For constructing the phylogenetic tree, single-nucleotide polymorphisms (SNPs) were identified from genome assemblies (BioProject accession nos. PRJNA290784 and PRJNA551684) following the North Arizona SNP Pipeline (NASP) pipeline (38) using E. coli strain EC958 (GenBank accession no. HG941718.1) as a reference. Duplicated regions of the reference genome, including repeat regions and multiple gene copies, were determined by aligning the reference sequence to itself using the NUCmer-3.23 (39). SNPs that fell within these duplicate regions were excluded from further analysis to avoid false SNP calls due to ambiguous read alignment. Each query genome assembly was aligned to the reference with NUCmer-3.23. The best SNPs in all genomes compared to the reference were concatenated in a matrix. Phylogenetic trees were inferred using the maximum likelihood (ML) method available in RAxML (40, 41). This returned the best-found tree from 100 replicates inferred using the general time-reversible substitution model.

For Inc typing, the plasmid sequences were queried against a locally downloaded version (retrieved 22 May 2018) of the PlasmidFinder database (, using the recommended percentage coverage threshold of 60% and a more stringent percentage identity and e-value thresholds of 90% and 0.00001, respectively (42). MOB typing was performed as previously described, and default value was chosen for all parameters (43). Specifically, the e-value threshold of each MOB type was chosen as follows: MOBC, 0.001; MOBF, 0.01; MOBH, 0.01; MOBP, 1; MOBQ, 0.0001; and MOBV, 0.01. In addition, 247,882 protein sequences were extracted from a National Center for Biotechnology Information dataset of 6952 complete Enterobacteriaceae plasmids (44) and were compiled into a database in combination with the sequences of 2423 antibiotic resistance proteins from ResFinder database (retrieved 22 May 2018) ( (45) as predicted by prodigal v2.6.3 (46). Genome assemblies were further annotated using Prokka v1.13 (47) against the above compiled protein database. For comparison, plasmids were aligned to one another via BRIG, displaying sequence matches with nucleotide identity ≥70% (48).

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