L-histidine metabolic reprogramming is associated with imipenem-relebactam resistance in carbapenem-resistant hypervirulent Klebsiella pneumoniae

Although the emergence of imipenem-relebactam (IMR)-resistant carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is known, the mechanisms underlying IMR resistance remain unclear. Here, we elucidated the evolutionary mechanisms underlying IMR resistance. Seven bla KPC-2 -carrying ST11 CR-hvKP strains were subjected to in vitro IMR selection, of which two acquired resistance. In these two resistant strains, the MICs of IMR were dynamically monitored. Galleria mellonella infection model, biofilm formation assays, and growth curve analyses were used for evaluation. Whole-genome sequencing, RT-qPCR, and transcriptome sequencing were performed to analyze the mechanisms. An exogenous L-histidine supplementation experiment was conducted to determine potential reversal of the IMR-resistant phenotype. Two in seven (i.e., KP520 and KP214) strains developed resistance at the 15th (KP520-15) and 20th (KP214-20) passages, respectively, following which, their biofilm-formation capacity and virulence were enhanced. The IMR-resistant strains exhibited changes in rearrangements of insertion sequences surrounding bla KPC-2 and inversion of the core transposon orientation, and significantly increased in the expressions of bla KPC-2 . The differentially expressed genes identified before and after IMR selected were predominantly enriched in metabolism-related pathways, among which L-histidine biosynthesis was the most significantly enriched pathways. Exogenous supplementation with 10 mmol/L L-histidine restored IMR susceptibility in resistant strains and significantly increased the expression of hisG , a key gene involved in L-histidine biosynthesis. Thus, in the two strains that acquired IMR resistance increased bla KPC-2 expression and metabolic changes involving L-histidine biosynthesis were associated with the resistant phenotype. In conclusion, prolonged IMR exposure may lead to the development of resistance in CR-hvKP, with enhanced biofilm formation and pathogenic potential. In CR-hvKP strains that acquired IMR resistance under in vitro selection, this resistance was associated with increased bla KPC-2 expression and transcriptional changes involving L-histidine biosynthesis.