Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Efflux pump inhibitory potential of indole derivatives as an arsenal against norA over-expressing Staphylococcus aureus.

NorA, an extensively studied efflux pump in Staphylococcus aureus, has been connected to fluoroquinolone, antiseptic, and disinfection resistance. Several studies have also emphasized how efflux pumps, including NorA, function as the first line of defense of S. aureus against antibiotics. In this study, we have screened some chemically synthesized indole derivatives for their activity as efflux pump inhibitors (EPIs). The derivative SMJ-5 was found to be a potent NorA efflux pump inhibitor among the screened indole derivatives, owing to increased ethidium bromide and norfloxacin accumulation in norA over-expressing S. aureus. The combination of SMJ-5 and ciprofloxacin demonstrated the eradication of S. aureus biofilm and prolonged the post-antibiotic effect more than ciprofloxacin alone. SMJ-5 was able to inhibit staphyloxanthin virulence. In in vitro time-kill trials and in vivo efficacy investigations, the combination enhanced the bactericidal activity of ciprofloxacin against S. aureus. Additionally, reverse transcription PCR results revealed that SMJ-5 also inhibits the NorA efflux pump indirectly at the transcriptional level. IMPORTANCE The NorA efflux pump is the most effective resistance mechanism in S. aureus. The clinical importance of NorA efflux pumps is demonstrated by the expression of pump genes in S. aureus strains in response to fluoroquinolones and biocides. Along with the repercussions of decreased fluoroquinolone sensitivity, increasing expression of efflux pump genes by their substrate necessitates the importance of efflux pump inhibitors. Reserpine and verapamil are clinically used to treat ailments and have proven NorA inhibitors, but, unfortunately, the concentration needed for these drugs to inhibit the pump is not safe in clinical settings. In the current study, we have screened some indole derivatives, and among them, SMJ-5 was reported to potentiate norfloxacin and ciprofloxacin at their sub-inhibitory concentration by inhibiting the norA gene transcriptionally. Here we highlight the promising points of this study, which could serve as a model to design a therapeutic EPI candidate against norA over-expressing S. aureus.

A Microbe-Derived Efflux Pump Inhibitor of the Resistance-Nodulation-Cell Division Protein Restores Antibiotic Susceptibility in Escherichia coli and Pseudomonas aeruginosa.

The use of efflux pump inhibitors (EPIs) as potentiators along with the traditional antibiotics assists in the warfare against antibiotic-resistant superbugs. Efflux pumps of the resistance-nodulation-cell division (RND) family play crucial roles in multidrug resistance in Escherichia coli and Pseudomonas aeruginosa. Despite several efforts, clinically useful inhibitors are not available at present. This study describes ethyl 4-bromopyrrole-2-carboxylate (RP1) isolation, an inhibitor of RND transporters from the library of 4000 microbial exudates. RP1 acts synergistically with antibiotics by reducing their minimum inhibitory concentration in strains overexpressing archetype RND transporters (AcrAB-TolC and MexAB-OprM). It also improves the accumulation of Hoechst 33342 and inhibits its efflux (a hallmark of EPI functionality). The antibiotic-RP1 combinations prolong the postantibiotic effects and reduce the mutation prevention concentration of antibiotics. Additionally, from Biolayer Interferometry spectra, it appears that RP1 is bound to AcrB. RP1 displays low mammalian cytotoxicity, no Ca2+ channel inhibitory effects, and reduces the intracellular invasion of E. coli and P. aeruginosa in macrophages. Furthermore, the RP1-levofloxacin combination is nontoxic, well-tolerated, and notably effective in a murine lung infection model. In sum, RP1 is a potent EPI and worthy of further consideration as a potentiator to improve the effectiveness of existing antibiotics.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus.

Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Antifungal Azoles as Tetracycline Resistance Modifiers in Staphylococcus aureus.

Staphylococcus aureus has developed resistance to antimicrobials since their first use. The S. aureus major facilitator superfamily (MFS) efflux pump Tet(K) contributes to resistance to tetracyclines. The efflux pump diminishes antibiotic accumulation, and biofilm hampers the diffusion of antibiotics. None of the currently known compounds have been approved as efflux pump inhibitors (EPIs) for clinical use. In the current study, we screened clinically approved drugs for possible Tet(K) efflux pump inhibition. By performing in silico docking followed by in vitro checkerboard assays, we identified five azoles (the fungal ergosterol synthesis inhibitors) showing putative EPI-like potential with a fractional inhibitory concentration index of ≤0.5, indicating synergism. The functionality of the azoles was confirmed using ethidium bromide (EtBr) accumulation and efflux inhibition assays. In time-kill kinetics, the combination treatment with butoconazole engendered a marked increase in the bactericidal capacity of tetracycline. When assessing the off-target effects of the azoles, we observed no disruption of bacterial membrane permeability and polarization. Finally, the combination of azoles with tetracycline led to a significant eradication of preformed mature biofilms. This study demonstrates that azoles can be repurposed as putative Tet(K) EPIs and to reduce biofilm formation at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus uses efflux pumps to transport antibiotics out of the cell and thus increases the dosage at which it endures antibiotics. Also, efflux pumps play a role in biofilm formation by the excretion of extracellular matrix molecules. One way to combat these pathogens may be to reduce the activity of efflux pumps and thereby increase pathogen sensitivity to existing antibiotics. We describe the in silico-based screen of clinically approved drugs that identified antifungal azoles inhibiting Tet(K), a pump that belongs to the major facilitator superfamily, and showed that these compounds bind to and block the activity of the Tet(K) pump. Azoles enhanced the susceptibility of tetracycline against S. aureus and its methicillin-resistant strains. The combination of azoles with tetracycline led to a significant reduction in preformed biofilms. Repurposing approved drugs may help solve the classical toxicity issues related to efflux pump inhibitors.

MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220.

MsrA, an efflux pump belonging to ATP-binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2-8-fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8-fold. The real-time fluorometry-based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post-antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.

Purification and biological activity of natural variants synthesized by tridecaptin M gene cluster and in vitro drug-kinetics of this antibiotic class.

The flexibility of the adenylation domains of non-ribosomal peptide synthetases (NRPSs) to different substrates creates a diversity of structurally similar peptides. In the present study, we investigated the antimicrobial activity of different natural variants synthesized by tridecaptin M gene cluster and performed the in vitro drug kinetics on this class. The natural variants were isolated and characterized using MALDI-MS and tandem mass spectrometry. All the peptides were studied for their antimicrobial activity in different pathogens, including colistin-resistant bacteria, and for haemolytic activity. Furthermore, in vitro drug kinetics was performed with tridecaptin M (or M1, the major product of the gene cluster). The natural variants displayed a varying degree of bioactivity with M11 showing the most potent antibacterial activity (MIC, 1-8 µg/ml), even against A. baumannii and P. aeruginosa strains. The in vitro kinetic studies revealed that tridecaptin M at a concentration of 16 µg/ml eradicated the bacteria completely in high-density culture. The compound demonstrated desirable post-antibiotic effect after two-hour exposure at MIC concentration. We also observed the reversal of resistance to this class of antibiotics in the presence of carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Altogether, the study demonstrated that tridecaptins are an excellent drug candidate against drug-resistant Gram-negative bacteria. Future studies are required to design a superior tridecaptin by investigating the interactions of different natural variants with the target.

Microbe-Derived Indole Metabolite Demonstrates Potent Multidrug Efflux Pump Inhibition in Staphylococcus aureus.

Efflux pumps are always at the forefront of bacterial multidrug resistance and account for the failure of antibiotics. The present study explored the potential of 2-(2-Aminophenyl) indole (RP2), an efflux pump inhibitor (EPI) isolated from the soil bacterium, to overcome the efflux-mediated resistance in Staphylococcus aureus. The RP2/antibiotic combination was tested against efflux pump over-expressed S. aureus strains. The compound was further examined for the ethidium bromide (EtBr) uptake and efflux inhibition assay (a hallmark of EPI functionality) and cytoplasmic membrane depolarization. The safety profile of RP2 was investigated using in vitro cytotoxicity assay and Ca2+ channel inhibitory effect. The in vivo efficacy of RP2 was studied in an animal model in combination with ciprofloxacin. RP2 exhibited the synergistic activity with several antibiotics in efflux pump over-expressed strains of S. aureus. In the mechanistic experiments, RP2 increased the accumulation of EtBr, and demonstrated the inhibition of its efflux. The antibiotic-EPI combinations resulted in extended post antibiotic effects as well as a decrease in mutation prevention concentration of antibiotics. Additionally, the in silico docking studies suggested the binding of RP2 to the active site of modeled structure of NorA efflux pump. The compound displayed low mammalian cytotoxicity and had no Ca2+ channel inhibitory effect. In ex vivo experiments, RP2 reduced the intracellular invasion of S. aureus in macrophages. Furthermore, the RP2/ciprofloxacin combination demonstrated remarkable efficacy in a murine thigh infection model. In conclusion, RP2 represents a promising candidate as bacterial EPI, which can be used in the form of a novel therapeutic regimen along with existing and upcoming antibiotics, for the eradication of S. aureus infections.