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In vitro activity of ciprofloxacin and ofloxacin against the Mycobacterium avium-intracellulare complex
DIAGNMICROBIOLINFECTDIS 1987;7:89-91
89
In Vitro Activity of Ciprofloxacin and Ofloxacin Against the Mycobacterium avium-intracellulare Complex Steven M. Johnson and Glenn D. Roberts
Detection and identification of mycobacteria in clinical specimens usually require several weeks, delaying selection of antimycobacterial drugs. Rapid methods would help to decrease such delays. The BACTEC detection system for mycobacteria (Johnston Laboratories, Towson, MD) is an automated radiometric system employing 14C-labeled palmitic acid in Middlebrook 7H12 medium. Growth is detected by measurement of 14C-labeled C02 liberated by bacteria after utilization of the labeled substrate. Average detection times using the BACTEC system for acid-fast smear-positive specimens have been reported to be two to three times faster than conventional methods. The BACTEC NAP (p-nitro-a-acetylamino-~-hydroxypropiophenone) differentiation test can distinguish Mycobacterium tuberculosis-Mycobacterium boris complex from other mycobacteria two to five days after initial growth is detected. Gas-liquid chromatography (GLC) for rapid identification of mycobacterial growth from solid medium can be performed in approximately two hours. Mycobacterial lipids are saponified, extracted, and the resulting hydrolysis products are analyzed by GLC. Finally, specific I2~I-DNA probes complementary to the ribosomal RNA of M. tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare are available far detection and identification of growth from solid or broth media within two hours (Gen-Probe, Inc., San Diego, CA). A suspension of the unknown organism is lysed, probe is added and will form a DNA:RNA hybrid if the complementary RNA is present. Unbound probe is separated from bound probe, the latter of which is counted in a gamma counter to calculate percentage of hybridization of the input probe. A procedure for the detection of mycobocteria directly from clinical specimens will be available soon. All of these methods have a place in the clinical microbiology laboratory. Members of the Mycobacterium a v i u m - i n t r a c e l l u l a r e c o m p l e x exhibit m a r k e d in vitro and in vivo resistance to the c o m m o n l y used antimycobacterial drugs (American Thoracic Society and the Centers for Disease Control, 1983; Davidson et al., 1981; Z i m m e r et al., 1982). A definite need exists for more effective agents. Ciprofloxacin, ofloxacin, and other fluoroquinolones are active against a broad spectrum of bacteria (Wolfson a n d Hooper, 1985). Previous in vitro trials have s h o w n variable activity of fluoroquinolones against the Mycobacterium avium-intracellulare c o m p l e x (Collins and Uttley, 1985; F e n l o n and Cynamon, 1985; Gay et al., 1984; Gaya and Chadwick, 1985; Mariuis and Legakis, 1985; Tsukamura, 1985). The present study was c o n d u c t e d to better d o c u m e n t the p r o p o r t i o n of isolates that have MICs less than the achievable blood levels of ciprofloxacin and ofloxacin. Patients whose isolates are in this more susceptible group m a y benefit from t h e r a p y with these agents. From the Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905. Address reprint requests to: Glenn D. Roberts, Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905. Received November 11, 1986; revised and accepted January 9, 1987.
© 1987 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017
0732-8893/87/$3.50
90
S.M. Johnson and G.D. Roberts
TABLE 1. Distribution of MICs of Mycobacterium avium-intracellulare Complex Isolates to Ciprofloxacin and Oflaxacin MIC (~.g/ml)
Antibiotic
Number of isolates
0.25
0.5
1
2
4
8
16
>16
Ciprofloxacin Ofloxacin
(100) (30)
4 0
9 0
13 1
12 3
9 6
12 1
22 2
19 17
One hundred strains of Mycobacterium avium-intracellulare complex taken from the Mayo Clinic stock culture collection were subcultured onto Middlebrook 7H10 agar plates. All subcultures were less than 1 mo old when tested for susceptibility. None of the isolates in our previous study (Gay et al., 1984) were included. Ciprofloxacin (Miles Pharmaceuticals, West Haven, CT) and ofloxacin (Ortho Pharmaceutical Corporation, Raritan, NJ) were provided as powders by the manufacturers. Solutions of these were made in sterile distilled water. Isolates were tested using the 1% proportion method as previously described (Gay et al., 1984). Appropriate amounts of antibiotic were added to Middlebrook 7Hll agar in quadrant plates. The concentrations of antibiotic used were log2 dilution steps from 0.25 to 16 p,g/ml. Suspensions of each isolate were tested in triplicate. Results were recorded after 2 wk of incubation at 35°C. Table I presents the distribution of MICs of ciprofloxacin and ofloxacin. The strains tested exhibit a spectrum of in vitro susceptibility. Although in vitro susceptibility testing is not always an accurate predictor of in vivo response of mycobacteria, particularly the M. a v i u m - i n t r a c e l l u l a r e complex, to antibiotics, it may be useful to compare the MICs obtained with the achievable serum levels of each drug. The peak serum level of ciprofloxacin achieved by suggested therapeutic regimens is approximately 3 ~g/ml (Davis et al., 1985; Hoffken et al., 1985; Hooper and Wolfson, 1985) and 10 ~,g/ml for ofloxacin (Fuchs et al., 1985; Lockley et al., 1984). Using these peak serum levels as guidelines, a substantial proportion of strains showed significant susceptibility. Thirty-eight of 100 strains had MICs of ~<2 wg/ml to ciprofloxacin and 26 of these had MICs of ~<1 ~.g/ml. Eleven of 30 strains had MICs of ~<8 ~,g/ml to ofloxacin. A large proportion of strains appeared resistant. The MICgo(concentration required to inhibit growth of 90% of strains tested) of both ciprofloxacin and ofloxacin was >16 wg/ml. This would indicate a somewhat greater degree of resistance of M. avium-intracellulare complex to ciprofloxacin than that found by Gay et al. in an earlier smaller series. In that series the MICso was 2 ~,g/ml and the MICgo was 16 wg/ml. The present larger series may more accurately reflect the extent of resistance. Investigators using different methods of susceptibility testing methods have found somewhat lower MICs for ciprofloxacin and ofloxacin (Fenlon and Cynamon, 1985). Despite the results obtained in any of these in vitro studies, they can only suggest whether or not patients with M. a v i u m - i n t r a c e l l u l a r e complex infection would benefit from a particular antibiotic. Susceptibility testing, however, may potentially guide selection of antibiotics. REFERENCES
American Thoracic Society and the Centers for Disease Control (1983) Treatment of tuberculosis and other mycobacterial diseases. Am Rev Resp Dis 127:790. Davidson PT, Khanijo V, Goble M, Moulding TS (1981) Treatment of disease due to Mycobacterium intracellulare. Rev. Infect Dis 3:1052.
Notes
91
Zimmer BL, DeYoung DR, Roberts GD (1982) In vitro synergistic activity of ethambutol, isoniazid, kanamycin, rifampin, and streptomycin against Mycobacterium avium-intracellulare complex. Antimicrab Agents Chemother 22:148. Wolfson JS, Hooper DC (1985) The fluoroquinolones: Structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrob Agents Chemother 28:581. Collins CH, Uttley AHC (1985) In-vitro susceptibility of mycobacteria to ciprofloxacin. J Antimicrob Chemother 16:575. Fenlon CH, Cynamon MH (1985) Comparative in vitro activities of ciprofloxacin and other 4quinolones against Mycobacterium tuberculosis and Mycobacterium intracellulare. Antimicrob Agents Chemother 29:386. Gay JD, DeYoung DR, Roberts GD (1984) In vitro activities of norfloxacin and ciprofloxacin against Mycobacterium tuberculosis, M. avium complex, M. chelonei, M. fortuitum, and M. kansasii. Antimicrob Agents Chemother 26:94. Gaya H, Chadwick MV (1985) In vitro activity of ciprofloxacin against mycobacteria. Eur J Clin Microbiol 4:345. Mariuis E, Legakis NJ (1985) In-vitro activity of ciprofloxacin against clinical isolates of mycobacteria resistant to antimycobacterial drugs. J Antimicrob Chemother 16:527. Tsukamura M (1985) In vitro antituberculosis activity of a new antibacterial substance ofloxacin (DL 8280). Am Rev Resp Dis 131:348. Davis RL, Koup JR, Williams-Warren J, Weber A, Smith AL (1985) Pharmacokinetics of three oral formulations of ciprofloxacin. Antimicrob Agents Chemother 28:74. Hoffken G, Lode H, Prinzing C, Borner K, Koeppe P (1985) Pharmacokinetics of ciprofloxacin after oral and parenteral administration. Antimicrob Agents Chemother 27:375. Hooper DC, Wolfson JS (1985) The fluoroquinolones:Pharmacology, clinical uses, and toxicities in humans. Antimicrob Agents Chemother 28:716. Fuchs PC, Barry AL, Jones RN, Thornsberry C (1985) Proposed disc diffusion susceptibility criteria for ofloxacin. J Clin Microbiol 22:310. Lockley MR, Wise R, Dent J (1984) The pharmacokinetics and tissue penetration of ofloxacin. J Antimicrob Chemother 14:647.
89
In Vitro Activity of Ciprofloxacin and Ofloxacin Against the Mycobacterium avium-intracellulare Complex Steven M. Johnson and Glenn D. Roberts
Detection and identification of mycobacteria in clinical specimens usually require several weeks, delaying selection of antimycobacterial drugs. Rapid methods would help to decrease such delays. The BACTEC detection system for mycobacteria (Johnston Laboratories, Towson, MD) is an automated radiometric system employing 14C-labeled palmitic acid in Middlebrook 7H12 medium. Growth is detected by measurement of 14C-labeled C02 liberated by bacteria after utilization of the labeled substrate. Average detection times using the BACTEC system for acid-fast smear-positive specimens have been reported to be two to three times faster than conventional methods. The BACTEC NAP (p-nitro-a-acetylamino-~-hydroxypropiophenone) differentiation test can distinguish Mycobacterium tuberculosis-Mycobacterium boris complex from other mycobacteria two to five days after initial growth is detected. Gas-liquid chromatography (GLC) for rapid identification of mycobacterial growth from solid medium can be performed in approximately two hours. Mycobacterial lipids are saponified, extracted, and the resulting hydrolysis products are analyzed by GLC. Finally, specific I2~I-DNA probes complementary to the ribosomal RNA of M. tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare are available far detection and identification of growth from solid or broth media within two hours (Gen-Probe, Inc., San Diego, CA). A suspension of the unknown organism is lysed, probe is added and will form a DNA:RNA hybrid if the complementary RNA is present. Unbound probe is separated from bound probe, the latter of which is counted in a gamma counter to calculate percentage of hybridization of the input probe. A procedure for the detection of mycobocteria directly from clinical specimens will be available soon. All of these methods have a place in the clinical microbiology laboratory. Members of the Mycobacterium a v i u m - i n t r a c e l l u l a r e c o m p l e x exhibit m a r k e d in vitro and in vivo resistance to the c o m m o n l y used antimycobacterial drugs (American Thoracic Society and the Centers for Disease Control, 1983; Davidson et al., 1981; Z i m m e r et al., 1982). A definite need exists for more effective agents. Ciprofloxacin, ofloxacin, and other fluoroquinolones are active against a broad spectrum of bacteria (Wolfson a n d Hooper, 1985). Previous in vitro trials have s h o w n variable activity of fluoroquinolones against the Mycobacterium avium-intracellulare c o m p l e x (Collins and Uttley, 1985; F e n l o n and Cynamon, 1985; Gay et al., 1984; Gaya and Chadwick, 1985; Mariuis and Legakis, 1985; Tsukamura, 1985). The present study was c o n d u c t e d to better d o c u m e n t the p r o p o r t i o n of isolates that have MICs less than the achievable blood levels of ciprofloxacin and ofloxacin. Patients whose isolates are in this more susceptible group m a y benefit from t h e r a p y with these agents. From the Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905. Address reprint requests to: Glenn D. Roberts, Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905. Received November 11, 1986; revised and accepted January 9, 1987.
© 1987 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017
0732-8893/87/$3.50
90
S.M. Johnson and G.D. Roberts
TABLE 1. Distribution of MICs of Mycobacterium avium-intracellulare Complex Isolates to Ciprofloxacin and Oflaxacin MIC (~.g/ml)
Antibiotic
Number of isolates
0.25
0.5
1
2
4
8
16
>16
Ciprofloxacin Ofloxacin
(100) (30)
4 0
9 0
13 1
12 3
9 6
12 1
22 2
19 17
One hundred strains of Mycobacterium avium-intracellulare complex taken from the Mayo Clinic stock culture collection were subcultured onto Middlebrook 7H10 agar plates. All subcultures were less than 1 mo old when tested for susceptibility. None of the isolates in our previous study (Gay et al., 1984) were included. Ciprofloxacin (Miles Pharmaceuticals, West Haven, CT) and ofloxacin (Ortho Pharmaceutical Corporation, Raritan, NJ) were provided as powders by the manufacturers. Solutions of these were made in sterile distilled water. Isolates were tested using the 1% proportion method as previously described (Gay et al., 1984). Appropriate amounts of antibiotic were added to Middlebrook 7Hll agar in quadrant plates. The concentrations of antibiotic used were log2 dilution steps from 0.25 to 16 p,g/ml. Suspensions of each isolate were tested in triplicate. Results were recorded after 2 wk of incubation at 35°C. Table I presents the distribution of MICs of ciprofloxacin and ofloxacin. The strains tested exhibit a spectrum of in vitro susceptibility. Although in vitro susceptibility testing is not always an accurate predictor of in vivo response of mycobacteria, particularly the M. a v i u m - i n t r a c e l l u l a r e complex, to antibiotics, it may be useful to compare the MICs obtained with the achievable serum levels of each drug. The peak serum level of ciprofloxacin achieved by suggested therapeutic regimens is approximately 3 ~g/ml (Davis et al., 1985; Hoffken et al., 1985; Hooper and Wolfson, 1985) and 10 ~,g/ml for ofloxacin (Fuchs et al., 1985; Lockley et al., 1984). Using these peak serum levels as guidelines, a substantial proportion of strains showed significant susceptibility. Thirty-eight of 100 strains had MICs of ~<2 wg/ml to ciprofloxacin and 26 of these had MICs of ~<1 ~.g/ml. Eleven of 30 strains had MICs of ~<8 ~,g/ml to ofloxacin. A large proportion of strains appeared resistant. The MICgo(concentration required to inhibit growth of 90% of strains tested) of both ciprofloxacin and ofloxacin was >16 wg/ml. This would indicate a somewhat greater degree of resistance of M. avium-intracellulare complex to ciprofloxacin than that found by Gay et al. in an earlier smaller series. In that series the MICso was 2 ~,g/ml and the MICgo was 16 wg/ml. The present larger series may more accurately reflect the extent of resistance. Investigators using different methods of susceptibility testing methods have found somewhat lower MICs for ciprofloxacin and ofloxacin (Fenlon and Cynamon, 1985). Despite the results obtained in any of these in vitro studies, they can only suggest whether or not patients with M. a v i u m - i n t r a c e l l u l a r e complex infection would benefit from a particular antibiotic. Susceptibility testing, however, may potentially guide selection of antibiotics. REFERENCES
American Thoracic Society and the Centers for Disease Control (1983) Treatment of tuberculosis and other mycobacterial diseases. Am Rev Resp Dis 127:790. Davidson PT, Khanijo V, Goble M, Moulding TS (1981) Treatment of disease due to Mycobacterium intracellulare. Rev. Infect Dis 3:1052.
Notes
91
Zimmer BL, DeYoung DR, Roberts GD (1982) In vitro synergistic activity of ethambutol, isoniazid, kanamycin, rifampin, and streptomycin against Mycobacterium avium-intracellulare complex. Antimicrab Agents Chemother 22:148. Wolfson JS, Hooper DC (1985) The fluoroquinolones: Structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrob Agents Chemother 28:581. Collins CH, Uttley AHC (1985) In-vitro susceptibility of mycobacteria to ciprofloxacin. J Antimicrob Chemother 16:575. Fenlon CH, Cynamon MH (1985) Comparative in vitro activities of ciprofloxacin and other 4quinolones against Mycobacterium tuberculosis and Mycobacterium intracellulare. Antimicrob Agents Chemother 29:386. Gay JD, DeYoung DR, Roberts GD (1984) In vitro activities of norfloxacin and ciprofloxacin against Mycobacterium tuberculosis, M. avium complex, M. chelonei, M. fortuitum, and M. kansasii. Antimicrob Agents Chemother 26:94. Gaya H, Chadwick MV (1985) In vitro activity of ciprofloxacin against mycobacteria. Eur J Clin Microbiol 4:345. Mariuis E, Legakis NJ (1985) In-vitro activity of ciprofloxacin against clinical isolates of mycobacteria resistant to antimycobacterial drugs. J Antimicrob Chemother 16:527. Tsukamura M (1985) In vitro antituberculosis activity of a new antibacterial substance ofloxacin (DL 8280). Am Rev Resp Dis 131:348. Davis RL, Koup JR, Williams-Warren J, Weber A, Smith AL (1985) Pharmacokinetics of three oral formulations of ciprofloxacin. Antimicrob Agents Chemother 28:74. Hoffken G, Lode H, Prinzing C, Borner K, Koeppe P (1985) Pharmacokinetics of ciprofloxacin after oral and parenteral administration. Antimicrob Agents Chemother 27:375. Hooper DC, Wolfson JS (1985) The fluoroquinolones:Pharmacology, clinical uses, and toxicities in humans. Antimicrob Agents Chemother 28:716. Fuchs PC, Barry AL, Jones RN, Thornsberry C (1985) Proposed disc diffusion susceptibility criteria for ofloxacin. J Clin Microbiol 22:310. Lockley MR, Wise R, Dent J (1984) The pharmacokinetics and tissue penetration of ofloxacin. J Antimicrob Chemother 14:647.