First identification of NDM-4-producing Escherichia coli ST410 in China

Dear Editor,

The worldwide dissemination of New Delhi metallo-β-lactamase 1 (NDM-1), an Ambler class B metallo-β-lactamase (MBL) conferring resistance to all β-lactams except monobactams, is of great concern for public health. NDM-4, which differs from NDM-1 by a single amino acid substitution (Met154Leu), was demonstrated to possess increased carbapenemase activity.¹ As an infrequent bla_NDM allele, only several sporadic cases of infections due to NDM-4-producing Escherichia coli have been described in India, Austria, and Europe.^{1, 2, 3} At present, only one NDM-4 plasmid (pJEG027; GenBank accession NO KM400601) has been characterized.⁴ To understand the epidemiology and control the spread of antibiotic-resistant pathogens, we have been investigating carbapenem-resistant Enterobacteriaceae from human patients in China. Previously, we reported a high incidence (33.3%) and endemic spread of NDM-1-positive Enterobacteriaceae in Henan Province.⁵ Here we describe the first NDM-4-producing E. coli ST410 isolate, which, to our knowledge, is the first case in China, and report the characterization of the bla_NDM-4 harboring IncX3 plasmid.

E. coli 14–55 was obtained from a blood culture of a 14-year-old girl hospitalized at The First Affiliated Hospital of Zhengzhou University, Zhengzhou city, Henan province, on 6 June 2014. The girl did not have a history of foreign travel. Susceptibility testing was initially performed by a Vitek 2 system (bioMérieux, Marcy l'Etoile, France), and Minimum Inhibitory Concentrations (MICs) were determined using the broth microdilution method and the agar dilution method (for fosfomycin) following the CLSI guidelines, and the MIC results were interpreted according to the CLSI breakpoints.⁶ The European Committee on Antimicrobial Susceptibility Testing breakpoints (available at http://www.eucast.org/clinical_breakpoints/) were used for colistin and tigecycline. E. coli 14–55 was resistant to all β-lactams, including imipenem and meropenem, fluoroquinolones, gentamicin and tetracycline, and was susceptible to amikacin, fosfomycin, tigecycline and colistin (Supplementary Table S1). The modified Hodge test and the imipenem-EDTA double-disk synergy test revealed that E. coli 14–55 was an MBL-producer.⁶ PCR and sequencing were performed to screen for the presence of MBL genes, including bla_IMP, bla_NDM, and bla_VIM; only bla_NDM was detected.⁵ The complete coding sequence (CDS) of bla_NDM was amplified with a pair of primers (NDM-F: 5′-GAA TTC GCC CCA TAT TTT TGC; NDM-R: 5′-AAC GCC TCT GTC ACA TCG AAA T) that flanked the bla_NDM gene, and DNA sequencing revealed the presence of bla_NDM-4 in E. coli 14–55. In addition, different β-lactamase encoding genes, including bla_CTX-M-15, bla_SHV-12, bla_TEM-1 and bla_CMY-61, together with the qnrB gene were identified in the NDM-4 positive isolate E. coli 14–55.⁵

E. coli phylogenetic group typing and multilocus sequence typing (MLST) showed that the isolate E. coli 14–55 belonged to phylogroup A and ST410.^{5, 7} At present, five E. coli STs (India: ST648; Italy and Denmark: ST405; Austria: ST167, ST4450, and ST101) have been identified carrying bla_NDM-4 worldwide.^{1, 2, 3} Thus, this study represents the first report of ST410 as a carrier of bla_NDM-4. Notably, recent publications from Germany and Italy described the clonal expansion of fluoroquinolone (FQ)-resistant CTX-M-15-producing E. coli ST410 in both human and animal populations.^{8, 9} Our previous and current studies also identified E. coli ST410 clinical isolates co-harboring multiple resistance determinants, including bla_NDM (bla_NDM-1/4), ESBL_S (bla_CTX-M-15, bla_SHV-12/bla_TEM-1) and ampC genes (bla_CMY-30/bla_CMY-61).⁵ In addition, NDM-5 producing E. coli ST410 was recently detected in Egypt.¹⁰ These findings suggest that E. coli ST410 might be an emerging pandemic clone similar to ST131 for the dissemination of CTX-M-15 or NDM encoding genes. This possibility remains to be examined from future surveillance work.

S1-PFGE and PCR-based plasmid replicon typing indicated E. coli 14–55 harbored three different plasmids that belonged to the IncX3, IncFIA and IncFIB incompatibility groups.^{5, 11} (Supplementary Figure S1). Southern hybridization using digoxigenin-labeled bla_NDM-1-specific probes (Roche Applied Sciences, Germany) showed that bla_NDM-4 was located on an approximately 50 kb plasmid in E. coli 14–55 (Supplementary Figure S1). Although the conjugal transfer of plasmids for E. coli 14–55 failed, the NDM-4 carrying plasmid belonging to the IncX3 incompatibility group was successfully transformed into E. coli DH5α Electro-Cells (TaKaRa, Dalian, China) by electroporation (Supplementary Figure S1). MICs for the transformant (named T55 in this study) are summarized in (Supplementary Table S1). To characterize the IncX3-type NDM plasmid, 12 pairs of primers were designed based on a reference IncX3 plasmid named pNDM-HN380 (NC_019162) for the mapping of the backbone regions (Supplementary Table S2), and the MDR regions were characterized by a primer walking method.

The bla_NDM-4 harboring plasmid in E. coli 14–55 (named pEC55-NDM-4) is 54 035 bp in size, with an average G+C content of 49.1%. The sequence data revealed that pEC55-NDM-4 is highly similar (>99.99%) to plasmid pNDM-HN380, which is the first characterized bla_NDM-1-harboring IncX3 plasmid of Klebsiella pneumoniae ST483 and was isolated in Hong Kong, with nucleotide changes from G to A at bp 4447, from T to G at 18 179 bp, from G to A at 21 656 bp, and from C to T at 23 844 bp (Figure 1).¹² The point mutation at position T18,179 G corresponds to the A460C mutation in bla_NDM-1, leading to the occurrence of the bla_NDM-4 allele. A comparative plasmid analysis between pEC55-NDM-4 and pJEG027, which is the only completely sequenced NDM-4 plasmid of a K. pneumoniae strain isolated in Austria, provided support for Espedido et al’s hypothesis that pJEG027 might have arisen from a pNDM-HN380-like plasmid ancestor (for example, pEC55-NDM-4) through the events of a different IS5 insertion and an IS26-mediated flanking deletion of cutA1-groL.⁴ Several recent studies revealed that the bla_NDM-4 gene was carried by plasmids belonging to different replicon types, including F, FII, L/M and X3 in E. coli and K. pneumoniae; however, only two partial sequences of IncFII plasmids (accession NO KP826711 and KP826707) and one partial sequence of an IncX3 plasmid (accession NO KP826709) except pJEG027 are available for the genetic context of bla_NDM-4.^{2, 3, 13} The second completely sequenced bla_NDM-4 carrying plasmid pEC55-NDM-4, in our study, is nearly identical to the first characterized bla_NDM-1-harboring IncX3 plasmid pNDM-HN380, further suggesting that bla_NDM-4 might have emerged on IncX3 plasmids via a point mutation in bla_NDM-1. The occurrence of a pNDM-HN380-like plasmid carrying bla_NDM-4, in this study, together with recent observations of IncX3-type plasmids carrying different bla_NDM alleles, including bla_NDM-1 (pNDM-HN380), bla_NDM-4 (pJEG027), bla_NDM-5 (pNDM_MGR194, pNDM5_0215 and pEc1929), and bla_NDM-7 (pEC50-NDM7), in different countries (Austria, India, China and Canada) and different species (K. pneumoniae, E. coli, Serratia marcescens, and Enterobacter hormaechei),^{4, 12, 14, 15} strongly indicates that IncX3 plasmids, which have a narrow host range in Enterobacteriaceae, might have played a major role in the rapid global dissemination of NDM-type MBLs among Enterobacteriaceae.

Figure 1 Comparison of linear plasmid maps of IncX3 plasmids harboring bla_NDM: pNDM-HN380 (bla_NDM-1, JX104760), pEC55-NDM-4 (bla_NDM-4, KX470734), pJE027 (bla_NDM-4, KM400601). Light gray shading indicates homologous regions, while dark gray shading indicates inversely displayed regions of homology. The different arrows indicate the positions, directions of transcription and predicted function of the genes.

In summary, our study represents the first report of a NDM-4 producing E. coli isolate recovered from a blood culture of a patient without a history of foreign travel. The FQ-resistant E. coli 14–55 with multiple β-lactamase encoding genes, including bla_NDM-4, bla_CTX-M-15, bla_SHV-12, bla_TEM-1 and bla_CMY-61, belonged to ST410, a potential international clone for the dissemination of CTX-M-15. Further surveillance is thus warranted to monitor the future dissemination of potentially endemic clones of ST410 that harbor bla_NDM. The bla_NDM-4 carrying IncX3 plasmid characterized in this study is nearly identical to pNDM-HN380 (with bla_NDM-1), which was reported in Hong Kong. This finding, together with recent observations of IncX3 plasmids carrying different bla_NDM alleles in different countries, further suggests that IncX3 plasmids might have become a common vehicle for the dissemination of different NDM alleles among Enterobacteriaceae worldwide.

This work was supported by grant 81501782 from the National Natural Science Foundation of China, grant 152300410034 from the Research Project of Basic and Advanced Technology of Henan Province, and grant 15B310014 from the Foundation of Henan Educational Committee. Nucleotide sequence accession number: The sequence of the bla_NDM-4 carrying plasmid pEC55-NDM-4 was submitted to the GenBank database under the accession number KX470734.

Supplementary Information for this article can be found on the Emerging Microbes & Infections website (http://www.nature.com/emi)