ATP. adenosine triphosphate (ATP) Nontuberculous Mycobacteria (NTM) ATP

164 ATP 1) 1) 1) 2) 2) 1) 1) 2) 16 4 8 16 9 7 adenosine triphosphate (ATP) Nontuberculous Mycobacteria (NTM) ATP MB ATCC 4 45 ATP 30 60 10 ATP 5 Relative light units (RLU) ratio 7 MB streptomycin, ethambutol, kanamycin, isoniazid, levofloxacin, ethionamide, amikacin 7 RLU ratio, rifampicin clarithromycin RLU ratio ATP MB Mycobacterium kansasii ATCC 12478 84.4 3 97.8 100 4 95 Mycobacterium intracellulare: 92.2, Mycobacterium avium:97.2, M. kansasii: 100 95.3 ATP NTM Key words: nontuberculous mycobacteria (NTM), adenosine triphosphate (ATP), minimum inhibitory concentration (MIC), bioluminescence (nontuberculous mycobacteria; NTM) 70 Mycobacterium avium complex (MAC) MAC MAC 2 MAC ( 565 0871) 2 15 TEL: 06 6879 6680 FAX: 06 6879 6683 E-mail: horikawa@hp-lab.med.osaka-u. ac.jp 2003 4 2003 1) MAC MAC AIDS MAC Mycobacterium kansasii 1997 MAC M. kansasii rifampicin (RFP) RFP NTM clarithromycin (CAM) azithromycin 36

ATP 165 RFP adenosine triphosphate (ATP) ATP ATP 2) 3) 4) 6) NTM ATP ATP MB I. A. American Type Culture Collection (ATCC) M. avium ATCC 700898, M. avium ATCC 25291, M. kansasii ATCC 12478 Mycobacterium intracellulare ATCC 13950 4 45 M. avium 20 M. intracellulare 20 M. kansasii 5 DDH (DNA DNA hybridization) B. ATP ATCC Middlebrook 7H9 broth McFarland No. 0.5 530 nm, 0.08 0 Middlebrook 7H9 broth 1/2, 1/10, 1/100 A(2-amino-2- methyl-1,3-propanediol) B (adenosine phosphate deaminase) 50 ml 0, 30, 60, 90, 120 ATP ATP MIC NTM 0 30 C. ATP ATP McFarland No. 0.5 (benzalkonium chloride) 50 ml 60, 70, 80 0, 5, 10, 15, 20 ATP D. ATP ATP ATP ATP (LP- 5000-kyokuto-1) 100 ml (Relative light units; RLU) E. 1. MIC MB MIC streptomycin (SM), ethambutol (EB), kanamycin (KM), isoniazid (INH), RFP, levofloxacin (LVFX), CAM, ethionamide (TH), amikacin (AMK) 9 2 SM, EB, KM 0.06 128 mg/ml 12 INH, RFP, LVFX, CAM 32 mg/ml 11 TH, AMK 0.5 16 mg/ml 6 MIC NTM 1 4 Middlebrook 7H9 broth 530 nm, 0.05 37 3 5 McFarland No. 0.5 0.08 0 McFarland No. 0.5 110 ml 11 ml NTM 100 ml 37 1 7 MIC 2. ATP ATP ATP MIC ATP MB ATP MIC 5 37

166 30 60, 10 ATP RLU ratio RLU RLU RLU ratio, RLU CAM 8 3 RLU CAM ph CAM RLU II. A. ATP Table 1 McFarland No. 0.5 1/ 100 RLU 302 506 1/10, 1/2 30 60 RLU ATP RLU 72,074 30 384 Table 2 M. avium, M. intracellulare 60 80 M. kansasii RLU 60 RLU 3 10 ATP 60, 10 B. 1. ATCC MB ATP 7 MIC MB 5 ATP RLU ratio ATP 5 1 Table 3 MB ATP RLU ratio RLU ratio RLU ratio M. avium ATCC 700898 INH 1 2 1 97.8 M. intracellulare ATCC 13950 1 M. avium ATCC 25291 ATP RFP, CAM 2 7 1 M. kansasii ATCC 12478 KM, AMK EB 84.4 RLU ratio M. avium ATCC 700898 EB LVFX 88.9 M. avium ATCC 25291 RLU ratio RFP, CAM KM AMK Table 1. E#ect of treatment time on the exogenous ATP. Dilution of organisms suspension Treatment time (min) 0 30 60 90 120 M. avium ATCC 25291 blank 95 48 41 49 76 1/100 265 329 302 292 345 1/ 10 2636 3981 4129 3779 4412 1/ 2 13052 26015 25566 26225 30611 M. intracellulare ATCC 13950 blank 165 51 54 46 114 1/100 329 506 499 476 461 1/ 10 1774 3987 4462 4133 4313 1/ 2 15867 40746 44716 54015 57430 M. kansasii ATCC 12478 blank 155 50 43 41 117 1/100 306 414 506 399 377 1/ 10 1284 3176 2720 3219 3382 1/ 2 18574 22077 24503 24090 23905 BrothMIC NTM blank 72074 384 NT NT NT NT: not tested 38

ATP 167 Table 2. E#ect of the temperature and incubation time on the extraction of ATP from nontuberculous mycobacteria. Temperature Extraction time (min) 0 5 10 15 20 M. avium ATCC 25291 room temperature 5643 7909 8949 9178 9483 60 5643 26090 26223 23592 30892 70 5643 24440 23807 28471 25906 80 5643 26967 26795 25016 24740 M. intracellulare ATCC 13950 room temperature 972 7359 7467 7323 7773 60 972 15368 17435 16998 17525 70 972 16982 18784 17432 18888 80 972 18823 18386 17160 18246 M. kansasii ATCC 12478 room temperature 661 1125 1321 1245 1211 60 661 8756 8608 8730 9305 70 661 6249 7178 5938 6579 80 661 5945 5945 5441 5472 2 M. kansasii ATCC 12478 RLU ratio RLU ratio KM 8 1 M. intracellulare ATCC 13950 LVFX 1 2 RLU ratio 4 RLU ratio SM, TH LVFX 100, EB INH 95 AMK KM 85 CAM RFP 75 RLU ratio CAM, TH 100, SM,INH RFP 95 LVFX 90, EB 85 AMK 80 KM 70 CAM RFP RLU ratio EB, KM AMK Table 4 MB 5 MIC ATP SM, EB, KM, INH, LVFX, TH, AMK 7 RLU ratio RFP, CAM 2 RLU ratio M. avium ATCC 700898 ATP INH 1 8 mg/ml MB 8 1 M. avium ATCC 25291 ATP RFP 8 1 M. kansasii ATCC 12478 ATP KM, AMK EB MB 84.4 RLU ratio 97.8 M. intracellulare ATCC 13950 ATP MB 1 4 95 2. MIC ATP MIC Table 5 MB ATP RLU ratio M. avium 93.9 RFP CAM INH 2 4 RLU ratio MB 78.3 RFP CAM 1 M. kansasii RLU ratio 1 RLU ratio 1 M. intracellulare 90.6 SM, KM, INH, RFP CAM MB RLU ratio EB RLU ratio 1 RLU ratio 3 20 11 CAM 1 RFP 39

168 Table 3. Concordance rate of the ATP method when the Microdilution (MB) method for reference strains is set as a basis. M. avium ATCC 700898 M. avium ATCC 25291 M. kansasii ATCC 12478 RLU ratio Discrepancy of MICs 3 2 1 0 1 2 3 Streptomycin 5 Ethambutol 5 Kanamycin 4 1 Isoniazid 1 3 1 Rifampicin 1 3 1 Levofloxacin 5 Clarithromycin 2 3 Ethionamide 4 1 Amikacin 3 2 Streptomycin 5 Ethambutol 2 2 1 Kanamycin 5 Isoniazid 1 1 2 1 Rifampicin 1 2 2 Levofloxacin 3 1 1 Clarithromycin 1 4 Ethionamide 4 1 Amikacin 4 1 Streptomycin 1 4 Ethambutol 1 1 3 Kanamycin 1 3 1 Isoniazid 1 4 Rifampicin 5 Levofloxacin 3 2 Clarithromycin 3 2 Ethionamide 4 1 Amikacin 1 1 3 Streptomycin 1 3 1 Ethambutol 1 4 Kanamycin 1 4 Isoniazid 5 Rifampicin 1 3 1 Levofloxacin 3 1 1 Clarithromycin 2 3 Ethionamide 4 1 Amikacin 1 3 1 Streptomycin 5 Ethambutol 1 4 Kanamycin 3 1 1 Isoniazid 1 4 Rifampicin 3 2 Levofloxacin 1 4 Clarithromycin 4 1 Ethionamide 1 4 Amikacin 3 2 Streptomycin 5 Ethambutol 1 3 1 Kanamycin 2 1 1 1 Isoniazid 1 4 Rifampicin 5 Levofloxacin 5 Clarithromycin 1 4 Ethionamide 5 Amikacin 1 4 Concordance rate ( ) 97.8 88.9 77.8 75.6 84.4 97.8 40

ATP 169 Table 3. continued M. intracellulare ATCC 13950 RLU ratio Discrepancy of MICs 3 2 1 0 1 2 3 Streptomycin 1 4 Ethambutol 2 3 Kanamycin 1 4 Isoniazid 4 1 Rifampicin 5 Levofloxacin 5 Clarithromycin 2 3 Ethionamide 1 4 Amikacin 5 Concordance rate ( ) 100 Streptomycin 1 4 Ethambutol 5 Kanamycin 1 4 Isoniazid 2 3 Rifampicin 5 Levofloxacin 4 1 Clarithromycin 5 Ethionamide 1 4 Amikacin 5 97.8 1 1 45 RLU ratio EM AMK 100, TH LVFX 97.8 SM, CAM KM 91.1, RFP 86.7, INH 82.2 RLU ratio CAM 100, RFP 97.8, SM 95.5, AMK 93.3 LVFX TH 84.4, KM 82.2, INH 80 EB 44.4 CAM RFP RLU ratio Table 6 MB ATP MIC ATP SM, EB, KM, INH, LVFX, TH, AMK 7 RLU ratio RFP, CAM RLU ratio M. avium INH TH MB ATP 97.2 M. kansasii 1 M. kansasii MIC RFP, CAM 0.25 mg/ml LVFX 0.25 mg/ml KM 8 mg/ml MIC M. intracellulare ATP SM, KM INH 92.2 45 ATP MB 95.3 M. kansasii ATCC 12478 ATP 5 SM, EB, KM, INH, LVFX, TH, AMK 7 RLU ratio RFP. CAM RLU ratio Tables 7 8 M. avium M. intracellulare MB ATP MIC M. avium MIC RFP, CAM 2 mg/ml LVFX 4 mg/ml M. intracellulare RFP, CAM MIC 0.25 mg/ml LVFX 2 mg/ml MIC 1997 4 MGIT 7) 41

170 Table 4. Concordance rate of the ATP method when the Microdilution (MB) method for reference strains is set as a basis. M. avium ATCC 700898 RLU ratio MIC range of MB method ( mg/ml) Discrepancy of MICs 3 2 1 0 1 2 3 Streptomycin 2 4 5 Ethambutol 4 8 5 Kanamycin 2 4 1 Isoniazid 32 32 1 3 1 Rifampicin 0.06 25 1 2 2 Levofloxacin 0.5 5 Clarithromycin 0.25 1 4 Ethionamide 2 8 4 1 Amikacin 1 2 3 2 Concordance rate ( ) 97.8 M. avium ATCC 25291 M. kansasii ATCC 12478 M. intracellulare ATCC 13950 Streptomycin 8 1 4 Ethambutol 4 8 1 1 3 Kanamycin 8 1 3 1 Isoniazid 32 1 4 Rifampicin 2 1 3 1 Levofloxacin 2 4 3 2 Clarithromycin 0.5 2 3 Ethionamide 4 8 4 1 Amikacin 4 8 1 1 3 Streptomycin 128 5 Ethambutol 4 8 1 4 Kanamycin 4 8 3 1 1 Isoniazid 4 8 1 4 Rifampicin 0.25 5 Levofloxacin 0.25 1 4 Clarithromycin 25 1 4 Ethionamide 1 1 4 Amikacin 2 4 3 2 Streptomycin 0.25 1 4 Ethambutol 1 2 3 Kanamycin 0.25 0.5 1 4 Isoniazid 4 8 4 1 Rifampicin 5 Levofloxacin 0.25 5 Clarithromycin 0.06 5 Ethionamide 16 16 1 4 Amikacin 0.5 5 97.8 84.4 100 MTB-1 8) MTB-SR NTM NTM NCCLS (National Committee for Clinical Laboratory Standards) 9) NTM MB ATP MIC ATP NTM 42

ATP 171 Table 5. Concordance rate of the ATP method when the Microdilution (MB) method for clinical isolates of M. avium, M. kansasii and M. intracellulare is set as a basis. M. avium (N 20) M. kansasii (N 5) M. intracellulare (N 20) RLU ratio Discrepancy of MICs 3 2 1 0 1 2 3 Streptomycin 8 11 1 Ethambutol 1 17 2 Kanamycin 5 15 Isoniazid 4 10 6 Rifampicin 1 3 9 7 Levofloxacin 4 10 Clarithromycin 1 1 8 10 Ethionamide 1 9 8 2 Amikacin 2 16 2 Streptomycin 2 9 7 2 Ethambutol 4 2 1 13 Kanamycin 6 9 4 1 Isoniazid 1 1 5 8 5 Rifampicin 6 14 Levofloxacin 11 6 3 Clarithromycin 3 11 6 Ethionamide 7 7 3 3 Amikacin 7 10 2 1 Streptomycin 2 3 Ethambutol 5 Kanamycin 5 Isoniazid 2 3 Rifampicin 1 4 Levofloxacin 3 2 Clarithromycin 1 4 Ethionamide 3 2 Amikacin 1 3 1 Streptomycin 1 3 1 Ethambutol 5 Kanamycin 1 3 1 Isoniazid 4 1 Rifampicin 1 4 Levofloxacin 4 1 Clarithromycin 3 2 Ethionamide 3 2 Amikacin 2 3 Streptomycin 4 5 11 Ethambutol 1 19 Kanamycin 4 7 9 Isoniazid 1 3 11 5 Rifampicin 2 3 15 Levofloxacin 7 12 1 Clarithromycin 1 1 10 8 Ethionamide 3 17 Amikacin 5 15 Streptomycin 5 12 3 Ethambutol 3 6 11 Kanamycin 1 3 13 2 1 Isoniazid 1 1 3 12 2 1 Rifampicin 1 1 15 3 Levofloxacin 4 4 8 3 1 Clarithromycin 7 10 3 Ethionamide 14 5 1 Amikacin 1 15 4 Concordance rate ( ) 93.9 78.3 100 97.8 90.6 87.8 43

172 Table 6. Concordance rate of the ATP method when the Microdilution (MB) method for clinical isolates of M. avium, M. kansasii and M. intracellulare is set as a basis. M. avium (N 20) M. kansasii (N 5) M. intracellulare (N 20) RLU ratio MIC range of MB method ( mg/ml) Discrepancy of MICs 3 2 1 0 1 2 3 Streptomycin 1 32 8 11 1 Ethambutol 4 128 1 17 2 Kanamycin 2 32 5 15 Isoniazid 4 32 4 10 6 Rifampicin 25 0.2 6 14 Levofloxacin 0.5 4 4 16 Clarithromycin 25 2 3 11 6 Ethionamide 4 16 1 9 8 2 Amikacin 1 16 2 16 2 Streptomycin 4 8 2 3 Ethambutol 2 4 5 Kanamycin 8 32 5 Isoniazid 2 4 2 3 Rifampicin 25 0.25 1 4 Levofloxacin 0.25 0.5 3 2 Clarithromycin 25 3 2 Ethionamide 1 2 3 2 Amikacin 2 8 1 3 1 Streptomycin 0.5 4 4 5 11 Ethambutol 2 32 1 19 Kanamycin 0.5 4 4 7 9 Isoniazid 2 32 1 3 11 5 Rifampicin 0.25 1 1 15 3 Levofloxacin 0.5 1 7 12 1 Clarithromycin 0.06 0.25 7 10 3 Ethionamide 4 16 3 17 Amikacin 0.5 4 5 15 Concordance rate ( ) 97.2 100 92.2 NTM McFarland No. 0.5 ATP 5 3 ATP RLU 10,000 RLU ratio 3 MB 60 91.1 ATP 3 RLU ratio 4) 6) ATP RLU ratio 0.5 NTM ATP RLU ratio M. avium ATCC 700898 M. intracellulare ATCC 13950 M. avium ATCC 25291 MB RFP, CAM 2 RLU ratio MB RLU ratio MB M. avium M. intracellulare RLU ratio RLU ratio 2 44

ATP 173 Table 7. Distributions of MICs of nine antibiotics obtained by Microdilution (MB) and ATP method against clinical isolates of M. avium (N 20). Method Minimum inhibitory concentration ( mg/ml) 0.06 25 0.25 0.5 1 2 4 8 16 32 64 128 128 Streptomycin Ethambutol Kanamycin MB method 1 6 7 3 3 ATP method 1 3 6 4 4 2 MB method 3 2 7 5 1 2 ATP method 3 2 7 4 1 1 2 MB method 2 3 6 7 2 ATP method 2 1 5 7 5 0.06 25 0.25 0.5 1 2 4 8 16 32 32 Isoniazid Rifampicin Levofloxacin MB method 1 8 4 6 1 ATP method 1 7 5 5 2 MB method 8 2 1 5 4 ATP method 3 5 2 4 2 4 MB method 4 6 6 4 ATP method 5 6 7 2 Clarithromycin MB method 2 1 8 5 4 ATP method 2 2 3 9 4 0.5 1 2 4 8 16 16 Ethionamide Amikacin MB method 7 6 6 1 ATP method 5 5 5 3 2 MB method 1 1 3 9 6 ATP method 1 1 5 6 6 1 RLU ratio M. kansasii ATCC 12478 RLU ratio RLU ratio RLU ratio 10) M. avium RFP CAM M. avium MIC ATP RLU ratio MB NTM trailing MIC MB MIC trailing 11) NTM MBC (minimum bactericidal concentration) MIC RLU ratio MB 12) MAC 3 MAC 3 trailing Middlebrook 7H10 45

174 Table 8. Distributions of MICs of nine antibiotics obtained by Microdilution (MB) and ATP method against clinical isolates of M. intracellulare (N 20). Method Minimum inhibitory concentration ( mg/ml) 0.06 25 0.25 0.5 1 2 4 8 16 32 64 128 128 Streptomycin Ethambutol Kanamycin MB method 3 8 7 2 ATP method 3 4 8 5 MB method 9 5 5 1 ATP method 1 8 5 5 1 MB method 1 2 10 7 ATP method 2 4 9 5 0.06 25 0.25 0.5 1 2 4 8 16 32 32 Isoniazid Rifampicin Levofloxacin MB method 4 4 5 3 3 1 ATP method 3 7 7 2 1 1 MB method 10 7 3 ATP method 10 2 3 5 MB method 6 14 ATP method 1 19 8 1 Clarithromycin MB method 4 10 6 ATP method 5 12 3 0.5 1 2 4 8 16 16 Ethionamide Amikacin MB method 6 3 7 4 ATP method 2 4 4 6 4 MB method 2 7 8 3 ATP method 5 4 10 1 NCCLS ATCC M. avium ATCC 700898 CAM ph 7.3 7.4 0.5 2 mg/ml MB 0.25 mg/ml, ATP 25 0.25 mg/ml NCCLS (ph 7.3 to 7.4) MIC 25 0.25 mg/ml MB ATP ATCC 3 11) 7 BrothMIC NTM MIC M. kansasii RFP NCCLS 1 mg/ml 25 0.25 mg/ml MIC 12) M. intracellulare M. avium RFP, SM KM M. avium M. intracellulare RFP, SM KM M. intracellulare M. avium 2 MIC M. avium MAC Heifets 13) M. intracellulare RFP CAM M. avium CAM RFP MB 55, ATP 70 MAC SM, EB, AMK MB 22.5 47.5, ATP 22.5 60 46

ATP 175 NTM NTM rifamycin caprazamycin-b, ketolides ATP MB NTM NTM ATP 1) 2003. 2003 78: 569 572. 2) 1997. ATP 45 146. 3) 1997. ATP 3 45 147. 4) 1998. Mycobacterium tuberculosis H37Rv 46: 834 840. 5) 1999. II 47: 170 175. 6) 2000. III filamentous cell treatment ATP 48: 167 173. 7) Palaci M, et al. 1996. Evaluation of Mycobacteria Growth Indicator Tube for recovery and drug susceptibility testing of Mycobacterium tuberculosis isolates from respiratory specimens. J. Clin. Microbiol. 34: 762 764. 8) 1998. Middlebrook 2 3 46: 719 727. 9) National Committee for Clinical Laboratory Standards (NCCLS). 2003. Susceptibility Testing of Mycobacteria, Nocardia, and Other Aerobic Actynomycetes; Approved Standard M24- A, NCCLS, Wayne, USA. 10) 2001. Rifalazil, clarithromycin levofloxacin 7HSF Mycobacterium avium 49: 649 652. 11) 2002. Middlebrook 4 Nontuberculous Mycobacteria BrothMIC NTM. 50: 381 391. 12) 2002. Mycobacterium intracellulare 50: 232 235. 13) Heifets. 1996. Susceptibility Testing of Mycobacterium avium Complex Isolates. Antimicrob. Agents Chemother. 40: 1759 1767. 47

176 Evaluation of Microdilution Susceptibility Test for Nontuberculous Mycobacteria Using the ATP Method Masayuki Horikawa 1),Isao Nishi 1),Masahiro Toyokawa 1),Naoki Sato 2), Yutaka Okazawa 2), Seishi Asari 1) 1) Clinical Laboratory, Osaka University Hospital 2) Kyokuto Pharmaceutical Industrial CO., LTD. We have developed a broth microdilution susceptibility test method for nontuberculous mycobacteria (NTM); in this test method, adenosine triphosphate (ATP) is measured through bioluminescence, with viable count as the indicator. We have conducted the basic analysis of the method and compared the method with the broth microdilution method (MB method) using four ATCC referece strains and 45 clinical isolates. As a result of the examination of the ATP-measuring method (ATP method), it has been shown that 30-minute pre-processing at room temperature and extraction at 60 for 10 minutes were appropriate steps. For the ATP method, judgment was made by measuring the relative light unit (RLU) ratio after culturing the microplate for 5 days, and comparison was made with the MIC value after culturing for 7 days. When the positive values for growth were set to an RLU ratio of for 7 drugs, that is, streptomycin, ethambutol, kanamycin, isoniazid, levofloxacin, ethionamide and amikacin, and to an RLU ratio of for rifampicin and clarithromycin, the rate of concordance with the MB method was rather lower (84.4 ) for Mycobacterium kansasii ATCC 12478, whereas 97.8 100 concordance rates were obtained for 3 ATCC reference strains. Regarding clinical isolates, the concordance rates obtained for Mycobacterium intracellulare, Mycobacterium avium, and M. kansasii were 92.2, 97.2 and 100, respectively. From the above results, we have concluded that the ATP method is useful as a method of testing drug susceptibility of NTM. 48