First Report of Complete Sequence of a blaNDM-13-Harboring Plasmid from an Escherichia coli ST5138 Clinical Isolate

Since the first report of blaNDM-1, 16 blaNDM variants have been identified among Gram-negative bacteria worldwide. Recently, a novel blaNDM variant, blaNDM-13, was identified in the chromosome of an ST101 Escherichia coli isolate from Nepal. Here we first reported plasmid-mediated blaNDM-13 in a carbapenem-resistant E. coli ST5138 clinical isolate associated with hospital-acquired urinary tract infection from China. blaNDM-13 and blaSHV-12 coexisted on the a ~54 Kb self-transferable plasmid. Compared with NDM-1, NDM-13, NDM-3, and NDM-4 had two amino acid substitutions (D95N and M154L), one amino acid substitution (D95N) and one amino acid substitutions (M154L), respectively. Complete plasmid sequencing showed that blaNDM-13-harboring plasmid (pNDM13-DC33) was highly similar to the blaNDM-1-harboring IncX3 plasmid pNDM-HN380, a common blaNDM-harboring vector circulating in China. In accordance with the structure of pNDM-HN380, pNDM13-DC33 consists of a 33-kb backbone encoding plasmid replication (repB), stability partitioning, and transfer (tra, trb, and pil) functions, and a 21-kb antimicrobial resistance region with high GC content between umuD and mpr genes. In conclusion, the present study is the first report of a plasmid-encoded blaNDM-13 and the complete sequence of a blaNDM-13-harboring plasmid (pNDM13-DC33). blaNDM-13 maybe originate from blaNDM-1 located on a pNDM-HN380-like plasmid by sequential mutations.


INTRODUCTION
Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumioniae, are common pathogens causing nosocomial infections. Carbapenems are the choice for the treatment of infections caused by multi-drug resistant Enterobacteriaceae, especially extended-spectrum β lactamase (ESBL)-and/or plasmid-mediated AmpC (pAmpC)-producing organisms (Tzouvelekis et al., 2012). The emergence of carbapenem-resistant K. pneumonia and E. coli producing carbapenemases (KPCs) and metallo-β-lactamases (MBLs) have become a major global health problem due to the limited number of effective antibiotic options to treat the infections caused by these multi-drug resistant Enterobacteriaceae (Tzouvelekis et al., 2012). In 2009, a novel MBL, named New Delhi metalloβ-lactamase-1 (NDM-1), was identified in a K. pneumoniae isolate from a Swedish patient who had returned from India with a urinary tract infection (Yong et al., 2009). Since then, NDM-1-producing Gram-negative isolates have emerged worldwide. NDM-1 was primarily identified in Enterobacteriaceae, especially in E. coli and K. pneumoniae, from the Indian subcontinent, Balkan states, the Arabian peninsula, and North Africa (Nordmann and Poirel, 2014). In China, NDM-1 was initially identified in 4 clonally unrelated Acinetobacter baumannii isolates in 2011 (Chen et al., 2011). Subsequently, this clinically important enzyme has spread among many species of Enterobacteriaceae in China (Hu et al., 2013;Liu et al., 2013;Zhang et al., 2013).
Since the first report of NDM-1, 16 NDM variants have been identified among Gram-negative bacteria worldwide (http:// www.ncbi.nlm.nih.gov/pathogens/submit_beta_lactamase/). Recently, a novel NDM variant, NDM-13, was reported in a multidrug-resistant E. coli clinical isolate in Nepal (Shrestha et al., 2015). The bla NDM-13 gene, interestingly, was found to locate within the chromosome of an E. coli ST101 isolate. The aim of the present study was to investigate whether bla NDM-13 was located on the plasmids of clinically isolated Enterobacteriaceae. We first found plasmid-mediated bla NDM-13 and completely sequenced a bla NDM-13 -harboring plasmid for the first time from a carbapenem-resistant E. coli ST5138 clinical isolate associated with hospital-acquired urinary tract infection in China.

Bacterial Strain
From Mar, 2014 to Oct, 2014, a total of 87 carbapenem-resistant Enterobacteriaceae (CRE) isolates causing clinical infections isolated from various specimens of patients at the First Affiliated Hospital of Wenzhou Medical University in Wenzhou, east China, were investigated for carbapenemase genes. The isolates were identified as E. coli by an automated microbiology analyzer (bioMe'rieux, Marcy l'Etoile, France) in accordance with the manufacturer's instructions.

Antimicrobial Susceptibility Testing
Gram-negative susceptibility (GNS) card on the Vitek system (bioMe'rieux, Marcy l'Etoile, France) was performed initially for antimicrobial susceptibility testing. Disk diffusion method was used for further confirmation and antimicrobial susceptibility results were interpreted according to the criteria recommended by Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2014). The E-test method was used for the determination of minimum inhibitory concentrations (MICs) of imipenem and meropenem for the E. coli isolate and its transconjugant. E. coli ATCC 25922 was used as control strain for antimicrobial susceptibility testing.

Detection of Carbapenemases and Extended-Spectrum β-Lactamases (ESBLs)
The modified Hodge test (MHT) was further performed on a Mueller-Hinton agar plate with ertapenem as substrate for the detection of carbapenemases as described previously (CLSI, 2014). MBLs were determined using a double-disc synergy test (Peleg et al., 2005). ESBLs were tested using the CLSIrecommended confirmatory double disk combination (CLSI, 2014).

Detection of Resistance Genes
The carbapenemase genes responsible for carbapenem resistance, including bla KPC , bla GES , bla SPM , bla IMP , bla VIM , bla SPM , and bla NDM , were detected using PCR and DNA sequencing as described previously (Queenan and Bush, 2007;Nordmann et al., 2011). ESBLs genes were detected in accordance with the method described previously (Andrade et al., 2010). PCR products were analyzed by electrophoresis in 1% agarose gels and were sequenced on both strands.

Transferability of Plasmids with Carbapenem Resistance
In order to determine whether carbapenem resistance was transferable in E.coli DC33 strain, filter mating conjugation was performed using E. coli 600 as the recipient as previously described (Wang et al., 2004). Plasmid DNA of E. coli DC33 strain was extracted with the QIAGENPlasmid Midi kit (Hilden, Germany) according to the manufacturer's instructions. The plasmid extracts were transferred into E. coli DH5α by using chemical transformation and transformants were selected on Luria-Bertani agar plates containing imipenem (0.5 µg/ml).

Determination of bla NDM-13 Location
The total bacterial DNA of E. coli DC33 was first prepared in agarose plugs, digested with S1 nuclease and further separated by pulsed-field gel electrophoresis (PFGE), as described previously (Chen et al., 2011). Then, the DNA bands were transferred horizontally to a nylon membrane (Millipore). A digoxigenin-labeled bla NDM-13 probe was used to hybridize with DNA bands and a nitro-blue tetrazolium/5-bromo-4-chloro-3 ′indolylphosphate color detection kit (Roche Applied Sciences) was applied to detected hybridization signals.

Sequencing a bla NDM-13 -Harboring Plasmid from the Transconjuguant of E. Coli DC33 Strain
In order to completely characterize the plasmid from the transconjugant of E. coli DC33 (designated as pNDM13-DC33), pNDM13-DC33 was isolated, purified, and sequenced using the Illumina MiSeq platform. The sequencing reads were de novo assembled, gaps between contigs were closed, open reading frames (ORFs) were predicted, and annotations were performed as described previously .

Carbapenemases and ESBLs Production and Detection of Resistance Genes
Among 87 CRE isolates, 7 were positive for bla NDM . After sequencing, E. coli strain DC33 was found to harbor bla NDM-13. E. coli strain DC33 was isolated from a urine culture of a 64year-old male hospitalized for prostatic hyperplasia in July, 2014. After hospitalized, the patient had the symptom of urinary tract infection. Subsequently, many white cells were found in urine sample under microscope. E. coli strain DC33 was isolated when the patient was hospitalized on day 8. E. coli DC33 was weakly positive for the MHT assay, but β-lactamase activity was inhibited by EDTA, indicating that E. coli DC33 produced a MBL. E. coli DC33 was also positive for CLSI-recommended confirmatory double disk combination test for detecting ESBLs. The results of detection of ESBL genes using PCR showed that E. coli DC33 was also positive for bla SHV while was negative for other resistance genes tested. After DNA sequencing, bla SHV was found to bla SHV-12.

MLST
MLST result showed E. coli DC33 belonged to ST5138, a single locus variant of ST617. Although ST5138 has been deposited in E. coli MLST database (http://mlst.warwick.ac.uk/mlst/dbs/ Ecoli), no study about E. coli ST5138 isolate is published. In our previous study, coexistence of bla NDM-1 and bla CMY-42 was found among E. coli ST167 clinical isolates in our hospital  (Zhang et al., 2013). As ST5138 was a single-locus variant of S167, we speculate that E. coli DC33 harboring bla NDM-13 is genetically related to E. coli ST 167 isolates carrying bla NDM-1 found in our previous study (Zhang et al., 2013). Recently, a Chinese study found an increasing prevalence of E. coli ST167 clinical isolates carrying both bla NDM-1 and bla NDM-5 on the conjugative IncX3 plasmid in various parts of China (Huang et al., 2016). Therefore, increasing emergence of bla NDM variants among E. coli ST167 and ST167 variants clinical isolates should be of concern. Up to now, bla NDM-13 was only reported in Nepal (Shrestha et al., 2015). The present study is the second report of this novel bla NDM variant.
Location of bla NDM-13 Gene and Transferability of Plasmids Carrying bla NDM-13 S1-PFGE result showed that a ∼54-Kb plasmid was found in E. coli DC33 (Figure 1). Subsequently, bla NDM-13 gene was found to be located on this plasmid, not on chromosome, which was confirmed by Southern-blot (Figure 1). The bla NDM-13harboring plasmid of E. coli DC33, designated as pNDM13-DC33, was successfully transferred into recipient E. coli 600 by filter mating conjugation. The antimicrobial resistance patterns of E. coli DC33 and its transconjugant were showed in Table 1. Shrestha et al found that bla NDM-13 was located within the chromosome (Shrestha et al., 2015). However, bla NDM-13 was first confirmed to be located on the plasmid in the present study.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org The chromosomal organization of the bla NDM-13 gene initially found in the E. coli isolate IOMTU558 from Nepal was similar to that in pNDM13-DC33, except for a 260-bp deletion in ISAba125 of 260 bp (353 to 94 bp upstream bla NDM-13 start codon). In contrast, the corresponding ISAba125 on pNDM13-DC33 was in full-length (1087 bp), but was interrupted by the insertion of an IS5 (at 265 bp upstream bla NDM-13 start codon) (Figure 1). A comparison of the chromosomal organization flanking bla NDM-13 in the E. coli isolate IOMTU558 (Shrestha et al., 2015) with plasmids harboring bla NDM identified the a set of ordered genes, tnpA-IS30-bla NDM-13 -bleMBL-trpF-dsbC-cutA-groES-groL, that were nearly identical in plasmid pPMK1 from Nepal, plasmidpKPX-1 from Taiwan, plasmid pNDM-MAR from Morocco, and in an Enterobacter hormaechei CCHB10892 plasmid from Brazil (Villa et al., 2012;Huang et al., 2013;Carvalho-Assef et al., 2014;Stoesser et al., 2014;Shrestha et al., 2015). This finding suggests that the chromosomal copy of bla NDM-13 may be the result of a rare integration event where a region of the plasmid recombined into the E. coli genome.
In conclusion, the present study is the first report of a plasmid-encoded bla NDM-13 and the complete sequence of a bla NDM-13 -harboring plasmid (pNDM13-DC33). bla NDM-13 maybe originate from bla NDM-1 located on a pNDM-HN380like plasmid by sequential mutations. The emergence of novel plasmid-mediated bla NDM variants, originating through the mutations in bla NDM from an epidemic plasmid, poses a concern that NDM variants with different β-lactamases hydrolytic activity will evolve.

Nucleotide Sequence Accession Number
The complete nucleotide sequences of plasmid pNDM13-DC33 has been deposited as GenBank accession no. KX094555.

ETHICAL APPROVAL
The Ethics Committee of the first Affiliated Hospital of Wenzhou Medical University exempted this study from review because the present study focused on bacteria.

AUTHOR CONTRIBUTIONS
JL, XQ, DZ, ZZ, YC, YG, and SW isolated bacteria and performed the laboratory measurements. FY and LW made substantial contributions to conception and design. LC, YT, and BK revised the manuscript critically for important intellectual content. LC and JL participated in experimental design and data analysis. FY drafted the manuscript. All authors read and approved the final manuscript.

FUNDING
This work was supported in part by National Institutes of Health (NIH) Grant R01AI090155 (to BK) and R21AI117338 (to LC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.