Droplet-based pico-reactors for high-throughput phenotyping and selection of biofilm-producing bacteria

Description

Biofilm formation is a natural strategy for many bacteria to survive in extreme environments. It is a protective layer consisting of extracellular DNA (eDNA), proteins, and polysaccharides, making them inaccessible to antibiotics or biocides. In recent years, microfluidic flow systems for biofilm studies have provided more precise environment control. However, the primary method for quantitative biofilm analysis relies heavily on microscopy, resulting in a low-throughput quantitative measurement with a limited image-scanning area. Not to mention, it is also a time-consuming process. Although the current advances allowed biofilm to be analysed at a higher level of detail by new and better hardware, the physical dissection and isolation of single cells and matrix components from living biofilms remain unsolved and unsatisfactory. Flow cytometry is an ideal method allowing a high-throughput quantitative analysis. Therefore, scientists propose to analyse biofilm formed in a microfluidic device, achieving both high-throughput analyses with precise biofilm formation control and high-throughput by integrating microfludics and flow cytometry. However, the reality is that flow cytometry is an end-point destructive measurement. The sample needs to be transferred from a microfluidic channel to a suspension for flow cytometry. Also, the biofilm is a complex matrix comprised of extracellular polymeric substances (EPS), making it impossible to be detected using the flow cytometry approach. Thus, we proposed a novel method that achieved a high-precise control of the environment for biofilm formation and a high-throughput quantitative analysis by integrating the microfluidic-droplet method and flow cytometry with fluorescence-activated cell sorting. We used Acinetobacter baumannii (A. baumannii) as our biological example as it is well-known organism to use biofilm production as a virulence factor. In this study, we successfully isolated A. baumannii based on their biofilm productivity. In addition, the isolates were recovered and vaible for future culture. We found that the biofilm phenotypes persisted after the cycle of isolation and recovery. This is to our best knowledge the first time that the use of microfluidic-droplet integrated with flow cytometry in biofilm study. We believe this method would provide new opportunities to link the biofilm phenotype to genotype, deepening the future biofilm studies.

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