The Viable but Nonculturable State for Bacteria

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The Viable but Nonculturable State for Bacteria: Status Update This dormant form of bacteria was first appreciated in 1982; now skeptics recognize this state as a bacterial response to stress and a strategy for survival James D. Oliver

The notion of microorganisms living in a viable but nonculturable (VBNC) state originates from studies by Rita Colwell at the University of Maryland and her collaborators, whose focus was on Vibrio cholerae. Here, I summarize the early history of this fıeld as well as some of the later studies on the genetics of the VBNC state, the role of quorum sensing in resuscitating dormant cells, and the effect global climate change is having on the spread and incidence of pathogenic vibrios. Much of this material was discussed during the symposium, “Healthy Waters, Healthy People: A Tribute to Rita Colwell,” convened during the 2015 ASM General Meeting in New Orleans, La. The VBNC state, now identifıed in numerous bacterial species, including important pathogens, as well as in some eukaryotes, is induced by a variety of environmental stresses. It is now widely believed that VBNC likely accounts for the seasonality of some bacteria in natural environments, and that these dormant cells resuscitate when the inducing stress is removed, possibly aided by the presence of other actively growing bacteria and the quorum sensing molecules that they produce. Further, VBNC cells typically are resistant to antibiotics as well other environmental stresses, allowing the dormant cells to persist despite these otherwise lethal stresses. Despite greatly reduced metabolic activity, which allows dormant cells to persist, these cells continue to express genes and show other signs of activity. We are also beginning to understand how the VBNC state and resuscitation from it is regulated genetically. Of special concern from a public health perspective is that global warming appears to be spreading Vibrio spp. and human vibriosis, adding yet another important concern related to global warming. The VBNC state, fırst

recognized by Colwell and her collaborators in 1982 and then applicable only to two bacterial species subsequently became a major area of research worldwide, leading to my collecting more than 600 publications on this topic during the course of several decades. Defining the Viable but Nonculturable State

The viable but nonculturable state refers to a microbial cell that fails to grow on nutrient media on which it would normally grow and develop into a colony but is alive and can return to a metabolically active and culturable state under appropriate circumstances. A variety of environmental stresses, ranging from suboptimum growth temperatures to the presence of heavy metals to elevated osmotic concentrations, can induce cells into the VBNC state, with the cells becoming fully nonculturable (Fig. 1a). However, a large percentage of the original

SUMMARY ➤ The viable but nonculturable (VBNC) state was recognized in 1982 by Rita Colwell of the University of Maryland and her collaborators. ➤ VBNC, now identified in numerous bacterial species, as well as in some eukaryotes, is induced by a variety of environmental stresses. ➤ Many different types of bacteria, including human pathogens, are capable of entering this state, maintaining cellular structure and biology, and continuing significant gene expression while otherwise being nonculturable by standard laboratory methods. ➤ Cells that enter the VBNC state eventually exit this state of dormancy to become fully culturable again, a shift called “resuscitation” that may be aided by quorum sensing signal molecules. ➤ Global climate change appears to be resuscitating dormant forms of Vibrio, leading Vibrio-related infections to increase worldwide.

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Microbe—Volume 11, Number 4, 2016

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FIGURE 1

Entrance of V. vulnificus into the VBNC state. (A) Cells were incubated in artificial seawater at 5°C and monitored by plate counts (䡩), direct microscopic counts (▫), or direct viable counts (●). (Adapted from JD Oliver, J. Microbiol. 43:93–100, 2005.) (B). Culturable Vibrio spp. (●) and water temperature (䡩) from estuarine waters of eastern North Carolina between August, 2000 and April, 2002. (Adapted from CS Pfeffer et al., Appl. Environ. Microbiol. 69:3526 –3531, 2003.)

population retains viability, demonstrating that culturability cannot be equated to viability. Indeed, while these cells exhibit greatly reduced but detectable metabolism, they can be resuscitated to a more metabolically active and culturable state. This phenomenon is not restricted to the laboratory, but can be seen among microbial cells in estuarine waters. It is most striking when measured across varying water temperatures (Fig. 1b), suggesting that seasonality for some microorganisms in natural environments is a result of the VBNC phenomenon. Huai-Shu Xu (1936 –2001) conducted the fırst VBNC studies between 1980 and 1982 in Colwell’s laboratory (Fig. 2). As pointed out in a tribute to him during the 2015 International Marine Microbiology Conference in China (http://immc2015.csp.escience.cn/dct/page /70002), “Professor Xu was one of the fırst Chinese scientists to visit the USA after the Cultural Revolution. During this visit at the University of Maryland during July 1980 to November 1982, he participated in marine microbiological research, and was instrumental in recognizing the viable but nonculturable (VBNC) state of Vibrio cholerae in the aquatic environment. This work re160 •


sulted in a landmark publication co-authored with Professor Rita Colwell.” Colwell and Xu’s proposal for a viable form of a serious pathogen that could not be cultured in the laboratory was “a revolutionary concept for its time, and the work polarized scientifıc opinion, which was then immersed in the dogma that culturability was akin to viability,” according to that tribute. Although this fırst published report about V. cholerae and Escherichia coli in 1982 did not use the term VBNC, it soon appeared in a 1985 report from Colwell and her collaborators when they described dormant cells. Subsiding Skepticism about the Reality and Importance of the VBNC State

Whereas some microbiologists remained skeptical about the VBNC state for many years, the phenomenon is now widely accepted as legitimate, reflecting in part extensive molecular studies that indicate such cells continue to express genes even after there are signifıcant decreases in overall metabolic activity. Many different types of bacteria, including human pathogens, are capa-


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FIGURE 2

Huai-Shu Xu (l) and Rita Colwell.

ble of entering this state, maintaining cellular structure and biology, and continuing signifıcant gene expression while otherwise being nonculturable by standard laboratory methods. According to UKEssays of Nottingham, United Kingdom: “That they can exit from this state, and become culturable again, is also undeniable. . .. The VBNC state plays a critical role in the survival of important human (and other) pathogens, and possibly in their ability to produce disease” (see www.ukessays.com/essays/biology/viable -but-non-culturable-bacteria-biology-essay .php). The number of bacterial types known to enter the VBNC state includes nearly 60 genera and more than 100 species. The list includes many human pathogens, including Mycobacterium tuberculosis, Helicobacter pylori, V. cholerae, Legionella pneumophila, Salmonella spp., and Campylobacter spp. As noted by Gengenbacher and Kaufmann of the Max Planck Institute for Infection Biology in Berlin, “Latent tuberculosis is the result of solid granulomas containing M. tuberculosis and because of decreased culturability, they are considered to be VBNC cells.” Moreover, “Chlamydia persistence is defıned as a long-term association between Chlamydia and their host cell in which these organisms remain in a viable but culture-negative state,” add Yasser AbdelRahman and Robert Belland of the University of

Tennessee Health Sciences Center. More recently, researchers reported the VBNC state in a number of eukaryotes, most notably the yeasts Saccharomyces cerevisiae and Brettanomyces spp. Studies examining in situ gene expression, along with providing defınitive evidence for the validity of the VBNC state, also provide evidence for why some cells appear less virulent while in the VBNC state. For example, we found that V. vulnifıcus no longer produces its antiphagocytic capsule when in a low temperature-induced VBNC state. This observation likely accounts, at least in part, for why this highly fatal pathogen causes so few infections during the winter. However, cells in the VBNC state are remarkably durable. For example, in this dormancy state V. vulnifıcus cells have an increased resistance to antibiotics, ethanol, high temperature, alkaline and acidic conditions, and heavy metals as well as to other potentially lethal stresses. Earlier, researchers did not understand the genetic basis for the VBNC state. More recently, researchers studying “persister” cells, another state of dormancy that we believe is related to the VBNC state, described the important role that toxin/antitoxin (T/AT) modules play during dormancy. Cells produce two proteins in T/AT systems, with the antitoxin protein inactivating the toxin protein. Stress leads to selective proteolysis of the antitoxin, freeing the toxin molecule and



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FIGURE 3

The VBNC state coincides with expression of hipA (A) and relE (B) toxins. Expression of hipA homologs hipA21, hipA2 and hipA3, and relE homologs relE1&2, relE3, relE4, and relE5, are elevated in VBNC cells relative to culturable cells in log phase. ****, P < 0.0001. (Adapted from M Ayrapetyan et al., Infect. Immun. 83:4194 – 4203, 2015.)

thus inhibiting cellular metabolism, including by blocking protein translation, which in turn leads to cell stasis. We found that both the hipA and relE systems, known to be involved in the persister response, occur in V. vulnifıcus. When cells are subject to low temperature stress, they induce the VBNC state following increases in activity of both these T/AT systems (Fig. 3). Resuscitating Cells from the VBNC State

Cells that enter the VBNC state eventually exit this state of dormancy to become fully culturable again, a shift called “resuscitation.” This phenomenon occurs in the laboratory, in vivo, and in situ. Indeed, entry into the VBNC state followed by resuscitation likely accounts for the fluctuating levels of individual species typically observed in natural environments (Fig. 1b). To resuscitate VBNC V. vulnifıcus, we use the membrane diffusion chambers that Gordon Mcfeters of Montana State University in Bozeman developed. These chambers allow full exchange of temperature, salinity, pH, nutrients, and bacteriophage, while retaining the bacterial cells within the chamber. Such studies show a cold temperature-induced loss of cell culturabil162 •


ity despite maintenance of high numbers of viable cells. At warmer temperatures, the cells quickly resuscitate to become culturable again. Many vibrios become nonculturable at cold temperatures, whether growing freely in water or within a shellfısh host. We routinely culture V. vulnifıcus from oysters, the primary vector for the potentially fatal food infections caused by this species during warm water months. However, we are unable to isolate V. vulnifıcus from oysters during winter months. Assuming these bacteria remain present in oysters but in the VBNC state, we reasoned that warming winter oysters would lead to increases in vibrio culturability as the VBNC cells underwent resuscitation. To our surprise, however, we saw no increase after we incubated such oysters for two months at 20oC. However, when we added other, genetically marked V. vulnifıcus cells to these oysters, the levels of background Vibrio spp. then increased signifıcantly. This new vibrio population, however, did not consist of the added V. vulnifıcus cells, but derived instead from resuscitated vibrios that were originally present in the oysters. Those results led us to wonder about the mechanism and signal that enabled the added


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FIGURE 4

Role of quorum sensing (AI-2) in resuscitation of V. vulnificus from the VBNC state. Cells were subjected to a room temperature upshift plus synthetic AI-2 or with cell free supernatants (CFS) taken from V. vulnificus, V. parahaemolyticus, or E. coli. Cells receiving any of these supplements responded by resuscitating within 3 hours, whereas cells subjected to room temperature upshift only (control) or upshift plus CFS from an E. coli mutant unable to produce AI-2 (⌬luxS) required >8h to resuscitate. (Adapted from M Ayrapetyan et al., Appl. Environ. Microbiol. 80:2478 –2483, 2014.)

bacteria to resuscitate the dormant V. vulnifıcus cells. A clue came from Slava Epstein of Northeastern University and his collaborators, who suggest that “scout cells” derived from the dormant cells trigger them to resuscitate. To test this possibility, we added cell-free supernatants (CFS) from known quorum sensingproducing cells to V. vulnifıcus cells in the VBNC state. Whereas we typically resuscitate V. vulnifıcus cells by shifting them to room temperature for about 8 hours, adding CFS along with that same temperature upshift led to an almost immediate increase in culturability (Fig. 4). The same was observed when using supernatant material from other quorum sensing cells, including V. parahaemolyticus and E. coli, or when the quorum sensing molecule AI-2 was added to the dormant cells. However, CFS from a mutant that cannot produce the AI-2 quorum sensing molecule failed to resuscitate the dormant cells. Together these fındings suggested to us that the dormant

microflora of the oysters sensed the A1–2 molecules, which were interpreted as a signal for the presence of a large population of actively metabolizing cells. The dormant cells responded by resuming full metabolic activity. Climate Change Seems To Be Accelerating Vibrio Resuscitation Rates

If temperature is so critical to the presence of free-living vibrio bacteria in water and in oysters, will increases in water temperature lead to increased geographic distribution and incidence of infections resulting from resuscitated cells? The answer to that question is not “if,” but “how soon?” Vibrio-related infections are indeed increasing worldwide, corresponding to rising global sea surface temperatures, according to Luigi Vezzulli and Carla Pruszzo of Università degli Studi di Genova in Genoa, Italy, and their collaborators. Indeed, as reported by Craig Bak-



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er-Austin and colleagues at the Cefas Laboratory in the UK, fatal cases of V. cholerae are being reported in northern Finland, and episodes of vibriosis occurred in Denmark following unusually warm summers. Such occurrences are likely because VBNC vibrio cells are being resuscitated in warmer waters. James D. Oliver is Bonnie E. Cone Distinguished Professor for Teaching in the Department of Biological Sciences, University of North Carolina at Charlotte.

Suggested Readings Ayrapetyan M, Williams TC, Oliver JD. 2014. Bridging the gap between viable but non-culturable and antibiotic persistent bacteria. Trends Microbiol. 23:7–13. Ayrapetyan M, Williams TC, Oliver JD. 2014. Interspecifıc quorum sensing mediates the resuscitation of viable but nonculturable vibrios. Appl. Environ. Microbiol. 80:2478 –2483. Baker-Austin, C., J. A. Trinanes, N. G. H. Taylor, R. Hartnell, A. Siitonen and J. Martinez-Urtaza. 2013. Emerging Vibrio risk at high latitudes in response to ocean warming. Nat. Clim. Change 3:73–77. Colwell RR, Brayton PR, Grimes DU, Rosazk DB, Huq SA, Palmer LM. 1985. Viable but non culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Nature Biotechnol. 3:817– 820.

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Li L, Mendis N, Trigui H, Oliver JD, Faucher SP. 2014. The importance of the viable but non-culturable state in human pathogens. Frontiers Microbiol. 5:72–100. Nowakowska J, Oliver JD. 2013. Resistance to environmental stresses by Vibrio vulnifıcus in the viable but nonculturable state. FEMS Microbiol. Ecol. 84:213– 222. Oliver JD. 2000. Public health signifıcance of viable but nonculturable bacteria. p. 277–300. In RR Colwell and DJ Grimes (ed.), Non- culturable microorganisms in the environment. ASM Press, Washington, D.C. Oliver JD. 2009. Recent fındings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol. Rev. 34:415– 425. Smith BE, Oliver JD. 2006. In situ and in vitro gene expression by Vibrio vulnifıcus during entry into, persistence within, and resuscitation from the viable but nonculturable state. Appl. Environ. Microbiol. 72: 1445–1451. Vezzulli L, Pezzati E, Brettar I, Höfle M, Pruzzo C. 2015. Effects of global warming on Vibrio ecology. Microbiol. Spectrum 3(3); doi.10.1128/microbiolspec. VE-0004 –2014. Xu H-S, Roberts N, Singleton FL, Attwell RW, Grimes DJ, Cowell RR. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol. 8:313–323.
