Campo-Pérez Víctor, Alcàcer-Almansa Júlia, Julián Esther, Torrents Eduard
Bacterial Infections and Antimicrobial Therapies Group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 15-21, 08028 Barcelona, Spain.
Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.
Microorganisms. 2023 Sep 6;11(9):2244. doi: 10.3390/microorganisms11092244.
Pathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the biomass by optical absorbance or fluorescence emission. However, using these methods to distinguish different species in dual-species or polymicrobial biofilms is currently impossible. Colony-forming unit counts from homogenized dual-species biofilms on selective agar medium allow species differentiation but are time-consuming for a high-throughput screening. Thus, reliable, feasible, and fast methods are urgently needed to study the behavior of polymicrobial and dual-species communities. This study shows that and strains expressing specific fluorescent or bioluminescent proteins permit the more efficient study of dual-species biofilms compared to other methods that rely on measuring the total biomass. Combining fluorescence and bioluminescence measurements allows an independent analysis of the different microbial species within the biofilm, indicating the degree of presence of each one over time during a dual-species biofilm growth. The quantitative strategies developed in this work are reproducible and recommended for dual-species biofilm studies with high-throughput microtiter plate approaches using strains that can constitutively express fluorescent or bioluminescent proteins.
致病细菌在感染过程中形成生物膜，而多微生物生物膜是最常见的表现形式。生物膜的附着、成熟和/或抗生素敏感性主要通过微量滴定板试验进行评估，在该试验中，细菌被染色以便通过光吸收或荧光发射对生物量进行定量。然而，目前使用这些方法无法区分双物种或多微生物生物膜中的不同物种。在选择性琼脂培养基上对均质化双物种生物膜进行菌落形成单位计数可实现物种区分，但对于高通量筛选来说耗时较长。因此，迫切需要可靠、可行且快速的方法来研究多微生物和双物种群落的行为。本研究表明，与其他依赖测量总生物量的方法相比，表达特定荧光或生物发光蛋白的菌株能更有效地研究双物种生物膜。结合荧光和生物发光测量可以对生物膜内不同的微生物物种进行独立分析，表明在双物种生物膜生长过程中，每种微生物随时间的存在程度。本研究中开发的定量策略具有可重复性，推荐用于使用能够组成型表达荧光或生物发光蛋白的菌株，通过高通量微量滴定板方法进行双物种生物膜研究。
