Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat-and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10 4 -to 10 5 -fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.

ABSTRACTLegionella pneumophilaserogroup (SG) 1 is the most frequent cause of legionellosis. This study analyzed environmental isolates ofL. pneumophilaSG 1 in Japan using monoclonal antibody (MAb) typing and sequence-based typing (SBT). Samples were analyzed from bathwater (BW;n= 50), cooling tower water (CT;n= 50), and soil (SO;n= 35). The distribution of MAb types varied by source, with the most prevalent types being Bellingham (42%), Oxford (72%), and OLDA (51%) in BW, CT, and SO, respectively. The ratios of MAb 3/1 positive isolates were 26, 2, and 14% from BW, CT, and SO, respectively. The environmental isolates from BW, CT, and SO were divided into 34 sequence types (STs; index of discrimination [IOD] = 0.973), 8 STs (IOD = 0.448), and 11 STs (IOD = 0.879), respectively. Genetic variation among CT isolates was smaller than seen in BW and SO. ST1 accounted for 74% of the CT isolates. The only common STs between (i) BW and CT, (ii) BW and SO, and (iii) CT and SO were ST1, ST129, and ST48, respectively, suggesting that each environment constitutes an independent habitat.

Thirty‐one epidemiologically unrelated Legionella pneumophila serogroup 1 isolates (10 from cooling towers, 10 from public spas and/or hot spring baths, and 11 from patients) were analyzed by pulsed‐field gel electrophoresis (PFGE) and sequence‐based typing (SBT) using 6 loci, flaA, pilE, asd, mip, mompS, and proA. The results of PFGE and SBT analysis indicated that all 10 isolates from cooling towers clustered into a unique type, which was distinct from strains of other environmental sources.

Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045

We collected 86 unrelated clinical Legionella pneumophila strains that were isolated in Japan during the period 1980 belonged to serogroup 1, followed by serogroups 5, 3 and 2. Interestingly, the patients with L. pneumophila serogroup 1 had a significantly higher male-to-female ratio (12.4) than the patients with other L. pneumophila serogroups (2.0) (OR, 10.5; 95 % CI, 2.5-44.5). When the serogroup 1 strains were analysed by monoclonal antibody (mAb) typing, the most prevalent subgroup was Benidorm (34.9 % of all isolates). Moreover, 79.7 % of the serogroup 1 isolates were bound by mAb 3/1, which recognizes the virulenceassociated epitope. When all 86 isolates were subjected to sequence-based typing (SBT) using seven loci, they could be divided into 53 sequence types (STs). The ST with the most isolates (seven) was ST1, to which most isolates from patients and environments around the world belong. However, six of the seven ST1 isolates were isolated before 1994. Other major STs were ST306 (n56), ST120 (n55) and ST138 (n55). All ST306 and ST138 isolates, except for one isolate (ST306), were suspected or confirmed to be derived from bath water, which suggests that these strains prefer bath habitats. The sources of all ST1 and ST120 isolates remain unclear. By combining the SBT and mAb data, the 86 isolates could be divided into 59 types (discrimination index, 0.984). This confirms the usefulness of this combination in epidemiological studies. INTRODUCTIONLegionellosis is caused by Legionella species, which are environmental Gram-negative bacteria. To date, 52 species of Legionella have been described (Kuroki et al., 2007). The species that is most commonly found in the environment and also causes the most disease is Legionella pneumophila (Yu et al., 2002). To aid the epidemiological surveillance of legionellosis, L. pneumophila isolates can be divided into serogroups by antisera that recognize differences on the In our previous study (Amemura-Maekawa et al., 2005), we analysed 27 epidemiologically unrelated L. pneumophila serogroup 1 isolates (ten from cooling towers, ten from public spas and/or hot spring baths, and seven from patients with public bath-related infections) from Japan by SBT using the six alleles proposed in 2005 by the EWGLI. The 27 isolates could be divided into 14 sequence types (STs). Notably, the 10 isolates from the cooling towers all showed the same allele types, namely flaA (1), pilE (4), asd (3), mip (1), mompS (1) and proA(1), whereas the public bath-derived isolates were more diverse.Here, to further characterize clinical L. pneumophila isolates from Japan and to confirm the usefulness of the mAb-and SBT-based classification methods, we analysed 86 isolates by both typing methods. METHODSL. pneumophila strains. We analysed 86 clinical isolates of L. pneumophila that were isolated in Japan during the period 1980-2008. Of these, 42 clinical Legionella isolates were from the Legionella Reference Center, which collects Legionella isolates obtained in six representative prefectural/municipa...