In this study, we aimed to associate the molecular typing of Legionella isolates with a culture technique during routine Legionella hospital environmental surveillance in hot water distribution systems (HWDSs) to develop a risk map able to be used to prevent nosocomial infections and formulate appropriate preventive measures. Hot water samples were cultured according to ISO 11731:2017. The isolates were serotyped using an agglutination test and genotyped by sequence-based typing (SBT) for Legionella pneumophila or macrophage infectivity potentiator (mip) gene sequencing for non-pneumophila Legionella species. The isolates' relationship was phylogenetically analyzed. The Legionella distribution and level of contamination were studied in relation to temperature and disinfectant residues. The culture technique detected 62.21% of Legionella positive samples, characterized by L. pneumophila serogroup 1, Legionella non-pneumophila, or both simultaneously. The SBT assigned two sequence types (STs): ST1, the most prevalent in Italy, and ST104, which had never been isolated before. The mip gene sequencing detected L. anisa and L. rubrilucens. The phylogenetic analysis showed distinct clusters for each species. The distribution of Legionella isolates showed significant differences between buildings, with a negative correlation between the measured level of contamination, disinfectant, and temperature. The Legionella molecular approach introduced in HWDSs environmental surveillance permits (i) a risk map to be outlined that can help formulate appropriate disinfection strategies and (ii) rapid epidemiological investigations to quickly identify the source of Legionella infections.
Keywords: Legionella pneumophila; culture technique; disinfection treatment; macrophage infectivity potentiator (mip) sequencing; non-pneumophila Legionella species; sequence types (STs); sequence-based typing (SBT); water safety plan.