Detection of Vibrio cholerae in environmental samples is made difficult by its ability to enter a dormant ‘viable but nonculturable’ (VBNC) state. VBNC cells will persist in an environment but will not grow on traditional growth media, allowing them to go undetected by conventional techniques. Recently, fluorescent antibody based microscopy has allowed identification of V.cholerae in samples from which it would previous have gone unnoticed. Antibiotic selection techniques, using knowledge of the antibiotic resistance of previously cultured strains from pandemics, also allows small numbers of VBNC cells to be identified, by suppressing the growth of other bacteria.
The danger of VBNC cells is not just that they are difficult to detect, they can be ‘resuscitated’, switching from dormancy to an active state that is capable of infection. The researches had in a previous study identified two autoinducer molecules that were upregulated in conditions of large cells densities to promote the production of Vibrio extracellular polysaccharide by active cells. Because autoinducers are only produced under conditions of high cell density, they reasoned that they might also signal to dormant VBNC cells that conditions are favourable for growth, causing resuscitation.
They therefore proceeded to investigate whether biologically or synthetically produced autoinducer molecules would induce resuscitation of VBNC V.cholerae in environmental samples from Bangladesh.
Antibiotic selection techniques were used to identify samples that contained active V.cholerae, only samples that did not already contain active cells were used. They found that samples containing no culturable V.cholerae, when treated with spent media from either autoinducer-producing V.cholerae or E.coli, produced resuscitated cells within only a few hours of treatment. Water from the same samples tested negative for culturable V.cholerae following treatment with spent media from the controls: a V.cholerae strain that had had its autoinducer genes deleted and E.coli that contained the cloning vector used to induce autoinducer production but without the genes required for their production. Furthermore, profiling of the culturable V.cholerae strains produced via this resuscitation showed their close similarity to strains that had caused recent epidemics in the area.
Having established the link between autoinducers and resuscitation, they proceeded to investigate whether a wild type strain of V.cholerae could initiate resuscitation of VBNC cells. It could not without the addition of a recombinant plasmid containing synthase genes. Wild strains that already contain this plasmid do show resuscitation activity.
Chemically synthesized versions of the two autoinducer molecules caused resuscitation when provided together and individually, showing that the presence of only one of them is required for dormant cells to become active. One of the autoinducers is narrowly produced by Vibrios only, however the other is produced by many bacterial groups. This study therefore suggests that resuscitation of VBNC V.cholerae could occur either outside or inside the human host as a result of high concentrations of autoinducer molecules produced by environmental bacteria or those of the human microbiome. They suggest that this may explain the seasonality of cholera, as the seasonal occurrence of heterologous bacteria that produce the same autoinducer molecule may trigger resuscitation of large numbers of dormant V.cholerae. Another model would be that the bacteria of the human microbiome cause resuscitation upon ingestion, or that faecal bacteria that enter the environment are favoured by seasonal conditions and so induce resuscitation in the environment.
Many potential models are suggested, however, the major contribution of this paper is not its speculations but the mechanism it has identified that links resuscitation of dormant V.cholerae cells to bacterial inter-species communication via autoinducer molecules.
Bari, S. N., Roky, M. K., Mohiuddin, M., Kamruzzaman, M., Mekalanos, J. J., & Faruque, S. M. (2013). Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples. Proceedings of the National Academy of Sciences, 110(24), 9926-9931.