Stepwise within-host evolution of a blaCTX-M-27-positive Klebsiella pneumoniae clone under changing antimicrobial pressures leading to enhancement of β-lactam resistance

Objective We consecutively obtained four bla CTX - M-27 -positive Klebsiella pneumoniae isolates (named KE411, KE412, KE413, and KE414) from a patient with acute leukemia who underwent sequential antimicrobial therapy. These isolates exhibited a stepwise increase in β-lactam resistance alongside changes in the antibiotic treatment and ultimately acquired resistance to all β-lactams examined, including carbapenems. This study aimed to identify the genetic changes between the isolates that led to increased antimicrobial resistance. Methods Complete genome sequences of the four isolates were compared to identify genetic differences between them. To characterize the genetic alterations, we profiled outer membrane proteins and quantified mRNA expression. Results Only one single-nucleotide polymorphism was found between the isolates, indicating their clonality. However, multiple genomic structural alterations associated with mobile genetic elements were detected, many of which contributed to differences in antibiotic susceptibility. There were two key genetic events in the evolutionary process: First, insertion of an IS Ecp1 -mediated transposition unit carrying bla CTX - M-27 into the 23S rRNA gene. This insertion was first detected in isolate KE412, obtained during cefepime therapy, and was shared by the later isolates, resulting in increased bla CTX - M-27 expression. Second, a 48-bp deletion that inactivated the porin-encoding ompK36 gene, detected in isolates KE413 and KE414 (obtained after shifting to doripenem therapy). Because the K. pneumoniae clone was deficient in OmpK35 production, this deletion led to the acquisition of carbapenem resistance due to dual deficiency of OmpK35 and OmpK36. Conclusion We reveal stepwise within-host evolution of a K. pneumoniae clone enabling its survival under changing antimicrobial pressures.