Members of the Legionella genus find suitable conditions for their growth and survival in nuclear power plant cooling circuits. To limit the proliferation of Legionella pathogenic bacteria in nuclear power plant cooling circuits, and ensure that levels remain below regulatory thresholds, monochloramine treatment can be used. Although the treatment is highly effective, i.e. it reduces Legionella numbers by over 99%, Legionella bacteria can still be detected at low concentrations and rapid re-colonisation of circuits can occur after the treatment has ceased. The aim of this study was to develop an in vitro methodology for determining the intrinsic susceptibility of L. pneumophila strains, collected from various nuclear power plant cooling circuits subjected to different treatment conditions. The methodology was developed by using an original approach based on response surface methodology (RSM) combined with a multifactorial experimental design. The susceptibility was evaluated by the Ct factor. The susceptibility of environmental strains varies widely and is, for some strains, greater than that of known tolerant species; however, strain susceptibility was not related to treatment conditions. Selection pressure induced by monochloramine use did not result in the selection of more tolerant Legionella strains and did not explain the detection of Legionella during treatment or the rapid re-colonisation of cooling circuits after disinfection has ceased.

Members of the pathogenic Legionella genus encounter suitable growth conditions in nuclear power plant cooling circuits. To limit its proliferation and ensure that levels remain below regulatory thresholds, chemical treatment with monochloramine can be used in continuous or sequential conditions. The aim of this study was to determine the impact of monochloramine on L. pneumophila subpopulations in the cooling circuits of a nuclear power plant. The chosen procedure involved monitoring the diversity and dynamics of L. pneumophila subpopulations every month over the course of a year in a nuclear power plant cooling circuit, which was treated for 2 months during the period under study. This study confirmed the effectiveness of monochloramine to limit L. pneumophila concentrations in cooling circuits. The culturable L. pneumophila community was strongly affected by the injection of monochloramine. Several subpopulations persisted during treatment at low concentrations (below the detection limit of standard methods), suggesting that the susceptibility of L. pneumophila is strain dependent. Although the composition of the subpopulations was not similar, the resilience of the community structure was observed. Indeed, the community eventually returned to its initial structure and presented a similar pattern of richness, diversity and uniformity to that seen before treatment.

Significance and Impact of the Study: IRS PCR, unlike PFGE and SBT, is a suitable tool for the ecological study of Legionella pneumophila in a large scale. It will be helpful to investigate diversity and dynamics of Leg. populations in water systems where these bacteria are strongly represented, as in cooling circuits.Keywords bacterial typing techniques, infrequent restriction site PCR, Legionella, pulsed-field gel electrophoresis, sequence-based typing. AbstractLegionella bacteria are ubiquitous in aquatic environments. Members of the species Legionella pneumophila are responsible for more than 98% of cases of Legionnaires' disease in France. Our objective was to validate a molecular typing method called infrequent restriction site PCR (IRS PCR), applied to the study of the ecology of Legionella and to compare this method with reference typing methods, pulsed-field gel electrophoresis (PFGE) and sequence-based Typing (SBT). PFGE and SBT are considered as gold methods for the epidemiological typing of Leg. pneumophila strains. However, these methods are not suitable to an ecological monitoring of Legionella in natural environments where a large number of strains has to be typed. Validation of IRS PCR method was performed by the identification of 45 Leg. pneumophila isolates from cooling circuits of thermal power plants by IRS PCR, PFGE and SBT. The parameters of each method were measured and compared to evaluate the effectiveness of IRS PCR. The results of this study showed that IRS PCR has a discriminating power similar or better than that of the reference methods and thus that, by its speed and low cost represents an appropriate tool for the study of the ecology of Legionella in cooling circuits.