Analysis of Bacterial Communities Using Droplets Based Millifluidics

Microbes typically form highly complex and diverse communities that account for a significant portion of life's genetic diversity. Analysis of living systems, e.g. bacterial or cell population, plays a significant role in detecting and identifying pathogens, testing antibiotic susceptibility, and the fundamental research of population diversity and evolution. This work focuses on the analysis of bacterial communities using droplets based millifluidics. To monitor the bacteria growth, we designed an optofluidic system, combining the encapsulation of bacteria in numerous emulsion droplets to monitor their long-term behavior and relationship in a co-culture environment using fluorescent signals. In the first part of this work, we co-encapsulated and cultured two isogenic strains of Escherichia coli (E. coli) in numerous emulsion droplets to reveal their competition and cooperation relationship. Since two strains of E. coli express blue and yellow fluorescent proteins (BFP and YFP, respectively), we quantified their growth by integrating a fluorescence detection system. We analyzed the following parameters: doubling time, population yield, final biomass ratio, correlation map of doubling time and competition coefficient to characterize and compare the bacterial growth kinetics and behavior in mono and co-cultures. In addition, the experimental observations were compared with the predictions from a single growth model. Finally, we employed the millifluidic device to verify the appearance of cross-protection between antibiotic-sensitive bacteria and antibiotic-resistant bacteria. It is one of the mechanisms by which different bacteria, sharing the same environment, protect each other to survive in the presence of antibiotics. For this purpose, the E.coli YFP strain was chosen as an antibiotic-sensitive group. Simultaneously, the E.coli BFP strain with β-lactam and its mutations were selected as resistant strains. Combining the millifluidic droplet reactor method with other detection strategies, e.g. fluorescence microscopy, fluorescence flow cytometry, and plate reader, we proved the appearance of cross-protection by detecting the filamentary cells, the fluorescence of cell-free media, viable cell rates, cell shape and size, as well as β-lactamase activity. All these results obtained by millifluidic devices proved that this strategy could be used in a high-throughput bacterial coexistence study. In addition, the research of these general fields, such as bacterial community and antibiotic impact, can help us to reveal the interaction between microbial species and determine the right dose of antibiotics to inhibit bacterial growth in a co-existent environment efficiently