Vanderbilt University Medical Scientist Training Program, Vanderbilt University School of Medicine, Nashville, TN, United States of America; Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN, United States of America; Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN, United States of America.
Department of Biostatistics, Vanderbilt University, Nashville, TN, United States of America.
J Microbiol Methods. 2023 Sep;212:106808. doi: 10.1016/j.mimet.2023.106808. Epub 2023 Aug 17.
Bacterial biofilms are critical to pathogenesis and infection. They are associated with rising rates of antimicrobial resistance. Biofilms are correlated with worse clinical outcomes, making them important to infectious diseases research. There is a gap in knowledge surrounding biofilm kinetics and dynamics which makes biofilm research difficult to translate from bench to bedside. To address this gap, this work employs a well-characterized crystal violet biomass accrual and planktonic cell density assay across a clinically relevant time course and expands statistical analysis to include kinetic information in a protocol termed the TMBL (Temporal Mapping of the Biofilm Lifecycle) assay. TMBL's statistical framework quantitatively compares biofilm communities across time, species, and media conditions in a 96-well format. Measurements from TMBL can reliably be condensed into response features that inform the time-dependent behavior of adherent biomass and planktonic cell populations. Staphylococcus aureus and Pseudomonas aeruginosa biofilms were grown in conditions of metal starvation in nutrient-variable media to demonstrate the rigor and translational potential of this strategy. Significant differences in single-species biofilm formation are seen in metal-deplete conditions as compared to their controls which is consistent with the consensus literature on nutritional immunity that metal availability drives transcriptomic and metabolomic changes in numerous pathogens. Taken together, these results suggest that kinetic analysis of biofilm by TMBL represents a statistically and biologically rigorous approach to studying the biofilm lifecycle as a time-dependent process. In addition to current methods to study the impact of microbe and environmental factors on the biofilm lifecycle, this kinetic assay can inform biological discovery in biofilm formation and maintenance.
细菌生物膜对于发病机制和感染至关重要。它们与抗菌药物耐药率的上升有关。生物膜与更差的临床结果相关，使其成为传染病研究的重要对象。生物膜动力学和动态学的知识差距使得生物膜研究难以从实验室转化为临床。为了弥补这一差距，本工作采用了一种经过充分表征的结晶紫生物量累积和浮游细胞密度测定法，跨越了一个临床相关的时间过程，并通过一种称为 TMBL（生物膜生命周期的时间图谱）测定法的方案扩展了统计分析，包括动力学信息。TMBL 的统计框架定量比较了不同时间、物种和培养基条件下的生物膜群落，以 96 孔格式进行。TMBL 的测量结果可以可靠地浓缩为响应特征，这些特征可以提供关于附着生物量和浮游细胞群体随时间变化的行为信息。在营养可变的培养基中进行金属饥饿条件下培养金黄色葡萄球菌和铜绿假单胞菌生物膜，以证明该策略的严谨性和转化潜力。与对照相比，在金属耗尽条件下观察到单种生物膜形成的显著差异，这与营养免疫的共识文献一致，即金属可用性驱动许多病原体的转录组和代谢组变化。总之，这些结果表明，TMBL 对生物膜的动力学分析代表了一种在统计学和生物学上严格的方法，用于研究生物膜生命周期作为一个随时间变化的过程。除了目前用于研究微生物和环境因素对生物膜生命周期影响的方法外，这种动力学测定法还可以为生物膜形成和维持的生物学发现提供信息。
