DIAGN MICROBIOLINFECTDIS 1992;15:367-370
367
In vitro Activity of DNA Gyrase Inhibitors, Singly and in Combination, Against Mycobacterium avium Complex Timothy J. Babinchak and Robert J. Fass
The in vitro activities of the DNA gyrase inhibitors ciprofloxacin, coumermycin, and novobiocin against 31 clinical isolates of Mycobacterium avium complex were studied using a microdilution technique. Minimal inhibitory concentrations (MICs) were determined in 4 days using Middlebrook 7H9 broth, and minimal bactericidal concentrations (MBCs) were determined by subculturing to Middlebrook 7H10 agar. MICs
were: ciprofloxacin, 0.5->16 (mean, 4.1) tzg/ml; novobiocin, 4->128 (mean, 54.7) tzg/ml; and coumermycin, 2->16 (mean, 17.5) izg/ml. MBCs were usually more than two dilution steps higher than MICs. Checkerboard studies failed to reveal synergistic or antagonistic inhibitory activity of DNA gyrase-A and DNA gyrase-B inhibitors in vitro.
Mycobacterium avium complex (MAC) is recognized
Hoffner et al., 1989; Young et al., 1987). These agents inhibit one subunit of bacterial DNA gyrase (gyrase A), whereas novobiocin and coumermycin, two older, structurally related antibiotics, inhibit the other subunit (gyrase B) (Hooper et al., 1982). In this study we used a broth microdilution method to determine the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of the DNA gyrase inhibitors against 31 clinical isolates of MAC. Synergy studies employing the checkerboard technique in microdilution were used to determine if a DNA gyrase-A inhibitor, ciprofloxacin, was synergistic with either of two DNA gyraseB inhibitors, novobiocin or coumermycin.
as the most common cause of disseminated bacterial infection in persons infected with h u m a n immunodeficiency virus (HIV) (Armstrong et al., 1985; Young, 1988). The number of persons infected with this virus continues to increase, and MAC infections are being recognized earlier in the clinical course of HIV infection. Treatment with available antimycobacterial agents, singly or in combination, has been disappointing, and the search for new agents active against MAC continues (Ahn et al., 1986; Hawkins et al., 1986; Wong et al., 1985; Young, 1988). The fluoroquinolones are a new group of antibacterial agents that demonstrate in vitro activity against MAC (Heifets and Lindholm-Levy, 1987; From the Division of InfectiousDiseases, Departmentof Internal Medicine, Ohio State UniversityCollegeof Medicine, Columbus, Ohio, USA. Address reprint requests to Dr. T.J. Babinchak,Allegheny General Hospital, Division of InfectiousDiseases, 320 East North Avenue, Pittsburgh, PA 15212, USA (present address). Received 2 April 1991; revised and accepted 19 August 1991. Results of this report were presented in preliminaryform at the 89th Annual Meeting of the American Societyfor Microbiology, 14-18 May 1989, New Orleans (TJ Babinchak, RJ Fass, Abst Annu Meet Am Soc Microbiol, 1989, C55, p 402). © 1992Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00
We obtained 31 unique clinical isolates of MAC from patients treated at the Ohio State University Hospitals. The isolates were defined as MAC by the clinical microbiology laboratory and further serotyped by Anna Tsang (National Jewish Center of Immunology and Respiratory Medicine, Denver, CO). Stock cultures were maintained on Lowenstein-Jensen slants (Difco Laboratories, Detroit, MI). Antimicrobial agents included ciprofloxacin (Miles Pharmaceuticals, West Haven, CT) and novobiocin (Upjohn, Kalamazoo, MI), prepared as stock solutions (1280 ~g/ml) using double-distilled sterile water as the diluent and stored at - 70°C according to man-
368
ufacturer instructions. Coumerycin (Hoffman-LaRoche, Nutley, NJ) was initially dissolved in dimethyl sulfoxide (DMSO) and diluted to final concentrations with double-distilled sterile water according to manufacturer instruction and stored at 4°C. For susceptibility testing, 96-well microdilution plates were prepared using a Dynatech MIC-2000 dispenser (Dynatech Laboratories, Alexandria, VA). Log2 dilutions of the antimicrobial agents were made in Middlebrook 7H9 broth with OADC enrichment (Difco). Concentrations tested were ciprofloxacin, 0.06-16.0 p,g/ml; novobiocin, 1.0-128 ~g/ml; and coumermycin, 0.25-32 ~g/ml. A standard inoculum was prepared for each mycobacterial isolate by growth in Middlebrook 7H9 broth with OADC enrichment at 35°C in 4% CO2 to a suspension equivalent to a 1.0 McFarland standard. An additional 103 dilution was made to yield a final inoculum of 1.5 x 105 colony-forming units (CFU)/ml as confirmed by colony counts. Each antimicrobial agent was tested in duplicate with positive and negative growth controls. Plates were sealed to minimize evaporation and incubated at 35°C in 4% CO2 for 14 days. Duplicate plates were incubated simultaneously at 35°C in room air. The minimum inhibitory concentration was read as the lowest concentration resulting in no visual turbidity after 4 days of incubation. Escherichia coli ATCC 25922 served as a control organism. MBCs were determined by subculturing 10 p,1 from all microdilution wells without visual turbidity and the positive and negative growth controls to Middlebrook 7H10 agar with OADC enrichment. Plates were incubated at 35°C in 4% CO2 for 3 weeks. The MBC was read as the lowest antimicrobial concentration resulting in a 99.9% reduction in colony count from the original inoculum. Synergy studies were performed in microdilution plates by the checkerboard method as previously described (Khardori et al., 1989). Ciprofloxacin (0.0332 p,g/ml) was tested in combination with both novobiocin (0.125-128~g/ml) and coumermycin (0.0116 p~g/ml). Synergy was determined using the fractional inhibitory concentration (FIC) defined as the minimum concentration of each antimicrobial agent that had an inhibitory effect when acting together, divided by the MIC of that drug alone. The sum of the FICs of both antibiotics was the FIC index. Synergism is defined as an FIC index ~0.5 and antagonism as an index >4.0. The MICs and MBCs for the 31 clinical isolates are shown in Table 1. MICs for all three drugs followed a normal unimodal distribution. For ciprofloxacin, 19 (61%) of 31 isolates were susceptible to concentrations achievable in vivo (MIC ~4 p~g/ml). Novobiocin and coumermycin were less active. There was
T.J. Babinchak and R.J. Fass
no significant difference in susceptibilities based on serotype. MICs read after 4 days of incubation did not increase with further incubation, up to 14 days. Results were the same after incubation in room air and CO2. Microdilution results were readily reproducible; of 26 MIC comparisons utilizing a single strain, 73.1% were identical and 26.9% differed by only a single dilution. MBCs of all three drugs were always greater than two dilution steps higher than MICs except for ciprofloxacin with three of 17 evaluable isolates. The checkerboard microdilution method failed to demonstrate synergy or antagonism between ciprofloxacin and either coumermycin or novobiocin. The FIC index was always >1 and ( 4 . There is no standard procedure for in vitro susceptibility testing of MAC organisms. Mycobacteria are usually tested by the 1% proportion method (Inderlied et al., 1987). This method compares mycobacterial growth between 7H10 agar with drug to a drugfree agar control. Testing of multiple drugs and dilutions with this method can be both cumbersome and time consuming. The BACTEC radiometric system (Johnston Laboratories, Towson, MD) has been shown to be effective in the isolation and susceptibility testing of mycobacterial specimens (Gill et al., 1985; Heifets et al., 1987; Hoffner et al., 1989; Kiehn and Cammarata, 1986; Tarrand and Groshel, 1985; Trimble et al., 1987). However, it requires the BACTEC machine, which is not used routinely for antimicrobial susceptibility testing. Results using broth microdilution with mycobacteria have been shown to be rapid, reliable, and reproducible (Khardori et al., 1989; Perronne et al., 1987; Trimble et al., 1987; Wallace et al., 1986; Yajko et al., 1987). In this study, we adapted the broth microdilution method to testing susceptibilities of MAC to gyrase inhibitors. It confirmed that ciprofloxacin has only moderate activity against members of the Mycobacterium avium complex. The mean MIC was 4 p,g/ml with 61% of the clinical isolates having MICs ~4 p,g/ml. The range of 0.5-16 ~g/ml is consistent with that reported using the standard 1% proportion method (Gay et al., 1984). Inderlied has reported strains of M. avium complex to be more resistant by agar dilution methods than by broth dilution (Inderlied et al., 1987). Other reports of ciprofloxacin activity using different methods of susceptibility testing, media, and inoculum size have produced similar results (Collins et al., 1985; Davies et al., 1987; Fenlon et al., 1986; Inderlied et al., 1987; Trimble et al., 1987). In our study, subculture of microdilution wells without visual turbidity resulted in growth of MAC organisms greater than two dilutions higher than the MICs in 82% of the cases. This is consistent with
Note
369
TABLE 1 Susceptibilities (~g/ml) of Mycobacterium avium Complex to DNA Gyrase Inhibitors Isolates
Ciprofloxacin
Novobiocin
Coumermycin
Serotype
n
MIC
MBC
MIC
MBC
MIC
MBC
1 4 8 Otheff Untypeable All
4 7 5 4 7 31
2-16 2-16 0.5-16 4-16 1-16 0.5-16
2->16 4->16 2->16 4->16 2->16 2->16
32-64 64-128 32-128 32->128 16-128 4->128
>128 >128 >128 >128 >128 >128
16->16 16->16 16->16 4->16 4->16 3->16
>32 >32 >32 >32 >32 >32
54.7
>128
17.5
>32
Geometric mean Escherichia coli ATCC 25922
4.1
~0.03
12.0
64
32
aTypes 2, 6, 16, and 18. MIC, minimalinhibitoryconcentration;and MBC, minimalbactericidalconcentration.
previous reports of MBCs of ciprofloxacin against MAC organisms (Heifets and Lindholm-Levy, 1987; Khardori et al., 1989). Ciprofloxacin has been shown to increase mycobacterial killing in vitro when it has been added to multiple drug regimens (Yajko et al., 1988). We postulated that the combination of a DNA gyrase-A inhibitor, ciprofloxacin, and a DNA gyrase-B inhibitor, novobiocin or coumermycin, would produce a synergistic effect on MAC organisms by inhibiting the DNA gyrase enzyme complex much as trimethaprim-sulfamethoxozole inhibits bacteria by acting at sequential steps in the folate metabolic pathway. However, synergy studies using the checkerboard technique failed to produce synergistic activity with these agents. Using similar techniques, synergy with
fluoroquinolones and coumermycin against Staphylococcus aureus was observed, although it was inconsistent (Neu et al., 1984). In time-kill experiments, ciprofloxacin and coumermycin were indifferent and the combination was less effective than ciprofloxacin alone in reducing bacterial counts in vegetations in rats with experimentally induced staphylococcal endocarditis (Perrone et al., 1987). It may be that gyrase-A and gyrase-B inhibitors each bind to both sites, exert their antibacterial effects at one, but block the effects of the alternate drug at the other. This study was supported in part by grant AI 25924 (AIDS Clinical Trials Group) from the National Institute of Allergy and Infectious Diseases.
REFERENCES Ahn CH, Ahn SS, Anderson RA, Murphy DT, Mammo A (1986) A four-drug regimen for initial treatment of cavitary disease caused by Mycobacterium avium complex. Am Rev Respir Dis 134:438-441. Armstrong D, Gold JWM, Dryjanski J, Whimbey E, Polsky B, Hawkins C, Brown AE, Bernard E, Kiehn TE (1985) Treatment of infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med 103:738743. Collins CH, Uttley AHC (1985) In vitro susceptibility of mycobacteria to ciprofloxacin. J Antimicrob Chemother 16:575-580. Davies S, Sparham PD, Spencer RC (1987) Comparative in vitro activity of five fluoroquinolones against mycobacteria. J Antimicrob Chemother 19:605-609. Fenlon CH, Cynamon MH (1986) Comparative in vitro activities of ciprofloxacin and other 4-quinolones against
Mycobacterium tuberculosis and Mycobactrium intracellulare. Antimicrob Agents Chemother 29:386-388.
Gay JD, DeYoung DR, Roberts GD (1984) In vitro activities of norfloxacin and ciprofloxacin against Mycobacterium tuberculosis, M. avium complex, M. chelonei, M. fortuiturn, and M. Kansasii. Antimicrob Agents Chemother 26:9496. Gill VJ, Park CH, Stock F, Gosey LL, Witebsky FG, Masur H (1985) Use of lysis-centrifugation (Isolator) and radiometric (BACTEC) blood culture systems for the detection of mycobacteria. ] Clin Microbiol 22:543546. Hawkins CC, Gold JWM, Whimbey E, Kiehn TE, Brannon P, Cammarata R, Brown AE, Armstrong D (1986) Mycobacterium avium complex infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med 105:184-188.
370
Heifets LB, Lindholm-Levy PJ (1987) Bacteriostatic and bactericidal activity of ciprofloxacin and ofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Tubercle 68:267-276. Heifets LB, Iseman MD, Lindholm-Levy PJ (1987) Determination of MICs of conventional and experimental drugs in liquid medium by the radiometric method against Mycobacterium avium complex. Drug Exp Clin Res 13:529-538. Hoffner SE, Kratz M, Olsson-Liljequist B, Svenson SB, Kallenius G (1989) In-vitro synergistic activity between ethambutol and fuorinated quinolones against Mycobacterium avium complex. J Antimicrob Chemother 24:317324. Hooper DC, Wolfson JS, McHugh GL, Winters MB, Swartz MN (1982) Effects of novobiocin, coumermycin A1, clorobiocin, and their analogs on Escherichia coli DNA gyrase and bacterial growth. Antimicrob Agents Chemother 22:662-671. Inderlied CB, Young LS, Yamada JK (1987) Determination of in vitro susceptibility of Mycobacterium avium complex isolates to antimycobacterial agents by various methods. Antimicrob Agents Chemother 31:1697-1702. Khardori N, Rolston K, Rosenbaum B, Hayat S, Bodey GP (1989) Comparative in-vitro activity of twenty antimicrobial agents against clinical isolates of Mycobacterium avium complex. J Antimicrob Chemother 24:667-673. Kiehn TE, Cammarata R (1986) Laboratory diagnosis of mycobacterial infections in patients with acquired immunodeficiency syndrome. J Clin Microbiol 24:708-711. Neu JC, Chin N, Labthavikul P (1984) Antibacterial activity of coumermycin alone and in combination with other antibiotics. Antimicrob Agents Chemother 35:687-689. Perrone CM, Malinverni R, Glauser MP (1987) Treatment of Staphylococcus aureus endocarditis in rats with cou-
T.J. Babinchak and R.J. Fass
mermycin A1 and ciprofloxacin, alone or in combination. Antimicrob Agents Chemother 31:539-543. Tarrand JJ, Groshel DHM (1985) Evaluation of the BACTEC radiometric method for detection of 1% resistant population of Mycobacterium tuberculosis. J Clin Microbiol 21:941-946. Trimble KA, Clark RB, Sanders WE Jr, Frankel JW, Cacciatore R, Valdez H (1987) Activity of dprofloxacin against mycobacteria in vitro: comparison of BACTECand macrobroth dilution methods. JAntimicrob Chemother 19:617622. Wallace RJ Jr, Nash DR, Steele LC, Steingrube V (1986) Susceptibility testing of slowly growing mycobacteria by a microdilution MIC method with 7H9 broth. J Clin Microbiol 24:976-981. Wong B, Edwards FF, Kiehn TE, Whimbey E, Donnelly H, Bernard EM, Gold JWM, Armstrong D (1985) Continuous high-grade Mycobacterium avium intracellulae bacteremia in patients with the acquired immune deficiency syndrome. Am ] Med 78:35-40. Yajko DM, Nassos PS, Hadley WK (1987) Broth microdilution testing of susceptibilities to 30 antimicrobial agents of Mycobacterium avium strains from patients with acquired immune deficiency syndrome. Antimicrob Agents Chemother 31:1579-1584. Yajko DM, Kirihara J, Sanders C, Nassos P, Hadley WK (1988) Antimicrobial synergism against Mycobacterium avium complex strains isolated from patients with acquired immune deficiency syndrome. Antimicrob Agents Chemother 32:1392-1395. Young LS (1988) Mycobacterium avium complex infection. J Infect Dis 157:863-867. Young LS, Berlin OGW, Inderlied CB (1987) Activity of ciprofloxacin and other fluorinated quinolones against mycobacteria. Am J Med (Suppl 4A):23-26.
367
In vitro Activity of DNA Gyrase Inhibitors, Singly and in Combination, Against Mycobacterium avium Complex Timothy J. Babinchak and Robert J. Fass
The in vitro activities of the DNA gyrase inhibitors ciprofloxacin, coumermycin, and novobiocin against 31 clinical isolates of Mycobacterium avium complex were studied using a microdilution technique. Minimal inhibitory concentrations (MICs) were determined in 4 days using Middlebrook 7H9 broth, and minimal bactericidal concentrations (MBCs) were determined by subculturing to Middlebrook 7H10 agar. MICs
were: ciprofloxacin, 0.5->16 (mean, 4.1) tzg/ml; novobiocin, 4->128 (mean, 54.7) tzg/ml; and coumermycin, 2->16 (mean, 17.5) izg/ml. MBCs were usually more than two dilution steps higher than MICs. Checkerboard studies failed to reveal synergistic or antagonistic inhibitory activity of DNA gyrase-A and DNA gyrase-B inhibitors in vitro.
Mycobacterium avium complex (MAC) is recognized
Hoffner et al., 1989; Young et al., 1987). These agents inhibit one subunit of bacterial DNA gyrase (gyrase A), whereas novobiocin and coumermycin, two older, structurally related antibiotics, inhibit the other subunit (gyrase B) (Hooper et al., 1982). In this study we used a broth microdilution method to determine the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of the DNA gyrase inhibitors against 31 clinical isolates of MAC. Synergy studies employing the checkerboard technique in microdilution were used to determine if a DNA gyrase-A inhibitor, ciprofloxacin, was synergistic with either of two DNA gyraseB inhibitors, novobiocin or coumermycin.
as the most common cause of disseminated bacterial infection in persons infected with h u m a n immunodeficiency virus (HIV) (Armstrong et al., 1985; Young, 1988). The number of persons infected with this virus continues to increase, and MAC infections are being recognized earlier in the clinical course of HIV infection. Treatment with available antimycobacterial agents, singly or in combination, has been disappointing, and the search for new agents active against MAC continues (Ahn et al., 1986; Hawkins et al., 1986; Wong et al., 1985; Young, 1988). The fluoroquinolones are a new group of antibacterial agents that demonstrate in vitro activity against MAC (Heifets and Lindholm-Levy, 1987; From the Division of InfectiousDiseases, Departmentof Internal Medicine, Ohio State UniversityCollegeof Medicine, Columbus, Ohio, USA. Address reprint requests to Dr. T.J. Babinchak,Allegheny General Hospital, Division of InfectiousDiseases, 320 East North Avenue, Pittsburgh, PA 15212, USA (present address). Received 2 April 1991; revised and accepted 19 August 1991. Results of this report were presented in preliminaryform at the 89th Annual Meeting of the American Societyfor Microbiology, 14-18 May 1989, New Orleans (TJ Babinchak, RJ Fass, Abst Annu Meet Am Soc Microbiol, 1989, C55, p 402). © 1992Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00
We obtained 31 unique clinical isolates of MAC from patients treated at the Ohio State University Hospitals. The isolates were defined as MAC by the clinical microbiology laboratory and further serotyped by Anna Tsang (National Jewish Center of Immunology and Respiratory Medicine, Denver, CO). Stock cultures were maintained on Lowenstein-Jensen slants (Difco Laboratories, Detroit, MI). Antimicrobial agents included ciprofloxacin (Miles Pharmaceuticals, West Haven, CT) and novobiocin (Upjohn, Kalamazoo, MI), prepared as stock solutions (1280 ~g/ml) using double-distilled sterile water as the diluent and stored at - 70°C according to man-
368
ufacturer instructions. Coumerycin (Hoffman-LaRoche, Nutley, NJ) was initially dissolved in dimethyl sulfoxide (DMSO) and diluted to final concentrations with double-distilled sterile water according to manufacturer instruction and stored at 4°C. For susceptibility testing, 96-well microdilution plates were prepared using a Dynatech MIC-2000 dispenser (Dynatech Laboratories, Alexandria, VA). Log2 dilutions of the antimicrobial agents were made in Middlebrook 7H9 broth with OADC enrichment (Difco). Concentrations tested were ciprofloxacin, 0.06-16.0 p,g/ml; novobiocin, 1.0-128 ~g/ml; and coumermycin, 0.25-32 ~g/ml. A standard inoculum was prepared for each mycobacterial isolate by growth in Middlebrook 7H9 broth with OADC enrichment at 35°C in 4% CO2 to a suspension equivalent to a 1.0 McFarland standard. An additional 103 dilution was made to yield a final inoculum of 1.5 x 105 colony-forming units (CFU)/ml as confirmed by colony counts. Each antimicrobial agent was tested in duplicate with positive and negative growth controls. Plates were sealed to minimize evaporation and incubated at 35°C in 4% CO2 for 14 days. Duplicate plates were incubated simultaneously at 35°C in room air. The minimum inhibitory concentration was read as the lowest concentration resulting in no visual turbidity after 4 days of incubation. Escherichia coli ATCC 25922 served as a control organism. MBCs were determined by subculturing 10 p,1 from all microdilution wells without visual turbidity and the positive and negative growth controls to Middlebrook 7H10 agar with OADC enrichment. Plates were incubated at 35°C in 4% CO2 for 3 weeks. The MBC was read as the lowest antimicrobial concentration resulting in a 99.9% reduction in colony count from the original inoculum. Synergy studies were performed in microdilution plates by the checkerboard method as previously described (Khardori et al., 1989). Ciprofloxacin (0.0332 p,g/ml) was tested in combination with both novobiocin (0.125-128~g/ml) and coumermycin (0.0116 p~g/ml). Synergy was determined using the fractional inhibitory concentration (FIC) defined as the minimum concentration of each antimicrobial agent that had an inhibitory effect when acting together, divided by the MIC of that drug alone. The sum of the FICs of both antibiotics was the FIC index. Synergism is defined as an FIC index ~0.5 and antagonism as an index >4.0. The MICs and MBCs for the 31 clinical isolates are shown in Table 1. MICs for all three drugs followed a normal unimodal distribution. For ciprofloxacin, 19 (61%) of 31 isolates were susceptible to concentrations achievable in vivo (MIC ~4 p~g/ml). Novobiocin and coumermycin were less active. There was
T.J. Babinchak and R.J. Fass
no significant difference in susceptibilities based on serotype. MICs read after 4 days of incubation did not increase with further incubation, up to 14 days. Results were the same after incubation in room air and CO2. Microdilution results were readily reproducible; of 26 MIC comparisons utilizing a single strain, 73.1% were identical and 26.9% differed by only a single dilution. MBCs of all three drugs were always greater than two dilution steps higher than MICs except for ciprofloxacin with three of 17 evaluable isolates. The checkerboard microdilution method failed to demonstrate synergy or antagonism between ciprofloxacin and either coumermycin or novobiocin. The FIC index was always >1 and ( 4 . There is no standard procedure for in vitro susceptibility testing of MAC organisms. Mycobacteria are usually tested by the 1% proportion method (Inderlied et al., 1987). This method compares mycobacterial growth between 7H10 agar with drug to a drugfree agar control. Testing of multiple drugs and dilutions with this method can be both cumbersome and time consuming. The BACTEC radiometric system (Johnston Laboratories, Towson, MD) has been shown to be effective in the isolation and susceptibility testing of mycobacterial specimens (Gill et al., 1985; Heifets et al., 1987; Hoffner et al., 1989; Kiehn and Cammarata, 1986; Tarrand and Groshel, 1985; Trimble et al., 1987). However, it requires the BACTEC machine, which is not used routinely for antimicrobial susceptibility testing. Results using broth microdilution with mycobacteria have been shown to be rapid, reliable, and reproducible (Khardori et al., 1989; Perronne et al., 1987; Trimble et al., 1987; Wallace et al., 1986; Yajko et al., 1987). In this study, we adapted the broth microdilution method to testing susceptibilities of MAC to gyrase inhibitors. It confirmed that ciprofloxacin has only moderate activity against members of the Mycobacterium avium complex. The mean MIC was 4 p,g/ml with 61% of the clinical isolates having MICs ~4 p,g/ml. The range of 0.5-16 ~g/ml is consistent with that reported using the standard 1% proportion method (Gay et al., 1984). Inderlied has reported strains of M. avium complex to be more resistant by agar dilution methods than by broth dilution (Inderlied et al., 1987). Other reports of ciprofloxacin activity using different methods of susceptibility testing, media, and inoculum size have produced similar results (Collins et al., 1985; Davies et al., 1987; Fenlon et al., 1986; Inderlied et al., 1987; Trimble et al., 1987). In our study, subculture of microdilution wells without visual turbidity resulted in growth of MAC organisms greater than two dilutions higher than the MICs in 82% of the cases. This is consistent with
Note
369
TABLE 1 Susceptibilities (~g/ml) of Mycobacterium avium Complex to DNA Gyrase Inhibitors Isolates
Ciprofloxacin
Novobiocin
Coumermycin
Serotype
n
MIC
MBC
MIC
MBC
MIC
MBC
1 4 8 Otheff Untypeable All
4 7 5 4 7 31
2-16 2-16 0.5-16 4-16 1-16 0.5-16
2->16 4->16 2->16 4->16 2->16 2->16
32-64 64-128 32-128 32->128 16-128 4->128
>128 >128 >128 >128 >128 >128
16->16 16->16 16->16 4->16 4->16 3->16
>32 >32 >32 >32 >32 >32
54.7
>128
17.5
>32
Geometric mean Escherichia coli ATCC 25922
4.1
~0.03
12.0
64
32
aTypes 2, 6, 16, and 18. MIC, minimalinhibitoryconcentration;and MBC, minimalbactericidalconcentration.
previous reports of MBCs of ciprofloxacin against MAC organisms (Heifets and Lindholm-Levy, 1987; Khardori et al., 1989). Ciprofloxacin has been shown to increase mycobacterial killing in vitro when it has been added to multiple drug regimens (Yajko et al., 1988). We postulated that the combination of a DNA gyrase-A inhibitor, ciprofloxacin, and a DNA gyrase-B inhibitor, novobiocin or coumermycin, would produce a synergistic effect on MAC organisms by inhibiting the DNA gyrase enzyme complex much as trimethaprim-sulfamethoxozole inhibits bacteria by acting at sequential steps in the folate metabolic pathway. However, synergy studies using the checkerboard technique failed to produce synergistic activity with these agents. Using similar techniques, synergy with
fluoroquinolones and coumermycin against Staphylococcus aureus was observed, although it was inconsistent (Neu et al., 1984). In time-kill experiments, ciprofloxacin and coumermycin were indifferent and the combination was less effective than ciprofloxacin alone in reducing bacterial counts in vegetations in rats with experimentally induced staphylococcal endocarditis (Perrone et al., 1987). It may be that gyrase-A and gyrase-B inhibitors each bind to both sites, exert their antibacterial effects at one, but block the effects of the alternate drug at the other. This study was supported in part by grant AI 25924 (AIDS Clinical Trials Group) from the National Institute of Allergy and Infectious Diseases.
REFERENCES Ahn CH, Ahn SS, Anderson RA, Murphy DT, Mammo A (1986) A four-drug regimen for initial treatment of cavitary disease caused by Mycobacterium avium complex. Am Rev Respir Dis 134:438-441. Armstrong D, Gold JWM, Dryjanski J, Whimbey E, Polsky B, Hawkins C, Brown AE, Bernard E, Kiehn TE (1985) Treatment of infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med 103:738743. Collins CH, Uttley AHC (1985) In vitro susceptibility of mycobacteria to ciprofloxacin. J Antimicrob Chemother 16:575-580. Davies S, Sparham PD, Spencer RC (1987) Comparative in vitro activity of five fluoroquinolones against mycobacteria. J Antimicrob Chemother 19:605-609. Fenlon CH, Cynamon MH (1986) Comparative in vitro activities of ciprofloxacin and other 4-quinolones against
Mycobacterium tuberculosis and Mycobactrium intracellulare. Antimicrob Agents Chemother 29:386-388.
Gay JD, DeYoung DR, Roberts GD (1984) In vitro activities of norfloxacin and ciprofloxacin against Mycobacterium tuberculosis, M. avium complex, M. chelonei, M. fortuiturn, and M. Kansasii. Antimicrob Agents Chemother 26:9496. Gill VJ, Park CH, Stock F, Gosey LL, Witebsky FG, Masur H (1985) Use of lysis-centrifugation (Isolator) and radiometric (BACTEC) blood culture systems for the detection of mycobacteria. ] Clin Microbiol 22:543546. Hawkins CC, Gold JWM, Whimbey E, Kiehn TE, Brannon P, Cammarata R, Brown AE, Armstrong D (1986) Mycobacterium avium complex infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med 105:184-188.
370
Heifets LB, Lindholm-Levy PJ (1987) Bacteriostatic and bactericidal activity of ciprofloxacin and ofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Tubercle 68:267-276. Heifets LB, Iseman MD, Lindholm-Levy PJ (1987) Determination of MICs of conventional and experimental drugs in liquid medium by the radiometric method against Mycobacterium avium complex. Drug Exp Clin Res 13:529-538. Hoffner SE, Kratz M, Olsson-Liljequist B, Svenson SB, Kallenius G (1989) In-vitro synergistic activity between ethambutol and fuorinated quinolones against Mycobacterium avium complex. J Antimicrob Chemother 24:317324. Hooper DC, Wolfson JS, McHugh GL, Winters MB, Swartz MN (1982) Effects of novobiocin, coumermycin A1, clorobiocin, and their analogs on Escherichia coli DNA gyrase and bacterial growth. Antimicrob Agents Chemother 22:662-671. Inderlied CB, Young LS, Yamada JK (1987) Determination of in vitro susceptibility of Mycobacterium avium complex isolates to antimycobacterial agents by various methods. Antimicrob Agents Chemother 31:1697-1702. Khardori N, Rolston K, Rosenbaum B, Hayat S, Bodey GP (1989) Comparative in-vitro activity of twenty antimicrobial agents against clinical isolates of Mycobacterium avium complex. J Antimicrob Chemother 24:667-673. Kiehn TE, Cammarata R (1986) Laboratory diagnosis of mycobacterial infections in patients with acquired immunodeficiency syndrome. J Clin Microbiol 24:708-711. Neu JC, Chin N, Labthavikul P (1984) Antibacterial activity of coumermycin alone and in combination with other antibiotics. Antimicrob Agents Chemother 35:687-689. Perrone CM, Malinverni R, Glauser MP (1987) Treatment of Staphylococcus aureus endocarditis in rats with cou-
T.J. Babinchak and R.J. Fass
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