Legionella is a Gram-negative, aerobic obligate bacterium, whose habitats are represented by both natural and artificial aquatic environments, in a free-living form, as parasites of protozoa and within biofilms (Bianchi et al. 2016). Colonization by Legionella occurs more easily if the artificial water network presents stagnation, biofilm, corrosion, limestone and temperatures between 25°C and 42°C.
Legionella, and particularly the species L.pneumophila (Lp), is the causative agent of Legionellosis, a generic term used to describe both pneumonic and non-pneumonic forms. Legionella is commonly transmitted by inhalation of contaminated aerosols, originating from air conditioning, cooling towers, hot and cold water systems, dental unit, humidifiers and whirlpool spas, but also by aspiration of contaminated water (Tesauro et al. 2018).
Legionellosis varies from a mild febrile illness (Pontiac fever) to a serious and sometimes fatal form of pneumonia (Legionnaires’ disease) with an approximate mortality rate of 10-30% (Italian guidelines). Elderly, immunocompromised, transplanted and onco-hematological patients are particularly at risk (Fields et al. 2002).
Some authors (Dusserre et al. 2008, Ducret et al. 2014, Li et al. 2014, Mansi et al. 2014, Casini et al. 2018) reports that Lp, if submitted to prolonged stress, such as continuous disinfection treatments, can enter in a state of low metabolic activity that makes it "viable but not cultivable" (VBNC). VBNC bacteria are not able to replicate in culture but regain virulence when environmental conditions return to be optimal, representing a risk for human health.
Laboratory methods currently used for both research and quantification of Lp in water are culture method and Real-Time PCR, both with strengths and limitations.
Culture method allows to detect and isolate all Legionella species and permits to correlate clinical strains with the environmental ones. Using this method, however, the results, expressed in Colony Forming Units (CFU), can be reported after long time, usually 11 days after the beginning of the analysis. In addition, an underestimation of Legionella species may occur because culture method detects only living bacteria and can be influenced by the presence of interfering bacterial flora (Leoni et al. 2001, Collins et al. 2015, Kirschner et al. 2016, Collins et al. 2017, De Giglio et al. 2020). Otherwise, qPCR strengths regard i) the ability to amplify and simultaneously quantify the DNA of the species of Legionella, expressing the results in Genomic Units (GU), ii) the rapid response times from the beginning of the analysis (hours instead of days) and iii) the capacity to also detect VBNC bacteria (Fittipaldi et al. 2011, Whiley and Taylor 2016, Boss et al. 2018). On the other hand, this technique does not have ISO validation and, as a substantial limit, it also detects the DNA of dead bacteria. Furthermore, at the moment, it is not possible to convert in a univocal way GU into CFU (Joly et al. 2006, Shih et al. 2006, Ditommaso et al. 2014, Collins et al. 2015, Collins et al. 2017, Reyneke et al. 2017, Whiley and Taylor 2016, Boss et al. 2018), as required by national and international guidelines.
To overcome this problem, some authors (Chang et al. 2009, Chang et al. 2010, Chen and Chang 2010, Qin et al. 2012, Mansi et al. 2014, Inoue et al. 2015a and 2015b, Reyneke et al. 2017) highlighted the possibility of adding to qPCR a pretreatment on the bacterial cells with Ethidium Monoazide Bromide (EMA), a fluorescent dye capable of crossing damaged cell membrane of dead bacteria and covalently binding to their DNA. Thereafter, exposing the bacterial colonies to a light of 500W, the dye is activated, with consequent induction of irreversible damage to the nucleic acids which cannot be amplified in a subsequent amplification.
Combination of qPCR and EMA pretreatment would therefore allow to amplify only the DNA of viable and VBNC bacteria, but also in this case there are limits. EMA, being toxic, could penetrate living cells and damage them, causing an underestimation of living bacteria (Reyneke et al. 2017, Boss et al. 2018). Furthermore, in case of high concentration of dead bacteria (>105 CFU/ml), the amount of EMA may not be enough to inactivate their DNA, with risk of having false positives (Fittipaldi et al. 2011, Qin et al. 2012). Subsequently, it would be necessary to optimize EMA concentration according to chlorine concentration possibly present in examined water samples. Finally, it is important to consider the rather significant cost of the method.
Aims of this work were i) to compare the culture and the qPCR methods on the same water samples and ii) to applicate and develop the EMA qPCR, using a commercial kit and modifying its protocol, in order to identify a rapid method able to speed up the procedures, detect the viable and “viable but not cultivable” bacteria (VBNC) and exclude dead bacteria, especially in situations where discriminating is particularly relevant, as in presence of clusters or outbreaks of Legionellosis.