Calcium phosphate nanoparticles as intrinsic inorganic antimicrobials: mechanism of action.

ABSTRACT

This is the final report of the study aimed at assessing the antimicrobial activity of calcium phosphate (CP) nanoparticles delivered in the form of hydroxyapatite (HAp) or amorphous CP (ACP) and understanding the fundamental principles behind their mechanisms of action. Not responding to propidium iodide and causing no gross morphological changes except moderate stress-induced filamentation in Escherichia coli (E. coli), CP nanoparticles were shown to be bacteriostatic, not bactericidal. Also, the lack of expression of genes involved in DNA repair indicated no genotoxic activity. In contrast, the softening of amide infrared bands and the partial dissociation of lipopolysaccharide structures comprising the membrane of Gram-negative Pseudomonas aeruginosa (P. aeruginosa) was detected in a vibrational spectroscopic analysis of the nanoparticle/bacterium interaction. Similarly, the inhibition of the growth of Staphylococcus aureus (S. aureus) was paralleled by a reduced integrated intensity and the softening of the C = O ester carbonyl stretch in lipoteichoic acid, a major component of the Gram-positive cell membrane. Electron microscopy analyses confirmed that changes to the cell membrane are a major mode of action of CP nanoparticles. While HAp got internalized by E. coli significantly more than ACP, the membrane damage was more pronounced in ACP-treated bacteria, which was explained by the higher surface reactivity of ACP. HAp nanoparticles decreased the activity of overexpressed efflux pumps in methicillin-resistant S. aureus, suggesting that they may hijack these pumps and use them to enter the cell without producing any visible damage to the membrane, thus acting on the cell from the inside out, as opposed to ACP, whose action is mostly external in mechanism. This may explain why HAp, unlike ACP, suppresses the mechanisms of resistance in methicillin- and multidrug-resistant S. aureus and P. aeruginosa, respectively. The findings of this study will be essential in the optimization of these nanoparticles for becoming an alternative to less biocompatible inorganics and small molecule antibiotics in the global effort to curb the rising resistance of bacterial pathogens to the existing therapies.