First, the 207 CRKP isolates from patients at 12 tertiary China teaching hospitals in eight Chinese provinces were collected From January 2015 to May 2021. And then the detached samples were cultured on blood agar plate and identified by MALDI-TOF MS. FK3006 was isolated from the sputum sample, for it is the only isolate harboring the co-existence of bla_OXA-232, bla_CTX-M-15, rmtF, and virulence plasmid analyzed by WGS. K. pneumoniae strain 3036 (FK3036) was used as control strain for the virulence-negative resistant strain (ST15, none Virulence factors) and NUTH-K2044(ST23) was used as a virulence-positive control (Supplementary Table S1). Plasmid conjugation was performed with Escherichia coli 600(EC600, rifampicin-resistant), which was used as the recipient strain.

The MICs of the FK3006, EC600, and transconjugants (FK3006JH-1, FK3006JH-2) were determined by standard broth microdilution method following the Clinical and Laboratory Standards Institute guidelines (CLSI, 2020), except for colistin and tigecycline, for which the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used (EUCAST, 2020). Each AST was independently repeated three times in our study. Escherichia coli ATCC 25922 was used as the quality control organism in MIC determination.

The horizontal transferability of bla_OXA-232, bla_CTX-M-15, rmtF, and iucA were examined using the conjugation assay with E. coli 600. The FK3006 was used as donor strain, and E. coli 600 was used as the recipient strain. The recipients and donors were cultured in Luria-Bertani broth (37°C) until logarithmic phase (OD600 = 0.4–0.6), mixed in a ratio of 2:1 (200 μl,100uL) in 4 ml LB broth for 24 h. And then the serial dilutions were plated on selective media with appropriate antibiotics (gentamicin, 8 μg/ml[rmtF]; ampicillin, 100 μg/ml[bla_CTX-M-15]; meropenem, 1 μg/ml[bla_OXA-232]; dipotassium tellurite, 8 μg/ml[iucA]; rifampicin, 600 μg/ml). The frequency of plasmid transfer was calculated as the number of transconjugants per recipient. The transconjugants acquiring gene were confirmed by PCR and primers are listed in Supplementary Table S2.

After the plasmid was confirmed to be obtained, we applied growth curve assays to investigate fitness. Transconjugants (FK3006JH-1 and FK3006JH-2) and EC600 were cultured in LB medium overnight, then diluted to an OD600 of 0.01 and grown at 37°C for 24 h. Culture densities were determined by measuring the OD600 every 1 h for the first 12 h and then 24 h (Liu et al., 2016).

Bacterial genomic DNA of FK3006 was isolated using the Qiagen DNA extraction Kit (Qiagen, Germany) and the genome sequencing was then performed by the PacBio Sequel platform and the Illumina NovaSeq 6,000 platform. CANU (version 1.7.1) software was applied to assemble the data acquired by PacBio platform sequencing. The ORF prediction was measured in SnapGene (version 4.2.4). Resistant plasmid replicons were predicted using the PlasmidFinder tool¹. To verify whether the plasmid was also a conjugative plasmid, VRprofile² and OriT Finder website³ were performed to analysis of the four conjugal modules in the plasmid, including the relaxase gene, the origin of transfer site (oriT), the type IV secretion system gene cluster (T4SS), and the type IV coupling protein (T4CP) gene. The transposons (Tns) and insertion sequences (ISs) were also annotated and determined through the VRprofile and ISFinder⁴. We used BLAST Ring Image Generator (BRIG) to determine similar plasmids by comparing their identities and coverages. The circular representation was performed by Proksee⁵. The gene environments surrounding the antibiotic resistance gene was analyzed by Easyfig software. VFDB⁶ was performed to annotate the virulence factors.

Briefly, bacterial clones were diluted with saline to a concentration of approximately 10⁸ CFU/ml, and then 1 μl bacterial clones were dropped on CAS and King’s B (2:1) plates. After incubation for 24 h at 37°C, siderophore production was determined by the presence of an orange halo around the bacterial colonies (Zhou et al., 2022).

Caterpillars of Galleria mellonella were stored at 4°C before use for ensuring their health and fitness. Caterpillars were selected weighing 200–250 mg each for the study. For the FK3006, EC600, FK3006JH-1 and FK3006JH-2 groups, the caterpillars were injected with 10 μl (~1 × 10⁶ CFU) bacterial suspension at the left proleg, while the control groups were injected with PBS or empty syringe. A minimum of 10 caterpillars was used in each treatment group; they then were kept in culture at 37°C and inspected for 144 h. The survival rates of each group were recorded for each day (Li et al., 2020).

Briefly, serum was separated from blood samples and stored at-80°C. An inoculum of 25 μl (~1 × 10⁶ CFU) bacteria prepared from the mid-log phase was incubated with 75 μl pooled human sera. The mixture was taken at 0, 1, 2, and 3 h, and then incubated on the MHA plate. The numbers of viable bacteria were determined at 24 h (Liu et al., 2019).

Approximately 1 × 10⁵ A549 human lung epithelial cells (ATCC CCL-185) were grown in each well of 24-well plates in DMEM medium containing 10% fetal calf serum at 37°C with 5% CO₂ for 12 h and then incubated for 21 h at 37°C with 2 × 10⁷ CFU bacteria (Gottig et al., 2016). After centrifugation (3,000 rpm, 5 min, 4°C), the LDH in the supernatant was measured using the LDH Cytotoxicity Assay kit according to the instructions (Solarbio BC0685).

The S1-Nuclease Pulsed Field gel electrophoresis (S1-PFGE) was used to determine the existence of plasmids profiles in donor strains, and recipient strains. In short, the isolates were embedded in 1% Seakem Gold agarose and digested with S1 nuclease (Takara, Dalian, China). PFGE analysis was carried out using a CHEF-Mapper system (Bio-Rad) for 19 h with a switch time 4.0–40 s (parameters: 14°C, voltage 6 V/cm, and electric field angle 120°). The XbaI digested DNA of Salmonella serotype Braenderup H9812 was considered as a molecular size marker (Ai et al., 2021).

Data analyses were executed with the software GraphPad Prism 8.0.2. Results are shown as a two-tailed non-parametric Student’s test. For in vivo and vitro experiments survival data were analyzed by the Log Rank test (Mantel-Cox). P < 0.05 was considered to be statistically significant.

The complete nucleotide sequences of the chromosome and p3006-2, p3006-3, p3006-7, and p3006-11 plasmid were deposited as GenBank accession numbers JANCTJ010000000 consists of sequences JANCTJ010000001-JANCTJ010000014.

To characterize the antibiotic-resistant phenotype of FK3006, the 18 antibiotics susceptibility was tested in this strain. Results showed that FK3006 was a multi-drug resistant strain, which not only exhibited high-level resistance to both aminoglycoside antibiotics and a series of β-lactam antibiotics, but also was resistant to ciprofloxacin, sulfamethoxazole, minocycline, and piperacillin-tazobactam (Table 1). Although it was sensitive to imipenem, it still exhibited low-level resistance to other carbapenems including meropenem and ertapenem.

According to the subsequent WGS-based analysis, we further found that FK3006 belonged to ST15-KL112 isolates, a typical MDR clone. Moreover, 13 resistant elements, three resistant plasmids, and one virulence plasmid were detected in this isolate (Table 2). In addition, three key resistance genes played an important role in the acquisition of resistance to aminoglycoside antibiotics (rmtF), β-lactam antibiotics (bla_CTX-M-15), and carbapenems (bla_OXA-232). Moreover, rmpA2 as a critical factor activating the expression of capsular polysaccharides (CPS) genes which is responsible for CPS biosynthesis was detected in the IncFIB(K)/HI1B plasmid of FK3006. The iucABCD-iutA operon which encoding proteins necessary for aerobactin siderophore biosynthesis was also found in p3006-2.

We have identified three key resistant plasmids, and one virulence plasmid in FK3006. As plasmids are often transmissible between bacteria, we made a detailed analysis of these plasmids, aiming to further clarify potential resistance and virulence dissemination threats of FK3006. In FK3006, we observed three resistant plasmids: p3006-3, p3006-7, and p3006-11. p3006-11 (bla_CTX-M-15) was a typical IncFII_K-type MDR plasmid and shared high identity with the mobilizable plasmid pKP7450-3(identity 99.95%, bla_CTX-M-15, IncFII(K), CP090471.1), as well as the mobilizable plasmid pMS3802-CTXM-vir (identity 99.59%, bla_CTX-M-15, IncHI1B(pNDM-MAR), CP068016.1; Figure 1A). p3006-11 harbored four conjugation modules, holding the potential to self-transfer. Hence, we applied conjugation assay to imitate and evaluate the dissemination ability of p3006-11 plasmid. We found the p3006-11 was successfully transferred from FK3006 to E. coli EC600 (1.1 × 10⁻⁶-9.7 × 10⁻⁵). Moreover, the S1-PFGE pattern (Figure 2A) and MICs of FK3006JH-1(EC600 harboring bla_CTX-M-15 plasmid) also confirmed that the resistance phenotype dissemination of FK3006 (Table 1). Notably, the obtain of p3006-11 did not affect the growth of E. coli 600, which ensuring the stable existence of the resistance plasmid (Supplementary Figure S1).

p3006-2 was a typical IncFIB(K)/HI1B type virulence plasmid and shared ~99% identity with pK2044 (CP026012.1) and pLVPK (AY378100) plasmids (Figure 1B). Notably, p3006-2 also harbored the core oriT site and T4CP, which were identical to pK2044 and pLVPK. Previous studies have confirmed the pK2044 and pLVPK virulence plasmid could transferred from hypervirulent K. pneumoniae (hvKP) to ST11 CRKP and E. coli strains with the help of a self-transferable IncFII_K plasmid. In this study, we also observed that the p3006-2 virulence plasmid could be induced transfer with the help of p3006-11 plasmid (8.3 × 10⁻⁸-3.2 × 10⁻⁷; Figure 2B).

p3006-3 plasmid harbored rmtF gene and belonged to IncFIB type plasmid (Figure 1C), which was nearly identical (99.98%) to the human K. pneumoniae plasmid pKP3295-3 (rmtF, IncFIB(pKPHS1) CP079728.1) and pWSD411-4 (rmtF, IncFIB(pKPHS1), CP045677.1) previously reported in Hangzhou, China. Meanwhile, p3006-3 plasmid was similar to the pPMK1-B(coverage 86%, identity 99.96%, IncFIB(pKPHS1), CP008931.1) which also could be self-transferred to the recipient strain except for a multidrug resistance region of p3006-3 (Shi et al., 2020).

p3006-7 plasmid was a 10,397 bp circular molecule, harboring bla_OXA-232 resistance element, and contained no necessary elements for transmission by bioinformatic analysis. The conjugation assay suggested that p3006-7 was not self-transferable. According to the genomic comparison, we found p3006-7 was almost identical to pKNICU5 plasmid (identity 99.93%, ColKP3, KY454616.1), isolated from the first OXA-232-producing K. pneumoniae strain in China (Figure 1D; Yin et al., 2017). As the self-transmissible modules were usually absent in such ColKP3 type plasmid, the bla_OXA-232 genes may be mainly clonal dissemination.

The existence of pLVPK-like plasmid p3006-2 and the hypermucoviscosity indicated that the FK3006 may be a hypervirulent strain, here we applied several experiments to confirm the virulent phenotype. We found that FK3006 strain have a survival of about 74% after 60 min of incubation with the serum, which was significantly higher than that of the hypervirulence-negative resistant strain FK3036 (ST15 CRKP; Figure 3A). Moreover, there was a significant increase of siderophore production in FK3006 (d = 25 mm), compared with FK3036 (d = 10 mm), which did not have the iuc operon (Figure 3B). A549 human lung epithelial cells were infected with FK3006 (LDH, 1.70 μmol/l) led to cell LDH release of 76.9% compared with the positive control strain NUTH-K2044 group (LDH, 2.21 μmol/l). And, the negative control FK3036 group only release 0.85 μmol/l LDH. We also applied G. mellonella infection model to analyze the virulence of FK3006 in vivo. Testing showed that the survival rate of FK3006 group was 20% at 72 h, which was almost identical to that of a virulent strain of NUTH-K2044 (Figure 3C). A whole genome BLAST search was performed against VFDB to validate virulence factors harbored by the FK3006 (Supplementary Table S3). These results were in accordance with the fact that FK3006 populations were typical hypervirulent strains.

Notably, according to the conjugation assay, we observed the virulence plasmid p3006-2 could successfully transferred to the recipient strain EC600, and obtained the transconjugant FK3006JH-2. We found that FK3006JH-2(iucA) strains have a survival of about 53% after 60 min of incubation with the serum, which was significantly higher than that of the recipient strain EC600 (Figure 3D). When the EC600 obtained the p3006-2 plasmid, siderophore production also increased with the diameter of the halo increased ~2-fold (Figure 3E). G. mellonella infection model showed that the survival rate of FK3006JH-2 group was significantly lower than that of EC600 group (Figure 3F). All these results indicated that the virulence features of the FK3006 could be transferred to the recipients through the key virulence plasmid p3006-2.

The plasmid not only contains genes that promote the survival of the host but also carries other MGEs, such as Tn and IS, which also make a significant contribution to the dissemination of resistance genes. To thoroughly analyze the dissemination potential of rmtF and bla_CTX-M-15 in FK3006, we also analyze each of the MGEs surrounding them. In p3006-11, the bla_CTX-M-15, together with other antibiotic resistance genes (sul2-aph(3″)-Ib-aph(6)-Id-blaTEM_-1B-dfrA14- qnrB1), was part of a large AMR of 37,526 bp that was bracketed by two opposite orientation copies of the IS26 (Figure 4A) and it was closely associated with the presence of class 1 integrons. ISEcp1, a member of the IS1380 family frequently upstream of the bla_CTX-M-15, seems to be a crucial element in the mobilization and dissemination of bla_CTX-M-15, as it not only located surrounding bla_CTX-M-15 in the p3006-11 plasmid, but also surrounding the bla_CTX-M-15 in the pE16K0288-1(CP052263.1, IncFIB(K), IncFII(K), Korea), pDA33141-217(CP029588.1, IncFIB(K), IncFII(K), Sweden) and pMS3802-CTX-M-Vir(CP068016.1, IncHI1B(pNDM-MAR), repB, Spain) (Upadhyay et al., 2015; Fu et al., 2020; Hernandez et al., 2021). Similar to pCRKP-1,215_1 (CP024839.1, IncFII(pKPX1), Korea) and pARLG-3,135-1(CP033947.1, IncFIB(pQil), IncFII(K), United States), the typical IS6100-rmtF-ARR-3 ARI was identified in p3006-3, containing class 1 integrons (Figure 4B). IS6100, like IS26, is a member of the IS6 family, and has previously been described as the most common IS element adjacent to rmtF (Mataseje et al., 2014). However, most of IS6100 appears in the vicinity of drug resistance genes alone unlike IS26 (Partridge et al., 2018).