Computational protocol: Genomic and functional characterisation of IncX3 plasmids encoding blaSHV-12 in Escherichia coli from human and animal origin

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[…] Four genetically and epidemiologically unrelated IncX3 plasmids encoding blaSHV-12 were randomly selected for further analysis from E. coli isolates belonging to the single ST of human origin (ST69) and diverse STs of animal origin, including the predominant animal-related ST (ST117). The relevant characteristics of the selected plasmids are specified in Table . Plasmid DNA from transformants was isolated using the QIAfilter Plasmid Midi Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s recommendations. Deep sequencing of the plasmid genomes was performed using 300-bp paired-end sequencing libraries (Nextera TAG-mentation sequencing kits [Epicentre]) on an Illumina MiSeq sequencer. High-quality filtered reads were subsequently assembled de novo using SPAdes algorithm (SPAdes version 3.7.1) for Illumina-derived reads and then manually curated to close the gaps. Putative open reading frames (ORFs) were identified by RAST version 2.0 and manually curated when necessary. BLASTP analyses of the putative ORFs against the NCBI non-redundant proteins (NR) database, Pfam, and Interpro scan were used to assess their putative functions by identification of structural features and motifs,. ResFinder (version 2.1), PlasmidFinder (version 1.3) and ISfinder were used to determine the presence of resistance genes, replicon types and insertion sequences, respectively–. Plasmid sequences were hierarchically clustered and displayed as a phenogram using the BioNJ algorithm, where the underlying distance matrix was calculated from the pairwise non-overlapping maximal unique matches (MUMs) using Nucmer version 3.07,. Relative pairwise distances were obtained by dividing the pairwise MUMs’ sum by the average genome size of the two paired genomes (MUMi genomic distance). BioNJ trees were generated from the MUMi distance matrix using SplitsTree4. BLAST analysis was used to assess sequence identity between the blaSHV-12-surrounding region and nucleotide sequences deposited to NCBI. […]

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