Coexistence of blaIMP−4 and blaSFO−1 in an IncHI5B plasmid harbored by tigecycline-non-susceptible Klebsiella variicola strain

Background Klebsiella variicola is considered a newly emerging human pathogen. Clinical isolates of carbapenemase and broad-spectrum β-lactamase-producing K. variicola remain relatively uncommon. A strain of K. variicola 4253 was isolated from a clinical sample, and was identified to carry the blaIMP−4 and blaSFO−1 genes. This study aims to discern its antibiotic resistance phenotype and genomic characteristics. Methods Species identification was conducted using MALDI-TOF/MS. PCR identification confirmed the presence of the blaIMP−4 and blaSFO−1 genes. Antibiotic resistance phenotype and genomic characteristics were detected by antimicrobial susceptibility testing and whole-genome sequencing. Plasmid characterization was carried out through S1-PFGE, conjugation experiments, Southern blot, and comparative genomic analysis. Results K. variicola 4253 belonged to ST347, and demonstrated resistance to broad-spectrum β-lactamase drugs and tigecycline while being insensitive to imipenem and meropenem. The blaIMP−4 and blaSFO−1 genes harbored on the plasmid p4253-imp. The replicon type of p4253-imp was identified as IncHI5B, representing a multidrug-resistant plasmid capable of horizontal transfer and mediating the dissemination of drug resistance. The blaIMP−4 gene was located on the In809-like integrative element (Intl1-blaIMP−4-aacA4-catB3), which circulates in Acinetobacter and Enterobacteriaceae. Conclusions This study reports the presence of a strain of K. variicola, which is insensitive to tigecycline, carrying a plasmid harboring blaIMP−4 and blaSFO−1. It is highly likely that the strain acquired this plasmid through horizontal transfer. The blaIMP−4 array (Intl1-blaIMP−4-aacA4-catB3) is also mobile in Acinetobacter and Enterobacteriaceae. So it is essential to enhance clinical awareness and conduct epidemiological surveillance on multidrug-resistant K. variicola, conjugative plasmids carrying blaIMP−4, and the In809 integrative element. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-024-00680-9.


Introduction
The Klebsiella pneumoniae complex is a member of the Klebsiella genus within the family Enterobacteriaceae, including Klebsiella pneumoniae, Klebsiella quasipneumoniae, and Klebsiella variicola.All members of this complex exhibit similar biochemical and phenotypic characteristics, making them indistinguishable using conventional microbiological methods [1,2].Furthermore, due to the inability of current microbiological automated detection systems to effectively differentiate Klebsiella species, there is a possibility of misidentifying certain isolates of K. variicola as K. pneumoniae in clinical settings [3][4][5], leading to an underestimation of the clinical prevalence of K. variicola.K. variicola was first described in 2004 and is considered a newly emerging pathogen in humans [6].It is a gram-negative, facultative anaerobic, non-spore-forming, and non-motile rod-shaped bacterium that forms circular, convex, and smooth colonies [7].In recent years, with the updating of the MALDI-TOF database and the widespread use of genomic sequencing, there has been an increasing number of reports on isolates of K. variicola, including the emergence of highly virulent K. variicola strains resistant to colistin [8], the presence of a hypervirulent conjugative plasmid in K. variicola [9], and the coexistence of bla NDM−1 and bla IMP−4 genes on one plasmid in K. variicola [10,11].Compared to K. pneumoniae, typically, K. variicola isolates exhibit lower antibiotic resistance rates [12], but studies have shown a high mortality rate associated with bloodstream infections caused by multidrug-resistant and extended-spectrum β-lactamaseproducing K. variicola [13,14].Therefore, it is crucial to pay close attention to and monitor multidrug-resistant and extended-spectrum β-lactamase-producing K. variicola.In this study, the K. variicola 4253 strain was isolated from a stool sample of a female leukemia patient in a teaching hospital.The strain was identified to carry the bla IMP−4 and bla SFO−1 genes through PCR, and its antibiotic resistance phenotype and genomic characteristics were extensively characterized.

Species identification and antimicrobial susceptibility testing
During routine monitoring targeting at carbapenemresistant Enterobacteriaceae (CRE) in Hangzhou, Zhejiang Province, China, K. variicola 4253 was collected from a stool sample of a female leukemia patient at the First Affiliated Hospital of Zhejiang University.Species identification was conducted using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonik GmbH, Bremen, Germany), while PCR was employed to identify the presence of the carbapenemase gene bla IMP−4 and the broad-spectrum β-lactamase gene bla SFO−1 (The primer sequences were showed in Table S1).
Agar dilution method and microbroth dilution method were utilized for antimicrobial susceptibility testing (AST), with Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 serving as the quality control strain.AST results were interpreted according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI 2020) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2020).

Plasmid characterization and conjugation experiment
The number and size of plasmids in K. variicola 4253 were assessed using S1-PFGE.The location of bla IMP−4 and bla SFO−1 on the plasmid was determined through Southern blotting and gene hybridization with a digoxigenin-labeled specific probe (The probe sequences were showed in Table S1).To investigate the transferability of the plasmid, E. coli J53 was employed as the recipient strain in conjugation experiments.Transconjugants were selected using Mueller-Hinton agar (OXOID, Hampshire, United Kingdom) supplemented with 200 mg/L NaN3 and 8 mg/L cefepime.Their identification as E. coli and the presence of the bla IMP−4 and bla SFO−1 genes were confirmed through PCR and further validated using MALDI-TOF/MS.

Whole genome sequencing and analysis
Genomic DNA was extracted using the Genomic DNA Isolation Kit (QIAGEN, Hilden, Germany), and sequencing was performed using the Illumina Novaseq6000 platform (Illumina, San Diego, CA, USA) and the Oxford Nanopore platforms (Oxford Nanopore Technologies, Oxford, United Kingdom).Following sequencing, the short and long reads were subjected to hybrid assembly using Unicycler v0.4.7 to obtain the complete genome sequence.Annotation was carried out using the RAST 2.0 and Prokka tools.Kleborate v2.0.1 and Kaptive v0.7.3 were utilized for subspecies, MLST, wzi allele, capsule (K), and O antigen (LPS) serotype analysis.Resistance genes were identified using Resfinder.Virulence genes were obtained from the VFDB website, while plasmid replicon types were determined using PlasmidFinder 2.1 and KpVR.Integrons and insertion sequence elements were disclosed using INTEGRALL and ISfinder, respectively.Comparative analysis of different plasmid genome sequences was performed using the BLAST Ring Image Generator (BRIG).Then, the genetic context encompassing the bla IMP−4 and bla SFO−1 genes was visualized using Easyfig 2.3.

Isolation of K. Variicola 4253 strain and antimicrobial susceptibility testing
The K. variicola 4253 was recovered from a fecal sample of a 22-year-old female patient with acute lymphocytic leukemia on November 8th, 2021.During hospitalization, the patient experienced high fever and received intravenous administration of cefoperazone sodium, sulbactam sodium, and vancomycin for treatment.After improvement of symptoms, the patient was discharged.The strain was identified as K. variicola using MALDI-TOF/MS, and further characterization through third-generation sequencing, using the Kleborate tool, confirmed it as K. variicola subsp.variicola.PCR and sequencing revealed the presence of the bla IMP−4 and bla SFO−1 genes.
As shown in Table 1, the antimicrobial susceptibility results demonstrated that both the strain and its transmissible conjugate exhibited resistance to broad-spectrum β-lactam drugs, including ceftriaxone, cefotaxime, ceftazidime, cefepime, aztreonam, piperacillin/tazobactam, and amoxicillin-clavulanic acid.However, they were susceptible to meropenem and imipenem.They also showed sensitivity to quinolones (levofloxacin, ciprofloxacin), sulfonamides (trimethoprim/sulfamethoxazole), and aminoglycosides (except for gentamicin, which showed resistance but amikacin showed sensitivity).Both the strain and its conjugate exhibited sensitivity to colistin.K. variicola 4253 demonstrated resistance to fosfomycin and tigecycline, while its conjugate remained susceptible to them.

Discussion
The antimicrobial susceptibility test indicated that K. variicola 4253 exhibited.insensitivity to tigecycline (MIC 8 mg/L).Through whole-genome analysis, no plasmid-mediated tet(X) gene encoding tetracycline inactivation enzyme or resistance-nodulation-division (RND) genes conferring tigecycline resistance were identified [15,16].Additionally, no conjugate conferring tigecycline insensitivity were obtained in the conjugation experiment.Therefore, it is postulated that the insensitivity of this strain to tigecycline is mediated by chromosomally carried genes.Previously reported mechanisms of tigecycline resistance in K. pneumoniae included mutations in the ribosomal protein S10 encoding gene rpsJ, which mediates tigecycline resistance [17], the involvement of the efflux pump OqxAB in low to moderate levels of tigecycline resistance [18], overexpression of the AcrAB efflux pump affecting sensitivity to tigecycline in K. pneumoniae [19][20][21], and mutations, insertions, deletions, and upregulation of transcriptional regulatory factors and related genes such as rarA, acrR, ramA, ramR, marA, and soxS, which can regulate the expression levels of the OqxAB and AcrAB efflux pumps and influence sensitivity to tigecycline [22][23][24].In this study, the chromosomal genome of the strain contained the efflux pump genes acrAB-tolC and oqxAB.However, whether the tigecycline-insensitive phenotype in this strain is caused by overexpression of the efflux pump or mutations in related genes remains to be determined and requires further experimentation.
Although the isolated strain K. variicola 4253 produces carbapenemase, the antimicrobial susceptibility test revealed its sensitivity to imipenem and meropenem (MIC 1 mg/L).Previous literature reports have shown heterogeneity in the sensitivity of Enterobacteriaceae isolates carrying bla IMP−4 gene to carbapenems.For instance, Yang et al. reported that most of the 25 IMPproducing K. pneumoniae (IMPKp) isolates (including 16 IMP-4 strains) collected from a teaching hospital in China between 2009 and 2016 exhibited low levels of resistance and susceptibility to various carbapenem drugs [25].Among them, three strains demonstrated intermediate to high-level resistance (MIC ≥ 8 mg/L) to imipenem and meropenem.Qiao et al. reported a Enterobacter hormaechei strain carrying bla IMP−4 , which showed intermediate susceptibility to imipenem and sensitivity to meropenem [26].Additionally, K. pneumoniae 1220, isolated from a three-month-old infant's blood specimen and carrying the bla IMP−4 gene on a plasmid, was capable of conjugation into E. coli EC600.The antimicrobial susceptibility results indicated that both the strain and its conjugative form exhibited resistance to imipenem and meropenem (MIC 8 mg/L) [27].It is speculated that IMP-4 hydrolyzing enzymes may have weak hydrolytic capabilities against carbapenems, and the production of IMP-4 is not the sole determinant for high-level carbapenem resistance.Other resistance mechanisms, including overexpression of efflux systems, porin protein deficiencies, and the presence of other broad-spectrum β-lactamases, may contribute to this process [28].Currently, the extent to which different resistance mechanisms impact the carbapenem-resistant phenotype in Enterobacteriaceae strains carrying bla IMP−4 remains unclear.
The genetic context of bla IMP−4 in K. variicola 4253 was represented by Intl1-bla IMP−4 -aacA4-catB3, which is similar to the integrative element In809 (Intl1-bla IMP−4 -qacG-aacA4-catB3).It is possible that the qacG gene cassette was lost during the dissemination process.In809 was first described in Acinetobacter species isolated from Hong Kong between 1997 and 2000 [29] and has been subsequently reported in various Acinetobacter [30]and Enterobacteriaceae isolates [11,25,31].This suggests that the bla IMP−4 array may be mobilized in Acinetobacter and Enterobacteriaceae through homologous recombination or plasmid-mediated horizontal transfer.Plasmid-mediated dissemination of carbapenemase-encoding genes plays a significant role in the emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE).Previously reported incompatible plasmid types carrying the bla IMP gene include HI2/HI5, FI/FII, L/M, A/C, P, and N [32,33].However, in this study, the plasmid carrying bla IMP−4 , p4253-imp, belonged to the IncHI5B type.IncHI plasmids are conjugative plasmids typically larger than 200 kb, serving as important vehicles for the dissemination of heavy metal resistance genes and antibiotic resistance genes.They are considered broad-host-range plasmids [34].The conjugation experiments in this study also confirmed that p4253-imp was a conjugative plasmid capable of mediating horizontal transfer of resistance genes.BLAST search revealed an extremely high similarity between p4253-imp and the plasmid pNDM-IMP-1 isolated from K. variicola SHET-01 in Shanghai [11].Given the geographic proximity between Shanghai and Hangzhou, it raises the possibility of clonal dissemination.Based on multilocus sequence typing (MLST), K. variicola SHET-01 belonged to ST3936, while K. variicola 4253 in this study belonged to ST347.Therefore, it is inferred that the strain isolated in this study is more likely to have been acquired through plasmid-mediated horizontal transfer.

Conclusion
We have reported the presence of a strain of K. variicola, which was insensitive to tigecycline, carrying a plasmid harboring bla IMP−4 and bla SFO−1 .We have provided a detailed exposition of its drug resistance phenotype and genomic characteristics.This strain was classified as a multidrug-resistant strain, and its one plasmid coexisting bla IMP−4 and bla SFO−1 , possessed the ability of horizontal transfer, thereby mediating the dissemination of drug resistance.Additionally, it is highly probable that K. variicola 4253 acquired this plasmid through plasmidmediated horizontal transfer.Furthermore, the bla IMP−4 array (Intl1-bla IMP−4 -aacA4-catB3) is also mobile in Acinetobacter and Enterobacteriaceae.Thus, it is imperative to enhance clinical awareness and conduct epidemiological surveillance in this regard.

Table 1
MIC values of antimicrobials for K. variicola 4253, transconjugant 4253-J53, and recipient strain J53 R: resistant; S: susceptible a Tazobactam at a fixed concentration of 4 mg/L

Table 2
Antibiotic resistance genes of K.variicola 4253