Antimicrobial peptides in combination with citronellal efficiently kills multidrug resistance bacteria

Antimicrobial peptides (AMPs) are considered as the most potential alternatives to antibiotics, but they have several drawbacks, including high cost, medium antimicrobial efficacy, poor cell selectivity, which limit clinical application. To overcome the above problems, combination therapy of AMPs with adjuvants might maximize the effectiveness of AMPs. We found that citronellal can substantially potentiate the ZY4R peptide efficacy against Escherichia coli ATCC25922. However, it is unclear whether ZY4R/citronellal combination poses synergistic antimicrobial effects against most bacteria, and their synergy mechanism has not been elucidated. To investigate synergistic antimicrobial efficacies, biosafety, and synergy mechanism of ZY4R/citronellal combination. Checkerboard, time-kill curves, cytotoxicity assays, and in vivo animal models were conducted to assess synergistic antimicrobial effects and biosafety of the ZY4R/citronellal combination. To evaluate their synergy mechanism, a series of cell-based assays and transcriptome analysis were performed. ZY4R/citronellal combination exhibited synergistic antimicrobial effects against 20 clinically significant pathogens, with the fractional inhibitory concentration index (FICI) ranging from 0.313 to 0.047. Meanwhile, ZY4R/citronellal combination enhanced antimicrobial efficacies without compromising cell selectivity, contributing to decreasing drug dosage and improving biosafety. Compared with ZY4R (4 mg/kg) and citronellal (25 mg/kg) alone, ZY4R (4 mg/kg)/citronellal (25 mg/kg) combination significantly decreased the bacterial load in peritoneal fluid, liver, and kidney (P < 0.05) and alleviated pathological damage of the organs of mice. Mechanistic studies showed that ZY4R allowed citronellal to pass through the outer membrane rapidly and acted on the inner membrane together with citronellal, causing more potent membrane damage. The membrane damage prompted the continuous accumulation of citronellal in cells, and citronellal further induced energy breakdown and inhibited exopolysaccharide (EPS) production, which aggravated ZY4R-induced outer membrane damage, thereby resulting in bacterial death. ZY4R/citronellal combination exhibited broad-spectrum synergy with a low resistance development and high biosafety. Their synergy mechanism acted on two important cellular targets (energy metabolism and membrane integrity). Combination therapy of ZY4R with citronellal may be a promising mixture to combat bacterial infections facing an antibiotic-resistance crisis.