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

Roberto Balbontín; Nelson Frazão; Isabel Gordo

In parallel, we performed identical propagations, but in which all strains lack RNase HI, as a proxy for optimal inhibition of RNase HI function. We observed that, in the presence of RNase HI, sensitive bacteria initially outcompete resistant clones but, as the propagation progresses, resistant bacteria increase in frequency -likely due to compensatory evolution -finally reaching coexistence ( Figure 5A Figure 5B ). Since the outcome of competitions under laboratory conditions can differ from those within the host (4,66), we next tested if inhibition of RNase HI would be an effective strategy in an animal model. We used the well-established mouse gut colonization model, which resembles the natural environment of many bacterial pathogens, including E. coli (67) . We colonized by oral gavage two cohorts of mice (each n=6): one with a 1:9 ratio mixture of CFP-tagged sensitive bacteria and a YFP-labeled double resistant strain [RpsL K43T RpoB H526Y , selected based on its ability to efficiently colonize the mammalian gut (68) ], and the other with an identical mixture but in which both strains are ΔrnhA. We then followed the frequency and absolute numbers [Colony Forming Units (CFUs) per gram of feces] of the resistant strain over time. Remarkably, when RNase HI function is intact, the double resistant strain persists in the gut of all animals at frequencies between 2.3 and 9.3% ( Figure 5C , blue lines) and high bacterial loads ( Figure 5D , blue lines) two weeks after gavage but, in the absence of RNase HI, resistant clones are rapidly outcompeted by sensitive bacteria ( Figure 5C and D, red lines). Indeed, resistant bacteria are driven to extremely low frequencies in as soon as 1 day postcolonization, and their extinction occurs in all the six mice in as soon as 5 days ( Figure 5C and D, red lines), demonstrating that RNase HI is a new target for efficiently eliminating resistant strains in the gut. Surprisingly, the absence of RNase HI does not lead to any detectable fitness defect in the sensitive bacteria, as the loads of E. coli are similar in the two cohorts of mice ( Figure 5E ). We then queried if the sequential application of an RNase HI inhibitor followed by an antibiotic could successfully eliminate the entire population of a potential gut pathogenic species composed of sensitive and resistant strains. In this scenario, we hypothesize that an antibiotic treatment should render completely different outcomes depending on the presence of RNase HI function. Indeed, a week-long treatment of streptomycin starting at day 16 after gavage enables resistant clones with intact RNase HI function to rebound and reach high loads (10 7 -10 8 CFUs/g feces), but it completely erradicates ΔrnhA bacteria in all the six mice ( Figure 5F ). These results demonstrate that RNAse HI function is not only a key determinant of the fitness of resistant bacteria, but also a potential co-adjuvant to eliminate undesirable bacteria in a natural environment for bacterial pathogens such as the mammalian gut. Altogether, these results show the enormous potential of targeting RNase HI as a promising antimicrobial therapy.