Biofilm development of an opportunistic model bacterium analysed at high spatiotemporal resolution in the framework of a precise flow cell

Abstract

Life of bacteria is governed by the physical dimensions of life in microscales, which is dominated by fast diffusion and flow at low Reynolds numbers. Microbial biofilms are structurally and functionally heterogeneous and their development is suggested to be interactively related to their microenvironments. In this study, we were guided by the challenging requirements of precise tools and engineered procedures to achieve reproducible experiments at high spatial and temporal resolutions. Here, we developed a robust precise engineering approach allowing for the quantification of real-time, high-content imaging of biofilm behaviour under well-controlled flow conditions. Through the merging of engineering and microbial ecology, we present a rigorous methodology to quantify biofilm development at resolutions of single micrometre and single minute, using a newly developed flow cell. We designed and fabricated a high-precision flow cell to create defined and reproducible flow conditions. We applied high-content confocal laser scanning microscopy and developed image quantification using a model biofilm of a defined opportunistic strain, Pseudomonas putida OUS82. We observed complex patterns in the early events of biofilm formation, which were followed by total dispersal. These patterns were closely related to the flow conditions. These biofilm behavioural phenomena were found to be highly reproducible, despite the heterogeneous nature of biofilm.

Single microcolony diffusion analysis in Pseudomonas aeruginosa biofilms

“…There is considerable diversity not only across different microcolonies within a biofilm but also within the different regions of a single microcolony. The architectural and biochemical heterogeneity arises due to chemical gradients, 9,10 stochastic gene expression 11 and genetic variation. 12 This inherent variation is reflected in the emergent properties that arise upon the conversion of planktonic cells into the biofilm state and can be quantified by measuring the abundance of various molecules, 13 by measuring gene expression levels, 14 respiratory activity, 15 stiffness of biofilms 16 or the diffusion and mobility of molecules within biofilms.…”

Machine-assisted cultivation and analysis of biofilms

“…While few studies already illustrate the advantage of automated methodologies for biofilm research 15–17 , several studies have applied microfluidic techniques to the field of biofilm analysis, primarily to investigate biofilm formation of model organisms that are often genetically modified in order to be traceable 17–22 . Most of these studies use singular approaches like optical microscopy or microbiological methods for the characterization of the cultured biofilms 23 .…”

Individual-based modeling for biofilm structure control

“…putida OUS82 has been monitored at high temporal (10 minutes) and spatial resolutions (1 µm) (Lim et al, 2016). Proper image processing could allow using the biofilm results to build the next generation of biofilm mathematical models.…”