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Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates.

Mycobacterium tuberculosis, the aetiological agent of tuberculosis (TB), has re-emerged as a killer pathogen in western countries after the increase of HIV-AIDS cases and development of resistance to various anti-tubercular drugs. This increase in the number of multi-drug resistant M. tuberculosis isolates has drawn the attention towards the identification of alternate drugs like fluoroquinolones (FQs) for the treatment of TB. It is known that M. tuberculosis commonly acquires drug resistant phenotype by accumulation of mutations in the structural genes encoding the drug target or the enzymes involved in drug activation. Other known cause of drug resistance in mycobacteria is efflux of drug molecules (1). The principal cellular target of the FQs is the DNA gyrase encoded by gyrA and gyrB genes. Mutation in the quinolone resistance determining region (QRDR) of gyrA was the most common cause of FQ resistance in various organisms (2,3). However, studies carried out in India have reported that only 11.7 (4) and 45 per cent5 of ofloxacin resistant M. tuberculosis isolates harbour mutations in their gyrA gene and no mutation was found in gyrB gene. As mutations in DNA gyrase alone do not account for the mechanism(s) of resistance in a significant proportion of FQs resistant M. tuberculosis isolates, it suggests the need to investigate the role of alternate mechanisms, like eflux pumps. The upregulation of eflux systems can significantly decrease the intracellular concentration of many antibiotics, reducing their clinical efficacy. For this reason attention has been focused on identifying inhibitors of the eflux systems of Gram-negative and Gram-positive bacteria that could potentially be used in combination with antibiotics to improve efficacy and abolish resistance (1). Banerjee and co-workers (6) observed that carbonyl cyanide m chlorophenyl hydrazone (CCCP), verapamil and 2,4-dinitro phenol (DNP) increased the accumulation of drug possibly due to inhibition of active eflux. Several mycobacterial eflux pumps associated with FQs resistance have been described. These eflux pumps include the pumps of Major Facilitator Superfamily (MFS) family (lfA, Rv1634 and Rv1258c) and ATP Binding Cassette (ABC) transporters (DrrAB, PstB and Rv2686c-2687c-2688c) (1). For better understanding of drug resistance and to find out the newer drugs and/or identify suitable drug targets for better treatment of TB, there is a need to understand the exact mechanism(s) of resistance to FQs in M. tuberculosis. In the present study we have studied the effect of certain eflux inhibitors on in vitro susceptibility levels in ofloxacin (OFL)-resistant clinical M. tuberculosis isolates.

Material & Methods

M. tuberculosis isolates: The study was performed in the National JALMA Institute for Leprosy & Other Mycobacterial Diseases, Agra. A total of 55 clinical isolates of M. tuberculosis along with M. tuberculosis reference strain [H.sub.37]Rv were included in the present study. Isolates were obtained from Mycobacterial Repository Centre of the Institute, which were deposited in the repository from July 2004 through January 2008. These included isolates from Agra (n=45), Delhi (n=3), Kanpur (n=3), Varanasi (n=2), Allahabad (n=1) and Jaipur (n=1). Oloxacin-resistant M. tuberculosis isolates (n=45) had ofloxacin MIC of [greater than or equal to] 4 mg/l tested by Lowenstein-Jensen (L-J) method. Of the 45 OFL-resistant isolates, 31 belonged to the MDR group. Ten M. tuberculosis isolates were oloxacin-sensitive with MIC <2-4 mg/l. All the M. tuberculosis isolates were biochemically identified (7).

Effect of efflux inhibitors on minimum inhibitory concentration (MIC) levels of OFL : To determine the extent of the efflux pump mediated ofloxacin resistance in M. tuberculosis isolates, MIC levels for oloxacin were determined using Resazurin microtitre assay (8) in the presence or absence of eflux pump inhibitors [CCCP and DNP and verapamil (Sigma, USA)]. CCCP and DNP are the proton motive force inhibitors whereas verapamil is a calcium channel blocker for ABC transporters (6). Stock solution of CCCP and DNP was prepared in DMSO while verapamil was dissolved in distilled water. Final concentrations used in Resazurin microtiter assay CCCP (1 mg/l), verapamil (5 mg/l) and DNP (20 mg/l).

A total of 100 [micro]l volume of Middlebrook 7H9 broth (Difco, USA) supplemented with 10 per cent oleic acid, albumin, dextrose and catalase (OADC) and 0.2 per cent glycerol was dispensed in the wells of a 96-well cell culture plate (Nunc, Denmark). Different concentrations of oloxacin prepared in Middlebrook 7H9 medium were: 0.25, 0.5, 1, 2, 4, 8, 16, 32 and 64 mg/l. M. tuberculosis growth was taken from L-J slope, homogenized bacterial suspension of No.1 McFarland standard was prepared and diluted to 1:20 in 7H9 broth. This diluted suspension (100 [micro]l) was used to inoculate each well of the plate. Plates were sealed and incubated at 37[degrees]C for one week. Resazurin dye (Sigma, USA) (0.02%, 25 ([micro]l) was added to each well; plates were reincubated for two more days. A change in colour from blue to pink indicated the growth of bacteria and the MIC was read as the minimum oloxacin concentration that prevented the colour change in resazurin dye.

DNA extraction, PCR, and DNA sequencing: Genomic DNA from mycobacterial isolate (log phase on L-J slant) was extracted using phsiochemical procedure as described by van Soolingen et al (9). A DNA fragment of 320 bp of quinolone resistant determining region (QRDR) of gyrA gene in M. tuberculosis isolates was amplified using the following primers: 5'CAG CTA CAT CGA CTA TGC GA 3' and 5'GGG CTT CGG TGT TAC CTC AT 3' as described earlier (4). Amplification reactions consisted of a denaturation step of 3 min at 95[degrees]C, followed by 35 cycles of 1 min at 94[degrees]C, 1 min at 51[degrees]C, and 2 min at 72[degrees]C, and a final extension step of 10 min at 72[degrees]C. The amplification product was used as the template in direct nucleotide sequencing. Out of 45, oloxacin-resistant isolates, eight were sequenced for QRDR of gyrA. Amplified PCR products were purified from 1 per cent agarose gel using QIAEX II Gel Extraction Kit (Qiagen, Germany). All purified PCR products were sequenced with ABI PRISM 310 automated DNA sequencer (Applied Biosystem, USA) as per manufacturer's instructions. Sequences generated were confirmed using BLAST tool (available online at and compared with M. tuberculosis H37Rv strain using ClustalW multiple sequence alignment ( clustalw2).


MICs of oloxacin determined in absence of eflux inhibitors were compared with those determined in presence of eflux inhibitors. Two fold or more reduction in MIC levels was considered as an indication of presence of eflux activity in oloxacin-resistant M. tuberculosis isolates (10). It was observed that the MIC levels of oloxacin decreased in 16 of 45 (35.5%) isolates in the presence of CCCP, in 24 (53.3%) isolates in the presence of verapamil and in 21 (46.6%) isolates in the presence of DNP. All three eflux inhibitors (CCCP, DNP and verapamil) showed the MIC reduction in 11 (24.5%) isolates (Table I). Eflux inhibitors did not have any effect on oloxacin MICs in 10 (100%) oloxacin-sensitive isolates. Of the 10 oloxacin sensitive isolates, three were inhibited at 1 [micro]/ml and seven were inhibited at 2 [micro]g/ml concentration.

In the presence of DNP, the MIC values for oloxacin were found to decrease 4-folds in 10 (22.2%) and 2-folds in 11 (24.4%) isolates, verapamil showed 4-folds inhibition in five (11.1%) isolates and 2-folds in 19 (42.2%) isolates. In presence of CCCP eflux inhibitor, MIC levels for ofloxacin were lowered 4-folds in three (6.6%) and 2-folds in 13 (28.8%) ofloxacin-resistant isolates (Table II).

Of the 30 oloxacin resistant isolates, eight were sequenced for QRDR of gyrA. It was observed that all eight isolates showed single mutation at codon Ser95Thr. Seven isolates (87.5%) showed the mutations at codon Asp94Tyr (JAL -445, JAL-297 and JAL -1423), Asp94Val (JAL -638 and JAL -584), Asp94Gly (JAL -559 and JAL -419), Gly88Arg (JAL -584) and Arg98Leu (JAL -1423) in ofloxacin resistant isolates. Mutations in gyrA gene and its relation to MIC levels of oloxacin were described in Table III. Two novel mutations in different codons (Arg98Leu and Gly88Arg) of gyrA were also found along with known mutations. Except mutation at Ser95thr codon, no other mutation was observed in one isolate (DAU-262) in gyrA gene.


Data from the present study revealed that the MICs of maximum number of isolates were affected in the presence of verapamil suggesting the importance of eflux pumps of ABC transporter family in oloxacin resistance in M. tuberculosis. It was also observed that MIC (0.5 mg/l) of H37Rv was not affected in presence of efflux pump inhibitors. Piddock and Ricci (11) had also reported that CCCP and reserpine do not change the MICs of FQs for the reference strain M. tuberculosis H37Rv.

Overall inhibitory effect of eflux inhibitors on ofloxacin MICs was observed in 66.6 per cent M. tuberculosis isolates showing the contribution of active eflux pumps in the development of oloxacin resistance in these isolates. In these isolates, MIC levels decreased two to eight fold after treatment with eflux pump inhibitors but only in 20 per cent isolates microbiological classification changed from resistant to sensitive (out of 6, 1 isolate was blocked by all the three inhibitors, 2 were blocked by DNP, 2 by DNP and verapamil whereas 1 isolate was blocked by verapamil). Hence pumps appear to be contributing to increase in the level of resistance majority of isolates. It has been reported that in the presence of reserpine and MC 207.110 efflux pump inhibitors two-fold reduction in MIC was seen in 57-100 per cent M. tuberculosis isolates resistant to FQs (ciproloxacin, moxiloxacin, levoloxacin, oloxacin, gatiloxacin) and 57 per cent to linezolid (10). Low activity of these inhibitors was also reported in ofloxacin-sensitive isolates (10). Reduction in MIC level in presence of CCCP, DNP and verapamil inhibitors provide evidence for the presence of both type proton motive force and ATP dependent extrusion system involved in FQs resistance.

From the earlier studies, DNA sequencing of gyrA showed that all the strains possessed a natural mutation at codon Ser95Thr (AGC [right arrow] ACC) polymorphism, which did not have a significant impact on fluoroquinolone susceptibility (12,13). The present study also revealed the same. Mutation at Ser95Thr codon was previously reported as a marker for evolutionary genetics and does not correlate with drug resistance (14) or it has no direct role in the development of drug resistance, as it also occurs in drug-sensitive strains (4,14). Seven of the eight OFL resistant isolates, sequenced possessed mutations other than Ser95Thr in the gyrA gene. Of which, two mutations at codon Gly88Arg (GGC [right arrow] CGC), Arg98Leu (CGC [right arrow] CTC) were novel and were not reported previously. In FQ resistant isolates, the mutations at codon 88 i.e., Gly88Cys and Gly88Ala mutations were reported earlier (15,16).

We also observed AGC [right arrow] ACC point mutation at codon Ser95Thr in one OFL resistant isolates (DAU-262). In this isolate only eflux mediated drug resistance mechanism (PMF and ATP dependent) were involved in which OFL MIC was decreased 2-fold (from 16 to 8 [micro]/ml) in presence of CCCP, DNP and verapamil inhibitors. Only one OFL resistant isolate (JAL-419) showed a point mutation at codon Asp94gly (GAC [right arrow] GTC) responsible for OFL resistance and OFL MIC level did not decrease in presence of tested eflux inhibitors. In the remaining six isolates both mechanisms i.e., point mutation and efflux pumps were involved in OFL resistance. The OFL MIC for these isolates was found to be between 8 and 32 [micro]g/ ml. Contribution to degree of quinolone resistance thus appears to be significant.

Gupta and co-workers (17) have also reported the reversal of resistance to all major anti-tuberculous drugs (rifampicin, isoniazid, streptomycin, oloxacin) in presence of eflux pump inhibitors (CCCP and verapamil) which vary in different mycobacterial species and isolates. Results of the present study provide further evidence that eflux mechanism plays an important role in the development of oloxacin resistance in M. tuberculosis isolates. It may further be speculated that as the drugs of FQ group show cross-resistance, these eflux pumps might be a common mechanism contributing to this phenomenon. As the isolates were not selected by any acceptable sampling procedure, it will not be appropriate to discuss the epidemiological relevance and statistical significance of these findings.

This preliminary study shows the role of eflux pumps inhibitor (CCCP, DNP and verapamil) in conferring oloxacin resistance phenotype in M. tuberculosis isolates. There is a need to further investigate the changes in mRNA levels of genes encoding efflux pumps as well as detection of mutations in gyrA prospectively by selecting the isolates using appropriate sampling procedure.


Authors acknowledge the Department of Biotechnology, and Central TB Division, New Delhi, for providing financial support and thank Shriyut Jitendra, Rajesh, Sanjay and Dhaniram for technical support. The first two authors (MS and GPSJ) acknowledge the Council of Scientific and Industrial Research, New Delhi for providing Senior Research Fellowship.

Received December 17, 2009


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Mradula Singh, G.P.S. Jadaun, Ramdas, K. Srivastava, Vipin Chauhan, Ritu Mishra, Kavita Gupta, Surya Nair, D.S. Chauhan, V.D. Sharma, K. Venkatesan * & V.M. Katoch ([dagger])

Departments of Microbiology & Molecular Biology & 'Biochemistry, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India

([dagger]) Present address: Secretary (Department of Health Research) & Director-General, Indian Council of Medical Research, V Ramalingaswami Bhawan, Ansari Nagar, New Delhi 110 029, India, e-mail: & Former Director, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Tajganj, Agra 282 001, India

Reprint requests: Dr V.M. Katoch, Secretary (Department of Health Research) & Director-General, Indian Council of Medical Research, V. Ramalingaswami Bhawan, Ansari Nagar, New Delhi 110 029, India

Table I. Summary of effect of efflux inhibitors on ofloxacin MICs
in resistant M. tuberculosis isolates

                                     No. of OFL resistant isolates
                                     showing OFL MIC ([micro]g/ml)
                                          in presence of

Oloxacin       No. of isolates
MIC            in which efflux   CCCP    Verapamil    DNP     V+D
([micro]g/     pumps inhibited    (C)       (V)       (D)

4                    1 *          --        --        --      --
8                   10 *           1         2         2       2
16                  10 *          --         3        --       1
32                   8 *           1        --         1       3
64                   1 *          --        --        --      --
Total No. of    30/45 (66.6%)      2         5         3       6

                  No. of OFL resistant    No reduction
                isolates showing OFL MIC     in MIC
                ([micro]g/ml) in presence
MIC             C+D     C+V    C+ V+ D

4               --      --        1             2
8               --      --        3             8
16              --       1        5             2
32               1       1        1             3
64              --      --        1             0
Total No. of     1       2        11      15/45 (33.3%)

In sensitive isolates, we could not observe the growth below the
MIC levels. Hence the eflux pump activity could not be determined.

* No. of ofloxacin resistant M. tuberculosis isolates in which
sensitivity was restored or reduction in MIC for ofloxacin was
observed in presence of eflux pump inhibitors

Table II. Fold changes in ofloxacin MIC of M. tuberculosis
isolates (n=45) in presence of efflux inhibitors (CCCP, DNP and

Efflux inhibitors     Fold changes in presence of eflux
(No. of isolates)     inhibitors in ofloxacin resistant
                                 isolates (%)

                        2            4            8

CCCP                13 (81.3)     2 (12.5)     1 (6.3)
(n = 16; 35.5%)
DNP                 11 (52.3)     5 (23.8)     5 (23.8)
(n = 21; 46.6%)
Verapamil           19 (79.2)     4 (16.6)     1 (4.2)
(n = 24; 53.3%)

Table III. Showing change in MIC levels for OFL in the presence
of inhibitors and presence of mutations in gyrA gene

                                    OFL MIC ([micro]g/ml)
                                       in presence of

S.No.   Isolate       OFL MIC
          code     ([micro]g/ml)   CCCP   Verapamil   DNP

1       JAL-559         32          8        16       32

2       JAL-638         32          32       16        8

3       JAL-445         16          8         8        2

4       JAL-297          8          8         4        8

5       JAL-419          8          8         8        8

6       JAL-1423        32          32       16        8

7       JAL-584         16          16        8        8

8       DAU-262         16          8         8        8

                Mutation in gyrA gene

         Amino            Nucleotide
          acid             changes

1       Ser95Thr   GAC [right arrow] GTC
        Asp94Gly   AGC [right arrow] ACC
2       Ser95Thr   AGC [right arrow] ACC
        Asp94Val   GAC [right arrow] GTC
3       Ser95Thr   AGC [right arrow] ACC
        Asp94Tyr   GAC [right arrow] TAC
4       Ser95Thr   AGC [right arrow] ACC
        Asp94Tyr   GAC [right arrow] TAC
5       Asp94gly   GAC [right arrow] GTC
        Ser95Thr   AGC [right arrow] ACC
6       Ser95Thr   AGC [right arrow] ACC
        Asp94Tyr   GAC [right arrow] TAC
        Arg98Leu   CGC [right arrow] CTC *
7       Asp94Val   GAC [right arrow] GCC
        Ser95Thr   AGC [right arrow] ACC
        Gly88Arg   GGC [right arrow] CGC *
8       Ser95Thr   AGC [right arrow] ACC

* Novel mutations in gyrA
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Author:Singh, Mradula; Jadaun, G.P.S.; Ramdas; Srivastava, K.; Chauhan, Vipin; Mishra, Ritu; Gupta, Kavita;
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:May 1, 2011
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