Olga Lomovskaya

Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents for Combination Therapy

ABSTRACT Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g., gyrA andparC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (1 μg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was ∼10−7 CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10−11). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones.

Tackling antibiotic resistance

The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable but can nevertheless be controlled, and it must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of 30 scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, USA, from 16 to 18 May 2011. From these discussions there emerged a priority list of steps that need to be taken to resolve this global crisis.

Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials

ABSTRACT Plant antimicrobials are not used as systemic antibiotics at present. The main reason for this is their low level of activity, especially against gram-negative bacteria. The reported MIC is often in the range of 100 to 1,000 μg/ml, orders of magnitude higher than those of common broad-spectrum antibiotics from bacteria or fungi. Major plant pathogens belong to the gram-negative bacteria, which makes the low level of activity of plant antimicrobials against this group of microorganisms puzzling. Gram-negative bacteria have an effective permeability barrier, comprised of the outer membrane, which restricts the penetration of amphipathic compounds, and multidrug resistance pumps (MDRs), which extrude toxins across this barrier. It is possible that the apparent ineffectiveness of plant antimicrobials is largely due to the permeability barrier. We tested this hypothesis in the present study by applying a combination of MDR mutants and MDR inhibitors. A panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant pathogens. The human pathogens Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhimurium were also tested. The results show that the activities of the majority of plant antimicrobials were considerably greater against the gram-positive bacteria Staphylococcus aureus and Bacillus megaterium and that disabling of the MDRs in gram-negative species leads to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb, was potentiated 100- to 2,000-fold (depending on the bacterial species) by disabling the MDRs. Comparable potentiation of activity was observed with plumbagin, resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine, a model plant antimicrobial, confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of gram-negative bacteria. These results suggest that plants might have developed means of delivering their antimicrobials into bacterial cells. These findings also suggest that plant antimicrobials might be developed into effective, broad-spectrum antibiotics in combination with inhibitors of MDRs.

Interplay between Efflux Pumps May Provide Either Additive or Multiplicative Effects on Drug Resistance

The effects of simultaneous expression of several efflux pumps on antibiotic resistance were investigated in Escherichia coli and Pseudomonas aeruginosa. Several combinations of efflux pumps have been studied: (i) simultaneous expression of a single-component efflux pump, which exports antibiotics into the periplasm, in combination with a multicomponent efflux pump that accomplishes efflux directly into the external medium; (ii) simultaneous expression of two single-component pumps; and (iii) simultaneous expression of two multicomponent pumps. It was found that when efflux pumps of different structural types were combined in the same cell (the first case), the observed antibiotic resistance was much higher than that conferred by each of the pumps expressed singly. Simultaneous expression of pairs of single-component or multicomponent efflux pumps (the second and third cases) did not produce strong increases in antibiotic resistance.

Waltzing transporters and 'the dance macabre' between humans and bacteria

Multidrug-resistance efflux pumps — in particular those belonging to the resistance-nodulation-cell-division (RND) family of transporters, with their unusually high degree of substrate promiscuity — significantly restrict the effectiveness of antibacterial therapy. Recent years have heralded remarkable insights into the structure and mechanisms of these fascinating molecular machines. Here, we review recent advances in the field and describe various approaches used in combating efflux-mediated resistance.

Use of an Efflux Pump Inhibitor To Determine the Prevalence of Efflux Pump-Mediated Fluoroquinolone Resistance and Multidrug Resistance in Pseudomonas aeruginosa

ABSTRACT Fluoroquinolone-resistance in Pseudomonas aeruginosa may be due to efflux pump overexpression (EPO) and/or target mutations. EPO can result in multidrug resistance (MDR) due to broad substrate specificity of the pumps. MC-04,124, an efflux pump inhibitor (EPI) shown to significantly potentiate activity of levofloxacin in P. aeruginosa, was used to examine the prevalence of EPO in clinical isolates. MICs were determined for ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin with or without EPI and for other antipseudomonal agents by using broth microdilution against P. aeruginosa isolates from adults (n = 119) and children (n = 24). The prevalence of the EPO phenotype (≥8-fold MIC decrease when tested with EPI) was compared among subgroups with different resistance profiles. The EPO phenotype was more prevalent among levofloxacin-resistant than levofloxacin-sensitive strains (61%, 48/79 versus 9%, 6/64). EPO was present in 60% of fluoroquinolone-resistant strains without cross-resistance, while it was present at variable frequencies among strains with cross-resistance to other agents: piperacillin-tazobactam (86%), ceftazidime (76%), cefepime (65%), imipenem (56%), gentamicin (55%), tobramycin (48%), and amikacin (27%). The magnitude of MIC decrease with an EPI paralleled the frequency of which the EPO phenotype was observed in different subgroups. EPI reduced the levofloxacin MIC by as much as 16-fold in eight strains for which MICs were 128 μg/ml. Efflux-mediated resistance appears to contribute significantly to fluoroquinolone resistance and MDR in P. aeruginosa. Our data support the fact that increased fluoroquinolone usage can negatively impact susceptibility of P. aeruginosa to multiple classes of antipseudomonal agents.

On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps : the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition

Tripartite efflux systems of Gram‐negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds. To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD–OprJ pump from Pseudomonas aeruginosa. These mutations enabled the pump to extrude the normally non‐transported β‐lactam antibiotic carbenicillin. All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD. Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8. These mutations also had a substantial impact on the MexCD–OprJ‐mediated transport of numerous other substrates. Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD–OprJ susceptible to inhibition by a thiol‐reactive agent, MIANS. Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu–Nap) substrates of the pump. Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates. These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.

Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa

Conformational restriction of the ornithine residue of the efflux pump inhibitor D-ornithine-D-homophenylalanine-3-aminoquinoline (MC-02,595, 2) furnished bioisosteric proline derivatives that were less toxic in vivo and as active as the lead in potentiating the activity of the fluoroquinolone levofloxacin via the inhibition of efflux pumps in Pseudomonas aeruginosa.

Peptidomimetics of Efflux Pump Inhibitors Potentiate the Activity of Levofloxacin in Pseudomonas aeruginosa

Several classes of peptidomimetics of the efflux pump inhibitor D-ornithine-D-homophenylalanine-3-aminoquinoline (MC-02,595) have been prepared and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin in Pseudomonas aeruginosa. A number of the new analogues were as active or more active than the lead, demonstrating that a peptide backbone is not essential for activity.

MexXY-OprM Efflux Pump Is Required for Antagonism of Aminoglycosides by Divalent Cations in Pseudomonas aeruginosa

ABSTRACT Antagonism of aminoglycosides by divalent cations is well documented for Pseudomonas aeruginosa and is regarded as one of the problems in aminoglycoside therapy. It is generally considered that divalent cations interfere with uptake of aminoglycosides at both the outer and inner membranes. It has been demonstrated recently that aminoglycosides can be removed from cells ofP. aeruginosa by the three-component multidrug resistance efflux pump MexXY-OprM. We sought to investigate the interplay between efflux and uptake in resistance to aminoglycosides inP. aeruginosa. To do so, we studied the effects of the divalent cations Mg2+ and Ca2+ on susceptibility to aminoglycosides in a wild-type strain of P. aeruginosa and in mutants either overexpressing or lacking the MexXY-OprM efflux pump. MICs of gentamicin, streptomycin, amikacin, apramycin, netilmicin, and arbekacin were determined in Mueller-Hinton broth in the presence of cations added at concentrations that varied from 0.125 to 8 mM. We found, unexpectedly, that while both Mg2+ and Ca2+ antagonized aminoglycosides (up to a 64-fold decrease in susceptibility at 8 mM), antagonism was seen only in the strains of P. aeruginosa that contained the functional MexXY-OprM efflux pump. Our results indicate that inhibition of the MexXY-OprM efflux pump should abolish the antagonism of aminoglycosides by divalent cations, regardless of its precise mechanism. This may significantly increase the therapeutic index of aminoglycosides and improve the clinical utility of this important class of antibiotics.