Viability PCR,a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.Legionella organisms are ubiquitous bacteria found in many types of water sources in the environment. Their growth is especially favored in human-made warm water systems, including cooling towers, hot tubs, showerheads, and spas (3, 14, 15, 38). Legionella bacteria replicate as intracellular parasites of amoebae and persist in the environment as free-living microbes or in biofilms. In aerosol form, they enter the lungs and can cause an acute form of pneumonia known as Legionnaires'' disease or a milder form of pulmonary infection called Pontiac fever. The species Legionella pneumophila is responsible for the vast majority of the most severe form of this atypical pneumonia (52, 70). Legionellosis outbreaks are associated with high mortality rates (15 to 20%) (15, 16, 38, 46), which can reach up to 50% for people with weakened immune systems (immunocompromised patients) (69). Legionella surveillance programs include regular monitoring of environmental water samples (9, 13, 66). It is generally acknowledged that Legionella represents a health risk to humans when cell densities are greater than 10⁴ to 10⁵ CFU per liter of water, and epidemiological data show that outbreaks of legionellosis occur at these concentrations (36, 47).The evaluation of the risk associated with Legionella has traditionally been performed using culture-based methods (1, 24). Culture is essential for identifying and typing Legionella strains during epidemics. However, Legionella culture requires long incubation times (up to 10 days) before results can be scored. This problem makes culture unsuitable for preventive actions and rapid response in emergency situations. Moreover, under certain conditions (i.e., low-nutrient environments, oxidative or osmotic stress, etc.), Legionella cells can lose the ability to be cultured, although they are still viable (7, 17, 20, 22, 39, 45, 67). These viable but nonculturable (VBNC) Legionella cells may still represent a public health hazard because they can regain their ability to grow in new, more favorable conditions (12, 19, 23, 61).Molecular approaches, such as quantitative real-time PCR (qPCR), are faster and can mitigate the main drawbacks of culture-based methods. qPCR is an alternative tool that offers rapid, sensitive, and specific detection of Legionella bacteria in environmental water samples (4, 5, 12, 26, 65, 68). PCR results can be obtained in hours instead of days, and VBNC Legionella cells can also be detected (12, 26). However, the major disadvantage of qPCR lies in its inability to evaluate viability due to the persistence of DNA in cells after death (27, 34). The monitoring of Legionella contamination levels by conventional qPCR may thus result in an overestimation of the risk of infection because false-positive results can be scored. However, the real risk from Legionella is limited to the live fraction of the total Legionella population. Only live or viable Legionella cells are able to replicate in pulmonary macrophages and cause severe pneumonia (14, 15). The development of more rapid, culture-independent methods capable of discriminating between live and dead cells is of major interest for measuring Legionella infection risks and preventing legionellosis. The nucleic acid-binding dye ethidium monoazide bromide (EMA), used in combination with qPCR, is an attractive alternative for selectively detecting and enumerating viable bacteria. EMA is particularly useful because it selectively penetrates cells with damaged membranes and covalently binds to DNA after photoactivation (21, 53). DNA-bound EMA molecules prevent PCR amplification and thereby lead to a strong signal reduction during qPCR. DNA from viable cells with intact cell membranes prevents EMA molecules from entering the cell and therefore can be amplified and quantified (56). Nocker et al. (41, 42) suggested that the signal reduction was due to a selective loss of genomic DNA from dead cells (rendered insoluble after cross-linkage) during the DNA extraction procedure rather than to PCR inhibition. However, Soejima et al. (59, 60) recently reported that treatment with EMA followed by visible light irradiation directly cleaves the chromosomal DNA of dead bacteria.In this study we optimized the EMA-staining procedure in conjunction with qPCR with pure cultures of L. pneumophila. We analyzed the potential for the EMA-qPCR method to discriminate Legionella cells with compromised or intact cell membranes. We optimized this EMA-qPCR technique, viability PCR, hereafter named v-PCR, and used it to quantify viable Legionella cells in environmental water samples. We compared our results with those obtained by conventional qPCR and culture methods. In addition, we evaluated the ability of v-PCR to monitor the efficacy of different disinfection strategies.