Coexistence of Two blaNDM–5 Genes Carried on IncX3 and IncFII Plasmids in an Escherichia coli Isolate Revealed by Illumina and Nanopore Sequencing

The emergence of carbapenem-resistant Enterobacteriaceae poses a significant threat to public health worldwide. Here, we reported a multidrug-resistant Escherichia coli strain with two different blaNDM–5-carrying plasmids from China. Illumina short-read and MinION long-read whole genome sequencing were performed. Genomic analysis found that one blaNDM–5 gene together with mphA was located on a 55-kb IncX3 plasmid, while the other blaNDM–5 gene was on a novel 68-kb IncFII plasmid. Susceptibility testing and quantitative reverse transcription PCR results further indicated that the transconjugants with the IncX3 plasmid exhibited higher-level carbapenem resistance and expression of blaNDM–5 than those with both plasmids or the IncFII plasmid. Two other β-lactamase genes (blaCTX–M–15 and blaOXA–1) were also detected on another 160-kb IncF plasmid. This is the first report of coexistence of two blaNDM–5-carrying plasmids in a single bacterial isolate, highlighting the genetic complexity of NDM-5 carbapenemase circulation, and the urgent need for continued active surveillance.


INTRODUCTION
Prevalence of carbapenemase-producing Enterobacteriaceae is a major concern around the world. NDM, one of the most important carbapenemases, represents a serious threat to patient care and public health. Since NDM-1 was first reported in 2009 (Yong et al., 2009), NDM-producing organisms has caused various types of clinical infections (Cornaglia et al., 2011). To date, 21 NDM variants have been discovered worldwide .
NDM-5, one of the most common variants encountered among Enterobacteriaceae, was first identified in an Escherichia coli isolate from a patient in the United Kingdom in 2011 with a recent history of hospitalization in India. It has two amino acid substitutions (Val88Leu and Met154Leu) in comparison with NDM-1, and confers increased resistance to extended-spectrum cephalosporins and carbapenems (Hornsey et al., 2011). Most strains usually harbored one single copy of the bla NDM gene. Coexistence of two copies of the bla NDM gene is rarely detected. Recent studies reported the presence of two tandem copies of bla NDM-1 in the chromosomes of an ST167 E. coli from China (Shen et al., 2017) and a Pseudomonas aeruginosa strain from Serbia (Jovcić et al., 2013). Duplication of the bla NDM-5 gene on an IncF plasmid was observed in an E. coli strain , which showed identical carbapenem resistance levels to those conferred by an IncX3 plasmid with a single copy. However, no studies have reported coexistence of two bla NDM-5 genes on different plasmids in the same strain. The effect of two bla NDM-5 -carrying plasmids on carbapenem resistance still remained unclear.
In the present study, we report an E. coli strain harboring two bla NDM-5 genes, which were carried by IncX3 and IncFII plasmids, respectively. Susceptibility testing indicated that the transconjugant with the IncX3 plasmid alone showed the highest imipenem and meropenem resistance among all the transconjugants. Illumina short-read and MinION long-read whole genome sequencing was performed to gain an insight into the genomic and plasmid features.

Bacterial Isolation and Identification
The bla NDM-5 -positive E. coli strain GZ04-0086 was recovered from the stool sample of a patient through routine surveillance in Guangzhou, China, in 2018. The species level identification was performed using Vitek 2 compact system (bioMérieux, France). Presence of common genes encoding carbapenemases and extended-spectrum β-lactamases was determined by PCR screening (Balm et al., 2012;Zong and Zhang, 2013;Elumalai et al., 2014). The complete bla NDM gene was amplified with previously described primers and confirmed by sequencing (Zou et al., 2015). Written informed consent was obtained from the patient. All experimental protocols were approved by the Institutional Ethics Committees of Academy of Military Medical Sciences.

Southern Blotting, Conjugation Experiment, and Antimicrobial Susceptibility Testing
Bacterial genomic DNA from strain GZ04-0086 was prepared in agarose plugs and digested with the S1 endonuclease (Takara, Dalian, China). DNA fragments were separated by pulsed-field gel electrophoresis (PFGE) through a CHEF-DR III system (Bio-Rad, Hercules, CA, United States) for 15 h at 6 V/cm, with initial and final pulse time of 0.22 s and 26.29 s, respectively.
The plasmid DNA was transferred to a positively charged nylon membrane (Solabio) and hybridized with the digoxigenin-labeled probe specific to bla NDM-5 .
Conjugation experiment was performed by filter mating using strain GZ04-0086 as donors and azide-resistant E. coli J53 as the recipient. The donor and recipient cultures were mixed at a ratio of 4:1 and collected on a membrane filter (pore size, 0.22 µm). The filter was placed on a BHI agar plate and incubated at 37 • C overnight without shaking. The mixture was transferred into BHI agar plates containing 4 µg/ml meropenem and 200 µg/ml sodium azide. The transconjugants were selected after 72 h of incubation. The plasmid transfer frequency was calculated as the ratio of transconjugants to recipient cells. Presence of either one or both of the bla NDM-5 -carrying plasmids in the transconjugants was further confirmed by S1-PFGE.
Antimicrobial susceptibility testing of strain GZ04-0086 and the transconjugants was performed using Vitek 2 compact system following the manufacturer's instructions. The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2017). E. coli ATCC 25922 was used as the control strain. Minimum inhibitory concentration (MIC) values of imipenem and meropenem were further determined using MIC test strips (MTS, Liofilchem, Italy).

Transcriptional Expression Analysis of bla NDM-5
Transcriptional expression of bla NDM-5 in strain GZ04-0086 and the transconjugants was determined by quantitative RT-PCR (qRT-PCR). Total RNA was isolated from bacterial cultures using RNApure Bacteria Kit (DNase I) (CWBio, China). RT-PCR was performed using UltraSYBR One Step RT-qPCR Kit (CWbio, China) in CFX96 Real-Time System (Bio-Rad, United States) with specific primers (NDM-RT-F 5 -GATTGCGACTTATGCCAATG-3 and NDM-RT-R 5 -TCGATCCCAACGGTGATATT-3 ) and 16S rRNA primers as previously described (Yu et al., 2019). Expression levels were normalized relative to the transcriptional level of the constitutive 16S rRNA and interpreted by the C t method (Livak and Schmittgen, 2001). Statistical analysis was performed using a student t test to compare the log-transformed expression levels of bla NDM-5 among strain GZ04-0086 and the transconjugants.

Illumina/MinION Sequencing and Analysis
Genomic DNA was extracted from cultured bacterium using High Pure PCR Template Preparation Kit (Roche). Whole genome sequencing was carried out using both the Illumina MiSeq platform and the Oxford Nanopore MinION platform. The de novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler (v0.4.8) (Wick et al., 2017). Genome sequences were annotated using RAST (Aziz et al., 2008). The sequence type was determined through the MLST web server (Larsen et al., 2012). Genome sequences of seven E. coli ST44 isolates provided by BacWGSTdb (Ruan and Feng, 2016) were used to construct a Maximum-Likelihood phylogenetic tree using RAxML (v8.2.4) (Stamatakis, 2014).
Plasmid replicon types and the sequence types of IncF plasmids were identified using PlasmidFinder and pMLST (Carattoli et al., 2014).

Nucleotide Sequence Accession Number
The complete sequences of the chromosome of strain GZ04-0086 and plasmids pCTXM-GZ04, pNDM5-GZ04_B, pNDM5-GZ04_A, and p13k-GZ04 have been deposited in GenBank under the accession numbers CP042336 to CP042340, respectively.

Susceptibility Testing and bla NDM-5 Expression Analysis of the Transconjugants
All the transconjugants acquired resistance to ampicillin, ampicillin/sulbactam, piperacillin, imipenem, meropenem, and cephalosporins. However, decreased MICs of piperacillin/tazobactam, cefepime, cefotetan and meropenem were observed in transconjugant J53-B compared with those of J53-A and J53-AB. Interestingly, susceptibility testing using MIC test strips revealed that J53-A had the highest MICs of imipenem (≥32 µg/ml) and meropenem (48 µg/ml) among all the transconjugants. J53-AB had MICs of 16 µg/ml for imipenem and meropenem, while J53-B had lower MICs (imipenem, 12 µg/ml; meropenem, 6 µg/ml). The results of qRT-PCR also indicated that the expression of bla NDM-5 in J53-A was 1.37-fold higher than that in J53-AB and 1.56-fold higher than that in J53-B (Figure 2). These slightly varied expression levels of bla NDM-5 were consistent with the imipenem and

Genomic Insights Through Short-Read and Long-Read Sequencing
Strain GZ04-0086 was subjected to whole genome sequencing using both the MiSeq and the MinION platforms. Complete sequences of a 4.81-Mb chromosome and four plasmids were obtained through a de novo hybrid assembly. GZ04-0086 belonged to phylogenetic group A and sequence type ST44. E. coli ST44 has been frequently isolated from clinical settings (Kanamori et al., 2017;Runcharoen et al., 2017;Sonda et al., 2018). A phylogenetic analysis of E. coli ST44 revealed that GZ04-0086 was closest to isolates DO40 from canine and RDK06_431D from a patient in Tanzania, with 51 and 54 SNPs, respectively (Supplementary Figure S2). The bla CTX−M-15 , bla OXA-1 , tet(B), tet(D), and aac (6 ) Genetic Characterization of bla NDM-5 -Carrying Plasmid pNDM5-GZ04_A The bla NDM-5 -carrying plasmid pNDM5-GZ04_A had 55,253 bp in length. The plasmid belonged to the incompatibility type IncX3. A BLAST search against NCBI revealed that pNDM5-GZ04_A presented 84% query coverage and 99% identity with plasmid pNDM_MGR194 in K. pneumoniae from India (Krishnaraju et al., 2015; Figure 3). Both plasmids shared the same backbone sequence containing genes for plasmid replication (repB), conjugation/T4SS (virB genes), maintenance (topB and hns) and partition (parA), and the genetic structure of the bla NDM-5 gene, which was organized as IS3000-ISAba125-IS5-bla NDM-5ble-trpF-tat-ctuA1-IS26. The accessory region between mpr and umuD genes of pNDM_MGR194 was composed of a Tn2 and the bla NDM-5 genetic structure, while that of pNDM-GZ04_A consisted of a Tn2 and an 18-kb novel composite transposon Tn6638. Tn6638 was flanked by two IS26 elements in the same orientation. In addition to the bla NDM-5 genetic structure, a 5.7-kb fragment of a Tn3 family transposon and a 3.2-kb remnant of an IS26-mphA-mrx-mphR-IS6100 region were also carried by Tn6638. The 5.7-kb fragment contained Tn3 tnpA, tnpR and an ABC-transporter-ATPase-encoding gene, which was previously present in a Tn3 family transposon in plasmid pH226B from E. coli (Zurfluh et al., 2016). The 3.2-kb remnant had undergone the deletion of the IS6100 and the truncation of the mphR gene compared with the prototype mphA-carrying region in plasmid pRSB101 (Szczepanowski et al., 2004).

DISCUSSION
Emergence of NDM-producing Enterobacteriaceae represents an additional challenge for clinical management and infection control. A variety of plasmids have contributed to the dissemination of the bla NDM gene among diverse Enterobacteriaceae species throughout the world. pNDM_MGR194-like IncX3 plasmids have been found to be a major vehicle for bla NDM-5 (Wu et al., 2019). It was likely that pNDM5-GZ04_A had evolved from these pNDM_MGR194like plasmids and undergone horizontal gene transfer which resulted in the additional insertion. Plasmids pNDM5-GZ04_A and pNDM5-GZ04_B had different backbones but the same bla NDM-5 genetic structure, suggesting possible occurrence of similar transposition events. It was noteworthy that a second bla NDM-5 gene could not be detected based on the Illumina short reads alone due to the repeated appearance of the same bla NDM-5 genetic context. MinION sequencing played a crucial role in characterizing the coexistence of two bla NDM-5 genes.
Despite the presence of bla NDM-5 , the transconjugant J53-B exhibited significantly lower carbapenem resistance than that of J53-A. This decrease might be associated with the lower copy number of pNDM5-GZ04_B. Our analysis also revealed a lower copy number of pNDM5-GZ04_B, which might be the key reason for the reduced expression of bla NDM-5 in J53-B. Previous studies demonstrated that two copies of bla NDM-5 in the same plasmid would not lead to stronger carbapenem resistances , while coexistence of two plasmids harboring bla NDM-1 and bla KPC-2 , respectively, conferred higher resistances (Wu et al., 2016, 1). However, GZ04-0086 and J53-AB also showed lower carbapenem resistance than that of J53-A. The coexistence of these two bla NDM-5 -carrying plasmids showed no additive effect on imipenem and meropenem resistance levels. The varied copy numbers and inter-plasmids competition seem to offset each other on carbapenem MICs and expression level of bla NDM-5 , which still need further investigation.

CONCLUSION
In summary, our study characterized a multidrug-resistant ST44 E. coli harboring two bla NDM-5 genes, which were carried by different plasmids. However, coexistence of the two bla NDM-5 -carrying plasmids did not lead to stronger carbapenem resistance. Both plasmids were transferable and might serve as new reservoirs for further dissemination of bla NDM-5 . Our findings highlighted the threat of NDM-5 carbapenemase circulation and the urgency of stringent surveillance and control measures.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the NCBI GenBank, CP042336, CP042337, CP042338, CP042339, and CP042340.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Institutional Ethics Committees of Academy of Military Medical Sciences. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
LY, YL, and LL performed the experiments. LY, MX, and HM analyzed the data. XG collected the samples. KW and PHL performed library construction and genome sequencing. HM and XD prepared the tables and figures. LY and PL prepared the manuscript. KQ, PL, and HS designed the study and revised the manuscript. All authors read and approved the final manuscript.