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1. EMA 5 DAD DNA B ERI ES SE nN EMA qPCR 2 C REDE 1998 2004 7 AES 7 1 VBNC NM ES Alfreider et al 2004 L pneumophila2. EMA qPCR Y I EMA qPCR 88 4 cr GIN 7 A cy cf J th x AT RA 3
3. 2 1 2 1 1 500ml pH pH HM 30S TOA DKK CM 40S TOA DKK Hi IM SS TOADKK 2 1 2 2 1 BCYEa Edelstein 1981 Pseudomonas aeruginosa 37 C
4. dg 2 Inoue H Iwasawa T Saruwatari Y and Agata K 2004 Improved acid pretreatment for the detection of Legionella species from environmental water samples using the plate culture method Biocontrol Sci 9 43 50 3 Inoue H Noda A Takama T lshima T and Agata K 2006 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 ARRE W 2014 CATa 42 349 351 4 BR 2004 Legionella pneumophila PCR 32 383 386 WE qu BE 2004 LAMP PCR
5. P aeruginosa 2 3 pH aeruginosa P aeruginosa pH 2 2 10 pH2 5 20 pH 3 0 XU pH 4 0 5 0 Bd 24 R 2 6 HCIKCI HCLKCI 161 32 32 39 5 G 1
6. HE CATa GVPC GVPC CATa AMO CATa 3 1 3 1 1 _ BETRE E 6 Legionella pneumophila Philadelphia 1 ATCC33152 Legionella bozemanii ATCC33217 Legionella longbeachae ATCC33462 Legionella micdadei ATCC33218 Legionella gormanii ATCC33297 Legionella anisa ATCC35292 BCYELF HHA AV YC 37 C CHE L 10 4 PDA 6
7. 80 6 2 No of No of Sample clones OTUs Chao1 Simpson Shannon Wiener Goods coverage analyzed obtained 0 1 H CTW A 51 11 15 7 87 2 16 92 2 CTW B 58 17 23 10 60 2 53 87 9 CTW C 62 16 28 13 32 2 63 87 1 CTW G 75 8 10 2 08 1 14 96 0 CTW H 66 19 30 9 71 2 45 84 8 CTW l 105 29 67 9 91 2 67 82 9 BW D 65 3 3 1 90 0 71 98 5 BW E 70 2 2 1 03 0 07 98 6 BW F 65 7 7 2 58 1 16 96 9 81 10 10 104 10 10 Amount of viable Legionella 16S rRNA genes copies 100 ml 10 CTW I 4 100 CTW C CTW G 8 100 gt ND ND e CTW B El 3 97 uw lt 4 e x BW E o 79 100 Pa CTW A 9 BW F 73 100 13 100 i e 4 4 10 10 103 10 10 Culturable Legionella counts CFU 100 ml 10 6 3 MHK pneumophila ATCC33152 EMA qPCR L pneumophila n 2 CTW A C G BW D F ID T
8. LAMP 1 51 R42 PCR Concentrations of residual free chlorine 10 mg L 1 mg L 0 2 mg L PCR culture PCR culture PCR culture CFU 100 ml CFU 100 ml CFU 100 ml Before treatment 1 7 x 107 1 7 x 107 1 7 x10 After 10 min lt 10 lt 10 FEF lt 10 30 min lt 10 lt 10 TEF lt 10 60 min lt 10 lt 10 lt 10 120 min lt 10 lt 10 HFF lt 10 180 min lt 10 lt 10 FF lt 10 360 min lt 10 lt 10 lt 10 720 min lt 10 lt 10 lt 10 1440 min lt 10 e 10 lt 10 Intensity of the amplified DNA band on the gel image is indicated in four grades bright clear dim but visible not visible 52 R43 PCR H 0 GA CMI BNPD PCR culture culture culture culture CFU 100 ml CFU 100 ml CFU 100 ml CFU 100 ml Before treatment 98x10 17x10 17x10 17x10 After 24h lt 10 l
9. 3 23 L pneumophila CATo B CATo Lk 3 3 4 pneumophila CATo 10 L pneumophila GVPCa 7 GVPAa 2 GVPTo 6 L pneumophila Fusarium sp M5 3 1 B CATo K 34 BCYEaHFHH GVPCa CATo GVPC
10. 1977 Feeley et McDade et al al 1978 Mueller Hinton IsoVitaleX r CYE Feeley et al 1979 Legion Legionella pneumophila Brenner et al 1979 fever 2 Legionnarres disease Pontiac EAEL TIE 15
11. LAMP 7 BLES Ethidium monoazide EMA 1 EMA DNA YORK EMA WEED APE R Etfi 2012 9 2012 12 95 SHEIK 111 25 1 ml 121C 20 min 500ml 512 78
12. Be Ethidium HERO DNA DNA polymerase DNA H Nogva et al 2003 V E HH FS Ht BLL VAR RD Ao Has BLU UV ART BER d B He B ER H E 5 PCR EMA WL pneumophila H PCR dg Hookey et al 1996 Adeleka et al 1996 Viable but nonculturable VBNC
13. 24 3 3 0 2 MHCLKCI pH 2 2 02M 02M KCl 0 2M pH 2 2 02M 0 2MKHzPO 02M AREN pH 2 2 02M Signa amp 0 2 MNa HPO pH pH 2 2 2 14 HCI KCI 200 ml 100 ml 0 1 M NaOH 100 pH 10 g 1000 ml 6000 mg L as CaCO 3000 2000 1000 500 250 mg L as CaCOs
14. 200 ul BCYEOHSHHICBE Lic 37C 6 2 1 6 QSO11731 400 ml 100 6400 xg 15 30 4ml 100 100 1 ml 10 200 ul 4f GVPC Dennis etal 1984a 37C 6 4 BCYEo 37C 2 BEELI BCYEo
15. 325 r 2 Inoue etal 2007 16S rRNA 5S rRNA mip ees infectivity potentiator PCR Polymerase chain reaction LAMP Loop mediated isothermal amplification Notomi et al 2000 DNA 1 8 H 0
16. LAMP 10 DNA LAMP 16S rRNA 1477 bp GenBank DDBJ EMBL BLAST Legionella sp L47 AB899895 95 42 7 _ 6 RIG CHK LC LAMP 4 8 500 mg L 1
17. H 4081 134 2002 CREA AL TWS EAN lt c He B ERI t CATw B DA E ES ze HF 89 30
18. RETRA 61 63 63 65 523 EMA 53 69 uli 7563 EMA qPCR YE 7 72 6 1 72 6 1 1 6 12 6 13 EMA DNA 6 14 7 pneumophila DNA 6 1 5 616 62 79 6 2 1 EMA qPCR 622 63 87 4 7 2 93 aie 101 102 iii 1 A 1 A HHH
19. DNA LAMP 1 DNA LAMP 12 LAMP 6
20. 2 3 16S 5S rRNA S DNA T ESSA S PCR PCR monoazide EMA
21. Nx AERO ISO11731 JIS K 0350 50 10 ISO11731 100 pH 2 2 0 2 M HCI KCI EZ Bopp et al 1981 3 1984a WYO H 6 1984 MWY Edelstein 1982 r BCYE L BCYE H E 50 Dennis et al 1984b
22. 5 Prince and Andrus 1992 100 mg L pie HE H Eo _ Eae eG L IN VU ARTZ He Fare 1 WECH 5 PCR PCR PCR Ng eg 1997 04 mg L E GRAN 1995a
23. 51 LAMP 22 43 PCR 20 39 LAMP 100 PCR 94 LAMP PCR VBNC 4 PCR 8g800 CFU 100 ml 1 DNA
24. PCR L pneumophila PCR LAMP DNA 7 pneumophila DNA At YORK D gt LAMP PCR pneumophila DNA RETRA 49 LAMP 4 3 EBMA qPCR 1 5 1 1 5 1 2 5 13 5 1 4 5 1 5 52 5 2 1 5 2 2 EMA DNA qPCR EMA
25. LOLI RAB POX BY FHETS OD EMIT Wery et al 2008 EMA qPCR DNA X L pneumophila DDNA pneumophila EMA PCR EMA qPCR 6 1 6 1 1 2012 11 2014 1
26. 2013 31 A 1 2 2 3 gt eG NEM ES 3 4 PCR LAMP VBNC DNA E Al q A A 1
27. H CATo 40 pg ml ifs 60 ugm 3 3 40 pg ml CATo GVPC H CAToa GVPC HEHE E bki GVPC 1 mg L CATodE HARE HOSS mg L B GVPC 80000 units L CATo 100000 units L H
28. pH 3000 mg L pH 222 HCI KCI MME VER 35 0 1 M pH 2 1 HCLKQ1 12 0 1M 65 ml EL V D pH 2 3 2 BYEo pH 22 BYBo pH 100 ml
29. mM i X a 32 481 487 3 Inoue H Takama T Yoshizaki M and Agata K 2015 Detection of Legionella species in environmental water by the quantitative PCR method in combination with ethidium monoazide treatment Biocontrol Sci 20 1 in press 6 Inoue H Fujimura R Agata K and Ohta H 2015 Molecular characterization of viable Legionella spp in cooling tower water samples by combined use of ethidium monoazide and PCR Microbes Environ 30 1 in press 102
30. B 400 ng ml Ph 322 5 GE 3 2 GVPo H GVPGa 10 GVPCa 2 MS MIO GVPCo GVPAg B GVPTa 3 3 2 B
31. 1 DNA E EE ke 7 BAE 500 mg L DNA DEEN Ja
32. Pine et al 1979 Hoffman etal 1983 Es ou BE 7 pneumophila L pneumophila elemental sulfur Inoue et al 2002 indole Inoue et al 2004
33. F CMI BNPD 48 PCR ET L pneumophila PCR DNA 48 DNA pneumophila 423 4 4 100 10 CFU 100 ml 31 GVPC 424 LAMP
34. 3 1 3 3 1 4 3 1 5 CATo 3 1 6 CATa 3 2 321 3 2 2 3 2 3 L pneumophila CATo n i 10 10 12 27 29 29 33 3 2 4 CAToa 3 25 CATOESHIO ACHE 33 BR 42 3 4 PCR LAMP 45 4 1 4 1 1 4 1 2 4 1 3 4 1 4 4 1 5 4 1 6 4 1 7 42 4 2 1 4 2 2 4 2 3 4 2 4 4 2 5 4 2 6 4 2 7 47 Ue E HH
35. VU ART BRAO CAT 43 5 y 0 8229x 0 2055 e o R2 0 6057 se e 65 de e be e ate 5 4 es MEC e P ai ro e a20 LE e e MA ee amp 2 3 m Bert aS gs cen 24 jT c 2 e ee o 5 L e se e c 90 e ee oF 2 ae sas a 8 ER e o S 00009 o e e i 0606009090 o oo oo e e 0000 e 1 e oe oo ocmee 1 1 2 3 4 5 Legionella counts by GVPC agar Log CFU 100 ml 3 3 GVPC CATo n 811 44 At srl 3 4 PCR LAMP LT D nob YORK
36. 161 58 36 6 2 1 2 2 5 2 6 161 63 HCI KCI R 0 93 2 7 100 pH pH 4 6 8 4 pH 2 2 2 4 13 2 1 Organism Legione
37. 30 8800 CFU 100 ml DNA PCR 3 10 20 60 CFU 100 ml 42 5 X 4 6 56 42 6 LAMP 2393 LAMP 4 7 LAMP 43 5 13 4 LAMP 67 4 LAMP 29 9
38. DNA PCR 4 1 3 LAMP LAMP Loopamp E 20 ul Bst DNA polymerase DNA 5 wl NC 25 Loopamp 65 60 min 60 min LAMP Ay _ 41 6 DNA PCR DNA 1 5 ml 50 ul DNA 5 3M 125 ul 45 U2 ul Pellet Paint Novagen
39. 34 3 2 Growth on Strain GVPa GVPCa GVPAa GVPGa GVPTa Penicillium sp M1 du Trichoderma sp M2 EF Aspergillus sp M3 Penicillium sp M4 FF Fusarium sp M5 stele Aspergillus sp M6 FF HF Aspergillus sp M7 Aspergillus sp M8 FF Penicillium sp M9 ab Penicillium sp M10 EF Tr Eo FF Growth growth inhibition no growth 35 3 3 pneumophila CFU of L pneumophila per plate on Strain GVPCa GVPAa GVPTa CATa Penicillium sp M1 23 42 OG 52 Trichoderma sp M2 42 26 39 35 Aspergillus sp M3 19 26 OG 41 Penicillium sp M4 OG 15 18 16 Fusarium sp M5 6 OG 12 44 Aspergillus sp M6 4 17 OG 39 Aspergillus sp M7 OG 22 OG 22 Aspergillus sp M8 OG 20 OG 31 Penicillium sp M9 OG 32 OG 35 Penicillium sp M10 OG OG 35 30 Overgrowth of the molds 36 34 BCYEa GVPCau CATo Recovery of Legionella cells on Initial cell Strain counts ny BCYE GVPCa CATo L pneumophila ATCC33152 2 x 10 100 74 78 L bozemanii ATCC33217 1x10 100 10 14 L longbeachae ATCC33462 3 x 10 100 85 83 L micdadei ATCC33218 6 x 10 100 71 83 L gormanii ATC
40. DNA 1 5 DNA CE Z H Fare d JR EB H H V Legionella lytica Legionella like amoebal pathogen LLAP E HG H He ES 7 RES 2 2 E 3 l E pneumophila PCR LAMP EMA qPCR B H H
41. qPCR EMA qPCR 5 2 1 12 PCR DNA 13 82 27 G3 T pneumophila 27 20 74 L pneumophila amp L quinlivanii 27 1 4 L pneumophila L gratiana 27 1 4 L feeleii 27 2 7 L busanensis 27 1 4 Legionella sp 27 2 7 qPCR 80 98 EMA qPCR 80 98 55 qPCR EMA qPCR 33 EMA qPCR EMA
42. 622 64 16S rRNA 616 bp OTU 6 3 617 417 200 99 OTU OTU 37 22 ctw A 9 AB858105 L pneumophila BW E 134 0 BW F 7 pneumophila 79 BW D pneumophila 3 7 A V 74 7 34 pneumophila CTW G H 0 1 11 bw D 15 AB857853 7 maceachernii OTU 43 7 BW D 65 BW E 1
43. 2007 4 2013 3 1184 53718 25 1ml 121C 20min 500ml 3 1 5 BF 6 1999 100 50C 30min 02M pH2 2 10 min GVPC MERCK CATo 200 pl 37C 6 AR
44. FAS 2002 MWY B YB CATo BRC GVPCodFHi 200 ug ml B E TA
45. CTW G CTW H 7 pneumophila CTW G 4 OLOTE Legionella sp LC2720 JN381005 CTW B 1 Legionella sp L 29 AB836218 16S rRNA 1500 bp 100 Legionella sp L 29 2006 9 AER BW D 20 L dumoffii EMA qPCR pneumophila DNA pneumophila L pneumophila D DNA re
46. 3837 3337 520 GVPC 42 CATo 1665 1459 206 1381 1155 226 T 811 3 3 mi CATo 3 CATo CATo
47. 1995 H 50 745 764 AI 100 HH Saw KRKE BW BOA FETT BGC Hc A Z Y APR IKE OO RR ELIZ B 4 E E
48. 1 ml 0 2 MHCI KCI 1ml pH 02M VEU LIEBE 0 2M ARENC OV C OUS UT E amp 0 pH BCYEo BYEo pH 200 ml 100 ml BYEox 0 2 M HCLKCI 282 100 ul POIL pH 02 M 02M pH RP 2 1 5 8 pH 1 ml 2 0 2 MHCI KCI pH 22 0 2 M pH 22 1 ml 10 pH 10
49. 3 5 EMA qPCR iE EMA PCR EMA qPCR iE HATE H ES EMA MLE BE OAR Jj PCR EMA 3 6 EMA qPCR K A BR B
50. BW F L feeei 13 2 L lytica 3 0 599 L dumofii 1 0 2 Ed6 5 pneumophila CTW A 287 8 CTW B 1 7 CTW C 11 3 CTW 1 0 pneumophila BW D 33 3 BW E 98 6 BWF 49 3 X 6 2 66
51. 65 523 EMA 48 EMA Control Test Kit 33 69 25 EMA EMA EMA Control Test Kit EMA 15 9 qPCR 66 5 1 PCR EMA qPCR PCR EMA PCR Legionella counts No of samples CFU 100 ml Positive Negative Positive Negative lt 10 81 73 0 56 65 9 25 96 2 23 46 990 58 93 596 10 40 14 12 690 13 15 396 1 3 896 10 20 4 4 6 590 50 90 2 1 8 2 24 0 0 2 41 0 0 100 990 11 9 990 11 12 996 0 0 11 22496 0 0 1000 9900 3 2790 3 3 590 0 0 3 6 1 0 0 Total 111 10096 85 100 26 10096 49 100 62 100 67 R52
52. ISO 11731 BOK LTA GL 0 45 um 6400Xg 30 min 100 500 u qPCR EMA qPCR 100 0 2m pH2 2 10 min GVPC MERCK 200 ul 37C fe 3 L 3 BCYEo 37C 2 ES 63 BCYEo
53. BCYEo 37 2 72 BCYEa SO0 100 DDH 16S rRNA ph 6 1 3 EMA DNA EMA Viable Legionella Selection Kit for PCR Ver 2 0 100 1 ml 1 5 ml 14000 x g 10 min 960 pl
54. HH eit 4 LL PCR LAMP LAMP 6 BKO DNA pneumophila DNA PCR 1 1 Acanthamoeba sp Legionella pneumophila ATCC33152 A Acanthamoeba sp pneumophila ATCC33152 B A B 5 Lm 1 2 A B 25C 0 20 40 60 80 100 cooling tower water n
55. pneumophila 4 2 PCR 4 1 02mgL 10 min PCR 10 mg L 30 mn 49 pneumophila 1 mg L 2 0 2mgyL 24 422 L pneumophila DNA 4 3 PCR 4 2 24 BNPD 60 CFU 100 ml 48 PCR 24 L DNA
56. 3 L 31 3 1 CATo ACES 10 g KOH 3g yeast extract 10 g a ketoglutaric acid 1g glycine 3g activated charcoal 2g agar 15g L cysteine hydrochloride 400 mg ferric pyrophosphate 250 mg vancomycin 5 mg polymyxin B 100000 units cycloheximide 80 mg amphotericin B 80 mg thiabendazole 40 mg deionized water up to 1000 ml pH 6 9 0 1 N 2 Acetamido 2 aminoethanesulfonic acid Aseptically added after autoclaved 32 32 fk 321 Legionella pneumophila ATCC33152 L bozemanii ATCC33217 L longbeachae ATCC33462 L micdadei ATCC33218 L gormanii ATCC 33297 L anisa ATCC35292 B MIC 23100 200 pg ml 100 ug ml
57. 2004 78 710 716 EX hi ALTEX 2002 Hoffman P S Pine L and Bell S 1983 Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila catalytic decomposition by charcoal Appl Environ Microbiol 45 784 791 Hookey J V Saunders N A Fry N K Birtles R J and Harrison T G 1996 Phylogeny of Legionellaceae based on small subunit ribosomal DNA sequence and proposal of Legionella lytica comb nov for Legionella like amoebal pathogens Int J Syst Bacteriol 46 526 531 Inoue H Kawano G Nagasawa H and Sakuda S 2002 Isolation of elemental sulfur as a self growth inhibiting substance produced by Legionella pneumophila Appl Environ Microbiol 68 4809 4811 Inoue H Yajima Y Kawano G Nagasawa H and Sakuda S 2004 Isolation of indole 3 aldehyde as a growth inhibitor of Legionella pneumophila from Diaion HP 20 resins used to culture the bacteria Biocontrol Sci 9 39 41 Inoue H Takama T Agawa Y Onodera J Ishima T Agata K Saitoh K and Furuhata K 2007 Evaluation of Duopath Legionella kit for the rapid identification of Legionella strains isolated from water sample
58. 6 G 6 1 25 1 ml 121C 20min 500ml 6 12 ISO 11731 GL f 0 45 um 6400Xg 30min 100 500 ul EMA qPCR PCR 100 0 2 M pH 22 10 min GVPC MERCK 200 ul 37C 6 AR 3 L S
59. 62 615 16S rRNA LBG 22SF LEG 858R PCR TaKaRa Ex Taq Hot Start Version 1 17 45 ul 3 pl 10 x Ex Taq buffer 2 4 ul dNTPs 2 5 mM 1 ul Forward primer 1 ul Reverse primer 0 15 ul Ex Taq polymerase 5 U ul DNA 5 30 ul Thermal Cycler SP 9 C 2min 95C 15 s 65C 30s 72C 60s 6 1 4 PCR 1 5 650 bp PCR QlAquick PCR purification kit Qiagen Mighty TA cloning kit
60. DNA EMA DNA PCR PCR DNA 2 2 C HCI KCI HCI KCI EG T 2 72
61. pMD20 T vector E coli DH5a Competent Cells DNA MI3 primer RV CAG GAA ACA GCT ATG ACC 3 M13 primer M4 5 GTT TTC CCA GTC ACG AC 3 BigDye Terminator Cycle Sequencing Ready Reaction Kit Applied Biosystems Applied Biosystems 3013xl genetic analyzer Applied Biosystems DNA MEGAS http www megasoftware net BLAST GenBank DDBJ EMBL DNA 74 OTU Mothur platform http www mothur org T 99
62. PCR DNA EMA EMA qPCR NI mi JU ON 69 Z y 0 4742x 0 4471 R2 0 3931 Legionella counts by culture Log CFU 100 ml 1 0 1 2 3 4 5 6 7 Concentration of Legionella 16S rRNA gene by qPCR Log copies 100 ml y 0 3532x 0 3917 R2 0 1955 Legionella counts by culture Log CFU 100 ml 1 0 1 2 3 4 5 6 7 Concentration of Legionella 16S rRNA gene by EMA qPCR Log copies 100 ml 5 1 qPCR EMA qPCR A EMA B EMA 70 o o ra pete TE M shes EE 9 Q9 i KR Or e d Ol oR O 6 p ro m a
63. pneumophila E EMA qPCR pneimophila pneumophila ND CTW H 10 CFU 100 ml L pneumophila CFU 16 S rRNA y 0 45x R 0 996 82 87 L lytica X97364 ctw C 17 AB858172 3 St Legionella sp strain LLAP2 U44909 Legionella sp strain LLAP3 X60080 ctw C 80 ctw H 36 1 L waltersii AF12288 Uncultured bacterium clone E9 HQ827915 gz _ ctw 1 90 AB934014 2 ctw H 19 Nera ctw H 16 AB933852 9 t L shakespearei 249736 84 ctw B 8 AB858163 1 bw F 74 2 Uncultured Legionella sp clone BFII B37 HQ112083 ctw C 8 AB858223 2 Ctw C 65 AB858217 1 ctw H 9 3 m deseen w C 1 ctw I ctw C 54 AB858207 1 ctw 1 88 1 99 ctw B 3 B85811 231 ctw B 24 AB858117 2 Uncultured Ed Sp clone BFIII B13 HQ112114 ctw I 104 AB933918 24 L drancourtii X97366 ctw A 59 eee al ctw A 14 AB858059 5 Uncultured Legionella sp clone S5 2 AY924148 ctw H 80 AB933894 5 L ctw B 29 TERE ctw C 58 1 ctw B 26 AB858118
64. MIC 29 3 13 100000 unit L B Hh GVPoa 80 mg L GVPGo GVPa 80 mg L GVPoa 40 mg L CATa GVPoa 80 mg L d 3 1 B 3 14 10 10 ml OPEL AL KIC GVPa GVPCodHh GVPAa GVPGod 10 CFU ml ORE 200 4 37C 8 8 37 C CHEE Lic 8 3 1 5 H 100 pl ORREN 3 f 3 CAT
65. 15 PCR DNA 0 55 v v pneumophila 15 PCR Bej et al 1991 AN FH H PCR Eid Cd Loop mediated isothermal amplification LAMP BH 2003 LAMP
66. DNA PCR 3 PCR PCR PCR LAMP 3 10 CFU 100 ml 61 PCR DNA LAMP LAMP 1 2 2393 29 10 80CFU 100 ml LAMP
67. Rowbotham 1980 XI 1 1 Tison etal 1980 1 2 Fields et al 2002 Legionellosis Fraser etal 1977 T FES DS BAD MUTE Brenner etal 1979
68. f Piao et al 2007 35 GRAD 1995b GER 156 2004 1998 1998 2001 4 2012 3 2013 1 3 2009 100 CFU 100 ml UE
69. 100 100 pl GVPC Dennis et al 1984a EL 37C 6 GVPC pneumophila DNA Beige et al 1995 DNA 2ml 100 14000 xg 10 min 4C 1960 ul 50 mM SOW 100C 15 min E 8 ul 1M Tris HCl MEX pH 7 Lik 14000 x g 10min 4C DNA PCR TaKaRa Ex Taq Hot Start Version 1 33 75 ul 5 ul 10 x Ex Taq buffer 4 ul dNTPs 2 5 mM 1 ul Forward primer 10 uM LEG 448 A 5 GAG GGT TGA TAG GTT AAG AGC 3 Yamamoto et al 1993 1 ul Reverse primer 10 uM LEG 854 B 5 CGG TCA ACT TAT CGC GTT TGC T 3 Yamamoto et al 1993 0 25 ul Ex Taq polymerase 5 U ul DNA 50 ul Lik GeneAm
70. E ES AM BE 2008 1 2012 12 HR TILE VAR BAO ES VOC UV AAT JUS ZIR OTIR Y pH E B POR S J
71. Bopp et al 1981 B REKO 27 PREDKO V Z7 I RARER IO BILLER CAD RE e LU CHBHE Y 28 3 HCHKCI 2
72. E Lt B ERI Yamamoto et al 1993 1997 Ng et al 1997 Wullings et al 2006 Wery et al 2008 Corsao et al 2010 Wullings et al 2011 7 pneumophila 90 BE OD EC AS E R 91 Total Legionella in Environmental water samples
73. EKO DNA EA OFC DNA PCR C PCR X Hi DNA DNA PCR PCR 100 LAMP PCR
74. 2 4 L pneumophila Legionella bozemanii Legionella micdadei Legionella longbeachae Legionella wadsworthii Legionella hackeliae Legionella birminghamensis Legionella cincinnatiensis Legionella tucsonensis Legionella lansingensis Legionella dumoffii Legionella gormanii Legionella jordanis Legionella oakridgensis Legionella feeleii Legionella parisiensis Legionella maceachernii Legionella anisa Legionella santicrucis Legionella sainthelensis Legionella nagasakiensis Legionella steelei Legionella cardiaca 23 Fields et al 2002 Yang et al 2012 Edelstein et al 2012 Pearce et al 2012 L pneumophila 00 N L5 4 6 5 20C 42C 36C 3 C
75. GVPCo PDA HEH h 27 LK Malloch 1981 3 1 2 7 B Sigma F Sigma DMSO BYE HVC 37C 500 ul 50 ml BYE 10 cells ml DMSO 5 u 48 L HB We LIL LV ART RAO 995 ul 37C C3 ERROR Z BEL
76. v i GVPC MERCK 0 2M BE U AERE pH 2 2 200 pl OILS MA a 6400 x g 30 min 100 EL 37C 3f 10 min LL TSO 11731 100 CATa 6 fee EMER EON i LSE HH BCYEo 37 C 2 Hik l 30 BCYEas HAT 3 1 6 CATo
77. 1ml 4ml 50 mM OY ZEE pH 7 pH 37C 6 Pseudomonas aeruginosa IAMIS14 10 CFU ml 1 ml 5 0 2M pH 2 2 2 5 3 0 4 0 50 1 ml 10 1 ml 4ml 50mM OY pH7 37C L 2 P aeruginosa 105 10 CFU ml 1ml 02M pH22 1ml 5 10 20
78. EMA PCR K 6 2 Good s coverage 82 9 98 6 6 3 EMA qPCR pneumophila 6 4 pneumophila 6 4 Bd 6 5 18 GEES 2013 CTW I
79. i i FEE 22 221 RE Vy le ER HCLKCI 2 2 MAK pH 2 3 HCI KCI pH QA 7 2 pH 24 2000 mg L pH 2 7 1000mg
80. pH 2000 mg L pH 2 2 BYEo 200 ul 10 ml BCYEo GVPC pH 6 8 Pine et al 1979 pneumophila A Stanier et al 1986 C 2 2 3 P aerug
81. 1 10 DDH 16S rRNA TT 5 1 3 EMA DNA EMA Viable Legionella Selection Kit for PCR Ver 2 0 100 1 ml 1 5 ml 14000 xg 10 min 960 u BCH A 10 2 B 5 15 min LED CrossLinker 12 15 min RIL DNA NucleoSpin Tissue XS 20 pl DNA EMA
82. PCR 1 4 6 PCR LAMP DNA LAMP PCR PCR ALSoud and R dstr m 2001 46 and Blackwe PCR Watson 11 2000 PCR 3 10 60 CFU 100 ml
83. fE Legionella sp L 47 Q9 9 DNA lysozyme proteinase K 2 30 DNA f DNA HBR h E CHRH LAMP L londiniensis 3 LAMP E L londiniensis LAMP 6 1 SE CHE LAMP 4 8
84. CTW I BW D BW E maceachernii 7 maceachernii 7 maceachernii Inoue et al 2007 SE L maceachernii L maceachernii VBNC pneumophila Legionella sp L 29 Legionella sp LC2720 390 63 Alfreider et al 2004 Wullings et al 2006 Wery et al 2008 Corsao et al 2010 Wullings et al 2011 99 30
85. PCR EMA qPCR PCR EMA PCR Legionella counts No of samples CFU 100 ml Positive Negative Positive Negative lt 10 55 67 1 53 66 2 2 100 53 66 2 2 10090 10 100 13 15 990 13 16 2 0 0 13 16 2 0 0 100 990 7 8 5 7 8 8 0 0 7 8 8 0 0 1000 9900 4 49 4 5 096 0 0 4 5 090 0 0 10000 99000 3 3 7 3 3 890 0 0 3 3 896 0 0 Total 82 100 80 100 2 10090 80 10096 2 10096 68 53 BB EMA qPCR EMA qPCR EMA EMA LAL Control Test Kit EMA 15 9 qPCR
86. 10 min 14000 xg 10 min 4C 70 500 25 ul O TES 10 mM Tris HCl 1 mM EDTA pH 8 0 DNA 417 PCR 14000 xg 10min 4 C x EDX 800 42 42 1 7 pneumophila DNA
87. 30 g 15500 mg L 50 40 mg 20 mg L CMI 10 20 mg CM 2 mg L BNPD 10 mg BNPD 10 mg L 24 4s 500 ml 500 ml PORK Lic 4 1 3 PB PKO ES KORE PCR pneumophila 500 ml FL 0 45 um 47 mm 50 ml 50 ml T 5ml 5 AREER C7 4 1 AE 1 ml 2ml PCR 47
88. d 6 3 EMA qPCR EMA qPCR pneumophila 100 ml 10 7 6 x 10 CFU EMA qPCR 16S rRNA 100 ml 6 0 x 10 24x10 6 4 CTW B C G D 16SrRNA 10 copies 100 ml 2 CTW A H 100 10 100 3 CTW A C D 10 CFU 100 ml Lk pneumophila ATCC33132 EMA qPCR x 045x 5 R 0 996
89. 45 Cic 1 2003 24 MEKOL OSTRI HS 2004 PCR PCR QR 4 D AKO PCR LAMP Mii 7c LAMP VRE BRITE AY RANE T 46 4 1 LAMP PCR Parameter LAMP method PCR method Polymerase Bst DNA polymerase Taq DNA polymerase Primers Four primers Two primers Reaction temperature Isothermal Three steps Reaction time Within 1 h About 2 h Detection method Turbidity Gel electrophoresis 4 1 4 1 1 5 3 pH7 4 5 Legionella pneumophila ATCC33152 10 CFU 100 ml 12 10 1 0 2 mg L 10 30 60 120 180 360
90. B Allen and Gottlieb 1970 K 34 GVPCa HE 7 CATO BCYEa 1984 B
91. RAT YRC OVC CHEE LE S A 101
92. OE TA EE aum kas ROW ERROR VN OCR i oo cc cai 2010 ZYE sz
93. PCR aw 100 LAMP PCR 4 5 31 LAMP 22 PCR 20 10 90 CFU 100 ml 24 LAMP PCR 21 3 100 CFU 100 ml 25 LAMP PCR 24 1 PCR 4 4 PCR DNA PCR 4 1
94. EMA qPCR 5 1 111 30 27 7 pneumophila 30 30 100 qPCR 85 77 EMA qPCR 49 44 DNA PCR 81 qPCR 56 EMA qPCR 23 EMA qPCR 56 23 Lic 30 1 10 CFU 100 m qPCR 4 10 20 20 30 CFU 100 ml EMA qPCR ELE qPCR 1 EMA qPCR 522
95. MEGAS Chaol Simpson Shannon Wiener Good s coverage Mothur platform 001 FastGroupll http fasteroup sdsu edu 99 6 1 6 16S rRNA DDBJ AB837847 AB858225 AB933772 AB934017 75 6 1 Sample ID Sampling site Treatment condition Sonpenranap Sampling date 4 PM of biocide PTS Cooling tower waters CTW A F buiding no treatment none November 6 2012 in Niigata CTW B a plant in Tokyo Pe supply lt 0 1 mg L CMI February 7 2013 a factory intermittent supply CTW C VERRE of CMI 0 1 mg L CMI February 14 2013 a factory continuous supply 1 L CMI D b 201 CIWS Sasana ofCMI D ensue cote a building continuous supply E L CMI 17 2014 CTW H ink anadaud of CMI 0 1 mg L CMI January 17 20 a factory continuous supply TW 2 L tota
96. pH 7 DNA DNA RNA RNase A NucleoSpin gDNA Clean up kit 100 ul DNA DNA GeneQuant pro amersham pharmacia biotech C DNA TE IM Tris HCl 1 mM EDTA 20pg ul TE 10 Cycleave PCR Legionella 16S rRNA Detection Kit 16SrRNA pneumophila DNA L pneumophila DNA qPCR 16S rRNA LEG 22SF 5 AAG ATT AGC CTG CGT CCGAT 3 LEG 858R 5 GTC AAC TTA 73 TCG CGT TTG CT 3 Miyamoto et al 1997 ELT L pneumophila mip LmipL920 SL GCT ACA GAC AAG GAT AAG TTG 3 LmipR15
97. A 10 pl 2 B 5 15 min LED CrossLinker 12 15 min JERR LHE DNA Beige et al 1995 1 ml 100 ARIRE OL 14000 x g 10 min 4C 950 ul 50 mwv 30 9 15 min 8 pl 1M Tris HCl 4ER PHD 14000xg 10min 4C DNA f DNA NucleoSpin gDNA Clean up kit 50 ul DNA 614 pneumophila DNA qPCR BCYEo G0 2 Hf 7 pneumophila ATCC33132 100 pl DIREZ AKIR L 100 ul 50 mM NaOH 9 1 min 16 u O 1M Tris HCl
98. GVPC Dennis et al 37C 100 ml 1 10 CFU 100 ml B DNA DNA r JVYC UY HAT BHO 14 2009 ETA 16S 3
99. HCL KCI HCI KCI pH REY ARERO v IVA ARENOR HCI K CILE REO bR pH R24 E pd 2 1 2000 mg L
100. L pneumophila lone SEC10 EU309487 W A 3 AB858072 10 bw F 43 24 59 Uncultured bacterium clone SBRO9C OTUSBR10 GU933694 ctw G 60 AB933815 9 ctw 1 158 1 m L nagasakiensis EU701006 L oakridgenesis Z32642 89 ctw B 70 AB8581 Sg ctw I 12 AB933932 10 Cluster 4 bw F 10 AB857987 2 Uncultured Legionella sp Clone BFIII B1 HO112102 Cluster 5 54 5 ctw I 61 AB933981 3 L geestiana 249723 L quinlivanii 249733 Uncultured Legionella T clone SEDO3 EU309480 Coxiella burnetii HM208383 0 01 6 4 83 6 4 16S rRNA 616 bp NJ 99 OTU 63 OTU OTU 60 Cluster 1 L anisa Z32635 L bozemanii Z49719 L wadsworthii Z49738 L gormanii Z3263
101. 20 Fields et al 2002 19 95 B B Fraser et al 1978 57 Fields et al 2002 Park et al 2003 Scola et al 2004 Kuroki et al 2007 L ck et al 2010 Yang et al 2012 Edelstein et al 2012 Pearce et al 2012 Campocasso et al 2012
102. EMA 5 qPCR 16S rRNA 5 1 Ed 5 2 qPCR EMA qPCR PCR DNA PCR EMA qPCR 10 30 CFU 100 ml 4 10 CFU 100 ml qPCR 10 CFU 100 ml
103. Non viable Legionella Viable Legionella Culturable Non culturable Legionella Legionella include VBNC state Non viable Legionella Culture lt gt Gene detection such as PCR Gene detection combined with EMA treatment 7 1 92 2009 3 1999 1994 International organization for standardization 1998 Water quality Detection and enumeration of Legionella 15011731 Jones G L and H bert G A ii iR 1981 JE LEGIONNAIRES the disease the bacterium and methodology Harrison T G and Taylor A G 1988 A laboratory manual for Legionella John Wiley amp Sons Ltd Adeleka A Pruckler J Benson R Rowbotham T Halablab M and Fields B 1996 Legionella like amoebal pathogens phylogenetic status and possible role in respiratory disease Emerging
104. R 0 74 325 CATo 1184 53718 3 3337 6 5 520 1 0 3 6 GVPC CATo CATo 6 5962 C 0 29 30 1 CATe 1 0 0 1 10 1
105. 16 697 699 Edelstein P H Edelstein M A Shephard L J Ward K W and Ratcliff R M 2012 Legionella steelei sp nov isolated from human respiratory specimens in California USA and south Australia Int J Syst Bacteriol 62 1766 1771 Feeley J C Gorman G W Weaver R E Mackel D C and Smith H W 1978 Primary isolation medium for Legionnaires disease bacterium J Clin Microbiol 8 320 325 Feeley J C Gibson R J Gorman G W Langford N C Rasheed J K Mackel D C and Baine W B 1979 Charcoal yeast extract agar Primary isolation medium for Legionella pneumophila J Clin Microbiol 10 437 441 Fields B S Benson R F and Besser R F 2002 Legionella and Legionnaires disease 25 year of investigation Clin Microbiol Rev 15 506 526 Fraser D W Tsai T R Orenstein W et al 1977 Legionnaires disease description of an epidemic of pneumonia N Engl J Med 297 1189 1197 Fraser D W Wachsmuth I K Bopp C Feeley J C and Tsai T F 1978 Antibiotic treatment of guinea pigs infected with agent of Legionnaires disease Lancet Jan 28 175 148 1998 15 1 9 95 DERES 30 555 561
106. 7 OTU ctw I 12 10 uncultured Legionella sp clone SEC03 Wery et al 2008 1009900 amp 5 amp ctw G 60 10 uncultured bacterium clone SBR09C OTUSBR10 GU933694 99 8 ctw A 64 4 uncultured bacterium clone 87 TO Ps 25C 20 Vlahos et al 2013 99 7 ctw H 39 2 uncultured Legionella sp clone SEC17 Wery et al 2008 99 7 KR ctw I 90 2 uncultured bacterium clone E9 HOQ827915 99 5
107. it RX E XT Shy tT E i en EON GM 1 AUN EJ re fH
108. 3 2 Uncultured Legionella sp clone Tsw5 3 AY924030 L worsliensis Z49739 ctw G 40 AB933796 2 L moravica 249729 gy ES L quateirensis 249732 ctw B 21 AB858114 2 Uncultured bacterium clone 7 DQ413067 ctw G 45 AB933800 4 se L pneumophila subsp fraseri HO287902 L pneumophila nep iscullei AF122885 ctw B 40 AB8581 sn L pneumophila subsp pneumophila NC002942 ee 1 73 w D 5 AB857889 1 L dumoffii 232637 Legionella sp L 29 AB856218 L parisiensis 249731 r Cluster 2 L steelei HQ398202 67 Legionella sp FM 3 661 Cd L ctw A 48 AB858088 1 m ctw C 67 AB858219 4 L tucsonensis Z32644 Legionella sp strain LLAP10 X97363 LL ctw G 91 AB933841 6 ctw I 1 AB933913 8 ctw H 20 AB933855 13 ctw I 27 AB933979 2 ctw C 9 jo geese L cherrii 249720 ctw H 100 AB933847 11 Legionella sp SO 0 aaa se ctw A 52 AB858093 i 92 ctw I 32 AB933982 15 76 R 139 Geert 3 Uncultured Legionella sp ne SEC17 EU309489 ctw H 39 AB933872 2 L massiliensis JF779685 L tunisiensis F779686 I1 72 L feeleii X73395 38 Ctw 1 10 AB933914 13 L dresdeniensis AM747393 L fairfieldensis 249722 m tw C 1 AB858164 3 ctw 1 87 1 bw F 50 2 L birminghamiensis 249717 Uncultu
109. BYEBo 0 5 ml DY Xv ARET BYEo pH 6 8 BYER HEO pH 100 ml BYEo 2ml BYEo pH 6 8 223 HCI KCI 3 2 5 X HCI KCI pH HCl KCI HCI KCI pH
110. D 2 1 0 0 5 10 15 20 Contact time min 2 3 Puseudomonas aeruginosa pH O pH 2 2 pH 2 5 PH3 0 Bl pH 4 0 A pH 5 0 23 4 E 3 LL O O 9 2 1 0 0 5 10 15 20 Contact time min 2 4 A 0 2 M pH 2 2 O L pneumophila ATCC33152 L micdadei ATCC33218 L gormanii ATCC35297 W L longbeachae ATCC33462 A L pneumophila serogroup 1 A L pneumophila serogroup 2 L pneumophila serogroup 3 24 4 E 3 LL O D 9 2 1 0 0 5 10 15 20 Contact time min 24 B 0 2 M pH 2 2 O L pneumophila serogroup 4 L pneumophila serogroup 5 L pneumophila serogroup 6 W L pneumophila serogroup 7 A L pneumophila serogroup 8 A L jordanis L dumoffii 25 aay y 1 1171x 2 2191 m R 2 0 93 e c E 2000 ca a u O e T 2p c d 1000 O 8 e e e a 500 v P 0 500 1000 1500 2000 2500 Legionella counts after HK treatment CFU plate 2 5 HCLKCI HK PB n 63 26 23
111. M5 Fusarium sp M5 L pneumophila ATCC33152 37 C 8 A GVPCo containing 80 ug ml of cycloheximide B GVPAo containing 80 ug ml of amphotericin B C GVPTo containing 40 ug ml of thiabendazole D CATo containing 80 ug ml of cycloheximide 80 ug ml of amphotericin B and 40 ug ml of thiabendazole 40 100 y 0 9623x 1 0251 R2 0 74 80 60 40 Legionella counts on CATa agar plate CFU plate e 0 20 40 60 80 100 Legionella counts on GVPCa agar plate CFU plate 3 2 GVPCo CATo n7 52 41 33 BR LOTKI ER H Lin et al 1999
112. 10 10 8x 10 10 10 1x10 10 2 6 HCLKCI Result HK PB Positive 52 58 Negative 104 101 Overgrowth 5 2 Total 161 161 Treatment with 0 2 M HCI KCI buffer pH 2 2 Treatment with 0 2 M acid phosphate buffer pH 2 2 Overgrowth of non target heterotrophic bacteria 19 2 7 HCLKCI pH pH NT HK PB Range 4 6 8 4 2 4 5 8 2 2 2 4 Mean 7 1 2 5 2 3 No treatment before treatment with the acid buffers P Treatment with 0 2 M HCI KCI buffer pH 2 2 Treatment with 0 2 M acid phosphate buffer pH 2 2 20 12 11 4 HC KCI 10 acid phosphate MEM ZZ ae Macllvaine 9 i 8 i zs i a 7 I 6 0 10 20 30 40 50 60 70 80 90 100 Addition of 0 1 M NaOH solution ml 2 1 0 1 M NaOH 21 7 0 6 8 6 6 I a 6 4 6 2 HCI KCI acid phosphate 60 3 e Macllvaine 0 1 2 3 4 5 Addition of acid buffer ml 2 2 BYEc 22 4 E 3 2 LL O
113. 720 1440 500 ml 500 ml DPD ik 4 12 31 50 CMI 2 3 10 BNPD 2 2 1 3 1 1 950 ml L pneumophila ATCC33152 10 CFU 100 ml E 31
114. Infectious Diseases 2 225 230 We 2010 DS 38 259 271 Alfreider A Vogt C and Babel W 2004 Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated ground water as revealed by 16S ribosomal DNA analysis Syst Appl Microbiol 25 232 240 Allen P M and Gottlieb D 1970 Mechanism of action of the fungicide thiabendazole 2 41 tiazolyl benzimidazole Appl Microbiol 20 919 926 Al Soud W A and Radstr m P 2001 Purification and characterization of PCR inhibitory components in blood cell J Clin Microbiol 39 485 493 2003 LAMP Legionella JARMAM 14 25 30 F 4H FIORI 13 19 25 2003 Loop mediated isothermal amplification LAMP en 93 TH 2004 LAMP Legionella BAORH 32 195 201 Beige J Loki
115. 2 EMA qPCR t HEDER DNA PCR EMA DNA EMA Chang et al 2009 Delgado Viscogliosi et al 2009 FRH Mie amp CHa BAEBCH Chang et al 2010 Chen et al 2010 Qin et al 2012 EMA DNA PCR EMA qPCR PCR GPCR f 1 5 1 1
116. 393 BOK 25 1 ml 500 ml 415 PCR IK LAMP EL 4 1 3 1ml 100 1 ml 0 2 M E H22 10 min 200 ul GVPC 37C 6 GVPC 3 S BCYEo 37C 2 48 BCYEo
117. 48 5 GTT TTG TAT GAC TTT AAT TCA 3 pneumophila Mahbubani et al 1990 PCR TaKaRa Ex Taq Hot Start Version 1 16 45 ul 3 10 x Ex Taq buffer 2 4 ul dNTPs 2 5mm 1 pl Forward primer 1 ul Reverse primer 1 ul SYBR Green I Lonza 1000 0 15 pl Ex Taq polymerase 5 U ul DNA TAKS 5 ul 30 Thermal Cycler Dice Real Time System I 9 2min 9 C 158 6 30 s 722706 60s 45 VEL 50C 9 10s 0 C 1 FAM DNA pneumophila DNA 6 1 L pneumophila PCR PCR Nishizawa et al 2008 qPCR 50
118. 77842 bath water n 61633 pool water n 2304 hot water supply n 3185 heat storage tank water n 1899 water for landscaping use n 596 m lt 10 CFU 100ml 10 90 100 990 m1000 9900 m gt 10000 munknown 1 3 2001 1 2012 12 2013 25 996 14 1 4 3 5 2 ERREKO 31 9 10 6 1 4 GVPC A GVPC B Samples E Viable Bacteria Dead Bacteria T or e DNA Extraction e of DNA EMA cannot penetrate cell membrane no amplification e ooo EMA can penetrate cell membrane modification of DNA with EMA 1 5 EMA PCR DNA EMA
119. 9 L steigerwaltii 249737 Cluster 2 L sainthelensi Z49734 L santicrucis HF558374 L longbeachae AY444740 L cicinatiensis Z49721 L gratiana Z49725 Cluster 3 L beliardensis AF122884 L busanensis AF424887 L gresilensis AF122883 Cluster 4 4 rubrilucens Z32643 L taurinensis DQ667196 L erythra 232638 Cluster 5 L brunensis Z32636 L cardiac JF831047 L hackliae M36028 L jamestownensis Z49726 L jordanis 232667 L spintensis M36030 84 Relative abundance 0 20 40 60 80 100 CTW A CTW B CTW C CTW G CTW H CTW I BW D BW E BW F m pneumophila m L lytica L feeleii m L maceachernii E L dumoffii m Undescribed culturable Legionella m Uncultured Legionella clone M65 85 OUT number 0 20 40 60 80 100 Clone number 6 6 86 6 3 EMA qPCR
120. Appl Microbiol 30 273 279 La Scola B Birtles R J Greub G Harrison T J Ratcliff R M and Raoult D 2004 Legionella drancourtii sp nov a strictly intracellular amoebal pathogen nt J Syst Evol Microbiol 54 699 703 Liebers D M Baltch A L Smith R P Hammer M C and Conroy J V 1989 Susceptibility of Legionella pneumophila to eight antimicrobial agents including four macrolides under different assay condition J Antimicrob Chemother 23 37 41 Lin A Stout J E Rihs J D Vickers R M and Yu V L 1999 Improved Legionella selective media by the addition of fluconazole results of in vitro testing and clinical evaluation Diagn Micribiol Infect Dis 34 173 175 Lick PC Jacobs E R ske I Schr ter Bobsin U Dumke R and Gronow S 2010 Legionella dresdenensis sp nov isolated from river water Int J Syst Evol Microbiol 60 2557 2562 McDade J E Shepard C C Fraser D W Tsai T R Redus M A and Dowdle W R 1977 Legionnaires disease isolation of a bacterium and demonstration of its role in other respiratory disease N Engl J Med 297 1197 1203 Mahbubani M H Bej A K Miller R Haff L DiCesare J and Atlas R M 1990 Detection of Legionella with polymerase chain reaction and gene probe methods Mol Cell Probes 4 175 187 Malloch David 1981 Moulds their isolation cultivation and identification University of Toronto press
121. C33297 4 x 10 100 4 3 L anisa ATCC35292 2x10 100 40 44 The value shows the percentage of CFU of Legionella two replicate plates when the CFU on BCYEa were 100 37 3 5 Number of samples Legionella counts Total CFU 100 ml Cooling tower water Bath water lt 10 3 012 72 3 46491 86 5 83503 79 696 10 90 4413 8 696 4587 8 590 9000 8 6 100 990 3206 6 396 1451 2 796 4657 4 4 1000 9900 2340 46 559 1 096 2899 2 8 gt 10000 876 1 790 110 0 2 986 0 996 Unknown 3337 6 5 520 1 0 3857 3 7 Total 51184 53718 104902 a u non target microorganisms 38 Unknown indicates that the presence of Legionella is unknown due to the overgrowth of F 3 6 Number of samples Pana DUE Cooling tower water Bath water CFU 100 ml GVPC CATo GVPC CATo lt 10 1532 45 9 2358 70 7 230 44296 349 07 196 10 90 298 8 9 382 11 4 37 7 196 77 14 896 100 990 177 5 396 258 7 796 14 2 796 27 5 2 1000 9900 123 3 796 155 4 6 9 1 7 11 2 196 gt 10000 63 1 996 88 2 6 7 1 3 6 1 2 Unknown 1144 34 396 96 2 996 223 42 9 50 9 696 Total 3337 3337 520 520 a non target microorganisms 39 Unknown indicates that the presence of Legionella is unknown due to the overgrowth of 3 1 ARA Fusarium sp
122. Canada Miyamoto H Yamamoto H Arima K Fujii J Maruta K Izu K Shiomori T and Yoshida S 1997 PCR method for detection of Legionella species and its application to surveillance of Legionellae in hospital cooling tower water Appl Environ Microbiol 63 2489 2494 Nogva H K Dromtorp S M Nissen H and Rudi K 2003 Ethidium monoazide for DNA based differentiation of viable and dead bacteria by 5 nuclease PCR Biotechniques 34 804 813 Ng D L K Koh B B Tay L and Heng B H 1997 Comparison of polymerase chain reaction and conventional culture for the detection of legionellae in cooling tower waters in Singapore Lett Appl Microbiol 24 214 216 97 Nishizawa T Komatsuzaki M Kaneko N and Ohta H 2008 Archaeal diversity of upland rice field soils assessed by the terminal restriction fragment length polymorphism method combined with real time quantitative PCR and a clone library analysis Microbes Environ 23 237 243 Notomi T Okayama H Masubuchi H Yonekawa T Watanabe K Amino N and Hase T 2000 Loop mediated isothermal amplification of DNA Nucleic Acids Res 28 e63 JE 1998 Legionella longbeachae 1 72 1076 1079 1984 Legionella
123. EMA DNA 514 qPCR H LE PCR Cycleave PCR Legionella 16S rRNA Detection Kit 0 2 ml PCR DNA ul 25 ul Thermal Cycler Dice Real Time System M H 95 C C 10 s 9 C 5 s 55C 10 s 72C 20s 45 2 FAM ROX FAM 16S rRNA ROX ROX PCR 5 15 EMA EMA Control Test Kit Vaiable bacteria selection EMA Viabl
124. HHHHHHHHHHHHHHI llHHHHHHHHHHHHHHHHHHHHHHHH Title OOU0000 00 Deveopment of improved methods for detection of Legiondla spp in artificial water environments Author s 0 0 00 Citation Issue Date 2015 03 16 URL http hdl handl e net 10636 549 Type Thesis or Dissertation Textversion ETD 201 3 1 1 PE 2 2 2 1 2 1 1 KRE 2 1 2 2 1 3 WRPEREETIE 2 1 4 2 1 5 2 1 6 2 2 2 2 1 222 223 23 3 3 1 3 1 1 3 12
125. ME pem eo N 1 1 1 xdi nS O LN NE MM N 1 1 1 1 CO ll 1 1 1 1 Na eo dP 4 del ied gt juu 00T n 607 gn InO q squnoo 5 900 27 Concentration of Legionella 16S rRNA gene by qPCR Log copies 100 ml 1 1 1 I l 1 1 1 L 1 dem gt s 1 e I l 1 1 I I 1 I 1 l 1 1 1 1 1 I 1 L 1 eee ae Suk os I 1 l I 1 I 1 4 1 I 1 I 1 f 1 I 4 I 1 I 1 I 1 I 1 juu 00T n 607 gln InO Ag squnoo 5 900 27 Concentration of Legionella 16S rRNA gene by EMA qPCR Log copies 100 ml A 5 2 qPCR EMA qPCR B EMA A EMA 71 6 EMA qPCR EMA PCR VBNC VBNC
126. OSG HIDE ins GVPA aZ Ht Hh GVPToa BYU GVPCo GVPAc GVPTo pneumophila OHH BCYEasTHH GVPCa CATa 5 OG GVPo BCYEo 3 gL 5 mgL GVPo GVPCadis 80 mg L F 80mgL B 40 mg L GVPTa d 100 ul DRR TEREN Z 5 37 C CHEE L Riz L md ATCC33152 Hh CATo 10 CFU ml 214 25 1 ml 121C 20min 500ml
127. Positive 292 29 Negative 750 1322 Total 1042 1351 57 Total 321 2072 2393 4 8 LAMP Before treatment After treatment Sample PAMP pus um aod md Bath system 1 4800 lt 10 Bath system 2 20 lt 10 Bath system 3 550 lt 10 Bath system 4 10 lt 10 Bath system 5 30 lt 10 Bath system 6 310 10 58 120 min 02 1 10 I M TIY 180 min 360 min 720 min 1440 min 0 2 1 10 0 2 1 10 02 1 10 0 2 1 10 500 bp 500 bp 4 1 L pneumophila PCR M 100 bp P N 59 500 bp 4 2 L pneumophila CMI BNPD PCR M 100 bp A B 15500 mg L C 20 mg L D CMI 2 mg L E BNPD 10 mg L 60 4 3 DNA 10 mg L
128. Wadowsky Yee Okuda WYO 58 1073 1082 Park M Y Ko K S Lee Park M S and Kook Y H 2003 Legionella busanensis sp nov isolated from cooling tower water in Korea nt J Syst Evol Microbiol 53 77 80 Pearce M M Theodoropoulos N Mandel M J Brown E Reed K D and Cianciotto N P 2012 Legionella cardiaca sp nov isolated from a case of native valve endocarditis in a human heart Int J Syst Bacteriol 62 2946 2954 Piao Z Sze C C Barysheva O lida K and Yoshida S 2006 Temperature regulated formation of mycelial mat like biofilms by Legionella pneumophila Appl Environ Microbiol 72 1613 1622 Pine L George J R Reeves M W and Harrell W K 1979 Development of a chemically defined liquid medium for growth of Legionella pneumophila J Clin Microbiol 9 615 626 Prince A M and Andrus L 1992 PCR How to kill unwanted DNA BioTechniques 12 358 360 Qin T Tian Z Ren H Hu G Zhou H Lu J Luo C Liu Z and Shao Z 2012 Applicationof EMA qPCR as a complementary tool for the detection and monitoring of Legionella in different water systems World J Microbiol Biotechnol 28 1881 1890 Rowbotham T J 1980 Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae J Clin Pathol 33 1179 1183 Stanier R Y Ing
129. a CATo 33 324 CATo CATo 3 3 34 LOT CATo GVPCo GVPCo CATo 214 GVPCo 29 13 6 CATo 4 1 9 3 2 214 52 GVPCa CATa
130. an Range HK 2 9 2 4 6 2 3 1 2 5 7 2 PB 2 3 2 2 2 4 2 3 2 3 2 5 MB 2 3 2 3 2 4 2 3 2 3 2 6 n 32 from different cooling towers n 13 from different spas Treatment with 0 2 M HCLKCI buffer pH 2 2 Treatment with 0 2 M acid phosphate buffer pH 2 2 Treatment with 0 2 M Macllvaine buffer pH 2 2 16 R24 pH TA HK PB 6000 7 4 5 8 3000 7 1 3 2 2000 6 9 2 7 1000 6 5 2 5 500 5 8 2 4 250 2 9 2 3 MB 3 4 2 9 27 2 6 2 5 24 Total alkalinity mg L as CaCO P Treatment with 0 2 M HCI KCI buffer pH 2 2 Treatment with 0 2 M acid phosphate buffer pH 2 2 Treatment with 0 2 M Macllvaine buffer pH 2 2 17 Fe 2 5 HCLKCI Sample Spa 1 Spa 2 Spa 3 Spa 4 Spa 5 CT6 CT7 CT8 Treatment with 0 2 M HCI KCI buffer pH 2 2 pH 8 0 8 5 7 7 8 0 9 0 7 5 8 5 8 2 No treatment MA SW 120 2 9 x 10 460 2 5 x 10 110 9 9 x 10 110 2 7 x 10 350 2 3 x 10 32 4 0 x 10 190 14 x 10 120 9 6 x 10 pH 2 6 5 5 2 6 2 6 3 8 2 5 2 7 2 6 Treatment with 0 2 M acid phosphate buffer pH 2 2 Total alkalinity mg L as CaCO 18 HK Plate count CFU ml 4 x 10 9 6 x 10 1 0 x 10 10 8x 10 10 1x10 10 24 2 3 2 3 24 2 3 2 3 2 3 PB Plate count CFU ml
131. e Legionella Selection Kit for PCR Ver 2 0 ATO Control DNA EMA Control DNA EMA PCR Control Test Kit Control DNA PCR EMA Control Test Kit 0 2 ml PCR 64 DNA YANK 5 wl 25 ul amp Thermal Cycler Dice Real Time System Z 9 C 10 s 95C 5 s 5 C 10 s 72 C 20 s 45 1 FAM 45 EMA DNA 48 52 WE 52 1 H LL lt qPCR
132. es J Schaberg T Finckh U Fischer M Mauch H Lode H K hler B and Rolfs A 1995 Clinical evaluation of a Mycobacterium tuberculosis PCR assay J Clin Microbiol 33 90 95 Bej A K Mahbubani M H and Atlas R M 1991 Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods Appl Environ Microbiol 577 597 600 Bopp C A Sumner J W Morris G K and Wells J G 1981 Isolation of Legionella spp from environmental water samples by low pH treatment and use of a selective medium J Clin Microbiol 13 714 719 Brenner D J Steigerwalt A G and McDade J E 1979 Classification of the Legionnaires disease bacterium Legionella pneumophila genus novum species nova of the family Legionellaceae familia nova Ann Intern Med 90 656 658 Campocasso A Boughhalmi M Fournous G Raoult D and La Scola B 2012 Legionella tunisiensis sp nov and Legionella massiliensis sp nov isolated from environmental water samples Int J Syst Evol Microbiol 62 3003 3006 Chang B Sugiyama K Taguri T Amemura Maekawa J Fumiaki K and Watanabe H 2009 Specific Detection of Viable Legionella cells by combined use of photoactivated ethidium monoazide PCR real time PCR Appl Environ Microbiol 75 147 153 Chang B Taguri T Sugiyama K Amemura Maekawa J Fumiaki K and Watanabe H 2010 Comparison
133. inosa pH SX ER EC M BC HCL KQI 4 N ES RELY DRRR E RLT HCLKCI HO pH 5 8 pH 2 4 ZeME ETE Z 652 HCl KC1
134. l chl N ber 29 2013 e in Fukuoka of stabilized chlorine VME oal chiondne NGM Bath waters Bw SO ouh continuous suPply 4 O mg L free chlorine March 21 2013 in Kanagawa of free chlorine BW E ahotelin Miyagi CON NUOUSSUPPIY no data free chlorine April 23 2013 of free chlorine a sports club continuous supply f L f hl May 15 201 BW F NUM iles chlorine 0 5 mg L free chlorine ay 15 2013 5 Chloro 2 methyl 4 isothiazolin 3 one The water treatment chemicals including CMI were batch fed as necessary 76 45 40 35 30 25 20 Threshold cycles 15 10 L pneumophila mip gene y 3 830Log x 30 33 R2 0 998 Legionella spp 16S rRNA gene y 3 472Log x 25 17 R2 0 998 0 01 0 1 1 10 100 Concn of Legionella pneumophila total DNA pg tube 6 1 16S rRNA gene mip gene PCR Legionella spp 16S rRNA gene pneumophila mip gene pneumophila DNA L pneumopnila qPCR value of L pneumophila qPCR value of Legionella SDD x 100 77 5096 of fluorescence plateau fee 1 Threshold cycles 6 2 PCR 78 62 f 62 EMA qPCR
135. lla pneumophila Legionella micdadei Legionella gormanii Legionella longbeachae L pneumophila L pneumophila L pneumophila L pneumophila L pneumophila L pneumophila L pneumophila L pneumophila Legionella jordanis Legionella dumoffii Pseudomonas aeruginosa American Type Culture Collection Serogroup 1 Isolated from cooling tower water samples Isolated from spa water samples Source ATCC33152 ATCC33218 ATCC33297 ATCC33462 CT Spa Spa Spa Spa Spa CT CT CT Spa IAM1514 9jAM Culture Collection Center for Cellular and Molecular Research Institute of Molecular and Cellular Bioscience The University of Tokyo Currently RIKEN Bioresource Center JCM 14 2 2 Parameter pH Electrical conductivity mS m Total hardness mg L as CaCO Total alkalinity mg L as CaCO3 Chloride mg L n 32 from different cooling towers P n 13 from different spas Cooling tower water Mean 82 80 250 180 77 15 Range 6 4 9 4 1 4 270 2 1300 2 560 2 570 Spa water Mean Range 7 0 2 0 8 8 540 37 3700 1100 19 9900 150 0 1700 1600 2 15000 2 3 pH Cooling tower water Spa water Buffer Mean Range Me
136. nella 69 151 157 199 b Legionella pneumophila serogroup 7 7 Pontiac fever T 69 654 665 WARTS 2004 78 90 98 1998 4 102 323 Yamamoto H Hashimoto Y and Ezaki T 1993 Comparison of detection methods for Legionella species in environmental water by colony isolation fluorescent antibody staining and polymerase chain reaction Microbiol Immunol 37 617 622 1997 Legionella AMADER amp STRIJE Microbes Environ 12 149 156 99 Yang G Benson R F Ratcliff R M Brown E W Steigerwalt A G Thacker W L Daneshvar M I Morey R E Saito A and Fields B S 2012 Legionella nagasakiensis sp nov isolated from water samples and from a patient with pneumonia Int J Syst Evol Microbiol 62 284 288 Ott
137. of ethidium monoazide and propidium monoazide for the selective detection of viable Legionella cells Jpn J Infect Dis 63 119 123 Chen N T and Chang C W 2010 Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real time quantitative PCR J Appl Microbiol 109 623 634 Corsaro D Pages G S Catalan V Loret J F and Greub G 2010 Biodiversity of amoebae and amoeba associated bacteria in water treatment plants nt J Hyg Environ Health 213 158 166 94 Dennis P J Bartlett C L R and Wright A E 1984a Comparison of isolation methods for Legionella spp Proceedings of the 2nd international symposium Washington DC Am Soc Microbiol 294 296 Dennis P J Green D and Jones B P C 1984b A note on the temperature tolerance of Legionella J Appl Bacteriol 56 349 350 Delgado Viscogliosi P Solignac L and Delattre J M 2009 Viability PCR a culture independent method for rapid and selective quantification of viable Legionella pneumophila cells in environmental water samples Appl Environ Microbiol 75 3502 3512 Edelstein P H 1981 Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens J Clin Microbiol 14 298 303 Edelstein P H 1982 Comparative study of selective media for isolation of Legionella pneumophila from portable water J Clin Microbiol
138. p 9700 Applied Biosystems 9 C 30 s 65C 30 8 72C 60 s 40 1 5 SYBR Green I BMP 254 nm 430 bp 414 2003 7 9 100 100 49 31 2004 6 2012 4 2
139. raham J L Wheelis M L and Painter P R 1986 The microbial world 5th edition Prentice Hall New Jersey 98 Tison D L Pope D H Cherry W B and Fliermans C B 1980 Growth of Legionella pneumophila in association with blue green algae cyanobacteria Appl Environ Microbiol 39 456 459 Vlahos N Kormas K A Pachiadaki M G Meziti A Hotos G N and Mente E 2013 Cange of bacterioplankton apparent species richness in two ornamental fish aquaria SpringerPlus 2 66 Watson R J and Blackwell B 2000 Purification and characterization of a common soil component which inhibits the polymerase chain reaction Can J Microbiol 46 633 642 Wery N Bru Adan V Minervini C Delg nes J P Garrelly L and Godon J J 2008 Dynamics of Legionella spp and bacterial populations during the proliferation of L pneumophila in a cooling tower facility Appl Environ Microbiol 74 3030 3037 Wullings B A and van der Kooij D 2006 Occurrence and genetic diversity of uncultured Legionella spp in drinking water treated at temperatures below 15 C Appl Environ Microbiol 72 157 166 Wullings B A Bakker G and van der Kooij D 2011 Concentration and diversity of uncultured Legionella spp in two unchlorinated drinking water supplies with different concentrations of natural organic matter Appl Environ Microbiol 77 634 641 MART E 055 omJIDH E 1995a Legio
140. red Legionella sp clone RWII A36 HQ111637 ctw A 1 ShwrB 43 0 B Ctw B 43 AB858136 4 Uncultured bacterium clone TO Ps 25C 20 JX105622 gt ctw A 64 AB858102 4 w B 48 AB858138 4 Uncultured Legionella sp clone SEC15 EU309488 95 ctw A 9 AB858105 4 ctw C 12 7 ctw 1 103 1 bw D 12 22 bw E 54 69 bw F 76 33 ctw A 2 AB858064 78 3 sl ctw C 29 AB858183 11 es 97L ctw C 6 AB858212 4 L impletisoli AB233209 eg L yabuuchiae AB233210 ctw I 16 AB933965 2 ctw H 63 AB933880 2 L adelaidensis 249716 L nautarum 249728 ctw B 62 AB858147 3 rm tw H 22 A8933857 2 L Legionella sp strain LLAP1 X97355 ctw B 61 AB858146 8 ctw 1 135 1 88 86 LL Uncultured Legionella sp cl 98 ct LL ctw G 1 51 AB933772 ctw C 18 AB 858173 2 ctw H 6 Uncultured bacterium clone GOUTB L lansingensis Z49727 ctw C 2 AB858175 7 ctw B 37 sss 4 Legionella sp L 47 __ mm Cluster 3 71 97 L maceachernii AF227161 f bw D 15 accor tm bw E 63 1 L micdadei AF227162 E ctw A 28 AB858070 5 LT L israelensis 232640 mL londiniensis 249730 AB899895
141. s Biocontrol Sci 12 155 158 GHA BR 2013 41 659 661 1999 un E ex Tl ca 73 25 34 R 2002 76 41 50 2003 0214004 2013 34 155 156 Dit Lu i M x BE du gt i ni H 96 Kuroki H Miyamoto H Fukuda K hhara H Kawamura Y Ogawa M Wang Y Ezaki T and Taniguchi H 2007 Legionella impletisoli sp nov and Legionella yabuuchiae sp nov isolated from soils contaminated with industrial in Japan Syst
142. t 10 lt 10 EEF 6x10 48h E 10 lt 10 sS 10 lt 10 Hydrogen peroxide 15500 mg liter Glutaraldehyde 20 mg liter 5 Chloro 2 methyl 4 isothiazolin 3 one 2 mg liter 2 Bromo 2 nitropropane 1 3 diol 10 mg liter Intensity of the amplified DNA band on the gel image is indicated in two grades bright not visible 53 4 4 Legionella counts CFU 100 ml lt 10 10 90 100 990 1000 9900 gt 10000 Total No of samples 51 24 16 100 54 Bath water source Spa water Tap water 22 44 9 29 56 9 12 24 5 12 23 5 11 22 4 5 9 8 4 8 2 3 5 9 0 0 2 3 9 49 51 R45 LAMP PCR LAMP PCR Legionella counts No of CFU 100 ml samples X i p f positive negative positive negative lt 10 51 22 29 20 31 10 90 24 24 0 21 3 100 990 16 16 0 16 0 1000 9900 7 7 0 7 0 gt 10000 2 2 0 2 0 Total 100 71 29 66 34 55 R46 MRKPOMaMROTRANER Element Composition 96 Fe 56 Si 23 S 8 Zn 6 Ca 1 The value shows the percentage of detected elements heavier than sodium 56 4 7 LAMP LAMP Culture Positive Negative
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