Centro de Malária e outras Doenças Tropicais, Instituto de Higiéne e Medicina Tropical, Universidade Nova de Lisboa, Portugal
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Abstract |
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Introduction |
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The integrity of the mycobacterial cell wall is affected by a variety of antibiotics that inhibit the synthesis of specific cell wall components. At sub-lethal concentrations, these antibiotics could reduce the effectiveness of the organism's cell wall as a barrier. With this consideration in mind, we have investigated whether cell-wall-inhibitory antibiotics can improve the activity of clarithromycin against the resistant M. tuberculosis to clinically useful levels.
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Materials and methods |
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MICs were determined using the procedure recommended by the manufacturers of the Bactec 460 radiometric system.25,26 The MIC, corresponding to the drug concentration resulting in >99% inhibition of the bacterial population, was defined as the lowest concentration for which the GI of the drug-containing vial was less than the
GI of the 1:100 control, obtained from the reading after the GI had reached 30.
Studies involving combinations of drugs were carried out using subinhibitory concentrations of cell wall inhibitors and clarithromycin, alone or in combination. The combined effects were estimated using the X/Y quotient, as described by others,11,13 except that the final quotient was expressed as the average value obtained from various Bactec 460 readings during exponential growth. In these calculations, X refers to the Bactec GI obtained with the drug combination and Y to the lowest GI obtained at the same time with either drug used alone. An X/Y value of 1 indicates that there was no interaction between the two drugs, an X/Y value of <0.5 indicates synergy and an X/Y value of >2.0 indicates antagonism.
Ethambutol, isoniazid, rifampicin and streptomycin were purchased and prepared according to the manufacturer's recommendations (Becton Dickinson). d-Cycloserine, cerulenin, vancomycin, bacitracin, polyoxyethylenesorbitan monooleate (Tween 80) and dimethyl sulphoxide (DMSO) were purchased from Sigma Aldrich Quimica SA (Madrid, Spain). Clarithromycin (Abbott Laboratories, North Chicago, IL, USA) was kindly supplied by the manufacturer. Stock solutions of d-cycloserine, vancomycin, bacitracin and Tween 80 were prepared in sterile distilled water. Cerulenin and clarithromycin were dissolved in ethanol. All stock solutions were sterilized using 0.2 mm pore size filters, except for DMSO, which was autoclaved.
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Results and discussion |
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Results obtained with the other inhibitors are shown in Table II. Results obtained with vancomycin were similar to those obtained with bacitracin. Like these inhibitors, d-cycloserine, which has not been tested before for its ability to potentiate antibiotics in M. tuberculosis, also showed synergy for some strains. Strain variability was seen for cerulenin, as reported in another investigation involving a similar approach.28
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Cefepime, which is not normally used in antituberculosis chemotherapy because of its high MICs for this pathogen, has appreciable activity in the presence of ethambutol.27 Other investigations in this field consider the potentiation of antibiotics already in use against M. tuberculosis, or the potentiation of antibiotics in strains having acquired resistance to these same drugs, by their association with ethambutol or non-antibiotic, non-antimycobacterium specific compounds such as cerulenin, trans-cinnamic acid or DMSO.28,35 In a contradictory report, it has been suggested that clarithromycin may itself be responsible for the enhanced in vitro activity of various first-line antibiotics, including ethambutol.36 Considering the intrinsic resistance of M. tuberculosis towards this antibiotic and its ribosome-specific site of activity,24 however, we suggest that this may be a result of a mechanism other than disruption of the lipid barrier of the cell wall.
Our results show that clarithromycin resistance in M. tuberculosis can be reversed by subinhibitory concentrations of cell wall inhibitors. Under these conditions, this antibiotic was highly effective against clinical isolates of M. tuberculosis, including strains resistant to three or four antibiotics. These results offer new insight into the use of macrolide antibiotics in antituberculosis chemotherapy.
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Acknowledgments |
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Notes |
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References |
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Received 15 December 1999; returned 3 April 2000; revised 19 April 2000; accepted 22 May 2000