Journal of Antimicrobial Chemotherapy (2004) 53, 441444 DOI: 10.

1093/jac/dkh119 Advance Access publication 12 February 2004

Mutant prevention concentration: comparison of fluoroquinolones and linezolid with Mycobacterium tuberculosis
J. C. Rodrguez1*, L. Cebrin1, M. Lpez1, M. Ruiz1, I. Jimnez2 and G. Royo1
1S.

Microbiology and 2S. Pharmacy, Hospital General Universitario de Elche, Elche, 03203 Spain
Received 8 October 2003; returned 25 November 2003; revised 9 December 2003; accepted 18 December 2003

Objectives: The mutant prevention concentration (MPC) has recently been defined to characterize the capacity for severely restricting the selection of resistant mutants during antibiotic treatment. We determined this parameter for ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin and linezolid in Mycobacterium tuberculosis clinical isolates in our setting. Methods: We determined the antibiotic concentration that prevents the selection of resistant mutants following inoculation with a high mycobacteria inoculum on Middlebrook 7H11 plates with serial dilutions of the antibiotics in 224 M. tuberculosis isolates. Results: Fifty percent of the strains exhibited values of MPC (MPC50) lower than 0.8, 0.6, 0.4, 0.4 and 0.6 mg/L for ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin and linezolid, respectively. If 90% of the strains are considered (MPC90), the values are seen to rise to 2, 1.8, 1, 1.2 and 1.2 mg/L, respectively. Conclusions: When we compare this parameter with the drug levels in serum and tissue, it can be seen that ciprofloxacin is the least useful of the fluoroquinolones studied, whereas moxifloxacin appears to be the most active. Linezolid exhibits excellent activity against this microorganism (MPC90 1.2 mg/L and AUC 140.3 mgh/L) and this makes us consider that its usefulness in the treatment of this pathology should be thoroughly evaluated.
Keywords: tuberculosis, drug activity, fluoroquinolones, oxazolidinones

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Introduction
In some cases, resistance to isoniazid and rifampicin may make it necessary to use new drugs in the treatment of tuberculosis. In order to design alternative guidelines for treatment,1 we calculated the mutant prevention concentration (MPC). This parameter was defined recently to characterize the capacity for preventing/severely restricting the emergence of drug-resistant mutants.2 We calculated this parameter for the fluoroquinolones and linezolid in Mycobacterium tuberculosis clinical isolates on the Mediterranean coast in the southeast of Spain.

studied.3,4 The strains were kept frozen at 70C. The strain Mt 14323 was processed for quality control.

Mutant prevention concentration (MPC)

The inocula were prepared from a culture in the Lowenstein medium; 5 L aliquots of a 2 McFarland suspension were inoculated on Middlebrook 7H11 plates (approximately 1010 microorganisms) with dilutions of antibiotics (mg/L) from 0.1 up to 4. The plates were incubated at 37C for 4 weeks. The MPCs were estimated by determining the concentration that allowed recovery of no colony.2

Antibiotics
The following antibiotics were used: ciprofloxacin (Bayer), levofloxacin (Hoechst), gatifloxacin (Bristol-Myers Squibb), moxifloxacin (Bayer) and linezolid (Pharmacia).

Materials and methods

Strains
Two hundred and twenty-four M. tuberculosis clinical isolates were obtained in the Elche health district between 1992 and 2003. For each strain, the following data were known: date of isolation, site of the disease, HIV serology of the patient and susceptibility to the antibiotics

Results
The MPC results are shown in Table 1. Fifty percent of the strains exhibited an MPC (MPC50) of less than 0.8, 0.6, 0.4, 0.4 and 0.6 mg/L for ciprofloxacin, levofloxacin,

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*Corresponding author. Tel: +34-6679493; Fax: +34-966679108; E-mail: micro_elx@gva.es

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J. C. Rodrguez et al.
Table 1. Distribution of the number of strains according to their MPC (mg/L) <0.1 CIP LVX GAT MXF LNZ 1 1 24 0 0 0.1 0 18 83 29 13 0.2 28 78 73 93 72 0.4 29 56 4 47 81 0.6 56 24 10 18 18 0.8 49 11 15 9 15 1.0 12 1 1 11 8 1.2 19 5 2 0 0 1.4 1 1 2 0 2 1.6 2 7 1 0 3 1.8 10 3 1 1 0 2.0 1 4 0 1 5 2.2 0 1 1 0 1 2.4 4 0 0 0 0 2.6 0 0 1 1 0 2.8 0 1 1 1 0 3.0 0 0 1 1 0 3.2 0 0 0 0 0 3.4 0 0 0 0 0 3.6 0 0 0 0 0 3.8 0 0 0 2 0 4 12 13 4 10 6

CIP, ciprofloxacin; LVX, levofloxacin; GAT, gatifloxacin; MXF, moxifloxacin; LNZ, linezolid.

Table 2. Comparison of maximum concentration reached in serum and area under the curve of the drugs studied Maximum concentration in serum (mg/L) 6.21 4.4 4.34 3.42 18.3 Total area under the curve, AUCtot (mgh/L) 4.830.8 44.8 4.4 30 3.8 39.3 5.35 140.3

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Antibiotic CIP10,13 LVX16 GAT8,21 MXF22 LNZ26,29

AUCtot/MPC50 638.5 74.7 75.0 98.2 233.8

AUCtot/MPC90 2.415.4 24.9 30 32.7 116.9

CIP, ciprofloxacin; LVX, levofloxacin; GAT, gatifloxacin; MXF, moxifloxacin; LNZ, linezolid.

gatifloxacin, moxifloxacin and linezolid, respectively. When 90% of the strains were considered (MPC90), the values rose to 2, 1.8, 1, 1.2 and 1.2 mg/L, respectively.

Conclusions
The mutant prevention concentration was more sensitive, since MIC measures the susceptibility of the dominant members of the population whereas MPC measures the susceptibility of mutant subpopulations. This new parameter has been reported to correlate more favourably with the genetic detection of mechanisms of resistance to fluoroquinolones in certain strains.5 It is very important to use sensitive tools to detect resistant individuals so that patients may be given the correct treatment and the true usefulness of an anti-tuberculous drug evaluated, since resistant mutants sometimes appear during treatment6 or relapses occur.7 This is probably related to the fact that in some types of tissue, the drug concentration is less than the mutant prevention concentration of the strains. Failure, relapses and the selection of resistant mutants during treatment are complex phenomena associated with the characteristics of the microorganism together with the characteristics and dosage of the drug used. Also, the drug levels reached vary depending on the type of tissue and patient concerned.8 Ciprofloxacin is the fluoroquinolone that has been most widely used in the treatment of tuberculosis. It has been used as a second line drug because it exhibits less activity than traditional treatments.9 However, the usefulness of the drug in the treatment of tuberculosis10

and its bactericidal activity11 have been seen to be dose-dependent, since the area under the curve varies between 4.8 and 30.8 mgh/L depending on the dose given. Ciprofloxacin has been reported12 to reach a maximum concentration in serum of 6.21 mg/L and a concentration between 1.7 and 7.1 times higher in pulmonary tissue. However, the serum and tissue levels decrease 812 h after the drug has been administered (0.13 mg/L and 0.67 mg/kg, respectively);13 therefore, it is important to take into consideration the period of time that the MPC is greater than the drug concentration in tissue.14 When we compare the activity of the different fluoroquinolones (Table 2), ciprofloxacin is the least active. This is shown by the low values of the quotient AUCtot/MPC and the fact that its alveolar macrophage levels are lower than those of other fluoroquinolones such as levofloxacin (13.8 versus 6.8 mg/L).15 The activity of the other three fluoroquinolones is comparable, although gatifloxacin and especially moxifloxacin appear to be the ones that exhibit the greatest activity and the lowest probability of resistant mutants being selected.16,17 Our findings are in agreement with those reported by Hu et al.,18 who point out that moxifloxacin and gatifloxacin exhibit a greater sterilizing capacity than levofloxacin in an in vitro model. Moreover, the fact that the fluoroquinolones penetrate the cells very well also contributes to the potential usefulness of these compounds in the treatment of tuberculosis.19 Twelve hours after the drugs were administered, the following concentrations of gatifloxacin and moxifloxacin (which were higher than the MPC for most of the strains) were achieved: in epithelial lining fluid (2.98 and 5.9 mg/L), alveolar macrophages (61.95 and

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Mycobacterium tuberculosis and mutant prevention concentration

54.1 mg/L) and bronchial mucosa (3.00 and 2.0 mg/kg), respectively.20,21 However, low doses may be found in certain types of tissue: in CSF 1 h after administration, the gatifloxacin concentration is between 0.46 and 1.84 mg/L depending on the dose given22 and 2 h after administration, the levofloxacin concentration is 1.99 mg/L.23 On the other hand, moxifloxacin reaches higher concentrations in CSF: in a rabbit model, between 9.5 and 1.8 mg/L is found depending on the dose given24 and in bone, the concentration reaches 6.3 mg/L.25 Our data confirm the good activity of moxifloxacin and gatifloxacin in murine models, where they are seen to exhibit similar bactericidal activity to that of isoniazid, although their activity is dependent on the dose administered.26,27 The data obtained on linezolid indicate excellent activity against the M. tuberculosis strains studied, as a result of the high drug concentration in serum and tissue (Table 2), with a concentration of 10.7 mg/kg being reached in bronchial mucosa, 8.1 mg/L in alveolar macrophages and 25.1 mg/L in epithelial lining fluid.28,29 These data confirm the results obtained with a murine model.30 The key to carrying out effective treatment and preventing the selection of resistant mutants is to choose the right antibiotic, the correct dose and complete the treatment, or to use two or more drugs.31,32 In this way, the drug concentration in serum and tissue will be above the MPC for the M. tuberculosis strain for the longest period possible. Therefore, it can be of help in determining the true usefulness of each individual drug in the treatment of different clinical situations, especially in cases of multidrug-resistant microorganisms or patients who find it difficult to adhere to treatment.33 Our findings indicate that levofloxacin, gatifloxacin and especially moxifloxacin are good alternative therapies and superior to ciprofloxacin. Also, linezolid appears to be a drug that achieves very good results and so its true usefulness in the treatment of tuberculosis should be evaluated.
9. Kennedy, N., Berger, L., Curram, J. et al. (1996). Randomized controlled trial of a drug regimen that includes ciprofloxacin for the treatment of pulmonary tuberculosis. Clinical Infectious Diseases 22, 82733. 10. Bergstermann, H. & Ruchardt, A. (1997). Ciprofloxacin once daily versus twice daily for the treatment of pulmonary tuberculosis. Infection 25, 22732. 11. Sirgel, F. A., Botha, F. J., Parkin, D. P. et al. (1997). The early bactericidal activity of ciprofloxacin in patients with pulmonary tuberculosis. American Journal of Respiratory and Critical Care Medicine 156, 9015. 12. Israel, D., Gillum, G., Turik, M. et al. (1993). Pharmacokinetics and serum bactericidal titers of ciprofloxacin and ofloxacin following multiple oral doses in healthy volunteers. Antimicrobial Agents and Chemotherapy 37, 21939. 13. Birmingham, M. C., Guarino, R., Heller, A. et al. (1999). Ciprofloxacin concentrations in lung tissue following a single 400 mg intravenous dose. Journal of Antimicrobial Chemotherapy 43, 438. 14. Hansen, G. T., Metzler, K., Drlica, K. et al. (2003). Mutant prevention concentration of gemifloxacin for clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 47, 4401. 15. Gotfried, M. H., Danziger, L. H. & Rodvold, K. A. (2001). Steadystate plasma and intrapulmonary concentrations of levofloxacin and ciprofloxacin in healthy adult subjects. Chest 119, 111422. 16. Firsov, A. A., Vostrov, S. N., Lubenko, I. Y. et al. (2003). In vitro pharmacodynamic evaluation of the mutant selection window hypothesis using four fluoroquinolones against Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 47, 160413. 17. Wise, R. (2003). Maximizing efficacy and reducing the emergence of resistance. Journal of Antimicrobial Chemotherapy 51, Suppl. S1, 3742. 18. Hu, Y., Coates, A. R. & Mitchison, D. A. (2003). Sterilizing activities of fluoroquinolones against rifampin-tolerant populations of Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 47, 6537. 19. Ballesta, S., Pascual, A., Garcia, I. et al. (1996). Penetracion de cinco fluoroquinolonas en polimorfonucleares humanos. Enfermedades Infecciosas y Microbiologa Clnica 14, 4703. 20. Lober, S., Ziege, S., Rau, M. et al. (1999). Pharmacokinetics of gatifloxacin and interaction with an antacid containing aluminum and magnesium. Antimicrobial Agents and Chemotherapy 43, 106771. 21. Soman, A., Honeybourne, D., Andrews, J. et al. (1999). Concentrations of moxifloxacin in serum and pulmonary compartments following a single 400 mg oral dose in patients undergoing fibre-optic bronchoscopy. Journal of Antimicrobial Chemotherapy 44, 8358. 22. Lutsar, I., Friedland, I. R., Wubbel, L. et al. (1998). Pharmacodynamics of gatifloxacin in cerebrospinal fluid in experimental cephalosporinresistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 42, 26505. 23. Scotton, P. G., Pea, F., Giobbia, M. et al. (2001). Cerebrospinal fluid penetration of levofloxacin in patients with spontaneous acute bacterial meningitis. Clinical Infectious Diseases 33, 10911. 24. Cottagnoud, P., Gerber, C. M., Acosta, F. et al. (2000). Linezolid against penicillin-sensitive and -resistant pneumococci in the rabbit meningitis model. Journal of Antimicrobial Chemotherapy 46, 9815. 25. Lovering, A. M., Zhang, J., Bannister, G. C. et al. (2002). Penetration of linezolid into bone, fat, muscle and haematoma of patients undergoing routine hip replacement. Journal of Antimicrobial Chemotherapy 50, 737. 26. Yoshimatsu, T., Nuermberger, E., Tyagi, S. et al. (2002). Bactericidal activity of increasing daily and weekly doses of moxifloxacin in murine tuberculosis. Antimicrobial Agents and Chemotherapy 46, 18759. 27. Alvirez Freites, E. J., Carter, J. L. & Cynamon, M. H. (2002). In vitro and in vivo activities of gatifloxacin against Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 46, 10225. 28. Gee, T., Ellis, R., Marshall, G. et al. (2001). Pharmacokinetics and tissue penetration of linezolid following multiple oral doses. Antimicrobial Agents and Chemotherapy 45, 18436. 29. Honeybourne, D., Tobin, C., Jevons, G. et al. (2003). Intrapulmonary penetration of linezolid. Journal of Antimicrobial Chemotherapy 51, 14314.

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