Development of a real-time PCR method for the detection and/or quantification of viable Legionella spp. and L. pneumophila in sanitary and thermal water samples

In 2017, about 8,000 cases of Legionnaires’ disease were reported in the EU and the number of unreported infections is probably higher. Monitoring programs must be conducted to assess the infectious risks in water networks. Preventive measures include the surveillance and control of water distribution systems and the improvement of the techniques for Legionella detection. A promising approach for detecting viable Legionella cells is viability-PCR (v-PCR), which is based on sample treatment with photoactivatable and cell membrane impermeant nucleic acid intercalating dyes prior to DNA extraction and PCR amplification. The aim of the present thesis is to develop and optimize a molecular method for the detection and/or quantification of live Legionella cells in sanitary and thermal water samples based on real-time PCR technology. Organization of the thesis and experimental approaches The first chapter is focused on the evaluation of four genomic DNA extraction methods, by comparing cell lysis efficiency and quality of DNA extract from complex water samples. The second chapter describes the development of a v-PCR protocol using PEMAX dye. In order to evaluate the matrix effect, the optimized protocol was tested on water samples artificially contaminated with variable concentrations of live and dead Legionella cells. Sanitary and thermal water samples were tested in parallel with the culture method ISO11731, v-PCR and qPCR. Results obtained highlighted the need to apply further strategies to increase the effectiveness: at first an immunomagnetic separation (IMS) of L. pneumophila cells was evaluated as a purification step before v-PCR to eliminate the competing microflora. A second method involved the use of Free DNA Removal Solution (FDRS) to remove free DNA from water samples prior to qPCR analysis. The third chapter evaluates a new approach called “nutritional stimulation” based on the culture-enrichment of filtered water samples in Legionella specific media to stimulate the growth of live Legionella cells, followed by DNA extraction and qPCR amplification. A shift between the cycle threshold (Ct) of an unstimulated and a stimulated aliquot of a sample interpreted as the presence of viable Legionella spp. cells. In this study a method for the detection of Legionella spp. and L. pneumophila was developed including an efficient DNA purification step that allows a high recovery of cells. Moreover, a v-PCR protocol was developed, and other strategies were evaluated for the selective quantification of live Legionella cells in water samples. We conclude from our study that PEMAX dye combined with qPCR cannot be reliably used to quantify viable Legionella in environmental samples. A very promising approach was developed based on nutritional stimulation of filtered water sample in BYEα and BYEα broth supplemented with GVPC for 48 h at 37°C, as it is easy to perform and in 51 hours from sampling provides information about the presence of viable Legionella spp. cells.