Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK
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Abstract |
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Introduction |
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KRM-1648 is a new broad-spectrum benzoxazinorifamycin antibiotic with good in vitro activity against mycobacteria, including M. tuberculosis.1 In a murine model, good activity of KRM-1648 has also been shown for M. tuberculosis.2 KRM-1648 inhibits the bacterial RNA polymerase activity of M. tuberculosis, presumably (as for rifampicin) by binding to the ß-subunit encoded by rpoB, and forming a stable drugenzyme complex,3 preventing transcription of RNA from the DNA template. Mutations giving rise to rifampicin resistance have been shown to cluster in rpoB. Clinical isolates of M. tuberculosis with mutations in rpoB are more susceptible to KRM-1648 than rifampicin, and some strains require <16 mg/L KRM-1648 for inhibition and may be considered clinically susceptible.4 KRM-1648 also accumulates rapidly within human macrophages, explaining in part, the improved therapeutic efficacy demonstrated in animal models of Mycobacterium avium and M. tuberculosis infection.5
We have already established a procedure for measuring accumulation of rifampicin by bacteria, including mycobacteria.6,7 The aim of this study was to investigate the accumulation of KRM-1648 by wild-type M. tuberculosis.
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Materials and methods |
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14C-labelled KRM-1648 (specific activity 15 µCi/mg) was generously provided by The Kaneka Corporation (Osaka, Japan). Radiochemical purity was determined by HPLC by Corning Hazleton and was found to be 94.1%. Biological activity of the radiolabelled KRM-1648 and non-radiolabelled drug was determined by measuring the MIC. All other agents were from Sigma (Poole, UK). KRM-1648 was dissolved in 10 mM TrisHCl, pH 3; ethambutol, Tween 80 and reserpine were prepared according to their manufacturer's instructions.
Bacterial strains, growth conditions and antibiotic susceptibility testing
Mycobacterium aurum A+ (Pasteur Institute, Paris, France) and M. tuberculosis H37Rv were maintained on LowensteinJensen slopes and cultured on Middlebrook 7H11 agar (Difco, West Molesey, UK) supplemented with 10% (v/v) OADC (oleic acid, albumin fraction V, dextrose and catalase) or Middlebrook 7H9 broth (Difco) supplemented with 10% (v/v) ADC (albumin fraction V, dextrose and catalase) and grown exactly as described previously.7 The MIC of each agent was determined as described previously.7 The plates were incubated for 48 h for M. aurum, and 21 days for M. tuberculosis. The MIC was defined as the lowest concentration of drug at which no visible growth was observed.
Measurement of KRM-1648 accumulation by mycobacteria
The concentration of 14C-labelled KRM-1648 accumulated was determined essentially as for rifampicin and as described previously.7
The effects of ethambutol, Tween 80 and reserpine on the accumulation of radiolabelled KRM-1648 by M. tuberculosis were also determined as described previously for rifampicin.7
Statistical analysis
The differences in the accumulation data obtained for each species were compared and the mean steady-state concentration values were analysed by Student's t test. A P value of <0.05 was considered significant.
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Results |
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A method to determine accumulation of 14C-labelled rifampicin was established for Staphylococcus aureus and mycobacteria including M. tuberculosis in two previous studies.6,7 Although the MIC of rifampicin for S. aureus and M. tuberculosis is 0.002 mg/L and 0.25 mg/L, respectively, the optimum concentration of radiolabelled rifampicin for accumulation studies was 2 mg/L with both species; in the time frame (~20 min) of the accumulation experiment this concentration had no deleterious effect upon cell viability or growth.6,7 Therefore, although the MIC of KRM-1648 was 0.25 mg/L for M. aurum and M. tuberculosis, all initial experiments were performed with 2 mg/L radiolabelled KRM-1648. The MIC values of the radiolabelled and unradiolabelled KRM-1648 were identical (data not shown). With 2 mg/L KRM-1648 and M. aurum a steady-state concentration of 21.3 ± 2.9 ng/mg cells 14C-labelled KRM-1648 was obtained within 5 min of exposure to 14C-labelled KRM-1648 (Figure 1).
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Effect of pH on accumulation of radiolabelled KRM-1648
KRM-1648 dissolves more readily at acid pH (Hidaka, personal communication), so the effect of measuring accumulation at acid versus neutral pH was determined. After exposure to 0.5 or 2 mg/L KRM-1648, slightly greater concentrations were accumulated at pH 4 than at pH 7 (Figure 2). The increase from 12.99 ± 0.4 ng/mg cells at pH 7 to 14.6 ± 0.3 ng/mg cells at pH 4 was statistically significant (P < 0.005).
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The MIC of KRM-1648 was reduced from 0.25 mg/L to 0.12 mg/L in the presence of 0.25 mg/L ethambutol (four-fold less than the MIC of ethambutol alone). The concentration of radiolabelled KRM-1648 accumulated by M. tuberculosis after exposure to 2 mg/L was increased in the presence of 0.25 mg/L ethambutol from 13.09 ± 0.6 ng/mg dry cells to 18.45 ± 0.9 ng/mg dry cells (Figure 2). This increase was statistically significant (P < 0.005).
Effect of Tween 80 on the accumulation of radiolabelled KRM-1648
The MIC of KRM-1648 was reduced from 0.25 mg/L to 0.12 mg/L in the presence of 0.05% Tween 80 (four-fold less than the MIC of Tween 80 alone). Although Tween 80 had a slight synergic effect on the antimicrobial activity of KRM-1648, the concentration of radiolabelled KRM-1648 accumulated by M. tuberculosis was unchanged in the presence of 0.05% Tween 80 (Figure 2). One explanation for these data is that growth in 0.05% Tween 80 gives rise to lower numbers of M. tuberculosis cells than growth in glycerol, and that it is this lower number that gives rise to the lower steady-state concentrations. To examine this, the viable count of M. tuberculosis was determined after growth in 0.05% Tween 80 with or without KRM-1648 compared with parallel cultures grown with glycerol. The viable count was slightly lower for the cultures grown in 0.05% Tween 80 (2.6 x 107 cfu/mL) than those grown with glycerol (2.7 x 107 cfu/mL) and in the presence of KRM 1648 plus Tween 80 it was further reduced (1.9 x 107 cfu/mL).
Effect of the efflux inhibitor reserpine on the concentration of radiolabelled KRM-1648 accumulated
Reserpine (20 mg/L) had no effect upon the MIC of KRM-1648 and a minimal effect upon accumulation, increasing the concentration of radiolabelled KRM-1648 accumulated by 0.5 ± 0.2 ng/mg dry cells. This was not statistically significant.
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Discussion |
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A well-established effect of ethambutol is to lower the MIC values of several antimycobacterial agents. We have previously shown that the concentration of rifampicin accumulated in the presence of ethambutol increased,7 supporting the hypothesis that ethambutol interacts with components of the mycobacterial cell wall increasing cell wall permeability.8 The data obtained for KRM-1648 further support the proposal that the synergy between antibiotics and ethambutol is a direct consequence of increased cell wall permeability to the drugs.
Tween 80 is a non-ionic, surface-active detergent often added to liquid media to obtain homogeneous cell suspensions of mycobacteria.9 It has been proposed that Tween 80 acts directly on the mycobacterial cell wall and subsequently alters its permeability.10 Despite this, in the study with rifampicin, and also in the present study with KRM-1648, it was found that Tween 80 had no effect upon the concentration of these antibiotics accumulated by M. tuberculosis. Although this is counter-intuitive to accepted dogma for the mechanism of action of Tween 80, we have also found that Tween 80 has slight bactericidal activity such that there are less viable cells present in an accumulation experiment and in our opinion thus giving rise to lower accumulation values. The antimicrobial synergy probably results from the additive effect of the bactericidal activity of Tween 80 plus that of KRM-1648. The mechanism of bactericidal action of the Tween 80 is unknown.
KRM-1648 dissolves more readily at acid pH, and was also found to accumulate to higher concentrations when the experiment was performed at pH 4. These data suggest that an acidic environment may enhance the activity of KRM-1648 against M. tuberculosis in vivo. As for rifampicin, there was a small, statistically insignificant, effect of the efflux inhibitor reserpine upon the concentration of KRM-1648 accumulated. The role of efflux in attenuating the activity of KRM-1648, if any, will need evaluating with a characterized efflux mutant of M. tuberculosis.
In conclusion, the good antimycobacterial activity of KRM-1648 probably results from the rapid influx of this agent combined with high affinity for the target enzyme.
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Acknowledgments |
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Notes |
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References |
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2 . Klemens, S. P., Grossi, M. A. & Cynamon, M. H. (1994). Activity of KRM-1648, a new benzoxazinorifamycin, against Mycobacterium tuberculosis in a murine model. Antimicrobial Agents and Chemotherapy 38, 22458.[Abstract]
3 . Fujii, K., Saito, H., Tomioka, H., Mae, T. & Hosoe, K. (1995). Mechanism of action of antimycobacterial activity of the new benzoxazinorifamycin KRM-1648. Antimicrobial Agents and Chemotherapy 39, 148992.[Abstract]
4 . Moghezeh, S. L., Pan, X., Arain, T., Stover, C. K., Musser, J. M. & Kreiswirth, B. N. (1996). Comparative antimycobacterial activities of rifampicin, rifapentine, and KRM-1648 against a collection of rifampin-resistant Mycobacterium tuberculosis isolates with known rpoB mutations. Antimicrobial Agents and Chemotherapy 40, 26557.[Abstract]
5 . Mor, N., Simon, B. & Heifets, L. (1996). Bacteriostatic and bactericidal activities of benzoxazinorifamycin KRM-1648 against Mycobacterium tuberculosis and Mycobacterium avium in human macrophages. Antimicrobial Agents and Chemotherapy 40, 14825.[Abstract]
6 . Williams, K. J. & Piddock, L. J. (1998). Accumulation of rifampicin by Escherichia coli and Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 42, 597603.[Abstract]
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Piddock, L. J., Williams, K. J. & Ricci, V. (1999). Accumulation of rifampicin by Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 45, 15965.
8 . Deng, L., Mikusova, K., Robuck, K. G., Scherman, M., Brennan, P. J. & McNeil, M. R. (1995). Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope. Antimicrobial Agents and Chemotherapy 39, 694701.[Abstract]
9 . Kuze, F., Kurasawa, T., Bando, K., Lee, Y. & Maekawa, N. (1981). In vitro and in vivo susceptibility of atypical mycobacteria to various drugs. Reviews in Infectious Diseases 3, 88597.[ISI][Medline]
10 . Masaki, S., Sugimori, G., Okamoto, A., Imose, J. & Hayashi, Y. (1990). Effect of Tween 80 on the growth of Mycobacterium avium complex. Microbiology and Immunology 34, 65363.[ISI][Medline]
Received 24 May 1999; returned 12 October 1999; revised 26 November 1999; accepted 10 December 1999