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

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1 164 ATP 1) 1) 1) 2) 2) 1) 1) 2) adenosine triphosphate (ATP) Nontuberculous Mycobacteria (NTM) ATP MB ATCC 4 45 ATP 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 Mycobacterium intracellulare: 92.2, Mycobacterium avium:97.2, M. kansasii: 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 ( ) 2 15 TEL: FAX: horikawa@hp-lab.med.osaka-u. ac.jp ) MAC MAC AIDS MAC Mycobacterium kansasii 1997 MAC M. kansasii rifampicin (RFP) RFP NTM clarithromycin (CAM) azithromycin 36

2 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 , M. avium ATCC 25291, M. kansasii ATCC Mycobacterium intracellulare ATCC M. avium 20 M. intracellulare 20 M. kansasii 5 DDH (DNA DNA hybridization) B. ATP ATCC Middlebrook 7H9 broth McFarland No nm, 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 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 mg/ml 12 INH, RFP, LVFX, CAM 32 mg/ml 11 TH, AMK mg/ml 6 MIC NTM 1 4 Middlebrook 7H9 broth 530 nm, McFarland No McFarland No ml 11 ml NTM 100 ml MIC 2. ATP ATP ATP MIC ATP MB ATP MIC 5 37

3 , 10 ATP RLU ratio RLU RLU RLU ratio, RLU CAM 8 3 RLU CAM ph CAM RLU II. A. ATP Table 1 McFarland No / 100 RLU /10, 1/ RLU ATP RLU 72, Table 2 M. avium, M. intracellulare 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 INH M. intracellulare ATCC M. avium ATCC ATP RFP, CAM M. kansasii ATCC KM, AMK EB 84.4 RLU ratio M. avium ATCC EB LVFX 88.9 M. avium ATCC RLU ratio RFP, CAM KM AMK Table 1. E#ect of treatment time on the exogenous ATP. Dilution of organisms suspension Treatment time (min) M. avium ATCC blank / / / M. intracellulare ATCC blank / / / M. kansasii ATCC blank / / / BrothMIC NTM blank NT NT NT NT: not tested 38

4 ATP 167 Table 2. E#ect of the temperature and incubation time on the extraction of ATP from nontuberculous mycobacteria. Temperature Extraction time (min) M. avium ATCC room temperature M. intracellulare ATCC room temperature M. kansasii ATCC room temperature M. kansasii ATCC RLU ratio RLU ratio KM 8 1 M. intracellulare ATCC 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 ATP INH 1 8 mg/ml MB 8 1 M. avium ATCC ATP RFP 8 1 M. kansasii ATCC ATP KM, AMK EB MB 84.4 RLU ratio 97.8 M. intracellulare ATCC ATP MB 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 CAM 1 RFP 39

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

6 ATP 169 Table 3. continued M. intracellulare ATCC RLU ratio Discrepancy of MICs 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 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 ATP MB 95.3 M. kansasii ATCC 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 MGIT 7) 41

7 170 Table 4. Concordance rate of the ATP method when the Microdilution (MB) method for reference strains is set as a basis. M. avium ATCC RLU ratio MIC range of MB method ( mg/ml) Discrepancy of MICs Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin Levofloxacin Clarithromycin Ethionamide Amikacin Concordance rate ( ) 97.8 M. avium ATCC M. kansasii ATCC M. intracellulare ATCC Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin Levofloxacin Clarithromycin Ethionamide Amikacin Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin Levofloxacin Clarithromycin Ethionamide Amikacin Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin 5 Levofloxacin Clarithromycin Ethionamide Amikacin MTB-1 8) MTB-SR NTM NTM NCCLS (National Committee for Clinical Laboratory Standards) 9) NTM MB ATP MIC ATP NTM 42

8 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 Streptomycin Ethambutol Kanamycin 5 15 Isoniazid Rifampicin Levofloxacin 4 10 Clarithromycin Ethionamide Amikacin Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin 6 14 Levofloxacin Clarithromycin Ethionamide Amikacin Streptomycin 2 3 Ethambutol 5 Kanamycin 5 Isoniazid 2 3 Rifampicin 1 4 Levofloxacin 3 2 Clarithromycin 1 4 Ethionamide 3 2 Amikacin Streptomycin Ethambutol 5 Kanamycin Isoniazid 4 1 Rifampicin 1 4 Levofloxacin 4 1 Clarithromycin 3 2 Ethionamide 3 2 Amikacin 2 3 Streptomycin Ethambutol 1 19 Kanamycin Isoniazid Rifampicin Levofloxacin Clarithromycin Ethionamide 3 17 Amikacin 5 15 Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin Levofloxacin Clarithromycin Ethionamide Amikacin Concordance rate ( )

9 172 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 Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin Levofloxacin Clarithromycin Ethionamide Amikacin Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin Levofloxacin Clarithromycin Ethionamide Amikacin Streptomycin Ethambutol Kanamycin Isoniazid Rifampicin Levofloxacin Clarithromycin Ethionamide Amikacin Concordance rate ( ) NTM McFarland No. 0.5 ATP 5 3 ATP RLU 10,000 RLU ratio 3 MB ATP 3 RLU ratio 4) 6) ATP RLU ratio 0.5 NTM ATP RLU ratio M. avium ATCC M. intracellulare ATCC M. avium ATCC MB RFP, CAM 2 RLU ratio MB RLU ratio MB M. avium M. intracellulare RLU ratio RLU ratio 2 44

10 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) Streptomycin Ethambutol Kanamycin MB method ATP method MB method ATP method MB method ATP method Isoniazid Rifampicin Levofloxacin MB method ATP method MB method ATP method MB method ATP method Clarithromycin MB method ATP method Ethionamide Amikacin MB method ATP method MB method ATP method RLU ratio M. kansasii ATCC 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

11 174 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) Streptomycin Ethambutol Kanamycin MB method ATP method MB method ATP method MB method ATP method Isoniazid Rifampicin Levofloxacin MB method ATP method MB method ATP method MB method 6 14 ATP method Clarithromycin MB method ATP method Ethionamide Amikacin MB method ATP method MB method ATP method NCCLS ATCC M. avium ATCC CAM ph mg/ml MB 0.25 mg/ml, ATP mg/ml NCCLS (ph 7.3 to 7.4) MIC mg/ml MB ATP ATCC 3 11) 7 BrothMIC NTM MIC M. kansasii RFP NCCLS 1 mg/ml 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 , ATP

12 ATP 175 NTM NTM rifamycin caprazamycin-b, ketolides ATP MB NTM NTM ATP 1) : ) ATP ) ATP ) Mycobacterium tuberculosis H37Rv 46: ) II 47: ) III filamentous cell treatment ATP 48: ) Palaci M, et al Evaluation of Mycobacteria Growth Indicator Tube for recovery and drug susceptibility testing of Mycobacterium tuberculosis isolates from respiratory specimens. J. Clin. Microbiol. 34: ) Middlebrook : ) National Committee for Clinical Laboratory Standards (NCCLS) Susceptibility Testing of Mycobacteria, Nocardia, and Other Aerobic Actynomycetes; Approved Standard M24- A, NCCLS, Wayne, USA. 10) Rifalazil, clarithromycin levofloxacin 7HSF Mycobacterium avium 49: ) Middlebrook 4 Nontuberculous Mycobacteria BrothMIC NTM. 50: ) Mycobacterium intracellulare 50: ) Heifets Susceptibility Testing of Mycobacterium avium Complex Isolates. Antimicrob. Agents Chemother. 40:

13 176 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 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

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