In vitro activity of C-8-methoxy fluoroquinolones against mycobacteria when combined with anti-tuberculosis agents

Tao Lu and Karl Drlica*

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To examine the effect of first-line and second-line anti-tuberculosis agents on the ability of fluoroquinolones to kill mycobacteria.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The fluoroquinolones are broad-spectrum, antibacterial agents that are occasionally considered for treatment of tuberculosis. Their use has been limited because resistance often arises to the readily available derivatives ofloxacin and ciprofloxacin. Newer fluoroquinolones containing a C-8-methoxy moiety exhibit greater activity, particularly against gyrase resistance mutants.13 This raises the possibility that new fluoroquinolones may restrict the selection of resistance.2,4 Two of the compounds, moxifloxacin and gatifloxacin, also display good activity in murine models of tuberculosis.5,6 However, mixed results were reported when fluoroquinolones were examined for intrinsic (in vitro) lethal activity in the presence of other anti-tuberculosis agents.7,8 Thus we have re-investigated the effect of commonly used agents on fluoroquinolone lethality.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains, culture methods and antibacterial agents

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% albumin–dextrose 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 3–4 days (M. smegmatis) or 4–5 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bactericidal activity of fluoroquinolone–rifampicin/isoniazid combinations

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 rifampicin–moxifloxacin 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 survival–concentration 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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Figure 1. Effect of anti-tuberculosis agents on the lethal activity of moxifloxacin. M. tuberculosis was treated for 6 days with moxifloxacin 2.5 mg/L plus the indicated concentrations of isoniazid (panel a), rifampicin (panel b), capreomycin (panel c), cycloserine (panel d) or ethambutol (panel e). Dilutions were prepared, and aliquots were applied to drug-free agar plates; after incubation for 4–5 weeks, colonies were counted, and the fraction of surviving cells was calculated. Open symbols: isoniazid, rifampicin, capreomycin, cycloserine or ethambutol alone; solid symbols: isoniazid, rifampicin, capreomycin, cycloserine or ethambutol plus moxifloxacin. Dotted lines indicate percentage survival when cells were treated only with moxifloxacin. Similar results were obtained from a duplicate experiment.

 


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Figure 2. Effect of rifampicin on the lethal activity of moxifloxacin on M. smegmatis. A liquid culture of exponentially growing M. smegmatis containing about 108 cfu/mL was treated with zero (open symbols) or 64 mg/L rifampicin (solid symbols) for 1 h and then with the indicated concentrations of moxifloxacin for an additional 18 h. Aliquots were removed and assayed for viable cells by growth of colonies on drug-free agar. Similar results were obtained from a duplicate experiment.

 
Effect of cycloserine and capreomycin on the bactericidal activity of moxifloxacin

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 fluoroquinolone–gyrase–DNA 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 10–3 to 4 x 10–3) and the fraction that were fluoroquinolone resistant by more than 10-fold (from 0.5 x 10–5 to 7 x 10–5).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present work examined agents singly and in combination for intrinsic (in vitro) ability to kill mycobacteria. The combination of moxifloxacin and isoniazid was slightly more lethal than either compound when used alone (Figure 1a), as was the combination of moxifloxacin and rifampicin at low rifampicin concentrations (Figure 1b). However, when rifampicin concentration increased, lethality dropped to that observed with rifampicin alone (Figure 1b). Follow-up experiments with M. smegmatis revealed that bacteriostatic concentrations of rifampicin inhibit the bactericidal effects of moxifloxacin at high concentration (Figure 2). Interference with quinolone lethality by rifampicin is a well known phenomenon with Escherichia coli. Presumably treatment with rifampicin, an inhibitor of RNA synthesis, or chloramphenicol, an inhibitor of protein synthesis, blocks the expression of a suicide protein involved in the lethal action of quinolones. Data in Figure 2, and the ability of chloramphenicol to inter-fere with the lethal action of ciprofloxacin during treatment of Mycobacterium bovis BCG, support the idea that a similar phenomenon occurs in mycobacteria.

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 10–40 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.


    Acknowledgements
 
We thank Marila Gennaro and X. Zhao for critical comments on the manuscript. The work was supported by NIH grant AI35257.


    Footnotes
 
* Corresponding author. Tel: +1-973-854-3360; E-mail: drlica{at}phri.org Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Dong, Y., Xu, C., Zhao, X. et al. (1998). Fluoroquinolone action against mycobacteria: effects of C8 substituents on bacterial growth, survival, and resistance. Antimicrobial Agents and Chemotherapy 42, 2978–84.[Abstract/Free Full Text]

2 . 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, 1756–8.[Abstract/Free Full Text]

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, 661–6.[Abstract/Free Full Text]

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, 2581–4.[Abstract/Free Full Text]

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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, 1610–16.[Abstract]

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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, 360–2.[Abstract/Free Full Text]

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, 51–5.[CrossRef][ISI][Medline]