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Plasmid-mediated colistin resistance encoded by the mcr-1 gene was first described in Enterobacteriaceae isolated from animals, raw meat and humans in China in November 2015 . Escherichia coli carrying a transferable colistin resistance plasmid have been reported from different host species and geographic areas. Here we report the first detection of mcr-1 in colistin-resistant extended-spectrum β-lactamase-producing E. coli (ESBL-E. coli) isolated from wild transboundary migratory waterfowl species Fulica atra from Asia.
In response to the threat of the emergence of plasmid-mediated colistin resistance, ESBL-E. coli isolates recovered from faecal swabs of long-range wild migratory birds in Pakistan were screened. During screening for additional drug resistance phenotypes among ESBL-E. coli isolates using the VITEK®2 compact system (AST-GN38 card; bioMérieux, Nürtingen, Germany), one ESBL-E. coli isolate (PK-13) was found that showed phenotypic resistance to polymyxin B. ESBL-E. coli PK-13 isolate exhibited multidrug resistance to fluoroquinolones, tetracycline and trimethoprim/sulfamethoxazole in addition to colistin and third-generation cephalosporins. The minimum inhibitory concentration (MIC) of colistin was determined using VITEK®2 (AST-N248 card) and was found to be 8 mg/L (Table 1). The isolate was found to be positive for the mcr-1 gene using a real-time PCR approach. The approximate size of the plasmid was determined by plasmid profile analysis, and the plasmid replicon type was found to be IncI2 as determined by PCR-based replicon typing (PBRT) using a PBRT kit (DIATHEVA, Fano, Italy) ( Table 1). To determine whether the colistin resistance was carried on a transferable plasmid, a conjugation experiment was performed with sodium azide-resistant E. coli K12-J53 as the recipient strain . Colistin resistance was successfully transferred to the host strain. Transconjugants were selected on LB agar plates supplemented with colistin (4 mg/L) and sodium azide (100 mg/L) and were subsequently confirmed by PCR for mcr-1. PBRT analysis of transconjugants showed the Inc2 replicon type in the transconjugants. Multilocus sequence typing (MLST) was carried out according to the MLST website (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). Sequences were analysed and computed using the software package Ridom SeqSphere 0.9.39 (http://www3.ridom.de/seqsphere). MLST revealed that E. coli isolate PK-13 belonged to ST354. Genotypic detection of common ESBL genes by PCR and sequencing showed the presence of the blaCTX-M-15 ESBL type (Table 1).