Elucidating adaptive compensatory tigecycline resistance mechanisms of RamA, RarA and SoxS in Klebsiella pneumoniae.

The molecular regulation of tigecycline resistance is complex, and the roles of RamA, RarA, and SoxS regulons in tigecycline resistance are not fully understood. Carbapenem-resistant (C248-ST11-blaKPC-2 and C182-ST11-blaNDM-1), colistin-resistant (HK1-ST562-mcr-1), and tigecycline-resistant (KP17) strains with single or double regulon gene knockouts were evolved in vitro to generate tigecycline-resistant mutants. CRISPR-Cas9 was used to create KP17 mutants lacking rarA, ramA, lon, or their combinations. The mutant prevention concentration (MPC) of tigecycline and mutation frequency of regulon mutants were assessed. Phenotypic differences between the mutants and parents were assessed using growth curves, in vitro competition growth, serum bactericidal activity, biofilm formation, and hydrogen peroxide resistance tests. Genetic and transcriptomic variations were analyzed using whole-genome and RNA sequencing. Acquired high-level tigecycline resistance in carbapenem-and colistin-resistant strains incurred fitness costs and reduced virulence. Colistin-resistant strains rapidly evolved high-level tigecycline resistance, with minimum inhibitory concentration of up to 256 mg/L. The RamRA-AcrAB/OqxAB pathway was pivotal for tigecycline resistance in carbapenem- and colistin resistant strains. Lon was not related to tigecycline resistance but appeared to be linked to oxidative stress. Although knocking out the key regulon genes RamA and/or RarA did not impede tigecycline resistance development, these knockouts influenced mutation frequencies and MPCs, with RarA knockout increasing the number of mutation sites. RarA and SoxS served as compensatory regulons in the absence of RamA, or in double knockouts. These findings improved our understanding of the mechanisms underlying tigecycline resistance in K. pneumoniae.