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Infection and Drug Resistance logoLink to Infection and Drug Resistance
. 2019 Jun 6;12:1555–1564. doi: 10.2147/IDR.S206211

Characterization of a NDM-7 carbapenemase-producing Escherichia coli ST410 clinical strain isolated from a urinary tract infection in China

Juan Xu 1, Fang He 2,
PMCID: PMC6559143  PMID: 31239731

Abstract

Purpose: The emergence of New Delhi metallo-beta-lactamase (NDM) carbapenemase-producing Escherichia coli leaves few therapeutic options. Infections due to NDM-7 carbapenemase-producing E. coli are infrequent. In this study, we report the whole-genome sequence of an NDM-7 carbapenemase-producing E. coli belonging to sequence type (ST) 410 isolated from a patient with a urinary tract infection in China.

Patients and methods: The NDM-7 producing E. coli strain EC25 was isolated from a urine sample of a male patient hospitalized in a tertiary hospital in Zhejiang Province of China. Susceptibility assay of antibiotics was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The whole genome of the strain was sequenced, and the blaNDM-7-harboring plasmid was analyzed. The genomic characterization and molecular epidemiology of the strain were further elucidated.

Results: E. coli EC25 was resistant to all antimicrobials tested, except tigecycline and colistin. The whole genome of E. coli EC25 was composed of one chromosomal DNA and five plasmids. Four virulence factors and twenty-five antimicrobial resistance genes, including blaNDM-7, were identified. Resistance genes were all located in an IncF-type plasmid (pEC25-1), except blaNDM-7, which was located in an individual IncX3-type plasmid (pEC25_NDM-7). Twenty-one phylogenetically related strains were identified. The phylogenetically related E. coli ST410 strains exist globally. The closest relative strain of EC25 was a strain isolated from Sichuan province of China in 2016, with a similar IncX3-type plasmid that encoded blaNDM-5.

Conclusion: Our study reports the emergence of an E. coli ST410 strain harboring blaNDM-7 in China. This strain may evolve as a successful epidemic clone of NDM-producing E. coli in China, and the blaNDM gene is prone to mutate during its dissemination.

Keywords: Escherichia coli, blaNDM-7, IncX3 type plasmid, carbapenem resistant, ST410

Introduction

Escherichia coli is a member of Enterobacteriaceae, and it is an important opportunistic pathogen. E. coli is the leading cause of urinary tract infections and the cause of a variety of other infections, including liver abscesses and bacteremia. E. coli that carry extended-spectrum β-lactamase (ESBL) are very common.1 Besides tigecycline and colistin, carbapenems are often the last resort for treating infections due to ESBL-producing E. coli. However, the emergence of carbapenemase has contributed to resistance to all β-lactams including carbapenems. New Delhi metallo-beta-lactamase (NDM) is one of the most important carbapenemases. Since the first report of blaNDM-1 in 2009,2 24 NDM variants (NDM-1 to NDM-24) were identified.3 NDM spread among Enterobacteriaceae and other Gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii,4,5 and E. coli is the predominant carrier of blaNDM.

NDM-7 differs from NDM-1 by the substitution of two amino acids at positions 130 (Asp→Asn) and 154 (Met→Leu). NDM-7 was first described in an E. coli strain in Germany in 2013, and it exhibited higher carbapenemase activity compared to NDM-1.6 NDM-7 was reported sporadically in many countries.713 NDM-7-producing E. coli was first reported in 2016 in China. blaNDM-7 was located on a conjugative IncX3-type plasmid in five clinically isolated E. coli strains of sequence types ST131 and ST650 in the Jiangxi Province.14 Yingying Hao et al recently reported an NDM-7-producing E. coli ST167 strain isolated from a urine sample in the Shandong Province of China, and blaNDM-7 was also located on a conjugative IncX3-type plasmid.15 IncX3-type plasmids likely play an important role in the distribution of blaNDM-7 in China. Considering the global emergence of NDM-7, an epidemiological survey and analysis of blaNDM-7-harboring strains are urgently needed to prevent its future prevalence.

The present study isolated an NDM-7-producing E. coli strain from a male patient hospitalized in a tertiary hospital in the Zhejiang Province of China. The entire genome of the strain was sequenced, and the blaNDM-7-harboring plasmid was analyzed. Genomic characterization and molecular epidemiology of the strain were further elucidated.

Material and methods

Patient and isolate

A 57-year-old male patient diagnosed with pulmonary infection was hospitalized in a tertiary hospital in the Zhejiang Province of China on August 14, 2017. The patient had a pulmonary infection and comorbidities, including hypoxic-ischemic encephalopathy, hypertension, and hypertensive heart disease. During his hospitalization, the patient received an indwelling urinary catheter. Routine urine tests suggested a urinary tract infection, and carbapenem-resistant E. coli was isolated from a urine sample on August 17, 2017. The strain was preliminarily identified using the VITEK MS system (bioMérieux, France) and further confirmed using 16S rRNA gene sequencing.

Antimicrobial susceptibility test

A susceptibility assay to antibiotics was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentrations (MICs) of tigecycline and colistin (Sigma-Aldrich, St. Louis, MO, USA) were determined using standard broth microdilution tests with fresh Mueller-Hinton broth (Cation-adjusted, Oxoid LTD, England). The MICs of other antimicrobial agents were determined using the Etest method. Antimicrobial susceptibility was determined using the breakpoints approved by the CLSI, and the US Food and Drug Administration (FDA) breakpoints were used for tigecycline.

Whole-genome sequencing

Genomic DNA of the isolate was extracted using a QIAamp DNA MiniKit (Qiagen, USA) and subjected to whole-genome sequencing (WGS) using the Illumina HiSeqTM 4000 platform (Illumina, San Diego, CA, USA). The short reads generated were de novo assembled into contigs using SPAdes. To obtain the complete sequence, EC25 was subjected to sequencing using the long-read MinION Sequencer (Nanopore, Oxford, UK). The MinION sequencing libraries were prepared using the rapid barcoding kit and loaded onto a single MinION R9.4 flow cell. The MinION reads were base-called with Albacore v2.0.2 and generated 2.4 Gbp of data. Raw and trimmed Illumina reads were assessed using FastQC v0.11.7, and quality trimming was performed using Trimmomatic v0.32. The MinION reads were demultiplexed and quality-trimmed using Porechop v0.2.3 with the default settings. The de novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler (v0.4.7; parameters: -min_component_size 500-min_dead_end_size 500-verbosity 1-mode conservative) under conservative mode for increased accuracy. Complete circular contigs generated were corrected using Pilon with Illumina reads for several rounds until no change was detected. We obtained one circular contig, which was represented by a complete chromosome with a size of 4,782,653 bp and five plasmids of 227,349 bp, 46,161 bp, 5,167 bp, 4,991 bp, and 3,373 bp. The whole-genome sequence was annotated in the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) server.

Identification of antimicrobial resistance genes

Acquired antimicrobial resistance genes were identified using ResFinder 3.0 with a 99% threshold for gene identification and a 100% minimum length via depositing of the entire genome sequence into the database.16 The carbapenem-resistant gene blaNDM-7 was confirmed using PCR and Sanger sequencing.

Genomic characterization

Multilocus sequence typing (MLST) of the isolate was analyzed using the BacWGSTdb server of the entire genome sequence.17 Virulence genes and plasmid replicon type were analyzed using VirulenceFinder 1.5 and PlasmidFinder 1.3.16 Further bioinformatics analysis, such as identification of insertion elements (IS), clustered regularly interspaced short palindromic repeat (CRISPR) sequences and secondary metabolite gene clusters, were predicted using applications of ISfinder, CRISPRFinder, and antiSMASH tools, respectively.18,19

Plasmid analysis

The plasmid sequences were annotated in the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) server. A graphical map of the blaNDM-7-carrying plasmid was converted in the CGView Server and completed with labels and footnotes.20

Phylogenetic relationship analysis

The phylogenetic relationship between EC25 and other E. coli strains was analyzed using the BacWGSTdb server and the entire genome sequence.17 The BacWGSTdb server offers SNP and genome MLST approaches to investigate the phylogenetic relationship of the uploaded genome sequence with sequences deposited in the BacWGSTdb. The database currently contains 10,545 E. coli strains, including 77 strains of ST410. Scheme cgMLST was used with a 200 threshold for phylogenetic analysis.

Nucleotide sequence accession numbers

The complete sequence of strain E. coli EC25 and plasmids were submitted to GenBank under accession number CP035123-CP035128.

Ethical approval

The urine sample and clinical isolate of E. coli EC25 were generated as part of routine hospital laboratory procedures. This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Zhejiang Provincial People’s Hospital, China. Written informed consent was obtained from the patient, which included publication of the case details.

Results and discussion

The MICs of strain EC25 to different antibiotics are presented in Table 1. The strain was resistant to all antimicrobials tested except tigecycline and colistin.

Table 1.

MICs of the antibiotics tested in E. coli EC25

Antibiotics MIC(mg/L)
Cefazolina >256
Ceftriaxonea >256
Cefotaximea >256
Cefepimea >256
Imipenema >32
Meropenema >32
Aztreonama >32
Amoxicillin/clavulanatea >256
Cefoperazone/sulbactama >256
Ciprofloxacina >32
Gentamicina >256
Tetracyclinea >256
Tigecyclineb 0.25
Colistinb 0.25

Notes: aTested by Etest method. bTested by standard broth microdilution tests.

The whole-genome sequence of E. coli EC25 was composed of one chromosomal DNA that comprised 4,782,653 bp and five plasmids of 227,349 bp, 46,161 bp, 5,167 bp, 4,991 bp, and 3,373 bp. The chromosomal DNA contained 88 tRNA genes, 3 rRNA operons, and 4,707 protein-coding sequences, which were identified using the PGAP server. The MLST scheme revealed that EC25 belonged to sequence type ST410. The genome contained two confirmed CRISPRs (CRISPR1 start position and end position: 1,042,955–1,043,471, CRISPR length: 516; CRISPR2 start position and end position: 1,065,855–1,066,433, CRISPR length: 578). Several IS elements were found in the genome, and most belonged to the IS3 and IS5 families. Four secondary metabolite regions, nrps-t1pks, thiopeptide, NRPS, and siderophore, were also identified.

Four virulence factors were found in the genome (Table 2), which were two copies of gad (glutamate decarboxylase), a single copy of iss (increased serum survival) and lpfA (long polar fimbriae). The distribution of the resistance genes in the genome of the strain EC25 is presented in Table 3. We identified the aminoglycoside resistance genes aph(3ʹʹ)-Ib, aph(6)-Id, aac(3)-IId, aadA16, aac(6ʹ)-Ib-cr, and aadA5, the beta-lactam resistance genes blaNDM-7, blaOXA-1, blaTEM-1B, and blaCTX-M-3, the fluoroquinolone resistance genes qnrB2, aac(6ʹ)Ib-cr and qnrS1, the rifampicin resistance gene arr-3, the macrolide, lincosamide and streptogramin B resistance gene mph(A), the sulfonamide resistance genes sul1and sul2, the tetracycline resistance gene tet(A), and the trimethoprim resistance genes dfrA27 and dfrA17. Except for blaNDM-7, all of the resistance genes were located in the IncF-type plasmid pEC25-1, including two copies of aac(6ʹ)Ib-cr and mph(A) and three copies of sul1. The carbapenem-resistant gene blaNDM-7 was located in an individual plasmid, pEC25_NDM-7.

Table 2.

Virulence genes in strain E. coli EC25

Virulence factor Identity Query/template length Contig Position in contig Protein function Accession number
gad 100 1401/1401 EC25 251,983..253,383 Glutamate decarboxylase AP010953
gad 99.79 1401/1401 EC25 2,444,792..2,446,192 Glutamate decarboxylase CP002967
iss 98.64 294/294 EC25 3,714,038..3,714,331 Increased serum survival CP004009
lpfA 100 573/573 EC25 4,749,714..4,750,286 Long polar fimbriae AY646923

Table 3.

Distribution of resistance genes in E. coli strain EC25

Resistance gene %identity HSP length/query Contig Position in contig Predicted phenotype Accession number
Aminoglycoside
aph(3ʹ’)-Ib 100 804/804 pEC25-1 94,244..95,047 Aminoglycoside resistance AF321551
aph(6)-Id 100 837/837 pEC25-1 95,047..95,883 Aminoglycoside resistance M28829
aac(3)-IId 99.88 861/861 pEC25-1 97,435..98,295 Aminoglycoside resistance EU022314
aadA16 99.65 846/846 pEC25-1 111,488..112,333 Aminoglycoside resistance EU675686
aac(6ʹ)-Ib-cr 100 600/600 pEC25-1 113,669..114,268 Fluoroquinolone and aminoglycoside resistance DQ303918
aadA5 100 789/789 pEC25-1 204,055.204,843 Aminoglycoside resistance AF137361
aac(6ʹ)-Ib-cr 100 600/600 pEC25-1 208,288.208,887 Fluoroquinolone and aminoglycoside resistance DQ303918
Beta-lactam
blaNDM-7 100 813/813 pEC25_NDM-7 44,052..44,864 Beta-lactam resistance JX262694
blaOXA-1 100 831/831 pEC25-1 209,018..209,848 Beta-lactam resistance HQ170510
blaTEM-1B 100 861/861 pEC25-1 214,606.215,466 Beta-lactam resistance AY458016
blaCTX-M-3 100 876/876 pEC25-1 216,248..217,123 Beta-lactam resistance Y10278
Fluoroquinolone
qnrB2 99.84 645/645 pEC25-1 105,800..106,444 Fluoroquinolone resistance DQ351242
aac(6ʹ)-Ib-cr 100 600/600 pEC25-1 113,669..114,268 Fluoroquinolone and aminoglycoside resistance DQ303918
aac(6ʹ)-Ib-cr 100 600/600 pEC25-1 208,288..208,887 Fluoroquinolone and aminoglycoside resistance DQ303918
qnrS1 100 657/657 pEC25-1 221,088..221,744 Fluoroquinolone resistance AB187515
Rifampicin
arr-3 100 453/453 pEC25-1 113,120..113,572 Rifampicin resistance JF806499
MLS - Macrolide, Lincosamide and Streptogramin B
mph(A) 100 906/906 pEC25-1 99,354..100,259 Macrolide resistance D16251
mph(A) 100 906/906 pEC25-1 196,453..197,358 Macrolide resistance D16251
Sulphonamide
sul2 100 816/816 pEC25-1 93,368..94,183 Sulphonamide resistance HQ840942
sul1 100 840/840 pEC25-1 104,470..105,309 Sulphonamide resistance U12338
sul1 100 840/840 pEC25-1 110,191..111,030 Sulphonamide resistance U12338
sul1 100 840/840 pEC25-1 202,669..203,508 Sulphonamide resistance U12338
Tetracycline
tet(A) 100 1200/1200 pEC25-1 118,110..119,309 Tetracycline resistance AJ517790
Trimethoprim
dfrA27 100 474/474 pEC25-1 112,514..112,987 Trimethoprim resistance FJ459817
dfrA17 100 474/474 pEC25-1 204,974..205,447 Trimethoprim resistance FJ460238

The plasmid profile of pEC25_NDM-7 is presented in Figure 1. It is an IncX3-type plasmid composed of 46,161 bp. The carbapenem-resistant gene blaNDM-7 was preceded by Tn3-IS3000-IS30-IS5 in the upstream region and followed by bleMBL-trpF-dsbC-cutA-IS26 in the downstream region. The similarity of pEC25_NDM-7 to other blaNDM-harboring plasmids was analyzed using Basic Local Alignment Search Tool (BLAST). pEC25_NDM-7 was 99% identical to several previously reported plasmids, namely, pJN05NDM7 (Accession No. MH523639), pAD-19R (Accession No. KX833071), pNDM-20 (Accession No. MF458176), pM216_X3 (Accession No. AP018146), and pKW53T-NDM (Accession No. KX214669). Plasmid pJN05NDM7 (carrying blaNDM-7) was identified in an E. coli ST167 clinical strain isolated from a patient with a urinary tract infection at a teaching hospital in the Shandong Province of China in 2015.15 Plasmid pAD-19R (carrying blaNDM-17) was identified in an E. coli ST48 strain isolated from a chicken at a commercial poultry farm in the Shandong Province of China in 2015.21 Plasmid pNDM-20 (carrying blaNDM-20) was identified in an E. coli ST1114 strain isolated from a swine faecal swab collected from a commercial pig farm in the Shandong province of China in 2016.22 Plasmid pM216_X3 (carrying blaNDM-4) was identified in an E. coli ST101 clinical strain isolated from a urine specimen at a tertiary care hospital in Yangon, Myanmar, in 2015.23 Plasmid pKW53T-NDM (carrying blaNDM-7) was identified in an E. coli ST448 clinical strain isolated from a urine sample in Kuwait in 2012.12 These plasmids encode distinct blaNDM variants, and it is likely that blaNDM mutates from blaNDM-1 to other blaNDM variants via nucleic acid replication in the 46,161 bp IncX3-type plasmid to evolve higher carbapenemase activity. Notably, the 46,161 bp IncX3-type plasmid does not carry antimicrobial resistance genes other than blaNDM, and the plasmid appeared in different ST type of E. coli strain across different countries in humans and animals. It seems the structure of the 46,161 bp plasmid is stable and suitable for the horizontal transfer of blaNDM, which plays an important role in horizontal transmission of NDM carbapenemase worldwide.

Figure 1.

Figure 1

Profile of the blaNDM-7-encoding plasmid pEC25_NDM-7.

The phylogenetic relationship between E. coli EC25 and other E. coli strains is presented in Table 4 and Figure 2. Twenty-one phylogenetically related strains were identified in the database, all of which belong to ST410. Most of these strains produce ESBL, and only six strains produced carbapenemase, including one KPC-2-producing strain, one VIM-4-producing strain, and four NDM-producing strains. The phylogenetically related E. coli ST410 strains are spread globally. Seven strains were isolated from Germany, five strains from the USA, four strains from China, two strains from Norway, and one strain each from Argentina, Brazil, and India. Of the four strains from China, two of the strains were isolated from the Zhejiang province, one strain was from the Jiangsu province, and one strain was from the Sichuan province. The strain closest to EC25 was E. coli SCEC020001, which was isolated from the Sichuan province of China in 2016. There are only 38 different alleles between E. coli EC25 and E. coli SCEC020001. E. coli SCEC020001 also harbors a 46,161 bp IncX3-type plasmid (pNDM5_020001, Accession No. CP032424) that encodes blaNDM-5. pNDM5_020001 was 99% identical to pEC25_NDM-7. The main difference between the two plasmids was the subtype of blaNDM; pNDM5_020001 encoded blaNDM-5 and pEC25_NDM-7 encoded blaNDM-7. There is a geographic distinction between the Zhejiang province and Sichuan province, and the patient did not have a travel history out of Zhejiang province in recent years, which suggests that the NDM-positive E. coli ST410 strain EC25 was locally acquired, and the blaNDM-7 subtype may evolve during local dissemination.

Table 4.

Information of close isolates (based on cgMLST strategy) to strain E. coli EC25

Isolate Accession number ST Host Isolation source Country state Collection year Major resistance genotype Different alleles Reference
EC25 CP035123 410 Homo sapiens Urine China: Zhejiang 2017 blaNDM-7 0 This study
SCEC020001 CP032426 410 Homo sapiens N/Aa China: Sichuan 2016 blaNDM-5 38 Unpublished
IMT31352 LJGF01 410 Dog Feces Germany 2013 ESBL 96 25
IMT28764 LJGC01 410 Mute swan Feces Germany 2012 ESBL 97 25
IMT33180 LJGI01 410 Homo sapiens Blood Germany 2009 ESBL 99 25
IMT28707 LJGB01 410 Mute swan Feces Germany 2012 ESBL 99 25
IMT33204 LJGK01 410 Homo sapiens Blood Germany 2010 ESBL 103 25
IMT33181 LJGJ01 410 Homo sapiens Blood Germany 2009 ESBL 118 25
Ecol_517 CP018965 410 Homo sapiens N/Aa Brazil: 2011 blaKPC-2 119 26
UCI_53 JMVS01 410 Homo sapiens Biliary drain USA 2013 ESBL 127 27
Swine70 LVOT01 410 Susscrofa Rectum China: Jiangsu 2012 - b 135 Unpublished
YD786 CP013112 410 Homo sapiens Urine USA 2012 ESBL 138 28
CoR-20 MKFO01 410 Homo sapiens Sputum China: Zhejiang 2014 ESBL 145 Unpublished
AST_82 LLXC01 410 N/Aa N/Aa USA 2015 AmpC 147 Unpublished
979 PIJB01 410 Homo sapiens Urine Argentina 2016 ESBL 150 Unpublished
CoR-35 NISA01 410 Homo sapiens Ascites China: Zhejiang 2015 ESBL 153 Unpublished
K71-77 LNGY01 410 Homo sapiens Blood Norway 2010 blaNDM-1 164 29
RL465 FLKU01 410 N/Aa N/Aa Germany N/Aa ESBL 174 30
AR434 CP029122 410 N/Aa N/Aa USA N/Aa - b 176 Unpublished
50,822,286 LNIK01 410 Homo sapiens Rectal swab Norway 2014 blaVIM-4 185 31
908,555 AXTR01 410 Homo sapiens N/Aa USA N/Aa - b 193 Unpublished
BV643 PVPJ01 410 Homo sapiens Blood India 2016 blaNDM-5 194 Unpublished

Notes: aData not available. bNo carbapenemase, ESBL, and AmpC-type ß-Lactamases can be found in the genome.

Figure 2.

Figure 2

Phylogenetic relationship between E. coli ST410 strain EC25 and other E. coli strains. Twenty-one phylogenetically related strains were identified in the BacWGSTdb server. All of these strains belonged to ST410. Seven strains were isolated from Germany, five from the USA, four from China, two from Norway, and one each from Argentina, Brazil, and India.

A variety of STs were found in NDM-positive E. coli strains worldwide, and there are no predominant STs. The number of large-scale studies examining the clonal background of NDM-positive E. coli strains is limited in China. Zhang R et al suggested that ST167 and ST410 were two of the most common sequence types of NDM-positive E. coli strains in China.24 We report the whole-genome sequence of a clinically isolated E. coli ST410 strain harboring blaNDM-7. Our study is the first report on fully sequenced E. coli ST410 strain carrying blaNDM-7 isolated from China because blaNDM-7 is rare. A similar NDM-positive E. coli ST410 strain was found in the BacWGSTdb server in other provinces of China. ST410 is more likely to emerge as a successful epidemic clone of NDM-producing E. coli in China, and the blaNDM gene is prone to mutate during its dissemination. Therefore, more studies are required to illuminate the epidemic clones of NDM-positive E. coli in China.

Conclusion

In summary, our study reports the first identification of a clinical carbapenem-resistant E. coli ST410 strain carrying blaNDM-7 recovered from a urinary tract infection in China. Our study highlights the potential transmission opportunity of carbapenem-resistant plasmid carriage in E. coli. Further studies involving more NDM-producing isolates are warranted to identify reservoirs and monitor the transmission dynamics of blaNDM genes in China.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (81702042), Natural Science Foundation of Zhejiang Province (LQ19H200003) and the Zhejiang Provincial Medical and Health Science and Technology Plan (2018KY344 and 2019KY311).

Disclosure

The authors report no conflicts of interest in this work.

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