DNA Extraction and Sequencing

From all food samples positive for ESBL/AmpC-producing E. coli or K. pneumoniae, a collection of isolates was chosen for WGS analysis in order to study the presence of AMR, virulence genes, and plasmid replicons, as well as to assess the multilocus sequence type (MLST). If applicable, a representative from each ESBL/AmpC enzyme type category (ESBL, AmpC, or ESBL together with AmpC) and bacterial species (E. coli or K. pneumoniae) was chosen from each positive food sample, excluding subsamples. Consequently, from one to three isolates were chosen for whole genome sequencing from each positive sample.

Bacterial DNA was extracted and purified with a PureLink Genomic DNA Mini Kit (Invitrogen by Thermo Fischer Scientific, Carlsbad, CA, United States) according to the manufacturer’s instructions. The assessment of DNA quality was carried out using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Wilmington, DE, United States) and DNA quantity was measured using a Qubit 2.0 fluorometer (Invitrogen, Life Technologies, Carlsbad, CA, United States). Library preparation was performed with an Illumina Nextera XT and sequencing with an Illumina Novaseq 6000 (Center for Genomics and Transcriptomics, Tübingen, Germany) with paired-end reads. Samples were sequenced with 100× coverage and 2× 100 bp read length.

Subsequently, seven short-read sequenced E. coli isolates were chosen for long-read sequencing in order to study beta-lactamase harboring plasmids in more depth. Isolates were chosen from short-read sequenced isolates to represent a wide selection of different beta-lactamases, MLST types, and plasmid replicons. DNA extraction and purification were performed as described in Section “Short-Read Sequencing.”

DNA extracts from three or two isolates at a time were multiplexed using a SQK-LSK109 ligation sequence kit (Oxford Nanopore Technologies, United Kingdom) according to the manufacturer’s protocol. Libraries were loaded onto FLO-FLG001 R9.4.1 Flongle flow cells (Oxford Nanopore Technologies, United Kingdom) used with the MinION Mk1B sequencing device and sequenced with MinKNOW software v19.06.8 for 20–24 h.

All raw sequences have been deposited at European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB37779¹. Accession numbers for each sequenced isolate are provided in Supplementary Table 2.