Detection of Two Copies of a bla(NDM-1)-Encoding Plasmid in Escherichia coli Isolates from a Pediatric Patient with Diarrhea

PURPOSE: To elucidate the contribution of a transferable plasmid harboring the bla(NDM-1) gene in an Escherichia coli clinical isolate to the spread of resistance determinants. METHODS: Nine extended-spectrum β-lactamase-producing E. coli were collected from diarrhea samples from a pediatric patient and genetic linkage was investigated through enterobacteriaceae repetitive intragenic consensus polymerase chain reaction (PCR). Bacterial species were identified by 16s rRNA sequencing, susceptibility testing with the use of a BD Phoenix(TM)-100 Automated Microbiology System, and assessment of virulence genes by PCR. The transferability of bla(NDM-1) in E. coli strain TCM3e1 was confirmed by conjugation experiments. Complete sequencing of E. coli strain TCM3e1 was determined with the PacBio and Illumina NovaSeq platforms and the characteristics were analyzed with bioinformatics software. RESULTS: The results showed that all nine E. coli strains were the same clone. E. coli strain TCM3e1 was resistant to 12 antimicrobial agents and carried the virulence gene EAST-1. Conjugation transfer analysis showed that bla(NDM-1) was carried on a self-transmissible plasmid. Two copies of the bla(NDM-1) gene were present on an IncC plasmid and some resistance genes with two or three copies were located downstream of the bla(NDM-1) gene and formed a tandem repeat fragment (bla(DNM-1)-bleo-sul1- aadA17- dfrA12). CONCLUSION: A transmissible plasmid harboring two copies of the bla(NDM-1) gene, including clonal dispersions of the bla(NDM-1) gene, was identified in clinical isolates. These findings emphasized the necessity of surveillance of the plasmid-borne bla(NDM-1) to prevent dissemination.