Characterization of Legionella pneumophila isolates from patients in Japan according to serogroups, monoclonal antibody subgroups and sequence types

Abstract

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...

Prevalence of Sequence Types among Clinical and Environmental Isolates of Legionella pneumophila Serogroup 1 in the United States from 1982 to 2012

“…According to the studies of the distribution of Lp1 STs in different regions of the world, it appears that the most prevalent ST among clinical and environmental populations of Lp1 is either ST1 (8,(27)(28)(29)(30)(31)(32) or an ST that is confined to a specific geographic region (33)(34)(35). For example, in Belgium and the United Kingdom, the most frequently detected ST among clinical isolates was ST47 (9,35).…”

Close Genetic Relationship between Legionella pneumophila Serogroup 1 Isolates from Sputum Specimens and Puddles on Roads, as Determined by Sequence-Based Typing

“…In Japan, the number of legionellosis cases peaked in July, the second half of the rainy season (32,33). To elucidate the possibility that splashed aerosols from puddles on a road, as well as public bath water, are a probable source of legionellosis, we need further investigation of the virulence of these strains by a combination of molecular typing profiles such as SBT, lag-1 allele typing, and monoclonal antibody subgrouping (9).…”

“…As with the case of the ST120 clinical isolate, the infection source of the remaining (26) 18 (15) 14 (11) 5 clinical isolates also remained unclear. For the identification of unrecognized sources of legionellosis, in our opinion, L. pneumophila strains should be isolated from clinical specimens and various environmental sources, including water from puddles on roads, and analyzed by molecular typing methods such as SBT and monoclonal antibody subgrouping (9). In samples from puddles, L. pneumophila SG 1, SG 5, and SG 8 were frequently detected.…”

“…Although 58 species and more than 70 serogroups (SG) of Legionella species have been identified (7), more than 90% of legionellosis cases are caused by L. pneumophila (8). Among 15 serogroups of L. pneumophila, most clinical strains (80%) in Japan belonged to SG 1 (9). Recently, in the United States, Kozak et al revealed that 75% of the L. pneumophila SG 1 clinical isolates but only 8% of environmental isolates harbored the lag-1 gene, which is required for the expression of the virulence-associated epitope recognized by monoclonal antibody 2 of the international standard panel (10).…”

The N -Acylneuraminate Cytidyltransferase Gene, neuA , Is Heterogenous in Legionella pneumophila Strains but Can Be Used as a Marker for Epidemiological Typing in the Consensus Sequence-Based Typing Scheme

“…By using the published neuA primers (15), standard SBT could be applied in all strains belonging to SG1 and to some other SGs. However, in some non-SG1 L. pneumophila strains, the neuA gene could not be amplified (1,11,16,20). It was considered likely therefore that this was due to either the heterogeneity of neuA DNA sequence preventing primer binding or the absence of the gene in some of these non-SG1 strains, as suggested by microarray analysis (3).…”