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ABSTRACT 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.

Objective: Efflux pumps are transmembrane proteins associated with bacterial resistance mechanisms. Bacteria use these proteins to actively transport antibiotics to the extracellular medium, preventing the pharmacological action of these drugs. This study aimed to evaluate in vitro the antibacterial activity of 1,8-naphthyridines sulfonamides, as well as their ability to inhibit efflux systems of Staphylococcus aureus strains expressing different levels of the NorA efflux pump. Methods: The broth microdilution test was performed to assess antibacterial activity. Efflux pump inhibition was evaluated in silico by molecular docking and in vitro by fluorometric tests, and the minimum inhibitory concentration (MIC) was determined. The MIC was determined in the association between 1,8-naphthyridine and norfloxacin or ethidium bromide. Results: The 1,8-naphthyridines did not show direct antibacterial activity. However, they effectively reduced the MIC of multidrug-resistant bacteria by associating with norfloxacin and ethidium bromide, in addition to increasing the fluorescence emission. In silico analysis addressing the binding between NorA and 1,8-naphthyridines suggests that hydrogen bonds and hydrophilic interactions represent the interactions with the most favourable binding energy, corroborating the experimental data. Conclusion: Our data suggest that 1,8-naphthyridines sulfonamides inhibit bacterial resistance through molecular mechanisms associated with inhibition of the NorA efflux pump in S. aureus strains.

ABSTRACT 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.

Cuminum cyminum L. (cumin) is valued for its aromatic and medicinal properties. There are several reports of antibacterial activity of C. cyminum essential oil (CcEO). Accordingly, the present study was conducted to investigate the mechanism(s) of action of the CcEO against multidrug-resistant (MDR) Staphylococcus aureus. Therefore, 10 S. aureus MDR isolates, obtained from different sources, were selected based on the antibiotic susceptibility patterns and the Clinical and Laboratory Standards Institute definition and subjected to the examinations. Our results exhibited promising bacteriostatic and bactericidal properties of the CcEO. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration values ranged from 5 to 10 and 10 to 20 μL ⋅ mL–1, respectively. Scanning electron microscope was used to assess the bacterial cell structure and morphology after the induction with 1/2 MIC concentration of the CcEO. The observed morphological changes appeared to be deformation of the cell membrane and destruction of the cells. In the case of quorum sensing inhibitory potential, treatment of S. aureus isolates with the sub-MIC concentrations (1/2 MIC) of the CcEO significantly reduced the hld expression (3.13-fold downregulation), which considerably controls S. aureus quorum-sensing accessory regulator system. Another virulence factor influenced by the CcEO was the polysaccharide intercellular adhesion production system, as an important component of cell–cell adhesion and biofilm formation. Consequently, the expression level of the intercellular adhesion (ica) locus in the S. aureus cells was examined following treatment with CcEO. The results showed significant decrease (−3.3-fold) in ica expression, indicating that the CcEO could potentially interfere with the process of biofilm formation. Using the ethidium bromide efflux inhibition assay, the S. aureus NorA efflux pump was phenotypically but not genotypically (in quantitative polymerase chain reaction assay) affected by the CcEO treatment. Using gas chromatography–mass spectrometry analysis, cuminic aldehyde (38.26%), α,β-dihydroxyethylbenzene (29.16%), 2-caren-10-al (11.20%), and γ-terpinene (6.49%) were the most detected compounds. The antibacterial and antivirulence action of the CcEO at sub-MIC concentrations means that no microbial resistance will be promoted and developed after the treatment with this agent. These findings revealed that the CcEO is a promising antibacterial agent to control infections caused by the MDR S. aureus strains.

Baki Vijaya Bhaskar,1 Tirumalasetty Muni Chandra Babu,1 Netala Vasudeva Reddy,2 Wudayagiri Rajendra1 1Division of Molecular Biology, Department of Zoology, 2Department of Biotechnology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India Abstract: Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds. Keywords: Staphylococcus aureus, NorA efflux pump, molecular dynamics, virtual screening, docking

Toxins and venoms produced by living organisms have exhibited a variety of biological activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm formation against 22 clinical isolates of Staphylococcus aureus. Bothrops moojeni venom exhibited anti staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC). Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC) and norfloxacin or ampicillin (both ½ x MIC) for 86.4% and 50% of the isolates, respectively. B. moojeni venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, both at ½ MIC, a reduction on the NorA efflux pump activity was observed. The detection of in vitro mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the search for new antimicrobial agents against resistant S. aureus.

Multidrug efflux systems play a prominent role in medicine, as they are important contributors to bacterial antibiotic resistance. NorA is an efflux pump transporter from the major facilitator superfamily that expels numerous drug compounds across the inner membrane of Staphylococcus aureus (S. aureus). The design of novel inhibitors to combat drug efflux could offer new opportunities to avoid the problem of antibiotic resistance. In this study, we performed molecular modeling studies in an effort to discover novel NorA efflux pump inhibitors. A group of over 673 compounds from the PubChem database with a high (>80%) level of similarity to the chemical structure of capsaicin was used to study the binding affinity of small molecule compounds for the NorA efflux pump. Ten potential lead compounds displayed a good druggability profile, with one in particular (CID 44330438) providing new insight into the molecular mechanism of the inhibition of major facilitator superfamily (MFS) efflux pump transporters. It is our hope that the overall strategy described in this study, and the structural information of the potential novel inhibitors thus identified, will stimulate others to pursue the development of better drugs to tackle multidrug resistance in S. aureus.

Staphylococcus aureus causes a wide variety of infections worldwide. Methicillin-resistant S. aureus is one of most common causes of nosocomial and community acquired infections. The fluoroquinolones are an important class of antibiotics that used to treat infections caused by S. aureus. Today, a significant increase in the rate of ciprofloxacin resistance in methicillin-resistant S. aureus strains is concerning. The norA efflux pump is considered as contributors to antibiotic resistance. Here, we aimed to evaluate the expression of norA efflux pump in the presence of hexahydroquinoline derivative in methicillin and ciprofloxacin resistant S. aureus. We were determined minimum inhibitory concentration of ciprofloxacin and hexahydroquinoline derivative and their combination by broth microdilution method against ciprofloxacin resistant S. aureus. The expression of the norA efflux pump gene was evaluated by quantitative Real-time PCR. This study showed that minimum inhibitory concentrations of ciprofloxacin in the presence of hexahydroquinoline derivative in comparison to ciprofloxacin were decreased. Quantitative Real-time PCR identified the increased expression of norA efflux pump gene in methicillin and ciprofloxacin resistant S. aureus strain. The increased expression of norA efflux pump gene may have resulted in the effort of S. aureus to survive. The results showed that the hexahydroquinoline derivative enhanced the antibacterial effect of ciprofloxacin against methicillin and ciprofloxacin resistant S. aureus. Therefore, the derivatives may be used as inhibitors of antibiotic resistance for combination therapy.