Growth Phase, Oxygen, Temperature, and Starvation Affect the Development of Viable but Non-culturable State of Vibrio cholerae.

Bin Wu,Weili Liang,Biao Kan

doi:10.3389/fmicb.2016.00404

Abstract

Vibrio cholerae can enter into a viable but non-culturable (VBNC) state in order to survive in unfavorable environments. In this study, we studied the roles of five physicochemical and microbiological factors or states, namely, different strains, growth phases, oxygen, temperature, and starvation, on the development of VBNC of V. cholerae in artificial sea water (ASW). Different strains of the organism, the growth phase, and oxygen levels affected the progress of VBNC development. It was found that the VBNC state was induced faster in V. cholerae serogroup O1 classical biotype strain O395 than in O1 El Tor biotype strains C6706 and N16961. When cells in different growth phases were used for VBNC induction, stationary-phase cells lost their culturability more quickly than exponential-phase cells, while induction of a totally non-culturable state took longer to achieve for stationary-phase cells in all three strains, suggesting that heterogeneity of cells should be considered. Aeration strongly accelerated the loss of culturability. During the development of the VBNC state, the culturable cell count under aeration conditions was almost 106-fold lower than under oxygen-limited conditions for all three strains. The other two factors, temperature and nutrients-rich environment, may prevent the induction of VBNC cells. At 22 or 37°C in ASW, most of the cells rapidly died and the culturable cell count reduced from about 108 to 106–105 CFU/mL. The total cell counts showed that cells that lost viability were decomposed, and the viable cell counts were the same as culturable cell counts, indicating that the cells did not reach the VBNC state. VBNC state development was blocked when ASW was supplied with Luria-Bertani broth (LB), but it was not affected in ASW with M9, suggesting that specific nutrients in LB may prevent the development of VBNC state. These results revealed that the five factors evaluated in this study had different roles during the progress of VBNC induction. Changing a single factor could influence and even block the development of the VBNC state. These findings provide new insight to help design further studies to better understand the mechanisms which trigger the development and regulation of the VBNC state.

Highlights

The viable but non-culturable (VBNC) state is defined as a state where bacteria are metabolically active but lack the ability to reproduce on routine culture media (Oliver, 2005)
To test if there were any differences in development of the VBNC state among the different strains, we analyzed the VBNC development curves for three V. cholerae strains, including the classical biotype strain O395 and the El Tor biotypes C6706 and N16961
These results suggested that the VBNC cells can remain viable in artificial sea water (ASW)-LuriaBertani broth (LB); the “viable” cells calculated by the PMA-qPCR in ASW-LB condition were not VBNC cells but dead cells that had gradually decomposed (Figure 5C)

Summary

Introduction

The viable but non-culturable (VBNC) state is defined as a state where bacteria are metabolically active but lack the ability to reproduce on routine culture media (Oliver, 2005). Studies have found that Vibrio cholerae cells in the VBNC state can be resuscitated by introduction in a rabbit ileal loop and in the intestines of human volunteers (Colwell et al, 1985; Kaper et al, 1995). For other pathogens such as Escherichia coli, V. vulnificus, and V. parahaemolyticus, the resuscitation can be achieved by a mere increase in the temperature (Oliver and Bockian, 1995; Wong et al, 2004; Pinto et al, 2011)

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