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Resistance evolution of hypervirulent carbapenem-resistant Klebsiella pneumoniae ST11 during treatment with tigecycline and polymyxin.

ABSTRACT: Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused increasing attention, especially ST11, the predominant CRKP clone in China. Here, we report a case of hv-CRKP-associated infection and reveal the in-host evolution of its mechanism of resistance to tigecycline and polymyxin under clinical therapy. A total of 11 K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from a male patient who suffered from continuous and multisite infections. String and antimicrobial susceptibility tests identified seven hypermucoviscous strains and three tigecycline-resistant and four colistin-resistant strains. Galleria mellonella larvae infection model confirmed the hypervirulence. Pulsed-field gel electrophoresis (PFGE) separated three PFGE clusters among all strains, and further Southern blotting detected that bla_KPC-2 was located on the same-sized plasmid. Whole-genome sequencing showed that all strains belonged to the hv-CRKP ST11-KL64 clone. Diverse hypervirulence factors and resistance genes were identified. Further sequencing with the Nanopore platform was performed on the CRKP-Urine1 strain, which contained one virulence plasmid (pVi-CRKP-Urine1) and two resistance plasmids (pKPC-CRKP-Urine1 and pqnrS1-CRKP-Urine1). The gene mutations responsible for tigecycline or colistin resistance were then amplified with PCR followed by sequencing, which indicated that mutations of ramR and lon were the potential loci for tigecycline resistance and that the pmrB, phoQ and mgrB genes for colistin resistance. A novel frameshift mutation of lon was identified in the high-level tigecycline-resistant strain (MIC, 128 mg/L). The results indicate that the hypervirulent ST11-KL64 clone is a potential threat to antiinfection treatment and is capable of rapid and diverse evolution of resistance during tigecycline and polymyxin treatment.

SUBMITTER: Jin X

PROVIDER: S-EPMC8205050 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:The increase in carbapenem-resistant Klebsiella pneumoniae (CRKP), especially the emergence of tigecycline-resistant K. pneumoniae (KP), is a serious public health concern. However, the underlying mechanism of tigecycline resistance is unclear. In this study, we evaluated the role of the CusS-CusR two-component system (TCS), which is associated with copper/silver resistance, in tigecycline resistance in CRKP. Methods:Following the in vitro evolution of tigecycline-resistant KP, the minimum inhibitory concentrations of tigecycline were determined using the micro-broth dilution method. RNA sequencing and data analysis were performed to identify differentially expressed genes. Quantitative PCR (qPCR) was performed to verify the genes of interest. Genes associated with tigecycline resistance, such as ramR, tex (T), and tet (A), were detected by PCR, and then mutants were confirmed by sequencing. Additionally, the efflux pump-associated genes soxS, oqxA, oqxB, acrE, and acrF were also analyzed by qPCR. CusR was deleted and complemented by the suicide vector pKO3-Km plasmid and pGEM-T-easy plasmid, respectively. Results:Nine strains of KP were evaluated in our study. Strains A2 and A3 were evolved from A1, B2, and B3 were evolved from B1, and C2 and C3 were evolved from C1. The tigecycline minimum inhibitory concentration for A1, B1, and C1 was 0.5 ?g/mL; that for A2, B2, and C3 was 16.0 ?g/mL; and that for A3, B3, and C3 was 32.0 ?g/mL. RNA-sequencing and qPCR confirmed that the differentially expressed genes cusE, cusS, cusR, cusC, cusF, cusB, and cusA showed higher expression in C2 and C3 than in C1. Genes related to the efflux pump AcrAB-TolC showed higher expression in B2 and B3 than in B1. No mutants of ramR, tex (T), or tet (A) were detected. SoxS, oqxA, oqxB, acrE, and acrF did not show increased expression in any group. After deletion and complementation of cusR among C3, the MIC of tigecycline decreased to 4 ?g/mL, and then recovered to 32 ?g/mL. The expression of cusFBCA, correspondingly decreased and increased significantly. Conclusion:In addition to its primary function in resistance to copper/silver, the CusS-CusR two-component system is associated with CRKP resistance to tigecycline.

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Resistance evolution of hypervirulent carbapenem-resistant Klebsiella pneumoniae ST11 during treatment with tigecycline and polymyxin.

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