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Mupirocin induced expression of genes encoding efflux pumps NorA and MepA as well as a yellow fluorescent protein (YFP) fluorescence reporter of NorA. Mupirocin exposure also produced reduced susceptibility to pump substrates ciprofloxacin and chlorhexidine, a change that was dependent on intact norA and mepA , respectively.

At present, the universal health problem with Staphylococcus aureus is the emergence of multidrug-resistant strains due to the overuse of antibiotics. Drug extrusion through efflux pumps is one of the bacterial mechanisms to neutralize the bactericidal effect of antibiotics. The antibacterial activity of silver nanoparticle as well as Fe 3 O 4 nanoparticle had been previously studied and widely described. Today, the development of green methods for nanomaterial synthesis is an important aspect of research in the field of nanotechnology. Here, we report the biosynthesis and characterization of Fe 3 O 4 @Ag nanocomposite by Spirulina platensis cyanobacterium and it impacts on the expression of efflux pump genes in ciprofloxacin-resistant S. aureus (CRSA). The physical properties of biosynthesized nanocomposite measured and confirmed by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and scanning and transmission electron microscopy. The minimum inhibitory concentration (MIC) of ciprofloxacin in CRSA strains was determined in the presence of Fe 3 O 4 @Ag nanoparticles by broth microdilution method. The effect of Fe 3 O 4 @Ag nanocomposite on the expression of norA and norB genes was evaluated by real-time PCR. The physical analysis confirmed well-dispersed, highly stable, and mostly spherical Fe 3 O 4 /Ag NPs with the average size of 30-68 nm. The results of antibacterial tests showed the synergistic effects of nanocomposite and antibiotics in MIC reduction. Additionally, in the presence of Fe 3 O 4 @Ag nanocomposite, the expression of norA and norB genes was decreased more than twofold compared to control. In conclusion, the Fe 3 O 4 /Ag nanocomposite can use as an effective inhibitor of antibiotic resistance in medicine.

The expression of the efflux pump systems is the most important mechanism of antibiotic resistance in bacteria, as it contributes to reduced concentration and the subsequent inactivity of administered antibiotics. NorA is one of the most studied antibacterial targets used as a model for efflux-mediated resistance. The present study evaluated shikimate pathway-derived phenolic acids against NorA (PDB ID: 1PW4) as a druggable target in antibacterial therapy using in silico modelling and in vitro methods. Of the 22 compounds evaluated, sinapic acid (−9.0 kcal/mol) and p-coumaric acid (−6.3 kcal/mol) had the best and most prominent affinity for NorA relative to ciprofloxacin, a reference standard (−4.9 kcal/mol). A further probe into the structural stability and flexibility of the resulting NorA-phenolic acids complexes through molecular dynamic simulations over a 100 ns period revealed p-coumaric acid as the best inhibitor of NorA relative to the reference standard. In addition, both phenolic acids formed H-bonds with TYR 76, a crucial residue implicated in NorA efflux pump inhibition. Furthermore, the phenolic acids demonstrated favourable drug likeliness and conformed to Lipinski's rule of five for ADME properties. For the in vitro evaluation, the phenolic acids had MIC values in the range 31.2 to 62.5 μg/mL against S. aureus, and E. coli, and there was an overall reduction in MIC following their combination with ciprofloxacin. Taken together, the findings from both the in silico and in vitro evaluations in this study have demonstrated high affinity of p-coumaric acid towards NorA and could be suggestive of its exploration as a novel NorA efflux pump inhibitor. [ABSTRACT FROM AUTHOR]

Background: Activity of norA efflux pump is one of the antibiotic resistance mechanisms in ciprofloxacin resistant Staphylococcus aureus. In this study, the effect of Artemisia ciniformis extract on reducing the expression of norA efflux pumps gene in ciprofloxacin-resistant Staphylococcus aureus isolates was studied. Materials and Methods: Ciprofloxacin resistant S. aureus isolates, were treated by different concentration of A. ciniformis extract. After extracting RNA and synthesizing cDNA, norA efflux pump expression was evaluated by Real Time PCR. Results: There was significant decrease of norA efflux pump expression in ciprofloxacin-resistant S. aureus isolates treated by A. ciniformis extract (P

The expression of the efflux pump systems is the most important mechanism of antibiotic resistance in bacteria, as it contributes to reduced concentration and the subsequent inactivity of administered antibiotics. NorA is one of the most studied antibacterial targets used as a model for efflux-mediated resistance. The present study evaluated shikimate pathway-derived phenolic acids against NorA (PDB ID: 1PW4) as a druggable target in antibacterial therapy using in silico modelling and in vitro methods. Of the 22 compounds evaluated, sinapic acid (−9.0 kcal/mol) and p-coumaric acid (−6.3 kcal/mol) had the best and most prominent affinity for NorA relative to ciprofloxacin, a reference standard (−4.9 kcal/mol). A further probe into the structural stability and flexibility of the resulting NorA-phenolic acids complexes through molecular dynamic simulations over a 100 ns period revealed p-coumaric acid as the best inhibitor of NorA relative to the reference standard. In addition, both phenolic acids formed H-bonds with TYR 76, a crucial residue implicated in NorA efflux pump inhibition. Furthermore, the phenolic acids demonstrated favourable drug likeliness and conformed to Lipinski’s rule of five for ADME properties. For the in vitro evaluation, the phenolic acids had MIC values in the range 31.2 to 62.5 μg/mL against S. aureus, and E. coli, and there was an overall reduction in MIC following their combination with ciprofloxacin. Taken together, the findings from both the in silico and in vitro evaluations in this study have demonstrated high affinity of p-coumaric acid towards NorA and could be suggestive of its exploration as a novel NorA efflux pump inhibitor.

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available about structure-activity relationship (SAR) around their nuclei and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover, compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against SA-1199B (norA+). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and showed an improvement in terms of "selectivity index" in comparison to 1. [ABSTRACT FROM AUTHOR]

One of the important pathogen with increasing resistance rate in hospitalized patients is Staphylococcus aureus. The useful results for antimicrobial activity of CCCP made it a proper candidate to enhance the inhibitory effect of some certain antibiotics like Ciprofloxacin. The aim of this study was to investigate the Inhibitory effects of CCCP on the gene expression of norA efflux pump and decrease Ciprofloxacin resistance in Staphylococcus aureus. Thirtyisolates ofS. aureus were acquired from different clinical specimens at the Baquba teaching hospital. Susceptibility test to ciprofloxacin was done by disk diffusion test and broth dilution method. Activity of the efflux pump was recognized using CCCP as an chemical efflux pump inhibotor. MIC of CCCP was evaluated with muller hinton Broth dilution method. Bacterial culture was performed and nor A gene expression was examined by Taqman qRT-PCR by one-step and .The expression of norA was significantly decreased in these isolates when were treated with CCCP & Cip. Our results showed that CCCPcan increase ciprofloxacin susceptibility through inhibition of the norA efflux pump. Combination of CCCP with ciprofloxacin can reduce the antibiotic resistance. [ABSTRACT FROM AUTHOR]

Background: Activity of norA efflux pump is one of the antibiotic resistance mechanisms in ciprofloxacin resistant Staphylococcus aureus. In this study, the effect of Artemisia ciniformis extract on reducing the expression of norA efflux pumps gene in ciprofloxacin-resistant Staphylococcus aureus isolates was studied. Materials & Methods: Ciprofloxacin resistant S .aureus isolates, were treated by different concentration of A. ciniformis extract. After extracting RNA and synthesizing cDNA, norA efflux pump expression was evaluated by Real Time PCR. Results: There was significant decrease of norA efflux pump expression in ciprofloxacin-resistant S. aureus isolates treated by A. ciniformis extract (P<0.05). Also, a different expression of norA efflux pump gene was reported. Conclusion: It seems A. ciniformis extract as a natural inhibitor, had potential for suppression of norA efflux pump activity. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus has acquired resistance to antibiotics since their first use. The S. aureus protein NorA, an efflux pump belonging to the major facilitator superfamily (MFS), contributes to resistance to fluoroquinolones (e.g., ciprofloxacin), biocides, dyes, quaternary ammonium compounds, and antiseptics. Different compounds have been identified as potential efflux pump inhibitors (EPIs) of NorA that result in increased intracellular concentration of antibiotics, restoring their antibacterial activity and cell susceptibility. However, none of the currently known EPIs have been approved for clinical use, probably due to their toxicity profiles. In the present study, we screened approved drugs for possible efflux pump inhibition. By screening a compound library of approximately 1200 different drugs, we identified nilotinib, a tyrosine kinase inhibitor, as showing the best efflux pump inhibitory activity, with a fractional inhibitory concentration index of 0.1875, indicating synergism with ciprofloxacin, and a minimum effective concentration as low as 0.195 μM. Moreover, at 0.39 μM, nilotinib, in combination with 8 μg/mL of ciprofloxacin, led to a significant reduction in biofilm formation and preformed mature biofilms. This is the first description of an approved drug that can be used as an efflux pump inhibitor and to reduce biofilms formation at clinically achievable concentrations. [ABSTRACT FROM AUTHOR]

One of the important pathogen with increasing resistance rate in hospitalized patients is Staphylococcus aureus. The useful results for antimicrobial activityof curcumin made it a proper candidate to enhance the inhibitory effect of some certain antibiotics like ciprofloxacin. The aim of this study was to investigate the Inhibitory effects of curcumin on the expression of NorA efflux pump and decrease ciprofloxacin resistance in Staphylococcus aureus. One hundred S. aureus isolates were acquired from different clinical specimens at the milad hospital (Tehran, iran). Susceptibility test to ciprofloxacin was done by Kirby-Bauer disk diffusion test and microdilution method, conforming to the CLSI guidelines. Activity of the efflux pump was recognized using CCCP as an chemical efflux pump inhibotor. MIC of curcumin was evaluated with Broth Microdilution method. Bacterial culture was performed near the curcumin and RNA Extraction was done. cDNA was synthesized and NorA gene expression was examined by Real-time PCR. The expression of NorA was significantly decreased in this isolated when it was treated with curcumin before RNA extraction compared with absent of curcumin. Our results showed that curcumin can increase ciprofloxacin susceptibility through inhibition of the NorA efflux pump. Combination of curcumin with ciprofloxacin can reduse the antibiotic resistanse. [ABSTRACT FROM AUTHOR]

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 µM. The study concluded that boeravinone B not only inhibits the NorAmediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) represents a global health issue threatening our social lifestyle and the world economy. Efflux pumps are widely involved in AMR by playing a primary role in the development of specific mechanisms of resistance. In addition, they seem to be involved in the process of biofilm formation and maintenance, contributing to enhance the risk of creating superbugs difficult to treat. Accordingly, the identification of non-antibiotic molecules able to block efflux pumps, namely efflux pump inhibitors (EPIs), could be a promising strategy to counteract AMR and restore the antimicrobial activity of ineffective antibiotics. Herein, we enlarge the knowledge about the structure-activity relationship of 2-phenylquinoline Staphylococcus aureus NorA EPIs by reporting a new series of very potent C-6 functionalized derivatives. Best compounds significantly inhibited ethidium bromide efflux in a NorA-overexpressing S. aureus strain (SA-1199B) and strongly synergized at very low concentrations (0.20–0.78 μg/mL) with ciprofloxacin (CPX) against CPX-resistant S. aureus strains (SA-1199B and SA-K2378), as proved by checkerboard and time-kill experiments. In addition, some of these EPIs (9b and 10a) produced a post-antibiotic effect of 1.2 h and strongly enhanced antibiofilm activity of CPX against SA-1199B strain. Interestingly, at the concentrations used to reach synergy with CPX against resistant S. aureus strains, most of the EPI compounds did not show any human cell toxicity. Finally, by exploiting the recent released crystal structure of NorA, we observed that best EPI 9b highlighted a favourable docking pose, establishing some interesting interactions with key residues. [Display omitted] • C-6 functionalized 2-phenylquinoline derivatives strongly inhibit NorA efflux pump. • Properly functionalized quinoline analogues potently synergize with ciprofloxacin. • Inhibition of NorA efflux pump strongly increases the activity of fluoroquinolones. • 9b and 10a boost the activity of ciprofloxacin against Staphylococcus aureus biofilm. • The new C-6 functionalized analogue 9b interacts with the key residue Asp307 of NorA. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Baicalein, the active constituent derived from Scutellaria baicalensis Georgi., has previously been shown to significantly restore the effectiveness of β-lactam antibiotics and tetracycline against methicillin-resistant Staphylococcus aureus (MRSA). With multiple therapeutic benefits, the antibacterial actions of baicalein may also be involved in overcoming other bacterial resistance mechanisms. The aim of the present study was to further investigate antibacterial activities of baicalein in association with various antibiotics against selected Staphylococcus aureus strains with known specific drug resistance mechanisms. Material and methods: A panel of clinical MRSA strains was used for further confirmation of the antibacterial activities of baicalein. The effect of baicalein on inhibiting the enzymatic activity of a newly discovered MRSA-specific pyruvate kinase (PK), which is essential for Staphylococcus aureus growth and survival was also examined. Results: In the checkerboard dilution test and time-kill assay, baicalein at 16μg/ml could synergistically restore the antibacterial actions of ciprofloxacin against the NorA efflux pump overexpressed SA-1199B, but not with the poor NorA substrate, pefloxacin. Moreover, synergistic effects were observed when baicalein was combined with ciprofloxacin against 12 out of 20 clinical ciprofloxacin resistant strains. For MRSA PK studies, baicalein alone could inhibit the enzymatic activity of MRSA PK in a dose-dependent manner. Conclusion: Our results demonstrated that baicalein could significantly reverse the ciprofloxacin resistance of MRSA possibly by inhibiting the NorA efflux pump in vitro. The inhibition of MRSA PK by baicalein could lead to a deficiency of ATP which might further contribute to the antibacterial actions of baicalein against MRSA. [Copyright &y& Elsevier]

Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones. [ABSTRACT FROM AUTHOR]