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The emergence of multidrug resistant microorganisms has triggered the impending need of developing effective antibacterial strategies. Staphylococcus epidermidis and the more virulent S. aureus are able to colonize and form biofilms on implanted medical devices causing clinical acute and chronic infections. The aim of the present work was to assess the effect of NorA efflux pump inhibitors (EPIs) on S. aureus and S. epidermidis biofilm formation by using known synthetic NorA EPIs and thus giving support to the hypothesis that efflux pumps could play an intriguing role in Staphylococcus biofilm formation. In particular, three 2-phenylquinolines (1a–c) and a pyrazolobenzothiazine (2), our previously reported NorA EPIs, at concentrations lower than MIC showed a good ability in reducing biofilm formation in S. aureus (ATCC 29213) and S. epidermidis (ATCC 35984). Then, to assess if biofilm formation inhibitors could be combined with an antibacterial to limit biofilm production, the two best compounds 1c and 2 were tested with ciprofloxacin (CPX) and the results showed an excellent synergistic effect against Staphylococcus strains with a biofilm formation inhibition over 60% at low concentrations. Further, to confirm the involvement of the NorA efflux pump in the biofilm production, NorA EPIs 1c and 2 were tested against a modified S. aureus strain overexpressing the NorA efflux pump (SA-1199B) and results highlighted a strong decrease in biofilm mass. In conclusion, cytotoxicity assays on HeLa and HepG2 cells showed that concentrations useful to inhibit biofilm formation in Staphylococcus strains in combination with CPX were lower than the toxic concentrations for human cells.

Investigation on the effect of known potent S. aureus NorA efflux pump inhibitors on the staphylococcal biofilm formation