Inhibiting Staphylococcus aureus NorA Efflux Pumps to Restore Ciprofloxacin Activity

Overexpression of the NorA efflux pump in Staphylococcus aureus yields a multidrug resistant mechanism that actively pumps antibiotics out of the cell. In order to strategically combat infections, an investigation of potential efflux pump inhibitors (EPI) was conducted to reinvigorate current antibiotic effectiveness. As part of a systematic screening of flavonoids, we found a compound (named flavonoid A) that synergizes with the antibiotic ciprofloxacin against S. aureus. The objective of this study is to characterize flavonoid A for this synergy. We hypothesize that flavonoid A reverses resistance to ciprofloxacin in S. aureus by inhibiting the NorA multidrug efflux pump. To test this hypothesis, three S. aureus (SA) strains were used: SA 1199 (wild type), ciprofloxacin resistant SA 1199B (NorA overexpressed) and SA K-1758 (knockout NorA). Thin layer chromatography (TLC) autobiography was performed to qualitatively assess a synergistic relationship between the flavonoid A and ciprofloxacin by observing growth inhibition of ciprofloxacin resistant SA 1199B on TLC plates with these compounds. The retention factor values for flavonoid A and the known EPI chlorpromazine were 0.29 and 0.28, respectively. Growth inhibition zones were observed on the TLC plate with ciprofloxacin and flavonoid A. A checkerboard assay was conducted to quantitatively examine the interaction between ciprofloxacin and flavonoid A. In the ciprofloxacin resistant SA 1199B, the ciprofloxacin minimal inhibitory concentration (MIC) was 8mg/L and with the flavonoid A (100mg/L) was 0.5mg/L for a 16 fold reduction. These results suggest that flavonoid A reverses ciprofloxacin resistance in the NorA overexpressed SA 1199B strain and synergism exist between these compounds. The ciprofloxacin MIC fold reduction in the presence of flavonoid A for the controls, SA K-1758 and SA 1199, were 0 and 4, respectively. An ethidium bromide (EtBr) efflux fluorescence assay was conducted to support that the active flavonoid A exhibits EPI activity. In the negative control, the EtBr retention was reduced to 31% after 30 minutes and in the presence of the known EPI, chlorpromazine was 100%. However at 200 mg/L of flavonoid A, the EtBr retention was 90%. These results suggest flavonoid A exhibits NorA inhibitory activity that is similar to chlorpromazine. A time to kill assay revealed the combination of flavonoid A and ciprofloxacin as bactericidal against S. aureus over a 24 hour period. By an In silico analysis, we built a NorA model, and docked flavonoid A or chlorpromazine to estimate the thermodynamic properties for this process. For chlorpromazine, the full fitness value and free binding energy to NorA was -1332.68 (kcal/mol), -8.08 (kcal/mol), and for flavonoid A was -1267.32 (kcal/mol), and -8.74 (kcal/mol), respectively. This analysis reveals flavonoid A reverses antibiotic resistance in S. aureus and synergizes with ciprofloxacin by acting as a NorA EPI. Further toxicological studies are pivotal for implementation in clinical trials.