The K. pneumoniae strain KPN710429 was isolated from a blood sample from a 69-year-old woman with duodenal cancer at the Second Hospital of Anhui Medical University in Anhui, China, in June 2020. This bloodstream infection was hospital-acquired, and the patient was not treated with polymyxin B or colistin before this strain was isolated. The species was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker, Bremen, Germany).

In vitro antimicrobial susceptibility tests (ASTs) were performed for amikacin, aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftazidime-avibactam, ceftriaxone, cefuroxime, ciprofloxacin, colistin, ertapenem, gentamicin, imipenem, levofloxacin, meropenem, piperacillin-tazobactam, ticarcillin-clavulanic acid, tigecycline, and tobramycin using the broth microdilution method. The susceptibility breakpoints of tigecycline and colistin were interpreted in according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint (EUCAST, 2020); all other antimicrobial agents were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2020). The standard Escherichia coli strain ATCC25922 was used for quality control.

The hypermucoviscous phenotype was detected using the string test as described previously (Mazloum et al., 2016). The virulence of KPN710429 were assessed by serum-killing and Galleria mellonella lethality assays according to previously described (Liu et al., 2021). For references, a hypervirulent K. pneumoniae (designated as KPN54798^H–ctrl) and classic K. pneumoniae (designated as KPN49^L–ctrl) were employed as hypervirulence and low-virulence control, respectively (Liu Z. et al., 2019b).

The total genomic DNA of strain KPN710429 was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Whole-genome sequencing (WGS) and assembly were performed as previously described (Liu et al., 2021). Virulence associated genes were predicted using VFDB¹ (Liu B. et al., 2019a). Antimicrobial resistance genes (ARGs) were identified by using Resfinder² (Bortolaia et al., 2020). The multilocus sequence type (MLST) was identified by submitting the genome sequence to MLST 2.0 (Larsen et al., 2012). Capsular serotyping was achieved by submitting the genome sequence to Kaptive Web³ (Wyres et al., 2016). Each plasmid identified in this study was analyzed using PlasmidFinder⁴ and pMLST⁵ to investigate the replicons (Carattoli et al., 2014), and sequences were compared using BLAST analysis and the BLAST Ring Image Generator (Alikhan et al., 2011). Sequence alignments for the genetic environments of bla_NDM–1 and mcr-9 were performed using BLAST and visualized with Easyfig v 2.2.3 (Sullivan et al., 2011).

The transferability of the bla_NDM–1 and mcr-9 genes was assessed using conjugation as previously described (Ai et al., 2021). Approximately 1 × 10⁸ colony-forming units (CFU)/mL of the donor strain (KPN710429) and the recipient strain (sodium azide-resistant E. coli J53) were mixed at 1:1 ratio and spotted onto filter membrane, which were placed on Muller-Hinton (MH) agar plates and incubated for 18–24 h at 37°C. Transconjugants were selected on MH agar containing both sodium azide (300 μg/mL) and ceftriaxone (32 μg/mL). Presence of bla_NDM–1 and mcr-9 in the transconjugants (designated as J53-KPN710429) was confirmed by PCR and sequencing analysis, Supplementary Table 1 lists the primers sequences. The minimum inhibitory concentrations (MICs) of antimicrobial agents for the transconjugants were assessed using the broth microdilution method.

Colistin induction assays were performed as previously described with some modifications (Kieffer et al., 2019). KPN710429 were inoculated in MH broth supplemented with colistin (0.5, 1.0, and 2.0 μg/mL) or without colistin at final bacterial suspensions of 1.0 MCF, respectively. After shaking at 37°C for 6 h, the strains were enriched by centrifugation and washed three times with sterile saline, then 0.5 MCF bacterial suspensions were prepared for colistin susceptibility testing and mRNA extraction. The relative expression level of mcr-9 and IncHI2-repA were determined by quantitative real-time (qRT)-PCR as described previously (Pfaffl, 2001). Expression of the K. pneumoniae rpoB housekeeping gene was used to normalize the transcript levels.

Additionally, the mgrB gene of induced strains was amplified and sequenced as previously described (Haeili et al., 2017), then the above sequences were aligned with that of the original strain to determine whether the mgrB was mutated during the colistin induction assay. Supplementary Table 1 lists the primers sequences.

The Shapiro–Wilk method was used to test normality. Normally data are presented as means ± standard deviations. Bliss method were used to calculated the 50% lethal dose (LD₅₀) of G. mellonella larvae (72 h after infection), and the LD₅₀ values were expressed as log₁₀(lg) transformed values. Statistical analysis was performed with GraphPad Prism (San Diego, CA, United States) using unpaired Student’s t-tests. P-values of <0.05 was considered statistically significant.

The K. pneumoniae KPN710429 genome sequence was submitted to GenBank under accession numbers CP073657 (plasmid pK710429_1), CP073658 (plasmid pK710429_2), CP073659 (plasmid pK710429_3), and CP073660 (chromosome of KPN710429).

The K. pneumoniae strain KPN710429 was isolated from a clinical specimen, generated as part of routine clinical laboratory procedure. The Ethics Committee of The Second Hospital of Anhui Medical University exempted this study from review because it focused only on bacteria.

Strain KPN710429 was identified as K. pneumoniae using MALDI-TOF-MS, and its string test was negative. In vitro AST results showed that strain KPN710429 was resistant to all cephalosporins, carbapenems, β-lactam/β-lactamase inhibitors, aminoglycosides, and quinolones, but susceptible to aztreonam, tigecycline and colistin (Table 1). The serum-killing assays demonstrated that KPN710429 was serum intermediate sensitive (grade 3) (Supplementary Figure 1). The lgLD₅₀ of G. mellonella larvae due to KPN710429 (5.54 ± 0.16) was higher than that due to KPN54798^H–ctrl (4.93 ± 0.08) (P = 0.0043), and there was no significant different from that due to KPN49^L–ctrl (5.86 ± 0.19) (P = 0.0907).

Whole-genome sequencing revealed that the genome of K. pneumoniae strain KPN710429 comprised a single chromosome (5,124,764 bp) and three plasmids ranging in size from 70,448 bp to 288,244 bp (Table 2). In silico analysis assigned this isolate to sequence type (ST) 1308 and serotype KL144. ResFinder analysis was performed to identify a number of ARGs. Among them, bla_OKP, fosA, oqxA, and oqxB were located on the chromosome, while all other ARGs, aac (6′)-Ib-cr, aadA2b, ant (2″)-Ia, bla_NDM–1, mcr-9, qacE, qnrA1, sul1, and tet(A), were harbored by the largest plasmid pK710429_2. Virulence gene analysis showed that the aerobactin receptor gene (iutA), enterobactin gene cluster (entABCDEFS/fepABCDG), salmochelin gene cluster (iroEN), type 1 fimbriae gene cluster (fimABCDEFHIK), type 3 fimbriae gene cluster (mrkABCDFHIJ), were present on the chromosome, while no virulence associated genes were detected in the three plasmids.

Sequence analysis showed that pK710429_2 was a large multidrug resistant (MDR) plasmid with a length of 288,244 bp and containing 302 coding genes. This plasmid belongs to the IncHI2/HI2A group with an average G + C content of 46.91%. With 81% query coverage and 99.97% identity in BLASTn, the sequence of pK55602_2 was partially consistent with that of plasmid p1575-1 (accession no. CP068288), a recently reported bla_NDM–1-mcr-9-co-positive-IncHI2 plasmid harbored by a clinical isolate of Enterobacter hormaechei (Ai et al., 2021; Figure 1). Further analyses revealed that the pK710429_2 sequence can be roughly divided into two distinct regions based on its similarity to different plasmids. The first mcr-9-harboring-region (accounted for 97% of the plasmid sequences) was highly similar to the sequences of several previously reported mcr-9-harboring-plasmids, including pCTXM9_020038 (99% query coverage and 99.99% identity, accession no. CP031724), pMRVIM0813 (98% query coverage and 99.99% identity, accession no. KP975077), and pME-1a (96% query coverage and 99.99% identity, accession no. CP041734). Seven ARGs [aac(6′)-Ib-cr, aadA2b, ant(2″)-Ia, qacE, qnrA1, sul1, and tet(A)] and two class 1 integrons were found clustered within a 39,671-bp-long array of IS26/IS4321-bounded gene cluster (positions 168, 917-176, 764 bp) in this region. The second bla_NDM–1-harboring-region, accounted for 3% of the plasmid sequences (positions 168, 917-176, 764 bp), was highly similar to the sequences of the Tn6696 region in plasmid pNDM1-CBG (100% query coverage and 99.99% identity, accession no. CP046118) (Figure 1).

Additionally, with 76% query coverage and 99.96% identity in BLASTn, the sequence of pK55602_2 was partially consistent with that of plasmid R478 (accession no. BX664015), the first prototype IncHI2 plasmid in which all the conjugative transfer genes were present in the Tra1 and Tra2 regions (Gilmour et al., 2004). Different from plasmid R478, the Tra1 region in pK710429_2 was divided into two parts at 101,547–111,838 bp and 161,470–167,880 bp, and the Tra2 region was split by an IS903B-like element (Figure 1). In the conjugation experiments, pK710429_2 was transferred to E. coli J53 at the frequency of 1.2 × 10^–6. PCR detection confirmed that the transconjugant (J53-KPN710429) was positive for bla_NDM–1 and mcr-9. Phenotypic testing of the transconjugant showed that it was resistance to all β-lactams except aztreonam (Table 1).

In plasmid pK710429_2, bla_NDM–1 gene was located on a region with the cassette structure of ΔIS3000-ISAba125-bla_NDM–1-ble-trpF-tat-cutA1-groES-groEL-ISKpn19, which was partly similar to that of Tn6696 in pNDM1-CBG. Parts of the Tar1 region were located upstream of this cassette, with an IS1 between them. Interestingly, the remaining parts of the Tar1 region were located downstream of mcr-9, also with an IS1 between them. The rcnR-rcnA-pcoE-pcoS-IS903 structure, locating upstream of mcr-9 in pK710429_2, was consistent with that of plasmid pCTXM9_020038, pME-1a, and pMRVIM0813 (Figure 2). However, qseB-qseC regulatory genes were not found in plasmid pK710429_2.

The inducibility of mcr-9 expression was detected using colistin induction assays. After pretreatment with 0.5, 1, and 2 μg/mL of colistin, the KPN710429 exhibited colistin MICs of 4, 16, and 32 μg/mL, respectively. The mcr-9 gene expressions were upregulated accordingly, while IncHI2 plasmid replicase gene repA expression remained unchanged (Figure 3). The mgrB gene was not mutated during the colistin induction assay (Supplementary Figure 2).