An NDM-1-producing Escherichia coli obtained in Denmark has a genetic profile similar to an NDM-1-producing E. coli isolate from the UK

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Sir, We read with interest the recent article by Mushtaq et al.1 on the phylogenetic diversity of 18 NDM-1 carbapenemase-producing Escherichia coli strains from England, Pakistan and India. The E. coli isolates producing NDM-1 belonged to six sequence types (ST101, ST405, ST648, ST90, ST410 and ST156) and three phylogroups (B1, D and A).1 Nevertheless, isolates of B1-ST101 accounted for half the collection, and included isolates from both England and Pakistan. Sixteen of 18 isolates had a group 1 CTX-M gene, 13 had a CIT-type acquired AmpC and 16 had either one or both of the armA and rmtC 16S rRNA methylase genes.1 Recently we have detected the first NDM-1-producing E. coli in Denmark. As with many of the NDM-1-producing isolates from other countries, our patient had a prior travel history to the Indian subcontinent. In our case the patient, an 89-year-old Danish woman of Pakistani descent, went to Pakistan on holiday. In Pakistan the patient was hospitalized due to dehydration. Six weeks after the patient returned to Denmark she was again hospitalized due to dehydration. As the patient had been hospitalized abroad, surveillance samples were collected to exclude multiresistant bacteria and an NDM-1-producing E. coli was isolated from a faecal sample. The MICs of the antimicrobial agents shown in Table 1 were determined by use of the microbroth dilution method as defined by the CLSI, with the exception of the MICs of tigecycline and mecillinam, which were determined using MIC test strips (Liofilchem, Rosetodegli Abruzzi, Italy). Results were interpreted according to EUCAST (www.EUCAST.org), except the results for nalidixic acid, tetracycline, streptomycin and sulfamethoxazole, which were interpreted according to CLSI.2 The reference strain E. coli ATCC 25922 was used for quality control. Description of the first NDM-1-producing E. coli isolate in Denmark, the recipient strain and two transformants Description of the first NDM-1-producing E. coli isolate in Denmark, the recipient strain and two transformants The blaNDM gene was identified by PCR using primers NDM-F (5′-GAAGCTGAGCACCGCATTAG-3′) and NDM-R (5′-TGCGGGCCGTATGAGTGATT-3′) to amplify an internal fragment of 761 bp.3 Furthermore, the isolate was screened for acquired AmpC genes (MOX, CIT, DHA, ACC, ENT and FOX) and for blaCTX-M, blaOXA and blaTEM by PCR using primers and conditions described previously.4 The isolate was screened for the 16S rRNA methylase genes armA and rmtC according to Galimand et al.5 and Mushtaq et al.,1 respectively. Our NDM-1-producing E. coli isolate was assigned to one of the major E. coli phylogenetic groups (A, B1, B2 and D) by multiplex PCR.6 Multilocus sequence typing (‘MLST’) was performed using seven conserved housekeeping genes (http://mlst.ucc.ie/mlst/dbs/Ecoli). Plasmid purification was performed using the Plasmid Mini AX kit (A&A Biotechnology, Poland). Plasmid profiling and restriction analysis using EcoRI revealed the presence of two large plasmids and one small plasmid. Plasmids were electroporated into electrocompetent E. coli (OneshotGeneHogs, Life Technology, Denmark) and transformants were selected on Luria-Bertani agar containing 2 mg/L cefotaxime. Screening of transformants identified strains transformed by either of the two large plasmids. PCR replicon typing of the original isolate and the two single plasmid transformants was performed.7 The isolate was resistant to all antimicrobial agents except colistin and tigecycline (Table 1). It tested positive by PCR for metallo-β-lactamase enzymes of the NDM group, ESBL enzymes belonging to group 1 CTX-M, OXA and TEM, and the CIT-type AmpC enzymes. PCR and further sequencing showed the following profile for the isolate: blaNDM-1, blaCTX-M-15, blaCMY-4, blaOXA- 2, blaTEM-1 and armA. The isolate was ST101 and belonged to phylogroup B1 and showed a similar genetic profile to one of the B1-ST101 isolates from the UK, which had a CIT AmpC enzyme, had a group 1 CTX-M-type enzyme and was positive for armA. NDM-1-producing B1-ST101 E. coli isolates have also been obtained from patients from Australia,8 Germany9 and Canada.10,11 Our study adds information to the global spread of NDM-1-producing B1-ST101 E. coli isolates. Characterization of the two large plasmids revealed blaNDM-1 to be located on an IncA/C plasmid together with blaCMY-4 and armA. The other large plasmid contained blaCTX-M-15 and blaTEM-1, and was not typeable by replicon typing. Recently Sekizuka et al.12 published a completely sequenced blaNDM-1 plasmid from an ST38 E. coli isolate from Japan. Similar to our NDM-1 plasmid, the NDM-1 plasmid in the Japanese isolate contained blaCMY-4 and armA in addition to blaNDM-1 and was IncA/C.12 This may suggest dissemination of similar plasmids into several different sequence types of E. coli. Further studies of NDM-1 isolates and their plasmids are needed to follow the global dissemination of this gene. This study was supported by the Danish Ministry of the Interior and Health as part of the Danish Integrated Antimicrobial Resistance and Research Programme (DANMAP). None to declare. We thank Karin Sixhøj Pedersen for technical assistance.