The Effect of Cross-Feeding on the Molecular Evolution of Antibiotic Tolerance
Antibiotic resistance is an urgent global public health threat, as resistant bacteria can overcome antibiotic stress by being able to grow despite treatment. Another recently-discovered strategy is antibiotic tolerance, which allows bacteria to die more slowly in antibiotics; and growth of bacteria can occur again when exposure to antibiotics is halted. Previous studies fail to consider that bacteria tend to live in microbial communities, often participating in mutualism through cross-feeding nutrient exchange. Although cross-feeding is ecologically important, its effects on the molecular evolution of antibiotic tolerance is unknown. I studied whether cross-feeding interactions affect the types and numbers of tolerance-associated mutations in Escherichia coli evolved in monoculture or in synthetic cross-feeding mutualism with Salmonella enterica. After 10 cycles of cyclical exposure to short-term ampicillin treatment and drug-free growth, the genomic DNA was extracted and whole-genome sequenced through Illumina NextSeq. Sequences were trimmed, aligned with the ancestor strain genome using BreSeq to identify mutations and compared to a parallel drug-free evolution experiment. I found that E. coli evolved 10-fold higher tolerance in both mono- and co-cultures at similar rates, but achieved tolerance by different mechanisms. I observed similar numbers of genes with tolerance-linked mutations between mono- and co-culture. However, preliminary mutation set enrichment assay indicated that the monoculture-evolved tolerance mutations are enriched in amino acid transport, while coculture-evolved ones are enriched in tRNA synthesis. This result suggests that ecological interactions can shape the molecular mechanisms underlying evolution of antibiotic tolerance, and future studies should address whether such mechanisms have clinical relevance.