High efflux pump activity and gene expression at baseline linked to poor tuberculosis treatment outcomes

Phenotypic TB drug resistance, also known as drug tolerance, has been previously attributed to slowed bacterial growth in vivo. The increased activity and expression of efflux systems can lower the intracellular concentration of many antibiotics thus reducing their efficacy. We hypothesized that efflux pump activation and expression could be a risk factor for TB drug tolerance in patients initiated on treatment. Analyses of gene expression levels of six select efflux pumps associated with drug tolerance in Mycobacterium tuberculosis and its correlation with the cell’s ability to efflux ethidium bromide (a common efflux substrate) were assayed. Efflux pump gene expression differed significantly between the strains from treatment failures and treatment successes. Efflux of ethidium bromide by M. tuberculosis isolates revealed that isolates from treatment failures rapidly efflux ethidium bromide more than isolates from treatment successes or the H37Rv control strains. The efflux pumps efpA, jefA (Rv2459c), Rv1258c, p55 and mmpL7 may have a role in TB drug tolerance. Quantifying the expression levels of M. tuberculosis efflux pump genes may be a new method to diagnose clinically persistent tuberculosis. High efflux pump activity and expression at baseline can be associated with tuberculosis treatment failure even when the Mycobacterium tuberculosis does not have established resistance mutations.Journal of Medical and Biomedical Sciences (2017) 6(1), 8-17Keywords: drug resistance, Efflux, Mycobacterium tuberculosis, expression, treatment outcome

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Novel epitopes identified from efflux pumps ofMycobacterium tuberculosiscould induce cytotoxic T lymphocyte response

PeerJ ◽

10.7717/peerj.1229 ◽

2015 ◽

Vol 3 ◽

pp. e1229 ◽

Cited By ~ 4

Author(s):

Ming-xia Zhai ◽

Fei Chen ◽

Yuan-yuan Zhao ◽

Ya-hong Wu ◽

Guo-dong Li ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Efflux Pump ◽

Cytotoxic T Lymphocyte ◽

Efflux Pumps ◽

Common Mechanism ◽

Antigenic Peptides ◽

Ifn Γ ◽

Cytotoxic T Lymphocyte Response

Overcoming drug-resistance is one of the major challenges to control tuberculosis (TB). The up-regulation of efflux pumps is one common mechanism that leads to drug-resistance. Therefore, immunotherapy targeting these efflux pump antigens could be promising strategy to be combined with current chemotherapy. Considering that CD8+ cytotoxic T lymphocytes (CTLs) induced by antigenic peptides (epitopes) could elicit HLA-restricted anti-TB immune response, efflux pumps from classical ABC family (Mycobacterium tuberculosis, Mtb) were chosen as target antigens to identify CTL epitopes. HLA-A2 restricted candidate peptides from Rv2937, Rv2686c and Rv2687c ofMycobacterium tuberculosiswere predicted, synthesized and tested. Five peptides could induce IFN-γ release and cytotoxic activity in PBMCs from HLA-A2+PPD+donors. Results from HLA-A2/Kbtransgenic mice immunization assay suggested that four peptides Rv2937-p168, Rv2937-p266, Rv2686c-p151, and Rv2686c-p181 could induce significant CTL responsein vivo. These results suggested that these novel epitopes could be used as immunotherapy candidates to TB drug-resistance.

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Novel epitopes identified from efflux pumps of Mycobacterium tuberculosis could induce cytotoxic T lymphocyte response

10.7287/peerj.preprints.987 ◽

2015 ◽

Author(s):

Mingxia Zhai ◽

Fei Chen ◽

Yuanyuan Zhao ◽

Yahong Wu ◽

Guodong Li ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Efflux Pump ◽

Cytotoxic T Lymphocyte ◽

Efflux Pumps ◽

Common Mechanism ◽

Antigenic Peptides ◽

Ifn Γ ◽

Cytotoxic T Lymphocyte Response

Overcoming drug-resistance is one of the major challenges to control tuberculosis (TB). The up-regulation of efflux pumps is one common mechanism that leads to drug-resistance. Therefore, immunotherapy targeting these efflux pump antigens could be promising strategy to be combined with current chemotherapy. Considering that CD8+ cytotoxic T lymphocytes (CTLs) induced by antigenic peptides (epitopes) could elicit HLA-restricted anti-TB immune response, efflux pumps from classical ABC family (Mycobacterium tuberculosis, Mtb) were chosen as target antigens to identify CTL epitopes. HLA-A2 restricted candidate peptides from Rv2937, Rv2686c and Rv2687c of Mycobacterium tuberculosis were predicted, synthesized and tested. Five peptides could induce IFN-γ release and cytotoxic activity in PBMCs from HLA-A2+ PPD+ donors. Results from HLA-A2/Kb transgenic mice immunization assay suggested that four peptides Rv2937-p168, Rv2937-p266, Rv2686c-p151, and Rv2686c-p181 could induce significant CTL response in vivo. These results suggested that these novel epitopes could be used as immunotherapy candidates to TB drug-resistance.

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Novel epitopes identified from efflux pumps of Mycobacterium tuberculosis could induce cytotoxic T lymphocyte response

10.7287/peerj.preprints.987v1 ◽

2015 ◽

Author(s):

Mingxia Zhai ◽

Fei Chen ◽

Yuanyuan Zhao ◽

Yahong Wu ◽

Guodong Li ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Efflux Pump ◽

Cytotoxic T Lymphocyte ◽

Efflux Pumps ◽

Common Mechanism ◽

Antigenic Peptides ◽

Ifn Γ ◽

Cytotoxic T Lymphocyte Response

Overcoming drug-resistance is one of the major challenges to control tuberculosis (TB). The up-regulation of efflux pumps is one common mechanism that leads to drug-resistance. Therefore, immunotherapy targeting these efflux pump antigens could be promising strategy to be combined with current chemotherapy. Considering that CD8+ cytotoxic T lymphocytes (CTLs) induced by antigenic peptides (epitopes) could elicit HLA-restricted anti-TB immune response, efflux pumps from classical ABC family (Mycobacterium tuberculosis, Mtb) were chosen as target antigens to identify CTL epitopes. HLA-A2 restricted candidate peptides from Rv2937, Rv2686c and Rv2687c of Mycobacterium tuberculosis were predicted, synthesized and tested. Five peptides could induce IFN-γ release and cytotoxic activity in PBMCs from HLA-A2+ PPD+ donors. Results from HLA-A2/Kb transgenic mice immunization assay suggested that four peptides Rv2937-p168, Rv2937-p266, Rv2686c-p151, and Rv2686c-p181 could induce significant CTL response in vivo. These results suggested that these novel epitopes could be used as immunotherapy candidates to TB drug-resistance.

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Role of the Mycobacterium tuberculosis P55 Efflux Pump in Intrinsic Drug Resistance, Oxidative Stress Responses, and Growth

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00550-09 ◽

2009 ◽

Vol 53 (9) ◽

pp. 3675-3682 ◽

Cited By ~ 77

Author(s):

Santiago Ramón-García ◽

Carlos Martín ◽

Charles J. Thompson ◽

José A. Aínsa

Keyword(s):

Oxidative Stress ◽

Drug Resistance ◽

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Stress Responses ◽

Efflux Pump ◽

Efflux Pumps ◽

Oxidative Stress Responses

ABSTRACT Bacterial efflux pumps have traditionally been studied as low-level drug resistance determinants. Recent insights have suggested that efflux systems are often involved with fundamental cellular physiological processes, suggesting that drug extrusion may be a secondary function. In Mycobacterium tuberculosis, little is known about the physiological or drug resistance roles of efflux pumps. Using Mycobacterium bovis BCG as a model system, we showed that deletion of the Rv1410c gene encoding the P55 efflux pump made the strain more susceptible to a range of toxic compounds, including rifampin (rifampicin) and clofazimine, which are first- and second-line antituberculosis drugs. The efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin inhibited the P55-determined drug resistance, suggesting the active export of the compounds by use of the transmembrane proton and electrochemical gradients as sources of energy. In addition, the P55 efflux pump mutant was more susceptible to redox compounds and displayed increased intracellular redox potential, suggesting an essential role of the efflux pump in detoxification processes coupled to oxidative balance within the cell. Finally, cells that lacked the p55 gene displayed smaller colony sizes and had a growth defect in liquid culture. This, together with an increased susceptibility to the cell wall-targeting compounds bacitracin and vancomycin, suggested that P55 is needed for proper cell wall assembly and normal growth in vitro. Thus, P55 plays a fundamental role in oxidative stress responses and in vitro cell growth, in addition to contributing to intrinsic antibiotic resistance. Inhibitors of the P55 efflux pump could help to improve current treatments for tuberculosis.

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A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

The Open Microbiology Journal ◽

10.2174/1874285801307010072 ◽

2013 ◽

Vol 7 (1) ◽

pp. 72-82 ◽

Cited By ~ 39

Author(s):

Marta Martins ◽

Matthew P McCusker ◽

Miguel Viveiros ◽

Isabel Couto ◽

Séamus Fanning ◽

...

Keyword(s):

Ethidium Bromide ◽

Efflux Pump ◽

Efflux Pumps ◽

Outer Cell ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Topoisomerase Iv ◽

Simple Method ◽

Gram Negative ◽

Over Expression

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

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A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01252-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5858-5866 ◽

Cited By ~ 23

Author(s):

Somanon Bhattacharya ◽

Jack D. Sobel ◽

Theodore C. White

Keyword(s):

Candida Albicans ◽

Efflux Pump ◽

Pathogenic Fungus ◽

Resistance Mechanism ◽

Efflux Pumps ◽

Resistant Isolate ◽

Vaginal Infections ◽

Content Type ◽

Qrt Pcr ◽

Pump Activity

ABSTRACTCandida albicansis a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginalC. albicansisolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginalC. albicansisolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginalC. albicansisolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine β-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginalC. albicansisolates.

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Evaluation of efflux pump gene expression among drug susceptible and drug resistant strains of Mycobacterium tuberculosis from Iran

Infection Genetics and Evolution ◽

10.1016/j.meegid.2015.08.036 ◽

2015 ◽

Vol 36 ◽

pp. 23-26 ◽

Cited By ~ 12

Author(s):

Jalil Kardan Yamchi ◽

Mehri Haeili ◽

Seifu Gizaw Feyisa ◽

Hossein Kazemian ◽

Abdolrazagh Hashemi Shahraki ◽

...

Keyword(s):

Gene Expression ◽

Mycobacterium Tuberculosis ◽

Efflux Pump ◽

Drug Resistant ◽

Resistant Strains ◽

Drug Resistant Strains

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Antimicrobial Efflux Pumps and Mycobacterium tuberculosis Drug Tolerance: Evolutionary Considerations

Current Topics in Microbiology and Immunology - Pathogenesis of Mycobacterium tuberculosis and its Interaction with the Host Organism ◽

10.1007/82_2012_300 ◽

2012 ◽

pp. 81-108 ◽

Cited By ~ 15

Author(s):

John D. Szumowski ◽

Kristin N. Adams ◽

Paul H. Edelstein ◽

Lalita Ramakrishnan

Keyword(s):

Mycobacterium Tuberculosis ◽

Efflux Pumps ◽

Drug Tolerance

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Efflux Pump-Mediated Intrinsic Drug Resistance in Mycobacterium smegmatis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.7.2415-2423.2004 ◽

2004 ◽

Vol 48 (7) ◽

pp. 2415-2423 ◽

Cited By ~ 140

Author(s):

Xian-Zhi Li ◽

Li Zhang ◽

Hiroshi Nikaido

Keyword(s):

Drug Resistance ◽

Ethidium Bromide ◽

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Upstream Region ◽

Drug Efflux ◽

Intact Cells ◽

Intrinsic Drug Resistance ◽

Multiple Drugs ◽

Increased Susceptibility

ABSTRACT The Mycobacterium smegmatis genome contains many genes encoding putative drug efflux pumps. Yet with the exception of lfrA, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism. We showed first by reverse transcription (RT)-PCR that several of these genes, including lfrA as well as the homologues of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA), and Rv3065 (mmr and emrE), were expressed at detectable levels in the strain mc2155. Null mutants each carrying an in-frame deletion of these genes were then constructed in M. smegmatis. The deletions of the lfrA gene or mmr homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs). The deletion of the efpA homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs). Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin. The upstream region of lfrA contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR. The deletion of lfrR elevated the expression of lfrA and produced higher resistance to multiple drugs. Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells. Finally, use of isogenic β-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain β-lactams in M. smegmatis.

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