Ferulic acid derivative inhibits NorA efflux and in combination with ciprofloxacin curtails growth of MRSA in vitro and in vivo

A series of ferulic acid (FA) derivatives were synthesized and evaluated for its ability to inhibit NorA efflux in methicillin resistant Staphylococcus aureus (MRSA), by in silico docking analysis. Based on prediction from glide scores and ability to reduce EtBr MIC, two of the ten derivatives S3- [4-((E)-2-(diethylcarbamoyl)vinyl)-2-methoxyphenyl acetate] and S6- [(E)-methyl 3-(4-((p-tolylcarbamoyl)methoxy)-3-methoxyphenyl)acrylate] were chosen as putative efflux pump inhibitors (EPI's). Time dependent accumulation studies revealed that S6 caused enhanced EtBr accumulation relative to standard NorA efflux inhibitor reserpine, in clinical isolate of MRSA (CIMRSA) and in NorA overexpressed strain of S. aureus (SA1199B). S6 also exhibited synergy with Ciprofloxacin (CPX) against NorA overexpressed strain (SA1199B) of S. aureus but not in NorA knock out strain (K1758). MIC reversal studies showed that S3 in CIMRSA and S6 in NorA overexpressed strain of S. aureus (SA1199B), caused a 4 fold reduction in CPX MIC. In vitro time kill studies revealed that both S3 and S6 with sub MIC of CPX caused a significant 4 log CFU decline in CIMRSA. A decline of >3 log fold CFU by time kill assay implies synergy between FA derivatives and CPX. When tested in vivo in infected muscle tissue of zebrafish both S3 and S6 with CPX caused >3.2 log decline in CIMRSA cell counts relative to CPX treatment alone. Of the two potent derivatives, S6 probably acts through NorA whereas S3 might exert its effect through pump other than NorA. Greater in vitro and in vivo efficiency of FA derivatives implies its potential to be used as an adjuvant along with CPX to curtail MRSA infection in higher animal models.