The hidden link between cefiderocol resistance and increased virulence in Klebsiella pneumoniae: insights from a transposon-directed insertion-site sequencing-based investigation

OBJECTIVES: The emergence of extensively drug-resistant Enterobacterales, particularly carbapenemase-producing Klebsiella pneumoniae, poses a serious global health threat. Cefiderocol (FDC), a sideromycin antibiotic, employs a 'Trojan horse' strategy by using bacterial iron transport systems to enter the cell and inhibit cell wall synthesis. Although initially promising, resistance to FDC has rapidly emerged in the clinics and in the environment, necessitating a comprehensive understanding of the underlying resistance mechanisms. METHODS: In this study, we used transposon-directed insertion-site sequencing in an extensively drug-resistant FDC-susceptible K. pneumoniae ST258 strain to systematically identify genetic networks that modulate FDC resistance. RESULTS: Our transposon-directed insertion-site sequencing analysis revealed 299 chromosomal genes significantly impacting FDC resistance, with 143 identified as conditionally resistant modulators (CRMs) and 156 as conditionally essential genes. CRMs notably included genes associated with FDC influx, such as the siderophore uptake genes cirA and tonB, and porin channels such as ompK36 and tolB. Importantly, we identified several CRMs involved in bacterial capsule synthesis and expression (e.g. csrD and glnD), suggesting that capsule overexpression is a novel resistance mechanism. Phenotypic characterization of cirA, csrD and glnD knockout mutants confirmed increased FDC resistance. Furthermore, disruption of csrD and glnD resulted in enhanced capsule production, increased resistance to human serum and, in particular, increased virulence in a Galleria mellonella infection model. CONCLUSIONS: These results emphasize the multifactorial nature of FDC resistance, involving both impaired drug entry and capsule-mediated protection, with potentially pleiotropic effects on bacterial virulence. Our study elucidates key genetic determinants of FDC resistance and highlights the complex interplay between antibiotic resistance and bacterial pathogenicity.