Computational protocol: Multiple ESBL-Producing Escherichia coli Sequence Types Carrying Quinolone and Aminoglycoside Resistance Genes Circulating in Companion and Domestic Farm Animals in Mwanza, Tanzania, Harbor Commonly Occurring Plasmids

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Protocol publication

[…] Twenty-five ESBL-producing E. coli isolates were chosen for whole-genome sequencing (WGS). The isolates were confirmed to be ESBL-producers using the VITEK®2 compact system (bioMérieux, Nürtingen, Germany). DNA was isolated using the Purelink Genome DNA Mini kit (Invitrogen, Darmstadt, Germany). WGS was carried out on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) using an Illumina Nextera XT library with 2x300-bp paired-end reads. The data was assembled using SPAdes (version 3.0) (). Contigs larger than 500-bp and a coverage higher than nine were ordered to E. coli MG1655 (accession number U00096.3) using MAUVE (). The contigs were concatenated to generate pseudogenomes. Whole-genome phylogeny was determined by the software package Harvest Suite using E. coli MG1655 as reference (). The phylogenetic tree was drawn using MEGA5 ().The raw data of the sequenced E. coli are available at the European Nucleotide Archive (ENA), under the project number PRJEB12335. [...] Sequences were analyzed for MLST (according to ), transferrable resistance genes, plasmid replicon types and pMLST using MLST 1.8, ResFinder, Plasmidfinder and pMLST software of the Center for Genomic epidemiology (; ; ). Phylogenetic groups were determined as previously described (). The presence of chuA, yjaA and TSPE4C.2 sequences was examined using blastN (). The location of blaCTX-M-15 was determined by analyzing the contigs harboring blaCTX-M-15 using blastN. Plasmid comparisons were carried out using the software BRIG (). […]

Pipeline specifications

Software tools SPAdes, Mauve, MEGA, pMLST, PlasmidFinder, BLASTN, BRIG
Databases ENA ResFinder
Applications Phylogenetics, WGS analysis, Nucleotide sequence alignment, Genome data visualization
Organisms Escherichia coli, Bacteria, Homo sapiens, Canis lupus familiaris, Sus scrofa
Chemicals Aminoglycosides