mrkA gene enhances the virulence phenotype of carbapenem-resistant Klebsiella pneumoniae both in vitro and in vivo

Carbapenem-resistant Klebsiella pneumoniae (CRKP) colonization in humans can lead to systemic infections. However, the primary virulence factor driving CRKP colonization to pathogenic infection remains unclear. We aimed to evaluate the role of the mrkA gene, a component of type 3 fimbriae in CRKP virulence both in vitro and in vivo. We examined its effects on capsule morphology (using transmission electron microscopy), siderophore assays, adhesion to host cells, serum resistance, and pathogenicity in mice. Comparisons were made between the wild-type Kp-mrkA strain, the mutant Kp-ΔmrkA, and the complementary Kp-CmrkA strains. Results showed that the Kp-ΔmrkA strain exhibited significantly reduced capsule production, biofilm, and siderophore formation, adherence to human epithelial cells, and human serum resistance compared to Kp-mrkA and Kp-CmrkA. Notably, the LD50 of Kp-mrkA demonstrated a 1000-fold increase in virulence compared to Kp-ΔmrkA, and survival curves indicated significantly higher mortality in mice infected with Kp-mrkA. Histopathological analysis revealed that Kp-mrkA infection caused severe tissue damage, including interstitial vascular congestion, edema, and inflammatory cell infiltration, which was more extensive than in Kp-ΔmrkA-infected mice. Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) showed elevated levels of multiple pro-inflammatory cytokines (TNF-α, IL-1β, INF-γ, and IL-6) in the serum and lungs of Kp-mrkA-infected mice compared to those infected with Kp-ΔmrkA. These findings suggest that the mrkA gene plays a critical role in CRKP pathogenicity by inducing a strong inflammatory response and structural damage, positioning it as a potential biomarker for managing CRKP colonization and infection.