Introduction

Klebsiella pneumoniae, as one of the most prevalent Gram-negative pathogens, is a significant source of both community-acquired and nosocomial infections.1,2 The widespread use of antibiotics has led to the emergence of multidrug resistance (MDR) in K. pneumoniae, particularly the rise of carbapenem-resistant K. pneumoniae strains (CRKP). This trend poses a substantial threat to human health, as the options for treating CRKP infections dwindle, given the diminishing efficacy of available antibiotics.3

The first reported instances of CRKP date back to the 1990s.4 Infection caused by carbapenem-resistant strains exhibit significantly higher mortality rates compared to those caused by carbapenem-susceptible strains.5 In the United States and European countries, K. pneumoniae ST258 (sequence type) contributes significantly to the spread of carbapenem resistance, while ST11 is predominant in China.6,7 The New Delhi metallo-β-lactamase-1 (NDM-1) was initially identified in K. pneumoniae and Escherichia coli in 2008 and has since become a global concern.8 Due to the presence of mobile genetic elements such as transposons and plasmids, K. pneumoniae often plays a pivotal role in facilitating the transfer of antimicrobial resistance genes between environmental strains and clinical strains.9 The IncX type plasmid is often associated with carbapenemase production, and the IncX3 plasmid is essential for the delivery of carbapenemase genes, especially blaNDM.10–13

This study focuses on the genomic characterization of a new sequence type, ST6758 CRKP strain, isolated from an inpatient at a teaching hospital in China, which carries blaNDM-1 on an IncX3 type plasmid. The complete genome sequence of this strain was determined, and a comprehensive analysis was conducted to examine its genomic features, plasmid characteristics, and resistance mechanisms. Furthermore, the transmission mechanisms of the carbapenemase gene blaNDM-1 in K. pneumoniae were investigated.

Material and MethodsPatient and Isolate

On May 27, 2019, a 69-year-old male was admitted to a tertiary hospital in Zhejiang Province, China, presenting with dysuria. Clinical evaluations led to the diagnosis of prostatic hyperplasia and urinary retention. K. pneumoniae strain CRKP331 was isolated from the patient’s urine sample on May 29. The strain was initially identified using the VITEK MS system (bioMérieux, France) and further validated by whole-genome sequencing. CRKP331 was susceptible to aminoglycoside antibiotics, and the aminoglycoside antibiotic isepamicin was used to treat the urinary tract infection. The patient eventually recovered.

Antimicrobial Susceptibility Test

Antimicrobial susceptibility testing was conducted using the VITEK 2 system (bioMérieux, France) with Gram-negative antimicrobial susceptibility testing cards (AST-GN13) and the standard broth microdilution test, adhering to the guidelines established by the Clinical and Laboratory Standards Institute (CLSI). The antimicrobial agents tested in this study included amikacin, ceftazidime, ceftriaxone, ampicillin/sulbactam, sulfamethoxazole/trimethoprim, cefepime, gentamicin, imipenem, meropenem, levofloxacin, and tobramycin. Breakpoints were interpreted in accordance with the recommendations outlined in the CLSI guidelines.14

Whole-Genome Sequencing

The complete genome sequence of the strain was determined utilizing the Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) in the 150-bp paired-end sequencing mode, with an average sequencing depth of ≥100×. Furthermore, long-read sequencing was conducted using a MinION sequencer (Nanopore, Oxford, UK). The short Illumina reads and long MinION reads were subjected to hybrid assembly using Unicycler (v0.4.7) in conservative mode. This resulted in the generation of complete circular contigs, which were then refined and corrected using Pilon with Illumina reads through multiple rounds of iteration until no further changes were detected. The resulting complete genome sequence was subsequently annotated automatically using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) server.

Genomic Analysis

A novel sequence type of K. pneumoniae was assigned utilizing the conserved segments of seven essential housekeeping genes (rpoB, gapA, mdh, pgi, phoE, infB and tonB) based on the BIGSdb-Pasteur MLST (Multilocus Sequence Typing) analysis (https://bigsdb.pasteur.fr/klebsiella/). The BacWGSTdb server was employed to investigate the strain’s antimicrobial resistance genes and plasmid replicons.15,16 A comparative analysis was conducted using circular representations, depicted as concentric rings, to assess the blaNDM-1-carrying plasmid and its resemblance to similar plasmids. This analysis was performed using the BLAST Ring Image Generator (BRIG).17

Phylogenetic Analysis

The phylogenetic tree was examined using CSI Phylogeny (version 1.4),18 which is based on a core-genome single-nucleotide polymorphism (SNP) strategy. CSI Phylogeny was employed to identify and filter K. pneumoniae CRKP331 SNPs, validate the SNPs, and construct a phylogeny based on a concatenated alignment of the high-quality SNPs. The maximum parsimony algorithm was then used to generate a phylogenetic tree from the resulting SNPs, which was subsequently visualised on the iTOL webpage.19

Nucleotide Sequence Accession Numbers

The whole genome results for strain CRKP331 have been deposited in DDBJ/EMBL/GenBank, and the accession number is SAMN40739512.

Ethical Approval

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Zhejiang Provincial People’s Hospital (Ethics approval number 2019KY244). In this case, the Ethics Committee of Zhejiang Provincial People’s Hospital granted an exemption from written informed consent because our study focused solely on bacteria. The clinical isolate K. pneumoniae CRKP331 was collected as part of the routine hospital laboratory procedures.

Results and Discussion

The minimum inhibitory concentrations (MICs) of the tested antibiotics are shown in Table S1. K. pneumoniae CRKP331 exhibited resistance to various antibiotics, including ceftazidime, ceftriaxone, cefazolin, cefepime, imipenem, meropenem, ampicillin/sulbactam, and sulfamethoxazole/trimethoprim. However, it remained susceptible to amikacin, gentamicin, and tobramycin, and was classified as intermediate to levofloxacin.

The genome sequence of K. pneumoniae CRKP331 consists of five contigs, with a total length of 5,765,149 bp. Of these, one is a chromosome, measuring 5,317,817 bp, while the remaining four are plasmids. Contig 2 is 206,059 bp, contig 3 is 111,193 bp, contig 4 is 82,257 bp, and pNDM-CRKP331 is 47,823 bp in length. The novel sequence type of CRKP331 was classified as ST6758 through MLST analysis. This classification was achieved by analyzing the conserved segments of seven essential housekeeping genes (rpoB4, gapA2, mdh1, pgi1, phoE4, infB4 and tonB174).

The antimicrobial resistance genes identified in the genome of the isolate are presented in Table 1. We identified the β-lactam resistance genes blaSHV-187, blaTEM-1B, blaCTX-M-3, and blaNDM-1; the fosfomycin resistance gene fosA; the quinolone resistance genes oqxB, oqxA, qnrS1 and aac(6′)-Ib-cr; the trimethoprim resistance gene dfrA27; the rifamycin resistance gene ARR-3; the sulfonamide resistance gene sul1; the aminoglycoside resistance gene addA16; the tetracycline resistance gene tet(A); the macrolide resistance gene mph(A) and the phenicol resistance gene floR.

Table 1 Antimicrobial Resistance Genes (ARGs) in Isolate K. Pneumoniae CRKP331

With the exception of oqxA, oqxB, blaSHV-187, and fosA, which are located on the chromosome, the remaining resistance genes are located on plasmids. A further analysis revealed the presence of aac(6′)-Ib-cr, ARR-3, dfrA27, aadA16, sul1, mph(A), tet(A), floR, blaTEM-1B, blaCTX-M-3 and qnrS1 on plasmid 2. In addition, the carbapenem resistance gene blaNDM-1 was found to be located on plasmid pNDM-CRKP331. The blaNDM-carrying plasmid pNDM-CRKP331 was designated as the IncX3-type plasmid. The similarity of pNDM-CRKP331 to other IncX3-type plasmids was analyzed using the basic local alignment search tool (Figure 1). These analogous plasmids were all derived from K. pneumoniae, including pNDM1_020049 (accession no. CP028786), pSHX180-NDM5 (accession no. CP094514), pKH1-4-NDM5 (accession no. CP102881), pOXA181-191773 (accession no. CP080367), pVA684-49 (accession no. CP093461), hvKP12-NDM (accession no. CP103320), pRIVM_C017275_2 (accession no. CP068868), pKP7728-NDM5 (accession no. CP092650). All the other plasmids, with the exception of pOXA181-191773 and pRIVM_C017275_2, carry the blaNDM gene. This suggests that the IncX3 plasmid was an important vector for blaNDM gene transfer in K. pneumoniae. A variety of blaNDM sub-type were found on these IncX3 plasmid, which highlights the possibility that the IncX3 plasmid was one of the major platforms for blaNDM gene evolution.20

Figure 1 Circular comparison between the blaNDM-1-carrying plasmid pNDM-CRKP331 and similar plasmids.

A simple annotation of the pNDM-CRKP331 plasmid was performed using the RAST Server. This revealed the presence of IS6 and ISKox3 upstream of blaNDM-1, while ISAba125 and IS3000 were identified just downstream of blaNDM-1. The ISFinder database was utilized to identify the IS element of pNDM-CRKP331. A total of twenty-two IS elements were identified in the plasmid, belonging to five IS families, including IS6, Tn3, ISKra4, ISL3, and IS30. Among these, IS6 (19/46) and Tn3 (9/46) were the most prevalent. This suggests that the blaNDM gene may act as an external gene and recombine into the IncX3 plasmid via insertion or transposition of IS elements.

Using the Basic Local Alignment Search Tool (BLAST) with a threshold of 85% plasmid length and 80% coverage, 41 strains of K. pneumoniae were retrieved from the NCBI database, harboring IncX3-type plasmid similar to pNDM-CRKP331 (Table 2). Among these strains, twenty-two sequence types (STs) were identified, including ST1, ST11, ST14, ST15, ST16, ST17, ST23, ST35, ST43, ST258, ST307, ST340, ST427, ST437, ST485, ST505, ST512, ST656, ST766, ST1383, ST4523, and ST6758. The most common sequence types were ST11 (6/42), ST37 (6/42), and ST512 (5/41). These strains were distributed across 11 countries, with the highest prevalence in China (22 strains, 22/42), followed by the USA (5/42) and Italy (5/42). Phylogenetic analysis of the 42 strains is shown in Figure 2. The carbapenemase genes carried by these strains were predominantly blaNDM (25/42). Additionally, blaKPC was identified in 18 strains, including blaKPC-2 and blaKPC-3. Notably, five strains carried both blaKPC and blaNDM. The CRKP331 strain demonstrates a unique spectrum of resistance genes. Nonetheless, it possesses a set of shared resistance genes with other strains, which confer resistance to β-lactams, aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and sulfonamides. These observations highlight the imperative for customized therapeutic approaches and underscore the evolving and complex nature of antimicrobial resistance within K. pneumoniae.

Table 2 Information of 41 Strains of K. Pneumoniae Retrieved from the NCBI Database That Harbouring Similar IncX3-Type Plasmid

Figure 2 Phylogenetic analyses of CRKP331 and 41 K. pneumoniae strains carrying an IncX3-type plasmid similar to pNDM-CRKP331. The antimicrobial resistance genes are represented by different colours in the cells, whereas the gene is absent in the empty cells. Each square colour indicates a specific sequence type.

We further investigated the IncX3-type plasmids in these strains. These plasmids predominantly carried single resistance genes (39/42), with a few (3/42) carrying two resistance genes. The resistance genes were primarily blaNDM (25/42), encompassing three subtypes: blaNDM-1, blaNDM-5, and blaNDM-7. Three strains carried blaKPC, all of which carried two resistance genes (blaKPC and blaSHV). Additionally, three strains carried only blaOXA-181. Some strains did not carry carbapenemase genes but instead carried an Extended Spectrum β-Lactamases (ESBLs) gene, such as blaSHV-12 (5/42) or blaSHV-182 (5/42). Notably, the five strains from Italy (all ST512) carried an IncX3 plasmid with only blaSHV-182, while the five strains from the USA (all ST258) carried an IncX3 plasmid, with four strains carrying only blaSHV-12 and one strain carrying both blaKPC-3 and blaSHV-12. Of the 22 strains from China, all carried the IncX3 plasmid, with 20 carrying blaNDM and the other two carrying blaOXA-181. The IncX3 plasmid carrying the blaNDM gene has been widely prevalent in K. pneumoniae in China, involving 15 ST types (ST1, ST11, ST14, ST15, ST17, ST23, ST35, ST340, ST485, ST505, ST656, ST766, ST1383, ST4523, ST6758).

In conclusion, we report a new ST-type CRKP strain, ST6758, which carries 16 antimicrobial resistance genes, including blaNDM. These resistance genes are primarily located on plasmids, with the carbapenemase gene blaNDM-1 specifically located on an IncX3-type plasmid. A total of 41 K. pneumoniae strains carrying similar IncX3-type plasmids were retrieved from the NCBI database, representing 20 ST types across 11 countries. The most common resistance gene carried by these IncX3-type plasmids is blaNDM, and all these plasmids carry only a single blaNDM gene. IncX3-type plasmids carrying the blaNDM gene are widely prevalent in K. pneumoniae in China, spanning 15 ST types. Measures should be taken to prevent the spread of these blaNDM-carrying IncX3-type plasmids. Our findings contribute to the understanding of the transmission mechanisms of carbapenemase genes in K. pneumoniae.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (82172314) and the Zhejiang Provincial Medical and Health Science and Technology plan (2022KY022 and 2023KY484).

Disclosure

The author reports no conflicts of interest in this work.

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