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.
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?
ReplyDeleteAs 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
DeleteDetecting 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?
ReplyDeleteThe 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
DeleteThe 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!
ReplyDeleteThe 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.
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.
DeleteThere 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.