101 Limited genetic diversity of blaCMY-2-containing IncI1-pST12 plasmids from Enterobacteriaceae the assembly graph using BANDAGE v0.8.1. (Wick et al. 2015). All extracted plasmid components were annotated using Prokka v1.13.3 (Seemann 2014). Using snippy v4.4.59 (https://github.com/tseemann/snippy), the number of single nucleotide polymorphisms (SNPs) was determined between the extracted plasmid components using a blaCMY2 gene containing IncI1–pST12 plasmid extracted from the GenBank (accession number: MH472638.1) as reference (Roer et al. 2019). A pan-genome was constructed, and a gene presence or absence was determined for all extracted plasmid components using roary v3.12 (Page et al. 2015). All extracted plasmids consisting of a single circular contig were aligned using GView 1.7 and progressiveMAUVE v2.4.0 to detect possible rearrangements (Petkau et al. 2010; Darling, Mau, and Perna 2010). If a hypervariable region is identified, the sequence of this region and its flanking regions are extracted using biopython v1.37. Moreover, segments (A, B, C, D) and flanking genes (PilV and rci) of a previously described hypervariable shufflon region of the IncI1 replicon containing plasmids (GenBank accession nr: AB027308.1) were BLAST searched in the extracted hypervariable regions (Brouwer et al. 2015; 2019). Classification of Pairwise Comparisons Pairwise comparisons of assembled plasmids were classified according to the known epidemiological link between the isolates: (i) same sample; (ii) same ward/flock but different sample; (iii) same location (hospital or farm) but different ward/flock and sample; (iv) same domain (human or broiler) but different location, ward/flock and sample; and (v) no known epidemiological link, i.e., different domain, location, ward/ floc, and sample. Ethical Statement The I-4-1-Healt study was judged to be beyond the scope of the Dutch Medical Research Involving Human Subjects Act and the Belgian Law on Experiments on Humans, dated 7 May 2004. Written or verbal informed consent for data collection and taking a faecal, perianal, or gastrointestinal stoma swab for microbiological culture is obtained from all participants or their legal representatives. For the veterinary domain, approval by an animal welfare body is not required. All human data are anonymized, i.e., data cannot be directly or indirectly related to their source. Data on institutions and farms are pseudonymized, i.e., identifying information is replaced by a code, and a key file that links this code to the identifying information is kept separate from the research data. 6
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