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.

Keywords: Carbapenem-resistant Klebsiella pneumoniae; colistin resistance; hypervirulence; in-host; tigecycline resistance; whole-genome sequencing.

Figure 1.

Medical history of the patient. Note: ^a UTI, urinary tract infection; BSI, bloodstream infection. ^{b, c} The CRKP isolates were not stored.

Figure 2.

Minimum-spanning tree of cgMLST profiles among eleven CRKP isolates. Note: The minimum-spanning tree was generated based on cgMLST analysis with 2358 conserved genome-wide genes. A cluster was defined at a distance of ≤15 alleles.

Figure 3.

Sequence alignment of bla_KPC-2 plasmids among CRKP-Urine1 and CRKP-Feces3, CRKP-Blood1 and CRKP-Urine2. Note: The resistance variation region was located at 4–19 kb of pKPC-CRKP-Urine1. Four copies of insertion sequence 26 (IS26) and resistance genes were identified in the resistance variation region. The bla_TEM-1B, rmtB and fosA3 genes were absent in CRKP-Urine2, whereas bla_CTX-M-65 was not found in CRKP-Blood1.

Figure 4.

In-host resistance evolution pathways against colistin and tigecycline.