In vitro activity of linezolid, clarithromycin and moxifloxacin against clinical isolates of Mycobacterium kansasii

Remedios Guna1,2, Carlos Muñoz1, Victoria Domínguez1, Ángeles García-García2, Jorge Gálvez2, Jesús-Vicente de Julián-Ortiz2 and Rafael Borrás1,*

1 Departamento de Microbiología, Facultad de Medicina y Hospital Clínico Universitario, Universidad de Valencia, Av. Blasco Ibáñez 17, 46010 Valencia, Spain; 2 Unidad de Investigación de Diseño de Fármacos y Conectividad Molecular, Departamento de Química Física, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain


* Corresponding author. Fax: +34-963-987-836; Email: rafael.borras{at}uv.es

Received 27 October 2004; returned 15 December 2004; revised 1 March 2005; accepted 4 March 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To compare the activity of linezolid with a range of drugs used in the treatment of Mycobacterium kansasii infections.

Results: The percentages of resistant isolates against isoniazid, rifampicin and ethambutol were 2.9%, 1.9% and 2.9%, respectively. All isolates were susceptible to clarithromycin and moxifloxacin both with MIC90 values of 0.125 mg/L. Linezolid was active against all isolates with MIC50 and MIC90 values of 0.5 and 1 mg/L, respectively, both below the susceptibility breakpoint established for mycobacteria.

Conclusion: Linezolid, clarithromycin or moxifloxacin, could be used as alternative drugs for treatment of infections due to rifampicin-resistant isolates as well as short-course or intermittent therapy of M. kansasii lung disease.

Keywords: non-tuberculous mycobacteria , antimycobacterial agents , broth microdilution method


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Mycobacterium kansasii is a non-tuberculous mycobacterium that can cause infections in both immunocompetent and immunodeficient patients with underlying pulmonary disease, AIDS or cancer. This organism is considered to be the second most common aetiological agent, after Mycobacterium avium complex, in both pulmonary and disseminated infections due to mycobacteria other than Mycobacterium tuberculosis in most parts of the world.13

The current treatment of pulmonary disease caused by M. kansasii in non-HIV-infected patients, according to the American Thoracic Society recommendations, includes isoniazid, rifampicin and ethambutol.3 Nevertheless, alternative drugs have been proposed for patients with M. kansasii infections resistant to rifampicin4,5 and AIDS patients treated with HIV protease inhibitors, because rifampicin accelerates hepatic metabolism of these drugs rendering them potentially ineffective.6

In this study, we evaluated the in vitro activity of linezolid, against M. kansasii clinical isolates and compared its activity with other drugs included clarithromycin and moxifloxacin. Linezolid belongs to a new class of antimicrobial agents, the oxazolidinones, inhibitors of protein synthesis at an early stage by inhibiting the formation of functional 70S initiation complex.7,8 Moreover, this antibiotic has been selected by molecular topology as an antimycobacterial drug,9 showing activity against both slowly and rapidly growing mycobacteria7,10– and has been used successfully in the treatment of a disseminated infection due to a Mycobacterium chelonae clarithromycin-resistant isolate.13


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates

One hundred and four primary clinical isolates of M. kansasii belonging to 104 consecutive patients diagnosed during the last 11 years, were selected from a total of 158 strains in our laboratory collection (Departamento de Microbiología, Hospital Clínico Universitario, Valencia, Spain). They were obtained from respiratory and non-respiratory sites, and identified by conventional methods14 and DNA hybridization probes (Accuprobe; Gen Probe Inc., San Diego, CA, USA). The frozen stocks were grown in modified 7H9 broth (Difco Laboratories, Detroit, MI, USA) supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment (Difco Laboratories) and then identified as M. kansasii genotype 1 by a DNA reverse hybridization method (INNO-LiPA Mycobacteria, Innogenetics, Ghent, Belgium).15

Source of drugs

Ethambutol, isoniazid and rifampicin were purchased from Sigma Chemical Co. (St Louis, MO, USA), and the remaining antibiotics were kindly supplied by different pharmaceutical laboratories (clarithromycin, Abbott; linezolid, Pharmacia-Upjohn; moxifloxacin, Bayer).

MIC determination

Antimicrobial susceptibility testing was performed with a microdilution method previously described,16,17 with minor modifications. Briefly, initial drug dilutions were prepared in either dimethyl-sulphoxide (Merck, Darmstadt, Germany) or deionized water, and subsequent 2-fold dilutions from 16 to 0.06 mg/L were carried out in clear-bottomed 96-well microplates containing 150 µL/well of modified 7H9 broth pH 6.8, or pH 7.4 for the clarithromycin assays, plus OADC (Difco Laboratories). The culture suspensions were prepared in the same medium to yield an absorbance equivalent to that of a 0.5 McFarland standard and 10 µL of inoculum was added to the wells. Plates were sealed with Parafilm M (Pechiney Plastic Packaging, Menasha, WI, USA) and were incubated at 37°C for 7 days. Starting at day 8 of incubation, 20 µL of resazurin (Sigma Chemical Co.) at 0.025% (w/v), as oxidation–reduction colorimetric indicator of bacterial growth, was added to the wells. The microplates were resealed and reincubated at 37°C for an additional period of 48–72 h. The MIC was defined as the lowest drug concentration without a colour change from blue to pink. MIC50 and MIC90 were defined as the lowest drug concentration that inhibits the growth of 50% and 90% of isolates, respectively.

The MIC of linezolid was determined following the criteria previously established for mycobacteria.10,12 As a result of the absence of criteria for moxifloxacin susceptibility assay interpretation, the ciprofloxacin breakpoint recommended by NCCLS18 for M. kansasii has been used, since it is similar to that described for 8-methoxyfluoroquinolones and Staphylococcus19 and tentatively suggested for non-tuberculous mycobacteria.18 For the remaining drugs, NCCLS criteria for this organism were used18 (Table 1).


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Table 1. Number and cumulative percentage of isolates inhibited at indicated MIC, MIC50 and MIC90 and percentage of resistant isolates

 
To establish the influence of resazurin on MICs, before the antimicrobial susceptibility assays were performed, a quality control strain (M. kansasii CECT 3030) was tested using the same method, with and without resazurin. In all assays, the quality control strain and clinical isolates were tested in duplicate and the resistant isolates were tested again.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The quality control strain (M. kansasii CECT 3030) was susceptible to all drugs. The MICs obtained in both assays, with and without resazurin, were identical and major differences were not found in intra- and inter-microplate assays (data not shown). Interpretation was easier with resazurin.

The drugs used in conventional therapy, isoniazid, rifampicin and ethambutol, showed a good activity with MIC90s of 4, 0.25 and 4 mg/L, respectively. Only five isolates showed resistance: three of them were resistant to one drug (isoniazid, rifampicin or ethambutol); one isolate was resistant to both isoniazid and ethambutol, and another one showed resistance to all three standard therapy drugs. The second-line drugs, clarithromycin and moxifloxacin, also showed an excellent in vitro activity against M. kansasii, both with MIC90s of 0.125 mg/L (Table 1).

Linezolid was active against isolates both resistant and susceptible to first-line drugs. All isolates were inhibited by antibiotic concentrations lower than the susceptible breakpoint established for mycobacteria (≤8 mg/L).10,12 The MIC range was between 0.125 and 4 mg/L, with MIC50 and MIC90 of 0.5 and 1 mg/L, respectively (Table 1).

The high activity of isoniazid, rifampicin and ethambutol found in this study is at odds with the M. kansasii wild-type susceptibility pattern previously described by other authors. These differences could be related to the method of sensitivity testing or the prevailing susceptibility pattern of M. kansasii isolates found in a geographical area. The proportion of isolates resistant to isoniazid described by Wallace et al.20 (77.8%) and Garrós García et al.21 (10%) by microdilution and proportion methods, respectively, using 1 mg/L isoniazid as breakpoint, is greater than those detected by us (2.9%) following the NCCLS criteria (5 mg/L).18 However, the rate of isolates (68.3%) resistant to isoniazid (>1 mg/L) is similar to that described by Wallace et al.20 but disagrees with the report of Garrós García et al.21 Rifampicin and ethambutol resistance detected is also lower than those reported in USA by Wallace et al.20 who cited resistances of 37% and 25.9%, respectively, using similar criteria. Nevertheless, our results are similar to those found by Garrós García et al.21 in Spain, who reported resistances to rifampicin of 3.3% and ethambutol of 6.6%.

The current treatment of pulmonary infection due to M. kansasii in patients not receiving HIV protease inhibitors, according to the American Thoracic Society recommendations includes three drugs: isoniazid, rifampicin and ethambutol, since the untreated isolates are inhibited by these and other drugs at serum concentrations achievable with usual therapeutic doses.3 Nevertheless, due to the common in vitro resistance of M. kansasii to isoniazid,20 the inclusion of this drug in the therapeutic regimen is controversial and its exclusion has been proposed.22

The low rate of resistance found to ethambutol and rifampicin in our health district supports the use of these drugs for the initial treatment of M. kansasii infections. Nevertheless, the good activity of isoniazid against M. kansasii detected in this study, the low frequency of relapses after treatment with isoniazid, rifampicin and ethambutol compared to the regimen with rifampicin and ethambutol,23,24 the synergic effect of isoniazid and clinical experience23 indicate the empirical use of triple therapy until susceptibility results are available.

The clarithromycin activity found in this study is similar to that described by other authors who reported MIC50 and MIC90 values between ≤0.125 and 0.25 mg/L, and 0.25 and 0.5 mg/L, respectively,4,25,26 but disagrees with the study of Yew et al.27 who report MIC90 of 1 mg/L. Likewise, the results obtained with moxifloxacin agree with the report of Gillespie and Billington28 who described MIC50 and MIC90 values of ≤0.06 and 0.06 mg/L, although they are lower than the data of Rodríguez Díaz et al.11 who by an agar dilution method obtained MIC50 and MIC90 values of 0.125 and 2 mg/L, respectively. These findings, together with the tissue distribution of both agents,29,30 support their use as alternative drugs in the treatment of M. kansasii infections in patients with therapy failure due to rifampicin resistance and in AIDS patients being treated with HIV protease inhibitors, as has been previously reported.4

Only two in vitro studies of linezolid activity against M. kansasii have been reported.10,11 One of them reported MIC50 and MIC90 values of 4 and >32 mg/L, respectively.11 However, our results (MIC50: 0.5 mg/L; MIC90: 1 mg/L) showed a good activity with 100% susceptible isolates, since they were inhibited at antibiotic concentrations lower than the susceptible breakpoint (≤8 mg/L) established for mycobacteria,12 previously described by Brown-Elliot et al.10 for this organism. Recently, the activity of this agent has been described in a murine experimental model of M. kansasii infection, although clarithromycin and rifampicin or their combination work better than linezolid, alone or in combination with clarithromycin.31 These findings, the pharmacokinetic features32 and the possibility to treat non-tuberculous mycobacterial infections using linezolid 600 mg once-daily dosing, as Brown-Elliot et al.10 cite, suggest that this antibiotic may be used in multi-drug therapy for the treatment of M. kansasii disease when rifampicin resistance is suspected.

The low MICs obtained in this study with clarithromycin, linezolid and moxifloxacin and the successful treatment of M. kansasii lung disease with a thrice-weekly clarithromycin-containing regimen reported by Griffith et al.33 raise the possibility of intermittent or short treatment regimens containing one of these drugs. Nevertheless, more studies are required to demonstrate the relationship between the microbiological data and the clinical effectiveness of linezolid in the treatment of M. kansasii infections.


    Acknowledgements
 
We acknowledge financial support for this research from the Spanish Ministry of Science and Technology (SAF2000-0223-C03-02).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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