The Impact of Protease during Recovery from Viable but Non-Culturable (VBNC) State in Vibrio cholerae.

Shin-Ichi Miyoshi,Anusuya Debnath

doi:10.3390/microorganisms9122618

Abstract

Vibrio cholerae can survive cold stress by entering into a viable but non-culturable (VBNC) state, and resuscitation can be induced either by temperature upshift only or the addition of an anti-dormancy stimulant such as resuscitation-promoting factors (Rpfs) at suitable temperature. In this study, the role of proteinase K was analyzed as an Rpf in V. cholerae. A VBNC state was induced in V. cholerae AN59 in artificial seawater (ASW) media at 4 °C, and recovery could be achieved in filtered VBNC microcosm, called spent ASW media, merely by a temperature upshift to 37 °C. The resuscitation ability of spent ASW was further enhanced by the addition of proteinase K. The mode of action of proteinase K was investigated by comparing its effect on the growth of the VBNC and culturable state of V. cholerae in ASW and spent ASW media. The presence of proteinase K allowed culturable cells to grow faster in ASW by reducing the generation time. However, this effect of proteinase K was more pronounced in stressed VBNC cells. Moreover, proteinase K-supplemented spent ASW could also accelerate the transition of VBNC into recovered cells followed by rapid growth. Additionally, we found that dead bacterial cells were the substrate on which proteinase K acts to support high growth in spent ASW. So, the conclusion is that the proteinase K could efficiently promote the recovery and growth of dormant VBNC cells at higher temperatures by decreasing the duration of the initial lag phase required for transitioning from the VBNC to recovery state and increasing the growth rate of these recovered cells.

Highlights

The viable but non-culturable state is a distinctive survival strategy adopted by numerous microbial species to habituate in unfavorable environmental conditions
The dormant viable but non-culturable (VBNC) state can be transitioned to an active culturable state by several methods such as high temperature, the addition of nutrients, catalase and direct interaction with eukaryotic cells [3,9,13,14]
This study showed that spent medium filtered from VBNC

Summary

Introduction

The viable but non-culturable state is a distinctive survival strategy adopted by numerous microbial species to habituate in unfavorable environmental conditions. The inability to detect various pathogenic bacteria in the VBNC state makes them a serious threat for food safety and public health because there is always a possibility of their resuscitation under appropriate conditions. Vibrio species are the predominant inhabitants of the marine ecosystem, and several of them were reported to be able to convert into the VBNC state when kept under low temperature and nutrient starvation conditions [1]. It has been reported that the mere transfer from low temperature to high temperature could induce recovery in Vibrio cholerae, V. parahaemolyticus and V. vulnificus [3,4,5,6]. Recovery from the VBNC state has been addressed different hypotheses; for example, according to Nilsson et al and Whitesides et al, all the population of V. vulnificus in a microcosm become VBNC and resuscitate without cell division.

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Conclusion