Spatial Structure Modulates Persister Formation in A Synthetic Cross-Feeding Bacterial Community

Abstract

Antibiotic persistence is an important mechanism that allows bacteria to survive antibiotic stress. Persistence also contributes to the evolution of antimicrobial resistance (AMR), which played a role in ~5-million death worldwide in 2019 alone. The role that microbial ecology plays in antibiotic persistence remains largely unknown. Here, I studied the effect that cross-feeding on agar surfaces has on antibiotic persistence. Using an obligate cross-feeding mutualism of engineered strains of Escherichia coli and Salmonella enterica, I discovered that in the spatially-structured environment, the antibiotic persister frequency in E. coli was ~100-fold higher in the cross-feeding coculture than in monoculture. This heightened E. coli persister frequency was removed (1) when E. coli’s metabolic dependency on S. enterica was broken through metabolite supplementation, and (2) when the growth environment was spatially homogeneous in the shaken liquid medium. Using high-throughput quantification of the E. coli growth physiology on agar, I found that average growth rate was not sufficient to explain the heightened E. coli antibiotic persistence in mutualism. By pairing the single-colony analysis with a PDE mathematical model on growth physiology, I found that the high persistence phenotype in the cross-feeding coculture is correlated with increased variability in both growth rate and lag time, and future effort will be needed to determine their relative contributions. Together, my thesis showed that the combination of cross-feeding and spatial structure is a novel mechanism which increased phenotypic heterogeneity in bacterial growth and persistence to antibiotics. Finally, my work implies the potential clinical threat of antibiotic persistence in spatially-structured polymicrobial infection sites. The experimental setup in this work is also foundational to incorporate spatial structure into the study of the highly debated relationship between mutualism and community stability.