Efflux pump insensitive rhodamine–jasplakinolide conjugates for G- and F-actin imaging in living cells

Fluorescent actin probes made of 6′-carbopyronines and jasplakinolide are insensitive to efflux pumps and stain F- and G-actin efficiently in living cells, allowing high quality 2D and 3D nanoscopy of dynamic actin structures.

Download Full-text

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.

Download Full-text

Effect of Titanium Dioxide Nanoparticles on the Expression of Efflux Pump and Quorum-Sensing Genes in MDR Pseudomonas aeruginosa Isolates

Antibiotics ◽

10.3390/antibiotics10060625 ◽

2021 ◽

Vol 10 (6) ◽

pp. 625

Author(s):

Fatma Y. Ahmed ◽

Usama Farghaly Aly ◽

Rehab Mahmoud Abd El-Baky ◽

Nancy G. F. M. Waly

Keyword(s):

Titanium Dioxide ◽

Quorum Sensing ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Titanium Dioxide Nanoparticles ◽

Sol Gel ◽

Efflux Pumps ◽

Antibiofilm Activity ◽

The Impact

Most of the infections caused by multi-drug resistant (MDR) P. aeruginosa strains are extremely difficult to be treated with conventional antibiotics. Biofilm formation and efflux pumps are recognized as the major antibiotic resistance mechanisms in MDR P. aeruginosa. Biofilm formation by P. aeruginosa depends mainly on the cell-to-cell communication quorum-sensing (QS) systems. Titanium dioxide nanoparticles (TDN) have been used as antimicrobial agents against several microorganisms but have not been reported as an anti-QS agent. This study aims to evaluate the impact of titanium dioxide nanoparticles (TDN) on QS and efflux pump genes expression in MDR P. aeruginosa isolates. The antimicrobial susceptibility of 25 P. aeruginosa isolates were performed by Kirby–Bauer disc diffusion. Titanium dioxide nanoparticles (TDN) were prepared by the sol gel method and characterized by different techniques (DLS, HR-TEM, XRD, and FTIR). The expression of efflux pumps in the MDR isolates was detected by the determination of MICs of different antibiotics in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Biofilm formation and the antibiofilm activity of TDN were determined using the tissue culture plate method. The effects of TDN on the expression of QS genes and efflux pump genes were tested using real-time polymerase chain reaction (RT-PCR). The average size of the TDNs was 64.77 nm. It was found that TDN showed a significant reduction in biofilm formation (96%) and represented superior antibacterial activity against P. aeruginosa strains in comparison to titanium dioxide powder. In addition, the use of TDN alone or in combination with antibiotics resulted in significant downregulation of the efflux pump genes (MexY, MexB, MexA) and QS-regulated genes (lasR, lasI, rhll, rhlR, pqsA, pqsR) in comparison to the untreated isolate. TDN can increase the therapeutic efficacy of traditional antibiotics by affecting efflux pump expression and quorum-sensing genes controlling biofilm production.

Download Full-text

Camphor and Eucalyptol—Anticandidal Spectrum, Antivirulence Effect, Efflux Pumps Interference and Cytotoxicity

International Journal of Molecular Sciences ◽

10.3390/ijms22020483 ◽

2021 ◽

Vol 22 (2) ◽

pp. 483

Author(s):

Marija Ivanov ◽

Abhilash Kannan ◽

Dejan S. Stojković ◽

Jasmina Glamočlija ◽

Ricardo C. Calhelha ◽

...

Keyword(s):

Fungal Pathogens ◽

Efflux Pump ◽

Efflux Pumps ◽

Liver Cells ◽

Ergosterol Biosynthesis ◽

Antifungal Compound ◽

Porcine Liver ◽

Fungal Virulence ◽

Genes Encoding ◽

Plant Constituents

Candidaalbicans represents one of the most common fungal pathogens. Due to its increasing incidence and the poor efficacy of available antifungals, finding novel antifungal molecules is of great importance. Camphor and eucalyptol are bioactive terpenoid plant constituents and their antifungal properties have been explored previously. In this study, we examined their ability to inhibit the growth of different Candida species in suspension and biofilm, to block hyphal transition along with their impact on genes encoding for efflux pumps (CDR1 and CDR2), ergosterol biosynthesis (ERG11), and cytotoxicity to primary liver cells. Camphor showed excellent antifungal activity with a minimal inhibitory concentration of 0.125–0.35 mg/mL while eucalyptol was active in the range of 2–23 mg/mL. The results showed camphor’s potential to reduce fungal virulence traits, that is, biofilm establishment and hyphae formation. On the other hand, camphor and eucalyptol treatments upregulated CDR1;CDR2 was positively regulated after eucalyptol application while camphor downregulated it. Neither had an impact on ERG11 expression. The beneficial antifungal activities of camphor were achieved with an amount that was non-toxic to porcine liver cells, making it a promising antifungal compound for future development. The antifungal concentration of eucalyptol caused cytotoxic effects and increased expression of efflux pump genes, which suggests that it is an unsuitable antifungal candidate.

Download Full-text

Contribution of Target Gene Mutations and Efflux to Decreased Susceptibility of Salmonella enterica Serovar Typhimurium to Fluoroquinolones and Other Antimicrobials

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00600-06 ◽

2006 ◽

Vol 51 (2) ◽

pp. 535-542 ◽

Cited By ~ 86

Author(s):

Sheng Chen ◽

Shenghui Cui ◽

Patrick F. McDermott ◽

Shaohua Zhao ◽

David G. White ◽

...

Keyword(s):

Salmonella Enterica ◽

Efflux Pump ◽

Critical Role ◽

Gene Mutations ◽

Efflux Pumps ◽

Fluoroquinolone Resistance ◽

Naturally Occurring ◽

Serovar Typhimurium ◽

Resistant Mutants ◽

Increased Susceptibility

ABSTRACT The mechanisms involved in fluoroquinolone resistance in Salmonella enterica include target alterations and overexpression of efflux pumps. The present study evaluated the role of known and putative multidrug resistance efflux pumps and mutations in topoisomerase genes among laboratory-selected and naturally occurring fluoroquinolone-resistant Salmonella enterica serovar Typhimurium strains. Strains with ciprofloxacin MICs of 0.25, 4, 32, and 256 μg/ml were derived in vitro using serovar Typhimurium S21. These mutants also showed decreased susceptibility or resistance to many nonfluoroquinolone antimicrobials, including tetracycline, chloramphenicol, and several β-lactams. The expression of efflux pump genes acrA, acrB, acrE, acrF, emrB, emrD, and mdlB were substantially increased (≥2-fold) among the fluoroquinolone-resistant mutants. Increased expression was also observed, but to a lesser extent, with three other putative efflux pumps: mdtB (yegN), mdtC (yegO), and emrA among mutants with ciprofloxacin MICs of ≥32 μg/ml. Deletion of acrAB or tolC in S21 and its fluoroquinolone-resistant mutants resulted in increased susceptibility to fluoroquinolones and other tested antimicrobials. In naturally occurring fluoroquinolone-resistant serovar Typhimurium strains, deletion of acrAB or tolC increased fluoroquinolone susceptibility 4-fold, whereas replacement of gyrA double mutations (S83F D87N) with wild-type gyrA increased susceptibility >500-fold. These results indicate that a combination of topoisomerase gene mutations, as well as enhanced antimicrobial efflux, plays a critical role in the development of fluoroquinolone resistance in both laboratory-derived and naturally occurring quinolone-resistant serovar Typhimurium strains.

Download Full-text

Two Distinct Major Facilitator Superfamily Drug Efflux Pumps Mediate Chloramphenicol Resistance in Streptomyces coelicolor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00853-09 ◽

2009 ◽

Vol 53 (11) ◽

pp. 4673-4677 ◽

Cited By ~ 24

Author(s):

James J. Vecchione ◽

Blair Alexander ◽

Jason K. Sello

Keyword(s):

Efflux Pump ◽

Streptomyces Coelicolor ◽

Efflux Pumps ◽

Major Facilitator Superfamily ◽

Close Relative ◽

Gram Positive ◽

Antibacterial Drugs ◽

Chloramphenicol Resistance ◽

Drug Activity ◽

Major Facilitator

ABSTRACT Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-β-naphthylamide, respectively. These compounds are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate drug activity against gram-positive bacteria, this is the first time that Phe-Arg-β-naphthylamide has been shown to potentiate drug activity against a gram-positive bacterium.

Download Full-text

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.

Download Full-text

Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01022-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 8

Author(s):

Raees A. Paul ◽

Shivaprakash M. Rudramurthy ◽

Manpreet Dhaliwal ◽

Pankaj Singh ◽

Anup K. Ghosh ◽

...

Keyword(s):

Aspergillus Flavus ◽

Efflux Pump ◽

Efflux Pumps ◽

Azole Resistance ◽

Wild Type ◽

Multidrug Efflux ◽

Environmental Isolates ◽

Content Type ◽

Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

Download Full-text

Object Metamorphosis Based on Generalized Morphology Transformation

International Journal of Virtual Reality ◽

10.20870/ijvr.2001.5.1.2671 ◽

2001 ◽

Vol 5 (1) ◽

pp. 92-103

Author(s):

Hua Li ◽

Wenyu Liu ◽

Guangxi Zhu ◽

Yaoting Zhu

Keyword(s):

Computer Animation ◽

Industrial Design ◽

Simple Computation ◽

Shape Transformation ◽

High Quality ◽

Growth Simulation ◽

Novel Approach ◽

Polyhedral Objects ◽

2D And 3D

A metamorphosis or a morphing is the process of continuously transforming one object into another, and are popular in computer animation, industrial design, and growth simulation. In this paper, a novel approach is presented for computing continuous shape transformation between polyhedral objects in this paper. Metamorphosis can be achieved by decomposing two objects into sets of individual convex sub-objects respectively and constructing the mapping between two sets, this method can solve the metamorphosis problem of two non-homotopic objects (including concave objects and holey objects). The results of object metamorphosis are discussed in this paper. The experiments show that this method can generate natural, high quality metamorphosis results with simple computation. This method can also be used in font composition and interpolation between two keyframes in 2D and 3D computer animation automatically.

Download Full-text

Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei

10.1101/2020.09.18.303271 ◽

2020 ◽

Author(s):

Megan E Grund ◽

Soo J Choi ◽

Dudley H McNitt ◽

Mariette Barbier ◽

Gangqing Hu ◽

...

Keyword(s):

Outer Membrane ◽

Burkholderia Pseudomallei ◽

Triple Helix ◽

Efflux Pump ◽

Fusaric Acid ◽

Efflux Pumps ◽

Resistant Bacteria ◽

Burkholderia Mallei ◽

Membrane Component ◽

Collagen Triple Helix

AbstractBacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the Tier one select agents Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using RT-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the MIC of FA 4-fold in its isogenic mutant. We furthermore showed that the Bucl8 pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8 pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.Author SummaryBurkholderia pseudomallei and Burkholderia mallei are highly infectious and multidrug resistant bacteria that are classified by the National Institute of Allergy and Infectious Diseases as Tier one select agents partly due to the intrinsic multidrug resistance associated with expression of the efflux pumps. To date, only few efflux pumps predicted in Burkholderia spp. have been studied in detail. In the current study we introduce Bucl8, an outer membrane component of an unreported putative efflux pump with a unique extended extracellular portion that forms a collagen triple helix and binds fibrinogen. We demonstrate Bucl8’s role in fusaric acid resistance by defining its operon via bioinformatic and transcriptional analyses, as well as by employing loss-of-function and gain-of-function genetic approaches. Our studies also implicate the Bucl8-associated pump in metabolic and physiologic homeostasis. Understanding how Bucl8 efflux pump contributes to Burkholderia pathology will foster development of pump inhibitors targeting transport mechanism or identifying potential surface-exposed vaccine targets.

Download Full-text

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.

Download Full-text