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.

这是该研究的最终报告，旨在评估以羟基磷灰石 (HAp) 或无定形 CP (ACP) 形式传递的磷酸钙 (CP) 纳米颗粒的抗菌活性，并了解其作用机制背后的基本原理。对碘化丙锭没有反应，除了在大肠杆菌( E. coli ) 中中度应力诱导的丝状化外，没有引起明显的形态变化，CP 纳米颗粒被证明是抑菌的，而不是杀菌的。此外，参与 DNA 修复的基因缺乏表达表明没有基因毒性活性。相反，酰胺红外波段的软化和包含革兰氏阴性的膜脂多糖结构的局部分解绿脓杆菌（铜绿假单胞菌）在纳米颗粒/细菌相互作用的振动光谱分析中被检测到。类似地，抑制金黄色葡萄球菌( S. aureus )的生长与降低积分强度和软化脂磷壁酸（革兰氏阳性细胞膜的主要成分）中的 C = O 酯羰基伸展相平行。电子显微镜分析证实，细胞膜的变化是 CP 纳米颗粒的主要作用方式。当 HAp 被大肠杆菌内化时与 ACP 相比，ACP 处理的细菌的膜损伤更明显，这可以通过 ACP 更高的表面反应性来解释。HAp 纳米颗粒降低了耐甲氧西林金黄色葡萄球菌中过表达的外排泵的活性，这表明它们可能劫持这些泵并利用它们进入细胞，而不会对膜产生任何可见的损伤，从而从内向外作用于细胞，与 ACP 不同，ACP 的作用主要是外部机制。这可以解释为什么 HAp 与 ACP 不同，会抑制耐甲氧西林和耐多药金黄色葡萄球菌和铜绿假单胞菌的耐药机制， 分别。这项研究的结果对于优化这些纳米粒子以成为生物相容性较差的无机物和小分子抗生素的替代品至关重要，以遏制细菌病原体对现有疗法的耐药性上升。