Identification and Characterization of the First Escherichia coli Strain Carrying NDM-1 Gene in China

New Delhi metallo-β-lactamase-1 (NDM-1), an acquired class B carbapenemase, is a significant clinical threat due to its extended hydrolysis of β-lactams including carbapenems. In this study, we identified the first confirmed clinical isolate of Escherichia coli BJ01 harboring bla NDM-1 in China. The isolate is highly resistant to all tested antimicrobials except polymyxin. bla NDM-1, bla CTX-M-57, and bla TEM-1 were identified in the isolate. bla NDM-1 was transferable to E. coli EC600 and DH5α in both plasmid conjugation experiments and plasmid transformation tests. BJ01 was identified as a new sequence type, ST224, by multilocus sequence typing. Analysis of genetic environment shows complex transposon-like structures surrounding the bla NDM-1 gene. Genetic analysis revealed that the region flanking bla NDM-1 was very similar to previously identified NDM-positive Acinetobacter spp. isolated in China. The findings of this study raise attention to the emergence and spread of NDM-1-carrying Enterobacteriaceae in China.


Introduction
Antimicrobial resistance is a growing global challenge to human health [1]. The emerging New Delhi metallo-b-lactamase (NDM-1), an acquired class B carbapenemase that was first clinically detected in a patient at a hospital in New Delhi, India, has brought up worldwide public attention again [2][3][4]. It confers resistance to a broad-spectrum of b-lactams, including carbapenems, which are the mainstream treatment for antibiotic-resistant bacterial infections [5]. Although current reports indicate that NDM-1 does not hydrolyze monobactams, most of NDM-1-carrying strains also express enzymes that could hydrolyze monobactams, making NDM-1-producers very difficult to control [6]. The rapid dissemination of NDM-1-producing gram-negative species also contributes to this major concern of public health. It has been reported in .50 countries across five continents, in the last 2 years [2,4].
NDM-1 is detected mainly in Escherichia coli and Klebsiella pneumonia but occasionally in Klebsiella oxytoca, Citrobacter freundii, Morganella morganii, Providencia spp., Enterobacter cloacae, Proteus spp., Acinetobacter baumannii, Stenotrophomonas maltophilia and Pseudomonas aeruginosa [3,7,8]. To our knowledge, NDM-1-producing strains have been seen only in Acinetobacter spp. [9][10][11][12] and Enterococcus faecium [13] in China. There is no evidence of the emergence of NDM-1producing Enterobacteriaceae in China at this point, although it was found in the stool samples of a 1-year-old infant and his mother in Hong Kong who once traveled to and were hospitalized in Hunan Province, China [14]. In the present study, we report the first confirmed case of NDM-1-producing E. coli infection in Beijing, China.

Patient Information
A 75-year-old Chinese patient with diabetes, who came from the Anhui province in southern China, was admitted for diabetesrelated foot complications comprising a swollen peak of the left foot and ulceration of the fourth and fifth toes on September, 2011. The NDM-1-producing E.coli, BJ01, strain was isolated from the ulcer secretion. Since the strain was found to be resistant to almost all tested antibiotics, a combination of intravenous levofloxacin and etimicin was given empirically. Amputation of the fifth metatarsal bones and an incisional drainage operation were performed. The wound started healing nicely. The patient was discharged from the hospital one month after admission. Neither the patient nor the patient's family members had a history of traveling to the Indian subcontinent or other countries. The patient in this manuscript has given written informed consent, as outlined in the PLOS consent form to publication of their case details.

Bacterial Isolate and Phenotypic Screening for MBL
The clinical isolate BJ01 of the metallo-b-lactamase(MBL)producing E. coli was derived from the ulcer secretion of the patient collected on the admission day. The strain with same phenotype was still positive on day 5 after the operation. The identification and drug susceptibility of the bacteria were tested by using Vitek 2 Compact (bioMerieux, Marcy I''Etoile, France). Minimal inhibitory concentrations (MICs) of 18 antibiotics and combinations of antimicrobials were determined by broth microdilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [15]. Most of antimicrobial agents were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), except for imipenem (Merck Sharp & Dohme, Whitehouse Station, USA), meropenem (Dainippon Sumitomo Pharma, Chuo-ku, Japan) and polymyxin (Sigma, St. Louis, USA). E.coli strain ATCC25922 was used as a quality control for MIC determination. MBL production was tested by comparing diameters of the zones of inhibition for meropenem (10 mg) and imipenem (10 mg) on Mueller-Hinton agar (MHA) versus MHA impregnated with EDTA, an MBL inhibitor. EDTA-impregnated MHA was prepared by spreading 2 mL of 5 mM EDTA on MHA [16].

Southern Blot
Southern blot was performed by using DIG High Prime DNA Labeling and Detection Starter Kit II (F. Hoffmann-La Roche Ltd., Basel, Swiss) following manufacturer's protocol. Briefly, genomic DNA was digested with restriction enzyme HindIII and BamHI. The digested DNA fragments were transferred to nylon membrane (Pharmacia & Biotech, Piscataway, USA), and hybridized with digoxigenin-labeled bla NDM-1 -specific probes and anti-DIG-AP antibody. The membrane was visualized by applying chemiluminescent substrate CSPD and X-ray exposure.

Conjugation and Transformation
The transfer of carbapenem resistance was tested using a conjugation test (broth mating method). E. coli EC600 (LacZ 2 Nal r Rif r ) was used as the recipient strain. Overnight cultures of the donor strain (300 mL) and recipient strain (100 mL) were mixed with 600 mL of fresh Mueller-Hinton broth and were incubated overnight at 35uC. The mixture was then inoculated on MHA plates containing rifampin (Sigma, St. Louis, USA, 100 mg/mL) plus ceftriaxone (F. Hoffmann-La Roche Ltd., Basel, Swiss, 4 mg/ mL) for 24 h at 35uC. The bacterial colonies were then transferred to a plate containing rifampin (Sigma, St. Louis, USA, 100 mg/ mL) plus imipenem (Merck Sharp & Dohme, Whitehouse Station, USA, 4 mg/mL) for 24 h at 35uC by replica plating, which maintains the original colony pattern.
Plasmid DNA was extracted using a Qiagen Mini Kit (Qiagen, Hilden, Germany) and was transformed into E. coli DH5acompetent cells (TAKARA, Shanghai, China). Transformants were selected on MHA plates containing IPM (4 mg/mL).
The identification and susceptibility of the transconjugant and transformant were confirmed via using the VITEK 2 system. PCR analysis was used to determine the presence of carbapenemase genes.

Multilocus Sequence Typing (MLST) and Sequencing of Genetic Environment
MLST with seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) was performed according to the protocol described on the E. coli MLST website (http://mlst.ucc.ie/mlst/dbs/Ecoli). The regions flanking the bla NDM-1 gene were sequenced by primer walking strategy, starting from each end of the bla NDM-1 gene in the DH5a NDM-1 transformant. The plasmid containing bla NDM-1 gene was used as a template for primer walking. The first set of primers was designed targeting each end of the bla NDM-1 sequence (59-GGTCGCCAGTTTCCATTTGC-39 and 59-TGCCGA-CACTGAGCACTAC-39). The amplification products were sequenced. Then new primers complementary to the known sequence were designed and synthesized for sequencing the unknown DNA sequence next to it. Totally, 13 pairs of primers were synthesized. Sequence files were assembled and aligned by using ContigExpress software.

MLST genotype analysis
MLST revealed that E. coli BJ01 belonged to a new sequence type 224 (ST224).

Genetic Environment of the bla NDM-1 gene
A 17,924-bp fragment was obtained from plasmid DNA of strain E. coli BJ01, and sequenced by primer walking. The sequence has been deposited in GenBank with accession No. JX296013. Sequencing of the bla NDM-1 upstream region identified the presence of an ISAba125 insertion sequence. Further sequence analysis revealed that the region flanking the ISAba125 was the IS3000 gene. The trpF gene flanked the 39 end of bla NDM-1 , followed by dsbC, truncated cut1 gene disrupted by IS26, umuD and a transposase gene. (Fig. 4)

Discussion
In contrast to most other countries where NDM has been mainly identified in Enterobacteriaceae, in China to date NDM has only been reported in Acinetobacter [9,11,12,24] and Enterococcus faecium [13]. However, other types of MBLs, such as IMP, and other classes of acquired carbapenemases, such a KPC, have been reported in Enterobacteriaceae in China [25][26][27][28][29], but isolates of NDM-1-producing E.coli have not previously been identified in China.
NDM-1-harboring strains could be highly multidrug resistant [3]. Previous reports on NDM-1-producing Acinetobacter spp. have demonstrated that strains resistant to all available antimicrobials except colistin were common in China [10,30]. Our results showed resistant to almost all tested antimicrobials of the identified NDM-1-producing E.coli isolate. The patient had a good general condition and the infection was localized. Ultimately, the patient survived the infection. But this case should have raised public concerns again over increasing incidence of highly multidrug resistant NDM-1-harboring strains in China.
Travelers contribute significantly to the global movement of microbes and resistance genes [31]. Although nosocomial trans-  First NDM-1-Producing E. coli Strain in China PLOS ONE | www.plosone.org mission of NDM-1 has occurred in many countries [26,32,33], medical tourism plays a significant role in the spread of NDM-1 [34], and traveling to the Indian subcontinent is a significant risk factor of infection with an NDM-1-producing bacterium [35]. Patients in China who were found to have bla NDM-1 -carrying bacteria had no history of traveling to the Indian subcontinent or another country [9,11,13,24]. In this study, the patient carrying the bla NDM-1 -positive E. coli strain had no foreign travel history.
The identified BJ01 strain demonstrated phenotypes of both ESBL and NDM-1 ( Table 1). This is consistent to previous reports on NDM-1-producers. For example, a study from China indicates that all 4 identified NDM-1-carrying A. baumannii isolates expressed both genes [30]. Data from the SMART study shows that bla NDM-1 positive enterobacteriaceae isolates often carry additional b-lactamase genes. Among 33 bla NDM-1 -carrying strains, bla CTX-M-15 is detected in 30 [36]. However, our data also suggested that ESBL and NDM-1 genes may be carried by distinct plasmids and could be transferred separately (Fig. 1). This has to be further confirmed by sequence analysis.
Analysis of the genetic environment of bla NDM-1 in BJ01 reveals that the region flanking bla NDM-1 is very similar to some Acinetobacter spp. isolated in China [10,11,24] and E. coli plasmid pNDM102337 (GenBank: JF714412) isolated in Canada, in which DsbC and cutI are located downstream of the bla NDM-1 . cutI is truncated by a transposon-like structure. In contrast with E. coli HK-01 (GenBank: HQ451074) and E. coli DVR22 (GenBank: JF922606.1), there was no bla DHA downstream of bla NDM-1 in BJ01 (Fig. 4). This finding indicates a potential link between Acinetobacter spp. and Enterobacteriaceae in term of the bla NDM-1 gene in China. More complex transposon-like structures are detected around bla NDM-1 . IS3000 and ISAba125 are located upstream of the bla NDM-1 , while IS26 and a transposase gene are located downstream of the bla NDM-1 . Transposons should play an important role in horizontal transfer of the bla NDM-1 between different species of bacteria, such as from Acinetobacter spp. to E. coli.
In conclusion, in mainland China, this is the first reported case of infection due to a NDM-1-producing E. coli, which is highly drug-resistant and comes with a new MLST genotype in E. coli. The patient carrying this strain does not have a travel history The detection of the NDM-1-positive E. coli isolate in our study indicates immediate importance of strengthening surveillance to prevent the nosocomial infection and dissemination of NDM-1 in China.