Global Emergence and Genomic Epidemiology of bla_NDM-Carrying Klebsiella variicola

Introduction

Klebsiella variicola is a gram-negative, facultative anaerobe, nonsporogenic, and nonmotile rod-shaped bacterium that forms round, convex, and smooth colonies.¹ Initially discovered in bananas in 2004, K. variicola is commonly present in agriculturally sourced soils, plants, freshwater, and sewage.² It frequently contributes to nitrogen fixation and promotes plant growth. Moreover, it is recognized as a commensal of insects and can act as a pathogen for both plants and animals.^3–5 Belonging to the Klebsiella genus, K. variicola has historically been prone to misidentification as Klebsiella pneumoniae through traditional biochemical methods. This challenge persisted until the adoption of advanced techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and whole-genome sequencing for accurate species identification.^6,7 The past misidentification, particularly within the K. pneumoniae complex, has hindered comprehensive research on K. variicola within the healthcare system.

K. variicola has emerged as a noteworthy human pathogen, witnessing a consistent rise in clinical infections in recent years.⁸ Reports have extensively documented infections involving isolates from various sources, including fecal, blood, sputum, vaginal, and urine samples.^9,10 Notably, hypermucoviscous strains of K. variicola were initially identified in 2015, intensifying the concerns surrounding its clinical impact.^11,12 Adding to these apprehensions, a study highlighted that among hospitalized adults with bloodstream infections, the mortality rate associated with K. variicola exceeded that linked to K. pneumoniae.¹³ This underscores the increasing significance of K. variicola as a clinically relevant pathogen, prompting a closer examination of its implications in healthcare settings.

In this investigation, the K. variicola strain 353, which carries the bla_NDM-1 gene, was isolated from a urinary tract infection in a male patient hospitalized in the department of rehabilitation of a teaching hospital in China. The isolate was preliminarily identified using the VITEK MS system (bioMérieux, France) and was further confirmed by whole-genome sequencing. Bioinformatics analysis was undertaken to delve into the genetic characteristics of both the strain and the plasmid carrying the bla_NDM-1 gene. The global transmission dynamics and genomic epidemiology of bla_NDM-carrying K. variicola were further investigated.

Materials and Methods

Antimicrobial Susceptibility Test

Antimicrobial susceptibility testing was performed using the VITEK 2 system (bioMérieux, France) with Gram-negative antimicrobial susceptibility testing cards (AST-GN13) and the Etest method. The testing procedures adhered to the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) M100, 33rd edition. Breakpoints were interpreted in accordance with the recommendations outlined in the CLSI guidelines. In cases where CLSI breakpoints were unavailable for colistin and tigecycline, interpretations for colistin minimum inhibitory concentration (MIC) followed the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), while standards set by the US Food and Drug Administration (FDA) were utilized for tigecycline.

Whole-Genome Sequencing

Whole-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×. Additionally, long-read sequencing was performed using a Nanopore MinION sequencer (Nanopore, Oxford, UK). Both short Illumina reads and long MinION reads underwent hybrid assembly using Unicycler (v0.4.7) in the conservative mode. This process resulted in complete circular contigs, which underwent further refinement and correction using Pilon with Illumina reads through multiple rounds of iteration until no further changes were detected. The resultant complete genome sequence was subsequently automatically annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) server.

Genomic Features and Plasmid Characterization

The investigation into the antimicrobial resistance genes and plasmid replicons of the strain was carried out using the BacWGSTdb server.^14,15 MLST (Multi Locus Sequence Typing) analysis was conducted utilizing the database available at http://mlstkv.insp.mx/.¹⁶ Circular comparisons, represented by concentric rings, were performed to examine the bla_NDM-1-carrying plasmid and its similarity to analogous plasmids. This comparative analysis was visualized using the BLAST Ring Image Generator (BRIG).¹⁷

Phylogenetic Analysis

The phylogenetic relationship between K. variicola 353 and other K. variicola strains obtained from the NCBI GenBank database was assessed using the BacWGSTdb server.^14,15 This server employs single nucleotide polymorphism (SNP) approaches to analyze the phylogenetic relationship of the uploaded genome sequence with sequences available in the database. The resulting phylogenetic tree was enhanced for visual clarity using iTOL.¹⁸

Nucleotide Sequence Accession Numbers

The complete genome sequence of K.variicola 353 has been submitted to the NCBI GenBank database and is assigned the accession number CP141632-CP141634.

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).

Results and Discussion

K. variicola strain 353 was isolated from a urine sample obtained from a male patient who was admitted to a tertiary hospital in China following a cerebral hemorrhage. The genome of the K. variicola strain 353 is composed of three contigs, totaling 6,213,895 bp. Notably, one of these contigs, designated as contig 1 and spanning 5,727,068 bp, is associated with the chromosome, while the remaining two contigs correspond to plasmids (contig pCRKP353-NDM1: 294,608 bp and contig 3: 192,219 bp). The genome harbors two distinct plasmid replicons: one on plasmid pCRKP353-NDM1 (IncU) and another on contig 3 (IncFIB(K)). Analysis conducted through the PGAP server yielded predictions for 6001 protein-coding sequences, 88 tRNA genes, and 25 rRNA operons.

The antibiotic susceptibility profiles, as depicted in Table S1, reveal that K. variicola 353 exhibited resistance to a broad spectrum of antibiotics, including ceftazidime, ceftriaxone, cefotetan, cefazolin, cefepime, ciprofloxacin, levofloxacin, ertapenem, imipenem, meropenem, ampicillin/sulbactam, and sulfamethoxazole/trimethoprim. However, it demonstrated susceptibility to amikacin, gentamicin, aztreonam, tigecycline, and colistin. Table 1 outlines the resistance genes identified in the genome of the isolate. Notably, β-lactam resistance genes bla_LEN-16 and bla_NDM-1 were identified, along with aminoglycoside resistance genes aph(3’)-Ia, aadA16, aadA2 and aac(6’)-Ib-cr, fosfomycin resistance gene fosA, quinolone resistance genes oqxB, oqxA, and qnrS1, trimethoprim resistance gene dfrA27, rifamycin resistance gene arr-3, and sulfonamide resistance gene sul1. Except for oqxA, oqxB, bla_LEN-16, and fosA, which are located on the chromosome, all other resistance genes, including bla_NDM-1, are situated on plasmid pCRKP353-NDM1.

Table 1 Antimicrobial Resistance Genes in Klebsiella Variicola 353

The plasmid pCRKP353-NDM1, carrying the bla_NDM gene, was identified as an IncU-type plasmid. Two copies of IS26 are situated upstream of bla_NDM-1, while two ISCR1 (insertion sequence common region 1) elements flank bla_NDM-1. Downstream of bla_NDM-1, an Intl1 (Class 1 Integron) is present, housing numerous antibiotic resistance genes such as sul1, aadA16, dfrA27, arr-3 and aac(6’)-Ib-cr. IS26 typically plays a pivotal role in disseminating antibiotic resistance genes within Gram-negative bacteria, contributing to their widespread distribution.^19–21 Additionally, ISCR1 is closely associated with various antibiotic resistance determinants, underscoring its significance in this context.²² This implies that both IS26 and ISCR1 are crucial players in the propagation of bla_NDM. The presence of Intl1 (Class 1 Integron) downstream of bla_NDM-1 enables the plasmid to harbor multiple drug-resistant genes, transforming it into a multi-drug-resistant plasmid. This emphasizes the role of Intl1 in facilitating the accumulation of diverse drug resistance determinants within the plasmid.

Comparative analysis of pCRKP353-NDM1 with similar plasmids obtained from the NCBI database was performed using the Basic Local Alignment Search Tool (Figure 1). These plasmids, all falling under the IncU type, include three that harbor the bla_NDM gene (Table S2). IncU plasmids are distinguished for their extensive host ranges, enabling the efficient transmission of antibiotic resistance genes owing to their robust binding and mobility features.²³ Notably, characterized by their high binding and mobility attributes, IncU plasmids demonstrate resilience and adept replication capabilities across diverse bacterial species. Consequently, they serve as crucial vectors in the dissemination of antibiotic resistance genes. It is noteworthy that IncU plasmids carrying bla_NDM-1 genes have begun to proliferate across diverse genera, encompassing Raoultella ornithinolytica, Enterobacter cloacae, and K. variicola.

Figure 1 Circular comparative analysis plasmid pCRKP353-NDM1 with similar plasmids retrieved from the NCBI database, including pKO41-SIM (Klebsiella michiganensis strain KM41, accession no. CP090080), pK92-qnrS (Klebsiella michiganensis strain K92, accession no. OL828743), pRes_C1672 (Klebsiella pneumoniae strain C1672, accession no. CP073918), pKp6001 (Klebsiella pneumoniae strain Kp6, accession no. CP082291), p7_SCLZS62 (Raoultella planticola strain SCLZS62, accession no. CP082175), pM27-NDM-363K (Raoultella ornithinolytica strain RoM27LC23, accession no. CP130154), pC5889_NDM (Enterobacter cloacae strain C5889, accession no. MZ532978), and pJNQH579-2 (Klebsiella variicola strain JNQH579, accession no. CP078148).

In order to gain a more profound understanding of global transmission dynamics and genomic epidemiology of bla_NDM-carrying K. variicola, an extensive search was conducted within the NCBI pathogen database for strains harboring the bla_NDM gene until December 1, 2023. A total of 72 bla_NDM-carrying K. variicola strains were identified from the NCBI database (Table 2). These strains were discovered in 12 countries worldwide, with the highest prevalence observed in the USA (32 strains), followed by China (18 strains), Bangladesh (4 strains), and three strains each from the United Kingdom, Switzerland, and South Korea. Additionally, two strains each were found in Australia and Brazil, as well as one strain each from Lebanon, Germany and Canada. This global distribution highlights the widespread dissemination of bla_NDM-carrying K. variicola. Among these strains, five sub-types of bla_NDM were identified: 38 strains carried bla_NDM-1, 13 strains carried bla_NDM-5, 11 strains carried bla_NDM-4, 3 strains carried bla_NDM-9, and 1 strain carried bla_NDM-18. According to the MLST (Multi Locus Sequence Typing) results available at http://mlstkv.insp.mx/,¹⁶ KV353 is assigned to a newly identified, unnamed sequence type (ST) distinguished by leuS60, pgi39, pgk1, phoE29, pyrG1, rpoB1 and fusA4. Among the 72 bla_NDM-carrying K. variicola strains, excluding 20 strains with unclassified sequence types, a total of 28 distinct ST types were identified (Figure 2). ST115 was the most prevalent, comprising 12 strains, followed by ST60 (4 strains), ST10 (3 strains), ST20 (3 strains), ST64 (3 strains), and ST277 (3 strains). Phylogenetic analysis revealed that K. variicola 353 belongs to a distinct clone. Furthermore, eleven strains originating from the United States were identified to be part of a single clone, exhibiting single nucleotide polymorphism (SNP) differences of ≤20. This observation suggests that clonal transmission, possibly associated with nosocomial infection, could be contributing to the epidemic of bla_NDM-carrying K. variicola.

Table 2 Clinical Metadata for 72 Klebsiella Variicola Strains Carrying blaNDM Retrieved from the NCBI Database

Figure 2 Phylogenetic tree of K. variicola 353, K. variicola ATCC BAA-830 and other blaNDM-carrying K. variicola strains retrieved from the NCBI database. Cells with different colors indicate the presence of different antimicrobial resistance genes, whereas blank cells indicate the absence of the gene. The color of each rectangular indicates a specific country.

Relevant analyses were conducted on the bla_NDM-carrying plasmid. Subsequent investigation into these bla_NDM-carrying strains, among which 34 had complete genome sequences, revealed that all bla_NDM-carrying plasmids were located on plasmids. Specifically, 13 were classified as IncX3 type, 10 as IncA/C2 type, 5 as IncFII type, 3 strains as IncHI1B type, with one each belonging to IncFIA, IncFIB, and IncN types. The occurrence of IncU type bla_NDM-carrying plasmid in K. variicola is uncommon.

Remarkably, seven strains from distinct regions were identified to carry multiple carbapenemases, with three strains co-harboring bla_NDM and bla_IMP, and four strains co-harboring bla_NDM and bla_KPC. Over the past decade, there has been a substantial increase in the proportion of K. pneumoniae strains carrying multiple-carbapenemase genes, presenting an elevated threat to public health.²⁴ The current study suggests that K. variicola strains carrying multiple-carbapenemase genes are also undergoing global dissemination. The existence and prevalence of K. variicola with multiple carbapenemases pose formidable challenges for clinical treatment.

Among the 72 strains of K. variicola obtained from NCBI, there was a notably higher prevalence in urine (18/72), blood (10/72), and sputum (6/72) samples. Additionally, K. variicola strains were detected in environmental sewage and fields. It is noteworthy, that one strain carrying the bla_NDM-1 gene was detected in hospital wastewater in China, and three strains carrying the bla_NDM-9 gene from South Korea were found in river water, while the remaining strains were of clinical origin. This implies that bacteria carrying the bla_NDM gene have transferred between clinical settings and the natural environment.

In summary, our findings reveal the global dissemination of K. variicola carrying the bla_NDM gene in both healthcare settings and natural environments. These data have unveiled a diverse array of antimicrobial resistance determinants in K. variicola, offering valuable insights that may contribute to the development of strategies for the prevention, diagnosis, and treatment of K. variicola infections.