A novel function of short cationic peptide FP-CATH9 without antimicrobial activity reverses resistance to minocycline in common multidrug-resistant gram-negative bacteria

The increase in bacterial resistance to minocycline and other tetracyclines poses a serious threat to global public health. Because the development of new antibiotics has proven problematic, antibiotic sensitization therapy is now an effective coping strategy. While antimicrobial peptides generally exhibit broad-spectrum antibacterial activity and good biocompatibility, naturally truncated portions of antimicrobial peptides (such as snake cathelicidin) often do not exhibit antimicrobial activity, and their function remains unknown. FP-CATH9 is a short cationic peptide derived from FP-CATH (snake cathelicidin antimicrobial peptide) with an amphiphilic α-helical structure and no discernible antibacterial activity. However, FP-CATH9 was previously found to significantly enhance the activity of minocycline against gram-negative bacteria. In the present paper, clinically relevant minocycline-resistant gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) were used as test bacteria for antibiotic sensitization screening. The sensitization activity of FP-CATH9 was found to be dose dependent in a double-dilution assay. The synergistic activity of FP-CATH9 on minocycline was subsequently determined using the checkerboard method. An ethidium bromide efflux test revealed that FP-CATH9 caused an accumulation of minocycline in bacteria. Additionally, FP-CATH9 exhibited low hemolytic activity on red blood cells and low cytotoxicity on Raw264.7 cells. In an in vivo model of bacterial infection, FP-CATH9 combined with minocycline exhibited an 80% protective effect on Galleria mellonella larvae infected with multidrug-resistant K. pneumoniae. In summary, FP-CATH9 is a new antibiotic adjuvant that reverses the resistance of gram-negative bacteria to minocycline by increasing intracellular accumulation of minocycline. This finding has broad application potential.IMPORTANCEThe existence of the efflux pump system enables bacteria to expel antibiotics, reduce the concentration of antibiotics in cells, and make antibiotics unable to effectively inhibit or kill bacteria, which is one of the main mechanisms of bacterial resistance to antibiotics. However, some efflux pumps are substrate specific, while others are with a wide range of substrates. In this study, FP-CATH9 as a new antibiotic adjuvant can specifically reverse the resistance of gram-negative bacteria to minocycline by increasing the intracellular accumulation of minocycline in bacteria and provides a new way to solve the increasing problem of bacterial drug resistance.