DNA breaks-mediated cost reveals RNase HI as a new target for selectively eliminating antibiotic resistance

Roberto Balbontín; Nelson Frazão; Isabel Gordo

In competitions between sensitive and resistant bacteria, we observed that addition of RHI001 to the medium increases the cost of most resistant mutants ( Figure 4B ), on average by 14%. RHI001 also reduces fitness of double resistant bacteria more than that of sensitive bacteria in the absence of competition ( Figure S4B and S4C). Chemical inhibition was not as effective as genetic removal (which increases cost on average by 28%) ( Figure 4C and D), as may be expected, since stability, diffusibility across the bacterial envelope, pharmacokinetics and pharmacodynamics of RHI001 in vivo are unknown, and potentially suboptimal. Nevertheless, these results suggest that inhibiting RNase HI may be a plausible strategy to select specifically against resistant strains coexisting with sensitive bacteria, as long as resistant strains fail to evolve adaptations that abrogate their extinction. In order to test this hypothesis, we propagated a mixture of sensitive bacteria (CFP-labeled) competing against a pool of five single resistant mutants (YFP-labeled RpsL K43N , RpsL K43T , RpoB H526L , RpoB H526Y , and RpoB S531F ) during 15 days, in the absence of antibiotics. We studied the frequency dynamics of resistant clones under both strong bottlenecks (1:1500 dilutions), where new adaptive mutations are less likely to spread, and weak bottlenecks (1:50 dilutions), where propagation of adapted clones is more probable.