2.5. Antibiotic Resistance Gene Identification

In this study we identified ARGs by annotating each whole genome assembly with the Comprehensive Antibiotic Resistance Database (CARD) version 3.0.9 using command line tool Resistance Gene Identifier (RGI) version 5.1.0 specifying input type contig (–t contig) with default parameters for BLAST alignment (–a BLAST) and strict and perfect hits only. In order to detect previously unknown homologs using detection models with curated similarity cut-offs while still ensuring the detected variant is a functional resistance gene rather than spurious partial hits, we used the strict algorithm rather than the algorithms that would select for exclusively perfect or loose hits [36]. Results from RGI were further quality controlled by removing any Antibiotic Resistance Ontology (ARO) hits defined as mutations or ARO hits with less than 50% coverage of the reference sequence unless cutoff on the edge of a contig. To determine if a hit was located on a chromosome or plasmid, contigs containing hits were run through blastn [37] in Geneious Prime version 2019.2.1 against bacteria (taxid = 2) from the RefSeq database. The top hit ranked by bit score was then used to determine if the contig was most similar to a known plasmid or chromosome sequence.