P-gp efflux pump inhibition potential of common environmental contaminants determined in vitro

ABSTRACT The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is slowly rising as a consequence of the increased use of fluoroquinolone antibiotics to treat community-acquired pneumonia. We tested the hypothesis that increased efflux pump (EP) expression by S. pneumoniae may facilitate the emergence of fluoroquinolone resistance. By using an in vitro pharmacodynamic infection system, a wild-type S. pneumoniae strain (Spn-058) and an isogenic strain with EP overexpression (Spn-RC2) were treated for 10 days with ciprofloxacin or levofloxacin in the presence or absence of the EP inhibitor reserpine to evaluate the effect of EP inhibition on the emergence of resistance. Cultures of Spn-058 and Spn-RC2 were exposed to concentration-time profiles simulating those in humans treated with a regimen of ciprofloxacin at 750 mg orally once every 12 h and with regimens of levofloxacin at 500 and 750 mg orally once daily (QD; with or without continuous infusions of 20 μg of reserpine/ml). The MICs of ciprofloxacin and levofloxacin for Spn-058 were both 1 μg/ml when susceptibility testing was conducted with each antibiotic alone and with each antibiotic in the presence of reserpine. For Spn-RC2, the MIC of levofloxacin alone and with reserpine was also 1 μg/ml; the MICs of ciprofloxacin were 2 and 1 μg/ml, respectively, when determined with ciprofloxacin alone and in combination with reserpine. Reserpine, alone, had no effect on the growth of Spn-058 and Spn-RC2. For Spn-058, simulated regimens of ciprofloxacin at 750 mg every 12 h or levofloxacin at 500 mg QD were associated with the emergence of fluoroquinolone resistance. However, the use of ciprofloxacin at 750 mg every 12 h and levofloxacin at 500 mg QD in combination with reserpine rapidly killed Spn-058 and prevented the emergence of resistance. For Spn-RC2, levofloxacin at 500 mg QD was associated with the emergence of resistance, but again, the resistance was prevented when this levofloxacin regimen was combined with reserpine. Ciprofloxacin at 750 mg every 12 h also rapidly selected for ciprofloxacin-resistant mutants of Spn-RC2. However, the addition of reserpine to ciprofloxacin therapy only delayed the emergence of resistance. Levofloxacin at 750 mg QD, with and without reserpine, effectively eradicated Spn-058 and Spn-RC2 without selecting for fluoroquinolone resistance. Ethidium bromide uptake and efflux studies demonstrated that, at the baseline, Spn-RC2 had greater EP expression than Spn-058. These studies also showed that ciprofloxacin was a better inducer of EP expression than levofloxacin in both Spn-058 and Spn-RC2. However, in these isolates, the increase in EP expression by short-term exposure to ciprofloxacin and levofloxacin was transient. Mutants of Spn-058 and Spn-RC2 that emerged under suboptimal antibiotic regimens had a stable increase in EP expression. Levofloxacin at 500 mg QD in combination with reserpine, an EP inhibitor, or at 750 mg QD alone killed wild-type S. pneumoniae and strains that overexpressed reserpine-inhibitable EPs and was highly effective in preventing the emergence of fluoroquinolone resistance in S. pneumoniae during therapy. Ciprofloxacin at 750 mg every 12 h, as monotherapy, was ineffective for the treatment of Spn-058 and Spn-RC2. Ciprofloxacin in combination with reserpine prevented the emergence of resistance in Spn-058 but not in Spn-RC2, the EP-overexpressing strain.

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Ketone- and Cyano-Selenoesters to Overcome Efflux Pump, Quorum-Sensing, and Biofilm-Mediated Resistance

Antibiotics ◽

10.3390/antibiotics9120896 ◽

2020 ◽

Vol 9 (12) ◽

pp. 896

Author(s):

Nikoletta Szemerédi ◽

Annamária Kincses ◽

Katerina Rehorova ◽

Lan Hoang ◽

Noemi Salardón-Jiménez ◽

...

Keyword(s):

Antibacterial Activity ◽

Quorum Sensing ◽

Efflux Pump ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Experimental Systems ◽

Efflux Pump Inhibition ◽

Target Molecules ◽

Vibrio Campbellii

The emergence of drug-resistant pathogens leads to a gradual decline in the efficacy of many antibacterial agents, which poses a serious problem for proper therapy. Multidrug resistance (MDR) mechanisms allow resistant bacteria to have limited uptake of drugs, modification of their target molecules, drug inactivation, or release of the drug into the extracellular space by efflux pumps (EPs). In previous studies, selenoesters have proved to be promising derivatives with a noteworthy antimicrobial activity. On the basis of these results, two series of novel selenoesters were synthesized to achieve more potent antibacterial activity on Gram-positive and Gram-negative bacteria. Fifteen selenoesters (eight ketone-selenoesters and seven cyano-selenoesters) were investigated with regards to their efflux pump-inhibiting, anti-quorum-sensing (QS), and anti-biofilm effects in vitro. According to the results of the antibacterial activity, the ketone-selenoesters proved to be more potent antibacterial compounds than the cyano-selenoesters. With regard to efflux pump inhibition, one cyano-selenoester on methicillin-resistant S. aureus and one ketone-selenoester on Salmonella Typhimurium were potent inhibitors. The biofilm inhibitory capacity and the ability of the derivatives to disrupt mature biofilms were noteworthy in all the experimental systems applied. Regarding QS inhibition, four ketone-selenoesters and three cyano-selenoesters exerted a noteworthy effect on Vibrio campbellii strains.

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Increased susceptibility of a triclabendazole (TCBZ)-resistant isolate of Fasciola hepatica to TCBZ following co-incubation in vitro with the P-glycoprotein inhibitor, R(+)-verapamil

Parasitology ◽

10.1017/s0031182013000759 ◽

2013 ◽

Vol 140 (10) ◽

pp. 1287-1303 ◽

Cited By ~ 15

Author(s):

M. MEANEY ◽

J. SAVAGE ◽

G. P. BRENNAN ◽

E. HOEY ◽

A. TRUDGETT ◽

...

Keyword(s):

Fasciola Hepatica ◽

Efflux Pump ◽

Morphological Changes ◽

Resistant Isolate ◽

Drug Efflux ◽

P Glycoprotein ◽

Efflux Pump Inhibition ◽

Inactivity Time ◽

Drug Efflux Pumps

SUMMARYA study was carried out to investigate whether the action of triclabendazole sulphoxide (TCBZ.SO) against the liver fluke, Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for this in vitro study and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. For experiments with the Oberon isolate, flukes were incubated for 24 h with either R(+)-VPL (1×10−4m) on its own, TCBZ.SO (15 μg mL−1) alone, a combination of R(+)-VPL (1×10−4m) plus TCBZ.SO (15 μg mL−1), TCBZ.SO (50 μg mL−1) on its own, or a combination of TCBZ.SO (50 μg mL−1) plus R(+)-VPL (1×10−4m). They were also incubated in TCBZ.SO (50 μg mL−1) alone or in combination with R(+)-VPL (1×10−4m) until they became inactive; and in TCBZ.SO (50 μg mL−1) alone for a time to match that of the combination inactivity time. Flukes from the Cullompton isolate were treated with either TCBZ.SO (50 μg mL−1) alone or in combination with R(+)-VPL (1×10−4m) until they became inactive, or with TCBZ.SO (50 μg mL−1) alone time-matched to the combination inactivity time. Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R(+)-VPL alone had a minimal effect on either isolate. TCBZ.SO treatment had a relatively greater impact on the TCBZ-susceptible Cullompton isolate. When R(+)-VPL was combined with TCBZ.SO in the incubation medium, however, the surface disruption to both isolates was more severe than that seen after TCBZ.SO treatment alone; also, the time taken to reach inactivity was shorter. More significantly, though, the potentiation of drug activity was greater in the Oberon isolate; also, it was more distinct at the higher concentration of TCBZ.SO. So, the Oberon isolate appears to be particularly sensitive to efflux pump inhibition. The results of this study suggest that enhanced drug efflux in the Oberon isolate may be involved in the mechanism of resistance to TCBZ.

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Curcumin-meropenem synergy in carbapenem resistant Klebsiella pneumoniae curcumin-meropenem synergy

Iranian Journal of Microbiology ◽

10.18502/ijm.v13i3.6397 ◽

2021 ◽

Author(s):

Dumrul Gülen ◽

Birol Şafak ◽

Berna Erdal ◽

Betül Günaydın

Keyword(s):

Klebsiella Pneumoniae ◽

Bacterial Infections ◽

Efflux Pump ◽

Synergistic Effects ◽

Treatment Options ◽

Resistance Pattern ◽

Carbapenem Resistant ◽

Efflux Pump Inhibition ◽

Disk Method

Background and Objectives: The frequency of multiple resistant bacterial infections, including carbapenems, is increasing worldwide. As the decrease in treatment options causes difficulties in treatment, interest in new antimicrobials is increasing. One of the promising natural ingredients is curcumin. It is known to be effective in bacteria such as Pseudomonas aeruginosa, Escherichia coli, Burkholderia pseudomallei through efflux pump inhibition, toxin inhibition and enzymes. However, because its bioavailability is poor, it seffectiveness occurs in combination with antibiotics. In the study, the interaction of meropenem and curcumin in carbapenemase producing strains of Klebsiella pneumoniae was tested. Materials and Methods: Thirty-nine Klebsiella pneumoniae isolates, resistant to meropenem, were used in this study. From those 15 MBL, 6 KPC, 17 OXA-48 and 1 AmpC resistance pattern were detected by combination disk method. Meropenem and Curcumin MIC values were determined by liquid microdilution. Checkerboard liquid microdilution was used to determine the synergy between meropenem and curcumin. Results: Synergistic effects were observed in 4 isolates producing MBL, 3 isolates producing KPC, 4 isolates producing OXA-48, and 1 isolates producing AmpC (totally 12 isolates) according to the calculated FICI. No antagonistic effects were observed in any isolates. Conclusion: Curcumin was thought to be an alternative antimicrobial in combination therapies that would positively contribute to the treatment of bacterial infection. The effectiveness of this combination should be confirmed by other in vitro and clinical studies.

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In vitro evaluation of glycol chitosan based formulations as oral delivery systems for efflux pump inhibition

Carbohydrate Polymers ◽

10.1016/j.carbpol.2017.02.096 ◽

2017 ◽

Vol 166 ◽

pp. 73-82 ◽

Cited By ~ 13

Author(s):

Delia Mandracchia ◽

Adriana Trapani ◽

Giuseppe Tripodo ◽

Maria Grazia Perrone ◽

Gaetano Giammona ◽

...

Keyword(s):

Oral Delivery ◽

Efflux Pump ◽

Delivery Systems ◽

Glycol Chitosan ◽

In Vitro Evaluation ◽

Efflux Pump Inhibition ◽

Oral Delivery Systems

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In Vitro Synergy of Ciprofloxacin and Gatifloxacin against Ciprofloxacin-Resistant Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.7.2959-2964.2005 ◽

2005 ◽

Vol 49 (7) ◽

pp. 2959-2964 ◽

Cited By ~ 51

Author(s):

George A. Pankey ◽

Deborah S. Ashcraft

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

In Vitro Study ◽

Multidrug Resistant ◽

Efflux Pump Inhibition ◽

Pump Mechanism ◽

Time Kill ◽

Synergy Testing ◽

Unique Plasmid

ABSTRACT Multidrug-resistant Pseudomonas aeruginosa with combined decreased susceptibility to ceftazidime, ciprofloxacin, imipenem, and piperacillin is increasingly being found as a cause of nosocomial infections. It is important to look for combinations of drugs that might be synergistic. Ciprofloxacin resistance by P. aeruginosa is mediated in part by an efflux pump mechanism. Gatifloxacin, an 8-methoxyfluoroquinolone, inhibits a staphylococcal efflux pump. An earlier in vitro study using an Etest synergy method and time-kill assay suggested synergy of ciprofloxacin and gatifloxacin against P. aeruginosa. Synergy testing was performed by Etest and time-kill assay for 31 clinically unique, plasmid DNA distinct, U.S. P. aeruginosa isolates. Etest MICs for ciprofloxacin were 4 to >32 μg/ml, and for gatifloxacin they were >32 μg/ml. Ciprofloxacin plus gatifloxacin showed synergy by the Etest method for 6 (19%) of the 31 P. aeruginosa isolates using a summation fractional inhibitory concentration of ≤0.5 for synergy. Synergy was demonstrated for 13/31 (42%) of isolates by time-kill assay. No antagonism was detected. The remaining isolates were indifferent to the combination. The Etest method and time-kill assay were 65% (20/31) concordant. The mechanism of the in vitro synergy may include P. aeruginosa ciprofloxacin efflux pump inhibition by gatifloxacin.

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Development of an Antibiotic Resistance Breaker to Resensitize Drug-Resistant Staphylococcus aureus: In Silico and In Vitro Approach

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.700198 ◽

2021 ◽

Vol 11 ◽

Author(s):

Gopalakrishnan Thamilselvan ◽

Hema Bhagavathi Sarveswari ◽

Sahana Vasudevan ◽

Alex Stanley ◽

Karthi Shanmugam ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Boronic Acid ◽

Bacterial Resistance ◽

Efflux Pump ◽

Parent Compound ◽

Efflux Pumps ◽

Online Tool ◽

Efflux Pump Inhibition ◽

Boronic Acid Derivatives

Efflux pumps are one of the predominant microbial resistant mechanisms leading to the development of multidrug resistance. In Staphylococcus aureus, overexpression of NorA protein enables the efflux of antibiotics belonging to the class of fluoroquinolones and, thus, makes S. aureus resistant. Hence, NorA efflux pumps are being extensively exploited as the potential drug target to evade bacterial resistance and resensitize bacteria to the existing antibiotics. Although several molecules are reported to inhibit NorA efflux pump effectively, boronic acid derivatives were shown to have promising NorA efflux pump inhibition. In this regard, the current study exploits 6-(3-phenylpropoxy)pyridine-3-boronic acid to further improve the activity and reduce cytotoxicity using the bioisostere approach, a classical medicinal chemistry concept. Using the SWISS-Bioisostere online tool, from the parent compound, 42 compounds were obtained upon the replacement of the boronic acid. The 42 compounds were docked with modeled NorA protein, and key molecular interactions of the prominent compounds were assessed. The top hit compounds were further analyzed for their drug-like properties using ADMET studies. The identified potent lead, 5-nitro-2-(3-phenylpropoxy)pyridine (5-NPPP), was synthesized, and in vitro efficacy studies have been proven to show enhanced efflux inhibition, thus acting as a potent antibiotic breaker to resensitize S. aureus without elucidating any cytotoxic effect to the host Hep-G2 cell lines.

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Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria

International Journal of Molecular Sciences ◽

10.3390/ijms22042062 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2062

Author(s):

Aneta Kaczor ◽

Karolina Witek ◽

Sabina Podlewska ◽

Veronique Sinou ◽

Joanna Czekajewska ◽

...

Keyword(s):

Molecular Modelling ◽

Efflux Pump ◽

Gram Negative Bacteria ◽

Potential Mechanism ◽

Multidrug Efflux ◽

Aromatic Rings ◽

Allosteric Site ◽

Multidrug Efflux Pump ◽

Dual Action

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

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