Silencing of an efflux pump coding gene decreases the efflux rate of pyrazinoic acid in Mycobacterium smegmatis

Background: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). The recommended treatment for TB is based on the use of first-line drugs, including pyrazinamide (PZA). PZA is also a drug used in the treatment of multidrug-resistant TB (MDR-TB) because of its main effect against the latent stage. The main cause of resistance to PZA is mutations in the pncA gene, which compromise the activity of the encoded enzyme pyrazinamidase (PZAse), which hydrolyzes PZA into POA, the active antituberculosis molecule. The mechanism of action of PZA requires that POA is expelled from the bacterium by an efflux mechanism. After that, if the extracellular medium is sufficiently acidic, POA is protonated and returns to the cytosol, releasing the proton and repeating the cycle, resulting lethal to the bacteria. The efflux pump responsible for extruding the POA to the extracellular environment is not yet known. Mycobacterium smegmatis is naturally resistant to PZA and has a 900-fold faster POA efflux rate than MTB, and has the advantage to be a faster growing mycobacterium. Methods: In the present study we have silenced the transcription of several genes encoding efflux pumps in M. smegmatis by CRISPRi (CRISPR interference). These genes (MSMEG_0250, MSMEG_3815, MSMEG_0241, MSMEG_5046 and MSMEG_0410) were homologous to efflux pump genes in MTB. POA efflux rate was measured, and a quantitative Wayne's test was performed after silencing each gene. Results: Silencing of MSMEG_0250, resulted in an approximately 5-fold decrease in the POA efflux rate in M. smegmatis (P<0.0001). None of the other silenced genes showed a notable decrease in the POA efflux rate.

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MmpL3 Is the Cellular Target of the Antitubercular Pyrrole Derivative BM212

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05270-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 324-331 ◽

Cited By ~ 141

Author(s):

Valentina La Rosa ◽

Giovanna Poce ◽

Julio Ortiz Canseco ◽

Silvia Buroni ◽

Maria Rosalia Pasca ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Protein A ◽

Multidrug Resistant ◽

Whole Genome ◽

Content Type ◽

Genomic Libraries ◽

Cellular Target ◽

Efflux Pump Inhibitors

ABSTRACTThe 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates andMycobacterium tuberculosisresiding within macrophages as well as againstMycobacterium aviumand other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. SpontaneousMycobacterium smegmatis,Mycobacterium bovisBCG, andM. tuberculosisH37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in themmpL3gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterialmembraneprotein,large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanidem-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [14C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.

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Farnesol abrogates biofilm- and efflux pump- associated genes in drug resistant Candida auris strains

Access Microbiology ◽

10.1099/acmi.cc2021.po0012 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Vartika Srivastava ◽

Aijaz Ahmad

Keyword(s):

Active Drug ◽

Efflux Pump ◽

Antifungal Susceptibility ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Major Facilitator Superfamily ◽

Drug Efflux ◽

Candida Auris ◽

Efflux Mechanism ◽

Major Facilitator

Background: Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and over expression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. Methods: In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. Rhodamine-6-G extracellular efflux and intracellular accumulation assays were used to study active drug efflux mechanism. We further studied the role of farnesol in modulating development of biofilms and drug efflux in C. auris. Down-regulation of biofilm- and efflux pump- associated genes by farnesol was also investigated. CLSM analysis for examining C. auris biofilm architecture among treated and untreated isolates. Results: Most of the isolates (twenty-two) were found resistant to FLZ whereas five were resistant to AmB. All the isolates were found capable of biofilm formation and ornamented with active drug efflux mechanism. The MIC for planktonic cells ranged from 62.5-125 mM and for sessile cells was 125 mM (0 h and 4 h biofilm) and 500 mM (12 h and 24 h biofilm), CLSM studies also confirmed these findings. Farnesol also blocked efflux pumps and down-regulated biofilm- and efflux pump- associated genes. Conclusion: Modulation of biofilm- and efflux pump- associated genes by farnesol represent a promising approach in combating C. auris infection.

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Targeting the multidrug efflux pump; Tap protein to reduce survival of Mycobacterium tuberculosis

10.21203/rs.3.rs-137844/v1 ◽

2020 ◽

Author(s):

Manish Dwivedi ◽

Sutanu Mukhopadhyay

Keyword(s):

Binding Affinity ◽

Efflux Pump ◽

Md Simulations ◽

Multidrug Resistant ◽

Target Protein ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Drug Molecules ◽

Mdr Tb ◽

Inhibitor Compounds

Abstract Tuberculosis (TB) is a serious communicative disease caused by a bacterium named Mycobacterium tuberculosis. Albeit there are vaccines and drugs available to treat the disease, Multidrug-resistant TB (MDR-TB) is still one of the most critical challenges for the investigators where the development of efflux systems makes them resistant to drugs. Tap is a multidrug efflux pump and proposed to have a significant role in the survival of M. tuberculosis making it drug-resistant. In the present study, we have utilized various In silico approaches to predict the applicability of FDA approved ion channel inhibitors and blockers as therapeutic leads against Tuberculosis. We have analysed 18 inhibitor compounds and eventually screened three ligands as drugs, Glibenclimide, Lubiprostone and Flecainde that have displayed the novel stable binding with Tap protein aiming to affect or inhibit its activity. Structure of Tap protein is predicted by Phyre2 server followed by its characterization by 10ns MD simulations using the CABS-flex 2.0 server and validation by Ramachandran plot. PyRx software presented the binding affinity varied in the range of -8.00 kcal/mol to -9.8 kcal/mol, implies that the drug molecules can spontaneously interact with the target protein. Amongst them, Glibenclimide shows the highest binding affinity with ΔG of -9.8 kcal/mol. This study proposed Tap protein as an interesting drug target and investigated drugs may show considerable effects on the target protein showing a novel therapeutic lead against Tuberculosis.

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Widespread Occurrence of Possible Antibiotic Efflux Mechanism in Multidrug Resistant Intestinal E. coli Strains Isolated from Healthy Human Subjects in Bangladesh

Bangladesh Journal of Microbiology ◽

10.3329/bjm.v30i1-2.28454 ◽

2016 ◽

Vol 30 (1-2) ◽

pp. 55-59

Author(s):

M Hasibur Rahman ◽

Jamil Mahmud ◽

Md Mahamudul Haque ◽

Farzana Tarannum Tuli ◽

Nazneen Jahan ◽

...

Keyword(s):

Ethidium Bromide ◽

Efflux Pump ◽

Human Subjects ◽

Agar Plate ◽

Multidrug Resistant ◽

Dilution Method ◽

Agar Dilution Method ◽

Healthy Human ◽

Efflux Mechanism ◽

High Level

A total of 50 isolates of Escherichia coli obtained from healthy adult human subjects were studied and tested for possible presence of efflux mechanism in resistance determination and possible correlation of plasmids with resistance.Minimal inhibitory concentration (MIC) of the antibiotics amoxicillin, azithromycin, ciprofloxacin, chloramphenicol and tetracycline were determined by agar dilution method with or without the H+/K+ proton pump inhibitor omeprazole. Plasmids were extracted by rapid alkaline plasmid extraction method and analyzed by agarose gel electrophoresis. Many strains showed 5 10 fold reduction of MIC values in the presence of omeprazole; a few strains showed up to 100-fold MIC reduction. Plasmid analysis of these 50 isolates revealed the presence of both plasmidless and plasmid containing strains, the latter with plasmid number varying from one to seven. However, the plasmids apparently had no relationship with high level antibiotic tolerance as indicated by the observation that some plasmidless strains had very high MIC values, while other strains containing several plasmids had very low MIC. Decrease in MIC in the presence of omeprazole apparently indicates existence of an efflux mechanism. Evidence of the efflux of ethidium bromide was noted in some strains that had been grown in ethidium bromide containing agar plate with and without omeprazole. These results suggest that reduction of MIC caused by omeprazole may be related to possible inhibition of efflux pump activity by omeprazole in the isolates studied.Bangladesh J Microbiol, Volume 30, Number 1-2,June-Dec 2013, pp 55-59

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Characterization of the Mycobacterial MSMEG-3762/63 Efflux Pump in Mycobacterium smegmatis Drug Efflux

Frontiers in Microbiology ◽

10.3389/fmicb.2020.575828 ◽

2020 ◽

Vol 11 ◽

Author(s):

Barbara De Siena ◽

Nicoletta Campolattano ◽

Gianluca D’Abrosca ◽

Luigi Russo ◽

Daire Cantillon ◽

...

Keyword(s):

Mycobacterium Smegmatis ◽

Nitrosative Stress ◽

Efflux Pump ◽

Drug Efflux ◽

Drug Resistant ◽

Antimicrobial Drugs ◽

Bioinformatics Analyses ◽

Major Health Problem ◽

Mdr Tb ◽

Biofilm Development

Multi-drug resistant tuberculosis (MDR-TB) represents a major health problem worldwide. Drug efflux and the activity of efflux transporters likely play important roles in the development of drug-tolerant and drug-resistant mycobacterial phenotypes. This study is focused on the action of a mycobacterial efflux pump as a mechanism of drug resistance. Previous studies demonstrated up-regulation of the TetR-like transcriptional regulator MSMEG_3765 in Mycobacterium smegmatis and its ortholog Rv1685c in Mycobacterium tuberculosis (Mtb) in acid-nitrosative stress conditions. MSMEG-3765 regulates the expression of the MSMEG_3762/63/65 operon, and of the orthologous region in Mtb (Rv1687c/86c/85c). MSMEG-3762 and Rv1687c are annotated as ATP-binding proteins, while MSMEG-3763 and Rv1686c are annotated as trans-membrane polypeptides, defining an ABC efflux pump in both M. smegmatis and Mtb. The two putative efflux systems share a high percentage of identity. To examine the role of the putative efflux system MSMEG-3762/63, we constructed and characterized a MSMEG-3763 deletion mutant in M. smegmatis (∆MSMEG_3763). By comparative analysis of wild type, knockout, and complemented strains, together with structural modeling and molecular docking bioinformatics analyses of the MSMEG-3763 trans-membrane protein, we define the protein complex MSMEG-3762/63 as an efflux pump. Moreover, we demonstrate involvement of this pump in biofilm development and in the extrusion of rifampicin and ciprofloxacin (CIP), antimicrobial drugs used in first- and second-line anti-TB therapies.

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Activity of Aztreonam in Combination with Avibactam, Clavulanate, Relebactam, and Vaborbactam against Multidrug-Resistant Stenotrophomonas maltophilia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00297-20 ◽

2020 ◽

Vol 64 (12) ◽

Cited By ~ 1

Author(s):

M. Biagi ◽

D. Lamm ◽

K. Meyer ◽

A. Vialichka ◽

M. Jurkovic ◽

...

Keyword(s):

Stenotrophomonas Maltophilia ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Treatment Strategies ◽

Multidrug Resistant ◽

Content Type ◽

Expression Of Genes ◽

Genes Encoding ◽

Novel Treatment Strategies

ABSTRACT The intrinsic L1 metallo- and L2 serine-β-lactamases in Stenotrophomonas maltophilia make it naturally multidrug resistant and difficult to treat. There is a need to identify novel treatment strategies for this pathogen, especially against isolates resistant to first-line agents. Aztreonam in combination with avibactam has demonstrated potential, although data on other aztreonam–β-lactamase inhibitor (BLI) combinations are lacking. Additionally, molecular mechanisms for reduced susceptibility to these combinations have not been explored. The objectives of this study were to evaluate and compare the in vitro activities and to understand the mechanisms of resistance to aztreonam in combination with avibactam, clavulanate, relebactam, and vaborbactam against S. maltophilia. A panel of 47 clinical S. maltophilia strains nonsusceptible to levofloxacin and/or trimethoprim-sulfamethoxazole were tested against each aztreonam-BLI combination via broth microdilution, and 6 isolates were then evaluated in time-kill analyses. Three isolates with various aztreonam-BLI MICs were subjected to whole-genome sequencing and quantitative reverse transcriptase PCR. Avibactam restored aztreonam susceptibility in 98% of aztreonam-resistant isolates, compared to 61, 71, and 15% with clavulanate, relebactam, and vaborbactam, respectively. The addition of avibactam to aztreonam resulted in a ≥2-log10-CFU/ml decrease at 24 h versus aztreonam alone against 5/6 isolates compared to 1/6 with clavulanate, 4/6 with relebactam, and 2/6 with vaborbactam. Molecular analyses revealed that decreased susceptibility to aztreonam-avibactam was associated with increased expression of genes encoding L1 and L2, as well as the efflux pump (smeABC). Aztreonam-avibactam is the most promising BLI-combination against multidrug-resistant S. maltophilia. Decreased susceptibility may be due to the combination of overexpressed β-lactamases and efflux pumps. Further studies evaluating this combination against S. maltophilia are warranted.

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Antimicrobial and Efflux Pump Inhibitory Activity of Carvotacetones from Sphaeranthus africanus Against Mycobacteria

Antibiotics ◽

10.3390/antibiotics9070390 ◽

2020 ◽

Vol 9 (7) ◽

pp. 390 ◽

Cited By ~ 2

Author(s):

Huyen Thi Tran ◽

Julia Solnier ◽

Eva-Maria Pferschy-Wenzig ◽

Olaf Kunert ◽

Liam Martin ◽

...

Keyword(s):

Mycobacterium Smegmatis ◽

Efflux Pump ◽

Microtiter Plate ◽

Antibacterial Effects ◽

Mycobacterial Infections ◽

Efflux Mechanism ◽

Inhibitory Potential ◽

Culture Growth ◽

Mycobacterium Aurum ◽

Microbroth Dilution

Carvotacetones (1–7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.

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The AcrAB-TolC Efflux Pump Contributes to Multidrug Resistance in the Nosocomial Pathogen Enterobacter aerogenes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.8.2640-2643.2002 ◽

2002 ◽

Vol 46 (8) ◽

pp. 2640-2643 ◽

Cited By ~ 98

Author(s):

Elizabeth Pradel ◽

Jean-Marie Pagès

Keyword(s):

Multidrug Resistance ◽

Sequence Analysis ◽

Parental Strain ◽

Efflux Pump ◽

Enterobacter Aerogenes ◽

Multidrug Resistant ◽

Resistant Isolate ◽

Nosocomial Pathogen ◽

Genes Encoding

ABSTRACT We identified the genes encoding the AcrA-AcrB-TolC efflux pump in Enterobacter aerogenes and constructed acrAB and tolC mutants from a multidrug-resistant isolate. Both derivatives were more susceptible to antibiotics than the parental strain. Sequence analysis and complementation experiments revealed that the multidrug-resistant isolate is an acrR mutant.

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The Global Consequence of Disruption of the AcrAB-TolC Efflux Pump in Salmonella enterica Includes Reduced Expression of SPI-1 and Other Attributes Required To Infect the Host

Journal of Bacteriology ◽

10.1128/jb.00363-09 ◽

2009 ◽

Vol 191 (13) ◽

pp. 4276-4285 ◽

Cited By ~ 70

Author(s):

Mark A. Webber ◽

Andrew M. Bailey ◽

Jessica M. A. Blair ◽

Eirwen Morgan ◽

Mark P. Stevens ◽

...

Keyword(s):

Salmonella Enterica ◽

Efflux Pump ◽

Multidrug Resistant ◽

Effector Proteins ◽

Wild Type ◽

Reverse Transcription Pcr ◽

Serovar Typhimurium ◽

Genes Encoding ◽

Global Consequence

ABSTRACT The mechanisms by which RND pumps contribute to pathogenicity are currently not understood. Using the AcrAB-TolC system as a paradigm multidrug-resistant efflux pump and Salmonella enterica serovar Typhimurium as a model pathogen, we have demonstrated that AcrA, AcrB, and TolC are each required for efficient adhesion to and invasion of epithelial cells and macrophages by Salmonella in vitro. In addition, AcrB and TolC are necessary for Salmonella to colonize poultry. Mutants lacking acrA, acrB, or tolC showed differential expression of major operons and proteins involved in pathogenesis. These included chemotaxis and motility genes, including cheWY and flgLMK and 14 Salmonella pathogenicity island (SPI)-1-encoded type III secretion system genes, including sopE, and associated effector proteins. Reverse transcription-PCR confirmed these data for identical mutants in two other S. Typhimurium backgrounds. Western blotting showed reduced production of SipA, SipB, and SipC. The absence of AcrB or TolC also caused widespread repression of chemotaxis and motility genes in these mutants, and for acrB::aph, this was associated with decreased motility. For mutants lacking a functional acrA or acrB gene, the nap and nir operons were repressed, and both mutants grew poorly in anaerobic conditions. All phenotypes were restored to that of the wild type by trans-complementation with the wild-type allele of the respective inactivated gene. These data explain how mutants lacking a component of AcrAB-TolC are attenuated and that this phenotype is a result of decreased expression of numerous genes encoding proteins involved in pathogenicity. The link between antibiotic resistance and pathogenicity establishes the AcrAB-TolC system as fundamental to the biology of Salmonella.

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Assessment of the Additional Value of Verapamil to a Moxifloxacin and Linezolid Combination Regimen in a Murine Tuberculosis Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01354-18 ◽

2018 ◽

Vol 62 (9) ◽

Cited By ~ 1

Author(s):

Elise D. Pieterman ◽

Lindsey H. M. te Brake ◽

Gerjo J. de Knegt ◽

Aart van der Meijden ◽

Jan-Willem C. Alffenaar ◽

...

Keyword(s):

Efflux Pump ◽

Multidrug Resistant ◽

Treatment Modalities ◽

Pharmacokinetic Parameters ◽

Combination Regimen ◽

Efflux Pump Inhibitor ◽

Content Type ◽

Resistant Tuberculosis ◽

Mdr Tb ◽

Additional Value

ABSTRACTThe favorable treatment outcome rate for multidrug-resistant tuberculosis (MDR-TB) is only 54%, and therefore new drug regimens are urgently needed. In this study, we evaluated the activity of the combination of moxifloxacin and linezolid as a possible new MDR-TB regimen in a murine TB model and the value of the addition of the efflux pump inhibitor verapamil to this backbone. BALB/c mice were infected with drug-sensitiveMycobacterium tuberculosisand were treated with human-equivalent doses of moxifloxacin (200 mg/kg of body weight) and linezolid (100 mg/kg) with or without verapamil (12.5 mg/kg) for 12 weeks. Pharmacokinetic parameters were collected during treatment at the steady state. After 12 weeks of treatment, a statistically significant decline in mycobacterial load in the lungs was observed with the moxifloxacin-linezolid regimen with and without verapamil (5.9 and 5.0 log CFU, respectively), but sterilization was not achieved yet. The spleens of all mice were culture negative after 12 weeks of treatment with both treatment modalities, and the addition of verapamil caused a significant reduction in relapse (14/14 positive spleens without versus 9/15 with verapamil,P= 0.017). In conclusion, treatment with a combination regimen of moxifloxacin and linezolid showed a strong decline in mycobacterial load in the mice. The addition of verapamil to this backbone had a modest additional effect in terms of reducing mycobacterial load in the lung as well as reducing the spleen relapse rate. These results warrant further studies on the role of efflux pump inhibition in improving the efficacy of MDR-TB backbone regimens.

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