Viable but Nonculturable Bacteria

Joshua R Stokell, The University of North Carolina at Charlotte, Charlotte, North Carolina, USA
Todd R Steck, The University of North Carolina at Charlotte, Charlotte, North Carolina, USA

Published online: October 2012

DOI: 10.1002/9780470015902.a0000407.pub2

Viable but nonculturable (VBNC) bacteria are not detected using standard microbiological assays, hence environmental samples thought to lack bacteria may in fact contain large numbers of these dormant-like cells. Though regrowth, or resuscitation, of bacteria remains the most definitive measure of the presence of VBNC cells, growth-independent viability assays using flow cytometry combined with LIVE/DEAD staining, RT-qPCR and microarray analysis have been developed to detect VBNC cells in environmental samples. Genes specific for the VBNC state have yet to be identified, however, several genes, including rpoS, are reported to be involved in the VBNC condition. Further elucidation of the genetic mechanism causing entry into and resuscitation from the VBNC state will likely coincide with advances in transcriptomic and proteomic technologies. Though controversial, the VBNC state can explain certain environmental microbial phenomena, such as the source of recurrent bacterial infections in plants and animals.

Key Concepts:

  • The VBNC state is thought to be a long-term survival mechanism that initiates in response to environmental stress.
  • A general, but not universal, acceptance of the VBNC condition exists even though the molecular mechanism involved has yet to be elucidated.
  • Resuscitation of the VBNC state has been observed in only some species of bacteria reported to become VBNC.
  • Several genes, including rpoS, mreB and rpf have been identified as being involved in the VBNC condition.
  • Bacteria in the VBNC state may retain pathogenicity.

Keywords: viable but nonculturable (VBNC); bacteria; resuscitation; dormancy; persister cells

Figure 1. Escherichia coli cells viewed via epifluorescent microscopy after staining with the LIVE/DEAD Bacterial Viability Kit. (a) Exponentially growing culture; (b) cells placed in boiling water bath for 1 min. Green fluorescing cells have an intact cell membrane and are tentatively identified as being viable; red fluorescing cells lack an intact cell membrane and are scored as dead.
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 References
    Anderson M, Bollinger D, Hagler A et al. (2004) Viable but nonculturable bacteria are present in mouse and human urine specimens. Journal of Clinical Microbiology 42(2): 753–758.
    Asakura H, Watarai M, Shirahata T and Makino S (2002) Viable but nonculturable Salmonella species recovery and systemic infection in morphine-treated mice. Journal of Infectious Diseases 186(10): 1526–1529.
    Aurass P, Prager R and Flieger A (2011) EHEC/EAEC O104:H4 strain linked with the 2011 German outbreak of haemolytic uremic syndrome enters into the viable but non-culturable state in response to various stresses and resuscitates upon stress relief. Environmental Microbiology 13(12): 3139–3148.
    Barcina I and Arana I (2009) The viable but nonculturable phenotype: A crossroads in the life-cycle of non-differentiating bacteria? Reviews in Environmental Science and Biotechnology 8(3): 245–255.
    Binsztein N, Costagliola MC, Pichel M et al. (2004) Viable but nonculturable Vibrio cholerae O1 in the aquatic environment of Argentina. Applied and Environmental Microbiology 70(12): 7481–7486.
    Boaretti M, Lleo MM, Bonato B, Signoretto C and Canepari P (2003) Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state. Environmental Microbiology 5(10): 986–996.
    Bogosian G, Aardema ND, Bourneuf EV, Morris PJ and O'Neil JP (2000) Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state. Journal of Bacteriology 182(18): 5070–5075.
    Bogosian G and Bourneuf EV (2001) A matter of bacterial life and death. EMBO Reports 2(9): 770–774.
    Chiu SW, Chen SY and Wong HC (2008) Localization and expression of MreB in Vibrio parahaemolyticus under different stresses. Applied and Environmental Microbiology 74(22): 7016–7022.
    Colwell RR, Brayton P, Herrington D et al. (1996) Viable but non-culturable Vibrio cholerae O1 revert to a cultivable state in the human intestine. World Journal of Microbiology and Biotechnology 12(1): 28–31.
    Coutard F, Lozach S, Pommepuy M and Hervio-Heath D (2007) Real-time reverse transcription-PCR for transcriptional expression analysis of virulence and housekeeping genes in viable but nonculturable Vibrio parahaemolyticus after recovery of culturability. Applied and Environmental Microbiology 73(16): 5183–5189.
    Dinu LD and Bach S (2011) Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor. Applied and Environmental Microbiology 77(23): 8295–8302.
    Fauvart M, De Groote VN and Michiels J (2011) Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. Journal of Medical Microbiology 60(Pt. 6): 699–709.
    Grey BE and Steck TR (2001) The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection. Applied and Environmental Microbiology 67(9): 3866–3872.
    Hayes CS, Aoki SK and Low DA (2010) Bacterial contact-dependent delivery systems. Annual Review of Genetics 44: 71–90.
    Hayes CS and Low DA (2009) Signals of growth regulation in bacteria. Current Opinion in Microbiology 12(6): 667–673.
    Heim S, Del Mar Lleo M, Bonato B, Guzman CA and Canepari P (2002) The viable but nonculturable state and starvation are different stress responses of Enterococcus faecalis, as determined by proteome analysis. Journal of Bacteriology 184(23): 6739–6745.
    Keer JT and Birch L (2003) Molecular methods for the assessment of bacterial viability. Journal of Microbiological Methods 53(2): 175–183.
    Khan MM, Pyle BH and Camper AK (2010) Specific and rapid enumeration of viable but nonculturable and viable-culturable gram-negative bacteria by using flow cytometry. Applied and Environmental Microbiology 76(15): 5088–5096.
    Kong IS, Bates TC, Hulsmann A et al. (2004) Role of catalase and oxyR in the viable but nonculturable state of Vibrio vulnificus. FEMS Microbiology Ecology 50(3): 133–142.
    Lewis K (2010) Persister cells. Annual Review of Microbiology 64(1): 357–372.
    Liu Y, Wang C, Fung C and Li XF (2010a) Quantification of viable but nonculturable Escherichia coli O157:H7 by targeting the rpoS mRNA. Analytical Chemistry 82(7): 2612–2615.
    Liu Y, Wang C, Tyrrell G and Li XF (2010b) Production of Shiga-like toxins in viable but nonculturable Escherichia coli O157:H7. Water Research 44(3): 711–718.
    Lleo MM, Benedetti D, Tafi MC, Signoretto C and Canepari P (2007) Inhibition of the resuscitation from the viable but non-culturable state in Enterococcus faecalis. Environmental Microbiology 9(9): 2313–2320.
    Mishra A, Taneja N and Sharma M (2012) Viability kinetics, induction, resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio cholerae O1 in freshwater microcosm. Journal of Applied Microbiology 112(5): 945–953.
    Mukamolova GV, Murzin AG, Salina EG et al. (2006) Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation. Molecular Microbiology 59(1): 84–98.
    Muresu R, Polone E, Sulas L et al. (2008) Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes. FEMS Microbiology Ecology 63(3): 383–400.
    Nocker A, Sossa-Fernandez P, Burr MD and Camper AK (2007) Use of propidium monoazide for live/dead distinction in microbial ecology. Applied and Environmental Microbiology 73(16): 5111–5117.
    Nyström T (2001) Not quite dead enough: on bacterial life, culturability, senescence, and death. Archives of Microbiology 176(3): 159–164.
    Nyström T (2005) Bacterial senescence, programmed death, and premeditated sterility. ASM News 71: 363–369.
    Oliver JD (2010) Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiology Reviews 34(4): 415–425.
    Oliver JD and Bockian R (1995) In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus. Applied and Environmental Microbiology 61(7): 2620–2623.
    Pinto D, Almeida V, Almeida Santos M and Chambel L (2011) Resuscitation of Escherichia coli VBNC cells depends on a variety of environmental or chemical stimuli. Journal of Applied Microbiology 110(6): 1601–1611.
    Rahman I, Shahamat M, Chowdhury MA and Colwell RR (1996) Potential virulence of viable but nonculturable Shigella dysenteriae type 1. Applied and Environmental Microbiology 62(1): 115–120.
    Rahman MH, Suzuki S and Kawai K (2001) Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus. Microbiological Research 156(1): 103–106.
    Rivers B and Steck TR (2001) Viable but nonculturable uropathogenic bacteria are present in the mouse urinary tract following urinary tract infection and antibiotic therapy. Urological Research 29(1): 60–66.
    Saha SK, Saha S and Sanyal SC (1991) Recovery of injured Campylobacter jejuni cells after animal passage. Applied and Environmental Microbiology 57(11): 3388–3389.
    Santander RD, Català-Senent JF, Marco-Noales E and Biosca EG (2012) In planta recovery of Erwinia amylovora viable but nonculturable cells. Trees 26(1): 75–82.
    Senoh M, Ghosh-Banerjee J, Ramamurthy T et al. (2012) Conversion of viable but nonculturable enteric bacteria to culturable by co-culture with eukaryotic cells. Microbiology and Immunology 56(5): 342–345.
    Shleeva MO, Bagramyan K, Telkov MV et al. (2002) Formation and resuscitation of “non-culturable” cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase. Microbiology 148(Pt. 5): 1581–1591.
    Signoretto C, Lleo MM and Canepari P (2002) Modification of the peptidoglycan of Escherichia coli in the viable but nonculturable state. Current Microbiology 44(2): 125–131.
    Smith B and Oliver JD (2006) In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state. Applied and Environmental Microbiology 72(2): 1445–1451.
    Trevors J and Masson L (2010) DNA technologies: What's next applied to microbiology research?. Antonie Van Leeuwenhoek 98(3): 249–262.
    Trevors JT (2011) Viable but non-culturable (VBNC) bacteria: Gene expression in planktonic and biofilm cells. Journal of Microbiological Methods 86(2): 266–273.
    Trevors JT (2012) Can dead bacterial cells be defined and are genes expressed after cell death? Journal of Microbiological Methods 90(1): 25–28.
    Vora GJ, Meador CE, Bird MM et al. (2005) Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp. Proceedings of the National Academy of Sciences of the USA 102(52): 19109–19114.
    Whitesides MD and Oliver JD (1997) Resuscitation of Vibrio vulnificus from the viable but nonculturable state. Applied and Environmental Microbiology 63(3): 1002–1005.
    Xu H-S, Roberts N, Singleton FL et al. (1982) Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microbial Ecology 8(4): 313–323.
 Further Reading
    Colwell RR (2000) Viable but nonculturable bacteria: A survival strategy. Journal of Infection and Chemotherapy 6(2): 121–125.
    Oliver JD (2005) The viable but nonculturable state in bacteria? Journal of Microbiology 43: 93–100.
    Poole K (2012) Stress responses as determinants of antimicrobial resistance in Gram-negative bacteria. Trends in Microbiology 20(5): 227–234.
    Sachidanandham R and Yew-Hoong Gin K (2009) A dormancy state in nonspore-forming bacteria. Applied Microbiology and Biotechnology 81(5): 927–941.
    Trevors J, Bej A, Mojib N, van Elsas J and Van Overbeek L (2012) Bacterial gene expression at low temperatures. Extremophiles 16(2): 167–176.

Related Sub-Topics:

Bacteria | Bacterial Culture, Growth and Development | Environmental Microbiology
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Article Title: Viable but Nonculturable Bacteria
 
How to Cite close
Stokell, Joshua R, and Steck, Todd R(Oct 2012) Viable but Nonculturable Bacteria. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000407.pub2]