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 blaIMP−4 and blaSFO−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.

Genomic characteristics of K. variicola 4253 were explored through whole-genome sequencing. The genomic sequence analysis revealed that the isolated strain belongs to ST347 (gapA-infB-mdh-pgi-phoE-rpoB-tonB, allele no. 16-24-21-27-47-22-67). The capsule serotype was identified as wzi269/KL25/K25, and the O antigen serotype was classified as OL5/O5. The strain possessed a circular chromosome with a size of 5,533,843 bp and three plasmids, namely p4253-imp, p4253-2, and p4253-3. The sizes of the plasmids were 334,271 bp, 175,117 bp, and 4,655 bp, respectively (p4253-3 is not visible in S1-PFGE due to its small size) (Fig. 1). The average G + C content of the chromosome and plasmids were 57.3%, 48.7%, 51.5%, and 42.8%, respectively. Notably, the blaIMP−4 and blaSFO−1 genes were located on the same plasmid (p4253-imp). There were 2,651 protein-coding genes, 171 tRNA genes, and 61 rRNA genes on the chromosome. (Table S2).

Plasmid profiles of K. variicola 4253. Plasmid size determination by S1-PFGE, with Salmonella enterica serotype Braenderup H9812 as the size marker. Southern blotting hybridization with the blaSFO−1-specific probe and blaIMP−4-specific probe

A total of nine different antimicrobial resistance genes were identified in the strain, including beta-lactamase genes (blaOXA−1, blaIMP−4, blaSFO−1, blaTEM−214, blaLEN−25), aminoglycoside resistance genes (aac [3]-IId, aac(6’)-Ib-cr, aac(6’)-Ib4, aph [6]-Id, and aph(3’’)-Ib), macrolide resistance genes (mphA, mphE, and msrE), phenicol resistance gene (catB3), rifampicin resistance gene (arr-3), sulfonamide resistance gene (sul1), tetracycline resistance gene (tet(D)), fosfomycin resistance gene (fosA), and efflux pump-associated genes (oqxA and oqxB), of which seven were encoded on plasmid p4253-imp (Table 2), indicating that p4253-imp is a multidrug-resistant plasmid.

Furthermore, a total of 56 virulence genes were identified in the strain (Table S3). The chromosome harbored genes such as type 3 fimbriae (mrkABCDFHIJ), type 1 fimbriae (fimABCDEFGHIK), fimbrial adherence determinants (stcB, stcC), efflux pump (acrA, acrB), aerobactin (iutA), enterobactin siderophore (entABCDEFS, fepABCDG, fes), salmochelin (iroE), magnesium uptake (mgtB), regulation (rcsA, rcsB), T6SS-I (tssJGFM), T6SS-II (clpV), T6SS-III (dotU, icmF, impJGHFA, ompA, sciN, vgrG). The plasmid p4253-2 carried the T6SS-I gene (tssH), while no virulence genes were found on other plasmids.

The size of the p4253-imp plasmid was 334,271 bp. It encoded 318 protein-coding genes and had a G + C content of 48.7%. No hits were found in the Plasmidfinder database for plasmid typing; however, analysis using the KpVR online tool revealed that the p4253-imp replicon belonged to the IncHI5B subtype. Based on this information, it can be inferred that the plasmid is a multidrug-resistant plasmid, carrying carbapenemase gene blaIMP−4 and broad-spectrum β-lactamase gene blaSFO−1. Consistent with the genomic features, S1-PFGE and Southern blot confirmed the size of the plasmid and the presence of blaIMP−4 and blaSFO−1 on the p4253-imp plasmid (Fig. 1). BLASTN search showed a high similarity between p4253-imp and pNDM-IMP-1 (CP050681.1), pKOX7525_1 (CP065475.1), pWH11 (ON882017.1), pIMP4-KP294 (CP083446.1), pKP1814-1 (KX839207.1), pA (CP068445.1), p2019SCSN059_tmexCD (ON169978.1), and pFK2020ZBJ35_tmexCD (ON169979.1) (More details about the 8 plasmids are showed in Table S4), with identity and coverage both exceeding 90%. Among these, p4253-imp exhibited higher similarity to pNDM-IMP-1, with 99% identity and 100% query coverage. pNDM-IMP-1 is a plasmid carrying blaIMP−4 from a urine catheter isolate of K. variicola SHET-01, obtained from a 2-year-old girl in the pediatric intensive care unit of a teaching hospital in Shanghai, China [11]. Gene comparison analysis (Fig. 2) revealed that p4253-imp and pNDM-IMP-1 share a similar plasmid backbone, including resistance determinants, insertion elements, conjugative transfer gene clusters, and related functional genes. Similar to the pNDM-IMP-1 plasmid, blaIMP−4 was carried by a Tn6406-like composite transposon, flanked by IS5075 elements, and containing an In809-like integron (Intl1-blaIMP−4-aacA4-catB3). The upstream sequence was identical to Tn5393-blaSFO−1-ampR-IS5075, where Tn5393 harbored aph [6]-Id and aph(3’’)-Ib resistance genes. Furthermore, conjugation experiments demonstrated the transferability of p4253-imp, and the antimicrobial susceptibility results indicated that this plasmid mediated the spread of resistance.

The genomic analysis of K. variicola 4253. (A) Comparative analysis of plasmid p4253-imp with other eight plasmids.. (B) The genetic context of blaSFO−1 and blaIMP−4 genes on p4253-imp, pNDM-IMP-1 and pKox7525_1