(565d) Identification of Resistance Determinants in Laboratory Evolved Mycobacterium Smegmatis evolved Under Temporally Variable Drug Profiles

Antibiotic resistance continues to be a global health concern with emergence of resistance in bacteria out-competing the antibiotic discovery rate. Adaptive Laboratory Evolution (ALE) is a useful approach to investigate factors that govern the evolution of resistance in bacteria in order to identify strategies to combat them.

In our work, we have studied the impact of temporally varying environments on resistance evolution in Mycobacterium smegmatis subjected to a range of constant and increasing antibiotic concentrations. Parallel adaptive evolution was carried out for a range of constant and step-wise increasing Norfloxacin concentrations up-to 32X WT MIC. Mutants with varying resistance levels were identified with resistance being proportional to selection pressure exposed during evolution. All the evolved populations also exhibited negligible intracellular accumulation of a fluorescent dye, Ethidium bromide (EtBr) with respect to WT strain. To identify resistance determining factors, Whole Genome Sequencing (WGS) was carried out for representative colonies from all the evolved populations.

No mutation in target gene (gyrA/B) was identified in all the populations evolved till 4X WT MIC irrespective of the evolution profile. In case of populations evolved under different constant drug pressure, mutations in various genes were identified throughout the genome with mutations majorly in metabolic genes in low resistance mutants and mutations in energy related pathways and essential genes in high resistance mutants. Target like mutations (helicase mutation) along with ribosomal mutations appeared in populations evolved under constant drug pressure of 4X MIC. Further, the number of unique mutations identified increased depending on the selection pressure during evolution. On the contrary, populations evolved under increasing drug pressure up-to 4X MIC had comparatively lower number of mutations with mutations in and around regulatory gene mutations being dominant.

All the evolved mutations harbored a mutation in a regulatory gene, lfrR known as a repressor of an efflux pump LfrA in M. smegmatis. The mutations were spread across the gene length, with a few mutations affecting the N-terminal domain while the majority affecting the C-terminal domain. Over-expression of lfrR gene in the evolved populations restored accumulation levels similar to WT levels. MIC of EtBr and Norfloxacin was also restored to WT levels. Similar restoration of WT accumulation and MIC level was obtained using Efflux Pump Inhibitor (CCCP) along with EtBr indicating efflux to be the primary defense mechanism for populations evolved under 4X MIC irrespective of the evolution profile.

In case of populations evolved under increasing Norfloxacin concentrations of up-to 32X MIC, mutation in target genes (gyrA and gyrB) were identified along with mutations in other regulatory genes including lfrR and crp1. Crp1 controls gene regulation under energy limiting and hypoxic conditions in M. smegmatis adjusting cellular machinery to match with the energy demands of the cell. These results suggest involvement of mutations beyond efflux related mutations under conditions threatening survival.

Overall, this work demonstrates multiple adaptive trajectories for populations evolved under diverse temporal profiles with conservation of certain mutations allowing survival and assisting in further accumulation of mutations. Also, it was demonstrated that efflux mutations in general precede target gene mutations. Therefore, including EPIs in drug regimen early on during treatment process would significantly impede resistance evolution and prevent high level resistance emergence.