**3. Discussion**

Several factors may hinder exact *Legionella* spp. quantification in environmental samples: (i) differences in the polycarbonate membrane characteristics (e.g., pore size, batches, fragility, crinkling and electrostatic interactions), (ii) different washing procedures to remove trapped bacteria from the membrane (e.g., shaker/vortex, ultrasound, finger and thumb scraping, or heat or acid treatment), which favor the detection of the microorganism but at the same time may reduce its concentration, and (iii) the choice of the culture medium [11–14].

As for the latter, the parameters affecting its quality are the following: (1) type and quantity of nutrients, (2) redox potential (Eh)—both after preparation and during incubation, (3) initial pH and buffering capacity, (4) water activity, and (5) type and activity of the antimicrobial agents—these can either be supplemented, already be present in the medium components, or accidentally form due to preparation errors, such as excessive heating [6,15]. Further evidence has highlighted several other deficiencies of those selective media that rely on a delicate balance between productive and selective mechanisms [4,8,16,17].

The quality of culture media has a dramatic effect on *Legionella* spp. recovery and counts. To evaluate the contribution of culture media, we checked relative recoveries of *Legionella* spp. from 148 environmental water samples because the response obtained from media plated with collection strains (i.e., quality control protocol) may vary when wild bacterial strains are present. It is known, for example, that virulent *L. pneumophila* are especially salt-sensitive, and that spontaneous mutations in stock strains may result in salt resistance [18]. Consequently, testing culture media using stock strains of bacteria may not always be a valid approach [5].

Although several different medium formulations are routinely used to detect *Legionella* spp. from environmental samples [10], there is paucity of studies assessing and comparing their abilities in growing *Legionella* spp. [4,5,9]. Since the quality of the culture medium strongly influences *Legionella* spp. detection and enumeration due to the presence of contaminating flora, here, we have assessed the recovery rates of *Legionella* spp. from hospital water samples using two different brands (i.e., Xebios and Oxoid) of either nonselective BCYEα or selective MWY medium.

Our analysis shows an excellent agreemen<sup>t</sup> between the recovery rates of the media from both companies (90.5%). Nonetheless, the quantitative recovery of *Legionella* colonies using Xebios media is significantly greater than that achieved by Oxoid media. Furthermore, the sensitivity of detection is significantly higher when samples are plated on MWY Xebios agar, while the selectivity of MWY appears to be the same regardless of the manufacturer. Moreover, there is a greater agreemen<sup>t</sup> between the two Xebios media compared to that between the two Oxoid media. Additionally, differences in colony size were apparent for the different agars (see supplementary materials). Specifically, the MWYXebios agar favored the growth of much larger colonies compared to MWYOxoid, and enhanced the recovery of non-*Legionella pneumophila* species.

As we used four different batches over a one-year period of study, it is highly unlikely that batch-to-batch variability may have played a role in the performance differences that we observed. We hypothesize that other factors such as the presence in the medium of toxic compounds (e.g., metals), growth-promoting factors, or high gel strengths may have influenced the growth and colony size on di fferent types of medium, as previously shown for *L. pneumophila* on BCYE α agar [19].

Collectively, these results highlight significant di fferences between the performances of media from di fferent manufacturers. Despite generating the same number of positive cultures, the Xebios media generally yielded greater numbers of *Legionella* spp. and larger colony sizes, allowing easier detection. Thus, the use of Xebios culture media is indicated to achieve the highest sensitivity and selectivity when detecting environmentally sampled *L. pneumophila*.
