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 log 10 copies/m 3 ); 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.
Pulsed-field gel electrophoresis (PFGE) is considered to be one of the most discriminative epidemiological methods for subtyping Legionella pneumophila strains and for elucidating the sources of infection. On the other hand, sequence-based typing (SBT), which was recently developed, is also a powerful epidemiological method. All L. pneumophila serogroup 1 isolates used in a study were independently obtained from a wide variety of Japanese locations. The primers used for SBT and the reaction mixture and conditions were the same as those used by Gaia et al. Putative novel variants found in this study were submitted to the curators of the European Working Group for Legionella Infections SBT database for verification and assignment of new allelic numbers according to the curators’ instructions. Both PFGE and SBT indicated that the spa-bath isolates from Japan were highly diverse. The water used in public spa baths in Japanese resorts is mostly obtained from hot springs. The first clinical isolate in Japan was not assigned to the cooling tower SB-type. Only four SB types were common between Europe and Japan. Although PFGE is the most widely used technique and is generally accepted to be highly effective in discriminating genomic differences, it may have certain drawbacks with regard to interlaboratory reproducibility. SBT appears to be less effective at discriminating between strains than PFGE. The authors have to consider the advantages and limitations of both methods and apply the most suitable method according to the requirements.
The Legionella Reference Center in Japan collected 427 Legionella clinical isolates between 2008 and 2016, including 7 representative isolates from corresponding outbreaks. The collection included 419 Legionella pneumophila isolates, of which 372 belonged to serogroup 1 (SG1) (87%) and the others belonged to SG2 to SG15 except for SG7 and SG11, and 8 isolates of other Legionella species (Legionella bozemanae, Legionella dumoffii, Legionella feeleii, Legionella longbeachae, Legionella londiniensis, and Legionella rubrilucens). L. pneumophila isolates were genotyped by sequence-based typing (SBT) and represented 187 sequence types (STs), of which 126 occurred in a single isolate (index of discrimination of 0.984). These STs were analyzed using minimum spanning tree analysis, resulting in the formation of 18 groups. The pattern of overall ST distribution among L. pneumophila isolates was diverse. In particular, some STs were frequently isolated and were suggested to be related to the infection sources. The major STs were ST23 (35 isolates), ST120 (20 isolates), and ST138 (16 isolates). ST23 was the most prevalent and most causative ST for outbreaks in Japan and Europe. ST138 has been observed only in Japan, where it has caused small-scale outbreaks; 81% of those strains (13 isolates) were suspected or confirmed to infect humans through bath water sources. On the other hand, 11 ST23 strains (31%) and 5 ST120 strains (25%) were suspected or confirmed to infect humans through bath water. These findings suggest that some ST strains frequently cause legionellosis in Japan and are found under different environmental conditions.IMPORTANCELegionella pneumophila serogroup 1 (SG1) is the most frequent cause of legionellosis. Our previous genetic analysis indicated that SG1 environmental isolates represented 8 major clonal complexes, consisting of 3 B groups, 2 C groups, and 3 S groups, which included major environmental isolates derived from bath water, cooling towers, and soil and puddles, respectively. Here, we surveyed clinical isolates collected from patients with legionellosis in Japan between 2008 and 2016. Most strains belonging to the B group were isolated from patients for whom bath water was the suspected or confirmed source of infection. Among the isolates derived from patients whose suspected infection source was soil or dust, most belonged to the S1 group and none belonged to the B or C groups. Additionally, the U group was discovered as a new group, which mainly included clinical isolates with unknown infection sources.
ABSTRACT We report the first case of neonatal Legionnaires' disease associated with water birth in a spa bath at home. Legionella pneumophila serogroup 6 was detected from postmortem lung tissue.
The ornithine-containing lipid (OL) and the serineglycine-containing lipid (SGL) of Flavobacterium activated and modulated the functions of human polymorphonuclear leukocytes (PMNs). The OL and the SGL strongly activated fMet-Leu-Phe- and interleukin-8-induced chemotaxis of PMNs at the concentration of 0.1 microg ml(-1), and a synthetic OL also activated the function of PMNs. Further, the OL strongly activated O(2)(-) production from PMNs. Although the OL and the SGL slightly modulated myeloperoxidase release from PMNs, inhibition effects of their component fatty acid analogues were observed. O(2)(-) production-inducing activity is a common biological activity between the OL and bacterial lipopolysaccharides, but OL and SGL, unlike lipopolysaccharide, are potent activators of PMN chemotaxis.
Legionellosis is caused by the inhalation of aerosolized water contaminated with Legionella bacteria. 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 amoebic co-culture, quantitative PCR, and 16S rRNA gene amplicon sequencing.Legionella species were not detected by amoebic co-culture. However, Legionella DNA was detected in 114/151 (75.5%) air samples collected near roads (geometric mean ± standard deviation: 1.80 ± 0.52 log10 copies/m3), which was comparable to the numbers collected from bathrooms [15/21 (71.4%), 1.82 ± 0.50] but higher than those collected from other indoor sites [11/30 (36.7%), 0.88 ± 0.56] (P < 0.05). The amount of Legionella DNA was correlated with the monthly total precipitation (r = 0.56, 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. 16S rRNA gene amplicon sequencing revealed that Legionella species were detected in 9/30 samples collected near roads (mean proportion of reads, 0.11%). At the species level, L. pneumophila was detected in 2/30 samples collected near roads (the proportion of reads, 0.09 and 0.11% of the total reads number in each positive sample). The three most abundant bacterial genera in the samples collected near roads were Sphingomonas, Streptococcus, and Methylobacterium (mean proportion of reads; 21.1%, 14.6%, and 1.6%, respectively). In addition, the bacterial diversity in outdoor environment was comparable to that in indoor environment which contains aerosol-generating features and higher than that in indoor environment without the features.DNA from Legionella species was widely present 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 not only in bathrooms but also in the areas surrounding asphalt roads. Therefore, the possibility of contracting legionellosis in daily life should be considered.
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