Genetic Context Analysis of Resistomes

The genetic context analysis was done in the host genomes of the latent genes in the core-resistomes. The analysis included 1429 unique resistance gene sequences corresponding to 136 ARGs. For each ARG, the closest known homolog was identified using BLASTx v2.10.1 [36 ] to align the gene sequences against the CARD database [26 ]. Here, CARD was used since it is more comprehensive than ResFinder and includes, in contrast to ResFinder, some genes that are not clinically relevant and/or mobile. Then, genetic regions of up to 10,000 base pairs upstream and downstream of the gene sequences were retrieved using GEnView v0.1.1 [42 (link)] and screened for the presence of genes associated with MGEs and integrons. The genetic regions were translated in all six reading frames using EMBOSS Transeq v6.5.7.0 [43 (link)] and searched with 124 HMMs from MacSyfinder Conjscan v2.0 representing genes involved in conjugation [44 (link)], using HMMER v3.1b2 [45 (link)]. Insertion sequences (ISs) and other mobile ARGs were identified by applying BLASTx v2.10.1 [36 ]. For IS elements, a reference database based on ISFinder [37 , 38 ] was used to find the best among overlapping hits, with the alignment criteria that hits should display $>50\%$ coverage and $>90\%$ amino acid identity to a known IS transposase, as well as being located within 1,000 base pairs of the latent resistance gene (upstream or downstream). For co-localized mobile ARGs, ResFinder v4.0 was used as a reference database [25 ], with the alignment criterion that hits should display an amino acid identity $>90\%$ to a known ARG. Finally, the genetic regions were searched for integrons using Integron Finder v1.5.1 [46 (link)]. After the screening, the genetic contexts were manually investigated and curated.