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Appl Environ Microbiol. Jun 2012; 78(12): 4263–4270.
PMCID: PMC3370504

Distribution of Monoclonal Antibody Subgroups and Sequence-Based Types among Legionella pneumophila Serogroup 1 Isolates Derived from Cooling Tower Water, Bathwater, and Soil in Japan

Junko Amemura-Maekawa,corresponding authora Kiyomi Kikukawa,b,* Jürgen H. Helbig,c Satoko Kaneko,d,* Atsuko Suzuki-Hashimoto,e Katsunori Furuhata,f Bin Chang,a Miyo Murai,b Masayuki Ichinose,e Makoto Ohnishi,a and Fumiaki Kuraa
aDepartment of Bacteriology I, National Institute of Infectious Diseases, Sinjuku-ku, Tokyo, Japan
bDepartment of Health Sciences, Saitama Prefectural University, Koshigaya-shi, Saitama, Japan
cInstitute of Medical Microbiology and Hygiene, TU Dresden, Dresden, Germany
dCenter for informational Biology, Ochanomizu University, Bukyo-ku, Tokyo, Japan
eTokyo Health Service Association, Shinjuku-ku, Tokyo, Japan
fSchool of Life and Environmental Science, Azabu University, Chuuo-ku, Sagamihara-shi, Kanagawa, Japan
and the Working Group for Legionella in Japan

Abstract

Legionella pneumophila serogroup (SG) 1 is the most frequent cause of legionellosis. This study analyzed environmental isolates of L. pneumophila SG 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.

INTRODUCTION

Legionella pneumophila serogroup (SG) 1 is the most common agent causing legionellosis found in patients; however, differences in SGs have been found both in isolates from patients and from soil and various freshwater environments (9), such as cooling towers and bathing facilities. In patients, most strains (80%) belonged to SG 1 in our previous study (2). In cooling tower water isolates of Japan, L. pneumophila SGs 1 and 7 accounted for 67 and 23%, respectively, with other SGs being rarely isolated. On the other hand, the isolates from bathwater and from soil were more serotypically diverse, but SG 1 was still dominant in both environments, at 31% (1) and 26% (10), respectively. L. pneumophila SG 1 can be divided based on having or not having the virulence-associated epitope recognized by monoclonal antibody (MAb) 3/1 (13). In England and Wales, of the clinical isolates, 91.6% were MAb 3/1 positive compared to only 8.3% of the environmental isolates (12).

L. pneumophila isolates can be characterized by sequence-based typing (SBT) using the seven loci (flaA, pilE, asd, mip, mompS, proA, and neuA) proposed by the European Working Group on Legionella Infections (EWGLI; http://www.ewgli.org/ [11, 21]). This is a separate classifier from serogroup or MAb subtyping and is generally more precise due to the mutability of the latter factors. It allows for phylogenetic studies and identification of isolates that are closely related. The variation in STs of clinical and environmental isolates of L. pneumophila worldwide is very diverse. The indices of discrimination (IODs) (14) of environmental isolates and clinical isolates were determined to be 0.888 and 0.964, respectively, in Canada and 0.822 and 0.946, respectively, in the United States (15, 22). In England and Wales, however, environmental isolates are more variable than clinical ones (IODs of 0.933 and 0.901, respectively [12]), but the diversity is comparably great.

When 69 SG1 clinical isolates from Japan were subjected to SBT, they could be divided into 41 sequence types (STs). The IOD was 0.979. The ST with the most isolates (n = 7) was ST1. This is the most common ST occurring in the environment and among patients worldwide. Other major STs were ST306 (n = 6), ST120 (n = 5), and ST138 (n = 5). All ST306 and ST138 isolates, with one exception (ST306), were derived from bathwater (or suspected to be), suggesting that these strains readily adapt to bathwater habitats. The source of all ST1 and ST120 isolates remains unclear (2). In Japan, data from the National Epidemiological Surveillance of Infectious Diseases indicate that hot springs and public baths are primary sources of L. pneumophila, rather than cooling towers; however, in most cases the source of the bacteria is unknown (19).

We analyzed here environmental isolates of L. pneumophila SG 1, which is the principal cause of legionellosis in bathwater (the main source of infection in Japan), soil (a potential source of contamination for various water systems), and cooling tower water (another major source of legionellosis). Isolates were identified using MAb typing and SBT and then compared to previous clinical isolates (2) to determine relations between isolates from different environments and from patients.

MATERIALS AND METHODS

L. pneumophila strains.

A total of 135 environmental strains of L. pneumophila SG 1, which were independently isolated and unrelated to cases of infection, were analyzed, including isolates from bathwater (BW; n = 50), cooling tower water (CT; n = 50), and soil (SO; n = 35). All of the CT and BW isolates were obtained from different facilities: 66% of the CT isolates and 42% of the BW isolates originated from the Kanto region in central Japan. The SO isolates, which were independently collected from across Japan, were obtained from topsoil samples from roadsides, farmlands, gardens, etc. (10).

MAb subgrouping.

A total of 135 environmental strains of L. pneumophila SG 1 were subtyped serologically, using MAbs as described previously, into nine subgroups named Allentown/France, Bellingham, Benidorm, Camperdown, Heysham, Knoxville, OLDA, Oxford, and Philadelphia (13).

SBT.

SBT was performed according to the EWGLI SBT protocol (http://www.ewgli.org/) as described previously (11, 21). The isolates that failed amplification of neuA (whose indicated allele number was “0”) were not given ST numbers but were allocated arbitrary numbers prefixed by J (2). A minimum-spanning tree that had categorical coefficients of similarity and the priority rule of the highest number of single-locus variants as parameters was used to indicate differences in the number of loci among operational taxonomic units (OTU). The neighbor-joining method was then used to find pairs of OTU that minimized the total branch lengths by number of base substitutions on flaA, pilE, asd, mip, mompS, proA, and neuA concatenated sequences (2,501 bp) at each stage of OTU clustering. Both trees were constructed using BioNumerics software (version 6.5; Applied Maths, Sint-Martens-Latem, Belgium).

RESULTS

MAb subgrouping.

The isolates examined here were comprised of nine MAb types in all. The BW isolates were comprised of all nine MAb types, the CT isolates were comprised only of four, and the SO isolates were comprised of six. The distributions of MAb subgroups in the environmental isolates differed from one another, and from that found in clinical isolates (Fig. 1). The most common MAb subgroup in BW isolates was the Bellingham subgroup (42%), whereas the Oxford subgroup was the most common in CT (72%) and the OLDA subgroup was the most common in SO (51%). Bellingham, Oxford, and OLDA are MAb 3/1-negative subgroups. On the other hand, the most common subgroup observed in Japanese clinical isolates was Benidorm (45%), which is MAb 3/1 positive (2). Benidorm was detected in 12% of isolates from bathwater and 3% of isolates from soil. Of the 135 environmental isolates, only 14% had the virulence-associated epitope recognized by MAb 3/1: Benidorm, Allentown/France, Philadelphia, and Knoxville (13). In BW, 26% of the isolates were MAb 3/1 positive, compared to 14% in SO and a mere 2% in CT.

Fig 1
Distributions of MAb types. (A) Isolates from bathwater (n = 50); (B) isolates from cooling tower water (n = 50); (C) isolates from soil (n = 35); (D) isolates from patients of legionellosis (n = 69 [2]). Allentown/France, Benidorm, Knoxville, and Philadelphia ...

SBT.

The 135 environmental isolates (with the exception of one SO isolate in which amplification of the neuA target failed) could be divided into 50 STs, including 33 singletons (IOD = 0.886; Tables 1 and and2).2). The ST with the largest number of isolates was ST1 (n = 43, 29%), followed by ST48 (n = 10, 6.7%), ST129 (n = 7, 4.7%), ST739 (n = 6, 4.0%), and ST22 (n = 5, 3.3%). Strains with indigenous STs were isolated from each environment. The only common STs across environments were ST1 (37 from CT and 6 from BW), ST48 (9 from SO and 1 from CT), and ST129 (5 from BW and 2 from SO).

Table 1
STs of Japanese environmental isolates of L. pneumophila serogroup 1
Table 2
STs and MAb subtypes of 135 Japanese environmental isolates of L. pneumophila serogroup 1a

There were no regional differences in the distribution of ST1 in either the CT or the BW isolates (76% [22/29] of CT isolates from the Kanto region and 71% [15/21] from other regions; 14% [3/21] of BW isolates from the Kanto region and 10% [3/29] from other regions). ST1 was not detected among the SO isolates.

The 50 CT isolates were divided into eight STs (IOD = 0.448). The 50 BW isolates were divided into 34 STs (IOD = 0.973). The 35 SO isolates (with one exception that failed neuA amplification) were divided into 11 STs (IOD = 0.879).

The minimum-spanning tree illustrates the distribution of the STs (Fig. 2). Thirty of the fifty STs obtained in this analysis were unique to Japan, according to data submitted to the EWGLI SBT database as of March 2012. Twenty of the fifty STs had already also been detected in clinical isolates in Japan and/or abroad, according to the same database. Most SO isolates formed three distinct groups (groups S1, S2, and S3 in Fig. 2). Group S2 had no linkage with other STs. CT isolates formed group C1 and group C2. The two groups were adjacent in the minimum-spanning tree, but even the most related STs (ST161 and ST150) that belonged to group C1 and group C2, respectively, differed in four loci. The BW isolates were dispersed, forming one major group (group B1) and two smaller groups. This finding was supported by neighbor-joining analysis based on a nucleotide sequence comparison of seven concatenated loci of SBT (2,501 bp) of the same isolates as in Fig. 2 (Fig. 3). Figure 3 shows that isolates belonging to each group found in the minimum-spanning tree were also clustered. However, the relationships observed between groups in the dendrogram were different from the minimum-spanning analysis, except for the cluster of group S1 and group C1. Two groups of isolates from cooling tower water (group C1 and group C2) were located distally (unlike Fig. 2). Groups C2, B2, and B3 shared many informative sites between groups, compared to groups C1, S1, S2, S3, and B1, as shown by the bootstrap support value of 74%.

Fig 2
Minimum-spanning tree showing how the L. pneumophila isolates, with seven determined alleles, are distributed in terms of their STs. The ST number is shown beside the circle. An underlined ST number indicates that the ST also has been reported abroad, ...
Fig 3
Phylogenetic tree of flaA, pilE, asd, mip, mompS, proA, and neuA concatenated sequences from L. pneumophila serogroup 1 isolates determined by the neighbor-joining method. Bootstrap support values for nodes outside groups higher than 50% are shown. The ...

Combining the sequence typing and the MAb subgrouping.

Some STs were composed of isolates belonging to different MAb subgroups (and vice versa). Thus, ST1 (n = 43) was composed of isolates belonging to the Oxford (n = 29) and OLDA (n = 14) subgroups. ST154 (n = 4) contained the Oxford (n = 2), OLDA (n = 1), and Philadelphia (n = 1) subgroups. ST448 (n = 3) consisted of OLDA, Oxford, and Benidorm isolates. In contrast, all ST48 (n = 9) and all ST129 (n = 5) isolates were Bellingham. By combining the data of SBT and MAb subgrouping, we could divide the 135 isolates into 58 types (IOD = 0.933; Tables 1 and and22).

DISCUSSION

We analyzed L. pneumophila SG 1 isolates from three distinct environments using MAb typing and SBT in Japan: cooling tower water, bathwater, and soil. The distributions of MAbs and STs of isolates differed both between the environments and from previous clinical isolates (2).

Of the SG 1 clinical isolates from Japan, 80% had the virulence-associated epitope recognized by MAb 3/1 (2). As for the analyzed 135 environmental isolates, MAb 3/1-positive isolates accounted for only 14%. Similar observations have also been made in studies conducted in other countries (i.e., Germany [3], England and Wales [12], and the United States [15]). Although these data indicated MAb 3/1 as the virulence-associated epitope, our study's MAb 3/1-positive isolates dispersed on the dendrogram by SBT (Fig. 3) in the three kinds of analyzed environments, suggesting the MAb 3/1 epitope is easily lost or gained during adaptation to environments when there is no pressure to retain human pathogenicity. Loss of the MAb 3/1 epitope may bring some advantage for fitness, as MAb 3/1-negative isolates dominated in each environment.

Although 30 of the 50 STs obtained in this analysis were unique to Japan, the EWGLI SBT database indicated that the majority of unique STs have single-locus variants abroad. Among the unique STs, only ST138 and ST162 in group B3, and ST141 have neither single-locus variants nor double-locus variants abroad. ST138 of the Benidorm subgroup is the primary clinical isolate associated with bathwater in Japan (2; unpublished results). Thus, a few STs might be unique to Japan, which is isolated by water.

All of the Japanese ST1 strains were of the MAb 3/1-negative OLDA or Oxford subgroups, whereas the ST1 strains in the EWGLI database are divided into nine MAb types. This distribution of MAb types within ST1 may be a regional difference. On the other hand, a regional difference did not always apply. All nine ST48 from our results were of the Bellingham subgroup, and according to the EWGLI SBT database prior to May 2011, all of the MAb-typed ST48 strains submitted thus far were Bellingham. Since May 2011, however, Camperdown and OLDA strains containing ST48 have been deposited. If more strains could be analyzed, we predict that different MAb types within many ST groups would be detected.

The isolates from soil were divided into three groups (Fig. 2) by the spanning tree analysis and only had two common STs that were detected in different environments: ST48 with an isolate from cooling tower water and ST129 with an isolate from bathwater. These findings indicate that these bacteria generally inhabit the soil but are able to contaminate water sources. Further investigations of more isolates from soil may identify STs that link the three groups or that have more corresponding STs with isolates from water environments. Nine of the 11 STs of soil isolates were also detected in clinical isolates, in contrast to only 11 of 34 in bathwater and 3 of 8 from cooling tower water. These findings support the possibility that soil is one of the infectious sources of legionellosis.

In Canada, the distribution of STs in strains from natural water sources was noted as significantly different compared to strains from a manufactured environment (22). We note a similar finding in the present study. The water of Japanese public baths is often derived from hot springs. The characteristics of hot spring water, namely, chemical features such as pH and temperature, are highly variable, whereas the water from hot or cold water systems and cooling towers tend to have rather similar characteristics due to similar water treatment procedures. In our results, STs and MAb types of isolates from bathwater both differed from and were more varied than those of cooling tower water. These features might be related to the kind of host amoebae, which adapt to and inhabit various environments (20, 23). It has been shown that the growth of L. pneumophila in some species of host amoebae depends on bacterial genetic background (4, 8). Some isolates with particular STs adapted for amoebae that live in bathwater may be infectious to humans.

L. pneumophila SG 1 isolates in cooling tower water in Japan were divided into two genetic groups (group C1 and group C2; Fig. 3). Recombination events may have occurred between members of group C1 and group C2. For example, ST161 (flaA11, pilE4, asd-3, mip-1, mompS1, proA1, and neuA1) was a recombinant between ST1 (flaA1, pilE4, asd-3, mip-1, mompS1, proA1, and neuA1) and ST154 (flaA11, pilE14, asd-16, mip-16, mompS15, proA13, and neuA2), which was a predicted primary founder, and ST150 (flaA11, pilE14, asd-16, mip-1, mompS15, proA13, and neuA1) was also a recombinant between ST1 (with adjacent alleles, mip-1 and neuA1) and ST154, shortening the distance between the two groups on the minimum-spanning tree (Fig. 2).

The IOD (0.886) of the 135 environmental isolates was lower than described in our previous report based on clinical isolates (0.979 [2]). These findings were similar to those reported in Canada and the United States (15, 22). The lower diversity observed among environmental isolates compared to clinical isolates may be due to the high prevalence of ST1 (22). ST1 is the most prevalent ST in the world (3, 67, 12, 1516, 22, 26). We have also shown that the majority of environmental isolates, especially from cooling tower waters in Japan (37/50, or 74%), are ST1. Similar results were shown in South Korea (46/68 [67.6%] of SG 1 isolates from cooling tower water were ST1), which is adjacent to Japan (16). In a Canadian study, 34.2% of L. pneumophila strains from manufactured environments and 7.7% of L. pneumophila isolates from natural water sources (lakes and hot springs) were SG 1 and ST1. Among the Canadian strains, five of six SG 1 isolates from cooling tower waters were ST1 (22). In a U.S. study, ST1 accounted for 40% of the L. pneumophila SG 1 environmental isolates; however, the types of environments were not indicated. In Singapore in tropical southeast Asia, the IOD of environmental L. pneumophila isolates was found to be 0.970, and three (two from a cooling tower and one from a water tank) of 16 SG 1 isolates were ST1 (17). In a study conducted in England and Wales, 154 of 276 L. pneumophila isolates, including 29 isolates derived from cooling tower water, were SG 1, and 54/154 (35%) SG 1 environmental isolates were ST1 (12). In our study, ST1 accounted for 74 and 12% of the environmental SG 1 isolates from cooling tower water and bathwater, respectively, whereas no ST1 was found in isolates from soil. Isolates with ST1 have adapted to water environments, especially in manufactured water systems such as cooling towers, and have been detected around the world. The ability of ST1 isolates to adapt to natural water sources such as lakes and hot springs might be rather low. Moreover, they might be unfit to survive well in soil environments. The predominant ST, ST1, of isolates from cooling tower water induced an insufficient IOD, 0.448, whereas the discrimination powers for isolates from bathwater and soil were sufficiently significant (0.973 and 0.879, respectively).

Handling of potting soil could be considered a risk factor for legionellosis. Surveys in several countries have detailed various Legionella species, including L. pneumophila SG1, that were isolated from potting soil or composted materials (5, 18, 25). SBT analysis on composted material isolates in United Kingdom revealed that their L. pneumophila SG1 isolates belonged to ST84 (18). Seven alleles belonging to ST84 were unshared by soil isolates in our study (except for one allele, flaA12, which was), although ST84 has been detected in clinical isolates in Japan (2) and other countries, according to the EWGLI database. Groups S1, S2, and S3, mainly formed by isolates derived from soil, were distant phylogenetically from groups of isolates derived from water environments. Only ST129 soil isolates shared the B1 group with isolates from a water environment. Although some isolates from cooling tower water and bathwater were included in groups S1, S2, and S3, this might imply that some part of these L. pneumophila subpopulations primarily inhabits soil, occasionally mutating and becoming fit to contaminate water environments. Recently, indigenous soil samples were collected in Thailand, and 115 Legionella isolates, including 2 L. pneumophila SG1 isolates, were identified (24; EWLGI database). One ST identified from the L. pneumophila SG1 soil sample isolates related to group S1 and the other to group S2, supporting the idea that most soil isolates belong to particular groups. It is also interesting that the most prevalent ST1 isolates from water samples were not isolated from soil in our study, suggesting the possibility of habitat segregation of L. pneumophila. To elucidate this possibility, we need to investigate more environmental isolates from both soil and water.

ACKNOWLEDGMENTS

We thank Norman K. Fry (Respiratory and Systemic Infection Laboratory, Health Protection Agency) for assigning the newly identified alleles.

This study was supported by the Health and Labor Sciences research grant H22-kenki-014 (to F.K.) and partially supported by Ministry of Education, Culture, Sports, Science, and Technology grant KAKENHI 23590530 (to J.A.-M).

The Working Group for Legionella in Japan included Mie Sasaki, Miyagi Prefectural Institute of Public Health and Environment; Mikako Hosoya, Niigata Prefectural Institute of Public Health and Environmental Sciences; Yuko Watanabe, Toshiro Kuroki, Kanagawa Prefectural Institute of Public Health; Masamichi Wada, Nagano Environmental Conservation Research Institute; Hitoshi Doi, Osaka Prefectural Institute of Public Health; and Koichi Murakami, Fukuoka Institute of Health and Environmental Sciences.

Footnotes

Published ahead of print 6 April 2012

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