Bisma, Muhammad Owais Khan, Usman Siddiq, Afshan Pervaiz, Syed Hasnain Ul Haque, Mehwish Imtiaz
Background: Multidrug-resistant bacterial infections are a growing challenge in tertiary-care hospitals, particularly where antimicrobial pressure is high and treatment options are limited. Amphibian skin secretions contain antimicrobial peptides that may provide novel leads for anti-infective development. Objective: This study aimed to isolate peptide-containing fractions from amphibian skin secretions and evaluate their in vitro antibacterial, antibiofilm, safety, and stability profiles against multidrug-resistant clinical isolates. Methods: An experimental laboratory-based study was conducted using 87 non-duplicate multidrug-resistant isolates, including MRSA, ESBL-producing Escherichia coli, ESBL-producing Klebsiella pneumoniae, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Acinetobacter baumannii. Amphibian skin secretions were processed into crude extract and purified by RP-HPLC to obtain two active fractions, AMP-1 and AMP-2. Antibacterial activity was assessed using MIC and MBC testing, time–kill kinetics, biofilm inhibition and disruption assays, hemolysis testing, mammalian-cell viability screening, and short-term serum stability assessment. Results: AMP-1 showed the strongest antibacterial profile, with median MICs of 4 µg/mL against MRSA, 8 µg/mL against ESBL-producing E. coli, and 16 µg/mL against carbapenem-resistant P. aeruginosa. MBCs were generally one dilution above MIC values. AMP-1 produced concentration-dependent killing against MRSA, inhibited biofilm formation more effectively than it disrupted mature biofilm, and showed lower hemolysis than AMP-2 at matched concentrations. AMP-1 retained 76% activity after 4 hours in 25% serum, compared with 68% for AMP-2. Conclusion: Amphibian-derived AMP fractions, particularly AMP-1, demonstrated promising in vitro activity and preliminary selectivity against MDR pathogens, supporting further peptide characterization, optimization, formulation development, and preclinical validation