Summary

Bacteria of the genus Legionella are widespread in many environments, both natural and artificial. The concentrations found in natural environments are very low. Contamination mainly occurs in artificial reservoirs with water temperatures ranging from 25 to 42 °C, where there is some stagnation along with the presence of deposits, biofilms and amoebae. Hot tubs, pools, evaporative condensers, misters and cooling towers are recognized sources of legionellosis outbreaks due to the substantial growth of bacteria of the genus Legionella in such environments.  

Bacteria of the genus Legionella cause two diseases: Pontiac fever, an influenza-like respiratory infection, and Legionnaire’s disease, a severe pneumonia. The United States Centers for Disease Control and Prevention (CDC) reported more than 6,800 cases of legionellosis in 2009-2010 and France identified 1,298 confirmed cases in 2012.

In July 2014, a new regulation amending the Safety Code to include provisions on maintaining a water cooling tower installation came into force in Quebec. This regulation requires the monitoring of Legionella pneumophila concentrations in cooling tower water. A cooling tower must not contain Legionella pneumophila concentrations considered to represent a public or occupational risk.

Following the legionellosis outbreak in the summer of 2012 in Quebec City, the Director of Public Health issued recommendations, one of which was to implement a method for “rapidly identifying the sources in an outbreak using sampling and culture analysis, q-PCR or any other methods to be developed based on their sensitivity and particular specificity.”

This study validated three molecular systems for detecting and quantifying the Legionella bacterium in water samples. The three systems were shown to be very specific for purposes of this analysis. The first system (JFP/JRP: LegLC) detects all species of Legionella, the second (PT69-PT70: lpneuFL) is specific for the Legionella pneumophila species and the third (P66/P65: Sg1) is highly specific for Legionella pneumophila belonging to serogroup 1 ¹⁸. In addition to being used to confirm identification by culture method, these systems can be applied to perform a rapid and specific analysis directly from water samples. The quality of the DNA extract is very important in the polymerase chain reaction (PCR). High quality DNA yields better quantifications due to greater control of inhibitors. Given the risk of PCR inhibition, it is essential to include the required controls for all samples. The extraction kits showed highly variable results. This step in the analytic process should be controlled and monitored in order to produce high quality extracts for PCR.

Although detection and quantification by PCR cannot yet replace the culture method recognized in the regulation, it can, in some critical situations, yield results much more quickly and produce analysis results for samples with an elevated heterotrophic microbial load that cannot be analyzed using the traditional culture method, as is currently the case in some Quebec industries. The set of controls incorporated into the analytical process increases confidence in the robustness of the results, even for these samples of very complex composition.

Although few correlations have been shown between quantitative PCR and culture results, it is important to note that all positive samples determined by culture method were also determined by PCR analysis, meaning that our systems yielded no false negatives. This favours its use in confirming the non-contamination of industrial cooling systems which are virtually impossible to analyze by culture method.

This study provides an analytical approach for characterizing three specification levels: genus (Legionella spp), species (Legionella pneumophila) and more specifically serogroup 1 of this species. The validation and optimization steps made it possible to identify and implement a reliable and robust analytical process for detecting the bacterium in most liquid samples.