Positive epistasis drives the acquisition of multidrug resistance

Abstract

The evolution of multiple antibiotic resistance is an increasing global problem. Resistance mutations are known to impair fitness, and the evolution of resistance to multiple drugs depends both on their costs individually and on how they interact--epistasis. Information on the level of epistasis between antibiotic resistance mutations is of key importance to understanding epistasis amongst deleterious alleles, a key theoretical question, and to improving public health measures. Here we show that in an antibiotic-free environment the cost of multiple resistance is smaller than expected, a signature of pervasive positive epistasis among alleles that confer resistance to antibiotics. Competition assays reveal that the cost of resistance to a given antibiotic is dependent on the presence of resistance alleles for other antibiotics. Surprisingly we find that a significant fraction of resistant mutations can be beneficial in certain resistant genetic backgrounds, that some double resistances entail no measurable cost, and that some allelic combinations are hotspots for rapid compensation. These results provide additional insight as to why multi-resistant bacteria are so prevalent and reveal an extra layer of complexity on epistatic patterns previously unrecognized, since it is hidden in genome-wide studies of genetic interactions using gene knockouts.

Figure 1. Distribution of fitness costs of single and double mutants indicative of positive epistasis.

(A) Distribution of fitness costs of clones carrying single point mutations that confer resistance to streptomycin (black), rifampicin (dark grey) and nalidixic acid (light grey). (B) Distribution of fitness costs of clones carrying double resistance to streptomycin/rifampicin (black), rifampicin/nalidixic acid (dark grey) and streptomycin/nalidixic acid (light grey). The mean fitness cost of double resistants is less than twice the mean cost of single resistance mutations.

Figure 2. Evidence for positive epistasis.

(A) Relation between the observed fitness of the double resistance genotypes and the expected fitness under the assumption of no epistasis. (B) Distribution of the epistasis level ε, whose median is 0.025 with bootstrap confidence interval [0.016, 0.032], showing positive epistasis. (C) Allelic dependence of epistasis between the rpsL, rpoB and gyrA (positive epistasis in light grey, negative dark grey and not significant in white. Black indicates combinations of alleles for which there was a low efficiency of transduction - synthetic sub-lethals).

Figure 3. Evidence for sign epistasis amongst alleles conferring resistance.

Sign epistasis occurs when the fitness of the double mutant (white bar) is greater than the fitness of at least one single mutant (dark grey and light grey bars). The genotypes of the double mutants where we found sign epistasis are indicated below the bars and the P values of the Wilcoxon test are indicated above the bars.

Figure 4. Mutational spectrum and frequency of spontaneous double resistance mutations.

Given the genetic background of the first mutation (column) the frequency of the second mutation (raw) appears as percentage in grey scale gradient. For a given background the percentage of mutations in the second locus adds up to 100%, in most cases, except when the second mutation occurred in a different gene. The percentage of the occurrence of each mutation in a wild-type background is also shown in the third column.