Differential Roles of Ag and ZnO in Bactericidal Activity of Ag/ZnO-Hydroxyapatite Nanocomposites against Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is a Gram-negative bacterium that thrives in diverse environments, including the human body and on inert surfaces such as medical devices. It is notorious for resistance to nearly all conventional antimicrobial agents, driven by intrinsic and acquired mechanisms, posing significant therapeutic challenges. Hydroxyapatite (HAp) nanocomposites are widely studied for antimicrobial properties, but their biomedical applications remain in early stages. Aim: This study characterized four Ag/ZnO-HAp nanocomposites with Ag:ZnO mole ratios of 0.005/0.005 (nanocomposite A), 0.01/0.005 (nanocomposite B), 0.005/0.01 (nanocomposite C), and 0.01/0.01 (nanocomposite D) to advance therapeutic strategies. Methods: We performed agar diffusion, bactericidal, and time-kill assays against P. aeruginosa PAO1. We also determined minimum bactericidal concentrations (MBCs) for eight multidrug-resistant P. aeruginosa clinical isolates. Results: Agar diffusion assays showed inhibition zones of 8, 11, 11, and 12 mm for nanocomposites A, B, C, and D, respectively, while the control HAp showed no inhibition zone. The bactericidal assay showed log reductions of 1.34, 5.07, 1.67, and 5.69 for nanocomposites A, B, C, and D, respectively, and time-kill kinetics showed specific death rates (min^？1) of 0.034, 0.07, 0.054, and 0.082 for the same nanocomposites, respectively. MBCs for clinical isolates ranged from <0.06 to >4 mg/mL, independent of their antibiotic susceptibility profiles. Conclusions: Overall, the results suggest that bactericidal efficacy follows the order D > B > C > A. Ag content primarily determines bactericidal activity, with ZnO contributing secondarily. However, higher ZnO levels inhibit Ag activity. Therefore, optimizing the Ag:ZnO ratio is crucial for maximizing bactericidal effects, and customizing the nanocomposites for specific bacterial strains is essential for effective biomedical applications.