Detection of Legionella Species and the Influence of Precipitation on the Amount of Legionella DNA Present in Aerosols from Outdoor Sites Near Asphalt Roads in Toyama Prefecture, Japan, with Microbiome Analysis

Abstract

Background: Legionellosis can be caused by the inhalation of aerosolized water contaminated with Legionella. In this study, we investigated the prevalence of Legionella species in aerosols collected from outdoor sites near asphalt roads, bathrooms in public bath facilities, and other indoor sites such as buildings and private homes using culture methods, quantitative PCR with ethidium monoazide treatment (EMA-qPCR), and 16S rRNA gene amplicon sequencing. Results: Legionella species were not detected in culture. However, Legionella DNA was detected in 114/151 (75.5%) air samples collected near roads (geometric mean ± standard deviation was 1.80 ± 0.52 log10 copies/m3); these numbers were comparable to those obtained from bathrooms [15/21 (71.4%), 1.82 ± 0.50] and higher than those obtained from other indoor sites [11/30 (36.7%), 0.88 ± 0.56] (P < 0.05). By EMA-qPCR, Legionella DNA was detected in 20/30 (66.7%) samples collected near roads, indicating the presence of membrane-intact Legionella cells in the air. The amount of Legionella DNA correlated with the monthly total precipitation (r = 0.25, P < 0.01). It was also directly and inversely correlated with the daily total precipitation for seven days (r = 0.21, P = 0.01) and one day (r = −0.29, P < 0.01) before the sampling day, respectively. In addition, 16S rRNA gene amplicon sequencing revealed that the three most abundant bacterial genera in the samples collected near roads were Sphingomonas (21.1%), Streptococcus (14.6%), and Methylobacterium (1.6%); Legionella species were detected in 9/30 samples (30%) collected near roads (mean proportion of reads, 0.11%). At the species level, L. pneumophila was detected in 2/30 samples collected near roads (mean proportion of reads, 0.11% and 0.09% in the detected samples).Conclusions: DNA from Legionella species, including Legionella pneumophila, were widely detected in aerosols collected from outdoor sites near asphalt roads, especially during the rainy season. Our findings suggest that there may be a risk of exposure to Legionella species in the areas surrounding asphalt roads.