Synergistic effect and resistance prevention activity of antimicrobial peptide MPX with Tulathromycin against Actinobacillus pleuropneumoniae

Introduction The emergence and dissemination of multidrug-resistant Actinobacillus pleuropneumoniae (APP) have significantly hindered the advancement of the swine industry. The combination of antimicrobial peptides (AMPs) with conventional antibiotics represents a promising strategy to combat drug-resistant bacterial infections. Tulathromycin (Tul), however, is prone to inducing resistance in APP due to its broad mutant selection window (MSW). Moreover, there is a paucity of data regarding the efficacy of AMP-macrolide combinations against APP. Consequently, this study aimed to evaluate the synergistic effects and resistance prevention of the AMP MPX in combination with Tul against APP. Methods To assess synergism, the minimum inhibitory concentrations (MICs) of MPX and Tul, both individually and in combination, were determined using the micro-broth dilution method and checkerboard assay. Time-kill assays were conducted to analyze antibacterial activity and kill rate by quantifying viable bacterial counts. The post-antibiotic effect (PAE) was calculated following exposure to 1−2 MIC of each drug alone and in combination. For resistance prevention analysis, serial passage experiments were performed over 30 generations under selective pressure at 1/4 MIC for each agent to monitor changes in MIC values. Additionally, the mutant prevention concentration (MPC) was measured to evaluate MPX’s capacity to narrow the MSW of Tul against APP. Finally, the antimicrobial activity of MPX and Tul was assessed against resistant APP strains. Results and discussion The fractional inhibitory concentration index (FICI) indicated an indifferent interaction (1.5) for susceptible strains and an additive effect (0.75) for resistant strains. Time-kill curves demonstrated that MPX significantly enhanced the antibacterial efficacy of Tul against APP. Specifically, the kill rate within 0−1 hour and the PAE at 1−2 MIC of Tul increased from 0.35−2.91 to 1.41−5.03 Log 10 CFU/mL/h and from 0.66−1.64 to 1.29−2.91 h, respectively, combination with MPX. Resistance induction assays revealed that MPX could restore or reduce the MIC of Tul against APP. Furthermore, MPX effectively narrowed the MSW of Tul, as evidenced by an MPC-based FICI of 0.375, confirming a synergistic interaction. The combination exhibited additive effects against resistant strains. These findings provide valuable insights into the potential application of MPX in combination with antimicrobial agents to prevent the emergence and dissemination of drug-resistant strains.