| Summary: | Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission. We compared IncI1–pST12 plasmids from both human and livestock origin and explored the degree of sequence similarity between plasmids from Enterobacteriaceae with different epidemiological links. Short-read sequence data of Enterobacteriaceae cultured from humans and broilers were screened for the presence of both a <i>bla</i><sub>CMY-2</sub> gene and an IncI1–pST12 replicon. Isolates were long-read sequenced on a MinION sequencer (OxfordNanopore Technologies). After plasmid reconstruction using hybrid assembly, pairwise single nucleotide polymorphisms (SNPs) were determined. The plasmids were annotated, and a pan-genome was constructed to compare genes variably present between the different plasmids. Nine <i>Escherichia coli</i> sequences of broiler origin, four <i>Escherichia coli </i>sequences, and one <i>Salmonella enterica</i> sequence of human origin were selected for the current analysis. A circular contig with the IncI1–pST12 replicon and <i>bla</i><sub>CMY-2</sub> gene was extracted from the assembly graph of all fourteen isolates. Analysis of the IncI1–pST12 plasmids revealed a low number of SNP differences (range of 0–9 SNPs). The range of SNP differences overlapped in isolates with different epidemiological links. One-hundred and twelve from a total of 113 genes of the pan-genome were present in all plasmid constructs. Next generation sequencing analysis of <i>bla</i><sub>CMY-2</sub>-containing IncI1–pST12 plasmids isolated from Enterobacteriaceae with different epidemiological links show a high degree of sequence similarity in terms of SNP differences and the number of shared genes. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution.
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