Clinical Characteristics and Whole-Genome Sequencing Analysis of Carbapenem- and Colistin-Resistant Hypervirulent Klebsiella pneumoniae

Background: Colistin‑resistant hypervirulent carbapenem‑resistant Klebsiella pneumoniae (ColR‑hv‑CRKP) has emerged as a critical clinical challenge, yet its clinical and genomic characteristics remain poorly understood. Methods: From 2021 to 2024, 326 non-duplicate hv-CRKP isolates were collected from patients with confirmed clinical infections at a tertiary hospital in China. Antimicrobial susceptibility was determined by broth microdilution. Clinical data were retrospectively analyzed, and logistic regression identified risk factors for ColR‑hv‑CRKP infection. Whole‑genome sequencing was performed to characterize molecular characteristics and colistin resistance-associated mutations. Results: Ninety‑two isolates (92/326, 28.22%) were colistin‑resistant by broth microdilutionby. Patients with ColR‑hv‑CRKP infection were more likely to have a history of ICU admission, prolonged hospitalization, and invasive procedures, were more likely to have isolates recovered from sputum specimens, and had more frequent prior exposure to colistin (all P < 0.05). Multivariable analysis identified prior colistin administration (OR = 4.58, 95% CI 2.41– 8.69) and sputum specimen source (OR = 2.44, 95% CI 1.35– 4.42) as independent risk factors for ColR-hv-CRKP infection. Whole‑genome sequencing revealed that most ColR‑hv‑CRKP isolates belonged to ST11 (90/92, 97.83%), mainly KL64 (52/92, 56.52%) or KL25 (36/92, 39.13%). Carbapenem resistance was primarily mediated by bla KPC-2 (91/92, 98.91%), while no mcr genes were detected. Whole-genome sequencing analysis showed that most ColR-hv-CRKP isolates (81/92, 88.04%) harbored colistin resistance‑associated mutations. pmrB alterations were the most frequent, detected in 45.65% (42/92) of isolates, predominantly Thr157Pro (18/42) and Ser205Pro (8/42) substitutions. mgrB mutations occurred in 35.87% (33/92) of isolates, predominantly due to insertional inactivation (27/33, 81.82%) mediated by ISKpn26 and IS903B elements. Phylogenetic analysis revealed that all ST11-KL25 ColR‑hv‑CRKP isolates clustered into a highly homogeneous group with minimal intra-lineage diversity and displayed significantly smaller pairwise SNP distances than ST11-KL64/KL107 isolates (P < 0.0001). Conclusion: Colistin resistance in hv‑CRKP was mainly associated with pmrB and mgrB mutations in the dominant ST11‑KL25 and ST11‑KL64 lineages. Prior colistin exposure was the key clinical risk factor, highlighting the selective pressure of colistin use and the urgent need for strengthened antimicrobial stewardship. Keywords: Klebsiella pneumoniae , colistin, resistance, whole-genome sequencing, clinical characteristics