Synergistic Antimicrobial Effect of Antimicrobial Peptides CATH-1, CATH-3, and PMAP-36 With Erythromycin Against Bacterial Pathogens

Abstract

With the increasing bacterial resistance to traditional antibiotics, there is an urgent need for the development of alternative drugs or adjuvants of antibiotics to enhance antibacterial efficiency. The combination of antimicrobial peptides (AMPs) and traditional antibiotics is a potential alternative to enhance antibacterial efficiency. In this study, we investigated the synergistic bactericidal effect of AMPs, including chicken (CATH-1,-2,-3, and -B1), mice (CRAMP), and porcine (PMAP-36 and PR-39) in combination with conventional antibiotics containing ampicillin, tetracycline, gentamicin, and erythromycin against Staphylococcus aureus, Salmonella enteritidis, and Escherichia coli. The results showed that the minimum bactericidal concentration (MBC) of CATH-1,-3 and PMAP-36 was lower than 10 μM, indicating that these three AMPs had good bacterial activity against S. aureus, S. enteritidis, and E. coli. Then, the synergistic antibacterial activity of AMPs and antibiotics combination was determined by the fractional bactericidal concentration index (FBCI). The results showed that the FBCI of AMPs (CATH-1,-3 and PMAP-36) and erythromycin was lower than 0.5 against bacterial pathogens, demonstrating that they had a synergistic bactericidal effect. Furthermore, the time-killing kinetics of AMPs (CATH-1,-3 and PMAP-36) in combination with erythromycin showed that they had a continuous killing effect on bacteria within 3 h. Notably, the combination showed lower hemolytic activity and cytotoxicity to mammal cells compared to erythromycin and peptide alone treatment. In addition, the antibacterial mechanism of CATH-1 and erythromycin combination against E. coli was studied. The results of the scanning electron microscope showed that CATH-1 enhanced the antibacterial activity of erythromycin by increasing the permeability of bacterial cell membrane. Moreover, the results of bacterial migration movement showed that the combination of CATH-1 and erythromycin significantly inhibits the migration of E. coli. Finally, drug resistance analysis was performed and the results showed that CATH-1 delayed the emergence of E. coli resistance to erythromycin. In conclusion, the combination of CATH-1 and erythromycin has synergistic antibacterial activity and reduces the emergence of bacterial drug resistance. Our study provides valuable information to develop AMPs as potential substitutes or adjuvants for traditional antibiotics.

Keywords: antibacterial mechanism; antibiotic resistance; antimicrobial peptides; synergistic antibacterial activity; traditional antibiotics.

Figure 1

Antibacterial effect of antimicrobial peptides combined with antibiotics against S. aureus, S. enteritidis, and E. coli. The isotope map shows the antibacterial effect of the combination of AMPs (CATH-1, CATH-3, and PMAP-36) and antibiotics (ampicillin, tetracycline, gentamicin, and erythromycin) against S. aureus (A), S. enteritidis (B), and E. coli (C). The synergic antibacterial activity was determined by the fractional bactericidal concentration index (FBCI). FBCI values were defined as synergy for FBCI < 0.5, additivity for 0.5 < FBCI ≤ 1, indifference for 1 < FBCI ≤ 2, and antagonism for FBCI > 2. In each contour map, the colors, including red, blue, and gray, represent synergy, additivity, and independence, respectively.

Figure 2

Time-killing kinetics of antimicrobial peptides combination with erythromycin. Time-killing kinetics of CATH-1, CATH-3, and PMAP-36 combination with erythromycin against S. aureus (A–C), S. enteritidis (D–F), and E. coli (G–I) are shown.

Figure 3

Hemolytic activity and cytotoxicity of antimicrobial peptides combination with erythromycin. Hemolytic activity of CATH-1, CATH-3, and PMAP-36 combination with erythromycin to the red blood cells of mice (A). Cell viability of peritoneal macrophages (B) and PK-15 cells (C) treated with CATH-1, CATH-3, and PMAP-36 combination with erythromycin according to WST-1 assay.

Figure 4

The effect of CATH-1 and erythromycin combination on E. coli morphology. E. coli cells were grown to the mid-logarithmic phase and then treated with CATH-1 and erythromycin. After treatment, SEM was performed to observe bacterial morphology. (A) Untreated E. coli. (B) 1/16 MBC CATH-1-treated E. coli. (C) 1/16 MBC erythromycin-treated E. coli. (D) 1/16 MBC CATH-1 + 1/16 MBC erythromycin-treated E. coli. Blue arrows represent rough cell membrane, white arrows represent micelles, and red arrows represent cell lysis.

Figure 5

The effect of CATH-1 and erythromycin combination on bacterial motility. Mid-logarithmic phase E. coli were treated with CATH-1, erythromycin, and CATH-1 + erythromycin, and then E. coli was inoculated in the center of the semisolid agar. The inoculated plates were incubated at 37°C for 18 h. The motility image is shown (A). The migration distance (mm) was measured (B). Asterisks (*) indicate significant difference (* p < 0.05, ** p < 0.01).

Figure 6

The effect of CATH-1 and erythromycin combination on bacterial drug resistance. The E. coli strain grown independently with CATH-1 (1/4 MIC), erythromycin (1/4 MIC), and CATH-1 (1/8 MIC) combination with erythromycin (1/16 MIC) at 37°C for consecutive 30 generations. A fixed concentration of CATH-1 (equivalent to 1/4 MIC) was added to the 2-fold increasing concentration of erythromycin. Bacteria from the highest concentration of drug combination were regrown, and MIC of erythromycin was measured, and then treated them with the drug combination again. The change in MIC was described by normalizing the MIC of n generation to the MIC of first generation.