Exploration into the origins and mobilization of di-hydrofolate reductase genes and the emergence of clinical resistance to trimethoprim

Abstract

Trimethoprim is a synthetic antibacterial agent that targets folate biosynthesis by competitively binding to the di-hydrofolate reductase enzyme (DHFR). Trimethoprim is often administered synergistically with sulfonamide, another chemotherapeutic agent targeting the di-hydropteroate synthase (DHPS) enzyme in the same pathway. Clinical resistance to both drugs is widespread and mediated by enzyme variants capable of performing their biological function without binding to these drugs. These mutant enzymes were assumed to have arisen after the discovery of these synthetic drugs, but recent work has shown that genes conferring resistance to sulfonamide were present in the bacterial pangenome millions of years ago. Here, we apply phylogenetics and comparative genomics methods to study the largest family of mobile trimethoprim-resistance genes (dfrA). We show that most of the dfrA genes identified to date map to two large clades that likely arose from independent mobilization events. In contrast to sulfonamide resistance (sul) genes, we find evidence of recurrent mobilization in dfrA genes. Phylogenetic evidence allows us to identify novel dfrA genes in the emerging pathogen Acinetobacter baumannii , and we confirm their resistance phenotype in vitro. We also identify a cluster of dfrA homologues in cryptic plasmid and phage genomes, but we show that these enzymes do not confer resistance to trimethoprim. Our methods also allow us to pinpoint the chromosomal origin of previously reported dfrA genes, and we show that many of these ancient chromosomal genes also confer resistance to trimethoprim. Our work reveals that trimethoprim resistance predated the clinical use of this chemotherapeutic agent, but that novel mutations have likely also arisen and become mobilized following its widespread use within and outside the clinic. Hence, this work confirms that resistance to novel drugs may already be present in the bacterial pangenome, and stresses the importance of rapid mobilization as a fundamental element in the emergence and global spread of resistance determinants.

Acinetobacter baumannii Antibiotic Resistance Mechanisms

“…Antifolate antibiotics exert their action by inhibiting purine metabolism, and thereby DNA and RNA synthesis. Trimethoprim is a dihydrofolate reductase (DHFR) inhibitor (blocking tetrahydrofolic acid formation by dihydrofolic acid, an essential step of folate biosynthesis, which in turn is involved in the generation of many nucleotides and amino acids), while sulfonamides are known dihydropteroate synthase (DHPS) inhibitors (halting the conversion of para-aminobenzoate or PABA to dihydropteroate; including sulfamethoxazole, commonly used in combination with trimethoprim, and sulfonamides) [ 185 ]. Resistance against diaminopyrimidines in A. baumannii infections is mainly conferred by trimethoprim-resistant dihydrofolate reductases DfrA1, DfrA5, DfrA7, DfrA10, DfrA12, DfrA14, DfrA16, DfrA17, DfrA19, DfrA20, DfrA27, and DfrB1 (encoded by dfrA1 , dfrA5 , dfrA7 , dfrA10 , dfrA12 , dfrA14 , dfrA16 , dfrA17 , dfrA19 , dfrA20 , dfrA27 , and dfrB1 , respectively) [ 97 ].…”

Genomic and phenotypic analyses of diverse non-clinical Acinetobacter baumannii strains reveals strain-specific virulence and resistance capacity

“…The strain E-072658 was resistant to sulfamethoxazole as it harbours the sul2 gene required for resistance to this drug [92]. Interestingly, all of the environmental isolates except for E-072658 were moderately or fully resistant to trimethoprim, despite lacking the trimethoprim resistance gene dfrA, which encodes a trimethoprim-insensitive homologue of the sensitive dihydrofolate reductase encoded by folA [93,94]. Four isolates (Ex003, B9, 10-3 and 10-4) were resistant to florfenicol and chloramphenicol, most likely due to the presence of genes encoding efflux-mediated resistance mechanisms, including major facilitator super family (MFS) transporters [95,96].…”

Development of antibacterial compounds that constrain evolutionary pathways to resistance

“…Factors associated with TMP resistance are far more complex in the clinical isolates. A recent study has shown the contribution of mobile dfrA genes to TMP resistance in the emerging pathogen Acinetobacter baumannii and its association to chromosomal folA genes in rapidly mobilizing novel mutations ( Sánchez-Osuna et al, 2020 ). The study shows how sulfonamide resistance in general extends to TMP with generalized chromosomal resistance determinants predating the origin of several genera and several clusters of resistance genes disseminated broadly among clinical isolates.…”