months. (a) Incubation for up to 14 days (time of study indicated in hours or days) at a water temperature of 11°C, and following resuscitation (‘R’; 24-h room temperature upshift of the 14-day VBNC cells). (b) In situ expression of tufA in V. vulnificus under the same conditions (11°C). (c) Incubation for up to 24 h in estuarine water at 21°C. Reproduced from Smith & Oliver (2006a, b). Copyright © American Society for Microbiology.

Concluding remarks

The exact role of the VBNC state in bacteria is yet to be elucidated. It is likely that its role and significance differ from bacterium to bacterium. Most investigators believe it to be a response to certain environmental stresses that allows the cell's survival. In some, for example V. vulnificus, entry into this dormancy state appears to be simply one aspect of the much-studied and complex ‘cold shock response’ that bacteria exhibit (Phadtare, 2004), with nonculturability being an ‘artifact’ of the sensitivity of such cells to the toxic peroxide present in laboratory media. Cells of V. vulnificus may be quite ‘content’ in their natural estuarine environment, albeit with decreased metabolic rates, as long as they are not subjected to laboratory culture! Or, as proposed by Barcina & Arana (2009), the VBNC state may be an intermediate in an altruistic death process that is part of a survival strategy. Or, as recently suggested by Epstein (2009), dormancy, and ‘waking up’ from this state, could be a method analogous to ‘sending out scouts’ to ‘test the environment’ for its suitability for growth of the entire population. In this scenario, if the resuscitating cells ‘detect’ that the previously stressful/adverse environment is now growth-permissive, they would signal the remaining cells to resuscitate.

Regardless of the role that the VBNC state plays, it is clear that a large number of non-spore-forming bacteria, most notably a large number of human pathogens, are capable of entering this state, maintaining cellular structure and biology and continuing significant gene expression while otherwise nonculturable by ‘standard’ laboratory methods. That they can exit from this state, and become culturable again, is also undeniable. Finally, it can no longer be questioned that the VBNC state plays a critical role in the survival of important human (and other) pathogens, and possibly in their ability to produce disease.

Acknowledgements

I would like to thank Brett Froelich and Tiffany Williams for critically reading this manuscript. This study was supported, in part, by a grant (2007-35201-18381) from the US Department of Agriculture.

References

Abe
A
Ohashi
E
Ren
H
Hayashi
T
Endo
H
(
2007
)
Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus
.
Microbiol Res
 
162
:
130
138
.
Adams
BL
Bates
TC
Oliver
JD
(
2003
)
Survival of Helicobacter pylori in a natural freshwater environment
.
Appl Environ Microb
 
69
:
7462
7466
.
Amel
BK-N
Amine
B
Amina
B
(
2008
)
Survival of Vibrio fluvialis in seawater under starvation conditions
.
Microbiol Res
 
163
:
323
328
.
Anderson
M
Bollinger
D
Agler
A
Hartwell
H
Rivers
B
Ward
K
Steck
TR
(
2004
)
Viable but nonculturable bacteria are present in mouse and human urine specimens
.
J Clin Microbiol
 
42
:
753
758
.
Anuchin
AM
Mulyukin
AL
Suzina
NE
Duda
VI
El-Registan
GI
Kaprelyants
AS
(
2009
)
Dormant forms of Mycobacterium smegmatis with distinct morphology
.
Microbiology
 
155
:
1071
1079
.
Asakura
H
Kawamoto
K
Haishima
Y
Igimi
S
Yamamoto
S
Makino
S-I
(
2008
)
Differential expression of the outer membrane protein W (OmpW) stress response in enterohemorrhagic Escherichia coli O157:H7 corresponds to the viable but non-culturable state
.
Res Microbiol
 
159
:
709
717
.
Baffone
W
Citterio
B
Vittoria
E
Casaroli
A
Campana
R
Falzano
L
Donelli
G
(
2003
)
Retention of virulence in viable but non-culturable halophilic Vibrio spp
.
Int J Food Microbiol
 
89
:
31
39
.
Banin
E
Israely
T
Kushmaro
A
Loga
Y
Orr
E
Rosenber
E
(
2000
)
Penetration of the coral-bleaching bacterium Vibrio shiloi into Oculina patagonica
.
Appl Environ Microb
 
66
:
3031
3036
.
Barcina
I
Arana
I
(
2009
)
The viable but nonculturable phenotype: a crossroads in the life-cycle of non-differentiating bacteria?
Rev Environ Sci Biotechnol
 
8
:
245
255
.
Barer
MR
Harwood
CR
(
1997
)
Viable but non-culturable and dormant bacteria: time to resolve an oxymoron and a misnomer?
J Med Microbiol
 
46
:
629
631
.
Barrett
T
(
1998
)
An investigation into the molecular basis of the viable but non-culturable response in bacteria. PhD Thesis, University of Aberdeen
 .
Bates
TC
Oliver
JD
(
2004
)
The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus
.
J Microbiol
 
42
:
74
79
.
Bates
TC
Adams
B
Oliver
JD
(
2003
) Survival of VBNC Helicobacter pylori cocci following antibiotic treatment. International Conference on Helicobacter pylori, Aarhus, Denmark.
Besnard
V
Federighi
M
Declerq
E
Jugiau
F
Cappelier
JM
(
2002
)
Environmental and physico-chemical factors induce VBNC state in Listeria monocytogenes
.
Vet Res
 
33
:
359
370
.
Biosca
EG
Amaro
C
Marco-Noales
E
Oliver
JD
(
1996
)
Effect of low temperature on starvation-survival of the eel pathogen Vibrio vulnificus biotype 2
.
Appl Environ Microb
 
62
:
450
455
.
Birbari
W
Wright
A
Rodrick
G
(
2000
)
Viable but non-culturable response for phase variants of Vibrio vulnificus in clams
.
J Shellfish Res
 
19
:
660
.
Boaretti
M
Del Mar Lleo
M
Bonato
B
Signoretto
C
Canepari
P
(
2003
)
Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state
.
Environ Microbiol
 
5
:
986
996
.
Bogosian
G
Bourneuf
EV
(
2001
)
A matter of bacterial life and death
.
EMBO Rep
 
9
:
770
774
.
Bunker
ST
Bates
TC
Oliver
JD
(
2004
)
Effects of temperature on detection of plasmid and chromosomally encoded gfp- and lux-labeled Pseudomonas fluorescens in soil
.
Environ Biosafety Res
 
3
:
1
8
.
Cappelier
JM
Magras
C
Jouve
JL
Federighi
M
(
1999a
)
Recovery of viable but nonculturable Campylobacter jejuni cells in two animal models
.
Food Microbiol
 
16
:
375
383
.
Cappelier
JM
Minet
J
Magras
C
Colwell
RR
Federichi
M
(
1999b
)
Recovery in embryonated eggs of viable but nonculturable Campylobacter jejuni cells and maintenance of ability to adhere to HeLa cells after resuscitation
.
Appl Environ Microb
 
65
:
5154
5157
.
Cappelier
JM
Besnard
V
Roche
S
Garrec
N
Zundel
E
Velge
P
Federighi
M
(
2005
)
Avirulence of viable but non-culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models
.
Vet Res
 
36
:
589
599
.
Cappelier
JM
Besnard
V
Roche
SM
Velge
P
Federighi
M
(
2007
)
Avirulent viable but non-culturable cells of Listeria monocytogenes need the presence of an embryo to be recovered in egg yolk and regain virulence after recovery
.
Vet Res
 
38
:
573
583
.
Colwell
RR
Grimes
DJ
(
2000
)
Semantics and strategies
.
Nonculturable Microorganisms in the Environment
  (
Colwell
RR
Grimes
DJ
, eds), pp.
1
6
.
ASM Press
, Washington, DC.
Colwell
RR
Brayton
PR
Herrington
D
Tall
BD
Huq
A
Levine
MM
(
1996
)
Viable but nonculturable Vibrio cholerae O1 revert to a culturable state in human intestine
.
World J Microb Biot
 
12
:
28
31
.
Conway
T
Schoolnik
GK
(
2003
)
Microarray expression profiling: capturing a genome-wide portrait of the transcriptome
.
Mol Microbiol
 
47
:
879
889
.
Cook
KL
Bolster
CH
(
2007
)
Survival of Campylobacter jejuni and Escherichia coli in groundwater during prolonged starvation at low temperatures
.
J Appl Microbiol
 
103
:
573
583
.
Costa
K
Bacher
G
Allmaier
G
et al. (
1999
)
The morphological transition of Helicobacter pylori cells from spiral to coccoid is preceded by a substantial modification of the cell wall
.
J Bacteriol
 
181
:
3710
3715
.
Cunningham
E
O'Byrne
C
Oliver
JD
(
2009
)
Effect of weak acids on Listeria monocytogenes survival: evidence for a viable but nonculturable state in response to low pH
.
Food Control
 
20
:
1141
1144
.
Day
AP
Oliver
JD
(
2004
)
Changes in membrane fatty acid composition during entry of Vibrio vulnificus in the viable but nonculturable state
.
J Microbiol
 
42
:
69
73
.
Del Campo
R
Russi
P
Mara
P
Mara
H
Peyrou
M
Ponce de Leon
I
Gaggero
C
(
2009
)
Xanthomonas axonopodis pv. citri enters the VBNC state after copper treatment and retains its virulence
.
FEMS Microbiol Lett
 
298
:
143
148
.
Del Mar Lleo
M
Benedetti
D
Tafi
MC
Signoretto
C
Canepari
P
(
2007
)
Inhibition of the resuscitation from the viable but non-culturable state in Enterococcus faecalis
.
Environ Microbiol
 
9
:
2313
2320
.
Dominque
GJ
Ghoniem
HM
Bost
KL
Fermin
C
Liset
GH
(
1995
)
Dormant microbes in interstitial cystitis
.
J Urologie
 
153
:
1321
1326
.
Du
M
Chen
J
Zhang
X
(
2007a
)
Characterization and resuscitation of viable but nonculturable Vibrio alginolyticus VIB283
.
Arch Microbiol
 
188
:
283
288
.
Du
M
Chen
J
Zhang
X
Li
A
Li
Y
Wang
Y
(
2007b
)
Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate
.
Appl Environ Microb
 
73
:
1349
1354
.
Duffy
LL
Dykes
GA
(
2009
)
The ability of Campylobacter jejuni cells to attach to stainless steel does not change as they become nonculturable
.
Foodborne Pathog Dis
 
6
:
631
634
.
Edwards
C
(
2000
)
Problems posed by natural environments for monitoring microorganisms
.
Mol Biotechnol
 
15
:
211
223
.
Ehrlich
GD
Veeh
R
Wang
X
Costerton
JW
Hayes
JD
Hu
FZ
Daigle
BJ
Ehrlich
MD
Post
JC
(
2002
)
Mucosal biofilm formation on middle-ear mucosa in the Chinchilla model of otitis media
.
Amer Med Assoc
 
287
:
1710
1715
.
Epstein
SS
(
2009
)
Microbial awakenings
.
Nature
 
457
:
1083
.
Freestone
PPE
Haigh
RD
Williams
PH
Lyte
M
(
1999
)
Stimulation of bacterial growth by heat-stable, norepinephrine-induced autoinducers
.
FEMS Microbiol Lett
 
172
:
53
60
.
Garcia
TM
Jones
S
Pelaz
C
Millar
RD
Kwaik
YA
(
2007
)
Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection
.
Environ Microbiol
 
9
:
1267
1277
.
Ghezzi
JI
Steck
TR
(
1999
)
Induction of the viable but nonculturable conditions in Xanthomonas campestris pv. campestris in liquid microcosms and sterile soil
.
FEMS Microbiol Ecol
 
30
:
203
208
.
Gourmelon
M
Cillard
J
Pommepuy
M
(
1994
)
Visible light damage to Escherichia coli in seawater: oxidative stress hypothesis
.
J Appl Bacteriol
 
77
:
105
112
.
Grey
BE
Steck
TR
(
2001
)
The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection
.
Appl Environ Microb
 
67
:
3866
3872
.
Gunasekera
TS
Sørensen
A
Attfield
PV
Sørensen
SJ
Veal
DA
(
2002
)
Inducible gene expression by nonculturable bacteria in milk after pasteurization
.
Appl Environ Microb
 
68
:
1988
1993
.
Gupte
AR
De Rezende
CLE
Joseph
SW
(
2003
)
Induction and resuscitation of viable but nonculturable Salmonella enterica serovar Typhimurium DT104
.
Appl Environ Microb
 
69
:
6669
6675
.
Heim
S
Lleo
MDM
Bonato
B
Guzman
CA
Canepari
P
(
2002
)
The viable but nonculturable state and starvation are different stress responses of Enterococcus faecalis, as determined by proteome analysis
.
J Bacteriol
 
184
:
6739
6745
.
Hett
EC
Chao
MC
Steyn
AJ
Fortune
SM
Deng
LL
Rubin
EJ
(
2007
)
A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis
.
Mol Microbiol
 
66
:
658
668
.
Imazaki
I
Nakaho
K
(
2008
)
Revivability of low temperature-inducible nonculturable cells in the plant pathogenic bacterium Ralstonia solancearum
.
Annu Meet Amer Soc Microbiol
 .
Islam
MS
Miah
MA
Hasan
MK
Sack
RB
Albert
MJ
(
1994
)
Detection of non-culturable Vibrio cholerae O1 associated with a cynaobacterium from an aquatic environment in Bangladesh
.
T Roy Soc Trop Med H
 
88
:
298
299
.
Israely
T
Banin
E
Rosenberg
E
(
2001
)
Growth, differentiation and death of Vibrio shiloi in coral tissue as a function of seawater temperature
.
Aquat Microb Ecol
 
24
:
1
8
.
Jones
MK
Oliver
JD
(
2009
)
Vibrio vulnificus: disease and pathogenesis
.
Infect Immun
 
77
:
1723
1733
.
Jones
MK
Warner
E
Oliver
JD
(
2008
)
Survival and in situ gene expression of Vibrio vulnificus at varying salinities in estuarine environments
.
Appl Environ Microb
 
74
:
182
187
.
Kana
BD
Gordhan
BG
Downing
KJ
et al. (
2008
)
The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro
.
Mol Microbiol
 
67
:
672
684
.
Kell
DB
Kaprelyants
AS
Weichart
DH
Harwood
CR
Barer
MR
(
1998
)
Viability and activity in readily culturable bacteria: a review and discussion of the practical issues
.
Antonie van Leeuwenhoek
 
73
:
169
187
.
Kong
I-S
Bates
TC
Hülsmann
A
Hassan
H
Oliver
JD
(
2004
)
Role of catalase and oxyR in the viable but nonculturable state of Vibrio vulnificus
.
FEMS Microbiol Ecol
 
50
:
133
142
.
Kusters
JG
Van Vliet
AHM
Kuipers
EJ
(
2006
)
Pathogenesis of Helicobacter pylori infection
.
Clin Microbiol Rev
 
19
:
449
490
.
Lahtinen
SJ
Ahokoski
H
Reinikainen
JP
Gueimonde
M
Nurmi
J
Ouwehand
AC
Salminen
SJ
(
2008
)
Degradation of 16S rRNA and attributes of viability of viable but nonculturable probiotic bacteria
.
Lett Appl Microbiol
 
46
:
693
698
.
Lai
C-J
Chen
S-Y
Lin
I-H
Chang
C-H
Wong
H-C
(
2009
)
Change of protein profiles in the induction of the viable but nonculturable state of Vibrio parahaemolyticus
.
Int J Food Microbiol
 
135
:
118
124
.
Lleò
MM
Pierobon
S
Tafi
MC
Signoreto
C
Canepari
P
(
2000
)
mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm
.
Appl Environ Microb
 
66
:
4564
4567
.
Lleò
MM
Bonato
B
Tafi
MC
Signoretto
C
Boaretti
M
Canepari
P
(
2001
)
Resuscitation rate in different enterococcal species in the viable but non-culturable state
.
J Appl Microbiol
 
91
:
1095
1102
.
Lleò
MM
Benedetti
D
Tafi
MC
Signoretto
C
Canepari
P
(
2007a
)
Inhibition of the resuscitation from the viable but non-culturable state in Enterococcus faecalis
.
Environ Microbiol
 
9
:
2313
2320
.
Lleò
MM
Bonato
B
Tafi
MC
Caburlotto
G
Benedetti
D
Canepari
P
(
2007b
)
Adhesion to medical device materials and biofilm formation capability of some species of enterococci in different physiological states
.
FEMS Microbiol Lett
 
274
:
232
237
.
Lyte
M
Frank
CD
Green
BT
(
1996
)
Production of an autoinducer of growth by norepinephrine cultured Escherichia coli O157:H7
.
FEMS Microbiol Lett
 
139
:
155
159
.
Maalej
S
Denis
M
Dukan
S
(
2004
)
Temperature and growth-phase effects on Aeromonas hydrophila survival in natural seawater microcosms: role of protein synthesis and nucleic acid content on viable but temporarily nonculturable response
.
Microbiology
 
150
:
181
187
.
Magariños
B
Romalde
JL
Barja
JL
Toranzo
AE
(
1994
)
Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment
.
Appl Environ Microb
 
60
:
180
186
.
Mason
DJ
Power
EGM
Talsania
H
Phillips
I
Gant
VA
(
1995
)
Antibacterial action of ciprofloxacin
.
Antimicrob Agents Ch
 
39
:
2752
2758
.
Masuda
Y
Tajima
K
Ezura
Y
(
2004
)
Resuscitation of Tenacibaculum sp., the causative bacterium of spotting disease of the sea urchin Strongylocentroutus intermedius, from the viable but non-culturable state
.
Fish Sci
 
70
:
277
284
.
McDougald
D
Rice
SA
Weichart
D
Kjelleberg
S
(
1998
)
Nonculturability: adaptation or debilitation?
FEMS Microbiol Ecol
 
25
:
1
9
.
McFeters
GA
Stuart
DG
(
1972
)
Survival of coliform bacteria in natural waters: field and laboratory studies with membrane filter chambers
.
Appl Microbiol
 
24
:
805
811
.
Mizunoe
Y
Wai
SN
Takade
A
Yoshida
S-i
(
1999
)
Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds
.
Arch Microbiol
 
172
:
63
67
.
Muela
A
Seco
C
Cmafeita
E
Arana
I
Orruòo
M
López
JA
Barcina
I
(
2008
)
Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state
.
FEMS Microbiol Ecol
 
64
:
28
36
.
Mukamolova
GV
Kaprelyants
AS
Young
DI
Young
M
Kell
DB
(
1998a
)
A bacterial cytokine
.
P Natl Acad Sci USA
 
95
:
8916
8921
.
Mukamolova
GV
Yanopolskaya
ND
Kell
DB
Kaprelyants
AS
(
1998b
)
On resuscitation from the dormant state of Micrococcus luteus
.
Antonie van Leeuwenhoek
 
73
:
237
243
.
Mukamolova
GV
Turapov
OA
Young
DI
Kaprelyants
AS
Kell
DB
Young
M
(
2002
)
A family of autocrine growth factors in Mycobacterium tuberculosis
.
Mol Microbiol
 
46
:
623
635
.
Mukamolova
GV
Murzin
AG
Salina
EG
Demina
GR
Kell
DB
Kaprelyants
AS
(
2006
)
Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation
.
Mol Microbiol
 
59
:
84
98
.
Mulvey
MA
Schilling
JD
Hultgren
SJ
(
2001
)
Establishment of a persistent Escherichia coli reservoir during the acute phase of a bladder infection
.
Infect Immun
 
69
:
4572
4579
.
Nilsson
H-O
Blom
J
Al-Soud
WA
Ljungh
Å
Andersen
LP
Wadström
T
(
2002
)
Effect of cold starvation, acid stress, and nutrients on metabolic activity of Helicobacter pylori
.
Appl Environ Microb
 
68
:
11
19
.
Nilsson
L
Oliver
JD
Kjelleberg
S
(
1991
)
Resuscitation of Vibrio vulnificus from the viable but nonculturable state
.
J Bacteriol
 
173
:
5054
5059
.
Nyström
T
(
2001
)
Not quite dead enough: on bacterial life, culturability, senescence, and death
.
Arch Microbiol
 
176
:
159
164
.
Nyström
T
(
2005
)
Bacterial senescence, programmed death, and premeditated sterility
.
ASM News
 
71
:
363
369
.
Oliver
JD
(
1995
)
The viable but nonculturable state in the human pathogen, Vibrio vulnificus (Minireview)
.
FEMS Microbiol Lett
 
133
:
203
208
.
Oliver
JD
(
2000
)
The public health significance of viable but nonculturable bacteria
.
Nonculturable Microorganisms in the Environment
  (
Colwell
RR
Grimes
DJ
, eds), pp.
277
299
.
ASM Press
, Washington, DC.
Oliver
JD
(
2005a
)
Viable but nonculturable bacteria in food environments
.
Food-Borne Pathogens: Microbiology and Molecular Biology
  (
Fratamico
PM
Bhunia
AK
Smith
JL
, eds), pp.
99
112
.
Caister Academic Press
, Norfolk, UK.
Oliver
JD
(
2005b
)
The viable but nonculturable state in bacteria
.
J Microbiol
 
43
:
93
100
.
Oliver
JD
(
2006
)
Vibrio vulnificus
.
Biology of Vibrios
  (
Thompson
FL
Austin
B
Swing
J
, eds), pp.
349
366
.
ASM Press
, Washington, DC.
Oliver
JD
Bockian
R
(
1995
)
In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus
.
Appl Environ Microb
 
61
:
2620
2623
.
Oliver
JD
Hite
F
McDougald
D
Andon
NL
Simpson
LM
(
1995
)
Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment
.
Appl Environ Microb
 
61
:
2624
2630
.
Ordax
M
Biosca
EG
Wimalajeewa
SC
Lopez
MM
Marco-Noales
E
(
2009
)
Survival of Erwinia amyulovora in mature apple fruit calyces through the viable but nonculturable state
.
J Appl Microbiol
 
107
:
106
116
.
Pai
SR
Actor
JK
Sepulveda
E
Hunter
RL
Jr
Jagannath
C
(
2000
)
Identification of viable non-viable Mycobacterium tuberculosis in mouse organs by directed RT-PCR for antigen 85B mRNA
.
Microb Pathogenesis
 
28
:
335
342
.
Phadtare
S
(
2004
)
Recent developments in bacterial cold-shock response
.
Curr Issues Mol Biol
 
6
:
125
136
.
Pommepuy
M
Butin
M
Derrien
A
Gourmelon
M
Colwell
RR
Cormier
M
(
1996
)
Retention of enteropathogenicity by viable but nonculturable Escherichia coli exposed to seawater and sunlight
.
Appl Environ Microb
 
62
:
4621
4626
.
Quirós
C
Herrero
M
Garcia
LA
Diaz
M
(
2009
)
Quantitative approach to determining the contribution of viable-but-nonculturable subpopulations of malolactic fermentation processes
.
Appl Environ Microb
 
75
:
2977
2981
.
Rahman
I
Shahamat
M
Kirchman
PA
Russek-Cohen
E
Colwell
RR
(
1994
)
Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae type 1
.
Appl Environ Microb
 
60
:
3573
3578
.
Rahman
M
Abd
H
Romling
U
Sandstrom
G
Mollby
R
(
2008
)
Aeromonas–Acanthamoeba interaction and early shift to a viable but nonculturable state of Aeromonas by Acanthamoeba
.
J Appl Microbiol
 
104
:
1449
1457
.
Rahman
MH
Suzuki
S
Kawai
K
(
2001
)
Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus
.
Microbiol Res
 
156
:
103
106
.
Reissbrodt
R
Rienaecker
I
Romanova
JM
Freestone
PPE
Haigh
RD
Lyte
M
Tschape
H
Williams
PH
(
2002
)
Resuscitation of Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat-stable enterobacterial autoinducer
.
Appl Environ Microb
 
68
:
4788
4794
.
Rivers
B
Steck
TR
(
2001
)
Viable but nonculturable uropathogenic bacteria are present in the mouse urinary tract following urinary tract infection and antibiotic treatment
.
Urol Res
 
29
:
60
66
.
Rowan
NJ
(
2004
)
Viable but non-culturable forms of food and waterborne bacteria: quo vadis?
Trends Food Sci Tech
 
15
:
462
467
.
Rowe
MT
Dunstall
G
Kirk
R
Loughney
CF
Cooke
JL
Brown
SR
(
1998
)
Development of an image system for the study of viable but non-culturable forms of Campylobacter jejuni and its use to determine their resistance to disinfectants
.
Food Microbiol
 
15
:
491
498
.
Sachidanandham
R
Gin
KY-H
(
2009
)
A dormancy state in nonspore-forming bacteria
.
Appl Microbiol Biot
 
81
:
927
941
.
Sardessai
YN
(
2005
)
Viable but non-culturable bacteria: their impact on public health
.
Curr Sci
 
89
:
1650
.
Saux
MF-L
Hervio-Heath
D
Loaec
S
Colwell
RR
Pommepuy
M
(
2002
)
Detection of cytotoxin-hemolysin mRNA in nonculturable populations of environmental and clinical Vibrio vulnificus strains in artificial seawater
.
Appl Environ Microb
 
68
:
5641
5646
.
Shleeva
M
Mukamolova
GV
Young
M
Williams
HD
Kaprelyants
AS
(
2004
)
Formation of ‘non-culturable’ cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation
.
Microbiology
 
150
:
1687
1697
.
Signoretto
C
Lleó
MM
Tafi
MC
Canepari
P
(
2000
)
Cell wall chemical composition of Enterococcus faecalis in the viable but nonculturable state
.
Appl Environ Microb
 
66
:
1953
1959
.
Signoretto
C
Lleó
MM
Candpari
P
(
2002
)
Modification of the peptidoglycan of Escherichia coli in viable but nonculturable state
.
Curr Microbiol
 
44
:
125
131
.
Signoretto
C
Burlacchini
G
Lleó
MM
et al. (
2004
)
Adhesion of Enterococcus faecalis in the nonculturable state to plankton is the main mechanism for persistence of this bacterium in both lake and seawater
.
Appl Environ Microb
 
70
:
6892
6896
.
Sinton
L
(
2006
)
‘Viable but non-culturable’ bacteria – menace or myth?
New Zeal Water Wastes Assoc J
 
143
:
31
38
.
Smith
BE
Oliver
JD
(
2006a
)
In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state
.
Appl Environ Microb
 
72
:
1445
1451
.
Smith
BE
Oliver
JD
(
2006b
)
In situ gene expression by Vibrio vulnificus
.
Appl Environ Microb
 
72
:
2244
2246
.
Sperandio
V
Torres
AG
Jarvis
B
Nataro
JP
Kaper
JB
(
2003
)
Bacteria–host communication: the language of hormones
.
P Natl Acad Sci USA
 
100
:
8951
8956
.
Steinert
M
Emody
L
Amann
R
Hacker
J
(
1997
)
Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii
.
Appl Environ Microb
 
63
:
2047
2053
.
Sun
F
Chen
J
Zhong
L
Zhang
X-H
Wang
R
Guo
Q
Dong
Y
(
2008
)
Characterization and virulence retention of viable but nonculturable Vibrio harveyi
.
FEMS Microbiol Ecol
 
64
:
37
44
.
Sussman
M
Loya
Y
Fine
M
Rosenberg
E
(
2003
)
The marine fireworm Hermodice carunculata is a winter reservoir and spring-summer vector for the coral-bleaching pathogen Vibrio shiloi
.
Environ Microbiol
 
5
:
250
255
.
Vora
GJ
Meador
CE
Bird
MM
Bopp
CA
Andreadis
JD
Stenger
DA
(
2005
)
Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp
.
P Natl Acad Sci USA
 
102
:
19109
19114
.
Weichart
D
Kjelleberg
S
(
1996
)
Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus
.
Microbiology
 
142
:
845
853
.
Whitesides
MD
Oliver
JD
(
1997
)
Resuscitation of Vibrio vulnificus from the viable but nonculturable state
.
Appl Environ Microb
 
64
:
3025
3028
.
Wolf
P
Oliver
JD
(
1992
)
Temperature effects on the viable but nonculturable state of Vibrio vulnificus
.
FEMS Microbiol Ecol
 
101
:
33
39
.
Wong
HC
Liu
SH
(
2008
)
Characterization of the low-salinity stress in Vibrio vulnificus
.
J Food Protect
 
71
:
416
419
.
Wong
H-C
Wang
P
(
2004
)
Induction of viable but nonculturable state in Vibrio parahaemolyticus and its susceptibility to environmental stresses
.
J Appl Microbiol
 
96
:
359
366
.
Wong
H-C
Wang
P
Chen
S-Y
Chiu
S-W
(
2004
)
Resuscitation of viable but non-culturable Vibrio parahaemolyticus in a minimum salt medium
.
FEMS Microbiol Lett
 
233
:
269
275
.
Xu
H-S
Roberts
N
Singleton
FL
Attwell
RW
Grimes
DJ
Colwell
RR
(
1982
)
Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment
.
Microb Ecol
 
8
:
313
323
.
Yamamoto
H
(
2000
)
Viable but nonculturable state as a general phenomenon of non-spore-forming bacteria, and its modeling
.
J Infect Chemother
 
6
:
112
114
.
Yaron
S
Matthews
K
(
2002
)
A reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes
.
J Appl Microbiol
 
92
:
633
640
.
Young
DB
Gideion
HP
Wilkinson
RJ
(
2009
)
Eliminating latent tuberculosis
.
Trends Microbiol
 
17
:
183
188
.
Editor: David Gutnick