1887

Abstract

can survive environmental adversities by entering into a viable but non-culturable (VBNC) state and is able to resuscitate under favourable conditions. In this study, an environmental strain of (AN59) showed a decrease in culturability from 4×10 to ≤ 3 c.f.u. ml in artificial seawater media at 4 °C within 35 days. During the course of VBNC progression, viability was confirmed by real-time RT-PCR which showed reduced but stable expression of molecular chaperones and . Resuscitation was induced in VBNC microcosm by a temperature increase from 4 to 37 °C for 24 h. The results obtained from resuscitation and growth experiments suggest that 10–10  c.f.u. ml of VBNC cells should recover upon temperature increase and grow to attain 10  c.f.u. ml . We used comparative proteomics to differentiate recovery from the VBNC state and selected 19 proteins whose expression was significantly variable between these two states. These proteins were mainly related to carbohydrate metabolism, phosphate utilization, stress response, transport and translation. The main difference in the proteome profile was higher protein expression in the recovery state compared to VBNC state. However, during recovery Pi-starvation led to expression of PhoX, PstB and Xds, which might help in utilization of extracellular DNA to promote growth after resuscitation. In addition, the expression of EctC suggests that osmotic adaptation is necessary to grow at high salinity. Detection of AhpC in the VBNC and recovery state indicates the significance of the oxidative stress response. A temperature-induced VBNC and recovery state is a combination of adaptive and survival responses under nutrient limitation.

Keyword(s): VBNC , proteomics , recovery and resuscitation
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/content/journal/micro/10.1099/mic.0.000798
2019-07-01
2019-10-18
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References

  1. Kaper JB, Morris JG, Jr, Levine MM. Cholera. Clin Microbiol Rev 1995;8:48–86 [CrossRef]
    [Google Scholar]
  2. Colwell RR. Global climate and infectious disease: the cholera paradigm. Science 1996;274:2025–2031 [CrossRef]
    [Google Scholar]
  3. Miller MB, Skorupski K, Lenz DH, Taylor RK, Bassler BL. Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. Cell 2002;110:303–314 [CrossRef]
    [Google Scholar]
  4. Faruque SM, Albert MJ, Mekalanos JJ. Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae. Mol Bio Rev 1998;62:1301–1314
    [Google Scholar]
  5. Ravel J, Knight IT, Monahan CE, Hill RT, Colwell RR. Temperature-induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation?. Microbiology 1995;141:377–383 [CrossRef]
    [Google Scholar]
  6. Pruzzo C, Tarsi R, Lleò MM, Signoretto C, Zampini M et al. Persistence of adhesive properties in Vibrio cholerae after long-term exposure to sea water. Environ Microbiol 2003;5:850–858 [CrossRef]
    [Google Scholar]
  7. Wai SN, Mizunoe Y, Yoshida S. How Vibrio cholerae survive during starvation. FEMS Microbiol Lett 1999;180:123–131 [CrossRef]
    [Google Scholar]
  8. Oliver JD. The viable but nonculturable state in bacteria. J Microbiol 2005;43:93–100 Review
    [Google Scholar]
  9. Li L, Mendis N, Trigui H, Oliver JD, Faucher SP. The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol 2014;5:258 [CrossRef]
    [Google Scholar]
  10. Asakura H, Watarai M, Shirahata T, Makino S-I. Viable but nonculturable Salmonella species recovery and systemic infection in morphine-treated mice. J Infect Dis 2002;186:1526–1529 [CrossRef]
    [Google Scholar]
  11. Baffone W, Citterio B, Vittoria E, Casaroli A, Campana R et al. Retention of virulence in viable but non-culturable halophilic Vibrio spp. Int J Food Microbiol 2003;89:31–39 [CrossRef]
    [Google Scholar]
  12. González-Escalona N, Fey A, Höfle MG, Espejo RT, A Guzmán C. Quantitative reverse transcription polymerase chain reaction analysis of Vibrio cholerae cells entering the viable but non-culturable state and starvation in response to cold shock. Environ Microbiol 2006;8:658–666 [CrossRef]
    [Google Scholar]
  13. Asakura H, Ishiwa A, Arakawa E, Makino S-ichi, Okada Y et al. Gene expression profile of Vibrio cholerae in the cold stress-induced viable but non-culturable state. Environ Microbiol 2007;9:869–879 [CrossRef]
    [Google Scholar]
  14. Wong HC, Shen CT, Chang CN, Lee YS, Oliver JD et al. Biochemical and virulence characterization of viable but nonculturable cells of Vibrio parahaemolyticus. J Food Prot 2004;67:2430–2435 [CrossRef]
    [Google Scholar]
  15. Lai CJ, Chen SY, Lin IH, Chang CH, Wong H-C. Change of protein profiles in the induction of the viable but nonculturable state of Vibrio parahaemolyticus. Int J Food Microbiol 2009;135:118–124 [CrossRef]
    [Google Scholar]
  16. Muela A, Seco C, Camafeita E, Arana I, Orruño M et al. Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state. FEMS Microbiol Ecol 2008;64:28–36 [CrossRef]
    [Google Scholar]
  17. Heim S, Lleo MM, Bonato B, Guzman CA, Canepari P. The viable but nonculturable state and starvation are different stress responses of Enterococcus faecalis, as determined by proteome analysis. J Bacteriol 2002;184:6739–6745 [CrossRef]
    [Google Scholar]
  18. Jia J, Li Z, Cao J, Jiang Y, Liang C et al. Proteomic analysis of protein expression in the induction of the viable but nonculturable state of Vibrio harveyi SF1. Curr Microbiol 2013;67:442–447 [CrossRef]
    [Google Scholar]
  19. Jiang X, Chai TJ. Survival of Vibrio parahaemolyticus at low temperatures under starvation conditions and subsequent resuscitation of viable, nonculturable cells. Appl Environ Microbiol 1996;62:1300–1305
    [Google Scholar]
  20. Nilsson L, Oliver JD, Kjelleberg S. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. J Bacteriol 1991;173:5054–5059 [CrossRef]
    [Google Scholar]
  21. Whitesides MD, Oliver JD. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. Appl Environ Microbiol 1997;63:1002–1005
    [Google Scholar]
  22. Weichart D, Oliver JD, Kjelleberg S. Low temperature induced non-culturability and killing of Vibrio vulnificus. FEMS Microbiol Lett 1992;100:205–210 [CrossRef]
    [Google Scholar]
  23. Wu B, Liang W, Kan B. Growth phase, oxygen, temperature, and starvation affect the development of viable but Non-culturable state of Vibrio cholerae. Front Microbiol 2016;7:404 [CrossRef]
    [Google Scholar]
  24. Coelho A, de Oliveira Santos E, Faria MLdaH, de Carvalho DP, Soares MR et al. A proteome reference map for Vibrio cholerae El Tor. Proteomics 2004;4:1491–1504 [CrossRef]
    [Google Scholar]
  25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–408 [CrossRef]
    [Google Scholar]
  26. Mongkolsuk S, Whangsuk W, Vattanaviboon P, Loprasert S, Fuangthong M. A Xanthomonas alkyl hydroperoxide reductase subunit C (ahpC) mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes. J Bacteriol 2000;182:6845–6849 [CrossRef]
    [Google Scholar]
  27. Miller VL, Mekalanos JJ. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 1988;170:2575–2583 [CrossRef]
    [Google Scholar]
  28. Sperandio V, Girón JA, Silveira WD, Kaper JB. The OmpU outer membrane protein, a potential adherence factor of Vibrio cholerae. Infect Immun 1995;63:4433–4438
    [Google Scholar]
  29. Mathur J, Davis BM, Waldor MK. Antimicrobial peptides activate the σE regulon through an OmpU-dependent signalling pathway. Mol Microbiol 2007;63:848–858 [CrossRef]
    [Google Scholar]
  30. Deuerling E, Patzelt H, Vorderwülbecke S, Rauch T, Kramer G et al. Trigger factor and DnaK possess overlapping substrate pools and binding specificities. Mol Microbiol 2003;47:1317–1328 [CrossRef]
    [Google Scholar]
  31. Liu Y, Beyer A, Aebersold R. On the dependency of cellular protein levels on mRNA abundance. Cell 2016;165:535–550 [CrossRef]
    [Google Scholar]
  32. Colwell RR, Brayton PR, Grimes DJ, Roszak DB, Huq SA et al. Viable but Non-Culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Nat Biotechnol 1985;3:817–820 [CrossRef]
    [Google Scholar]
  33. Coutard F, Crassous P, Droguet M, Gobin E, Colwell RR et al. Recovery in culture of viable but nonculturable Vibrio parahaemolyticus: regrowth or resuscitation?. ISME J 2007;1:111–120 [CrossRef]
    [Google Scholar]
  34. Seper A, Fengler VHI, Roier S, Wolinski H, Kohlwein SD et al. Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation. Mol Microbiol 2011;82:1015–1037 [CrossRef]
    [Google Scholar]
  35. McDonough E, Kamp H, Camilli A. Vibrio cholerae phosphatases required for the utilization of nucleotides and extracellular DNA as phosphate sources. Mol Microbiol 2016;99:453–469 [CrossRef]
    [Google Scholar]
  36. Wang HW, Chung CH, Ma TY, Wong H-chung, Wong HC. Roles of alkyl hydroperoxide reductase subunit C (AhpC) in viable but nonculturable Vibrio parahaemolyticus. Appl Environ Microbiol 2013;79:3734–3743 [CrossRef]
    [Google Scholar]
  37. Mykytczuk NCS, Trevors JT, Foote SJ, Leduc LG, Ferroni GD et al. Proteomic insights into cold adaptation of psychrotrophic and mesophilic Acidithiobacillus ferrooxidans strains. Antonie van Leeuwenhoek 2011;100:259–277 [CrossRef]
    [Google Scholar]
  38. Leblanc L, Leboeuf C, Leroi F, Hartke A, Auffray Y. Comparison between NaCl tolerance response and acclimation to cold temperature in Shewanella putrefaciens. Curr Microbiol 2003;46:157–162 [CrossRef]
    [Google Scholar]
  39. Chuang MH, Wu MS, Lo WL, Lin J-T, Wong C-H et al. The antioxidant protein alkylhydroperoxide reductase of Helicobacter pylori switches from a peroxide reductase to a molecular chaperone function. Proc Natl Acad Sci U S A 2006;103:2552–2557 [CrossRef]
    [Google Scholar]
  40. Ongagna-Yhombi SY, Boyd EF. Biosynthesis of the osmoprotectant ectoine, but not glycine betaine, is critical for survival of osmotically stressed Vibrio parahaemolyticus cells. Appl Environ Microbiol 2013;79:5038–5049 [CrossRef]
    [Google Scholar]
  41. Kapfhammer D, Karatan E, Pflughoeft KJ, Watnick PI. Role for glycine betaine transport in Vibrio cholerae osmoadaptation and biofilm formation within microbial communities. Appl Environ Microbiol 2005;71:3840–3847 [CrossRef]
    [Google Scholar]
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