Butyrophenone Antipsychotics Reverse Ciprofloxacin Resistance in Staphylococcus aureus

Bacterial antibiotic resistance is a growing and major threat to public health.  The Center for Disease Control estimates that at least 2 million people a year will acquire a serious infection with bacteria resistant to one or more antibiotics.  Efflux-related multidrug resistance involves transmembrane pumps that efflux out antibiotics.  In Staphylococcus aureus, the membrane protein NorA has been found to mediate the efflux of hydrophilic fluoroquinolone antibiotics, such as ciprofloxacin.  A mutation in the promoter region of norA increases the expression of the NorA efflux pump in S. aureus strain 1199B resulting in resistance to ciprofloxacin.  A great effort is underway to discover novel efflux pump inhibitors (EPI) that may reverse resistance and improve the effectiveness of current antibiotics as combinatorial drug therapy.  Phenothiazine antipsychotic drugs such as chlorpromazine have been found to inhibit efflux pump activity in S. aureus.  The objective of our research was to determine the effectiveness of butyrophenone antipsychotic drugs; haloperidol, droperidol and spiperone, as NorA efflux pump inhibitors that reverse ciprofloxacin resistance in S. aureus 1199B.  Combinations of concentrations of butyrophenones or control EPI (chlorpromazine) along with ciprofloxacin (CIP) were utilized in checkerboard assays to determine their minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC).  For all of our butyrophenone compounds we observed a reduction in the MIC of ciprofloxacin in S. aureus 1199B reversing its susceptibility from resistant to susceptible.  The MICs for all three butyrophenones, was greater than 200 ug/mL in both strains, demonstrating that on their own, these drugs would be considered non-antibiotics.  Checkerboard assays resulted in average FIC for droperidol, haloperidol, and spiperone of 0.195, 0.2 and 0.172 respectively, demonstrating a synergistic mechanism with ciprofloxacin for all three compounds (FIC ≤ 0.5).  Ethidium bromide efflux assays in S. aureus 1199B were used to evaluate these butyrophenones as inhibitors of the NorA efflux pump.  The efflux assays demonstrated that butyrophenones were able to reduce the loss of ethidium bromide fluorescence over time, confirming a blockage of the NorA efflux pump.  A complete inhibition of ethidium bromide efflux was achieved at a concentration of 200 ug/mL for haloperidol and droperidol while only 100 ug/mL of spiperone was needed to achieve the same effect.  All together these results show that butyrophenones are inhibitors of the NorA efflux pump, and its derivatives have potential for treating ciprofloxacin resistant S. aureus infections in combination therapy.