Sequel 1 platform

DNA from fresh bacterial culture (colonies growing 48 h) was extracted according to the method described by Rossi et al. (2018 (link)). Sequel I platform (Pacific Biosciences, California, USA) was used for whole-genome sequencing. Library preparation was done following the microbial multiplexing protocol according to the manufacturer instructions for sheared DNA. Shearing was performed using g-TUBEs (Covaris, USA), and no size selection was done during the library preparations. Open reading frame (ORF) was predicted using RAST 2.0 (Brettin et al. 2015 (link)) with default parameters combined with BLASTP/BLASTN (Boutet et al. 2013 (link)), and insertion sequences were identified using the ISfinder database (http://www-is.biotoul.fr/). Comparative genome alignments were performed using Mauve (version 2.3.1).

The MICs, AMR profile and dendrogram from PFGE suggested that the five isolates were indistinguishable; hence, the genomic DNA of two E. coli strains (chosen as representatives, for comparison purposes) was extracted using NucleoSpin Microbial DNA Kit (Macherey-Nagel, Germany). Sequel I platform (Pacific Biosciences, CA, United States) was used for sequencing. Library preparation was done following the microbial multiplexing protocol according to the manufacturer’s instructions for sheared DNA. Shearing was performed using g-tubes (Covaris, United States), and no size selection was done during the library preparations. HGAP4 was used to perform the assemblies of the genomes with minimum seed coverage of 30. ResFInder 3.2³ (Zankari et al., 2012 (link)), PlasmidFInder⁴ (Carattoli et al., 2014 (link)), VirulenceFinder 2.0⁵ (Joensen et al., 2014 (link)) ISfinder database⁶, MLST 2.0⁷ (Larsen et al., 2012 (link)), and CHTyper 1.0⁸ (Camacho et al., 2009 (link)) were utilized to detect resistance genes, plasmid replicon type, virulence genes, mobile elements, Sequence type (ST) and FimH/FumC type, respectively. Open reading frames (ORF) were predicted using RAST 2.0 (Brettin et al., 2015 ) with default parameters combined with BLASTP/BLASTN. Comparative genome alignments were performed using the Mauve (version 2.3.1). Gene organization and diagrams were sketched using Inkscape 0.92.4⁹.

Based on the results of short-read sequencing (see below), twenty-five KPC producers were selected to be sequenced using long-read sequencing technology, to help close the whole plasmid sequences. These isolates were selected as representatives of all different hospitals, bacterial species, STs, replicon profiles and KPC alleles.
Genomic DNA was extracted from the clinical isolates using NucleoSpin Microbial DNA kit (Macherey–Nagel, Germany). Whole genome sequencing (WGS) was performed on the Sequel I platform (Pacific biosciences, Menlo Park, CA, United States). Microbial multiplexing protocol was used for the library preparation according to the manufacturer instructions for Sheared DNA. DNA shearing was performed using the Megaruptor 2 (Diagenode, Liege, Belgium) using long hydropores producing 10 kb long inserts. No size selection was performed during the library preparation. The Microbial Assembly pipeline offered by the SMRT Link v9.0 software was used to perform the assembly and circularization with minimum seed coverage of 30X. Assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).

The genomic DNA of the two E. coli strains was extracted using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany). Sequel I platform (Pacific Biosciences, Menlo Park, CA, United States) was used for sequencing. Library preparation was done following the microbial multiplexing protocol according to the manufacture instructions for sheared DNA. Shearing was performed using g-tubes (Covaris, United States), and no size selection was done during the library preparations. HGAP4 was used to perform the assemblies of the genomes with minimum seed coverage of 30. ResFinder 3.2³ (Zankari et al., 2012 (link)), PlasmidFinder⁴ (Carattoli et al., 2014 (link)), VirulenceFinder 2.0⁵ (Joensen et al., 2014 (link)) ISfinder database⁶, MLST 2.0⁷ (Larsen et al., 2012 (link)), and CHTyper 1.0⁸ (Camacho et al., 2009 (link)) were utilized to detect resistance genes, plasmid replicon type, virulence genes, mobile elements, multilocus sequence types (STs) (according to Oxford and Pasteur schemes) and FimH/FumC type respectively. Open reading frame (ORF) were predicted using RAST 2.0 (Brettin et al., 2015 (link)) with default parameters combined with BLASTP/BLASTN. Comparative genome alignments were performed using the Mauve (version 2.3.1). Gene organization and diagrams were sketched using Inkscape 0.92.4⁹.

Genomic DNA was extracted from the four clinical isolates using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany). Whole genome sequencing (WGS) was performed on the Sequel I platform (Pacific biosciences, Menlo Park, CA, United States). Microbial multiplexing protocol was used for the library preparation according to the manufacturer instructions for Sheared DNA. DNA shearing was performed using the Megaruptor 2 (Diagenode, Liege, Belgium) using long hydropores producing 15kb long inserts. No size selection was performed during the library preparation. Microbial Assembly pipeline offered by the SMRT Link v8.0 software was used to perform the assembly and circularization with minimum seed coverage of 30×. Assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Antibiotic resistant genes, plasmid replicons, mobile elements and multilocus sequence types (MLST) were determined through uploading the assembled sequences to ResFinder 4.1 and CARD (Zankari et al., 2012 ; Alcock et al., 2020 (link)), PlasmidFinder (Carattoli et al., 2014 ), ISfinder (Siguier et al., 2006 (link)), and MLST 2.0 (Larsen et al., 2012 ), respectively. Comparative genome alignment was done using Mauve v.2.3.1.² and BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011 (link)). Diagrams and gene organization were sketched using Inkscape 0.92.4³.

For genomic characterization, genomic DNA was extracted using NucleoSpin Microbial DNA kit (Macherey-Nagel, Duren, Germany) and sheared using the Hydropore-long on Megaruptor 2 (Diagenode). Microbial multiplexing library preparation was performed without size selection according to the manufacturer’s instructions. The multiplexed library was sequenced using long reads sequencing technology using the Sequel I platform (Pacific Biosciences, Menlo Park, CA, USA) for a 10 h movie run.