Novel Trimethoprim Resistance Gene Identified in Riemerella anatipestifer from China.

Resistance to trimethoprim is mainly mediated by the acquisition of mobile genes, and most of them were discovered in Enterobacteriales. A total of 139 Riemerella anatipestifer isolates were collected from different farms in China during 2014 to 2020. Whole genome sequencing (WGS) and genome analysis of R. anatipestifer isolates revealed a 504-bp open reading frame (ORF) encoding a putative gene. This DfrA variant shared 66.47% amino acid sequence identity with DfrA36 and shared ≤51.20% identity with any other previously identified DfrA proteins. The novel gene, designated , conferred trimethoprim (TMP) resistance when cloned into Escherichia coli BL21(DE3). Thirty -positive isolates were identified from Jiangsu and Guangdong province (5/38, 13.16%, and 25/101, 24.75%, respectively). Five of the 38 isolates had obtained the complete genome sequences. Genomic analysis showed that the gene was located on chromosomes or a plasmid (four of them were on chromosomes and one was located on a plasmid). The plasmid p20190305E2-2_2 carried , , , , (88.36% identity with ), Δ, and (X18). Further research indicated that usually coexisted with in R. anatipestifer. In this study, a novel trimethoprim resistance gene, , was identified and characterized in chromosome and plasmid sequences from R. anatipestifer using WGS and bioinformatic methods. It further expands knowledge about the pool of mobile genes that confer resistance to trimethoprim and provides information about antibiotic resistance genes in R. anatipestifer, where the resistance gene pool circulating is not well understood. Trimethoprim is a synthetic antimicrobial agent inhibiting dihydrofolate reductase (DHFR), which is encoded by the gene. Acquired genes that confer trimethoprim resistance due to mutations in the gene are designated and divided into two main families including and . Resistance to trimethoprim is mainly mediated by the acquisition of mobile genes, and most of them were discovered in Enterobacteriales. R. anatipestifer belongs to the Flavobacteriaceae family, and the reservoir of resistance genes in R. anatipestifer has not been fully investigated. A novel trimethoprim resistance gene, , which was identified and characterized in chromosome and plasmid sequences in this study, increased the MIC of TMP (>256-fold) in E. coli BL21(DE3). Our study expands knowledge about the pool of mobile genes that confer resistance to trimethoprim and broadens the understanding of the host spectrum of family genes.