Genotypic and Phenotypic Characterization of IncX3 Plasmid Carrying blaNDM-7 in Escherichia coli Sequence Type 167 Isolated From a Patient With Urinary Tract Infection

Infections due to New Delhi metallo-beta lactamase (NDM)-7-producing Escherichia coli are infrequent and sporadic. In this study, we report one case of recurrent urinary tract infection caused by blaNDM-7-producing E. coli belonging to phylogenetic group A, sequence type (ST) 167. In this study, we aimed to describe the genotype and phenotype of blaNDM-7-producing E. coli in China. The isolate exhibited resistance to β-lactam antimicrobials, trimethoprim-sulfamethoxazole, quinolones, and aminoglycosides. blaNDM-7 is located on a conjugative plasmid designated pJN05NDM-7 belonging to type IncX3. pJN05NDM-7 was fully sequenced and compared with all publicly available blaNDM-7-harboring plasmids. pJN05NDM-7 is almost identical to pKpN01-NDM7 and pKW53T, although the plasmids are geographically unrelated. The comparison of IncX3 plasmids harboring blaNDM in China showed high similarity, with genetic differences within insertion fragments. Notably, the differences in plasmids of animal and human origin were insignificant, because only one plasmid showed deletion inside the ISAba125 region compared with pJN05NDM7. Our study demonstrates that E. coli carrying IncX3 plasmids play an important role as a reservoir and in the spread of blaNDM. Further studies should be performed to control the dissemination of blaNDM among food animals.


INTRODUCTION
New Delhi metallo-beta-lactamase (NDM)-producing bacteria are spread worldwide and pose a serious threat to public health, and is highly disseminated in China Liu et al., 2018). The surveillance for carbapenem-resistant Enterobacteriaceae (CRE) showed that bla NDM production was the second major mechanism of carbapenem resistance in Escherichia coli, and bla NDM−1 was the most frequent variant (Khan et al., 2017). Since the first report on NDM-1 in 2009, 20 variants of NDM have been assigned in the Lahey Clinic database . NDM-7, which differs from NDM-1 by two point mutations corresponding to amino acid substitutions, was described in 2013 with increased carbapenemase activity compared with NDM-1 (Cuzon et al., 2013). bla NDM−7 is infrequently detected, and sporadic cases of infections due to bla NDM−7 -producing enterobacteria have been reported in France, India, the United States of America, and Japan (Cuzon et al., 2013;Chen et al., 2015;Wang et al., 2016;Devanga Ragupathi et al., 2017;Pal et al., 2017;Sugawara et al., 2017;Espinal et al., 2018). In China, bla NDM−7producing E. coli ST131 was first reported in 2016; however, the genetic content of bla NDM−7 -harboring plasmids was not clearly described (Wang et al., 2016). Considering its global distribution, increasing attention should be paid to epidemiological survey of bla NDM−7 .
In this study, we detected a bla NDM−7 -producing E. coli isolate from a patient without a history of traveling admitted in a Chinese Hospital. To elucidate the molecular epidemiology and evolutionary dynamics involved in the dissemination of bla NDM , the genomic content and in-depth molecular characterization of the strains was determined in this study.

Bacterial Strains
The carbapenem-resistant E. coli strain JN05 was recovered from urine sample of a 61-year-old woman with recurrent urinary tract infection at a teaching hospital in Shandong Province of China in 2015. The patient was diagnosed with vesicovaginal fistula secondary to cervical cancer after chemotherapy and electrocautery surgery in 2009. According to the abdominal ultrasonography, the patient was diagnosed with hydronephrosis and hydroureter of upper segment on admission. After the treatment with multiple antibiotics failed, nephrostomy was performed to improve hydronephrosis. There was no history of traveling abroad. Informed consent was signed by 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 strain obtained from the patient was identified as E. coli by using Vitek-2 compact system and confirmed by Vitek-MS system (BioMérieux, France). Phenotypic detection of carbapenemases was performed using carbapenem inactivation method (CIM) and EDTA-modified CIM (eCIM) test.

Antibiotic Susceptibility Assay
Susceptibility assay of antibiotics was performed on Mueller-Hinton (MH) agar plates using E test strips ( Table 1). Susceptibility assay results were interpreted by Clinical Laboratory Standards Institute (CLSI) breakpoints (CLSI, 2017), with the exception of tigecycline, polymyxin B, and fosfomycin, which were interpreted by EUCAST breakpoints (EUCAST, 2017).

Molecular Typing
Multilocus sequence typing (MLST) and phylogenetic typing was performed for molecular typing of the isolate as previous described (Wirth et al., 2006;Wang et al., 2016). The virulence factors of extraintestinal pathogenic E. coli (ExPEC)-associated genes were screened by PCR-based assays (Wang et al., 2016).

Analysis of bla NDM -Carrying Plasmids
Conjugation test was performed by mixed broth method using E. coli J53Azi R as the recipient strain. Transconjugants were selected on MH agar plates containing 6 µg/mL ceftazidime and 100 µg/mL sodium azide. The antimicrobial susceptibility test of the transconjugant was carried out as antibiotic susceptibility assay of clinical strain. The size and amounts of plasmids carried by the clinical isolate and transconjugant were evaluated by S1-pulsed-field gel electrophoresis (PFGE) as previously described .

Plasmid Sequencing
The plasmid pJN05NDM carrying bla NDM−7 (present in strain JN05) was extracted and sequenced using an Illumina Hiseq platform and assembled by SOAPdenovo at the MajorBio Co (Shanghai, China). The gaps were closed through PCR and Sanger Sequencing at Sangon Biotech (Shanghai, China). The plasmid sequences were annotated by BLAST against the non-redundant protein database. PlasmidFinder was used for detection and typing of the plasmid.

Molecular Grouping, Resistance Genotyping, and Virulence Genotyping
The E. coli strain JN05 was assigned to ST167 and belonged to phylogenetic group A. It carried papG II, which may play an important role in the pathogenic process. Multiple antimicrobial resistance genes, including bla NDM−7 , bla CTX−M−3 , bla CTX−M−14 , bla TEM−1 , qnrS, armA, and acc(6 )-Ib genes, are responsible for the resistance profile of strain JN05.

Analysis of the Plasmid Harboring NDM
New Delhi metallo-beta-lactamase -harboring plasmid of strain JN05 was successfully transferred into E. coli J53Azi R by conjugation experiment. The presence of NDM-7 in the transconjugant was confirmed using PCR, and MLST was used to distinguish the transconjugants from the clinical strain. The transconjugant J05 was susceptible to aztreonam, quinolones, and aminoglycosides, but resistant to carbapenems and cephalosporin. S1-PFGE showed that the clinical strain JN05 harbored six plasmids, and the transconjugant J05 contained a single plasmid, which was approximately 46 Kb (Supplemental Figure S1). pJN05NDM-7 is a 46,161-bp plasmid belonging to the IncX3 incompatibility group. The complete sequence of plasmid pJN05NDM-7 was submitted to GenBank under accession number MH523639. In pJN05NDM-7, bla NDM−7 was preceded by IS3000-ISAba125-IS5 in the upstream region and followed by ble-trpF-dsbC-IS26-∆umuD in the downstream region. This bla NDM genetic structure was common in Enterobacteriaceae for the horizontal transfer of bla NDM (Pal et al., 2017).
Multiple NDM variants were harbored in the plasmids, including NDM-1, . We observed that the IncX3 plasmids carrying bla NDM−5 originating from different provinces of China showed high similarity, except three plasmids with various lengths of insertion sequences (pP744, pRJA274, and pZHDC40). Six IncX3 plasmids originating from pigs and one plasmid from chicken were identical to pNDM-HN380, thus confirming that this mobile NDM vector is widespread in China (He et al., 2017;Ho et al., 2018).
As obvious differences were observed among sequences of pJN05NDM-7, pP744, pRJA274, and pZHDC40, linear structural comparison of whole genome sequences of pJN05NDM with the above plasmids was performed (Figure 2). The backbone of these plasmids showed high degrees of conservation and similarity, with sequence polymorphism at the region of additional insertion around the NDM gene. The plasmids did not carry any resistance genes other than NDM, except pRJA274. pRJA274 is a 53,134-bp circular IncX3 type plasmid haboring two resistance genes including bla NDM−1 and bla SHV−12 . pRJA274 is almost identical to pJN05NDM-7, but the ISAba125 element (935-bp) between IS3000 and IS5 at the nucleotide position 40,753 was missing. In addition, the backbone of pRJA274 shared identity with plasmid pIncX-SHV. Compared with pJN05NDM-7, the inserted sequence of pP744 was different, with a deletion of 543 bp at ISAba125 located downstream of IS5. In pZHDC40,

DISCUSSION
In this study, we aimed to evaluate the genotype and phenotype of bla NDM−7 -producing E. coli in China and found that E. coli carrying IncX3 plasmids play an important role as a reservoir and in the spread of bla NDM .
Although ST131 is the most prevalent strain type of E. coli worldwide, ST167 is considered to be related to clinical infections in China (Yang et al., 2014). In this study, JN05 assigned to ST167 was isolated from a 61-year-old woman with recurrent urinary tract infection. The isolate JN05 belonged to phylogroup A and was positive for papG II, which increased the ability of P-fimbriae adhesin (Wang et al., 2016). In addition, this isolate contained multiple resistance genes, including bla TEM−1 ,bla CTX−M−3 , bla CTX−M−14 , bla TEM−1 , qnrS, armA, and acc(6 )-Ib genes; therefore, showed multidrug resistance and increased resistance to β-lactam drugs.
According to the surveillance of CRE strains in China, bla NDM was mainly responsible for carbapenemase resistance in E. coli, while bla NDM−7 was relatively uncommon. Since the first report on clinical infection due to bla NDM−7 in France, this is the first report on fully sequenced plasmid carrying bla NDM−7 isolated from China.
Escherichia coli isolates carrying bla NDM−7 belonging to different STs were sporadic reported worldwide (Cuzon et al., 2013;Wang et al., 2016;Devanga Ragupathi et al., 2017;Pal et al., 2017;Espinal et al., 2018). According to previous reports, bla NDM−7 gene can be carried by several Enterobacter species and multiple types of plasmids including IncX3, IncF, and IncA/C groups, with sizes ranging from 37 to > 100 kb. IncX3, a selfconjugative plasmid, was most frequently observed to be the carrier of bla NDM−7 .
Interestingly, the plasmid pJN05 was identical to the plasmid pKW53T-NDM-7 isolated in Kuwait. Geographical contiguity or travel history could not be considered as a cause of resistance gene transmission, suggesting that the plasmids maybe native and not imported. We proved that plasmids harboring bla NDM were hidden in the environment and in the human gut worldwide long before we identified them. It is possible that IncX3 plasmids carrying different variants originated from the same plasmid, but point mutations during transmission and evolution generated the differences. Exposure to carbapenem agents speed up the evolution of plasmids carrying bla NDM variants and enhance enzyme activity toward carbapenems.
Notably, bla NDM -producing isolates of animal origin increased, indicating that food animals have become the reservoir of bla NDM (He et al., 2017;Kong et al., 2017). To understand the geographical distribution and gene polymorphism among the plasmids originating from different region, IncX3 plasmids carrying bla NDM isolated from different areas were compared. Six plasmids harboring bla NDM originating from pigs or chickens showed high similarity (> 99%) to those from patients. Because carbapenems were not approved for use in food animals in China, we assumed that the NDM-producing isolates were introduced to the farm via contaminated feed and water. The food animals contaminated by bacteria harboring bla NDM accelerated the spread of resistance genes among healthy population. Nonetheless, more data are needed to explain the dissemination of bla NDM among animals and humans.

CONCLUSION
In conclusion, this study identified self-transmissible IncX3 plasmids carrying bla NDM , which were disseminated in geographically segregated areas in China and other countries in the world. This study emphasizes the important role of IncX3 plasmids in transmission of bla NDM in China. Effective measures should be taken to monitor and control the rapid dissemination of bla NDM .

AUTHOR CONTRIBUTIONS
YH and YJ contributed to experiment conception, design, and wrote the paper. CS and YB performed data analysis.

FUNDING
The study was supported by grants from Focus on Research and Development Plan in Shandong Province (2016GSF201078) and the Medical and Health Development Plan of Shandong Province (2017WS471).

ACKNOWLEDGMENTS
We thank Professor Zhiming Lu for helpful discussion and continuous encouragement.