Murky waters – how bacteria survive in artificial water systems

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  • We use water for so many processes beyond hygiene and clinical applications that its inherent dangers are often not obvious. Any wet environment may serve as a refuge for bacteria, before moving on to a new host organism such as humans. Dr Sébastien Faucher from McGill University in Montreal has set out to unravel the survival strategies of bacteria in water with a view to developing improved microbial management approaches.

    For obvious reasons, a lot of thought and debate is spent on microbial contaminations in clinical environments or the food industry. However, we hear much less about bacteria in other man-made systems such as air conditioning, showers or sprinklers. Anywhere where we find water, we will probably also find bacteria.

    Human cultured macrophages infected with Legionella pneumophila expressing the green fluorescent protein. Accumulation of L. pneumophila inside cells produces green dots.

    Dr Sébastien Faucher is Associate Professor at the Faculty of Agricultural and Environmental Sciences at the McGill University in Montreal. His research studies genetic factors governing the survival of bacterial pathogens in non-clinical environments.

    Water as a refuge
    Bacteria are not always transferred directly from one host to the next and instead, they often need to find a temporary refuge. Niches with little circulation in man-made water systems are ideal for bacteria like Legionella pneumophila – the microorganism that causes the potentially fatal Legionnaires’ disease by infecting the lung. Legionella pneumophila can, for example, be transmitted by inhaling the aerosol from a shower that has not been used in a while because the bacteria have had time to multiply in the stagnant water. Cooling towers are frequently reported as the source of outbreaks.

    There have been numerous fatal Legionella outbreaks over the past decades, and it is vital we increase our understanding of these ubiquitous bacteriaQuote_brain

    Environmental factors
    Bacterial survival in water systems depends on a variety of environmental factors, such as availability of nutrients and trace metals, pH or temperature. Dr Faucher’s team has examined these factors systematically, finding that temperature and pH clearly impact Legionella survival in water. These bacteria die at an elevated temperature and low pH, with their preference appearing to be in an environment at 15°C and a neutral pH. Interestingly as well, absence of trace metals does not seem to harm Legionella. This is an important insight as previously it was assumed that the presence of Legionella correlated with the presence of trace metals, particularly iron.

    Surviving starvation
    Dr Faucher’s research has demonstrated that Legionella can sense and adapt to poor nutrient supply. This allows it to wait for more than six months, without any source of food, for prey cells to arrive in the system. Starving Legionella bacteria shrink in size, leverage intracellular energy stores and shut down regular metabolic processes to prevent wasting the scarce available resources in water. If they fail to do so due to mutations in key genes, bacteria have less of a chance of survival.

    Legionella pneumophila wild-type strain (left panel) and a rpoS mutant strain (right panel) coloured with a viability stain after suspension in water for 24h. Live cells are coloured green, dead cells are coloured red. Note the presence of dead cells in the right panel.

    The exact molecular chain of signalling events remains to be elucidated. According to Dr Faucher’s current insights, specific messenger molecules are produced when the bacterium experiences a lack of essential nutrients, for example amino acids. These trigger a general alarm signal that activates a key protein called RpoS. This regulator then causes a wide range of changes to gene expression through its regulatory network. One of the genes regulated is bdhA, which allows Legionella to use internal energy reserves; without it, Legionella cannot survive in water.

    Gimme danger
    In addition to fending off starvation, Legionella bacteria also become more infectious in water. Dr Faucher noted an increased expression of genes associated with resistance to antibiotics, host invasion and motility. Such genes code for transporters that can expel drugs from the cell, proteins that facilitate entry into host cells or structural components of flagella. In laboratory experiments, Legionella has indeed also shown increased resistance to some common antibiotics upon exposure to water.

    Schematic of a cooling tower. Biofilm are shown in green.

    The team also showed that Legionella remain infectious after six months in water. This highlights the importance behind preventing water stagnation, through regular flushing or removal of dead ends in pipework.

    Microbial communities
    Whilst water is a pre-requisite for microbial life, it does not always provide ideal conditions. When faced with hostile environments, microbes generally find strength in numbers and, in such cases, bacteria can form biofilms. In other words, the bacteria can group together and produce an extracellular matrix which fixes them in their location. Different microorganisms may cooperate in such a slime-like assembly.

    It is intriguing to imagine a probiotic strategy that uses benign microorganisms to counteract Legionella colonisation of cooling towersQuote_brain

    Amoeba, unicellular organisms, may enter infested areas to feed on bacteria. However, Legionella has the capability to exploit this behaviour and infect amoeba or other small organisms. It can effectively live as a parasite and multiply inside these organisms.

    Both of the above strategies help shield bacteria from chemicals that we apply in an attempt to sterilise the system.

    Introducing competition
    Dr Faucher’s more recent research interests revolve around the impact of microbial communities on Legionella survival. Why are some locations more prone to Legionella contamination than others? The answer might lie in the varying composition of microbial communities at different locations. It appears that some microorganisms are permissive to Legionella growth whilst others inhibit it.

    Cooling towers are an excellent study ground for such investigations. These are basically water reservoirs used to chill water circulating through air conditioning or industrial systems. Managing the microbial contamination in cooling towers can be challenging though and, as such, novel approaches are needed. It is intriguing to imagine a probiotic strategy that uses benign microorganisms to counteract Legionella colonisation.

    Dr Faucher’s research is steadily filling our knowledge gap in a niche of microbiology that is often only talked about when disaster strikes. There have been numerous fatal Legionella outbreaks in various countries over the past decades; it is vital that we increase our understanding of these ubiquitous bacteria to find improved microbial management strategies to prevent future tragedies.

  • Do you think current Legionella risk management legislation and official guidance is effective?
    In 2012, an outbreak of Legionnaires’ disease struck Quebec City. Legislation at the time was clearly insufficient to prevent outbreaks: proper maintenance of cooling towers was not mandatory. A few years later, we now have strict guidelines: a maintenance plan must be in effect and cooling towers must be routinely tested for the presence of Legionella. When the population of Legionella reaches 1,000,000 per litre, the cooling tower must be shut down and shock treatment applied. This helps prevent outbreaks from happening.

    What needs to be improved regarding the management of Legionella?
    There is still a need to improve the detection methods and treatment. The current detection procedure can take up to two weeks to complete. During that time, a cooling tower has the time to grow enough Legionella to cause an outbreak. Faster techniques are being developed, such as quantitative real-time PCR and biosensors. Chlorine is the treatment of choice now, but some cooling towers still harbour low levels of Legionella even when constantly treated. The problem is the biofilm, which protects the microbes from the disinfectant.

    Microbes are extremely resistant and are able to adapt and evolve to find solutions to whatever we throw at themQuote_brain

    How would you prevent accumulation of biofilm in a cooling tower?
    I believe that it is not possible to do so. Biofilm are extremely hard to remove from cooling towers. Microbes are extremely resistant and are able to adapt and evolve to find solutions to whatever we throw at them. Since we can’t exterminate them, it might be better to use them to our advantage. The idea here is to find, or develop, a population of microbes that inhibits growth of Legionella, and let this population colonise the cooling tower, which could, in theory, protect it from Legionella.

    Which other bacteria does your laboratory study?
    We are studying Campylobacter jejuni, a major cause of gastroenteritis in humans. Campylobacter normally colonise chickens and from there can travel to water streams and contaminate crops. We found that exposure to water improves the resistance of some strains to disinfectants. We have also studied its interactions with an aquatic ciliate named Tetrahymena – a unicellular organism that feeds on bacteria. When presented with Campylobacter, Tetrahymena pick them up and package them in many layers of membranes before excreting them out. These packages are called fecal pellets and increase the survival of Campylobacter in water.

    Are your research findings relevant to other man-made environments such as hospitals or food manufacturing plants?
    Many pathogens are transmitted to humans from the environment – not from other humans. This includes food-borne pathogens. In general, a lot is known about the virulence systems of pathogens, but a lot less is known about their behaviour outside the host, such as in water and in food. The current antibiotics usually target major metabolic pathways of pathogens, including cell wall synthesis, translation, transcription, etc. Since those pathways are also required outside the host, it adds pressure to evolved resistance. It might be better to target systems exclusively used during infection, but to do so, we need to know how bacteria survive outside the host.

  • Research Objectives
    Dr Faucher’s research focuses on improving our understanding of how water-borne pathogens survive in water systems, especially in terms of understanding the behavioural, genetic and environmental factors involved.

    Funding

    • Canadian Institutes of Health Research (CIHR)
    • NSERC Discovery Grant
    • FRQ_NT Team grant
    • Programme Innov’Action Agroalimentaire

    Collaborators

    • Laam Li
    • Hana Trigui
    • Nilmini Mendis
    • Kiran Paranjape
    • Émilie Bédard
    • Michèle Prévost
    • Steve Charette

    Bio
    Dr Sébastien Faucher obtained his PhD from U. Montreal in 2007. As a postdoctoral fellow at Columbia University, between 2007–2010, he studied the virulence of Legionella pneumophila. In 2010, he then moved to McGill University to study the host side of infectious disease. He joined the Faculty of Agricultural and Environmental Sciences in July 2011 when he was appointed Assistant Professor.

    Contact
    Dr Sébastien P. Faucher
    McGill University
    Faculty of Agricultural and Environmental Sciences
    Department of Natural Resource Sciences
    Macdonald-Stewart Building
    21111 Lakeshore Road
    Ste. Anne de Bellevue, Quebec
    H9X 3V9
    Canada

    E: sebastien.faucher2@mcgill.ca
    T: 514-398-7886
    W: www.faucherlab.com
    Twitter: @faucher_sp

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