Evaluation of Recombinant Ib-AMP<sub>4</sub> and Oncorhyncin II Peptides in Synergy for Treating <i>Acinetobacter baumannii</i> Under <i>In Vitro</i> Conditions

Background: The rising prevalence of microbial resistance in Acinetobacter baumannii infections has become a significant public health concern. Researchers are exploring antimicrobial peptides (AMPs) as promising alternatives to traditional antibiotics. Among these, Ib-AMP4 and Oncorhyncin II have shown notable antibacterial properties. Objectives: The primary objective of this study was to produce recombinant peptides for Ib-AMP4 and Oncorhyncin II. Additionally, the study aimed to investigate their combined effects against A. baumannii (ATCC 19606). Methods: The study focused on the self-directed expression of synthetic genes encoding Ib-AMP4 and Oncorhyncin II within the Escherichia coliBL21 (DE3) pLysS host expression system. Following purification and optimization, the peptides' biological effectiveness against A. baumannii (ATCC 19606) was evaluated through various assays, including minimum inhibitory concentration (MIC), time-kill, and growth kinetic assessments. Results: The MIC assay confirmed the ability of Ib-AMP4 and Oncorhyncin II to inhibit the growth of A. baumannii, with effective concentrations at 111 µg/mL and 80.25 µg/mL, respectively. Moreover, a synergistic effect was observed when Ib-AMP4 and Oncorhyncin II were combined at 2 × MIC, as demonstrated by the checkerboard method, leading to a rapid reduction in viable cell count. Growth kinetic analysis further revealed a 95% reduction in bacterial turbidity within 5 hours of exposure to the peptide combination. Conclusions: The production of recombinant Ib-AMP4 and Oncorhyncin II peptides showed significant efficacy against A. baumannii. Checkerboard, time-kill, and growth kinetic assays consistently demonstrated their synergistic effects. These findings highlight the potential of combining these peptides as an effective strategy to combat A. baumannii infections.