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FEMS Microbiology Letters 251 (2005) 19–22

In vitro and ex vivo antimycobacterial potential of azole drugs againstMycobacterium tuberculosis H37Rv

Zahoor Ahmad, Sadhna Sharma, G.K. Khuller *

Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India

Received 4 April 2005; received in revised form 7 July 2005; accepted 18 July 2005

First published online 8 August 2005

Edited by R.S. Buxton

Abstract

The antimycobacterial activity of two clinically approved antifungal azole drugs, clotrimazole and econazole, was evaluatedagainst Mycobacterium tuberculosis H37Rv under in vitro and ex vivo conditions. The minimum inhibitory concentration(MIC90) was 0.120 lg ml�1, whereas the minimum bactericidal concentration and effective concentration was 0.125 lg ml�1 for boththe drugs demonstrating their potent antimycobacterial activity. Further, the azole drugs exhibited a synergistic activity with eitherrifampicin or isoniazid as evaluated on the basis of reduction of colony forming units. The results suggest that azole compoundsbear the potential to enhance the efficacy of currently prescribed antitubercular drugs.� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Clotrimazole; Econazole; Antitubercular drugs; Mycobacteria

1. Introduction

The fact that any new drug development, right fromits inception to manufacturing, entails a tremendous in-put in terms of research efforts, time and cost, need notbe over emphasized. The rising incidence of tuberculosis(TB), especially the multidrug resistant form (MDR-TB), calls for development of new drugs with potentantimycobacterial activity. It is rather unfortunate thatapart from the long acting rifamycins, no significantaddition has been made to the armamentarium of anti-tubercular drugs in the recent years. However, the de-tailed analysis of the genome of Mycobacterium

tuberculosis has offered some insights and the potentialopportunities in this direction [1]. It has been demon-strated that 14a-demethylase is an important mycobac-

0378-1097/$22.00 � 2005 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2005.07.022

* Corresponding author. Tel.: +91 0172 2755175/2747585; fax: +910172 2744 401/2745 078.

E-mail address: gkkhuller@yahoo.co.in (G.K. Khuller).

terial protein, which is probably required for sterolbiosynthesis [2]. A similar enzyme (truly involved insterol synthesis) is known to be present in fungi and isinhibited by azole compounds or their derivatives, whichexplains their antifungal activity [3]. Therefore, it istempting to speculate that azoles might exhibit a similarinhibitory activity in mycobacteria. Indeed, experimentshave shown that the growth of Mycobacterium smegma-tis, which is a saprophytic strain of mycobacteria, wasinhibited by azoles [4]. However, the compounds meritevaluation against virulent strains of mycobacteria suchas M. tuberculosis H37Rv in order to substantiate theiruse as antitubercular drugs and this issue has been ad-dressed in the present study. Two clinically approvedazole compounds, i.e., clotrimazole and econazole weretested for this purpose under in vitro and ex vivo condi-tions. In addition, the effect of combining azoles withkey frontline antitubercular drugs, rifampicin andisoniazid, was also assessed and the results are beingcommunicated here.

. Published by Elsevier B.V. All rights reserved.

20 Z. Ahmad et al. / FEMS Microbiology Letters 251 (2005) 19–22

2. Materials and methods

2.1. Chemicals and drugs

Econazole, clotrimazole, isoniazid, rifampicin andRPMI 1640 were obtained from Sigma Chemical Co.(St. Louis, MO, USA). Middlebrook 7H10 agar andOADC were obtained from Beckton and Dickinson,USA. All other reagents were of analytical gradeobtained from standard companies

2.2. Animals

Laca mice of either sex weighing 20–25 g obtainedfrom the Central Animal House, Post Graduate Insti-tute of Medical Education and Research, Chandigarh(India) were used in the study. Animals were housedin biosafety cabinets (Nuaire Instruments, NU 605-600E, Series 6) and were given pellet diet and waterad libitum. The study was approved by the Institute�sAnimal Ethics Committee.

2.3. Culture

Mycobacterium tuberculosis H37Rv, originallyobtained from National Collection of Type Cultures(NCTC, London) was maintained on modifiedYouman�s medium.

2.4. Effect of econazole and clotrimazole on the growth ofM. tuberculosis H37Rv

Mycobacterium tuberculosis H37Rv was grown at37 �C under shaking conditions in Youman�s mediumin presence of either clotrimazole or econazole at vary-ing concentrations ranging from 0.00 to 0.15 lg ml�1

for up to 8 days (a similar time period was required toattain maximum growth in case of drug free medium).50 ll of undiluted and 1:100 diluted cultures were platedon Middlebrook 7H10 agar plates supplemented withOADC. The plates were incubated at 37 �C for 28 daysfollowed by the enumeration of colony forming units(cfu). The minimum inhibitory concentration (MIC90)was defined as the lowest drug concentration at which90% of growth was inhibited on the basis of cfuenumeration.

Minimum bactericidal concentration (MBC), i.e., thelowest drug concentration that causes total cell deathwas evaluated as described by Colin et al. [4]. Youman�smedium (100 ml) containing either clotrimazole or econ-azole at varying concentrations (0.01–0.25 lg ml�1) wasinoculated with M. tuberculosis H37Rv at a density of1 · 105 cells ml�1. The cultures were incubated at 37 �Cfor 8 days to allow full growth. Afterwards, the cultureswith no visible growth were sub-cultured in drug freebroth (5 ml aliquots were taken to inoculate 100 ml

broth). The conditions and time of incubation was sameas described above. Later on 50 ll of undiluted/1:100 di-luted aliquots were plated on Middlebrook 7H10 agarplates for enumeration of cfu. The MBC was consideredto be the lowest drug concentration at which no growthwas observed (potentially equalivent to 99.99%inhibition).

2.5. Effect of azole drugs in combination with conventional

antitubercular drugs on the growth of M. tuberculosis

H37Rv

The synergistic/additive/antagonist effects of azoleswith antitubercular drugs were evaluated by growingM. tuberculosis H37Rv in presence of different combina-tions of azoles + antitubercular drugs, i.e., (i) econazole;(ii) clotrimazole; (iii) isoniazid; (iv) rifampicin; (v) clo-trimazole + rifampicin; (vi) clotrimazole + isoniazid;(vii) econazole + rifampicin; (viii) econazole + isoniazid(ix) drug free controls. In each experiment involving twoantibiotics, the drugs were mixed to produce an initialconcentration in the medium, which is equal to 1 + 1MIC of each. Subsequently, 3 serial dilutions were madeto obtain 1/2, 1/4 and 1/8 MIC concentrations. Inocula-tion of the organism and the determination of MIC ofeach drug alone and in combination were conductedas described above. The fractional inhibitory concentra-tion (FIC) index was calculated by the standard formulaas described earlier [5]:

FIC ¼ MIC combinationa

MIC aloneaþMIC combinationb

MIC aloneb;

where a and b are two antimicrobial agents.

2.6. Ex vivo studies

Mice were infected via the lateral tail vein with1.5 · 105 viable bacilli of M. tuberculosis H37Rv in0.1 ml of 0.9% NaCl. Fifteen days later, the mice weresacrificed, the spleens were removed and the spleno-cytes were isolated under sterile conditions [6]. Thecells were distributed in 24 – well tissue culture platesso as to give a concentration of 106 cells/well. Theazole drugs were added at either lowest experimentalconcentration/MBC concentration separately to tripli-cate wells. The petri plates were kept in a CO2 incu-bator maintained at 37 �C and 5% CO2. At day 4and day 8 post-incubation, 50 ll aliquots from eachwell were plated on Middlebrook medium for cfu enu-meration. Colonies were counted on day 28-post inoc-ulation. The lowest drug concentration that showed99% reduction in cfu compared to the drug – free in-fected splenocytes was considered to be the effectiveconcentration (EC) of the drug under ex vivoconditions.

Z. Ahmad et al. / FEMS Microbiology Letters 251 (2005) 19–22 21

3. Results and discussion

Table 1 shows the percent growth inhibition ofM. tuberculosis H37Rv in the presence of clotrimazole/econazole on the basis of cfu enumeration. Both thedrugs inhibited the growth in a concentration dependentmanner. Fifty percent reduction in growth was observedat lowest experimental concentration (0.05 lg ml�1) ofeconazole as well as clotrimazole whereas 90%inhibition (MIC90) was observed at 0.12 lg ml�1 of eachdrug (Table 1). Further, no growth was observed indrug-free medium inoculated from drug-treated culturescontaining 0.125 lg ml�1 of azole drugs. Hence,0.125 lg ml�1 was considered to be the MBC for clo-trimazole as well ass econazole. These results demon-strate that econazole and clotrimazole are potentinhibitors of M. tuberculosis H37Rv with low MIC/MBC values. In fact, they were more potent inhibitorsin comparison to the known antitubercular drug rifam-picin, whose MIC was found to be 0.2 lg ml�1. The ob-served MIC and MBC values are in agreement with thevalues reported for these drugs againstM. smegmatis [2].However, in the present study, isoniazid was found to bemore potent against M. tuberculosis whereas the drug isknown to have a low potency against M. smegmatis.This difference can be explained on the basis that M.

smegmatis is generally resistant to isoniazid as comparedto M. tuberculosis. Further, the bactericidal action ofazole(s) has been attributed to its multiple targets inM. tuberculosis as supported by the presence of a largenumber of CYP450s in this organism and the in vitrotight binding of azoles drugs to CYP121 [2]. Whatevermay be the cellular target(s), our observations demon-strate the strong antimycobacterial potential of azoledrugs against M. tuberculosis.

As tuberculosis requires multidrug administration, itbecomes essential to evaluate the interaction index orfractional inhibitory concentration of any compoundto be used as an antitubercular drug along with existingdrugs [9]. The combined effects of econazole/clotrima-

Table 1Concentration dependent effect of azoles on growth of Mycobacterium

tuberculosis H37Rv

Drugconcentration(lg ml�1)

% Inhibition

Clotrimazole Econazole

0 0 (5.8 ± 0.1) 0 (5.8 ± 0.1)0.025 28.43 (5.64 ± 0.07) 26.56 (5.64 ± 0.07)0.050 48.13 (5.5 ± 0.05) 46.56 (5.53 ± 0.09)0.075 71.25 (5.26 ± 0.1) 70.93 (5.27 ± 0.08)0.100 81.56 (5.06 ± 0.1) 81.87 (5.06 ± 0.12)0.11 86.46 (4.85 ± 0.04) 86.59 (4.84 ± 0.02)0.12 92.97 (4.57 ± 0.01) 93.99 (4.49 ± 0.1)0.125 >99.99 (undetectable) >99.99 (undetectable)

Values are mean ± SD of three independent experiments. Numbers inparenthesis indicate log10 cfu.

zole plus rifampicin/isoniazid on M. tuberculosis

H37Rv are presented in Table 2. These results indicatemarginal synergistic inhibitory activity and the effectwas evident even at 1/4th MIC of drugs when used incombination. This observation is important because itis known that the reduction of MIC/MBC values ofantibiotics increases their effective periods underin vivo conditions and this may help in reducing thedrug dosing frequency [7].

The requirement of prolonged chemotherapy to pre-vent relapse in TB patients suggests that in addition tothe population of actively growing mycobacteria, thereis a subset of non-dividing organisms (persisters) in theinfected tissue that remain relatively resistant to the ac-tion of drugs [8]. Therefore, the antimycobacterial activ-ity of any new molecule under in vitro conditions maylead to false conclusions unless evaluated under the con-ditions that resemble the in vivo conditions. To addressthis problem, azole antifungals were evaluated in anex vivo culture model that mimics the complex in vivoenvironment encountered by M. tuberculosis duringinfection. There was a significant reduction in cfu in in-fected splenocytes following exposure to econazole orclotrimazole as compared to controls. At the lowestexperimental concentration, the reduction in cfu was al-most 50% and at MBC the reduction was >99% as com-pared to controls (Table 3). Furthermore, it wasobserved that neither clotrimazole nor econazole hadany effect on the viability of splenocytes, at the concen-trations reported here. These results indicate that thereduction in colony counts was due to the bactericidalactivity of azole drug (a true ex vivo effect) and notdue to any direct lethal effect on the splenocytes. Thenon-toxicity of these drugs at the concentrations usedin the present study is in confirmation with previousstudies [10]. It is also emphasized that the ratio of EC/MIC, which is a measure of intracellular bioavailabilityand drug metabolism, was found to be unity demon-strating the potent antibacterial activity of clotrimazoleand econazole under ex vivo conditions. An EC/MIC

Table 2Synergistic activity of azoles with antitubercular drugs

Drug MIC90 (lg ml�1) FIC index

Alone Combination

Econazole 0.120 0.031a 0.5Isoniazid 0.100 0.025b 0.5Clotrimazole 0.120 0.031c 0.5Rifampicin 0.200 0.050d 0.5

Three different replicates yielded the same values. The individual MICwas: econazole/clotrimazole 0.120 lg ml�1, RIF 0.2 lg ml�1 and INH0.1 lg ml�1.a Econazole (0.031) + isoniazid (0.025)/rifampicin (0.050).b Isoniazid (0.025) + econazole (0.031)/clotrimazole (0.031).c Clotrimazole (0.031) + isoniazid (0.025)/rifampicin (0.050).d Rifampicin (0.050) + clotrimazole (0.031)/econazole (0.031). An

FIC index 60.5 indicates synergistic activity.

Table 3Ex vivo bactericidal activity of econazole and clotrimazole

Drug concentration (lg ml�1) Log cfu ml�1 after 4 days of incubation Log cfu ml�1 after 8 days of incubation

Control 4.07 ± 0.06 4.09 ± 0.02Econazole (0.06) 3.69 ± 0.06* 3.66 ± 0.06**Econazole (0.125) Undetectable UndetectableClotrimazole (0.06) 3.73 ± 0.06* 3.65 ± 0.07**Clotrimazole (0.125) Undetectable Undetectable

Values are mean ± SD of three independent experiments. *p < 0.01, **p < 0.001 as compared to control. Initial load of bacteria was 4.02 ± 0.03 logcfu ml�1.

22 Z. Ahmad et al. / FEMS Microbiology Letters 251 (2005) 19–22

ratio of <16 has been correlated with excellent intracel-lular antimycobacterial activity [7].

The present communication is the first report demon-strating the antimycobacterial potential of azole drugsagainst M. tuberculosis H37Rv under in vitro andex vivo conditions. The study also highlights their syner-gistic activity with isoniazid and rifampicin, two of thekey front line antitubercular drugs. Further studies car-ried out in this direction may clearly substantiate the po-tential of azole drugs to enhance the efficacy ofconventional antimycobacterial agents.

References

[1] Guardiola-Diaz, H.M., Foster, L.A., Mushrush, D. and Vaz,A.D.N. (2001) Azole-antifungal binding to a novel cytochromeP450 from Mycobacterium tuberculosis. Biochemical Pharmacol-ogy 61, 1463–1470.

[2] McLean, K.J., Marshall, K.R., Richmond, A., Hunter, I.S.,Fowler, K., Kieser, T., Gurcha, S.S., Besra, G.S. and Munro,A.W. (2002) Azole antifungals are potent inhibitors of cyto-chrome P450 mono-oxygenases and bacterial growth in myco-bacteria and streptomycetes. Microbiology 148, 2937–2949.

[3] Hartman, P.G. (1997) Inhibitors of ergosterol biosynthesis asantifungal agents. Current Pharmaceutical Design 3, 177–208.

[4] Colin, J., Jackson, D.C., Lamb, Kelly, D.E. and Kelly, S.L. (2000)Bactericidal and inhibitory effects of azole antifungal compoundson Mycobacterium smegmatis. FEMS Microbiol. Lett. 192, 159–162.

[5] Kalita, A., Verma, I. and Khuller, G.K. (2004) Role of humanNeutrophil peptide-1 as a possible adjunct to antituberculosischemotherapy. J. Infec. Dis. 190, 1476–1480.

[6] Turner, D.J., Hoyle, S.L., Snewin, V.A., Marie-Pierre, G.,Brown, I.N. and Young, D.B. (2002) An ex vivoculture model for screening drug activity against in vivophenotypes of Mycobacterium tuberculosis. Microbiology 148,2929–2936.

[7] Michael, J.H. and Michael, H.C. (2004) Design and synthesis ofantitubercular: preparation and evaluation against Mycobacte-rium tuberculosis of an isoniazid Schiff base. J. Antimicrob.Chemother. 53, 185–191.

[8] Betts, J.C., Lukey, P.T. and Robb, L.C. (2002) Evaluation of anutrient starvation model of Mycobacterium tuberculosis persis-tence by gene and protein expression profiling. Mol. Microbiol.43, 717–731.

[9] Leonid, B.H., Michael, D.I. and Pamela, J.L. (1998) Combi-nations of rifampin or rifabutin plus ethambutol againstMycobacterium avium complex. Am. Rev. Resp. Dis. 137,711–715.

[10] Benko, F., Hernadi, A., Megyeri, A., Kiss, G., Somogyi, Z.,Tegyey, F., Kraicsovits and Kovacs, P. (1999) Comparison of thetoxicity of fluconazole and other azole antifungal drugs to murineand human granulocytes-macrophage progenitor cells in vitro. J.Antimicrob. Chemother. 43, 675–681.