Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Some fluoroquinolones, including ofloxacin, levofloxacin, moxifloxacin and ciprofloxacin show in vitro activity against M. tuberculosis.14 As M. tuberculosis possesses only the genes gyrA and gyrB, which encode the topoisomerase II enzyme DNA gyrase, it is assumed that this is the single target of this class of drugs as there are no parC/parE genes that encode the second target enzyme (topoisomerase IV) found in other bacterial species.5 This hypothesis is supported by the finding that only gyrA mutations have been reported in fluoroquinolone-resistant M. tuberculosis clinical isolates.69 Although the major form of quinolone resistance is altered DNA gyrase, energy-dependent quinolone efflux has been shown to be a contributory factor in other bacteria.10 Active efflux of norfloxacin has been reported in a quinolone-resistant strain of Mycobacterium smegmatis mc2155, and the gene encoding this system, lfrA, has been cloned.11 The LfrA efflux pump is homologous to QacA from Staphylococcus aureus, but not to NorA. QacA confers resistance to ethidium and other organic cations, such as chlorhexidine, via PMF-dependent efflux.12 However, as for NorA, LfrA preferentially pumps out hydrophilic quinolones.
Owing to moderate in vivo activity and increasing fluoroquinolone resistance, these agents are generally considered second-line agents against tuberculosis.13,14 However, with the increasing incidence of MDR TB, and the increased use of fluoroquinolones in combination with other agents to treat TB, much attention has focused on the therapeutic value of the fluoroquinolones for the treatment of TB and other mycobacterial diseases.13,14 As DNA gyrase is intracellular, in order to exert their antibacterial effect the fluoroquinolones must cross the bacterial cell wall. However, there have only been a few studies measuring the accumulation/transport of antituberculous agents by M. tuberculosis, and hence the role permeability plays, if any, in this species' drug resistance has not been determined.
We have previously used the modified fluorescence method to measure accumulation of norfloxacin by mycobacteria15 and shown for Mycobacterium aurum and M. smegmatis that similar kinetics of accumulation were obtained to those found for fluoroquinolones and other bacteria. Of interest, there was no effect of Tween-80 or subinhibitory concentrations of ethambutol or of the proton motive force (efflux pump) inhibitor 2,4-dinitrophenol (DNP), whether added before or after norfloxacin, on the final steady-state concentration (SSC) accumulated. As there was no effect of DNP on norfloxacin accumulation by these mycobacteria, significant activity of a quinolone efflux system in these wild-type mycobacteria was not suspected. Therefore, the aim of the present study was to investigate the accumulation of fluoroquinolones with antibacterial activity for mycobacteria by wild-type fluoroquinolone-susceptible M. tuberculosis H37Rv.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fluoroquinolones (ciprofloxacin and moxifloxacin, Bayer AG, Wuppertal, Germany; norfloxacin, Sigma, Poole, UK; levofloxacin and ofloxacin, Aventis, Romainville, France), ethambutol, Tween-80, CCCP (carbonyl cyanide m-chlorophenyl-hydrazone) and reserpine were prepared according to the manufacturers' instructions. All other agents were from Sigma.
Bacterial strains, growth conditions and antibiotic susceptibility testing
All experiments were performed in a class I cabinet within a category III facility. M. tuberculosis H37Rv was maintained on LowensteinJensen slopes and cultured on Middlebrook 7H11 agar (Difco, West Molsey, 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.16,17 The MIC of each agent was also determined as described previously.16,17 The plates were incubated for 21 days and the MIC was defined as the lowest concentration of drug at which no visible growth was observed. The effect of ethambutol, Tween-80, CCCP and reserpine on the susceptibility of M. tuberculosis was also determined. The growth kinetics and estimation of cell dry weight were also as described previously.17
Measurement of fluoroquinolone accumulation
A modified fluorometric method, adapted to accommodate the growth characteristics of mycobacteria, was used.15 Cells were grown to mid-exponential phase, OD550 of 11.2, and harvested by centrifugation in a Sigma 6K10 centrifuge (supplied by Phillip Harris) at 3003g for 20 min at 15°C. The cells were washed in 10 mL of 50 mM sodium phosphate buffer (pH 7) and concentrated with the same buffer to give the suspension of M. tuberculosis an OD550 of 8. This suspension was placed in a 37°C water bath and left for 10 min to equilibrate. Fluoroquinolone was added to a final concentration of 10 mg/L and 1 mL samples were removed at timed intervals. The cells were centrifuged immediately at 12 000g (Sigma 6K10) for 3 min at 4°C and the cell pellets washed once with ice-cold sodium phosphate buffer (50 mM, pH 7) and resuspended in 1 mL of 0.1 M glycine hydrochloride (pH 3). The samples were left overnight, at room temperature with agitation, to lyse. The following day the samples were centrifuged at 12 000g (Eppendorf 5403) and the fluorescence of the supernatants determined at the appropriate excitation and emission wavelengths for each agent, and the data expressed as ng fluoroquinolone/mg dry weight cells. All experiments were performed at least three times and mean values ± s.ds are shown. The SSC values were plotted against the molecular size of the free form of each agent and the partition coefficient (Papp). The Papp was calculated by determining the concentration of agent in the aqueous phase (0.1 M sodium phosphate buffer pH 7) and organic phase (n-octanol) of each agent as described previously.18
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The most active fluoroquinolones were moxifloxacin and sparfloxacin with MICs of 0.5 mg/L, ciprofloxacin, clinafloxacin and grepafloxacin all had the same MIC, 1 mg/L, and gatifloxacin and norfloxacin each had an MIC of 2 mg/L. The MICs of various inhibitors were: ethambutol >0.5 mg/L, 0.25% Tween-80, reserpine 128 mg/L, 64 µM DNP and 32 µM CCCP. Addition of ethambutol (0.25 mg/L), Tween-80 (0.05%), CCCP (32 µM) and reserpine (20 mg/L) had no effect upon the MIC of any agent.
Accumulation of fluoroquinolones
A method to determine accumulation of norfloxacin was established for mycobacteria in a previous study.15 Despite the MIC of norfloxacin for M. tuberculosis being 2 mg/L, due to the fluorescence of the fluoroquinolones studied, the optimum concentration for accumulation studies was 10 mg/L, and in the time frame of the accumulation experiment (20 min) had no deleterious effect upon cell viability or growth (data not shown).
The time to achieve an SSC of each agent in M. tuberculosis was 60240 s (Table and Figure
). Of all five agents examined, moxifloxacin accumulated the lowest concentration, 31.5 ± 1.9 ng/mg dry weight cells, which was followed by levofloxacin, norfloxacin, ofloxacin and ciprofloxacin (97.7 ± 7.5 ng/mg dry weight cells) (Table
and Figure
). However, ciprofloxacin took longer to achieve an SSC than the other four agents; levofloxacin reached steady state in the least time (60 s). There was only a weak correlation between molecular size and MIC (r = -0.59). However, a stronger correlation was found between molecular size and SSC (r = -0.76), and initial rate (r = -0.86); larger molecules accumulated to the lowest concentration and more slowly. However, these molecules (moxifloxacin and levofloxacin) were also the most active. Although all five agents had low hydrophobicity values (Papp
0.11), those with the lowest values accumulated to the higher concentrations. As for molecular size, there was a correlation between the Papp values and MIC (r = -0.86), SSC (r = -0.64) and initial rate (r = -0.68). There was no correlation between MIC and either SSC (r = 0.24) or initial rate (r = 0.41).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, accumulation of fluoroquinolones with activity for M. tuberculosis was investigated to determine whether the concentration accumulated reflected activity. Sparfloxacin was not available in radiolabelled form for this study and as it fluoresces poorly it was not studied. Likewise we have found that clinafloxacin and grepafloxacin also fluoresce poorly, so these agents were not examined further. Moxifloxacin, levofloxacin and ofloxacin had the same MIC for this strain of M. tuberculosis H37Rv; moxifloxacin and levofloxacin also accumulated to the same concentration. However, levofloxacin accumulated at a greater rate than moxifloxacin, and ofloxacin accumulated slowly. It has been postulated previously16,17 that the rates of influx of rifampicin and KRM-1648 were associated with activity and not the SSC. It was further postulated that as the concentration accumulated mirrored the IC50 value for inhibition of the target enzyme, the SSC values reflected binding of the drug to its target. The same may also be true for fluoroquinolones, and the rate of influx is sufficiently rapid to ensure that moxifloxacin reaches its target protein(s) to give rise to an MIC similar to that of levofloxacin. For strains of M. tuberculosis with greater susceptibility to fluoroquinolones than those investigated in the present study, it is likely that the target enzyme(s) is more susceptible to the drug.
A well-established effect of ethambutol is to lower the MICs of several antimycobacterial agents. We have previously shown that the concentrations of rifampicin and KRM-1648 accumulated in the presence of ethambutol increased,16,17 supporting the hypothesis that ethambutol interacts with components of the mycobacterial cell wall increasing cell wall permeability.21 However, as ethambutol had no effect on the MIC of any agent in this study, no accumulation experiments were performed. It may be that as most fluoroquinolones are zwitterions and enter the cell rapidly, any permeabilizing effect of ethambutol is irrelevant.
Tween-80 is a non-ionic surface-active detergent often added to liquid media to obtain homogeneous cell suspensions of mycobacteria.22 It has been proposed that Tween-80 acts directly on the mycobacterial cell wall and subsequently alters its permeability.23 Despite this, in studies in this laboratory with rifampicin and KRM-1648,16,17 Tween-80 had no effect on the concentration of these norfloxacin accumulated by M. aurum. There was also no antimicrobial synergy between Tween-80 and any fluoroquinolone tested against M. tuberculosis H37Rv and so no accumulation experiments were performed.
As there was no effect upon the activity of each agent in the presence of the efflux pump inhibitors CCCP, DNP and reserpine, and our studies with M. smegmatis and M. aurum demonstrated no effect upon the concentration of norfloxacin accumulated, no accumulation experiments in the presence of these inhibitors were performed with M. tuberculosis.
In conclusion, the antimycobacterial activities of fluoroquinolones are probably due to the rapid influx of these agents combined with good affinity for the target enzyme.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Berlin, O. G. W., Young, L. S. & Bruckner, D. A. (1987). In vitro activity of six fluorinated quinolones against Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 19, 6115.[Abstract]
3
.
Gillespie, S. H. & Billington, O. (1999). Activity of moxifloxacin against mycobacteria. Journal of Antimicrobial Chemotherapy 44, 3935.
4 . Hoffner, S. E., Gezelius, L. & Olsson-Liljequist, B. (1997). In-vitro activity of fluorinated quinolones and macrolides against drugresistant Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 40, 8858.[Abstract]
5
.
Guillemin, I., Jarlier, V. & Cambau, E. (1998). Correlation between quinolone susceptibility patterns and sequences in the A and B subunits of DNA gyrase in mycobacteria. Antimicrobial Agents and Chemotherapy 42, 20848.
6 . Alangaden, G. J., Manavathu, E. K., Vakulenko, S. B., Zvonok, N. M. & Lerner, S. A. (1995). Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients. Antimicrobial Agents and Chemotherapy 39, 17003.[Abstract]
7 . Cambau, E., Sougakoff, W., Besson, M., Truffot-Pernot, C., Grosset, J. & Jarlier, V. (1994). Selection of a gyrA mutant of Mycobacterium tuberculosis resistant to fluoroquinolones during treatment with ofloxacin. Journal of Infectious Diseases 170, 47983.[ISI][Medline]
8 . Williams, K. J. & Piddock, L. J. V. (1996). gyrA of ofloxacinresistant clinical isolates of Mycobacterium tuberculosis from Hong Kong. Journal of Antimicrobial Chemotherapy 37, 10324.[ISI][Medline]
9 . Takiff, H. E., Salazaar, L., Guerrero, C., Philipp, W., Huang, W. M., Kreiswirth, B. et al. (1994). Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrobial Agents and Chemotherapy 38, 77380.[Abstract]
10 . Piddock, L. J. V. (1999). Mechanisms of fluoroquinolone resistance: an update 19941998. Drugs 58 ,Suppl. 2, 118.[ISI][Medline]
11 . Liu, J., Takiff, H. E. & Nikaido, H. (1996). Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump. Journal of Bacteriology 178, 37915.[Abstract]
12 . Paulsen, I. T., Brown, M. H. & Skurray, R. A. (1996). Proton-dependent multidrug efflux systems. Microbiology Reviews 60, 575608.
13 . Alangaden, G. J. & Lerner, S. A. (1997). The clinical use of fluoroquinolones for the treatment of mycobacterial diseases. Clinical Infectious Diseases 25, 121321.[ISI][Medline]
14 . Jacobs, M. R. (1999). Activity of quinolones against mycobacteria. Drugs 58 ,Suppl. 2, 1922.
15
.
Williams, K. J. & Piddock, L. J. V. (1998). Accumulation of norfloxacin by Mycobacterium aurum and Mycobacterium smegmatis. Antimicrobial Agents and Chemotherapy 42, 795800.
16
.
Piddock, L. J. V., Williams, K. J. & Ricci, V. (2000). Accumulation of rifampicin by Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 45, 15965.
17
.
Piddock, L. J. V. & Ricci, V. (2000). Accumulation of KRM-1648 by Mycobacterium aurum and Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 45, 6814.
18 . Mortimer, P. G. S. & Piddock, L. J. V. (1991). A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 28, 63953.[Abstract]
19
.
Piddock, L. J. V., Jin, Y. F., Ricci, V. & Asuquo, A. (1999). Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Journal of Antimicrobial Chemotherapy 43, 6170.
20 . Kocagoz, T., Hackbarth, C. J., Unsal, I., Rosenberg, E. Y., Nikaido, H. & Chambers, H. F. (1996).Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra.Antimicrobial Agents and Chemotherapy 40, 176874.[Abstract]
21 . 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]
22 . 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]
23 . Masuki, S., Sugimori, G., Okamoto, A., Imose, J. & Hayashi, Y. (1990). Effect of Tween 80 on the growth of Mycobacterium avium complex.Microbiology and Immunity 34, 65363.
Received 14 May 2001; returned 11 August 2001; revised 10 September 2001; accepted 17 September 2001