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2 BCYEα 2 BCYEα 2 1 acid treatment 2 heat treatment Gimenez stain

3 1 PCR L. pneumophila 2 DNA-DNA (DNA) (PFGE) 2 Sequence Based Typing (SBT) 3 Multiple-Locus Variable number tandem repeat (VNTR) Analysis (MLVA) 1. Legionella sp L. pneumophila 1 2 L. cherrii

4 Legionella ,2) ) 1976 the Legion Legionella pneumophila Legionellaceae Legionella pneumophila 3 fraseri, pascullei, pneumophila L. lytica L. drancourtii Legionella Legionella Fluoribacter Tatlockia P2 BSL2 2

5 ml 25% 1/500 ph

6 PCR DNA PCR DNA Legionella Legionella pneumophila

7 PCR

8 L- 1 BCYEα buffered charcoal-yeast extract agar with 0.1 α-ketoglutalate ph 6.90± ± BCYEα BCYEα

9 100 ml (CFU/100 ml) 10 L- L- PCR (1) BCYEα ACES Buffer α- A Bacto agar L- L- 10 g 10 g 2.8 g 1 g 2 g 17 g 980 ml 0.4 g 10 ml 0.25 g 10 ml μm L- 15 ml 20 ml (2) BCYEα BCYEα 1

10 BMPAα GVPC MWY WYO CCVC PAV B B B B B 80,000 U 4 mg 80 mg 80,000 U 1 mg 3 g 80 mg 50,000 U 1 mg 3 g 80 mg 10 mg 10 mg 100,000 U 5 mg 3 g 80 mg 4 mg 16 mg 0.5 mg 80 mg 40,000 U 80 mg 0.5 mg 50 BCYEα

11 1 BCYEα WYOα GVPC MWY CCVC PAC(BMPAα) PAV OXOID BD OXOID OXOID BD 4 BD 4 BD 4 Oxoid BD Difco BBL Oxoid BD Difco BMPAα MWY GVPN Oxoid GVPN GVPC Difco ACES Buffer g 4 3

12 2 Bacillus (1) acid treatment ph M 5.3 ml 0.2M 25 ml 100 ml M 23.0 g/l 0.1M 21.0 g/l ph 2.2 (2) heat treatment

13 3 L- BCYE 5 BCYEα IsoVital X L. jordanis L. spiritensis L. oakridgensis L ml 3,000 g 5,000 7,000 rpm ml ml 47 mm μm 5 ml 5 10 voltex mixer KHz 130 W 24

14 5) 100 μl μl μl 20 KHz 130 W ~3 100 μl ~35 2

16 2. Gimenez Warthin-Starry 1 Gimenez stain a. 1 g ml b. 1 ml 24 ml c. 65 ml a b c a. 0.2M 2.84 g 100 ml b. 0.2M 2.76 g 100 ml a 3.5 ml b 15.5 ml 19.0 ml 1 ml 0.1M 9 ml 0.8 g 100 ml

17 fluorescent isothiocyanate FITC IFA IFA L. pneumophila 6 PBS ph7.2 NaH 2 PO 4 2H 2 O 9 g Na 2 HPO 4 12H 2 O g NaCl 160 g 20 L ph ph 9.0 9

18 0.2M Na 2 HPO IFA PBS ph 7.2 PBS FITC 4

19 3. 1 L. pneumophila DNA-DNA hybridization mip (macrophage infectivity potentiator gene) L. pneumophila 1 L. pneumophila 15 1 L. bozemanae L. longbeachae L. feeleii L. hackeliae L. quinlivanii L. sainthelensi L. spiritensis L. erythra 2 L. pneumophila 1 6 L. bozemanae 1 L. gormanii L. dumoffii L. micdadei 10 L. pneumophila 7 15 L. longbeachae 1 L. longbeachae 2 L. hacckeliae 1&2 L. feeleii 1 L. feeleii 2 Oxoid L. pneumophila 1 L. pneumophila 2-14 L. longbeachae 1 2 L. bozemanae 1 2 L. dumoffii L. gormanii L. jordanis L. micdadei L. anisa 60 L. pneumophila 1 L. pneumophila BCYEα McFarland No /ml ,000 3,500 rpm

20 5 L. bozemanae L. dumoffii L. gormanii L. micdadei DDH 2 Duopath Legionella 30 L. pneumophila 3) (1) PCR L. pneumophila L. pneumophila PCR LEG Lmip PCR LEG (genus Legionella 16S rrna gene) Lmip (L. pneumophila macrophage infectivity potentiator gene) PCR L. pneumophila LEG Legionella sp. LEG LEG 448A 5 -GAGGGTTGATAGGTTAAGAGC-3 LEG 854B 5 -CGGTCAACTTATCGCGTTTGCT-3 Lmip Mahbubani Lmip L GCTACAGACAAGGATAAGTTG-3 Lmip R GTTTTGTATGACTTTAATTCA-3 BCYE-α 1 μl 50 μl ,000 rpm 5 2 μl LEG PCR (2 46 μl) 10 reaction buffer 5.0 μl dntp mixture 4.0 μl ultra-pure water μl LEG F (20 μm)0.5 μl LEG R (20 μm)0.5 μl Taq polymerase (5 U/μl) 0.25 μl 200 μl PCR 23 μl

21 2 μl PCR % 430 bp Lmip PCR 650 bp EmviroAmp Legionella Kit PCR 5S rrna : Forward primer (5-29) 5'-GGCGACTATAGCGPTTTGGAA-3' Reverse primer (91-112) 5'-GCGATGACCTACTTTCPCATGA-3' mip L. pneumophila : Forward primer ( ) 5'-GCATTGGTGCCGATTTGG-3' Reverse primer ( ) 5'-GRTTTGCCATCAAATCTTTRTGAA-3' *P=A/G, R=C/T DNA 20 µl spin down 10,000 rpm PCR 25 µl Forward primer 10 pmol/µl 1 µl Reverse primer 10 pmol/µl 1 µl 10 Reaction buffer 2.5 µl 2.5 mm dntp mixture 2 µl Chromosomal DNA 0.5 µl Ultra-pure water 14.8 µl AmpliTaq DNA polymerase 0.2 µl Perkin Elmer Model

22 % 5 µl 5S rrna 108 bp mip 168 bp 5S rrna mip PCR mip PCR LAMP Loop-mediated Isothermal Amplification L. londiniensis LAMP (2) DNA-DNA DNA 70% DNA-DNA L. pneumophila 25 DNA DDH DNA-DNA 4 L. busanensis L. gresilensis L. londiniensis L. natarum L. quinlivanii L. geestiana

23 (3) 16S rrna PCR DNA 20 μl spin down 10,000 rpm 27f Forward primer (8-27) 5'-AGAGTTTGATCCTGGCTCAG-3' 1429r Reverse primer ( ) 5'-GGCTACCTTGTTACGACTT-3' PCR 25 μl Forward primer 2 pmol/μl 2.5 μl Reverse primer 2 pmol/μl 2.5 μl 10 Reaction buffer 2.5 μl 2.5 mm dntp mixture 2.5 μl Chromosomal DNA 2.5 μl Ultra-pure water μl AmpliTaq DNA polymerase 0.25 μl PCR QIAquick PCR Purification Kit QIAGEN 1 PCR 5 PB buffer 2 2 ml collection tube QIAquick spin column

24 3 10,000 g 60 4 collection tube QIAquick spin column ml PE buffer QIAquick spin column 6 10,000 g 60 7 collection tube QIAquick spin column 8 PE buffer 10,000 g ml tube QIAquick spin column 10 EB buffer 50 μl QIAquick spin column ,000 g r1l Reverse primer ( ) 5'- GTA TTA CCG CGG CTG CTG G -3' r2l Reverse primer ( ) 5'- CAT CGT TTA CGG CGT GGA C -3' r2l Reverse primer ( ) 5'- GAC TAC CAG GGT ATC TAA TC -3' r3l Reverse primer ( ) 5'- TTG CGC TCG TTG CGG GAC T -3' r4l Reverse primer ( ) 5'- ACG GGC GGT GTG TAC AAG -3' re1l Forward primer ( ) 5'-GTA GGA GTC TGG ACC GTG T-3' f1l Forward primer (9-27) 5'- GAG TTT GAT CCT GGC TCA G-3' f2l Forward primer ( ) 5'- CCA GCA GCC GCG GTA ATA C-3' 926f Forward primer ( ) 5'- AAA CTC AAA GGA ATT GAC GG-3' f3l Forward primer ( ) 5'- GTC CCG CAA CGA GCG CAA C -3' BigDye Terminator v3.1 Cycle Sequencing Kit

25 Applied Biosystems 3130/3130xl Genetic Analyzer 500 bp DNA Data Bank of Japan BLAST version S rrna mip 16S rrna L. londiniensis L. nautarum L. nautarum

26 4 1) % L. pneumophila 1 80% 100% BinaxNow Q ELISA 4 HRP (horseradish peroxidase) L. pneumophila 1 1 L. pneumophila L. pneumophila 1 PCR 37

27 5. DNA 1) PCR PCR LAMP DNA L. pneumophila mip 16S rrna 5S rrna LAMP PCR LAMP mip L. pneumophila PCR (1)DNA 1 ml 0.1 ml lysozyme (5 mg/ml ) ml proteinase K (1 mg/ml) 0.1 ml 20%SDS 55 DNA 50 µl 10 µl 1st step PCR 2nd step PCR 1 µl 1st step PCR (2) 1st step primers: LmipL920 5'-GCTACAGACAAGGATAAGTTG-3' LmipR1548 5'-GTTTTGTATGACTTTAATTCA-3' 2nd step primers: LmipL997 5'-TAATCCGGAAGCAATGGCTA-3' LmipR1466 5'-GGGCCAATAGGTCCGCCAAC-3' (3) 100 µl 100 mm Tris-HCl (ph8.3) 1.5 mm MgCl µm dntp 40 µm primers 2.5 U AmpliTaq (4)

28 PCR 489 bp DNA DNA 2) PCR PCR LAMP DNA (1) 500 ml 5 ml 2 ml 2 ml 13,000 15,000 rpm μl (2) DNA 2 TE 1 M NaCl 10% TritonX μl 100 μl 2 90 μl 20 mg/ml Proteinase K 10 μl ,000 rpm μl (3) DNA Buffer AL 200 μl 200 μl DNA

29 5,000 rpm ml Buffer AW1 300 μl 10,000 rpm ml Buffer AW2 300 μl 12,000 15,000 rpm ml Buffer AE 50 μl 2 10,000 rpm μl DNA DNA (4) DNA 5 μl DNA 5 μl 10 ml L. londiniensis 16S rrna 5S rrna mip PCR LAMP (5) DNA 3 DNA 5 μl PCR ml

30 6. ELISA L. pneumophila 1-6 1) BCYEα BCYE 2 1% PBS ph ml 1 3,000 rpm % PBS ph7.2 McFarlandNo.4 5 Egg Yolk Enrichment 50% (Becton, Dickinson) /ml PBS ph % IFA 18 5 mmφ UV

31 3 U 25 μl 1 BSA PBS ph μl μl μl 16 1,024 4 IFA 2 IFA 10 μl PBS ph 7.2 PBS FITC IgG IgM IgA 10 μl PBS PBS 5 4 ProLong Gold Antifade Reagent (Invitrogen)

32 mm IgG IgM L. pneumophila 1-6 L. bozemanae L. dumoffii L. gormanii L. micdadei 1 BCYEα ,000 rpm w/v% PBS /ml μl μl BSA 0.1% 350 μl 50 μl 1: U μl 1:8 25 μl

33 1:16 25 μl 2 25 μl : :128 1:256

34 7. PFGE Sequence Based Typing SBT Multiple-Locus Variable number tandem repeat (VNTR) Analysis (MLVA) IOD (Index of Discriminationn) 51) 1) PFGE PFGE 4 2 (1) BCYEα μl % SeaKem R Gold Agarose plug mold (Bio-Rad (2) (0.1mg/ml proteinase K, 1% N-lauroylsarcosine, 0.5 M EDTA, ph 8.0) 1 ml 50 1 (3) 4 mm Pefabloc SC(Roche Diagnostics) TE 10 mm Tris-HCl, 1 mm EDTA, ph 8.0 1ml mM Pefabloc SC TE TE 1 ml 30 (50 mm NaCl, 10 mm Tris-HCl, 10 mm MgCl2, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin) 200 μl 30 (4) 50 units/sample SfiI Roche 100 μl TBE Tris-borate 45 mm, EDTA 1 mm 4 (5) 1% SeaKem R Gold Agarose CHEF DR System (Bio-Rad) 0.5 TBE V/cm μg/ml 30 3

35 2) Sequence Based Typing SBT SBT L. pneumophila 7 flaa pile asd mip momps proa neua L. pneumophila EWGLI ( Multiple-Locus Variable number tandem repeat VNTR Analysis MLVA MLVA VNTR VNTR PCR MLVA SBT PFGE

36 1.,,,,,,,,,,, : Legionella pneumophila serogroup 7 Pontiac fever..., 69: , Glick TH, Gregg MB, Berman B, Mallison G, Rhodes WW Jr, and Kassanoff I: Pontiac fever. An epidemic of unknown etiology in a health department: I. Clinical and epidemiologic aspects. Am. J. Epidemiol., 107: , ,,,,,,,,, : Legionnaires disease ( )., 55: , : 5. : 3,,, :., 25:8-13, :., 58: 51-54, : 3. BCYEα. 20, , ,,,,,,,,,, : 2. 20, , ,, : Legionella : Wadowsky-Yee-Okuda (WYO)., 58: , ,,,, : Legionella., 65: , Reinthaler FF, Sattler J, Schaffler-Dullnig K, Weinmayr B, and Marth E: Comparative study of procedures for isolation and cultivation of Legionella pneumophila from tap water in

37 hospitals. J. Clin. Microbiol., 31: , ,,, :., 73: 25-34, ,,,, :., 76: 41-50, Lin A, Stout JE, Rihs JD, Vickers RM, and Yu VL: Improved Legionella selective media by the addition of fluconazole: results of in vitro testing and clinical evaluation. Diagn. Microbiol. Infect. Dis., 34: , Inoue H, Noda A, Takama T, Ishima T, Agata K: Enhanced antifungal effect of the selective medium for the detection of Legionella species by a combination of cycloheximide, amphotericin B and thiabendazole. Biocontrol Sci., 11: 69-74, ,,,,,,, : , , ,, : Legionella., 7: 21-25, ,,,,,, :., 76: , Water quality -- Detection and enumeration of Legionella: ISO 11731, 1998(E). 21.,,, :., 61,, ,,,,, : Legionella., 72: , ,,, :., 59: 73-74, Kura F, Amemura-Maekawa J, Yagita K, Endo T, Ikeno M, Tsuji H, Taguchi M, Kobayashi K, Ishii E, and Watanabe H: Outbreak of Legionnaires' disease on a cruise ship linked to spa-bath filter stones contaminated with Legionella pneumophila serogroup 5. Epidemiol. Infect., 134: , 2006.

38 25. Brown SL, Bibb WF, and McKinney RM: Retrospective examination of lung tissue specimens for the presence of Legionella organisms: comparison of an indirect fluorescent-antibody system with direct fluorescent-antibody testing. J. Clin. Microbiol., 19: , Nagai T, Sobajima H, Iwasa M, Tsuzuki T, Kura F, Amemura-Maekawa J, and Watanabe H: Neonatal sudden death due to Legionella pneumonia associated with water birth in a domestic spa bath. J. Clin. Microbiol., 41: , Benson RF, and Fields BS: Classification of the genus Legionella. Semin. Respir. Infect., 13:90-99, Amemura-Maekawa J, Kura F, Chang B, Suzuki-Hashimoto A, Ichinose M, Endo T, and Watanabe H: Distinct difference of flaa genotypes of Legionella pneumophila betweenisolates from bath water and cooling tower water, Microbiol. Immunol., 52: , : PCR Legionella., 50 : , Mahbubani MH, Bej AK, Miller R, Haff L, DiCesare J, and Atlas RM Detection of Legionella with polymerase chain reaction and gene probe methods. Molecular and Cellular Probes, 4: , Perkin Elmer: Package insert, EnviroAmp Legionella Kits. Perkin Elmer Corporation Ezaki T, Hashimoto Y, and Yabuuchi E: Fluorometric deoxyribonucleic acid -deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol., 39: , , : DNA-DNA. 21, , :, 48-61, Hiraishi A: Direct automated sequencing of 16S rdna amplified by polymerase chain reaction from bacterial cultures without DNA purification. Lett. Appl. Microbiol. 15:

39 36. Hiraishi A Shin YK Ueda Y, and Sugiyama J: Automated sequencing of PCR-amplified 16S rdna on Hydrolink gels. J. Microbiol. Methods, 19: , Janda JM, and Abbott SL: 16S rrna gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J. Clin. Microbiol., 45: , Ratcliff RM, Lanser JA, Manning PA, and Heuzenroeder MW: Sequence-based classification scheme for the genus Legionella targeting the mip gene. J. Clin. Microbiol., 36: , KohlerRB, Zimmerman SE, Wilson E, Allen SD, Edelstein PH, Wheat LJ, and White A: Rapid radioimmunoassay diagnosis of Legionnaires disease. Ann. Inter. Med., 94: , , 29: , ( 41.,,,,,, :., 43: 1-4, , II. 81: , ,,,,,,,, : Two step polymerase chain reaction Legionella pneumophila., 67: , Aoki S, Hirakata Y, Miyazaki Y, Izumikawa K, Yanagihara K, Tomono K, Yamada Y, Tashiro T, Kohno S, and Kamihira S: Detection of Legionella DNA by PCR of whole-blood samples in a mouse model. J. Med. Microbiol., 52: , Koide M, Higa F, Tateyama M, Nakasone I, Yamane, N, and Fujita J: Detection oflegionella species in clinical samples: Comparison of polymerase chain reaction and urinary antigen detection kits. Infection. 34: , Morozumi M, Nakayama E, Iwata S, Aoki Y, Hasegawa K, Kobayashi R, Chiba N, Tajima T, and Ubukata K: Simultaneous detection of pathogens in clinical samples from patients with

40 community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes. J. Clin. Microbiol., 44: , ,,,,,,,,,,,. 19, 23-36, ,, : DNA. 20, 25-35, ,,,,,, :., 71: , Hunter PR, Gaston MA: Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol., 26: , Chang B, Amemura-Maekawa J, Watanabe H. An improved protocol for the preparation and restriction enzyme digestion of pulsed-field gel electrophoresis agarose plugs for the analysis of Legionella isolates. Jpn. J. Infect. Dis., 62:54-56, Amemura-Maekawa J, Kura F, Helbig JH, Chang B, Kaneko A, Watanabe Y, Isobe J, Nukina M, Nakajima H, Kawano K, Tada Y, Watanabe H, and Working Group for Legionella in Japan: Characterization of Legionella pneumophila isolates from patients in Japan according to serogroups, monoclonal antibody subgroups and sequence types. J. Med. Microbiol., 59: , Amemura-Maekawa J, Kura F, Chang B, and Watanabe H: Legionella pneumophila serogroup 1 isolates from cooling towers in Japan form a distinct genetic cluster. Microbiol. Immunol., 49: , ,, Legionella pneumophila MLVA. 22, , 2011.

42 1. Legionella sp.

43 L. pneumophila 1 2 L. cherrii 4.

2

2 3 4 TTT TCT TAT TGT TTC TCC TAC TGC TTA TCA TAA TGA TTG TCG TAG TGG CTT CCT CAT CGT CTC CCC CAC CGC CTA CCA CAA CGA CTG CCG CAG CGG ATT ACT AAT AGT ATC ACC AAC AGC ATA ACA AAA AGA ATG ACG AAG AGG GTT