Cerium, copper, gallium, manganese and silver substituted hydroxyapatite: cytotoxicity and antibacterial activity toward multidrug-resistant clinical isolates

• Evaluation of antibacterial activity of HAp doped with Ag + , Ce 3+ , Cu 2+ , Ga 3+ , Mn 2+ . • All HAp materials are non-cytotoxic, except Cu-doped HAp. • Ag + -doped HAp showed significant antibacterial activity against all pathogens. • Ga 3+ and Mn 2+ doping inhibited bacterial attachment and survival. • Proposed standardized methods for antibacterial testing of HAps. Certain bacterial species are a persistent issue in hospital-acquired infections due to their fast and potent development of multi-drug resistance. This study aimed to evaluate the antibacterial activity of mono-substituted HAp, incorporating potentially antibacterial ions (Ag + , Ce 3+ , Cu 2+ , Ga 3+ , and Mn 2+ ), against clinically-relevant bacteria Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae , and Pseudomonas aeruginosa . Samples were analyzed using X -ray diffraction, Rietveld refinement analysis, Fourier Transform Infrared Spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy and zeta potential, alongside the cytotoxicity assays and antibacterial activity determined by disc diffusion and broth microdilution methods. All materials were non-cytotoxic to cells, except for Cu-substituted HAp. HAp substituted with Ag + showed significant antibacterial activity against all tested bacteria. Ce-substituted HAp showed no antibacterial effect, while Ga- and Mn-substitution indicated potential antibacterial properties towards certain bacterial strains by inhibiting bacterial attachment to its surface. Judging by the SEM analysis, the gram-negative bacteria attached to the HAp surface in a lesser amount then the gram-positive ones. Given the variability in methodologies used for evaluating the antibacterial activity of HAp reported in the literature, we propose a standardized testing protocol that incorporates minimum bactericidal concentration (MBC) assessments and disc diffusion tests on finished materials or coatings, coupled with SEM analysis.