Synergistic antibacterial effects of pinaverium bromide and oxacillin against Staphylococcus epidermidis

Staphylococcus epidermidis (S. epidermidis) adheres to the surface of medical devices, forming highly drug-resistant biofilms, which has made the development of novel antibacterial agents against S. epidermidis and its biofilms a key research focus. By drug repurposing, this study aims to explore the combinational antimicrobial effects between pinaverium bromide (PVB), a L-type calcium channel blocker, and oxacillin (OXA) against S. epidermidis.

Clinical isolates of S. epidermidis were collected from January to September 2022 at the Department of Clinical Laboratory of the Third Xiangya Hospital, Central South University. The minimal inhibitory concentrations (MICs) of PVB and OXA were determined using the broth microdilution method. Checkerboard assays and time-kill curves were performed to assess the fractional inhibitory concentration index and synergistic bactericidal efficiency of the drug combination. Resistance selection assays evaluated PVB's ability to inhibit the development of OXA resistance. Biofilm eradication assays, combined with confocal laser scanning microscopy (CLSM) and the persister cell quantification, were conducted to evaluate the effect of PVB and OXA on drug-resistant biofilms and persister cells. The mechanisms of PVB action were further investigated using transmission electronic microscopy (TEM), reactive oxygen species (ROS) quantification, and ATP quantification.

The MICs of PVB and OXA against the standard strain S. epidermidis RP62A were both 8 μg/mL. Checkerboard assays showed that the fractional inhibitory concentration index (FICI) for the combination was 0.250 0 for RP62A and ranged from 0.187 5 to 0.500 0 for clinical isolates, indicating synergistic effects. Resistance selection assays demonstrated that PVB not only failed to induce resistance but also effectively inhibited the development of OXA resistance. The combination of 1×MIC of PVB and OXA reduced biofilm biomass (A_{570 nm}) from (2.36±0.46) to (1.12±0.39) (t=3.504, P=0.02). CLSM revealed significant biofilm structural disruption and an increased proportion of dead bacteria. Additionally, after 4 hours of treatment, the total persister cell count was reduced from lg(7.73±0.21) to lg(2.79±0.43) (t=4.143, P=0.014). Synergistic biofilm eradication was further confirmed in clinical isolates. TEM revealed that PVB caused significant bacterial structural damage. The combination of OXA and PVB significantly induced ROS production, increasing the relative fluorescence intensity from (30 000.00±2 000.00) to (45 666.67±2 081.67) (t=10.68, P<0.001), and markedly reduced ATP generation, lowering the relative fluorescence intensity form (565.00±33.18) to (205.67±35.23) (t=4.932, P=0.003).

The combination of PVB and OXA exhibits significant synergistic antimicrobial activity against S. epidermidis, its biofilms, and persister cells. This combination holds promise as a potential alternative therapy for biofilm-associated infections caused by S. epidermidis.