Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

• Published in: Journal of applied microbiology 2011-08, Vol.111 (2), p.499-510
• Main Authors: Touron-Bodilis, A, Pougnard, C, Frenkiel-Lebossé, H, Hallier-Soulier, S
• Format: Article
• Language: English
• Subjects: bacteria; biocides; Biological and medical sciences; Colony Count, Microbial; cooling systems; culture; Disinfectants; DNA, Bacterial - genetics; Environmental Monitoring - methods; Fundamental and applied biological sciences. Psychology; industrial cooling systems; L. pneumophila; Legionella - genetics; Legionella - isolation & purification; Legionella pneumophila; Legionella pneumophila - genetics; Legionella pneumophila - isolation & purification; Legionella spp; Limit of Detection; Microbiology; monitoring; nucleic acids; Polymerase Chain Reaction - methods; quantitative polymerase chain reaction; real‐time PCR; river water; Rivers - microbiology; Water Microbiology; Water Supply
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2011.05063.x

Aims: This study was designed to evaluate the usefulness of quantification by real-time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90-431). Methods and Results: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10⁵ GU l⁻¹) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57-100% of the samples. Conclusions: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real-time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. Significance and Impact of the Study: This study shows the possibility of using real-time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.