Epidemiological characteristics an outbreak of ST11 multidrug-resistant and hypervirulent Klebsiella pneumoniae in Anhui, China

Klebsiella pneumoniae has become a primary threat to global health because of its virulence and resistance. In 2015, China reported multidrug-resistant (MDR) and hypervirulent K. pneumoniae (hvKp) isolates. The emergence of MDR-hvKp poses a significant threat to public health. We collected 76 MDR K. pneumoniae isolates from the same hospital, of which there were a total of six MDR-hvKp isolates. We performed multilocus sequence typing (MLST) and capsular typing, whole genome sequencing, comparative genome analysis, and phylogenetic analysis as well as phenotypic experiments, including growth curves, mucoviscosity assay, Galleria mellonella infection model, human whole blood survival, and human neutrophil bactericidal assay to further characterize the samples. We identified six large plasmids carrying extended spectrum β-lactamase (ESBL) genes or carbapenemase genes (blaCTX–M–65, blaKPC–2, blaSHV–12, blaSHV–158), 9 plasmids containing other drug resistance genes, and 7 hypervirulence plasmids carrying rmpA and rmpA2 in ST11 MDR-hvKp isolates. Some of these plasmids were identical, whereas others differed only by insertion elements. In addition, we identified a plasmid, p21080534_1, that carries hypervirulence genes (iucABCD, iutA, rmpA2), a carbapenemase gene (blaKPC–2), and an ESBL gene (blaSHV–12), as well as MDR-hvKp 21072329, which did not carry rmpA or rmpA2, but exhibited hypervirulence and hypermucoviscosity. ST11 MDR-hvKp derived from hypervirulence and multidrug resistance plasmids not only causes significant treatment difficulties, but also represents an unprecedented challenge to public health. Therefore, urgent measures are needed to limit further spread.


Introduction
Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that causes various diseases including pneumonias, sepsis, and urinary tract infections (Podschun and Ullmann, 1998;Paczosa and Mecsas, 2016). Generally, K. pneumoniae colonizes the mucosal surface of the host without symptoms and primarily spreads through contact (Jarvis et al., 1985). For hosts with weakened immunity, such as the elderly, children, and critically ill patients, K. pneumoniae may result in serious hospital-acquired infections and community-acquired infections (CAI) (Paczosa and Mecsas, 2016). In recent years, infections caused by K. pneumoniae have steadily increased, particularly from multi-drug-resistant (MDR) strains, such as carbapenem-resistant K. pneumoniae (CRKP), extended spectrum β-lactamase (ESBL)-producing K. pneumoniae, and other MDR strains (Paterson et al., 2004;Munoz-Price et al., 2013). The US Centers for Disease Control and Prevention (CDC)'s 2019 report on antimicrobial resistance (AMR) listed Carbapenem-resistant Enterobacterales as a major threat (CDC, 2019); thus, the emergence of MDR K. pneumoniae has become a burden to the medical community.
With the continuous spread of hypervirulence and MDR plasmids worldwide, some cKps acquired hypervirulence plasmids to form hypervirulent strains, whereas hvKps acquired MDR plasmids to form MDR hypervirulence strains. The K-types and MLST of hvKps are not limited to just a few types. In 2015, five cases of ST23-KL1 and ST1797-KL1 MDR-hvKps carrying Klebsiella pneumoniae carbapenemase (KPC)-2 occurred in the ICU of two hospitals in Zhejiang Province, China, which resulted in fatal outcomes . The following year, five additional infections caused by ST11-KL47 MDR-hvKps carrying bla KPC−2 , bla CTX−M−65 , and bla TEM−1 were reported at the same hospital (Gu et al., 2018). Recently, China reported cases of ST11-KL47 and ST11-KL64 hypervirulent, carbapenem-resistant K. pneumoniae types, which caused liver abscess (Yang et al., 2020). There have also been reports of MDR-hvKps in other parts of Asia and throughout the world (Tang et al., 2020;Wyres et al., 2020a;Dong et al., 2022). In 2012, a case was identified in France caused by ST86-KL2 MDR-hvKp carrying bla CTX−M−3 in a patient who only resided in France and Algeria (Surgers et al., 2016). The following year, a death caused by ST23 MDR-hvKp carrying bla KPC−2 (Cejas et al., 2014) was reported in Argentina. In 2016, the United States experienced a case caused by ST23-KL1 carrying bla SHV−36 in a patient who had traveled to South America (Karlsson et al., 2019). Due to the frequent identification of MDR-hvKp in multiple countries around the world, it is obvious that this pathogen is a serious threat to global health.
In this study, we collected 76 MDR K. pneumoniae isolates from a tertiary hospital in Anhui, China between May 2020 and January 2022. They were characterized as harboring ESBL genes. Of these, we identified 6 isolates of MDR hvKp and 2 isolates of polymyxin-resistant ST11 K. pneumoniae by genomic information and phenotypic experiments. These hvKps were classified as ST11-KL64, rather than the classical ST23-KL1. We performed phenotypic experiments, whole genome sequencing, and a comparative genome analysis of these strains to determine the genetic background and evolutionary mechanisms of ST11 MDR-hvKp.

Materials and methods
Bacterial strains and growth conditions A total of 76 clinical MDR K. pneumoniae isolates were collected from different patients between May 2020 and January 2022 (Supplementary Table 1). Isolates were plated on blood agar plates and cultured at 37 • C for 24 h to isolate the bacterial clones. A VITEK 2 Compact System (bioMérieux, France) was used to identify the positive culture strains. All isolates were stored in 40% (v/v) glycerol broth at -80 • C until use. A total of 4,972 isolates of K. pneumoniae in China as of 2021 were collected from the NCBI database (including whole genome sequences and whole genome shotgun sequences). 1

Determination of minimum inhibitory concentration
Antibiotic susceptibility of all strains was performed by broth microdilution as recommended by the Clinical and Laboratory Standards Institute (CLSI). K. pneumoniae was cultured overnight in LB liquid medium at 37 • C and 220 rpm. 0.5 µL of K. pneumoniae solution was streaked onto LB plates and incubated at 37 • C for 24 h. Several monoclonal strains were selected to adjust the concentration of the bacteria in MH (Mueller-Hinton Broth) medium (Wiegand et al., 2008). The final inoculum size for broth dilution was 5 × 10 5 colonyforming units (CFUs)/well, after inoculating into MH medium with various concentrations of Polymyxin B. Each concentration gradient was divided into three parallel groups and grown at 37 • C and 220 rpm with shaking for 24 and 48 h. The experiment was repeated three times independently.

Growth curves
Klebsiella pneumoniae growth curves were established in LB medium manually. Overnight cultures were diluted to an OD 600 of 0.02 and grown in 96-well plates at 37 • C and 220 1 https://www.ncbi.nlm.nih.gov/pathogens/ rpm with shaking. The absorbance of the culture solution at 600 nm (OD 600 ) was measured every 0.5 h until it had peaked and was flat.

Mucoviscosity assay
K. pneumoniae viscosity was determined using the string test (Shon et al., 2013). Strains that formed strings 5 mm or longer after stretching with the tip of a sterile inoculation loop were considered to have a hypermucoviscosity phenotype. K. pneumoniae was cultured overnight in LB liquid medium at 37 • C and 220 rpm. The cultures were diluted the following day to an OD 600 of 1 and centrifuged at 2,350 g for 5 min, and the OD 600 of the supernatant was measured every minute.

Galleria mellonella infection model
The virulence of K. pneumoniae isolates was evaluated using the G. mellonella infection model. Larvae (0.3-0.4 g) were stored in the dark and used within 3 days after shipment (Tianjin Huiyude Biotechnology Co., Ltd.). Prior to injection, the bacterial pellet was washed with sterile saline and diluted to 1 × 10 8 CFU/mL. Using a 1 mL insulin syringe (Shanghai Kindly Ent Dev), 10 µL of the bacterial suspension was injected into the center of the second gastropod of the larvae. A group of 10 larvae were randomly selected for injection. Each treatment was performed in triplicate with 30 larvae. After injection, the larvae were incubated at room temperature and survival was monitored daily for 3 days. Death was recorded when the larvae no longer responded to touch. The larvae without or injected with 10 µL sterile saline were used as negative controls. In all cases, no dead larvae were observed in the negative control groups.

Human whole blood survival
Peripheral blood was collected from healthy volunteers (the author himself) and stored in heparin sodium anticoagulant tubes. Logarithmic phase K. pneumoniae was collected and suspended in PBS at 10 8 CFU/mL. Then, 10 µL of bacteria and 90 µL of blood were mixed in a 96-well plate and incubated for 1 h at 37 • C in 5% CO 2 . After 1 h, the blood cells were lyzed using cell lysis buffer (10 mM EDTA, 0.25% Triton-X100 in PBS) and plated on TSB agar. The following day, the surviving bacterial cells were determined using a CFU assay.

Isolation and bactericidal assay of human neutrophils
Human neutrophils were isolated from the venous blood of healthy volunteers using a human peripheral blood Frontiers in Microbiology 03 frontiersin.org neutrophil isolation kit (Solarbio, China) according to the manufacturer's instructions (Gao et al., 2019). Logarithmic phase K. pneumoniae was collected and suspended in PBS at 10 8 CFUs/mL. Then, 20 µL of bacteria were mixed with 100 µL of neutrophils (10 6 cells/ml) in a 96-well plate. The total volume was adjusted to 150 µL with PBS and they were incubated for 1 h at 37 • C in 5% CO 2 . Neutrophils were lyzed after 1 h using cell lysis buffer and plating on TSB agar. The following day, the surviving bacterial cells were assessed by CFU assay.

Whole genome sequencing, assembly, and annotation
A total of 39 isolates were sequenced, 7 isolates were sequenced by the third-generation sequencing, and the remaining 32 isolates were sequenced by the secondgeneration sequencing. Briefly, whole genome sequencing of K. pneumoniae was performed using a PacBio RS II and an Illumina HiSeq 4000 platform at the Beijing Genomics Institute (BGI, Shenzhen, China). Four SMRT cells Zero-Mode Waveguide arrays for sequencing were used with the PacBio platform to generate the subread set. PacBio subreads (length < 1 kb) were removed. The Pbdagcon program 2 was used for self-correction. Draft genomic unitigs, which are uncontested groups of fragments, were assembled using the Celera Assembler against a high-quality corrected circular consensus sequence subreads set. To improve the accuracy of the genomic sequences, GATK 3 and SOAP tool packages (SOAP2, SOAPsnp, SOAPindel) were used to make single-base corrections. De novo hybrid assembly of short Illumina reads and long PacBio reads was performed using Unicycler v0.4.8 (Wick et al., 2017) and annotated using the rapid prokaryotic genome annotation tool, Prokka 1.14.6 (Seemann, 2014). The plasmid map was drawn using BRIG 0.95 and Easyfig 2.2.5 (Alikhan et al., 2011;Sullivan et al., 2011).

Genome profiling and comparative genomics analysis
Acquired antimicrobial resistance genes (ARGs) were identified using ABRicate version 1.0.1 4 by aligning the genomic sequences to the ResFinder database and NCBI database (Zankari et al., 2012). The virulence factors of the isolates were identified using Kleborate and ABRicate by aligning the genomic sequences to the VFDB database (Zankari et al., 2012;Wyres et al., 2016a). Multilocus sequence typing (MLST) 2 https://github.com/PacificBiosciences/pbdagcon 3 https://www.broadinstitute.org/gatk/ 4 https://github.com/tseemann/abricate was performed by MLST 2.1 5 (Jolley and Maiden, 2010). Capsule typing was performed by Kleborate (Wyres et al., 2016b;Lam et al., 2021). The HarvestTools kit (Parsnp, Gingr, and HarvestTools) and BacWGSTdb were used to perform a comparative genomic analysis and phylogenetic analysis on the different isolates and to construct phylogenetic trees based on Single nucleotide polymorphisms (SNPs) in all isolates using the maximum likelihood method. The interactive tree of life (iTOL) v5 6 was used to draw a phylogenetic tree (Treangen et al., 2014;Letunic and Bork, 2019;Feng et al., 2021). SNPs were called from the completed genome of 21,072,329 using Snippy 7 and the genes in which SNPs were located were annotated and functionally classified using eggNOG 5.0 (Huerta-Cepas et al., 2019). Whole genome sequencing data were deposited in the NCBI database 8 and are publicly available under BioProject: PRJNA669320, PRJNA685326, PRJNA823907, and PRJNA838703.

Statistical analyses
All analyses were performed using Prism software (GraphPad Software, La Jolla, CA, United States). Error bars represent SEM. All experiments were repeated at least three times.

Results
ST11 Klebsiella pneumoniae isolates were the most prevalent and exhibit hypervirulence characteristics We performed MLST and capsular typing tests on 76 MDR isolates (Supplementary Table 1), they were resistant to at least three antimicrobial classes (Magiorakos et al., 2012). And all isolates were resistant to cefuroxime axetil, cefuroxime sodium, and levofloxacin (Supplementary  Table 4). Of the ST11 isolates, nine were KL64, five were KL47, and one was KL15. All ST15 isolates were KL19, whereas all ST307 isolates were KL102. We also identified an isolate which was a single locus variant of ST11, KP2000557 (ST751-KL64). Compared with ST11, it contained a mutation in the 1,716 DNA sequence of rpoB (c. 1716T > G, p. Ile572Met), suggesting that it may be derived from an ST11 mutation.
To evaluate the virulence of these isolates and to identify hvKp, we performed growth curves, Galleria mellonella infection model, and mucoviscosity assay (Walker et al., 2019) using NTUH-K2044 (the first classical hypervirulent K. pneumoniae) as a hvKp control (Wu et al., 2009). NTUH-K2044 exhibited the highest growth rate (Supplementary Figure 1). The growth rate of the ST307 isolates was similar to that of NTUH-K2044, that of the ST11 and ST15 isolates were lower compared with that of NTUH-K2044. Eleven ST11 isolates exhibited high lethality toward G. mellonella and were similar or more lethal than NTUH-K2044 (Figure 1). However, only two ST15 isolates and one ST307 isolate had higher lethality compared with NTUH-K2044 against G. mellonella (ST15: 21,073,066, 21,091,216; ST307: 21,072,123) (Supplementary Figure 2). The OD 600 of the isolates was adjusted to 1 and centrifuged at 2,350 g for 5 min (Walker et al., 2019). The supernatant of the classical hvKp NTUH-K2044 was completely transparent after centrifugation for 3 min (Figure 2 and Supplementary Figure 3), whereas that of 21,072,329, 21,080,534, and KP2000557 remained opaque, and it was difficult to centrifuge them completely (Figures 2A-C). However, in the string test, only KP2000557 could form strings above 1 cm. Nonetheless, we identified K. pneumoniae isolates carrying rmpA and rmpA2 and having a higher or similar lethality to G. mellonella than NTUH-K2044 as hvKp. All 6 isolates of MDR-hvKp were ST11 isolates; thus, we focused our study on ST11. We performed human whole blood survival assays and human neutrophil bactericidal assays. Of note, 10 6 CFU of MDR-hvKp KP2000557 did not survive in healthy human whole blood but resisted human neutrophil killing (Supplementary Figure 4). 10 6 CFU of 21,072,928, 21,111,661, and KP2000557 were unable to survive in healthy human whole blood.

FIGURE 3
Phylogenetic tree based on core genome single nucleotide polymorphisms (SNPs) and distribution of resistance genes, virulence factors of K. pneumoniae isolates (ST11, ST15, ST307). Isolates with different MLST and capsule types are shown in different colors using iTOL v4 (Letunic and Bork, 2019). Carrying resistance genes are colored in purple, and carrying virulence factors are colored in green. Scale bar indicates the number of nucleotide substitutions per site.
The sequences around rmpA2 carried by all isolates were completely identical, namely IS630-iucA-iucB-iucC-iucD-iutA-rmpA2-ISEc16 (Figures 5A-C). Meanwhile, the RmpA2 of all isolates were identical (Supplementary Figure 8A). The rmpA on p21080937_2 inserted a guanine (G) into the 286th nucleotide sequence, resulting in a frameshift mutation of rmpA (Supplementary Figure 8B). And a missense mutation (418C > A, Leu140Ile) occurred in rmpA on pKP2000557_2. There were mainly two types of sequences around rmpA in each isolate, the presence or absence of the downstream salmochelin operon iro ( Figure 5D). By comparing the gene clusters of different isolates, we found that the original rmpADC gene cluster was rmpA-rmpC-rmpD-iroN-iroD-iroC-iroB. Because of the insertion of ISKpn26 into iroN, the sequence of the gene cluster was changed, resulting in the insertion mutation. ISs-mediated transfer may lead to rmpADC detachment from iroBCDN, resulting in rmpADC gene cluster sequences on p21080237_1, 21080534_4, pFRPDR_1 and pKP2000557_2 (Supplementary Figure 9).
Through comparison, we found that some plasmids carried by hvKp have extremely high similarity. Compared with p21080237_2, p21091025_1 carried an ESBL gene, bla CTX−M−65 , which was absent from p21080237_2 ( Figure 4C). NCBI database comparison revealed that p21072329_3 exhibited highly similar to the pMTY12128 from Japan (Supplementary Figure 10A). pMTY12128 had only an additional insertion of ISKpn8 than p21072329_3; otherwise, they share 100% homology. Furthermore, p21072329_4 shared 100% homology with p21080237_6 (Supplementary Figure 10B). All isolates, except 21,080,237, contained a ColRNAI plasmid, p21072328_5 (5,596 bp), which carries genes of unknown function (Supplementary Figure 11C).

Phylogenetic analysis of ST11 Klebsiella pneumoniae
To determine the relatedness of our isolates to other Chinese isolates, we collected 4,973 whole genome sequences of all K. pneumoniae isolates from China contained in the NCBI database as of 2021. Of these isolates, 2,524 (50.75%) ST11 isolates were detected. We conducted a comparative genomics analysis of the ST11 and ST751 isolates (Supplementary Figure 12A). Fifteen ST11 isolates and one ST751 isolate constituted three clades based on the phylogeny which also corresponds to the K-types (Supplementary Figure 12B). The lowest SNP among all ST11 strains was 119 (21,080,237 and 21,080,631) and the highest was 3,797 (21,072,329 and 21,101,834) (Supplementary Figure 13). 21,080,237 and 21,080,631 shared high homology and they belonged to the same clade as the isolates from Zhejiang. 21,080,534 and 21,083,181 were similar to the isolates from Nanjing (KPN 02 and KPN08, etc.).
rmpADC and rmpA2. Furthermore, we did not detect mcrlike genes, such as mcr-1, in the genome or plasmid of the ST11 polymyxin-resistant isolates, FRPDR, and 21,091,025. In addition, mgrB had no IS insertion.
Using 20,172,329 as a reference genome, we identified 6,283 non-repetitive mutations. Of these, 5,508 mutations were in coding sequences (CDS), 773 mutations were in non-coding regions, and 2 mutations were in tRNA coding regions. Among the mutations in the CDS, there were 4,015 synonymous mutations, 1,390 missense mutations, 64 frameshift mutations, 30 nonsense mutations, and 4 intragenic mutations. The putative functions of these mutated CDSs were classified using the eggNOG database ( Figure 6A). Although "cell wall/membrane/envelop biogenesis" exhibited the highest number of mutations, most of the mutations were synonymous mutations (276) and there were only 83 missense mutations, less compared with "carbohydrate metabolism and transport" (138) and "transcription" (99). In addition, there were many genes related to "cell wall/membrane/envelop biogenesis" (260) (Figure 6B), which does not indicate that this function is prone to mutation.
Isolates 21,091,025 and FRPDR did not carry mcr-like genes, we hypothesized that the mutation may be located in the chromosome-mediated polymyxin resistance genes arnT, eptA (pmrC), mgrB, phoPQ, pmrAB, pmrD, and ugd. The SNPs of colistin-related resistance genes were counted and 28 SNPs were found, including 4 missense mutations and 24 synonymous mutations ( Figure 6C). In 21,091,025, the 572nd nucleotide of phoP was mutated from adenine (A) to cytosine (C), resulting in a mutation of the 191st amino acid of the encoded PhoP from Asp to Ala. Both phoE and pmrA of FRPDR were mutated. The 273rd nucleotide of phoE is mutated from cytosine (C) to adenine (A), causing the mutation of the 91st amino acid of its encoded PhoE from Ser to Arg. The 157th nucleotide of pmrA is a guanine (G) mutated to a thymine (T), resulting in a mutation of the 53rd amino acid of PmrA encoded by Gly to Cys. These mutations may lead to polymyxin resistance. In addition, the 115th amino acid of PhoP of 21,120,240 was also mutated (Leu115Pro), however, this did not result in polymyxin resistance. We analyzed the SNPs of 21,072,329 to explore the reasons for its hypervirulence. K. pneumoniae with different capsule types differs greatly in the gene cluster. Thus, we only analyzed the mutations present in 21,072,329 compared with other ST11-KL47 isolates, including ST11-KL47 K. pneumoniae 19PDR22 that we previously obtained (He et al., 2022). Through a comparison of different ST11-KL47 isolates, we found two SNPs that were present only in 21,072,329 and located in wzc (221T > G, Leu74Arg) and gene 23445 (845G > T, Gly282Val) ( Figure 6D). Wzc is a tyrosine kinase involved in capsular polysaccharide (CPS) synthesis and assembly, and 23445 encodes the LysR family transcriptional regulator. However, it remains to be determined whether their mutation leads to the generation of a hypervirulence phenotype.

Discussion
It is generally considered that highly virulent pathogens, including hvKp, rarely develop resistance to antibiotics. Thus, the higher the virulence, the weaker the resistance. The emergence of MDR-hvKp challenges this idea. Since the twentyfirst century, hvKps have gradually spread globally, resulting in significant medical and health issues. In 2015, the first MDR-hvKp was identified in China, and its MLST was primarily ST23 . Subsequently, China reported a variety of other MLST MDR-hvKps, in particular, ST11 (Gu et al., 2018;Yang et al., 2020;Zheng et al., 2020). According to the existing research (Russo et al., 2018), we identified hvKp carrying both rmpA and rmpA2 in this study. Among the 76 MDR K. pneumoniae isolates we collected, 16 represented ST11 and its variants, 5 were ST11 MDR-hvKps, and one was ST11 variant MDR-hvKp. These MDR-hvKps were resistant to at least 13 of the 16 tested antibiotics and carried at least two β-lactamases and seven resistance genes. These MDR-hvKps exhibited hypermucoviscosity and hypervirulence. However, the results of the string test showed that only KP2000557 could form strings above 1 cm, which was inconsistent with the results of the mucoviscosity assay. Some studies have shown that the string test is not a method that can accurately identify hvKp, and it may be better to identify hvKp with multiple methods. In addition, patients with MDR-hvKp 21,080,237 and 21,080,534 were both around 30 years old. This indicates that the most dangerous MLST of K. pneumoniae in China remains ST11, and it could be evolved from an MDR-KP to an MDR-hvKp, which poses a serious threat to public health.
Except for the classical ST23-KL1 hvKp, most of the hvKps of other MLSTs exhibit hypervirulence phenotypes resulting from the acquisition of hypervirulence plasmids (Yang et al., 2021). The generation of ST11 MDR-hvKps is caused by the transfer of multidrug resistance and hypervirulence plasmids (Xu et al., 2021). We detected rmpA and rmpA2 in 5 isolates of MDR-hvKp and they were all located in hypervirulence plasmids. RmpA2 was identical in all strains, but there were differences in RmpA. The surrounding sequences of rmpA on these hypervirulence plasmids are quite different; however, rmpA2 shows less of a difference. In addition, iucABCD and iutA are always downstream of rmpA2. Some isolates have iroBCDN downstream of rmpA, whereas some isolates have only ISs. Upon comparison, the reason for the deletion of iroBCDN downstream of rmpA could be the insertion of an IS into iroN, causing a mutation in iroN. Moreover, ISs mediate the transfer of rmpA, which separates rmpA from iroBCDN. The initially reported hypervirulence plasmids, pK2044 and pLVPK, carried iroBCDN, iucABCD, iutA, rmpA, and rmpA2 (Chen et al., 2004;Chou et al., 2004). We speculated that the hypervirulence plasmid may have evolved from pK2044 and pLVPK with the insertion of ISs.
We found that some plasmids showed only minor differences, including differences in SNPs, deletions, or IS insertions. This suggests the existence of transfers between plasmids. With the exception of 21,080,237, we found a ColRNAI plasmid with unknown function in other isolates. This plasmid only carried mbeB, mbeB, mobC, the gene encoding toxin, and many genes of unknown function. This plasmid may be related to bacterial plasmid mobilization and adaptability, but its detailed function needs to be determined. Of note, we found that the IncFII/IncR plasmid, p21080534_1 of MDR-hvKp 21,080,534, carried the carbapenemase gene (bla KPC−2 ), ESBL gene (bla SHV−12 ), hypervirulence genes (iucABCD, iutA, and rmpA2), and mercury resistance genes (merACPT). This is a rare plasmid carrying both hypervirulence and multidrug resistance genes, which may be derived from the hypervirulence plasmid and IS carrying multidrug resistance genes. Besides p21080534_1, other plasmids carrying both hypervirulence genes and multidrug resistance genes have been recently reported in China (Shen et al., 2019). These plasmids facilitate the transformation of cKp into MDR-hvKp and pose unprecedented challenges in controlling the spread of MDR-hvKp.
Polymyxins were once considered the last line of defense against MDR Gram-negative bacteria (Li et al., 2006); however, with the continuous abuse of antibiotics, bacteria have developed a variety of polymyxin resistance mechanisms, including plasmid-mediated colistin resistance gene mcr-1, two-component system PhoP/Q, PmrA/B, and pmrHFIJKLM operon (Groisman, 2001;Gunn, 2008;Olaitan et al., 2014;Liu et al., 2016). ST11 isolates, FRPDR (MDR-hvKp) and 21,091,025, are resistant to polymyxin, but we did not find mcr-like genes. Moreover, we found missense mutations in phoP (21,091,025: 572A > C, Asp191Ala), phoE (FRPDR: 273C > A, Ser91Arg), and pmrA (FRPDR: 157G > T, Gly53Cys) on chromosome 21,091,025. The mutation of the 53rd amino acid of PmrA has been reported (Sun et al., 2009) and we speculate that these mutations may be responsible for polymyxin resistance. We did not detect rmpA or rmpA2 in 21,072,329, which only carried one siderophore (yersiniabactin). Interestingly, 21,072,329 showed a phenotype of hypervirulence and hypermucoviscosity, and its virulence and viscosity were higher compared with NTUH-K2044. By aligning the genome of 21,072,329 with other isolates, we found two unique SNPs (wzc: 221T > G, Leu74Arg, 23445: 845T > G, Val282Gly). Wzc is involved in the synthesis and assembly of K. pneumoniae CPS. The hyperviscous phenotype of hvKp is largely due to overproduction of CPS. Therefore, the mutation of wzc may cause 21,072,329 to exhibit the characteristics of hvKp. Gene 23445 encodes the LysR family transcriptional regulator. It cannot be ruled out that there are other factors that lead to its hypervirulence and hypermucoviscosity phenotype, and could also be related to an epistatic phenomenon with a combination of mutations. This study still has some limitations, namely a single medical institution was sampled over a short time period. Thus, the results of this study need to be further corroborated. Therefore, the results of this study need to be further confirmed by constructing mutant genes in cKp or constructing reverse mutations in 21,072,329.
In conclusion, we performed a genome-wide analysis of ST11, ST15, and ST307 that broke out in Hefei, Anhui, especially ST11 MDR-hvKp. We identified the mechanisms of plasmid transmission for ST11 MDR-hvKp and the correlation between isolates. In addition, we also reported the dangerous hypervirulence plasmid, p21080534_1, which not only carried the hypervirulence genes, rmpA2, iucABCD, and iutA, but also the ESBL gene, bla SHV−12 , and the carbapenemase gene, bla KPC−2 . Currently, the spread of hypervirulence and multidrug resistance plasmids carried by K. pneumoniae may have exceeded our expectations. It is urgent to limit the spread of MDR-hvKp and their associated plasmids.

Ethics statement
Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Author contributions
BS and ZH designed the project. ZH, WX, and HZ performed the experiments. ZH, YD, HL, LZ, and CZ collected isolates. ZH analyzed the data and wrote the manuscript. YL and BS critically revised the manuscript. All authors read and approved the final manuscript.