Compensatory evolution in populations with fixed or non-fixed antibiotic resistance

The manner in which costs associated with antibiotic resistance mutations are alleviated through the acquisition of compensatory mutations has bearing on the dynamics of resistance accumulation and spread. Compensatory evolution has been broadly studied in the lab in populations in which resistance is entirely fixed. Yet within natural environments, resistance may often exist within populations in which resistant and susceptible bacteria co-exist. Focusing on a single Rifampicin resistance mutation, carrying a substantial yet non-extreme cost to fitness, we examined the manner in which compensation is achieved, both when resistance is fixed, and when initial majorities of resistant cells are grown alongside susceptible cells. We found that in all cases compensatory evolution occurs, although compensatory mutations never fully alleviate the initial costs of resistance. In contrast to prevalent assumptions, compensatory mutations mostly occur outside of the target gene of the antibiotic. The pathways of compensation are quite convergent, meaning that across independent populations we often observe compensatory mutations to the same loci, and sometimes even observe the precise same compensatory mutations, across populations. The target size for compensatory mutations appears to be very high, as great variation exists in compensatory mutations, even within a single population, with fixed-resistance populations displaying higher variation than populations where resistance is not fixed.