Resistance to Environmental Stress by Vibrio vulnificus in VBNC State

In order to survive rapidly changing marine environments microorganisms must firstly identify these changes and then respond accordingly, by either adapting physiology or utilising complex survival mechanisms. The Viable But Not Culturable (VBNC) state is one such mechanism wherein microorganisms enter a state of low metabolic activity in which they can no longer grow on media but retain viability and, once environmental conditions permit, they can return to a culturable state. This allows certain microorganisms to survive when seasonal or short term variations in biotic or abiotic factors occur. One such microorganism is the bacteria Vibrio vulnificus which is found in estuarine conditions and so is already adapted to a highly variable environment. This pathogenic bacterium is associated with 95% of all seafood related deaths in the US and, when in the VBNC state, is protected against potentially lethal environmental factors whilst maintaining its pathogenic viability. Nowakowska and Oliver (2013) set out to determine whether cells of V. vulnificus in the VBNC state were resistant to different stressors than those in the culturable state and to examine whether any genetic regulators were responsible.

2 strains of vulnificus were used in this study: the C-genotype (clinical) and the E-genotype (environmental). All strains were grown overnight at room temperature and then grown to an early log phase after which they were inoculated at a 1:100 dilution in ½ strength seawater. The cells were then either exposed to various chemical or physical stressors, or placed at 5˚C to induce the VBNC state. To test for culturability the cells were plated on agar and incubated at 37˚C overnight and to test for viability the Live/Dead BacLight bacterial assay was used before and immediately after exposure.  Resuscitation of VBNC cells was achieved by incubating at 37˚C overnight.  The following physical and chemical challenges were applied to both the VBNC and culturable vulnificus; heat challenge, oxidative challenge (exposure to H₂0₂), osmotic challenge (exposure to NaCl), ethanol challenge, pH challenge, antibiotic challenge (exposure to ampicillin and chloramphenicol) and a heavy metal challenge (zinc and copper exposure). For each of the stressors the time was adjusted such that 99.99% loss in culturability of the culturable cells was achieved, providing a control for the VBNC cells. Finally RNA extraction and RT-PCR was used to isolate spoT, relA and rpoS (global stress regulator gene) genes for examination of possible gene regulation mechanisms.

Both the C and G genotypes of the culturable cells of vulnificus showed equal sensitivity to all stressors, both groups showing a reduction of 99.99% culturability. When in the VBNC state however both groups (C and E) demonstrated the same ability to withstand heat and antibiotic resistance. The E genotype in the VBNC state showed significantly greater resistance to low and high pH, hydrogen peroxide, high salinity and ethanol when compared to the C-genotype. In terms of gene regulation, there was no significant difference in the expression of relA and spoT between the culturable and VBNC state of the C- genotype of vulnificus. There was a significant difference in the expression of relA between the culturable and VBNC cells in genotype-E with an up regulation with decreased temperature observed. This up regulation could have some role in allowing the E-genotype to be more equipped to deal with acute stressors.

This paper set out to answer a question and I feel it did so successfully and builds from previous work in this field. I think the paper was very easy to follow, set out well and didn’t make any unwarranted or sensationalist claims. Finally, the authors emphasise the importance of coming up with better ways to detect and destroy vulnificus in shellfish production and this could provide the basis for further work in the field.

Paper reviewed

Nowakowska, Joanna and James D. Oliver. (2013). Resistance to environmental stresses by vibrio Vulnificus in the viable but nonculturable state. FEMS Microbiology Ecology 84.1 213-222