A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability
| Autor: | Maëlle Molmeret, Sylvie Hallier-Soulier, Eric Dusserre, Jerome Etienne, Christophe Ginevra, Sophie Jarraud, Gabriel Festoc, François Vandenesch |
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| Rok vydání: | 2008 |
| Předmět: |
DNA
Bacterial Legionella Microorganism Polymerase Chain Reaction Applied Microbiology and Biotechnology Legionella pneumophila Microbiology law.invention law Environmental Microbiology medicine Humans Polymerase chain reaction Ecology biology Pontiac fever biology.organism_classification Isolation (microbiology) medicine.disease Real-time polymerase chain reaction Chlorine Legionnaires' Disease Water Microbiology Bacteria Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 74:4817-4824 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.02899-07 |
| Popis: | Legionella pneumophila, the bacterium responsible for Legionnaires' disease and Pontiac fever, is ubiquitous in natural and man-made aqueous environments and requires free-living amoebae for its intracellular replication (1, 15, 31). Under appropriate conditions, L. pneumophila can also survive for long periods as a free organism in low-nutrient environments (4, 30). Regular monitoring of potentially contaminated water sources is essential to prevent legionellosis outbreaks (21, 27). Culture with selective media is the standard method for the detection, isolation, and identification of L. pneumophila in clinical and environmental samples (18, 19), but it can take more than 7 days. Cost-effective and reliable real-time quantitative PCR methods have been developed for rapid detection/quantification of Legionella DNA in water samples and are often used as a routine monitoring tool (14, 36). The results are expressed as the number of genome units (GU) per liter, but the precise equivalence with the number of CFU has not been established. Culture and PCR agree well on samples from hot water systems but not from cooling towers. Culture is always less sensitive than PCR (2, 23, 36). Discrepancies between PCR and culture results can be explained by several factors. Legionella growth can be inhibited or masked by overgrowth of contaminating microorganisms (18). Furthermore, L. pneumophila can enter a viable but noncultivable (VBNC) state, from which it can recover after passage in amoebae (12, 30). These VBNC legionellae may be detected by PCR, along with dead bacteria, possibly explaining, at least in part, why PCR values are usually higher than those obtained by culture. In order to understand the significance of positive L. pneumophila PCR results for water samples, we prepared L. pneumophila-containing water samples, with and without chlorination, and tested them comparatively by (i) conventional culture, (ii) a real-time PCR assay for total L. pneumophila DNA, (iii) a fluorescence assay for viable bacteria (VBNC and cultivable bacteria), and (iv) immunodetection of all living and dead intact L. pneumophila cells. Potential VBNC bacteria detected by the fluorescence assay were tested for infectivity and recultivability in a coculture technique with amoebae. |
| Databáze: | OpenAIRE |
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