Antibiotic usage promotes the evolution of resistance against gepotidacin, a novel multi-targeting drug

Abstract

Multi-targeting antibiotics, i.e. single compounds capable to inhibit two or more bacterial targets offer a promising therapeutic strategy, but information on resistance evolution against such drugs is scarce.Gepotidacin is 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 over 2000-fold increment in resistance, while individually none of these mutations affect resistance significantly. Alarmingly, gepotidacin-resistant strains are found to be as virulent as the wild-type K. pneumoniae strain in a murine model, and extensive crossresistance was demonstrated between gepotidacin and ciprofloxacin, a fluoroquinolone antibiotic widely employed in clinical practice. This suggests that numerous fluoroquinolone-resistant pathogenic isolates carry mutations which would promote the evolution of clinically significant resistance against gepotidacin in the future. We conclude that prolonged antibiotic usage could select for mutations that serve as stepping-stones towards resistance against antimicrobial compounds still under development. More generally, our research indicates that even balanced multi-targeting antibiotics are prone to resistance evolution.

High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through 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 in the following discussion 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).…”

Fighting antibiotic resistance—strategies and (pre)clinical developments to find new antibacterials

“…Compared with fluoroquinolones, gepotidacin has a unique binding site on GyrA and topoisomerase IV (subunit ParC). Nevertheless, early findings indicate that fluoroquinolone resistance might be a stepping stone for gepotidacin resistance as the target sites on topoisomerase IV are in close proximity to each other (preprint: Szili et al , 2018 ; World Health Organization, 2022 ).…”

Improved bacterial recombineering by parallelized protein discovery

“…genome editing (i.e., MAGE and DIvERGE) holds the promise of enabling rapid analysis of genotype-to-phenotype relationships and predicting future mechanisms of antimicrobial resistance (13,46). In contrast, C. freundii is an intriguing biomanufacturing host in which the optimization of metabolic pathways has remained challenging (47,48).…”

“…138474) were constructed by replacing the Redβ ORF of pORTMAGE311B plasmid (Addgene accession no. 120418) (46) with PapRecT and CspRecT, respectively. pORTMAGE-Pa1 was constructed in many steps: 1) The Kanamycin resistance cassette and the RSF1010 origin-of-replication on pORTMAGE312B with Gentamicin resistance marker and pBBR1 origin-of-replication, amplified from pSEVA631 (75) (gift from Victor de Lorenzo, Consejo Superior de Investigaciones Cientificas, Madrid, Spain); 2) optimization of RBSs in pORTMAGE-Pa1 was done by designing a 30-nt optimal RBS in front of the SSAP ORF and in between the SSAP and MutL ORFs with an automated design program, De Novo DNA (76); 3) PaMutL was amplified from P. aeruginosa genomic DNA and cloned in place of EcMutL_E32K; and finally 4) PaMutL was mutated by site-directed mutagenesis to encode E36K.…”