Acinetobacter baumannii has recently been recognised as one of the critically important pathogens owing to its high levels of antibiotic resistance1. A. baumannii has been shown to cause 10% of hospital-acquired infections2, 60% of the fatality rate for community-acquired pneumonia3 and 35% of ventilator-related pneumonia and bloodstream infections4. can heighten its resistance levels by forming biofilms, a matrix structure consisting of extracellular products, such as proteins, polysaccharides and extracellular DNAs. The biofilm matrix is a natural protective layer preventing antibiotics from direct contact with living bacteria inside5. Most current advances studying biofilm development primarily focus on assessing bulk-culture levels. Due to the heterogeneity of A. baumannii, such methods overlook the potential contribution of minority behaviours within cell populations. Therefore, performing single-cell level assays for biofilm production in A. baumannii will play a significant role in analysing the biofilm behaviour of the medically significant organism.
To fill this gap, we successfully performed reliable and high-throughput phenotypic assays of the biofilm-producing population of A. baumannii using single-cell containing droplets. Firstly, we used microfluidic droplets (double emulsions, DEs) to encapsulate single A. baumannii cells with rich media containing YoYo-1, a fluorescent dye staining the extracellular DNA that correlates with biofilm level. We cultured cells for 21 hours and collected DEs droplets with relatively high or low levels of fluorescence using FACS, to allow detection of biofilm production or lack thereof. Post-sorting microscopic data confirmed the accuracy of the sorting results; the biofilm and the non-biofilm forming populations in droplets were distinguished. The variation of biofilm levels observed had each originated from a single cell seeding event, indicating population heterogeneity. The work opens a new opportunity to accurately select phenotypes of interest, including those that are not easily distinguishable or selectable from an agar plate.