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Objective: This study aimed to reveal the prevalence and fitness of qnrS1-carrying plasmids in hypervirulent Klebsiella pneumoniae (hvKP) isolates. Materials and Methods: Two hundred ninety-nine hvKP strains carrying qnrS1 were collected and screened for resistance genes using PCR and sequencing. The location of qnrS1 and rmpA2 was identified by Southern blotting. The transferability and fitness of qnrS1-carrying plasmids were analyzed by conjugation experiments and plasmid stability assay. Result: In 299 hvKP isolates, the most frequently detected capsular serotype was K64 (81.9%, 245/299), followed by K1 (4.7%, 14/299) and K2 (3.7%, 11/299). All K64-hvKP were sequence type (ST) 11. The qnrS1 and rmpA2 gene mainly was located on the ∼70–210 kb IncFrepB and ∼170–220 kb IncFIB plasmid, respectively. QnrS1-carrying plasmids could be transferred into Escherichia coli J53. However, the plasmid was transferred at a low rate of 13.4% (40/299). The 40 donor isolates belong to 4 STs—ST11, ST700, ST592, and ST86, and none contains the CRISPR-Cas loci. CRISPR-Cas loci were mainly found in ST23 K. pneumoniae. The relative fitness (RF) of qnrS1-carrying plasmids in ST86 and ST11 (cotransfer with blaTEM-1 genes) was more than one and enhanced during cultivation, especially in ST86. However, the RF of qnrS1-carrying plasmids in ST592 and ST700 showed a high fitness cost. Whole-genome sequencing showed that the qnrS1-carrying plasmids in ST86 harbored more maintenance modules (SOS inhibitor protein psiB, parA, and parB partition systems) and insertion sequence (IS) elements (IS91, IS481-like, IS1380), indicating that the qnrS1-carrying plasmid in ST86 is more stable than the other types of qnrS1-carrying plasmids. Conclusion:QnrS1-carrying IncFrepB plasmids were highly prevalent and show polymorphism in hvKP strains. The qnrS1-carrying IncFrepB plasmid in ST86 hvKP should be highlighted due to its remarkable adaptability advantages. [ABSTRACT FROM AUTHOR]