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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 6  |  Issue : 2  |  Page : 177-183

Contribution of putative efflux pump genes to isoniazid resistance in clinical isolates of Mycobacterium tuberculosis


Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi, India

Date of Web Publication19-May-2017

Correspondence Address:
Mandira Varma-Basil
Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi - 110 007
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_26_17

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  Abstract 

Background: Isoniazid (INH) resistance in Mycobacterium tuberculosis has been mainly attributed to mutations in katG (64%) and inhA (19%). However, 20%–30% resistance to INH cannot be explained by mutations alone. Hence, other mechanisms besides mutations may play a significant role in providing drug resistance. Here, we explored the role of 24 putative efflux pump genes conferring INH-resistance in M. tuberculosis. Materials and Methods: Real-time expression profiling of the efflux pump genes was performed in five INH-susceptible and six high-level INH-resistant clinical isolates of M. tuberculosis exposed to the drug. Isolates were also analyzed for mutations in katG and inhA. Results: Four high-level INH-resistant isolates (minimum inhibitory concentration [MIC] ≥2.5 mg/L) with mutations at codon 315 (AGC-ACC) of katG showed upregulation of one of the efflux genes Rv1634, Rv0849, efpA, or p55. Another high-level INH-resistant isolate (MIC 1.5 mg/L), with no mutations at katG or inhA overexpressed 8/24 efflux genes, namely, Rv1273c, Rv0194, Rv1634, Rv1250, Rv3823c, Rv0507, jefA, and p55. Five of these, namely, Rv0194, Rv1634, Rv1250, Rv0507, and p55 were induced only in resistant isolates. Conclusion: The high number of efflux genes overexpressed in an INH-resistant isolate with no known INH resistance associated mutations, suggests a role for efflux pumps in resistance to this antituberculous agent, with the role of Rv0194 and Rv0507 in INH resistance being reported for the first time.

Keywords: Efflux pumps, isoniazid resistance, Mycobacterium tuberculosis


How to cite this article:
Narang A, Giri A, Gupta S, Garima K, Bose M, Varma-Basil M. Contribution of putative efflux pump genes to isoniazid resistance in clinical isolates of Mycobacterium tuberculosis. Int J Mycobacteriol 2017;6:177-83

How to cite this URL:
Narang A, Giri A, Gupta S, Garima K, Bose M, Varma-Basil M. Contribution of putative efflux pump genes to isoniazid resistance in clinical isolates of Mycobacterium tuberculosis. Int J Mycobacteriol [serial online] 2017 [cited 2019 Sep 18];6:177-83. Available from: http://www.ijmyco.org/text.asp?2017/6/2/177/206598




  Introduction Top


Despite the availability of a battery of drugs to fight tuberculosis (TB), the disease claims a larger number of lives than any other infectious disease. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB has added a new challenge to this old and persistent disease. In Mycobacterium tuberculosis, drug resistance is an outcome of multiple mechanisms operating simultaneously.[1] Resistance to isoniazid (INH), an important first-line anti-TB agent, is also a complex process. Mutations in several genes, including katG, ahpC, inhA, kasA, and ndh, have all been associated with INH resistance. Of these, mutations in katG and inhA are considered to be the main cause of INH resistance.[2],[3],[4] Apart from this most common resistance mechanism, a wide array of efflux mechanisms mediated by several proteins contributes in a major way to intrinsic resistance to drugs, including INH.[5],[6]

A number of efflux pumps have been shown to be involved in INH resistance.[7],[8],[9],[10] It has been demonstrated that efflux systems could be induced by prolonged exposure of M. tuberculosis to INH, resulting in an increased resistance phenotype.[11] Further, gene knockout experiments have shown that the iniA gene is essential for the activity of an efflux pump that confers resistance to INH and ethambutol (EMB) in M. tuberculosis.[12] Gene expression studies by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with clinical isolates of M. tuberculosis showed that various major facilitator superfamily (MFS) efflux pump genes (efpA[Rv2846c], Rv1258c, jefA [Rv2459], and P55 [Rv1410c]) and ATP-binding cassette (ABC) superfamily transporters (Rv1819c and pstB [Rv0933]) were overexpressed in the presence of INH.[7],[8],[9],[10] Collectively, these results suggest that INH-resistance in M. tuberculosis may be attributed to mutations in known genes and may also be influenced or modulated by efflux-related mechanisms. However, all cases of INH resistance can still not be explained.

In the present study, we investigated the expression levels of 24 membrane transporter genes, annotated as probable/hypothetical in the M. tuberculosis genome [13] and available at http://www.tuberculist.epfl.ch/, in INH-susceptible and -resistant M. tuberculosis clinical isolates under INH stress. The efflux activity in the clinical isolates was also studied in the presence of efflux pump inhibitors verapamil, which is shown to effect several bacterial ABC efflux pumps [14],[15] and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which not only inhibits the activity of efflux pumps of class MFS [14],[16],[17] but also leads to inhibition of activity of adenosine triphosphate synthase and affects ABC superfamily pumps.[18] For a holistic view on INH resistance, we also investigated the clinical isolates for mutations known to be responsible for 60%–90% of INH resistance,[2],[3],[4] in regions of katG and inhA. The aim was to further the existing knowledge about the role of efflux pumps in INH resistance in M. tuberculosis.


  Materials and Methods Top


Selection of putative efflux genes and synthesis of primers

Ten putative efflux genes predicted as membrane transporters of M. tuberculosis H37Rv and selected on the basis of the presence of high-confidence MDR mutations (HCM) in a previous study from our laboratory, were included in the study.[19] The HCM were predicted after comparative analysis with the gene sequence of an MDR strain of M. tuberculosis, available at the Open Source Drug Discovery (www.osdd.net).[20] Additional ten genes, bioinformatically identified to be putative drug transporters (http://tuberculist.epfl.ch/)[21] and four efflux pump genes previously shown to respond with higher expression under INH stress, i.e., jefA, efpA, p55, and pstB were also included [Table 1].[9],[10],[11],[12] The selected genes represented the three classes of efflux pumps, i.e., ABC superfamily, MFS, and resistance nodulation division (RND) superfamily [Table 1].
Table 1: Primer sequences of the genes used in the quantitative reverse transcription.polymerase chain reaction assays

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Sequences of putative efflux genes, namely, Rv1272c, Rv1456c, Rv1457c, Rv1686c, Rv1687c, Rv0842, Rv0849, Rv0876c, Rv2265, Rv2456c, jefA, and the housekeeping gene rrs were retrieved from http://genolist.pasteur.fr/Tuberculist/.[21] The primers were designed using Gene Runner Version 3.01 Software (Copyright: √Frank Buquicchio and Michael Spruyt) [Table 1]. Previously published primer sequences were used for efflux pumps Rv1273c, Rv1458c, Rv0194, Rv1819c, Rv1634, Rv1250, Rv1877, Rv0676c, Rv3823c, Rv0507, efpA, p55, pstB,[19],[22] and the housekeeping gene sigA.[19] The desired sequences were synthesized by Sigma-Aldrich (India).

Bacterial strains, growth conditions, and reagents

The reference strain M. tuberculosis H37Rv and 11 clinical isolates of M. tuberculosis were obtained from Department of Microbiology at Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi, India. The isolates were characterized by niacin, nitrate and catalase tests,[23] PCR restriction analysis,[24] IS6110 typing,[25] and mycobacterial interspersed repetitive unit-variable-number of tandem repeat typing.[26]

The mycobacterial strains were grown in Middlebrook 7H9 Broth (Difco Laboratories, Detroit, MI, USA) supplemented with oleic acid, albumin bovine fraction V, dextrose, catalase (Difco), and 0.2% glycerol at 37°C. Lowenstein–Jensen (LJ) medium was used for maintenance of cultures and drug susceptibility assays. Streptomycin (SM), INH, rifampicin (RIF), EMB, verapamil, and CCCP were obtained from Sigma-Aldrich (St. Louis, MO, USA). The drugs and efflux pump inhibitors were freshly prepared and filter sterilized before use.

Drug susceptibility profile of clinical isolates

The drug susceptibility profile of the isolates to SM, INH, RIF, and EMB was evaluated by 1% proportion drug susceptibility testing (PDST) using LJ medium with the following concentrations: SM, 4 μg/mL; INH, 0.2 μg/mL; RIF, 40 μg/mL; and EMB, 2 μg/mL.[27]

Minimum inhibitory concentration determination

Minimum inhibitory concentrations (MICs) of INH and the efflux pump inhibitors, for the clinical isolates under study, were determined by microplate alamar blue assay (MABA) performed in 96-well U-bottom plates as described previously, with minor modifications.[28] The concentration for MIC testing ranged from 0.1–20 mg/L for INH; 5–120 mg/L for verapamil; and 0.1–1 mg/L for CCCP. Stock solutions of INH and verapamil were prepared in deionized water and CCCP in dimethyl sulfoxide. Growth controls (medium containing inoculum but no antibiotic) and sterility controls (medium without inoculum and antibiotic) were also included in the assay. Each concentration of drug was tested in triplicates, and the procedure was repeated a minimum of three times.

The MICs of INH were also calculated in the presence of subinhibitory concentrations (½ MIC) of verapamil and CCCP, as described previously.[12]

In vitro drug exposure of Mycobacterium tuberculosis clinical strains and H37Rv to isoniazid

M. tuberculosis H37Rv and the clinical isolates were cultured in flasks containing Middlebrook 7H9 medium supplemented with 0.2% glycerol and 0.05% Tween 80. The freshly growing culture of each isolate at mid-log phase (OD600-0.5) was exposed to a subinhibitory concentration of INH (½ MIC)[19] [Table 2] and incubated at 37°C for 24 h as described previously.[29] The unexposed culture was taken as control. The cells were harvested by centrifugation at room temperature at 8000 rpm for 10 min, and total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions and subsequently treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA, USA). The quantity and quality of the RNA extracted from each condition were assessed by virtual gel electrophoresis on the DNR Bio-Imaging Systems (MiniLumi) as well as by spectrophotometric measurement of the A260/A280 ratio. PCR amplification of the hsp65 gene was performed as an additional quality control measure to assess possible DNA contamination, using the total RNA as a template. Potential amplified products were assessed by gel electrophoresis on ethidium bromide-stained 1.5% agarose gel. Only RNA extracts with no visible amplified product were used for subsequent experiments. The cDNA was synthesized using random hexamer primers provided with the first strand cDNA synthesis kit (Fermentas Life Sciences, Lithuania) as per the manufacturer's instructions.
Table 2: Minimum inhibitory concentration of isoniazid in the presence and absence of efflux pump inhibitors

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Expression study of putative efflux pump genes by quantitative reverse transcription-polymerase chain reaction

Real-time PCR was performed to quantify the expression of putative drug efflux genes using QuantiTect SYBR Green Master Mix Kit (Roche Applied Science, Indianapolis, USA) in a LightCycler 480 II Real-Time PCR System (Roche Applied Science, Indianapolis, USA). The housekeeping sigma factor gene sigA and 16S ribosomal RNA gene rrs were used as internal controls in qRT-PCR assays.[30],[31] Melting curve analysis was performed after each run in a LightCycler 480 II instrument to confirm the specificity of the primers. Each qRT-PCR experiment was performed on duplicate biological samples which were further assayed in triplicates. The starting amounts of cDNA for the amplification of efflux genes and the reference genes were equalized to 0.15 ng/μl for each sample. Relative quantification in clinical isolates was performed to determine overexpression of efflux genes in cultures exposed to drug stress as compared to unexposed cultures, by 2-ΔΔCt method.[32] The data were analyzed using the built-in quantification software. A relative expression equal to one indicated that the expression level was identical to the control and a fold change ≥2.5 was considered as significant overexpression.

Sequencing for mutation analysis

The resistance determining regions of katG and inhA were sequenced in the clinical isolates studied (n = 11). Primer sequences used for amplification of regions of katG and inhA were as given by Hazbón et al.[4] PCR amplicons of all clinical isolates were commercially sequenced by M/s I st Base Asia, Malaysia.


  Results Top


Drug susceptibility profile of clinical isolates

For the study, 11 clinical isolates were used. Of these, five isolates (EP2-S1-15 to EP2-S5-15) were pan-susceptible, five isolates (EP2-R1-15 to EP2-R5-15) were resistant to all the first-line drugs, i.e., SM, INH, RIF, and EMB, and one isolate (EP2-R6-15) was monoresistant to SM.

Minimum inhibitory concentrations of isoniazid and the effect of efflux pump inhibitors on minimum inhibitory concentrations of the clinical isolates

The MICs to INH ranged from 0.007 to 0.2 mg/L in the pan-susceptible isolates (n = 5). In 4/5 of the susceptible isolates (EP2-S1-15, EP2-S3-15 to EP2-S5-15), the presence of verapamil led to 2–8-fold decrease in the MICs of INH. The addition of CCCP resulted into 2–16-fold decrease in all the susceptible isolates [Table 2].

In the resistant isolates, MICs to INH varied from 2.5 - 10 mg/L. The MICs of INH decreased 2-fold in the presence of verapamil, in all the resistant isolates. Two- to eight-fold reduction was observed in the MICs of INH in 3/5 resistant isolates (EP2-R1-15, EP2-R4-15, EP2-R5-15) when CCCP was supplemented [Table 2].

For isolate EP2-R6-15 (found INH-susceptible by PDST on repeated testing), MIC of INH was 1.25 mg/L [Table 2]. MIC of INH in this isolate decreased 2-fold in the presence of verapamil and 8-fold in the presence of CCCP.

Mutations in clinical isolates

Regions of katG and inhA were sequenced for mutations related to INH resistance. None of the susceptible isolates had mutations at katG or inhA. All the five MDR isolates studied, had a mutation at codon 315 (AGC-ACC) in katG [Table 3], there were no mutations in inhA. Although isolate EP2-R6-15 had relatively higher MIC for INH (1.25 mg/L) than the susceptible isolates, it did not have any mutations in katG or inhA.
Table 3: Correlation between minimum inhibitory concentrations of isoniazid, mutations in katG and inhA, and efflux genes overexpressed in Mycobacterium tuberculosis clinical isolates

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Efflux pump expression in Mycobacterium tuberculosis clinical isolates

All the INH-susceptible isolates overexpressed one or more putative efflux pump genes under INH stress [Table 3]. The efflux pump gene efpA alone was overexpressed by two INH-susceptible isolates (EP2-S1-15 and EP2-S3-15). The remaining INH-susceptible isolates (n = 3) overexpressed ≥3 efflux pumps.

Of the INH-resistant isolates, four INH-resistant isolates (EP2-R1-15, EP2-R3-15 to EP2-R5-15) overexpressed only one efflux pump gene, i.e., Rv1634, Rv0849, efpA, or p55. The isolate EP2-R6-15 overexpressed 8 of the 24 efflux pump genes. Of these, the genes Rv0194, Rv1634, Rv1250, Rv0507, and pstB had not been overexpressed in any of the susceptible isolates [Table 3].


  Discussion Top


The intrinsic resistance of M. tuberculosis to most antibiotics is generally attributed to the low permeability of the mycobacterial cell wall because of its specific lipid-rich composition and structure. This low permeability, which limits drug uptake, seems to be one of the main factors involved in drug resistance.[33] In addition to the unique structure of mycobacteria, mutations of the drug target genes are known as important causes of drug resistance in M. tuberculosis. Velayati et al. have also highlighted the possibility of sequential mutations in MDR-TB strains that showed resistance to second-line drugs.[34]

Mutations in katG and inhA or more frequently in its promoter region have been shown to be the main cause for INH resistance.[2],[3],[4] Globally, 64%[35] and in India 52%–91%[36],[37],[38],[39],[40],[41] of all observed phenotypic INH resistance were associated with the katG 315 mutation. inhA mutation (-15C→T) is the second most frequently observed mutation worldwide, reported among 19% of phenotypically resistant isolates leading to low-level INH resistance.[35] This mutation has been found to be associated with INH resistance in 9.1% of INH-resistant cases in an Indian study.[39] Other less common mutations related to INH resistance, in the genes kasA, ahpC, and ndh, are either accompanied by other common mutations or are also found in susceptible isolates.[2],[3],[4],[42],[43],[44],[45] Although mutations are the common cause of resistance, it is not possible to explain all cases of clinically observed multiple-drug resistance with mutations in the target genes.

Since the first evidence of active efflux being involved in antibiotic resistance,[46] efflux mechanisms have been recognized to be major players in bacterial drug resistance,[47],[48] which is a great cause of concern in the numerous pathogenic strains that have developed MDR phenotypes.[48] The ability of MDR transporters to pump drugs out of the cell helps bacteria to escape conventional antibiotic therapies. They, therefore, constitute potentially valuable targets in the search for new inhibitors restoring the efficacy of conventional treatment.[49]

In the present study, we compared the expression levels of 24 genes encoding putative drug efflux transporters under INH stress, in five INH-sensitive and six INH-resistant M. tuberculosis isolates. Of the six INH-resistant isolates, five showed upregulation of 1–8 efflux pump genes. Each of these isolates showed high-level resistance to INH (MIC 1.25–10 mg/L). Mutation analysis of the genes katG and inhA showed the presence of mutation 315 (AGC-ACC) in the katG gene of 4/5 (80%) of these isolates. Interestingly, the four isolates with mutations in katG gene showed upregulation of only one efflux pump each and the isolate without any mutation at katG or inhA (EP2-R6-15) showed upregulation of eight efflux pump genes.

One of the high-level INH-resistant isolates (MIC: 10 mg/L) from our panel (EP2-R5-15) showed overexpression of Rv0849. However, Rv0849 was also overexpressed in one of the INH-susceptible isolate (EP2-S5-15). Both these isolates showed a 2-fold reduction in MIC of INH in the presence of verapamil and CCCP. Overexpression of Rv0849 has also been observed in an earlier study, in an isolate having defined mutations for INH resistance, namely, katG 298 (TTG-TGG) and inhA-15 (C-T).[22] In the same study, Li et al. showed that Rv1634 was overexpressed in a wild-type strain. Contrary to their observation, in the present study, Rv1634 was found overexpressed only in high-level INH-resistant isolates (n = 2). One of these, with MIC 2.5 mg/L had a mutation at codon 315 (AGC-ACC) of the katG gene (EP2-R1-15). In the presence of verapamil and CCCP, MIC of INH was reduced 2-fold in this isolate, confirming the role of efflux pumps in INH resistance in the isolate. The second INH-resistant isolate (EP2-R6-15) with overexpression of Rv1634, did not have mutations at katG or inhA. MIC of INH in this isolate was reduced 2-fold in the presence of verapamil, and a drastic 8-fold reduction was seen in the presence of CCCP. Similarly, upregulation of Rv1634 has been reported in XDR-TB strains rather than drug-susceptible strains by Kanji et al.[50] The same group of investigators also reported three XDR-TB isolates wild type for rpsL, rrs, gidB, or drrA, having a mutation at position 1839306 in Rv1634.[51]

The isolate EP2-R3-15 showed upregulation of the efflux gene p55 and a 2-fold reduction in the MIC of INH, in the presence of verapamil. A susceptible isolate EP2-S5-15 also showed an increase in the expression of p55 and a 2-fold reduction in the MIC of INH, in the presence of verapamil and CCCP. The gene p55 (Rv1410c) encodes for a multidrug efflux pump of the MFS family in M. tuberculosis and Mycobacterium bovis and has been observed to confer resistance to SM and tetracycline.[3] In the study by Li et al., the expression of p55 was found higher in MDR isolates than pansusceptible isolates.[22] However, similar to our study, Rodrigues et al. found p55 to be upregulated in M. tuberculosis susceptible isolates.[52]

In our study, efpA was overexpressed in one INH-resistant isolate (EP2-R4-15) and three INH-susceptible isolates (EP2-S1-15, EP2-S3-15, and EP2-S5-15). Contrary to our findings, Gupta et al.[53] emphasized the increase in expression of efpA under INH stress only in INH-resistant isolates.

Surprisingly, the isolate EP2-R6-15, with high-level resistance to INH by MABA (MIC 1.25 mg/L), though susceptible to INH by PDST and no mutations at katG or inhA, showed overexpression of eight efflux genes, i.e., Rv1273c, Rv0194, Rv1634, Rv1250, Rv3823, Rv0507, jefA, and pstB, in response to INH stress. Of these, Rv0194, Rv1634, Rv1250, Rv0507, and pstB were overexpressed only in INH-resistant isolates [Table 3]. The study by Li et al.[22] also demonstrated that under INH stress, efflux pumps Rv1250, and pstB were overexpressed in addition to jefA (Rv2459) and p55 in one MDR isolate that did not have mutations associated with INH resistance.


  Conclusions Top


This study associated efflux genes Rv0194, Rv1634, Rv1250, Rv0507, and pstB with INH resistance. Of these, the role of Rv0194 and Rv0507 in INH resistance has been observed for the first time.

Financial support and sponsorship

This work was supported by a grant from the Department of Biotechnology, Government of India: No. BT/PR1597/MED/29/308/2011. Senior Research Fellowship to Anshika Narang was provided by Indian Council of Medical Research, Delhi, from the Project No. 80/697/2011-ECD-I.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3]


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