Bacterial single-cell RNA sequencing captures biofilm transcriptional heterogeneity and differential responses to immune pressure

Biofilm formation is an important mechanism of survival and persistence for many bacterial pathogens. These multicellular communities contain subpopulations of cells that display vast metabolic and transcriptional diversity along with high recalcitrance to antibiotics and host immune defenses. Investigating the complex heterogeneity within biofilm has been hindered by the lack of a sensitive and high-throughput method to assess stochastic transcriptional activity and regulation between bacterial subpopulations, which requires single-cell resolution. We have developed an optimized bacterial single-cell RNA sequencing method, BaSSSh-seq, to study Staphylococcus aureus diversity during biofilm growth and transcriptional adaptations following immune cell exposure. BaSSSh-seq was first applied to compare transcriptional profiles of S. aureus biofilm and planktonic growth. BaSSSh-seq was next applied to S. aureus biofilms following direct co-culture with the major leukocyte infiltrates associated with biofilm infection: macrophages, granulocytic myeloid-derived supressor cells, and neutrophils.