Computational protocol: Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance

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Protocol publication

[…] 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.) 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 ) 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 ; Fu et al.), 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 ) 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 ) with default parameters. Taxonomy was identified based on annotations in the NCBI non-redundant nucleotide database (NCBI Resource Coordinators ).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. […]

Pipeline specifications

Software tools CD-HIT, MUSCLE, FastTree
Databases ARDB ARDB
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Homo sapiens, Escherichia coli