Scarless excision of an insertion sequence in the OmpK36 promoter restores meropenem susceptibility in a non-carbapenemase-producing <i>Klebsiella pneumoniae</i>

Carbapenem-resistant <i>Klebsiella pneumoniae</i> (CRKP) poses a significant global health challenge due to its limited treatment options and high mortality rates. Meanwhile, the prevalence of non-carbapenemase-producing CRKP (NC-CRKP) strains is increasing, but their resistance mechanisms remain less understood compared to those of carbapenemase-producing CRKP (CP-CRKP). In this study, KP-469, an NC-CRKP strain, was found to lack the major porins OmpK35 and Ompk36 but possessed OmpK37, coexisting with ESBL resistance genes CTX-M and SHV. Membrane porin coding sequence alignment revealed a minor deletion in Ompk35 and a 768 bp insertion sequence in the promoter region (IS-PR) of Ompk36, located between the -10 region and the ribosome-binding site (RBS). In the KO-469 strain with scarless excision of IS-PR and the constructed pHSG396-promoter-Ompk36 strain that incorporated wild-type Ompk36 promoter into KP-469, the transcription levels of Ompk36 were significantly higher than that in KP-469 strain, and His-tag antibody quantification further confirmed the regular expression of Ompk36 in KO-469. These results demonstrated that IS-PR markedly reduced the transcriptional and translational efficiency of Ompk36 in the KP-469 strain, leading to decreased permeability to meropenem. Moreover, the restored susceptibility to meropenem in the KO-469 strain was validated by <i>in vitro</i> antimicrobial susceptibility tests and an <i>in vivo</i> intraperitoneal infection model constructed in neutrophil-depleted mice. The novel carbapenem resistance mechanism of NC-CRKP caused by the insertion sequence in the OmpK36 promoter will facilitate the development of antibacterial regimens for treating NC-CRKP infections.