Genomic and phylogenetic analysis of Klebsiella michiganens

Introduction

Multidrug-resistant Enterobacteriaceae infection is a severe threat to public health that is worsening globally and is associated with increasing mortality rates.1 Klebsiella pneumoniae is a significant pathogen that contributes to the dissemination of multidrug-resistant strains in healthcare environments.2 K. michiganensis is a novel specie of pathogenic Klebsiella genus. Since its first discovery in 2013, K. michiganensis has continued to expand in the clinical setting and has been discovered colonizing patients and causing nosocomial epidemics.3,4 Carbapenems play a critical role in managing severe infections caused by multidrug-resistant Gram-negative bacteria. Currently, the increased incidence of carbapenem-resistant Enterobacteriaceae (CRE), particularly E. coli and K. pneumoniae isolates, is overburdening worldwide healthcare systems.5,6

The carbapenem resistance mechanism in Gram-negative bacteria is primarily attributed to the expression of carbapenemase enzymes, such as KPC, VIM, OXA-48, and NDM. These β-lactamase genes, which are found on mobile genetic elements and conjugative plasmids, are associated with the widespread dissemination of carbapenem resistance.7 K. michiganensis producing blaKPC,4,8,9 blaVIM,10 blaNDM11,12 and blaOXA-4812 have been found and they have been distributed in countries, such as China,4 Canada,8 Italy,9 Japan,11 Switzerland,10 and South Africa.12 However, only sporadic cases have been reported, and the prevalence and transmission patterns of multidrug-resistant K. michiganensis are poorly understood. Further research is required to investigate the genomic traits and antimicrobial resistant characteristics exhibited by the K. michiganensis.

Here, we report the genomic traits of a multidrug-resistant ST43 K. michiganensis strain discovered in China. In addition, we performed a comprehensive genomic epidemiology analysis to date with all the available genome sequences of ST43 K. michiganensis isolates.

Materials and Methods

The K. michiganensis strain 2563 was isolated in a sputum sample collected from a woman upon admission to the hospital with pulmonary infection. The culture of K. michiganensis 2563 was obtained from the sputum sample within 24 hours of collection. The isolate was first identified by VITEK 2 (bioMérieux, Marcy-l’Étoile, France) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS, Bruker, Billerica, MA, USA). K. michiganensis 2563 was tested for antimicrobial susceptibility with different antimicrobial agents, including piperacillin, gentamicin, imipenem, amoxicillin-clavulanic acid, cefotaxime, cefepime, ceftazidime, amikacin, ciprofloxacin, levofloxacin, moxifloxacin, trimethoprim-sulfamethoxazole, aztreonam, meropenem, piperacillin-tazobactam, ampicillin/sulbactam, cefazolin, chloramphenicol, and tetracycline. The Clinical and Laboratory Standards Institute (CLSI) 2021 guidelines were used for interpretation of the results. Antimicrobial susceptibility testing was quality-controlled using Escherichia coli (ATCC 25922).

The genome of K. michiganensis strain 2563 was sequenced using the 150  bp paired-end protocol on the HiSeq X10 platform (Illumina, San Diego, CA, USA) and the MinION platform (Nanopore, Oxford, UK). Unicycler v 0.5.013 was utilized for the hybrid assembly of both Illumina short-reads and Nanopore long-reads. The genome annotation was conducted employing the NCBI Prokaryotic Genome Annotation Pipeline. On January 19, 2023, we retrieved genome sequences and strain metadata of 577 K. michiganensis from the National Center for Biotechnology Information database. We conducted in silico multilocus sequence typing (MLST) analysis using our BacWGSTdb server.14 The NCBI AMRFinderPlus tool15 and VFDB database (www.mgc.ac.cn/VFs/) were utilized to conduct a search for antimicrobial resistance genes and virulence genes. Easyfig16 was utilized in order to conduct an analysis of the genetic context of antimicrobial resistance genes, while BRIG17 was used to compare the complete sequences of blaNDM-carrying plasmids. The global distributed K. michiganensis strains share the same sequence type with the K. michiganensis strain 2563 were selected for the phylogenetic analysis (Table S1). Snippy was used to construct phylogenetic trees based on recombination-free core genome SNPs (github.com/tseemann/snippy). rhierBAPS18 was used to define clusters of global ST43 K. michiganensis isolates. The interactive Tree of Life (iTOL) V5 web server19 was used to visualize and interpret the phylogenetic trees and display the presence/absence of different classes of antimicrobial resistance genes.

Results

The genome of K. michiganensis strain 2563 was assembled into six contigs, with a total length of 6,720,981 bp and an overall G+C content of 55.46%. The contig 1 consisting of 5,961,931 bp was recognized as the chromosome, while the remaining six contigs were associated with different plasmids at 362,448 bp, 183,276 bp, 105,902 bp, 54,035 bp and 53,269 bp, respectively. A total of 6130 protein-coding sequences, 84 tRNA genes, 118 rRNA genes, and one CRISPR array were identified by the PGAP server. K. michiganensis 2563 displayed a high degree of antimicrobial resistance to multiple β-lactam antibiotics, including piperacillin, amoxicillin-clavulanic acid, aztreonam, meropenem, imipenem, ampicillin, ampicillin/sulbactam, cefotaxime, cefazolin, cefepime, piperacillin-tazobactam and ceftazidime. However, it remained susceptible to amikacin, gentamicin, levofloxacin, ciprofloxacin, moxifloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline (Table 1). K. michiganensis 2563 harbored multiple antimicrobial resistance genes, including aminoglycosides [aph(3’)-Ia], carbapenem [blaNDM-1], ESBL [blaSHV-12], other β-lactams [blaOXY-1 and bleMBL], fosfomycin [fosA], and fluoroquinolones [oqxA10 and oqxB9]. Furthermore, multiple virulence genes have been identified on the chromosome of this strain, including entA, entB, fyuA, irp1, irp2, mgtB, ompA, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU and ybtX.

Table 1 Minimal Inhibitory Concentrations (MICs) to Different Antimicrobial Agents of K. michiganensis 2563

The blaNDM-1 gene was located on an IncX3 type plasmid (p2563-NDM). In addition to the resistance gene blaNDM-1, the IncX3 plasmid harbored the ESBL resistance gene blaSHV-12 and other β-lactam gene bleMBL (Figure 1A). Five insertion sequences (ISs), namely Tn3, IS3000, ISAba125, IS5 and IS30, were identified upstream of the blaNDM-1 gene, and the bleMBL, trpF, tat, IS91 and IS26 genes were detected downstream. Sequence comparison using BLAST showed that the p2563-NDM plasmid exhibited 100% query coverage and 100% identity with various blaNDM-bearing plasmids, such as pCQ17X3 from an E. cloacae strain CQ17, which was isolated from a sputum sample in China (NCBI GenBank accession number: CP103970.1); pNDM1_020049 in K. pneumoniae strain SCKP020049 from a human pus sample in China (NCBI GenBank accession number: CP028786.2) and pC46-NDM11 was identified in C. freundii strain C46 isolated from urine in China (NCBI GenBank accession number: MW269623.1) (Figure 1B).

Figure 1 Genetic organization of plasmids harboring the blaNDM gene. (A) Genetic environment of the blaNDM-1 flanking region in p2563-NDM. The red arrows represent the β-lactam resistance genes, the Orange arrows represent additional coding sequences (CDSs). (B) Circular comparison of p2563-NDM and IncX3 blaNDM carrying plasmid. The homology between these plasmids is shown by the percentage identity in the figure legend, whereas the absence of or a similarity value of <50% is indicated on the circular map as a white gap. The red text highlighted in the figure represent antimicrobial resistance genes.

According to an in silico MLST investigation, K. michiganensis strain 2563 belongs to ST43. To investigate the global genomic epidemiology of K. michiganensis strains, we analyze the phylogenetic relationships between K. michiganensis 2563 and other 27 ST43 K. michiganensis strains retrieved from the NCBI GenBank database. ST43 K. michiganensis isolated from Japan (n=7), Switzerland (n=6), Australia (n=5), China (n=5), the United States (n=4), and Germany (n=1). The most prevalent source of ST43 K. michiganensis (n=23, 82.14%) was clinical samples, followed by several samples from sink (n=2), livestock (n=1), soil (n=1), and serpentes (n=1). The isolation rate showed an increasing trend yearly, from only one strain in 2013 to ten strains in 2018 (Figure 2A). Phylogenetic analysis suggested that the global ST43 K. michiganensis could be divided into four clusters, and the strain 2563 isolated was located in cluster 1. The average SNP difference between these strains was 291 SNPs, whereas 16 pairs had differences of less than 21 SNPs (Figure 2B). These highly homologous strains appeared in the Chinese sink and Japanese human origins, but did not occur transnational or cross-host transmission. The closest relative strain of ST43 K. michiganensis strain 2563 was strain 12,084, which recovered from clinical specimens in China in 2013, with 171 SNPs differences. This strain only carried five antimicrobial resistance genes, ie, aph(3’)-Ia,blaOXY-1, fosA, oqxA10, and oqxB9. The range of antimicrobial resistance genes harbored by ST43 K. michiganensis ranged between 4 and 19, including aminoglycoside [ant(2”)-Ia (7.14%), aph(3’)-Ia (96.42%), aadA2 (28.57%), aph(3”)-Ib (3.57%), aac(6’)-31 (21.43%), aac(6’)-Ib (10.71%), aadA5 (3.57%), aph(6)-Id (3.57%), and aac(3)-IId (3.57%)], rifampin [arr-3 (3.57%)], ESBL [blaCTX-M-3 (10.71%), blaSHV-12 (10.71%)], carbapenems [blaIMP-1 (25%), blaIMP-4 (3.57%), blaKPC-2 (7.14%), and blaNDM-1 (10.71%)], other β-lactamase [blaCARB-2 (7.14%), blaFOX-5 (7.14%), blaOXA-1 (3.57%), blaOXY-1 (100%), blaTEM-1 (10.71%), bleMBL (10.71%)], chloramphenicol [catB11 (7.14%), catB3 (14.29%), cmlA6 (7.14%)], trimethoprim [dfrA19 (7.14%)], fosfomycin [fosA (100%)], macrolide [mph(E) (7.14%), msr(E) (7.14%)], quinolone [oqxA10 (100%), oqxB9 (100%), qnrA1 (7.14%), and qnrS1 (3.57%)], and sulfonamide [sul1 (42.96%), sul2 (3.57%)] (Figure 2A). All ST43 K. michiganensis strains carried the same virulence genes, namely entA, entB, fyuA, irp1, irp2, mgtB, ompA, yagZ/ecpA, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU and ybtX, except for strain SCKM090630 which lacks entA and entB.

Figure 2 Phylogenetic relationship of global ST43 K. michiganensis strains. (A) The phylogenetic tree was generated based on the differences of core genome single nucleotide polymorphisms, annotated by iTOL webserver. The figure provides information regarding the antimicrobial resistance genes, isolation source, time, and country of these strains. (B) Visualization heat map of the differences of core genome single nucleotide polymorphisms among global ST43 K. michiganensis strain. The number in the square indicates the number of core genome single nucleotide polymorphisms between strains. The source, country and cluster of strains are also colored.

Discussion

CRE is a serious challenge in the hospital settings, with high frequency of treatment failures due to a high resistance rate to various antimicrobial agents.20 In our investigation, we identified a blaNDM-1-carrying K. michiganensis strain and characterized its genomic characteristics. In addition, we conducted a global genomic epidemiological investigation of ST43 K. michiganensis to aid health departments in their efforts to monitor carbapenem-resistant pathogens more effectively. K. michiganensis 2563 was a multidrug-resistant strain, which was resistant to β-lactam antimicrobial agents, including meropenem and imipenem. Fortunately, our findings indicated that amikacin, gentamicin, levofloxacin, ciprofloxacin, moxifloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline can still be effectively used to treat the infection. A previous study indicated that despite harboring the fosA gene, the strain remained susceptible to fosfomycin, which was consistent with our findings.21 The carbapenemase gene can be transferred horizontally between antimicrobial-resistant and -susceptible strains via transposable element and conjugative plasmids. This is the primary factor contributing to the rising incidence of CRE infections in both hospital and community settings.22 The blaNDM-1 is located on an IncX3-type plasmid in K. michiganensis 2563, which is the main replicon type of blaNDM-carrying and blaKPC-carrying plasmid in China.23,24 We also investigated the genetic surroundings of blaNDM-1 and discovered an abundance of mobile genetic elements, which shared a highly conserved sequence structure to those described before.25,26 We discovered that the blaNDM-carrying IncX3 plasmid was highly similar among different species, indicating that the antimicrobial resistance determinant carried by the IncX3 plasmid may be capable of horizontal transmission between species, posing a significant danger to the prevention and management of nosocomial infection. Moreover, multiple virulence genes were also identified in K. michiganensis 2563, including enterobactin and yersiniabactin, which act as siderophores to promote bacterial invasion and infection of the host.27

Whole-genome sequencing offers useful data for epidemiological surveillance on account of its strong discriminatory power and low cost.28 Through phylogenetic analysis, we found that the global population of ST43 K. michiganensis population was expanding continuously since it was first discovered in a case reported in 2013. We hypothesized that for highly probable local transmission, the differences between isolates were ≤21 SNPs.7 The clonal transmission of ST43 K. michiganensis has occurred locally in Japan and China, which should attract the attention to avoid the expansion and development of transcontinental or trans-host transmission. K. michiganensis 2563 is most closely related to strain 12,084, which lacks the carbapenem resistance gene and the IncX3-type plasmid, implying that the acquisition of the blaNDM-1 gene by K. michiganensis 2563 may have occurred through horizontal transmission.

In summary, we characterized the genomic features of a K. michiganensis strain carrying blaNDM-1 from China and conducted an epidemiological study of ST43 K. michiganensis at the global scale. The implementation of an active surveillance programs to control the further transmission of this emerging multidrug resistant pathogen is an absolute requirement.

Data Sharing Statement

The complete genome sequence of K. michiganensis 2563 has been deposited into NCBI GenBank database under the accession numbers CP116214-CP116219. The data supporting the findings of this study are available within the article or its Supplementary Materials.

Institutional Review Board Statement

The study was performed in compliance with the Declaration of Helsinki and was approved by the Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (Approval No.:2022-227). Written informed consent was waived by the Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine as the study exclusively focused on bacteria.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This study was funded by grants from the Zhejiang Provincial Medical and Health Science and Technology plan (2022RC299, 2023KY119 and 2023XY088).

Disclosure

The authors declare no conflict of interest.

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