Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA
Received 9 May 2003; returned 5 August 2003; revised 10 September 2003; accepted 25 September 2003
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Abstract |
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Methods: A clinical isolate of Mycobacterium tuberculosis and a laboratory strain of Mycobacterium smegmatis were grown in liquid medium and treated with a fluoroquinolone in the presence or absence of anti-tuberculosis agents. Bacterial survival was determined by viable colony counts on agar medium.
Results: When moxifloxacin activity was examined in two-drug combinations containing traditional anti-tuberculosis agents, activity was greater than either compound alone with isoniazid, capreomycin and low, but not high, concentrations of rifampicin. Cycloserine contributed no additional activity, and ethambutol interfered with the lethal action of moxifloxacin and gatifloxacin. Experiments with M. smegmatis confirmed that both rifampicin and ethambutol reduce fluoroquinolone lethality. Moreover, ethambutol increased the recovery of fluoroquinolone-resistant mutants newly created by ethyl methanesulphonate treatment.
Conclusions: The intrinsic bactericidal activity of C-8-methoxy fluoroquinolones can be adversely affected by some agents currently used for treatment of tuberculosis.
Keywords: moxifloxacin, gatifloxacin, rifampicin, ethambutol
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Introduction |
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Materials and methods |
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Mycobacterium tuberculosis strain TN6515, which we have described previously,4 is a pan-susceptible member of the W family, a group of strains that contains many multidrug-resistant members. Mycobacterium smegmatis mc2155 was provided by Dr Stewart Cole of the Institut Pasteur. Both mycobacteria were grown in Middlebrook 7H9 medium and on Middlebrook 7H10 agar plates (supplemented with 10% albumindextrose complex and 0.05% Tween 80). Gatifloxacin and AM1121 (Bristol-Myers Squibb, Wallingford, CT, USA), moxifloxacin (Bayer AG, West Haven, CT, USA) and levofloxacin (RW Johnson Pharmaceutical Research Institute, Spring House, PA, USA) were dissolved in 0.1 N NaOH to yield a final concentration of 10 g/L. Other compounds were obtained from Sigma Biochemicals (St Louis, MO, USA). Except for rifampicin, which was dissolved in 95% ethanol, these compounds were dissolved and diluted in distilled water.
Measurement of bacterial susceptibility
MIC(99), the drug concentration required to inhibit colony formation by 99%, was determined by diluting stationary phase cells and then spotting 10 µL aliquots on agar plates containing linear increments of fluoroquinolone, or no drug. Colonies were counted after incubation at 37°C for 34 days (M. smegmatis) or 45 weeks (M. tuberculosis). The number of colonies recovered was plotted against drug concentration to determine the MIC(99) by interpolation. To measure a wide range of bactericidal activity, cells were grown to 108 cfu/mL by shaking (M. smegmatis) or by rolling-culture incubation (M. tuberculosis). Cultures were distributed into tubes containing liquid medium and various concentrations of drugs. Incubation was continued for 18 h (M. smegmatis) or 6 days (M. tuberculosis). Serial dilutions, which eliminated drug carryover, were prepared, and aliquots from the dilutions were then spotted on drug-free agar plates for colony number determination.
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Results |
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Preliminary experiments with M. tuberculosis showed that at low concentrations, moxifloxacin killed M. tuberculosis more extensively than did gatifloxacin; both compounds were more lethal than levofloxacin (the dose that reduced survival by 90%, LD90, for moxifloxacin, gatifloxacin and levofloxacin was 0.29, 0.45 and 0.65 mg/L, respectively). Lethal activity of moxifloxacin or gatifloxacin was then measured in the presence of isoniazid or rifampicin, two first-line anti-tuberculosis agents. In previous work, we proposed that a fluoroquinolone concentration exists above which resistant mutants are rarely selected.2,4 This threshold, called the mutant prevention concentration (MPC), may serve as a minimum value for dosing. Thus we set fluoroquinolone concentrations at MPC and varied the concentration of other agents, none of which attain a serum concentration that exceeds MPC at recommended doses.4
The combination of isoniazid and moxifloxacin exhibited more bactericidal effect than either compound alone (Figure 1a). The bactericidal effect of isoniazid changed little between concentrations of 0.2 mg/L and 2 mg/L, concentrations that are commonly reached in humans (Cmax = 7.6 mg/L). The rifampicinmoxifloxacin combination was more lethal than rifampicin alone, but only if the rifampicin concentration was low (Figure 1b). To examine in more detail the loss of additivity between moxifloxacin and rifampicin, we treated M. smegmatis with rifampicin at eight times its MIC(99) (64 mg/L) for 1 h and then added moxifloxacin at various concentrations for an additional 18 h. Surviving cells were determined by plating on drug-free agar. As shown in Figure 2, the presence of rifampicin interfered with the lethal action of moxifloxacin if it was at a high concentration. The shoulder seen in the survivalconcentration curve (Figure 2) is characteristic of some but not all fluoroquinolones; the increase in survival observed at very high quinolone concentration is characteristic of most quinolones. Neither feature is currently explained.
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The effect of cycloserine and capreomycin on moxifloxacin lethality was measured by setting the moxifloxacin concentration at the MPC and then varying the concentration of either of the other two compounds. Capreomycin plus moxifloxacin exhibited greater activity than either alone (Figure 1c). Cycloserine had little effect on moxifloxacin activity (Figure 1d).
Effect of ethambutol on fluoroquinolone activity
Ethambutol alone exhibited little lethal activity on M. tuberculosis (Figure 1e), and it reduced the lethal activity of moxifloxacin by about 80%. Ethambutol also interfered with the bacteriostatic activity of fluoroquinolones. When its concentration was fixed at 0.5 mg/L, half its MIC(99), ethambutol raised the MIC(99) of both moxifloxacin (from 0.037 to 0.055 mg/L) and gatifloxacin (from 0.03 to 0.052 mg/L).
With M. smegmatis, ethambutol 0.5 mg/L had little effect on the bacteriostatic activities of moxifloxacin and gatifloxacin (MIC(99) dropped slightly from 0.048 to 0.045 mg/L and from 0.068 to 0.065 mg/L, respectively, for the two fluoroquinolones in the presence of ethambutol). However, ethambutol at 0.5 mg/L interfered with the lethal activities of both fluoroquinolones (not shown), suggesting that the effects of ethambutol could occur after formation of fluoroquinolonegyraseDNA complexes. Interference was also seen with AM1121, a C-8-hydrogen derivative of gatifloxacin (not shown). Thus, the reduction of fluoroquinolone lethality by ethambutol is not restricted to compounds having a C-8-methoxy group, or to M. tuberculosis.
Since quinolone resistance due to gyrA (DNA gyrase) mutations is genetically recessive, interference with fluoroquinolone lethality is expected to increase the recovery of newly formed resistant mutants (lethal activity is expected to eliminate mutant cells before all sensitive gyrase is replaced by the resistant form). To test this idea, we treated M. smegmatis with the mutagen ethyl methanesulphonate (0.2% v/v) for 4 h and then selectively enriched fluoroquinolone-resistant mutants by incubation with moxifloxacin 2.5 mg/L, or a combination of moxifloxacin 2.5 mg/L plus ethambutol (2 mg/L). Ethambutol increased the fraction of surviving cells by two-fold (from 2 x 103 to 4 x 103) and the fraction that were fluoroquinolone resistant by more than 10-fold (from 0.5 x 105 to 7 x 105).
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Discussion |
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In spite of the interference of rifampicin with moxifloxacin lethality, three-drug combinations containing moxifloxacin (2.5 mg/L) or gatifloxacin (1.5 mg/L) plus isoniazid (2 mg/L) and rifampicin (0.5 mg/L) reduced the number of viable M. tuberculosis cells by six-to-seven orders of magnitude during a 6 day treatment (not shown). This was a four- to 10-fold improvement over the two-drug combination of isoniazid and rifampicin. A similar but smaller effect was seen in a murine model of tuberculosis.5 Thus the C-8-methoxy fluoroquinolones contribute lethal activity to combination treatments.
We also examined the effect of several second-line agents on moxifloxacin activity. Capreomycin at 1040 mg/L reduced colony-forming units by about three orders of magnitude; when combined with moxifloxacin, survival was about one-third that observed with either compound alone (Figure 1c). Cycloserine reduced survival to several percent at the concentrations tested, but it showed little effect on moxifloxacin lethality (Figure 1d). In a murine model of tuberculosis, neither capreomycin nor cycloserine affected the activity of moxifloxacin.9 Ethambutol interfered with the intrinsic lethality of moxifloxacin (Figure 1e) and gatifloxacin (not shown). With M. smegmatis, ethambutol had little effect on the bacteriostatic activity of fluoroquinolones under conditions in which lethal action was reduced by 50%80%. This result shows that the effect of ethambutol on fluoroquinolone activity is not limited to M. tuberculosis. Also consistent with reduced lethal activity was the enhancing effect of ethambutol on the selective enrichment of fluoroquinolone-resistant mutants arising from treatment with ethyl methanesulphonate. Understanding why ethambutol exhibits interference with fluoroquinolone action in vitro but not in macrophage or murine models of tuberculosis5,10 requires additional study.
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Acknowledgements |
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Footnotes |
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References |
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2
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Dong, Y., Zhao, X., Domagala, J. et al. (1999). Effect of fluoroquinolone concentration on selection of resistant mutants of Mycobacterium bovis BCG and Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43, 17568.
3
.
Zhao, B.-Y., Pine, R., Domagala, J. et al. (1999). Fluoroquinolone action against clinical isolates of Mycobacterium tuberculosis: effects of a C8-methoxyl group on survival in liquid media and in human macrophages. Antimicrobial Agents and Chemotherapy 43, 6616.
4
.
Dong, Y., Zhao, X., Kreiswirth, B. et al. (2000). Mutant prevention concentration as a measure of antibiotic potency: studies with clinical isolates of Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 44, 25814.
5
.
Alvirez-Freites, E., Carter, J. & Cynamon, M. (2002). In vitro and in vivo activities of gatifloxacin against Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 46, 10225.
6
.
Lounis, N., Bentoucha, A., Truffot-Pernot, C. et al. (2001). Effectiveness of once-weekly rifapentine and moxifloxacin regimens against Mycobacterium tuberculosis in mice. Antimicrobial Agents and Chemotherapy 45, 34826.
7 . Rastogi, N., Goh, K., Bryskier, A. et al. (1996). In vitro activities of levofloxacin used alone and in combination with first- and second-line antituberculosis drugs against Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 40, 161016.[Abstract]
8 . Tomioka, H., Sato, K., Shimizu, T. et al. (2002). Anti-Mycobacterium tuberculosis activities of new fluoroquinolones in combination with other antituberculosis drugs. Journal of Infection 44, 1605.[CrossRef][ISI][Medline]
9
.
Fattorini, L., Tan, D., Iona, E. et al. (2003). Activities of moxifloxacin alone and in combination with other antimicrobial agents against multidrug-resistant Mycobacterium tuberculosis infection in BALB/c mice. Antimicrobial Agents and Chemotherapy 47, 3602.
10 . Kaur, D. & Khuller, G. (2001). In vitro, ex-vivo and in vivo activities of ethambutol and sparfloxacin alone and in combination against mycobacteria. International Journal of Antimicrobial Agents 17, 515.[CrossRef][ISI][Medline]