Phylogenetic analysis of erm genes

Gene sequencing sharing high similarity to ermA, ermB, ermC and ermF were obtained from publicly available databases. The Ribosomal Database Project Fungene Repository (Fish et al.2013) was used to obtain ermB- and ermC-associated sequences. It was required that sequences share 97% amino acid sequence coverage to established HMM protein models for Fungene gene families “Resfam_ermA”, “Resfam_ermB” and “Resfam_ermC” (Version 8.8). Additionally, ermF gene nucleotide sequences were obtained from proteins listed in the ARDB-Antibiotic Resistance Genes Database (version 1.1, July 3, 2009) (Liu and Pop 2009) and associated with the annotation “ermF”. All erm-associated sequences were combined and clustered at 99% nucleotide similarity using CD-HIT (v4.6.1c) (Li and Godzik 2006; Fu et al.2012), resulting in 66 unique clusters. One representative sequence for each cluster was identified by CD-HIT and was aligned using Muscle (v3.8.31) (Edgar 2004) with the following parameters: gap open –400, gap extend 0, clustering method UPGMB. A maximum-likelihood phylogenetic tree was constructed from this alignment using FastTree (v2.1.8) (Price, Dehal and Arkin 2010) with default parameters. Taxonomy was identified based on annotations in the NCBI non-redundant nucleotide database (NCBI Resource Coordinators 2017).

To consider an erm gene sequence to be associated with a previously targeted PCR primer sequence, both forward and reverse primers were required to share 100% nucleotide similarity over a minimum of 17 bp of the primer length.