Genotypic and Phenotypic Characterization of blaNDM–7-Harboring IncX3 Plasmid in a ST11 Klebsiella pneumoniae Isolated From a Pediatric Patient in China

NDM-7, a variant of New Delhi metallo-beta-lactamases (NDM), has the highest carbapenem-hydrolyzing activity. NDM-7-producing enterobacteria have been reported in many countries. In this study, we reported NDM-7 production in ST11 Klebsiella pneumoniae isolated from a boy hospitalized in the pediatric intensive care unit of a teaching hospital in China. The isolate exhibited resistance to β-lactam antimicrobials, quinolones, and trimethoprim/sulfamethoxazole, and it harbored blaNDM–7, blaCTX–M–15, qnrA, qnrB, and qnrS. The serotype of the isolated K. pneumoniae was assigned as K1, and it contained three virulence genes, including kfuBC, uge, and fim. The blaNDM–7 gene was located on a conjugative IncX3 plasmid designated as pB14NDM-7. This plasmid was fully sequenced and compared with the available blaNDM–7-harboring IncX3 plasmids. pB14NDM-7 contained a conserved genetic context of ISkox3-umuD-IS26-ΔTn125-IS5-ΔTn125-IS3000-ΔTn2. pB14NDM-7 showed 99% nucleotide identity and the same genetic context with three blaNDM–7-harboring IncX3 plasmids obtained from Escherichia coli in China. Our results indicate that IncX3 plasmid may contribute to the prevalence of blaNDM–7 in China. The high prevalence of NDM variants worldwide highlights the critical need for careful monitoring and control of the rapid dissemination of blaNDM.


INTRODUCTION
The prevalence of carbapenem-resistant Klebsiella pneumoniae has increased and becomes a serious public health threat since the early 2000s. Several mechanisms are responsible for carbapenem resistance of K. pneumoniae, among which carbapenemase production remains the most clinically relevant (Pitout et al., 2015). The most common metallo-β-lactamases (MBLs) identified in K. pneumoniae are New Delhi metallo-beta-lactamases (NDM), while other metallolactamases are relatively rare in this species (Gregson et al., 2016). NDM was first described in Sweden in 2009 in a patient who had received medical care in India. Thereafter, it has been reported in more than 40 countries worldwide, even in cases with no epidemiological links with India, such as in the Balkan states or the Middle East (Yong et al., 2009;Pérez-Vázquez et al., 2019).
The gene encoding NDM is often carried by plasmids and is easily transferred to other microorganisms through horizontal gene transfer. Owing to this reason, the emergence of carbapenem-resistant strains of pathogenic microorganisms has increased rapidly (Rolain et al., 2010). NDM-1 is the most described NDM and has emerged as a global health threat. Similar to other MBLs, NDM-1 can hydrolyze all beta-lactams except aztreonam (Yong et al., 2009). Many NDM variants have evolved in enterobacteriaceae by single and/or double amino acid residue substitutions at different positions, and a total of 24 known variants of NDM have been identified so far (Khalid et al., 2020). NDM-7 was first discovered in Escherichia coli in Germany in 2013 (Cuzon et al., 2013). NDM-7 contains Asp-130-Asn and Met-154-Leu substitutions and has the greatest carbapenemhydrolyzing activity (Yoon et al., 2018). To date, NDM-7producing enterobacteria have been reported in more than 10 countries (Solgi et al., 2017;Ahmad et al., 2018;Yoon et al., 2018;Mouftah et al., 2019). In China, NDM-7 has been detected and described in E. coli but not in K. pneumoniae Bi et al., 2018;Hao et al., 2018;Xu and He, 2019). In this study, we isolated a ST11 K. pneumoniae harboring bla NDM-7 from a boy hospitalized in the pediatric intensive care unit of a Chinese Hospital. Genotypic and phenotypic characterization of the IncX3 plasmid carrying bla NDM-7 gene was performed. In 2015, an IncX3 plasmid carrying bla NDM-7 was isolated from E. coli in the same hospital (Hao et al., 2018). To analyze the evolution of IncX3 plasmid carrying bla NDM-7 , we constructed a phylogenetic tree from 16 plasmids based on homologous proteins.

Bacterial Strains
Carbapenem-resistant K. pneumoniae was isolated from a 4year-old boy who was diagnosed with autoimmune encephalitis and hospitalized in the pediatric intensive care unit of a teaching hospital in Shandong Province of China in 2018. We designated this isolate as B14. During hospitalization, arterial catheterization was performed. B14 was obtained from blood cultures 2 weeks after hospitalization; however, no pathogen was isolated from the sputum, cerebrospinal fluid, and ascites. The patient was treated with ceftriaxone before blood culture, following which his condition improved, and he was discharged before the antibiotic sensitivity results were obtained.
The patient had no history of traveling abroad. Informed consent was signed by the family member of the patient involved in this study. The methods in this study were approved by the Ethics Committee of Shandong Provincial Hospital and were carried out in accordance with the approved guidelines. The isolate was identified as K. pneumoniae using Vitek-2 compact system and confirmed using Vitek-MS system (BioMérieux, France). Phenotypic detection of carbapenemases was performed using carbapenem inactivation method (CIM) and EDTAmodified CIM (eCIM) test.

Multilocus Sequence Typing
Multilocus sequence typing (MLST) of K. pneumoniae was performed according to protocols available on the MLST Pasteur website 2 . Seven conserved housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were amplified, sequenced, and compared with those in the MLST databases.

Analysis of bla NDM -Carrying Plasmids
Conjugation was performed using the mixed broth method. Briefly, E. coli J53 Azi R was used as the recipient strain, and B14 served as the donor. Transconjugants were selected on Mueller Hinton agar supplemented with meropenem (0.5 µg/ml) and sodium azide (100 µg/ml). Antimicrobial susceptibility test of the transconjugant was carried out as described for the clinical strain. In order to evaluate the stability of the plasmid in the recipient E. coli J53 after conjugation, antibiotic susceptibility test was performed after the recipient cells containing the plasmid were sequentially subcultured 20 times on blood agar plate without any antibiotics. The size and amount of plasmids carried by the clinical isolate and transconjugant were evaluated using S1-pulsed-field gel electrophoresis (S1-PFGE) as previously described (Hao et al., 2018). The genome of Salmonella H9812 digested with XbaI was used as the marker.

Plasmid Sequencing
The plasmid carrying bla NDM-7 was defined as pB14NDM-7. To better understand the characteristics of pB14NDM-7, its complete sequence was determined. The plasmid was extracted and sequenced using an Illumina Hiseq platform and assembled using SOAPdenovo at MajorBio Co., (Shanghai, China). The gaps were closed through PCR and Sanger Sequencing at Sangon Biotech (Shanghai, China). The plasmid sequences were annotated using basic local alignment search tool (BLAST) against the non-redundant protein database. Plasmid Finder was used for detection and typing of the plasmid.

Phylogenetic Tree Construction
The plasmids carrying bla NDM-7 were retrieved from National Center for Biotechnology Information (NCBI) database for the query "NDM-7 and plasmid." Out of 49 plasmids, 15 belonged to the IncX3 incompatibility group. The sequence of pB14NDM-7 was compared with the 15 bla NDM-7 -harboring IncX3 plasmids. The plasmid sequences were annotated for encoded proteins using Prodigal, a prokaryote genome annotation tool 3 . The STAG algorithm of OrthoFinder was used to construct a phylogenetic tree from 16 plasmids based on homologous proteins.
3 http://compbio.ornl.gov/prodigal/ MLST, Serotype, Resistance, and Virulence Genotyping PCR amplification and sequencing confirmed that B14 harbored bla NDM-7 and bla CTX-M-15 . These two genes were responsible for the resistance to β-lactam antibiotics. In addition, B14 carried qnrA, qnrB, and qnrS, which may lead to quinolones resistance. However, high-level fluoroquinolone resistance was probably mediated by chromosomal mutations in gyrA and/or parC genes. MLST revealed that the sequence type of B14 was ST11. Based on PCR amplification results, the serotype of B14 was determined to be K1. Furthermore, B14 contained three virulence genes, which included kfuBC, uge, and fim.

Transferability and Stability of Plasmid
bla NDM-7 -harboring plasmid of B14 was successfully transferred into E. coli J53 by conjugation. The transconjugant was identified as E. coli using Vitek-2 compact system and confirmed using Vitek-MS system. The presence of bla NDM-7 in the transconjugant was confirmed using PCR. The transconjugant was resistant to carbapenems and cephalosporin but susceptible to aztreonam, aminoglycosides, quinolones, colistin, and tigecycline. After 20 passages, the antibiotic susceptibility patterns of the transconjugant showed no changes. S1-pulsedfield gel electrophoresis showed that B14 harbored two plasmids, and the transconjugant J14 contained a single plasmid, which was approximately 46 kb (Supplementary Figure S1).
Plasmids pB14NDM-7 and pJN05NDM-7 were from the same hospital and differed only in gene dsbC. pJN05NDM-7 is a 46,161-bp circular IncX3-type plasmid. In dsbC gene, there was a 60-bp repetitive sequence in the region between the positions 8084 and 8144, corresponding to the 8023-8083 regions in plasmid pJN05NDM-7. In addition, there was a 88-bp deletion in pB14NDM-7 corresponding to the 8085-8172 region in gene dsbC of plasmid pJN05NDM-7.

DISCUSSION
NDM-7-producing K. pneumoniae are relatively rare as compared to NDM-7-producing E. coli. NDM-7 was first reported in K. pneumoniae in 2014 in Minnesota (Lee et al., 2014). Herein, we described the emergence of ST11 K. pneumoniae carrying bla NDM-7 on IncX3 plasmid in China. Although NDM-7 production has been reported in diverse clones of K. pneumoniae, including ST138, ST273, ST278, ST437, and ST654 (Shankar et al., 2019), our study is the first to report the presence of bla NDM-7 in ST11 K. pneumoniae in China. This strain was isolated from a 4-year-old boy with no history of traveling abroad.
The K serotypes and phenotypes are responsible for the invasive nature of certain K. pneumoniae. Serotypes K1, K2, K4, and K5 are highly virulent and may cause severe infections in humans (Wang et al., 2017). The serotype of B14 was found to be K1. The carbapenem-resistant K1 K. pneumoniae can cause infections at multiple sites, including liver abscesses, pneumonia, meningitis, and blood stream infections. Additionally, B14 contained three virulence genes, which included kfuBC, uge, and fim. ST11 is the major sequence type of hypervirulent carbapenem-resistant K. pneumoniae from Asia, especially China (Zhao et al., 2019). Therefore, it is speculated that B14 has certain virulence traits. However, it is gratifying that the patient's condition improved, following which he was discharged. As this study was retrospective, we could not perform in-depth analysis of the patient's surrounding environment and the source of the strain.
B14 was highly resistant to broad spectrum cephalosporins, monobactam, and carbapenems. However, it is known that NDM does not decompose aztreonam (Paul et al., 2017). Therefore, resistance of B14 to aztreonam in the present study might be due to the coexistence of bla CTX-M-15 gene that encodes extended spectrum β-lactamase. In addition, PCR results showed that B14 also carried quinolone-resistance genes, including qnrA, qnrB, and qnrS. Conjugation test revealed that only bla NDM-7 could be horizontally transferred, and the sequencing results showed that plasmid IncX3 harbored by B14 does not carry antimicrobial resistance genes other than bla NDM-7 . This finding corroborates the results of Hao et al. (2018).
IncX3 plasmids carrying bla NDM variants have been increasingly reported all over the world in recent years. A previous study has proved that IncX3 plasmid can transfer bla NDM between different enterobacterial species over a wide range of temperatures . IncX3 has also been reported to be an important carrier of bla NDM-7 (Paul et al., 2017). In order to further understand the evolutionary relationship of bla NDM-7 -harboring IncX3 plasmid, we downloaded the nucleotide sequences of 15 IncX3 plasmids from NCBI and analyzed their homology with pB14NDM-7. The results showed that all the 16 plasmids were closely related to each other, suggesting parallel evolution of these plasmids. It should be noted that pB14NDM-7 showed 99% nucleotide identity with three plasmids obtained from E. coli in China, which suggests that IncX3-type plasmids are popular vectors in mediating dissemination of bla NDM-7 in China Bi et al., 2018).
In plasmids, genes are frequently associated with mobile genetic elements such as transposons (Tn) and insertion sequences (IS). pB14NDM-7 contained a conserved genetic context of ISkox3-umuD-IS26-Tn125-IS5-Tn125-IS3000-Tn2. The bla NDM genetic structure is common in enterobacteriaceae for the horizontal transfer of bla NDM and has been reported in the transmission of bla NDM-7 and bla NDM-5 (Zhang et al., 2016;Guo et al., 2019). In addition, plasmids pB14NDM-7 and pJN05NDM-7 were from the same hospital. However, pJN05NDM-7 was isolated from E. coli, and the two patients were not in the same hospital area. These two plasmids differed only in gene dsbC. Disulfide bond formation is a crucial step in the folding process of a protein and is catalyzed by bacterial proteins of the Dsb system. DsbC is responsible for rearranging incorrect disulfides introduced between cysteine residues (Banaś et al., 2020).

CONCLUSION
In summary, our study described the emergence of bla NDM-7 in ST11 K. pneumoniae in China for the first time. Our results indicate that IncX3 plasmid may contribute to the prevalence of bla NDM-7 in China. The high prevalence of NDM variants worldwide highlights the critical need for careful monitoring and control of the rapid dissemination of bla NDM .

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of Shandong Provincial Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
YJ designed the experiments and revised the manuscript. CS carried out the experiments and wrote the manuscript. YH analyzed the data. YW and MJ contributed to experiment conception. All authors contributed to the article and approved the submitted version.

FUNDING
This study is funded by the National Natural Science Foundation of China (nos. 81401696 and 81902119).