A comparison of methods to assess the antimicrobial activity of nanoparticle combinations on bacterial cells

Background
Bacterial cell quantification after exposure to antimicrobial compounds varies widely throughout industry and healthcare. Numerous methods are employed to quantify these antimicrobial effects. With increasing demand for new preventative methods for disease control, we aimed to compare and assess common analytical methods used to determine antimicrobial effects of novel nanoparticle combinations on two different pathogens.
Methods
Plate counts of total viable cells, flow cytometry (LIVE/DEAD BacLight viability assay) and qPCR (viability qPCR) were used to assess the antimicrobial activity of engineered nanoparticle combinations (NPCs) on Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria at different concentrations (0.05, 0.10 and 0.25 w/v%). Results were analysed using linear models to assess the effectiveness of different treatments.
Results
Strong antimicrobial effects of the three NPCs (AMNP0–2) on both pathogens could be quantified using the plate count method and flow cytometry. The plate count method showed a high log reduction (>8-log) for bacteria exposed to high NPC concentrations. We found similar antimicrobial results using the flow cytometry live/dead assay. Viability qPCR analysis of antimicrobial activity could not be quantified due to interference of NPCs with qPCR amplification.
Conclusion
Flow cytometry was determined to be the best method to measure antimicrobial activity of the novel NPCs due to high-throughput, rapid and quantifiable results.

【背景】在工业与医疗卫生领域，针对暴露于抗菌化合物后的细菌细胞定量分析方法差异显著。目前已有多种手段用于量化此类抗菌效应。随着疾病防控新型预防方案的需求持续攀升，本研究旨在对比并评估常用分析方法，以检测新型纳米颗粒组合（novel nanoparticle combinations, NPCs）对两种不同病原菌的抗菌活性。
【方法】本研究采用总活菌平板菌落计数法、流式细胞术（flow cytometry，LIVE/DEAD BacLight活菌检测试剂盒）以及定量PCR（qPCR，活菌qPCR）三种方法，评估工程化纳米颗粒组合（engineered nanoparticle combinations, NPCs）在质量体积百分比浓度分别为0.05、0.10与0.25 w/v%条件下，对革兰氏阳性菌——金黄色葡萄球菌（Staphylococcus aureus）与革兰氏阴性菌——铜绿假单胞菌（Pseudomonas aeruginosa）的抗菌活性。采用线性模型对实验结果进行分析，以评估不同处理方式的效果。
【结果】三种NPCs（AMNP0–2）对两种病原菌均表现出显著抗菌效应，可通过平板菌落计数法与流式细胞术完成量化检测。当细菌暴露于高浓度NPCs时，平板菌落计数法可实现大于8个数量级的菌落对数杀灭值。流式细胞术活/死菌检测试剂盒也得到了与之一致的抗菌活性检测结果。但由于NPCs会干扰qPCR扩增过程，无法通过活菌定量PCR分析完成抗菌活性的量化检测。
【结论】流式细胞术因具备高通量、快速且可量化的检测优势，被确定为检测新型NPCs抗菌活性的最优方法。