Resistome Analysis of Antibiotic Resistant Bacteria

To investigate the selection on ESBL production, DNA was isolated from 1 ml of feces and 1 ml of manure slurry of all nine tubes after experiment 2 and 3, using the protocol as described for the EFFORT-project⁵³ . Illumina sequencing was performed as described by the manufacturer using 150 bp paired end Illumina Novaseq sequencing Libraries were created using the Ilumina Nextera XT DNA Library Preparation Kit according to the manufacturer’s protocol⁵⁴ . Resulting DNA reads were processed using FastDeME according to the default settings⁵⁵ . Reads were processed using FastP to remove poor quality reads, sequencing adapters and barcodes^{56 (link)}. To investigate the resistome, Kaiju was used to profile the reads for bacterial species identification and the tool KMA was used to detect allignments to known antimicrobial resistance genes with the default Resfinder database^{57 (link),58 (link)}. The sequence depth of each resistance gene was adjusted for sequencing depth to depth per gigabase. Resistance classes were obtained from the Resfinder database⁵⁹ and hits to each resistance gene were summed per antimicrobial class. Sequence data is available under accession PRJEB49007 at the sequence read archive (SRA). Results were visualized using Microsoft Excel.