Sunday, 9 February 2014

Dormant V.cholerae cells are resuscitated by quorum sensing molecules

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 Sciences110(24), 9926-9931.


  1. It is interesting that Vibrio cholera responds in such a way to the autoinducer molecules. Whilst I think it stands that they may influence the change to an active state from a VBNC state, I think other environmental parameters also have an influence. Many physical and chemical stressors can induce bacteria to enter the VBNC state (Oliver, 2005). VBNC bacteria undergo morphological changes which protect them from environmental conditions with are otherwise potentially lethal to active bacterial cells. As such, I doubt that the autoinducer molecules would initiate the change from VBNC to active if the environmental conditions weren’t right. Would the molecules even be produced in such conditions? Are they dependant on a similar environmental range, and their presence is indicator to such bacteria of a ‘safe’ environment in which the active bacteria could tolerate?

    1. As they are only produced in high cell densities they would not be produced unless conditions were advantageous. So perhaps the correct conditions permit growth of active cells to sufficient densities to produce these autoinducer molecules and trigger resuscitation. That could be the mechanism by which conditions induce change from VBNC cells to an active state

  2. Detecting VBNC bacteria is highly problematic and difficult. Whilst sampling for harmful species, I wonder if applying a range of synthetic autoinducers to the samples could allow easier detection of bacteria by increasing their activity?

    1. The problem with synthetic autoinducers is that they may produce active, potentially infectious cells in the environment. I think the fluorescent antibody techniques are judged to be less risky

  3. The discovery of bacterial social communication (quorum sensing) has always attracted my attention. Consistent discoveries of this phenomenon in microbes from a range of environments (such as marine environment, surface of corals, light organ of bobtail squid, human body etc.) show how widespread quorum sensing is!
    The other reason that makes it very interesting area of future research is its potential as an alternative to antibiotics.
    Many posts on this blog have highlighted the problem of antibiotics and fear of development of superbugs (multidrug resistant bacteria) because of continuous use of antibiotics.
    In this scenario manipulating quorum sensing of pathogens (quorum quenching) is seen as exciting new area of research and development of new alternative drugs.
    For example, see Faloon et al. 2014
    Faloon, P., Weiner, W. S., Matharu, D. S., Neuenswander, B., Porubsky, P., Youngsaye, W., ... & Schreiber, S. L. (2014). Discovery of ML 370, an inhibitor of Vibrio cholerae Quorum Sensing Acting via the LuxO response regulator.

    1. Yes I have read about quorum quenching, and I am sure that whilst writing one of my more recent blog posts I read it quorum-sensing blockers were being considered as an alternative to aquaculture antibiotics. As it does not kill the bacteria directly, it should not be as strong a selection pressure for resistant strains. However, if preventing quorum sensing does affect the bacteria's ability to reproduce then I think resistant bacteria would still actually be selected for on a population scale.
      There is also the issue that as many non-specific quorum sensing molecules are used by many species, it may be difficult to target specific pathogenic bacteria, blocking their quorum sensing at the cost of beneficial species might not be advantageous.