Characterization of the IncX3 Plasmid Producing blaNDM–7 From Klebsiella pneumoniae ST34

Carbapenemase-producing Klebsiella pneumoniae has been a major clinical threat worldwide because therapeutic options are limited. Although New Delhi metallo-β-lactamase (NDM) is an important carbapenemase responsible for carbapenem resistance, it is uncommon in carbapenemase-producing K. pneumoniae in China. In this study, we described strain HZW25, an NDM-7-producing K. pneumoniae strain belonging to sequence type 34 (ST34). HZW25 exhibited resistance to all β-lactams tested but was susceptible to aminoglycosides and fluoroquinolones. The whole genome of HZW25 was sequenced with Pacific Biosciences RSII SMRT technology. HZW25 was composed of one chromosomal DNA and four plasmids, and the resistance genes of HZW25 were all located on the chromosome, except blaNDM–7 was located on a conjugative plasmid belonging to type IncX3 designated P4. The results of conjugation and transformation experiments showed that blaNDM–7 could be horizontally transferred successfully from the donor strain, HZW25, to the recipient strains, E. coli J53 and E. coli DH5α. The NDM variant transposable elements of the blaNDM–7-harboring plasmid P4 were the ISL3 and IS3000 families. The upstream region of blaNDM–7 contained ΔISAba125, which was inserted near the IS5 or ΔIS5 sequence. Our study is the first report of metallo-β-lactamase NDM-7 in a carbapenemase-producing K. pneumoniae strain with ST34 in China. The emergence of NDM-producing K. pneumoniae would be troublesome during treatment using ceftazidime-avibactam. Therefore, the rapid and accurate identification of carbapenemase-producing K. pneumoniae is necessary.


INTRODUCTION
Clinical treatment of carbapenem-resistant Enterobacteriaceae (CRE) is a critical challenge (Lee et al., 2016). Carbapenemase, which can be found as serine proteases and metalloproteinases, is responsible for carbapenem resistance. New Delhi metallo-β-lactamase (NDM) is a metalloβ-lactamase able to hydrolyze carbapenem (Khan et al., 2017). Although NDM-producing CRE has increased globally recently, the worldwide distribution and prevalence of NDM-positive strains appear to be variable between different countries and regions (Findlay et al., 2017;Khan et al., 2017;Lazaro-Perona et al., 2017;Wang et al., 2018;Perez-Vazquez et al., 2019;Wu et al., 2019). NDM-producing CRE strains have mainly spread in South Asia, the Baltans, North Africa and the Middle East (Dortet et al., 2014;Wu et al., 2019). Chinese national surveillance of carbapenem-resistant CRE in China has shown that NDM-producing CRE are less common than KPCproducing CRE, and NDM-positive strains are mainly E. coli (Zhang et al., 2017(Zhang et al., , 2018Wang et al., 2018).

Patient and Bacterial Strain
Carbapenem-resistant K. pneumoniae was isolated from a bile sample collected from a young woman in Hangzhou First People's Hospital in March 2017. The strain was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS, Bruker MALDI-TOF Microflex LT/SH, Bruker Diagnostics, Germany) according to the manufacturer's instructions. The patient had no history of traveling abroad. Informed consent was obtained for this study. The methods in this study were approved by the institutional ethics committee of Hangzhou First People's Hospital and were carried out in accordance with the approval guidelines.

Antimicrobial Susceptibility Testing and Detection of Carbapenemase
Antimicrobial susceptibility was determined by the VITEK compact II automated microbiology system and interpreted according to the Clinical and Laboratory Standards Institute guidelines in 2018 (CLSI). The modified carbapenemase inactivation method (mCIM) and EDTA-modified mCIM (eCIM) were used to detect carbapenemase and metalloβ-lactamase as recommended by the CLSI in 2018 (CLSI, 2018).

Detection of Carbapenemase Genes
PCR was performed to screen carbapenem resistance genes, including bla KPC , bla IMP , bla VIM , bla NDM , and bla OXA-48 . The amplicons were sequenced by Sanger sequencing.

Conjugation and Transformation Experiments
To evaluate the horizontal transferability of bla NDM , mixed broth mating was used as described in a previous study (Du et al., 2017). Sodium azide-resistant E. coli J53 was used as the recipient strain (donated by Professor YU, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University). The transconjugants were selected on MacConkey agar containing 100 mg/liter sodium azide and 2 mg/liter meropenem for 24 h at 37 • C. The electrotransformation assay was also performed to evaluate the dissemination of bla NDM using DH5α as the recipient strain as in a previous study (Findlay et al., 2017). The presumptive transconjugants were selected on MH agar plates supplemented with 2 mg/liter meropenem. All successful transformants were confirmed for the presence of bla NDM by PCR, and an antimicrobial susceptibility test was performed using the E-test method in parallel with the original strains and donor strains.

Whole Genome Sequencing
The DNA of HZW25 was extracted according to the manufacturer's instructions and sequenced with Pacific Biosciences RSII SMRT technology SMRT technology (Menlo Park, CA, United States). Sequence reads were assembled using a hierarchical genome assembly process (HGAP) compiled specifically for quality trimming and de novo assembly. The graphical maps of the whole genome were converted by BLAST Ring Image Generator (BRIG). The whole-genome sequence was annotated using the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) server available at NCBI. 1 Multilocus sequence typing (MLST) analysis of HZW25 and cgMLST phylogenetic relationship analysis of all public sequences of ST34 were performed using the BacWGSTdb server with the entire genome sequence (Ruan and Feng, 2016). The antibiotic resistance genes were determined using ResFinder 3.0 with > 97% gene identity threshold (exception of β-lactamase variants with 100% identity) and 100% gene length and the comprehensive antibiotic resistance database (CARD) at https://card.mcmaster.ca/ with resistance gene identifier of > 90% identity. The virulence factors were detected using the virulence database of K. pneumoniae 2 and a virulence factor database. 3

Plasmid Analysis
All plasmid sequences were annotated using the PGAAP server. The plasmid replicon type was determined by PlasmidFinder 4 with a 95% threshold for identity and 100% coverage. The bla NDM-7 -carrying plasmid was compared to the publicly available plasmid references using BLAST at GenBank (www. ncbi.nlm.nih.gov/GenBank/). The plasmid comparisons were generated by Easyfig according to the online protocol, 5 and presentations were generated by EdrawMax. The genetic environment of bla NDM-7 was analyzed and compared that of bla NDM variants.

Nucleotide Sequence Accession Number
The complete nucleotide sequence of HZW25 and four plasmids were deposited under the GenBank accession numbers CP025211-CP025215.

Susceptibility Test
HZW25 was resistant to all β-lactams tested, while it was susceptible to aminoglycosides and fluoroquinolones. Carbapenemase activity was positive with mCIM and eCIM tests, suggesting the production of metallo-β-lactamase (MBL).

Antimicrobial Resistance Genes and Transfer Experiments
The acquired antibiotic resistance genes, including bla NDM-7 , bla SHV−26 , fosA, oqxA, and oqxB, were responsible for the resistance profile of HZW25. In addition, intrinsic antibiotic resistance genes with CARD resistance gene identifiers were also identified, including antibiotic efflux pumps of the major facilitator superfamily (MFS) (KpnE, KpnF, KpnG, KpnH, and ermR), an ATP-binding cassette (ABC) antibiotic efflux pump (msbA), regulators of the efflux pump (marA and marR), the porin membrane protein OmpK35 and a bleomycin resistance gene (BRP). There were no mutations in resistance genes on the chromosome. Only bla NDM-7 was located on the plasmid, while the other resistance genes were located on the chromosome. bla NDM-7 was successfully transferred to E. coli AzR J53 by conjugation and to E. coli DH5α by electroporation, and the transconjugants displayed resistance to broad-spectrum cephalosporins and carbapenems ( Table 1). The presence of the bla NDM-7 gene in transconjugants was confirmed by PCR.

Molecular Grouping and Whole Genome Sequencing
Approximately 1.2 Gb clean data was generated after whole genome sequencing with Pacific Biosciences RSII SMRT technology, providing a 221.0-fold average coverage of the genome. The data were assembled into five contigs, which contained one chromosome and four plasmids (Figure 1). HZW25 belonged to ST34 with the MLST sequence 2-3-6-1-9-7-4. The isolate was negative for the string test due to the deficiency of the rmpA and rmpA2 genes. HZW25 belonged to the K73 capsular type as determined by the wzi gene encoding the outer membrane protein of the cluster. Virulence genes were present in HZW25, including the mrkABCDFHIJ operon encoding type 3 fimbriae for biofilm formation, the fimABCDEFGHIK operon encoding type 1 fimbriae for adherence, the iutA gene encoding aerobactin, the entABCDEFS operon and the fepABCDG operon encoding enterobactin siderophore, and the iroE and iroN genes encoding salmochelin. HZW25 also had a secretion system including the clpV gene encoding T6SS-II, dotU, the impAFGHJ operon, ompA and sciN encoding T6SS-III. The four plasmids were designated P1, P2, P3 and P4, and they belonged to IncFIB(K), IncR, IncFII(pKPX1), and IncX3, respectively. The plasmids were characterized, and the results are shown in Table 2. Only P4 carried a resistance gene with bla NDM-7 , and the other plasmids harbored no resistance genes. To date, there have been 57 K. pneumoniae strains belonging to ST34 worldwide, including Japan (n = 31), the United States (n = 10), China (n = 2), the United Kingdom (n = 2) and other countries (n = 12). Almost all strains were collected from humans and carried the bla SHV−26 , fosA, oqxA, and oqxB resistance genes, but only HZW25 had bla NDM variants of bla NDM-7 . Thirty-one strains from Japan carried bla IMP−1 and bla CTX−M−2 in addition to bla SHV−26 (Supplementary 1). All chromosomes of the ST34 K. pneumoniae strains harbored virulence clusters, such as the type I fimbriae cluster fim operon, type III fimbriae cluster mrk operon, and enterobactin siderophore cluster fep and ent operon (Supplementary 1). The phylogenetic trees of all ST34 K. pneumoniae strains in GenBank revealed that HZW25 was in a separate cluster (Figure 2), suggesting that the HZW25 strain had a long evolutionary distance from the others.

Characterization and Genetic Context of the bla NDM-7 Gene
The bla NDM-7 gene was localized in plasmid P4 and could be horizontally transferred successfully from the donor strain HZW25 to the recipient strains E. coli J53 and E. coli DH5α. P4 was 68 637 bp in size with a G + C content of 45.69% and 93 open reading frames. Comparative DNA sequence analysis showed that P4 possessed an IncX3-type backbone. A comparison of the whole region between P4, pEC25_NMD-7, pNDM5_020001, and pEh1A showed that the genetic context of the regions flanking the NDM variants was similar, and the backbone of plasmids showed high degrees of conversation and similarity. The NDM variant transposable elements of P4, pEC25_NMD-7 and pNDM5_020001 were highly similar with the ISL3 family  and IS3000 transposons. ISAba125 was upstream of the bla NDM variants and was inserted by the IS5 or IS5 sequence (Figure 3).

DISCUSSION
Carbapenemase-resistant K. pneumoniae (CRKP) has been a significant clinical problem with very limited therapeutic options (Lee et al., 2016). Due to the high-level resistance of CRKP to almost all antibiotics and the high mortality rate of CRKP infections, carbapenemase-producing carbapenem resistance is a public health threat (Lee et al., 2016;Zhang et al., 2018). In China, CRE strains are mainly composed of KPCproducing K. pneumoniae and NDM-producing E. coli (Zhang et al., 2017(Zhang et al., , 2018. NDM-7 has been reported in E. coli strains (Hao et al., 2018). Recently, NDM-7 carrying the IncA/C2 type plasmid harbored by K. pneumoniae strain ST147 was reported (Shankar et al., 2019). In our study, bla NDM-7 was first reported in ST34 K. pneumoniae. HZW25 was isolated from a young woman with acute cholangitis infection; based on multilocus sequence typing, HZW25 belongs to ST34 and contains several resistance genes responsible for beta-lactam resistance. The bla NDM-7 gene was located on an IncX3 plasmid, and the genetic environments and characteristics of the bla NDM-7 -producing plasmid P4 were analyzed. The module of NDM-7 transposable elements was  highly similar to the bla NDM variants harboring the IncX3type plasmid, suggesting that this NDM variant module could disseminate among different clones. In recent years, NDM variants, including NDM-7, have been increasingly reported in CRE isolates (Khan et al., 2017;Wu et al., 2019). In contrast to foreign countries, such as India, Spain and Canada, where NDM-7-producing strains are mainly K. pneumoniae strains (Chen et al., 2015;Seara et al., 2015;Lynch et al., 2016;Lazaro-Perona et al., 2017), in China, NDM variants are more common in carbapenemase-producing E. coli (Bi et al., 2018;Wang et al., 2018;Zhang et al., 2018). Wang L reported NDM-7-producing uropathogenic E. coli from a patient with bacteriuria in 2016 (Wang et al., 2016). Bi R reported a high prevalence of bla NDM variants in CRE E. coli strains, and bla NDM-7 ranked third among those variants (Bi et al., 2018). Hao Y analyzed the genotypic and phenotypic characterization of bla NDM-7 in E. coli from a patient with a urinary tract infection (Hao et al., 2018). Recently, Xu J and He F also reported NDM-7producing E. coli from a urinary sample (Xu and He, 2019).
Although bla NDM variants are both located on bacterial chromosomes and plasmids, bla NDM variants positioned on plasmids play a vital role in the dissemination of resistance genes (Bonnin et al., 2012;Ho et al., 2012). bla NDM variants harboring plasmids are mainly of the IncFII, IncX3, and IncC(IncA/C2) types (Perez-Vazquez et al., 2019). IncX3 is the most common type of plasmid carrying bla NDM , and most IncX3 plasmids are present in E. coli (Paul et al., 2017;Bi et al., 2018). From the worldwide distribution of bla NDM -carrying plasmids in Enterobacteriaceae, IncX3 plasmids may serve as an important vehicle in the dissemination of NDM in East Asia, particularly in China (Ho et al., 2012). Hao Y compared the backbones of plasmids carrying NDM variants and collected from human and food animal origin, and the results showed that all plasmids were highly similar (> 99%) among patients (Hao et al., 2018). This suggested that IncX3 plasmids may serve as one of the major platforms on which bla NDM genes evolve with the generation of new NDM variants. Lee CS isolated NDM-7-producing K. pneumoniae and E. coli simultaneously from a patient, suggesting that bla NDM-7 might be transferred between K. pneumoniae and E. coli in vivo (Lee et al., 2014).
In our study, bla NDM-7 was in a 68 637 bp IncX3-type plasmid. bla NDM-7 was successfully transferred to E. coli AzR J53 by conjugation and to E. coli DH5a by electroporation. Compared to the common genetic contexts of bla NDM variants, bla NDM-7 had a similar surrounding environment, with ISAba125 (intact or truncated) upstream and ble MBL downstream; these elements are located in the transposon-like structure flanked by IS3000 and IS26, responsible for horizontal transfer of bla NDM-7 among Enterobacteriaceae.
The bla NDM-7 gene can be carried by different K. pneumoniae strains of different STs, including ST147, ST138, ST273, ST437, and ST278 (Lee et al., 2014;Chen et al., 2015;Seara et al., 2015;Chou et al., 2016;Lynch et al., 2016;Perez-Vazquez et al., 2019;Shankar et al., 2019). Although these strains were isolated in different countries and regions, these strains contain plasmids carrying bla NDM-7 with similar surroundings and are characterized by horizontal gene transmission (Chen et al., 2015). Chen described bla NDM -carrying plasmids from different Enterobacteriaceae isolates with identical structures, suggesting that very effective horizontal transfer events had occurred (Chen et al., 2015). Seara N reported the interhospital spread of NDM-7-producing K. pneumoniae in Spain (Seara et al., 2015). In our study, ST34 K. pneumoniae carrying bla NDM-7 was reported for the first time, and the evolutionary distance from known ST34 K. pneumoniae strains showed that the strain we isolated was far from the other isolated strains.
Ceftazidime-avibactam is an effective antibiotic for CRE. It has good antibacterial activity against KPC-producing K. pneumoniae but not NDM-producing K. pneumoniae or E. coli (van Duin and Bonomo, 2016;Shirley, 2018). The emergence of NDM-producing K. pneumoniae would be troublesome in CRE treatment using ceftazidime-avibactam. Therefore, it is important to screen carbapenemase types before ceftazidime-avibactam therapy, and it is necessary to detect serine proteases and metalloproteinases in epidemiological investigations.

CONCLUSION
This study describes bla NDM-7 in an ST34 K. pneumoniae strain for the first time. The bla NDM-7 gene is located on a conjugated and horizontally transmitted IncX3-type plasmid. The potential dissemination of bla NDM -like genes in IncX3type plasmids should be considered. Before treatment with ceftazidime-avibactam, it is necessary to determine the types of carbapenemase in carbapenemase-resistant K. pneumoniae.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
MW, XW, and DY provided assistance and guidance in the research. QC and JZ wrote the manuscript. SW and YY assisted the manuscript checking. All authors checked the manuscript and submitted the final version.