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Distribution of Sequence-Based Types of Legionella pneumophilaSerogroup 1 Strains Isolated from Cooling Towers, Hot Springs, andPotable Water Systems in China
Tian Qin,a,b Haijian Zhou,a,b Hongyu Ren,a,b Hong Guan,a Machao Li,a,b Bingqing Zhu,a,b Zhujun Shaoa,b
National Institute for Communicable Disease Control and Prevention and State Key Laboratory for Infectious Disease Prevention and Control, Chinese Centre for DiseaseControl and Prevention, Beijing, Chinaa; Collaborative Innovation Centre for Diagnosis and Treatment of Infectious Diseases, Hangzhou, Chinab
Legionella pneumophila serogroup 1 causes Legionnaires’ disease. Water systems contaminated with Legionella are the impli-cated sources of Legionnaires’ disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-basedtyping. Strains were isolated from cooling towers (n � 96), hot springs (n � 42), and potable water systems (n � 26). Isolatesfrom cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination[IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolateswas lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of ana-lyzed strains (n � 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1.In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. Therewere differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strainsisolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these coun-tries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environ-mental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.
Bacteria of the genus Legionella, widely present in water, con-tribute to human diseases. To date, more than 50 Legionella
species have been characterized, and 25 species are known to causehuman disease (1, 2). Legionella was first reported as a pathogenfollowing a pneumonia outbreak in 1976 (3). Since then, severalcommunity-, hospital-, and travel-associated Legionella outbreakshave been reported. The most severe form of pneumonia causedby Legionella is known as Legionnaires’ disease; however, there isalso a milder, flu-like form known as Pontiac fever (4). Legionellapneumophila is responsible for approximately 90% of human in-fections (5). L. pneumophila is divided into 15 serogroups, ofwhich serogroup 1 is the most prevalent disease-causing variant.Few outbreaks by other Legionella species have been reported (5,6). Transmission of bacteria from the environment to humansoccurs via inhalation or aspiration of Legionella-containing aero-sols (7, 8). Cooling towers (9, 10), hot springs (11, 12), and potablewater systems (13, 14) in large facilities, hotels, hospitals, and pub-lic baths that are contaminated with Legionella are the implicatedsources of outbreaks and sporadic cases of Legionnaires’ disease.
The water systems of several countries are highly contaminatedwith Legionella. In our previous study that compared the efficacyof different Legionella detection methods, we isolated Legionella atrates of 26.39%, 54.44%, and 18.94% from cooling towers, hotsprings, and piped water systems, respectively, with Legionellaconcentrations of �1,000 CFU/liter (15). Studies have reportedthat L. pneumophila serogroup 1 is the most frequently isolatedspecies in cooling towers and hot springs (9, 11).
Several subtyping techniques have been used to identify and char-acterize L. pneumophila strains. Sequence-based typing (SBT),which is based on multilocus sequence typing (MLST), is cur-rently used for L. pneumophila. According to the European Work-ing Group on Legionella Infections (EWGLI; http://www.ewgli.org/), L. pneumophila isolates can be characterized by SBT using
seven loci, including five genes associated with virulence (fliC,pilE, mip, proA, and mompS) and two housekeeping genes (asdand neuA) (16, 17). SBT is a highly discriminatory method thatallows the rapid identification of isolates that are closely related. Inthis study, we used SBT to assess the genetic characteristics of L.pneumophila strains isolated from cooling towers, hot springs, andpotable water systems in China. Furthermore, the SBT results ofChinese strains were compared with those of Japan and SouthKorea.
MATERIALS AND METHODSBacterial isolates and culture conditions. A total of 164 environmental L.pneumophila serogroup 1 strains were included in this study. These strainswere isolated during regular surveillance events performed in the cities ofShanghai, Beijing, Shenzhen, Wuxi, Jinan, and Shijiazhuang between2005 and 2012. In this surveillance, water samples from central air condi-tioning cooling towers and potable water systems in public buildings andhospitals and from indoor and outdoor hot springs of public baths werecollected for a Legionella survey. The cooling towers were closed, and thehot springs were the sulfuric acid type. The temperature of the watersamples from cooling towers and potable water systems was about 25°C,and that of the hot spring samples was between 31°C and 49°C. The pH ofthe water systems ranged from 6.0 to 8.0. Sampling was conducted via
Received 19 November 2013 Accepted 22 January 2014
Published ahead of print 24 January 2014
Editor: C. A. Elkins
Address correspondence to Tian Qin, [email protected], or Zhujun Shao,[email protected].
T.Q. and H.Z. contributed equally to this article.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.03844-13
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identical sampling protocols in all geographic regions. A total of 500 ml ofeach water sample from cooling towers, hot springs, or faucets was col-lected in sterile, screw-cap containers. All strains used in this study be-longed to L. pneumophila serogroup 1. Strains were stored at �80°C incharcoal yeast extract (CYE) with 20% sterile glycerol. The water sourcesconsisted of cooling towers (96 isolates), hot springs (42 isolates), andpotable water systems (26 isolates) of public buildings, hospitals, andpublic baths (Table 1). The bacteria were streaked onto buffered charcoalyeast extract (BCYE) agar plates. Colonies were identified by serotypingafter they were inoculated onto BCYE agar plates and incubated at 35°C in2.5% CO2 for 48 h.
DNA extraction, PCR amplification, and sequencing. GenomicDNA was extracted using a QIAamp DNA minikit (Qiagen, Dusseldorf,Germany). PCR was performed in a T100 Thermal Cycler (Bio-Rad Lab-oratories, CA, USA) using the primers for seven genes (flaA, pilE, asd, mip,mompS, proA, and neuA) in SBT analysis (16, 17). The PCRs were pre-pared in a reaction volume of 50 �l with 5 �l of 10� PCR buffer (TaKaRa,Dalian, China), 1 unit of Taq polymerase (TaKaRa), a 200 �M concentra-tion of the deoxynucleosides (dNTPs; TaKaRa), 0.4 �M each primer set,20 ng of the DNA template, and filtered sterile water. PCR was performedwith an initial denaturation step at 94°C for 5 min followed by 30 cycles,each consisting of an initial denaturation at 94°C for 40 s followed byannealing and extension steps. The primers and corresponding annealingtemperatures used were previously described (16, 17). DNA sequencingwas performed using an ABI BigDye Terminator cycle sequencing kit(Applied Biosystems, CA, USA); the products were analyzed on a 3130xlABI Prism genetic analyzer (Applied Biosystems) at Tsingke Biotechnol-ogy Co., Ltd. (Beijing, China).
SBT. SBT was performed via the EWGLI method using seven genes(flaA, pilE, asd, mip, mompS, proA, and neuA) (16, 17). The SBT database,available on the EWGLI website, was used for nucleotide analysis. Thesequences were compared with those in the SBT database, which were alsoavailable on the EWGLI website (http://www.hpa-bioinformatics.org.uk/legionella/legionella_sbt/php/sbt_homepage.php). The index of dis-crimination (IOD) was calculated using the following formula: IOD �1 � �[nj (nj � 1)]/[N(N � 1)], where nj is the number of strains belongingto the jth pattern, and N is the number of strains in the population (18).Diversity of SBT loci was calculated by Nei’s index: 1 � � (allele fre-quency)2 (19).
BioNumerics software (version 5.10; Applied Maths, Kortrijk, Bel-gium) was used to create minimum spanning trees (MST). In MST, thefounder sequence type (ST) is defined as the ST with the highest numberof single-locus variants. Clusters of related STs that descend from a com-mon ancestor are defined as clone groups (complexes); single genotypesthat do not correspond to any clone groups are defined as singletons.Types are represented by circles; the size of a circle indicates the number ofstrains of this particular type. Heavy solid lines connect two types thatdiffer within a single locus, light solid lines connect double-locus variants,
heavy dotted lines connect triple-locus variants, light dotted lines connectquadruple-locus variants (see Fig. 3 and 4).
Statistical analyses. All calculations were conducted using SPSS, ver-sion 13.0, software (SPSS, Inc., Chicago, IL). A chi-square test was used tocompare the proportional distributions of STs according to different re-gions and environmental water source types.
Nucleotide sequence accession numbers. The sequences of the DNAfragments encompassing the seven genes flaA, pilE, asd, mip, mompS,proA, and neuA as determined by SBT analysis were deposited in theGenBank database under accession numbers KJ160781 to KJ161159.
RESULTSSBT analysis. In this study, 164 Legionella isolates were analyzedby SBT and divided into 42 STs (IOD, 0.749): 16 STs were associ-ated with more than one strain, and 26 STs were identified withone single strain (Fig. 1). Most of the STs were city specific; how-ever, ST1, ST154, ST630, ST752, and ST1471 were present in morethan one city. Querying the EWGLI SBT database (available athttp://www.ewgli.org) found that 23 of the 42 STs were unique toChina and 11 (ST1556 to ST1566) were identified for the firsttime. Furthermore, of the 16 STs that were identified with morethan one strain, 5 STs (ST149, ST155, ST160, ST1471, andST1479) had been previously identified in China, and the other 11STs were prevalent throughout the world, according to theEWGLI SBT database. Only one ST (ST59) had been isolated inclinical cases in China, and 10 STs had been isolated in clinicalcases abroad, according to the EWGLI SBT database.
ST1 (1,4,3,1,1,1,1) (the sequence of numbers represents theallelic profile, respectively, of the seven genes flaA, pilE, asd, mip,mompS, proA, and neuA, as shown in Fig. 1), which was identifiedwith 49.4% of the analyzed strains (n � 81), was present in allcities. Based on the results, 53.1% (51/96), 14.3% (6/42), and92.3% (24/26) of cooling tower, hot spring, and potable waterisolates belonged to ST1. There were no regional differences in thedistribution of ST1 in potable water systems (75.0% [6/8] fromJinan and 100% [18/18] from Shanghai; �2 � 4.875, P � 0.086);however, the distribution of ST1 in cooling towers was regionallydifferent (Fig. 2). For cooling tower water isolates, 12.5% (2/16),20.0% (4/20), 40.0% (2/5), 65.4% (17/26), 75.0% (9/12), and100% (17/17) of the isolates from Wuxi, Shenzhen, Jinan, Shang-hai, Beijing, and Shijiazhuang, respectively, belonged to ST1 (�2 �38.687, P 0.001). Additionally, 40% (6/15) of the hot springisolates from Shenzhen belonged to ST1; however, no ST1 strainswere isolated from hot springs in Beijing (�2 � 6.618, P � 0.018).
ST154 was the second most predominant ST. In this study,ST154 isolates were from two water sources: the cooling tower inShenzhen and the hot spring in Beijing. According to the EWGLISBT database, accessed on 8 January 2014, there were 33 isolatesbelonging to ST154. These strains were isolated from seven coun-tries between 1982 and 2013. Among them, 10 strains were clini-cal, and the others were environmental.
Strains from different sources showed different levels of diver-sity. The 42 hot spring isolates were divided into 19 STs (IOD,0.934), the 96 cooling tower isolates were divided into 25 STs(IOD, 0.711), and the 26 potable water isolates were divided intothree STs (IOD, 0.151). With the exception of two strains, allpotable water isolates (92.3%) belonged to ST1. In contrast,53.1% (51/96) and 14.3% (6/42) of cooling tower and hot springisolates, respectively, belonged to ST1 (�2 � 40.401, P 0.001).For the seven SBT loci, Nei’s index values were 0.739 to 0.847,0.482 to 0.572, and 0.148 to 0.151 for hot spring, cooling tower,
TABLE 1 Water sources of strains used in this study
Year City Facility type Water sourceNo. of strainsanalyzed
2005 Beijing Building Cooling tower 122005 Shenzhen Building Cooling tower 202005 Beijing Public bath Hot spring 132008 Shanghai Hospital Cooling tower 262008 Shijiazhuang Hospital Cooling tower 172008 Shanghai Hospital Potable water 182009 Jinan Building Potable water 82009 Jinan Building Cooling tower 52010 Wuxi Hospital Cooling tower 162011 Beijing Public bath Hot spring 142012 Shenzhen Public bath Hot spring 15
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FIG 1 Clustering results of data obtained by SBT analysis of 164 L. pneumophila serogroup 1 isolates. These strains were isolated during regular surveillance indifferent water systems of China between 2005 and 2012. Clustering was created using the unweighted pair group method with average linkages. The ST, city,surveillance year, number of isolates, ST complex, and alleles used in the SBT analysis are shown. The asterisks indicate STs that were identified for the first timeaccording to the EWGLI SBT database (accessed 5 September 2013).
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and potable water isolates, respectively (Table 2), which revealed ahigh diversity of hot spring isolates and a low diversity of potablewater isolates.
Population structure analyses. MST illustrates the distribu-tion of STs. As shown in Fig. 3, 28 STs were predicted to form five
clone groups (complexes), whereas 14 STs, which differed fromevery other ST in four or more genes, were identified as singletons.The biggest clone group was the ST1 complex, containing 91 iso-lates belonging to six STs. In the ST1 complex, ST1 was predictedas the putative ancestor and contained the highest number of iso-
FIG 2 Distribution of STs of L. pneumophila serogroup 1 strains isolated during regular surveillance in six cities in China. The city name, surveillance year, watersource, and number of L. pneumophila serogroup 1 strains are shown in each box.
TABLE 2 Diversity of seven SBT loci of L. pneumophila serogroup 1 isolates from three water sources
Locus
Cooling tower (n � 96)a Hot spring (n � 42) Potable water (n � 26)
No. oftypes
No. ofisolates/type
Nei’sindex
No. oftypes
No. ofisolates/type
Nei’sindex
No. oftypes
No. ofisolates/type
Nei’sindex
flaA 7 13.7 0.552 9 4.7 0.847 3 8.7 0.151pilE 4 24.0 0.547 5 8.4 0.765 3 8.7 0.151asd 9 10.7 0.570 5 8.4 0.768 2 13 0.148mip 10 9.6 0.482 5 8.4 0.770 3 8.7 0.151mompS 8 12.0 0.565 8 5.3 0.804 3 8.7 0.151proA 9 10.7 0.572 7 6.0 0.739 3 8.7 0.151neuA 7 13.7 0.552 7 6.0 0.772 3 8.7 0.151a n, number of isolates.
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lates (81/91, 89.0%). The other five STs of the ST1 complex werefour single-locus variants and one double-locus variant of ST1and were identified with one to four strains each.
The second clone group was the ST154 complex (11 STs and33 isolates), followed by the ST149 complex (5 STs and 9 iso-lates), the ST454 complex (4 STs and 4 isolates), and theST1565 complex (2 STs and 2 isolates). There were differencesin the distribution of clone groups based on the water source.With the exception of one cooling tower isolate, all strains ofthe ST149 complex were obtained from hot springs. In con-trast, all ST454 and ST27 complex strains were isolated fromcooling towers. However, the ST1 complex was present in allthree water sources, and the ST154 complex was present incooling towers and hot springs.
Comparison of isolates obtained from China, Japan, andSouth Korea. The 164 isolates obtained in this study were com-pared with L. pneumophila serogroup 1 isolates from Japan andSouth Korea (Fig. 4). The SBT data of 135 Japanese and 104 SouthKorean isolates were obtained from other studies (20, 21). These403 isolates were divided into 107 STs. The majority of the STs(100/107, 93.5%) were exclusive to one country; four STs (ST1,ST150, ST154, and ST159) were present in all three countries, andthree STs (ST22, ST45, and ST59) were present in two countries(Fig. 4 and 5). In the MST dendrogram, eight clone groups(groups I to VIII) were identified: three clone groups (groups I toIII) were relatively large and contained isolates from three coun-
tries, and five clone groups (groups IV to VIII) were small andcontained isolates from one country.
Group I, which consisted of the ST1 complex in this study andof groups C1 (20) and CG1 (21) in previous studies, was the largestclone group. ST1 was identified as the potential founder of groupI. Group II was mainly composed of three STs: ST154, ST150, andST159. ST150 and ST159 were single-locus and double-locus vari-ants of ST154, respectively. Group II consisted of the ST154 com-plex in this study and of groups C2 and CG3 in previous studies(20, 21).
Group III contained strains of five clone groups previouslyidentified in this study (ST149 and ST454 complexes) and otherstudies (groups B1, B2, and CG2). These previously identifiedclone groups were independently distributed in group III andlinked to each other. South Korean CG2 (ST59, ST363, STK1, andSTK11) and Japanese group B2 (ST86, ST128, ST603, and ST605)were clustered together and linked to the Chinese ST149 complex(ST149, ST155, ST156, ST199, and ST1476), Japanese group B1,and the Chinese ST454 complex. A few singletons identified in thisstudy and other studies, such as ST1564, STK9, and STK10, weresimilarly clustered in group III.
The other five clone groups mainly contained STs and strainsexclusive to one country. Group IV mainly contained ChineseST27 complex, group V mainly contained Japanese group S1,group VI mainly contained Japanese group S2, group VII mainly
FIG 3 Minimum spanning tree analysis of 164 L. pneumophila serogroup 1 strains isolated in China. STs are shown as circles. The size of each circle correspondsto the number of isolates within this particular type; the STs and the number of isolates in each ST are shown in the circles. The shading simply links STs or setsof STs within an ST complex.
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contained Japanese group B3, and group VIII contained Japanesegroup S3.
DISCUSSION
In this study, we analyzed L. pneumophila serogroup 1 isolates bySBT from three water sources in China: cooling towers, hotsprings, and potable water systems. Furthermore, isolates fromChina were compared with isolates from Japan and South Korea.Our findings revealed that STs had several unique allelic profilesand that ST1 of L. pneumophila serogroup 1 was the most preva-lent sequence type in China. Additionally, the distributions ofisolate STs differed among the water sources and cities.
Even though 23 of the 42 STs obtained in this study wereunique to China, the EWGLI SBT database indicated that therewere single-locus variants abroad of the majority of the STs.Therefore, a few STs might be unique to China. In our previous
study, we reported four Legionnaires’ disease cases attributed toST36 and ST346 strains (22). However, no isolates belonging tothese two STs were isolated in this study. In 2011, we detected acase of Legionnaires’ disease caused by an L. pneumophila sero-group 1 strain of ST59 (unpublished data) in Beijing. The patientwas a 70-year-old man. An L. pneumophila serogroup 1 strainbelonging to ST59 was isolated from bronchoalveolar lavage fluid(BALF) after typical symptoms of Legionnaires’ disease emergedin him. However, no ST59 strain was found upon epidemiologicalinvestigations. In this study, two ST59 strains were isolated fromhot springs, which suggests that hot springs are potential sourcesof legionellosis.
In this study, the distribution of STs from natural watersources (e.g., hot springs) was significantly different from thatfrom artificial water sources (e.g., cooling towers and potable wa-ter systems). This result, which is in accordance with previousstudies conducted in Japan and Canada (20, 23), might be attrib-uted to differences in the characteristics of the water sources (e.g.,temperature and pH). The characteristics of hot springs are highlyvariable, whereas those of cooling towers and potable water sys-tems tend to have similar characteristics due to similar water treat-ments. Legionella has high prevalence, rapid intracellular growthrates, and wide genetic diversity in hot springs (11). In this study,STs of isolates from hot springs, which had higher genetic diver-sity, were significantly different from those from cooling towersand potable water systems. These results might be due to hostamoebae, which adapt to and inhabit different environments (24,25). It has been reported that the growth of L. pneumophila in hostamoebae depends on the bacterial genetic background (26, 27).Hot spring isolates, which have unique STs adapted to amoebae,may be infectious to humans.
ST1, which is the most prevalent ST worldwide (20, 21, 23,
FIG 4 Minimum spanning tree analysis of 403 environmental L. pneumophila serogroup 1 isolates from China (n � 164), Japan (n � 135), and South Korea (n �104). STs are shown as circles. The size of each circle indicates the number of isolates within this particular type; the STs and the number of isolates in each STare shown in the circles. The shading simply links STs or sets of STs within an ST complex. Eight clonal groups were identified (groups I to VIII). Group namesin parentheses were from previous studies (1, 21).
FIG 5 Venn diagram of the number of STs shared among different countriesor subsets of countries.
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28–32), was the most dominant ST in this study. Similar resultshave been reported in other countries. In Japan, the majority(29%) of environmental isolates, especially from cooling towerwaters (37/50, 74%), are ST1 (20). In a recent study conducted inthe United States, ST1 was the most frequent ST found in bothsporadic and environmental isolates, accounting for 25% and49% of the total number of isolates, respectively (32). In a studyconducted in England and Wales, 54 of 154 (35%) of L. pneumo-phila serogroup 1 environmental isolates belonged to ST1 (31).Additionally, this study revealed that the prevalence rates of ST1 inartificial water systems were higher than those in natural watersources. In this study, ST1 accounted for 92.3% and 53.1% of theenvironmental serogroup 1 isolates from potable water systemsand cooling towers, respectively, whereas only 14.3% of hot springserogroup 1 isolates belonged to ST1. This result is consistent withthe results of a study conducted in Japan (20), where ST1 ac-counted for 74% and 12% of the environmental serogroup 1 iso-lates from cooling towers and hot springs, respectively, whereasno ST1 strain was isolated from soil. ST1 isolates have adapted towater environments, especially to artificial water systems such aspotable water systems and cooling towers, and have been isolatedworldwide. The ability of ST1 isolates to adapt to natural watersources such as hot springs and soil might be rather low.
Potable water isolates were divided among three STs, and mostof them belonged to ST1. Potable water ST1 isolates had a low IOD(0.151), whereas hot spring and cooling tower isolates had highIODs (0.934 and 0.711, respectively). Two other potable waterisolates belonged to ST354 and ST1556: ST354 was detected inclinical isolates in Japan and Canada, and ST1556 was a novel STidentified in this study. These STs did not have the same alleles inthe seven genes used for SBT subtyping. Further investigations ofpotable water isolates may identify STs that link the three STs orthat indicate similar STs with isolates from water environments.ST1 and ST354 were identified in clinical isolates; therefore, pota-ble water systems might represent an infectious source of legion-ellosis.
China, Japan, and South Korea belong to East Asia. Three ma-jor clone groups (groups I through III) were identified amongenvironmental L. pneumophila serogroup 1 isolates from thesethree countries. Groups I and II were identified in each country.Recombination events that shortened the distance between thegroups I and II of the MST may have occurred (Fig. 4). ST150(11,14,16,1,15,13,1), ST161 (11,4,3,1,1,1,1), and STK14 (11,14,3,1,15,13,1) are recombinants between ST1 (1,4,3,1,1,1,1) andST154 (11,14,16,16,15,13,2), which are predicted primary found-ers of group I and group II, respectively. Group III consists ofseveral clone groups previously identified in each country. Re-combination STs between different clone groups linked them to-gether and formed group III in the MST. Another five clonegroups mainly contained STs and strains exclusive to one country.This result suggested that certain clones of environmental L. pneu-mophila serogroup 1 isolates colonized different regions. Overall,there were similar alleles between STs of different groups in theMST. Four alleles were shared by ST161 and STK14, which be-longed to group I and group II, respectively; four alleles wereshared by STK8 and ST27, which belonged to group I and groupIV, respectively; and three alleles were shared by ST52 and ST1558,which belonged to group I and group III, respectively. These re-sults suggest that these groups diverged in the recent past and that
if more isolates had been included in the analysis, all of the STsmight be contained in one clone group.
In conclusion, there is a complex population structure and biasdistribution of genotypes of environmental L. pneumophila sero-group 1 isolates in China. The comparative analysis among strainsisolated from China, Japan, and South Korea revealed that similarclones exist in different countries and that certain clones colo-nized different regions. The findings of this study highlight theimportance of understanding the epidemiology and ecology of L.pneumophila from public facilities in terms of public health. As aresult of the potential for water sources to harbor and disseminateLegionella, improved control and prevention strategies are ur-gently needed.
ACKNOWLEDGMENTS
We thank Norman K. Fry (Respiratory and Systemic Infection Labora-tory, Health Protection Agency Centre for Infections, London, UnitedKingdom) for assigning the newly identified alleles and sequence types.
This study was supported by grants from the National Natural ScienceFoundation of China (grant number 81201251), the Science Foundationfor the State Key Laboratory for Infectious Disease Prevention and Con-trol of China (grant number 2011SKLID202), and the Priority Project onInfectious Disease Control and Prevention (grants 2012ZX10004215,2013ZX10004221, and 2013ZX10004610) of the Ministry of Health,China.
REFERENCES1. Diederen BMW. 2008. Legionella spp. and Legionnaires’ disease. J. Infect.
56:1–12. http://dx.doi.org/10.1016/j.jinf.2007.09.010.2. Fields BS, Benson RF, Besser RE. 2002. Legionella and Legionnaires’
disease: 25 years of investigation. Clin. Microbiol. Rev. 15:506 –526. http://dx.doi.org/10.1128/CMR.15.3.506-526.2002.
3. Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ, SharrarRG, Harris J, Mallison GF, Martin SM, McDade JE, Shepard CC,Brachman PS. 1977. Legionnaires’ disease: description of an epidemic ofpneumonia. N. Engl. J. Med. 297:1189 –1197. http://dx.doi.org/10.1056/NEJM197712012972201.
4. Glick TH, Gregg MB, Berman B, Mallison G, Rhodes WW, Jr, Kassan-off I. 1978. Pontiac fever. An epidemic of unknown etiology in a healthdepartment: I. Clinical and epidemiologic aspects. Am. J. Epidemiol. 107:149 –160.
5. Yu VL, Plouffe JF, Castellani-Pastoris M, Stout JE, Schousboe M,Widmer A, Summersgill J, File T, Heath CM, Paterson DL, ChereshskyA. 2002. Distribution of Legionella species and serogroups isolated byculture in patients with sporadic community-acquired legionellosis: aninternational collaborative survey. J. Infect. Dis. 186:127–128. http://dx.doi.org/10.1086/341087.
6. Sopena N, Sabria M, Pedro-Botet M, Manterola JM, Matas L, Domín-guez J, Modol JM, Tudela P, Ausina V, Foz M. 1999. Prospective studyof community-acquired pneumonia of bacterial etiology in adults. Eur. J.Clin. Microbiol. Infect. Dis. 18:852– 858. http://dx.doi.org/10.1007/s100960050419.
7. Blatt SP, Parkinson MD, Pace E, Hoffman P, Dolan D, Lauderdale P,Zajac RA, Melcher GP. 1993. Nosocomial Legionnaires’ disease: aspira-tion as a primary mode of disease acquisition. Am. J. Med. 95:16 –22.http://dx.doi.org/10.1016/0002-9343(93)90227-G.
8. Breiman RF, Cozen W, Fields BS, Mastro TD, Carr SJ, Spika JS,Mascola L. 1990. Role of air sampling in investigation of an outbreak ofLegionnaires’ disease associated with exposure to aerosols from an evap-orative condenser. J. Infect. Dis. 161:1257–1261. http://dx.doi.org/10.1093/infdis/161.6.1257.
9. Lin H, Xu B, Chen Y, Wang W. 2009. Legionella pollution in coolingtower water of air-conditioning systems in Shanghai, China. J. Appl.Microbiol. 106:606 – 612. http://dx.doi.org/10.1111/j.1365-2672.2008.04031.x.
10. Dondero TJ, Jr, Rendtorff RC, Mallison GF, Weeks RM, Levy JS, WongEW, Schaffner W. 1980. An outbreak of Legionnaires’ disease associatedwith a contaminated air-conditioning cooling tower. N. Engl. J. Med.302:365–370. http://dx.doi.org/10.1056/NEJM198002143020703.
Qin et al.
2156 aem.asm.org Applied and Environmental Microbiology
on June 26, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
11. Qin T, Yan G, Ren H, Zhou H, Wang H, Xu Y, Zhao M, Guan H, Li M,Shao Z. 2013. High prevalence, genetic diversity and intracellular growthability of Legionella in hot spring environments. PLoS One 8:e59018. http://dx.doi.org/10.1371/journal.pone.0059018.
12. Su HP, Tseng LR, Tzeng SC, Chou CY, Chung TC. 2006. A legionellosiscase due to contaminated spa water and confirmed by genomic identifi-cation in Taiwan. Microbiol. Immunol. 50:371–377. http://dx.doi.org/10.1111/j.1348-0421.2006.tb03803.x.
13. Garcia-Nuñez M, Sopena N, Ragull S, Pedro-Botet ML, Morera J,Sabria M. 2008. Persistence of Legionella in hospital water supplies andnosocomial Legionnaires’ disease. FEMS Immunol. Med. Microbiol. 52:202–206. http://dx.doi.org/10.1111/j.1574-695X.2007.00362.x.
14. Shands KN, Ho JL, Meyer RD, Gorman GW, Edelstein PH, MallisonGF, Finegold SM, Fraser DW. 1985. Potable water as a source of Legion-naires’ disease. JAMA 253:1412–1416. http://dx.doi.org/10.1001/jama.1985.03350340064018.
15. Qin T, Tian Z, Ren H, Hu G, Zhou H, Lu J, Luo C, Liu Z, Shao Z. 2012.Application of EMA-qPCR as a complementary tool for the detection andmonitoring of Legionella in different water systems. World J. Microbiol.Biotechnol. 28:1881–1890. http://dx.doi.org/10.1007/s11274-011-0986-x.
16. Gaia V, Fry NK, Afshar B, Lück PC, Meugnier H, Etienne J, Peduzzi R,Harrison TG. 2005. Consensus sequence-based scheme for epidemiolog-ical typing of clinical and environmental isolates of Legionella pneumo-phila. J. Clin. Microbiol. 43:2047–2052. http://dx.doi.org/10.1128/JCM.43.5.2047-2052.2005.
17. Ratzow S, Gaia V, Helbig JH, Fry NK, Lück PC. 2007. Addition of neuA,the gene encoding N-acylneuraminate cytidylyl transferase, increases thediscriminatory ability of the consensus sequence-based scheme for typingLegionella pneumophila serogroup 1 strains. J. Clin. Microbiol. 45:1965–1968. http://dx.doi.org/10.1128/JCM.00261-07.
18. Hunter PR, Gaston MA. 1988. Numerical index of the discriminatoryability of typing systems: an application of Simpson’s index of diversity. J.Clin. Microbiol. 26:2465–2466.
19. Malorny B, Junker E, Helmuth R. 2008. Multi-locus variable-numbertandem repeat analysis for outbreak studies of Salmonella enterica sero-type Enteritidis. BMC Microbiol. 8:84. http://dx.doi.org/10.1186/1471-2180-8-84.
20. Amemura-Maekawa J, Kikukawa K, Helbig JH, Kaneko S, Suzuki-Hashimoto A, Furuhata K, Chang B, Murai M, Ichinose M, Ohnishi M,Kura F, Working Group for Legionella in Japan. 2012. Distribution ofmonoclonal antibody subgroups and sequence-based types among Legio-nella pneumophila serogroup 1 isolates derived from cooling tower water,bathwater, and soil in Japan. Appl. Environ. Microbiol. 78:4263– 4270.http://dx.doi.org/10.1128/AEM.06869-11.
21. Lee HK, Shim JI, Kim HE, Yu JY, Kang YH. 2010. Distribution ofLegionella species from environmental water sources of public facilitiesand genetic diversity of L. pneumophila serogroup 1 in South Korea. Appl.Environ. Microbiol. 76:6547– 6554. http://dx.doi.org/10.1128/AEM.00422-10.
22. Qin T, Xia J, Ren H, Zhou H, Tang B, Shao Z. 2012. Liver cirrhosis as
a predisposing condition for Legionnaires’ disease: a report of four labo-ratory confirmed cases from China. J. Med. Microbiol. 61:1023–1028.http://dx.doi.org/10.1099/jmm.0.040170-0.
23. Reimer AR, Au S, Schindle S, Bernard KA. 2010. Legionella pneumophilamonoclonal antibody subgroups and DNA sequence types isolated inCanada between 1981 and 2009: laboratory component of national sur-veillance. Eur. J. Clin. Microbiol. Infect. Dis. 29:191–205. http://dx.doi.org/10.1007/s10096-009-0840-3.
24. Patterson WJ, Hay J, Seal DV, McLuckie JC. 1997. Colonization oftransplant unit water supplies with Legionella and protozoa: precautionsrequired to reduce the risk of legionellosis. J. Hosp. Infect. 37:7–17. http://dx.doi.org/10.1016/S0195-6701(97)90068-2.
25. Stockman LJ, Wright CJ, Visvesvara GS, Fields BS, Beach MJ. 2011.Prevalence of Acanthamoeba spp. and other free-living amoebae in house-hold water, Ohio, USA—1990 –1992. Parasitol. Res. 108:621– 627. http://dx.doi.org/10.1007/s00436-010-2120-7.
26. Buse HY, Ashbolt NJ. 2011. Differential growth of Legionella pneumo-phila strains within a range of amoebae at various temperatures associatedwith in-premise plumbing. Lett. Appl. Microbiol. 53:217–224. http://dx.doi.org/10.1111/j.1472-765X.2011.03094.x.
27. Dey R, Bodennec J, Mameri MO, Pernin P. 2009. Free-living freshwateramoebae differ in their susceptibility to the pathogenic bacterium Legion-ella pneumophila. FEMS Microbiol. Lett. 290:10 –17. http://dx.doi.org/10.1111/j.1574-6968.2008.01387.x.
28. Borchardt J, Helbig JH, Lück PC. 2008. Occurrence and distribution ofsequence types among Legionella pneumophila strains isolated from pa-tients in Germany: common features and differences to other regions ofthe world. Eur. J. Clin. Microbiol. Infect. Dis. 27:29 –36. http://dx.doi.org/10.1007/s10096-007-0392-3.
29. Cazalet C, Jarraud S, Ghavi-Helm Y, Kunst F, Glaser P, Etienne J,Buchrieser C. 2008. Multigenome analysis identifies a worldwide distrib-uted epidemic Legionella pneumophila clone that emerged within a highlydiverse species. Genome Res. 18:431– 441. http://dx.doi.org/10.1101/gr.7229808.
30. Chasqueira MJ, Rodrigues L, Nascimento M, Marques T. 2009. Se-quence-based and monoclonal antibody typing of Legionella pneumophilaisolated from patients in Portugal during 1987–2008. Euro Surveill.14(28):pii19271. http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId�19271.
31. Harrison TG, Afshar B, Doshi N, Fry NK, Lee JV. 2009. Distribution ofLegionella pneumophila serogroups, monoclonal antibody subgroups andDNA sequence types in recent clinical and environmental isolates fromEngland and Wales (2000 –2008). Eur. J. Clin. Microbiol. Infect. Dis. 28:781–791. http://dx.doi.org/10.1007/s10096-009-0705-9.
32. Kozak-Muiznieks NA, Lucas CE, Brown E, Pondo T, Taylor TH, Jr,Frace M, Miskowski D, Winchell JM. 2014. Prevalence of sequence typesamong clinical and environmental isolates of Legionella pneumophila se-rogroup 1 in the United States from 1982 to 2012. J. Clin. Microbiol.52:201–211. http://dx.doi.org/10.1128/JCM.01973-13.
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