Centre for Medical Microbiology, University College London, Hampstead Campus, Rowland Hill St., London NW3 2PF, UK
Received 31 January 2005; returned 2 April 2005; revised 14 April 2005; accepted 12 May 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: The mutation rate in Mycobacterium fortuitum to ciprofloxacin, levofloxacin, moxifloxacin, rifampicin, erythromycin and gentamicin resistance was determined when grown with and without various sub-MIC concentrations of ciprofloxacin.
Results: M. fortuitum exposed to MIC ciprofloxacin had an increase in the mutation rate of between 72- and 120-fold when selected on quinolones or other antimycobacterial antibiotics. Smaller, but significant increases in mutation rate were seen when the organism was exposed to lower concentrations (
MIC and
MIC).
Conclusions: These data show that sub-MIC concentrations of fluoroquinolone significantly increase mutation rates and these data suggest that care must be taken to ensure that bacteria are not exposed to subinhibitory concentrations when adding quinolones to a regimen used to treat mycobacterial infection.
Keywords: fluoroquinolones , mycobacteria , quinolones , M. fortuitum
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of quinolone antibiotics have been shown to have activity against many mycobacterial species57 and this has been confirmed in animal models of infection.5 It has also been reported that the bactericidal activity demonstrated in vitro and in animal models can also be replicated during short monotherapy clinical trials.8,9 Some larger scale studies have suggested that regimens containing fluoroquinolone antibiotics are effective.10,11 Despite this, fluoroquinolones have not established themselves as first line agents in chemotherapy. Rather they are used when patients cannot tolerate the standard regimen of rifampicin, isoniazid, pyrazinamide and ethambutol and in the management of patients with multiple drug resistance.12,13
Fluoroquinolones exert their antibacterial effect on mycobacteria by disrupting the action of the DNA gyrase system which results in double stranded DNA breaks.14 As a result of this action, they trigger the SOS response, a mechanism which enables bacteria to survive in the face of threats to the integrity of their genome.14,15 The SOS response is usually triggered when the organism is exposed to DNA damaging agents such as fluoroquinolones, ultraviolet light, reactive oxygen intermediates or salicylic acid.14,16 The SOS response mediates survival of the organism by allowing DNA replication to continue past breaks that would normally block it. In exchange for this survival advantage there is an increased mutation rate as the polymerases that perform the repair are prone to error.17,18 Recent studies have shown that error prone polymerase activation occurs at the end of stationary phase and in starvation.19 Previous studies in Escherichia coli and methicillin-resistant Staphylococcus aureus have suggested that bacteria growing in the presence of sub-lethal concentrations of fluoroquinolones have an increased mutation rate to antibiotic resistance.14,15,20 Thus it is important to determine whether fluoroquinolones also exert this effect on mycobacteria and to quantify it as these drugs are used for the management of lower respiratory tract infections and this may expose mycobacteria to these agents.21
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ciprofloxacin and moxifloxacin were supplied by Bayer, gentamicin, erythromycin and rifampicin were purchased from Sigma Chemical Co. and levofloxacin was supplied by Roussel Laboratories. The drugs were dissolved according to the manufacturers' instructions. A clinical strain of M. fortuitum (MF01332) isolated from a specimen submitted to the Department of Medical Microbiology at the Royal Free Hospital was used for these experiments. The MICs of ciprofloxacin, moxifloxacin, levofloxacin, rifampicin, erythromycin and gentamicin for MF01332 were determined by the Etest method (AB Biodisk, Solna, Sweden) using the manufacturer's instructions. These data were used to determine the concentration of antibiotic added to either broth culture or selective plates (see below).
Mutation induction
Fresh cultures of M. fortuitum were grown in MuellerHinton (Oxoid, Basingstoke, UK) broth to which ciprofloxacin had been added at concentrations equivalent to MIC (0.06 mg/L),
MIC (0.03 mg/L) and
MIC (0.015 mg/L). To determine the effect of ciprofloxacin on mutation rate, the organism was grown in a parallel broth with no ciprofloxacin added. Following inoculation of
105 cfu/mL, cultures were incubated at 37°C aerobically for 48 h without shaking. Following incubation, viable counts were estimated by the method of Miles and Misra adapted for M. tuberculosis.22 Briefly, samples were mixed by brief vortexing and log dilutions to 106 in sterile distilled water were set up. Twenty microlitres of each dilution was spotted onto a blood agar plate and dried. The plates were incubated for 48 h at 37°C then the number of colonies counted. Each determination was made in triplicate and expressed as mean colony forming units (cfu) per mL.
Mutation rate estimation
After incubation for 48 h, mutation rate estimation was performed as follows: bacteria were harvested by centrifugation (3000g) for 10 min, supernatants were removed, and pellets were then resuspended in a measured volume. The total volume of the resuspended pellet was noted and recorded for future calculation of mutation rates. The MICs of the various antibiotics used for mutant selection were as follows: ciprofloxacin 0.12 mg/L, moxifloxacin 0.06 mg/L, levofloxacin 0.12 mg/L, erythromycin 24 mg/L, rifampicin 4.0 mg/L, gentamicin 0.75 mg/L for MF 01332. Proportions of each pellet were then spread onto antibiotic-containing Iso-Sensitest (Oxoid, Basingstoke, UK) agar plates at 2x MIC of each antibiotic.23 Plates were then incubated for 72 h at 37°C and examined on a daily basis and the numbers of colonies were counted and recorded. Mutation rates were estimated using the median mutation method of Drake described by Rosche and Foster.24 A total of five pairs of median mutation rate estimations was performed for each selection antibiotic consisting of five paired mutation frequency experiments (a total of 25 selection experiments per data point).23,24 As mutation rate experiments are subject to considerable variation between experiments, this batch to batch variation was controlled by calculating the ratio of the median mutation rate of cultures grown in drug-free medium and the median mutation rate grown in the presence of differing concentrations of ciprofloxacin. Experiments were repeated using rifampicin MIC (8 mg/L) as a control to determine whether the effect was specific to ciprofloxacin.
Statistical assessment
The differences between the mean mutation rate ratios were compared for each concentration of ciprofloxacin and each selecting agent by a one-way analysis of variance (ANOVA) using the KruskalWallis non-parametric method. This was calculated using GraphPad Instat (Graph Pad Software, CA, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutation frequencies take no account of the growth of the organisms nor the possibility of a jackpot mutation.25 Since culturing in the presence of subinhibitory concentrations significantly affects the growth of the bacteria, it is especially important to calculate a mutation rate by a method which takes account of the reduced growth of the organisms and allows for the possibility of a jackpot mutation.24 The rate of mutation to resistance to six different antibiotics was tested by selecting against each of these agents after growth in the presence of different subinhibitory concentrations of ciprofloxacin (,
and
MIC). When the bacteria are exposed to
MIC ciprofloxacin in the broths, the mutation rate is increased for all of the antibiotics in selection experiments. The mutation rate estimation data for each of the drugs, including drug-free media are listed in Table 1.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our experiments show that when M. fortuitum is grown in the presence of a fluoroquinolone, in this instance ciprofloxacin, there is an increase in the rate at which mutations occur. The greatest increase in the mutation rate, in comparison with organisms grown in the absence of antibiotic was up to 120-fold when the antibiotic concentration was the equivalent of MIC. The effect also appeared to be dose-dependent as smaller, but significant increases in the mutation rates were also seen at lower ciprofloxacin concentrations. Previous authors have used a difference of 10-fold to distinguish hypermutators from normal bacteria by differences in mutation rates of as little as sevenfold, indicating that the increase in mutation rate that we have demonstrated must be considered both statistically and biologically significant.3335
The increases in mutation rate were found irrespective of selecting agent (the antibiotic incorporated into the plates): ciprofloxacin, levofloxacin, moxifloxacin, erythromycin, gentamicin and rifampicin. This is an important finding as it suggests that the effect of the fluoroquinolone affects the whole genome since, to become resistant to the antibiotics tested, mutations must occur in a wide range of different genes.4
The concentrations of quinolone that are responsible for the increase in mutation rate demonstrated in this paper are clinically relevant and likely to occur between doses of antibiotics. Fluoroquinolones are often used in the management of non-tuberculosis mycobacteria26,27 and multiple drug-resistant M. tuberculosis disease and are combined with other second line agents.36,37 Mutation to resistance occurs at a higher rate for second line anti-tuberculosis agents than isoniazid and rifampicin. For example, the mutation rate for rifampicin is between 108 and 109/cell division but 106/cell division for ethambutol.2 This means that any increase in mutation rate may have a significant effect on the speed at which resistance may emerge to other second line agents in a regimen. This throws into question the common practice of adding a fluoroquinolone to a mycobacterial treatment regimen when a resistant strain is isolated. In such circumstances, the capacity of isoniazid and rifampicin to prevent the emergence of resistant mutants is lost. This is especially likely in those patients with cavitatory disease, who have intestinal malabsorption or do not adhere closely to the prescribed regimen.38,39 This suggests that when fluoroquinolones are used, care must be taken to ensure that a regimen is prescribed that minimizes the risk of exposing bacteria to subinhibitory concentrations of quinolone. The data presented in this paper require to be confirmed in M. tuberculosis and these experiments, coupled with DNA array analysis are currently under way in our laboratory.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. David HL. Probability distribution of drug-resistant mutants in unselected populations of Mycobacterium tuberculosis. Appl Microbiol 1970; 20: 8104.
3. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 1998; 79: 329.[CrossRef][Medline]
4.
Gillespie SH. Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob Agents Chemother 2002; 46: 26774.
5.
Ji B, Lounis N, Maslo C et al. In vitro and in vivo activities of moxifloxacin and clinafloxacin against Mycobacterium tuberculosis. Antimicrob Agents Chemother 1998; 42: 20669.
6.
Gillespie SH, Morrissey I, Everett D. A comparison of the bactericidal activity of quinolone antibiotics in a Mycobacterium fortuitum model. J Med Microbiol 2001; 50: 56570.
7.
Gillespie SH, Billington O. Activity of moxifloxacin against mycobacteria. J Antimicrob Chemother 1999; 44: 3935.
8. Kennedy N, Fox R, Kisyombe GM et al. Early bactericidal and sterilizing activities of ciprofloxacin in pulmonary tuberculosis. Am Rev Respir Dis 1993; 148: 154751.[ISI][Medline]
9.
Gosling RD, Uiso LO, Sam NE et al. The bactericidal activity of moxifloxacin in patients with pulmonary tuberculosis. Am J Respir Crit Care Med 2003; 168: 13425.
10. Kennedy N, Berger L, Curram J et al. Randomized controlled trial of a drug regimen that includes ciprofloxacin for the treatment of pulmonary tuberculosis. Clin Infect Dis 1996; 22: 82733.[ISI][Medline]
11. Tuberculosis Research Centre. Shortening short course chemotherapy: a randomised clinical trial for treatment of smear positive pulmonary tuberculosis with regimens using ofloxacin in the intensive phase. Indian J Tuberc 2002; 49: 2738.
12. Yew WW, Lee J, Wong PC et al. Tolerance of ofloxacin in the treatment of pulmonary tuberculosis in presence of hepatic dysfunction. Int J Clin Pharmacol Res 1992; 12: 1738.[ISI][Medline]
13.
Yew WW, Chan CK, Leung CC et al. Comparative roles of levofloxacin and ofloxacin in the treatment of multidrug-resistant tuberculosis: preliminary results of a retrospective study from Hong Kong. Chest 2003; 124: 147681.
14.
Drlica K, Zhao X. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol Mol Biol Rev 1997; 61: 37792.
15. Phillips I, Culebras E, Moreno F et al. Induction of the SOS response by new 4-quinolones. J Antimicrob Chemother 1987; 20: 6318.[Abstract]
16.
Gustafson JE, Candelaria PV, Fisher SA et al. Growth in the presence of salicylate increases fluoroquinolone resistance in Staphylococcus aureus. Antimicrob Agents Chemother 1999; 43: 9902.
17. Boshoff HI, Reed MB, Barry CE, III et al. DNAE2 polymerase contributes to in vitro survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 2003; 113: 193.[CrossRef]
18. Mizrahi V, Andersen SJ. DNA repair in Mycobacterium tuberculosis. What have we learnt from the genome sequence? Mol Microbiol 1998; 29: 13319.[CrossRef][ISI][Medline]
19. Layton JC, Foster PL. Error-prone DNA polymerase IV is controlled by the stress-response sigma factor, RpoS, in Escherichia coli. Mol Microbiol 2003; 50: 54961.[CrossRef][ISI][Medline]
20. Fung-Tomc J, Kolek B, Bonner DP. Ciprofloxacin-induced, low-level resistance to structurally unrelated antibiotics in Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 1993; 37: 128996.[Abstract]
21.
Finch R, Schurmann D, Collins O et al. Randomized controlled trial of sequential intravenous (i.v.) and oral moxifloxacin compared with sequential i.v. and oral co-amoxiclav with or without clarithromycin in patients with community-acquired pneumonia requiring initial parenteral treatment. Antimicrob Agents Chemother 2002; 46: 174654.
22.
Billington OJ, McHugh TD, Gillespie SH. Physiological cost of rifampin resistance induced in vitro in Mycobacterium tuberculosis. Antimicrob Agents Chemother 1999; 43: 18669.
23. Gillespie SH, Dickens A, Voelker L. Evolution of fluoroquinolone resistance in Streptococcus pneumoniae. Microb Drug Resist 2002; 8: 7984.[CrossRef][ISI][Medline]
24. Rosche WA, Foster PL. Determining mutation rates in bacterial populations. Methods 2000; 20: 417.[CrossRef][ISI][Medline]
25.
Luria SE, Delbruck M. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 1943; 28: 491511.
26.
Joint Tuberculosis Committee. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1997. Thorax 2000; 55: 2108.
27. American Thoracic Society. Diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am J Respir Crit Care Med 1997; 156: S1S25.[ISI][Medline]
28. Klopman G, Fercu D, Renau TE et al. N-1-tert-butyl-substituted quinolones: in vitro anti-Mycobacterium avium activities and structureactivity relationship studies. Antimicrob Agents Chemother 1996; 40: 263743.[Abstract]
29. Renau TE, Gage JW, Dever JA et al. Structureactivity relationships of quinolone agents against mycobacteria: effect of structural modifications at the 8 position. Antimicrob Agents Chemother 1996; 40: 23638.[Abstract]
30. Renau TE, Sanchez JP, Gage JW et al. Structureactivity relationships of the quinolone antibacterials against mycobacteria: effect of structural changes at N-1 and C-7. J Med Chem 1996; 39: 72935.[CrossRef][ISI][Medline]
31. Renau TE, Sanchez JP, Shapiro MA et al. Effect of lipophilicity at N-1 on activity of fluoroquinolones against mycobacteria. J Med Chem 1995; 38: 29747.[CrossRef][ISI][Medline]
32.
Andries K, Verhasselt P, Guillemont J et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 2005; 307: 2237.
33.
Oliver A, Canton R, Campo P et al. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 2000; 288: 12514.
34.
Denamur E, Bonacorsi S, Giraud A et al. High frequency of mutator strains among human uropathogenic Escherichia coli isolates. J Bacteriol 2002; 184: 6059.
35.
Matic I, Radman M, Taddei F et al. Highly variable mutation rates in commensal and pathogenic Escherichia coli. Science 1997; 277: 18334.
36.
Blumberg HM, Burman WJ, Chaisson RE et al. American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: treatment of tuberculosis. Am J Respir Crit Care Med 2003; 167: 60362.
37. Gillespie SH, Kennedy N. Fluoroquinolones: a new treatment for tuberculosis? Int J Tuberc Lung Dis 1998; 2: 26571.[ISI][Medline]
38. Lipsitch M, Levin BR. Population dynamics of tuberculosis treatment: mathematical models of the roles of non-compliance and bacterial heterogeneity in the evolution of drug resistance. Int J Tuberc Lung Dis 1998; 2: 18799.[ISI][Medline]
39. Elliott AM, Berning SE, Iseman MD et al. Failure of drug penetration and acquisition of drug resistance in chronic tuberculous empyema. Tuber Lung Dis 1995; 76: 4637.[CrossRef][ISI][Medline]