Marcelo Gonçalo A, Galhano Joana, Duarte Maria Paula, Capelo-Martínez José Luis, Lodeiro Carlos, Oliveira Elisabete
BIOSCOPE Group, LAQV-REQUIMTE, Chemistry Department, NOVA School of Science and Technology, FCT NOVA, NOVA University Lisbon, 2829-516 Caparica, Portugal.
MEtRICs, NOVA School of Science and Technology, NOVA University Lisbon, 2829-516 Caparica, Portugal.
Nanomaterials (Basel). 2022 Jun 23;12(13):2164. doi: 10.3390/nano12132164.
The use of nanoparticles in multiple industries has raised concerned voices about the assessment of their toxicity/antimicrobial activity and the development of standardized handling protocols. Issues emerge during the antimicrobial assaying of multiple cargo, colorimetric, colloidal nanoformulations, as standard protocols often rely on visual evaluations, or optical density (OD) measurements, leading to high variance inhibitory concentrations (MIC). Thus, a fast, luminescence-based assay for the effective assessment of the antimicrobial activity of nanoparticles is herein reported, using the bioluminescence of an in-house ATCC 8739 construct with the pMV306G13 + Lux plasmid ( Lux). The new strain's sensitivity to ofloxacin as a standard antibiotic was confirmed, and the methodology robustness verified against multiple nanoparticles and colorimetric drugs. The reduction of incubation from 24 to only 8 h, and the sole use of luminescence (LUX) to accurately determine and distinguish MIC and MIC, are two main advantages of the method. By discarding OD measurements, one can avoid turbidity and color interferences when calculating bacterial growth. This approach is an important tool that contributes to the standardization of methods, reducing samples' background interference and focusing on luminescence as a direct probe for bacterial metabolic activity, growth and, most importantly, the correct assessment of nanomaterials' antimicrobial activity.
纳米颗粒在多个行业的应用引发了人们对其毒性/抗菌活性评估以及标准化处理方案制定的担忧。在对多种载药、比色、胶体纳米制剂进行抗菌检测时出现了问题，因为标准方案通常依赖于视觉评估或光密度（OD）测量，导致最低抑菌浓度（MIC）的差异很大。因此，本文报道了一种基于发光的快速检测方法，用于有效评估纳米颗粒的抗菌活性，该方法利用了携带pMV306G13 + Lux质粒（Lux）的内部构建的ATCC 8739菌株的生物发光。确认了新菌株对作为标准抗生素的氧氟沙星的敏感性，并针对多种纳米颗粒和比色药物验证了该方法的稳健性。将孵育时间从24小时缩短至仅8小时，以及仅使用发光（LUX）来准确确定和区分MIC和最小杀菌浓度（MBC），是该方法的两个主要优点。通过摒弃OD测量，在计算细菌生长时可以避免浊度和颜色干扰。这种方法是一种重要工具，有助于方法的标准化，减少样品的背景干扰，并将发光作为细菌代谢活性、生长以及最重要的纳米材料抗菌活性正确评估的直接探针。
