Intrinsic resistance of Mycobacterium tuberculosis to clarithromycin is effectively reversed by subinhibitory concentrations of cell wall inhibitors

Suzana Bosne-David*, Vanessa Barros, Sandra Cabo Verde, Clara Portugal and Hugo L. David

Centro de Malária e outras Doenças Tropicais, Instituto de Higiéne e Medicina Tropical, Universidade Nova de Lisboa, Portugal


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Subinhibitory concentrations of bacitracin, vancomycin and other inhibitors of cell wall synthesis reversed to varying extents the intrinsic resistance of Mycobacterium tuberculosis to clarithromycin. Ethambutol reversed clarithromycin resistance in all of the M. tuberculosis strains studied regardless of their susceptibility to this drug.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Natural or intrinsic multiple drug resistance of Mycobacterium spp. has been attributed to the organization and chemical nature of lipids in the outer layer of the tripartite cell wall.16 The effectiveness of this barrier can be modified by non-lethal biosynthetic inhibitors that can disrupt the organization of the outer layer or by compounds containing hydrophobic branches, which can interact with the amphipathic components of the outer layer.715 Many of these studies have been carried out on Mycobacterium avium since this species is characteristically resistant to the antibiotics normally active against Mycobacterium tuberculosis. In contrast, clarithromycin, a 6-O-methyl hydrophobic derivative of erythromycin, is active against M. avium,16,17 and so is used to manage this and other non-tubercular mycobacterial infections,18,19 whereas it is effective against M. tuberculosis only at very high concentrations.2023 The fact that it does have an inhibitory effect against both species suggests that M. avium and M. tuberculosis have a common site where clarithromycin is active and that the resistance of the latter might result from a target having a much lower affinity for clarithromycin or from the cell wall of the organism acting as a substantial barrier to clarithromycin. Recently, Doucet-Populaire and collaborators24 reported that the affinity of clarithromycin for its ribosomal target did not differ significantly between M. avium and the intrinsically resistant Mycobacterium smegmatis. Thus, whichever situation limits the effectiveness of clarithromycin against M. tuberculosis, high concentrations of the drug would bring about the observed inhibition of growth.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
M. tuberculosis H37Rv and 22 clinical isolates were used. Their susceptibility to first-line antibiotics was determined using Bactec 460 radiometric criteria (Becton Dickinson, Sparks, MD, USA). A control was prepared containing a 102-fold dilution of the initial innoculum used in the drug-containing vials (‘1:100 control’). Incubation was continued for a further day after the growth index (GI) of the 1:100 control vial had reached 30; the GI was then read after this additional day of incubation. The difference in the GI values ({triangleup}GI) was calculated for these 2 days. In this method, a drug is considered effective if it reduces bacterial growth by >=99% compared with a vial inoculated with the same initial inoculum but no drugs. Thus if the {triangleup}GI of the drug-containing vial is less than the {triangleup}GI of the 1:100 control, bacteria are considered susceptible, while if it is greater they are considered resistant.

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 {triangleup}GI of the drug-containing vial was less than the {triangleup}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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The susceptibility of each M. tuberculosis strain to isoniazid, rifampicin, streptomycin and ethambutol is summarized in Table IGo. The MIC of clarithromycin was >4.0 mg/L for all the strains tested while the MIC of ethambutol varied. A combination of ethambutol and clarithromycin at individual concentrations that were below their MICs yielded significant inhibition in the growth of all strains regardless of their susceptibility to ethambutol, as can be seen by the X/Y values obtained (Table IGo). Bacterial counts (cfu/mL) at the end of the experiments showed that the bacterial population in vials containing the drug combination was 1 log lower than that in vials with either drug alone, corresponding to 90% inhibition (not shown). Antagonism was not apparent in these experiments (Table IGo) or when ethambutol at concentrations above the MIC was combined with clarithromycin (not shown).


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Table I. Results of combined action of subinhibitory concentrations of ethambutol and clarithromycin against M. tuberculosis H37Rv and ethambutol-sensitive and -resistant clinical isolates of tubercle bacilli
 
Ethambutol has shown promise as a potentiator of antibiotic activity in M. tuberculosis27,28 and is a key drug for this purpose against the M. avium complex.11 It affects the arabinogalactan structure of the mycobacterial cell wall, and probably inhibits arabinan biosynthesis.29,30 From our results, we suspect that, besides this target, ethambutol may have other, non-lethal, mechanisms of activity that can alter cell wall permeability. Others have also suggested that ethambutol may have secondary mechanisms of action,3133 including detergent properties.34 Our results support these propositions: we found synergy between clarithromycin 2 mg/L and 0.05% Tween 80 (X/Y = 0.27 ± 0.02) and between clarithromycin 2 mg/L and 2.5% DMSO (X/Y = 0.093 ± 0.04) against M. tuberculosis H37Rv.

Results obtained with the other inhibitors are shown in Table IIGo. 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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Table II. Results of drug combination studies with cell-wall-inhibiting compounds and clarithromycin for five clinical strains of M. tuberculosis and H37Rv
 
Our observations suggest that the mechanism of resistance to clarithromycin in M. tuberculosis involves a permeability barrier, supporting the results of an independent investigation of clarithromycin's mechanism of action in M. smegmatis.24 Also significant was a clarithromycin MIC of 0.2 mg/L for one M. tuberculosis strain from our collection, strain 23797 (this very sensitive strain was not tested for enhancement to this drug).

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.


    Acknowledgments
 
We thank Núria Martín-Casabona, Vall d'Hebron Hospital, Barcelona, for some of the strains used in this study. We also thank Leonard Amaral for his helpful advice in the preparation of this manuscript. These investigations were supported financially by the Gulbenkian Foundation, the Comissão Nacional de Luta Contra a SIDA and the Fundação para a Ciência e Tecnologia (Portugal).


    Notes
 
* Corresponding author. Tel. and Fax: +351-(1)362-24-58; E-mail: suzanadavid{at}ihmt.unl.pt Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received 15 December 1999; returned 3 April 2000; revised 19 April 2000; accepted 22 May 2000