Molecular characteristics and antimicrobial susceptibility of carbapenem-resistant Klebsiella pneumoniae in a multicenter study in Ningbo, China

Abstract

Objective: To analyze the molecular epidemiology and antimicrobial resistance profiles of carbapenem-resistant Klebsiella pneumoniae (CR-KP) isolates in Ningbo, with the aim of providing a theoretical basis for hospital infection control strategies and the implementation of precise clinical diagnosis and treatment protocols.

Methods: During the period from April 30, 2023 to June 30, 2024, clinical isolates of Klebsiella pneumoniae were collected from multiple centers in Ningbo, including The Affiliated Li Huili Hospital (Yinzhou District, Ningbo), Xiangshan Red Cross Taiwan Compatriot Hospital Medical and Health Group (Xiangshan, Ningbo), and the Second Hospital of Ninghai County (Ninghai, Ningbo). A total of 81 CR-KP strains were identified using the broth dilution method for carbapenem resistance screening. These isolates were submitted to Beijing Novo gene Co., Ltd. for sequencing analysis. The sequencing data were analyzed using online tools (https://bigsdb.pasteur.fr/ and http://genepi.food.dtu.dk/resfinder) to obtain information on multilocus sequence typing (MLST), capsular serotype (KL type), virulence genes, and resistance genes. Phylogenetic relationships were constructed using SNP software. For plasmid characterization, the PlasmidFinder online tool (https://cge.food.dtu.dk/services/PlasmidFinder/) was utilized to identify plasmid replicon genes and perform Inc. typing analysis. Furthermore, to conduct a comprehensive collinearity analysis of the bla _KPC-2 resistance plasmid gene, gene cluster maps were constructed using Bakta v1.11.0 and Clinker v0.0.28 software packages.

Results: Among the 81 CR-KP isolates, MLST typing revealed that ST11 was the predominant sequence type, accounting for 66.67% (54/81), with KL64 being the dominant capsular type. Among the non-ST11 CR-KP isolates, the ST15 type accounted for 48.15% (13/27), with KL19 being the predominant capsular serotype. The carriage rate of virulence genes-including rmpA2, fyuA, and 10 other genes-was significantly higher in ST11 CR-KP compared to non-ST11 CR-KP (p < 0.05). Analysis of resistance genes revealed that ST11 CR-KP primarily carried bla _KPC-2 (100%, 54/54), whereas the resistance gene profiles among non-ST11 CR-KP isolates were more diverse, including bla_NDM , bla _IMP, and bla _OXA. Plasmid typing indicated that ST11 CR-KP predominantly harbored IncFII (98.15%, 53/54) and RepB (72.22%, 39/54) plasmid types. In contrast, non-ST11 CR-KP isolates exhibited a wider range of plasmid types, including IncX3 (33.33%, 9/27), RepB (25.93%, 7/27), IncFII (25.93%, 7/27), IncFIB (7.41%, 2/27), and both ColKP3 and Col440II (7.41%, 2/27). Antimicrobial susceptibility testing demonstrated high resistance rates to commonly used antibiotics in both ST11 and non-ST11 CR-KP isolates. ST11 CR-KP exhibited 100% resistance to six antibiotics, including ceftriaxone (CRO), cefotetan (CTT), and cefepime (FEP), and showed susceptibility only to gentamicin (GEN), aztreonam/avibactam (AZA), ceftazidime/avibactam (CZA), polymyxin B (POL), and tigecycline (TGC). Non-ST11 CR-KP showed a significantly higher resistance rate to gentamicin (GEN) and ceftazidime/avibactam (CZA) than ST11 CR-KP (p < 0.05), but lower resistance rates to cefotetan (74.07%), all of which were statistically significant (p < 0.05).

Conclusion: In the Ningbo region, CR-KP is predominantly of the ST11-KL64 type, exhibiting both strong antimicrobial resistance and high virulence characteristics. Non-ST11 CR-KP isolates carry genetically diverse carbapenemase genes and mobile genetic elements (e.g., IncX3, ColKP3). ST11 CR-KP strains demonstrate significantly stronger resistance profiles compared to non-ST11 strains. Therefore, stringent control over the use of carbapenem antibiotics is essential, along with measures to prevent the spread of resistance plasmids and the continuous improvement of hospital infection control strategies.

Keywords: Klebsiella pneumoniae; carbapenem resistance; plasmids introduction; resistance genes; virulence genes.

Figure 1

Basic clinical data of the strains.

Figure 2

Minimum spanning tree based on MLST. (A) Distribution of STs of CR-KP in different KL isolates, with different colors representing different KL types. (B) Distribution of STs of CR-KP in isolates from different sample types, with different colors representing different sample types. (C) Distribution of STs of CR-KP in isolates from different collection sites, with different colors indicating different collection sites. Circles represent ST types, and the size of the circles indicates the number of strains in each ST.

Figure 3

Phylogenetic of virulence-associated genes, antibiotic resistance, and plasmids. A core genome SNP-based phylogenetic analysis was performed to assess genetic relationships among 81 Klebsiella pneumoniae clinical isolates. The resulting phylogenetic tree illustrates evolutionary divergence through branch lengths, with terminal clades representing individual strains. An adjacent annotation panel employs color-coded blocks (arranged left-to-right) to visualize the distribution of: (1) antimicrobial resistance determinants, (2) plasmid replicon types, and (3) virulence-associated genes. Color gradients correspond to specific genetic elements, while blank spaces indicate their absence in respective isolates.

Figure 4

Sankey diagram of antibiotic resistance genes in CR-KP. A Sankey diagram also known as a Sankey energy flow chart, is used here. The left nodes represent non-ST11 and ST11 types of CR-KP, while the right nodes represent five antimicrobial classes (including 22 kinds of antibiotic resistance genes) the width of the branches correlates with number of resistance genes.

Figure 5

Comparative genomic architecture of bla_KPC-2 loci in CR-KP. In the annotated genetic map, colored arrows represent predicted genes or open reading frames (ORFs), with arrow directionality indicating transcriptional orientation and color gradients distinguishing functional gene categories. All 61 isolates carrying the bla_KPC-2 gene were analyzed using Illumina short-read sequencing due to budget constraints.