**1. Introduction**

*Legionella* is a genus consisting of fastidious waterborne pathogens responsible for a severe form of pneumonia named Legionnaires' disease (LD) and for a flu-like infection known as Pontiac fever (PF) [1]. *Legionella* is widespread in natural freshwater environments, where it can be found free-living or intracellularly in hosts such as amoebae [2]. Among the 62 species known to date, *L*. *pneumophila* is

the species most frequently found in cases of infection, amounting in 2018 to approximately 94.1% of the culture-confirmed LD cases in notified in EU/EEA (European Legionnaires' disease Surveillance Network annual meeting 2019, unpublished data). However, just under half of the known species cause illness and, in several countries such as New Zealand, soil-born *L. longbeachae* is the primary cause of LD [1]. Infection is acquired through inhalation of contaminated aerosols produced by various man-made water systems, such as showers, spa pools, fountains and cooling towers of air conditioning systems [3]. When *Legionella* colonizes the water systems, it often finds favorable conditions for growth, such as temperatures between 25 ◦C and 45 ◦C or the presence of biofilm, reaching high concentrations and becoming a serious risk for human health. After the first LD outbreak occurred in Philadelphia in 1976, numerous other outbreaks and sporadic cases have been reported worldwide [4–10]. In Italy in 2018, the incidence of Legionnaires' disease was 4.9 cases per million inhabitants, 2964 notified cases [11], and in the European network for Legionnaires' disease surveillance, Italy ranked first in terms of the number of reported cases [12]. In addition, in 2018 two important outbreaks occurred in Italy that required increased environmental monitoring ([13]; data unpublished).

Most European countries have adopted a preventive approach, implementing actions for prevention and control of *Legionella* contamination. Monitoring *Legionella* contamination of potable water systems is of paramount importance for risk assessment. To this end, the plate culture method, performed using specific media (buffered charcoal yeas<sup>t</sup> extract, BCYE), usually supplemented with different combinations of antimicrobial selective substances, is considered the gold standard for detection and enumeration of *Legionella* in water samples [14]. Culture can also be performed in accordance with ISO 11731:2017, an updated norm which replaced both ISO 11731:1998 (used in this study) and ISO 11731-2:2004. [15–17]. Although plate culture methods are specific for *Legionella*, they have high variability in enumeration, are time-consuming and require significant experience in recognizing *Legionella* colonies [18]. In addition, the enumeration of a *Legionella* concentration may be under-estimated due to the inability to detect viable but not-culturable bacteria or *Legionella* within amoebae [1]. Molecular methods based on the detection of *Legionella* DNA have been demonstrated to be highly specific and sensitive, are able to discriminate between species and serogroup and can detect viable but nonculturable bacteria, but are not considered fully suitable to enumerate *Legionella* in water samples because they are unable to reliably discriminate whether DNA detected is from live or dead organisms [19,20]. A promising alternative method is the Legiolert test (IDEXX Laboratories, Westbrook, ME, USA), a liquid culture method based on bacterial enzyme detection technology, which determines the most probable number (MPN) of exclusively *L. pneumophila* species present in water samples. The presence of *L. pneumophila* is visualized through the utilization of a substrate present in the Legiolert reagent. In published studies, Legiolert has shown equal performance to traditional plate culture methods, providing results in seven days with simplified sample preparation and analysis [21].

In this study, the detection and enumeration of *L. pneumophila* were determined in both 10 mL and 100 mL of potable water samples using the Legiolert method. Data obtained were compared with those obtained by the traditional plate culture method, performed according to the ISO 11731:1998 using 1 L of potable water, in order to evaluate the possibility of using the Legiolert method as a valid alternative to traditional plate culture. The results of this study are encouraging for the adoption of the Legiolert method for *L. pneumophila* enumeration in water samples.
