Rapid Evolution of Reduced Susceptibility against a Balanced Dual-Targeting Antibiotic through Stepping-Stone Mutations

Abstract

Multitargeting antibiotics, i.e., single compounds capable of inhibiting two or more bacterial targets, are generally considered to be a promising therapeutic strategy against resistance evolution. The rationale for this theory is that multitargeting antibiotics demand the simultaneous acquisition of multiple mutations at their respective target genes to achieve significant resistance. The theory presumes that individual mutations provide little or no benefit to the bacterial host. Here, we propose that such individual stepping-stone mutations can be prevalent in clinical bacterial isolates, as they provide significant resistance to other antimicrobial agents. To test this possibility, we focused on gepotidacin, an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific mutations in these target proteins provide an >2,000-fold reduction in susceptibility, while individually, none of these mutations affect resistance significantly. Alarmingly, strains with decreased susceptibility against gepotidacin are found to be as virulent as the wild-type Klebsiella pneumoniae strain in a murine model. Moreover, numerous pathogenic isolates carry mutations which could promote the evolution of clinically significant reduction of susceptibility against gepotidacin in the future. As might be expected, prolonged exposure to ciprofloxacin, a clinically widely employed gyrase inhibitor, coselected for reduced susceptibility against gepotidacin. We conclude that extensive antibiotic usage could select for mutations that serve as stepping-stones toward resistance against antimicrobial compounds still under development. Our research indicates that even balanced multitargeting antibiotics are prone to resistance evolution.

Rational design of balanced dual-targeting antibiotics with limited resistance

“…The frequency-of-resistance assays have indicated that spontaneous resistance evolution to ULD1 and ULD2 is rare and is responsible for only a modest decrease in compound susceptibility of S. aureus. However, a prior study suggests that combination of multiple, specific mutations at all drug targets, in the long run, can eventually render even multitargeting antibiotics ineffective [8]. To test this possibility, we have repeated the frequency-of-resistance assays with PLOS BIOLOGY 2 ULD1-resistant S. aureus VISA laboratory isolates, both of which carried a single mutation at GyrB, the primary target of ULD1/ULD2.…”

“…To this aim, we have initiated adaptive laboratory evolution experiments under ULD1, ULD2, and novobiocin stresses against VISA. We have employed a previously established protocol that aims to maximize the level of drug resistance in the evolving bacterial populations [8,55]. To accurately assess potential resistance mechanisms, 10 parallel evolving populations have been exposed to gradually increasing concentrations of each compound.…”

“…Although it is a major focus of the pharmaceutical industry [4,5,7], designing multitargeting antibiotics remains challenging. Indeed, only a handful of such antibiotic candidates display a balanced inhibition at multiple microbial targets [8][9][10][11].…”

“…Second, low level of resistance against these antibiotics can readily emerge by mutations in efflux pumps and transcription-translation [18]. Even worse, fluoroquinolone resistance mutations also promote the acquisition of resistance in other antibiotic candidates currently under clinical development [8,[19][20].…”

High-efficiency multi-site genomic editing (HEMSE) ofPseudomonas putidathrough thermoinducible ssDNA recombineering

“…By playing with a stringent expression system for rec2 , applying multiple cycles of recombinase production/ oligonucleotide transformation and reversibly inhibiting the MMR system during a limited time window we report below high-fidelity recombination frequencies that approach those achieved with the archetypal Red-based system (Datsenko and Wanner, 2000). This opens genome-editing possibilities in this environmental bacterium that were thus far limited to strains of E. coli , closely related enteric species (Nyerges et al, 2018; Szili et al, 2019) and some lactic acid bacteria (van Pijkeren et al, 2012).…”