Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance

Abstract

Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a Ͼ2,000-fold increased trimethoprim resistance on overexpression in E. coli. Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.KEYWORDS type II dihydrofolate reductase, trimethoprim resistance, E. coli clinical isolates, dfrB4, antibiotic-resistant genes, class I integron, urinary tract infection P ublic health agencies worldwide rank trimethoprim (TMP) a broad-spectrum antibiotic of importance in human medicine (1). Widely used as a result of its low cost and effectiveness, TMP inhibits the activity of many microbial chromosomal dihydrofolate reductases (DHFRs); thus, DHFRs have long served as prioritized targets of antiproliferative drugs (2). Although the majority of living cells harbor a chromosomal member of the type I DHFR family, encoded by a dfrA homolog, the dfrB genes encode a family of plasmid-borne type II DHFRs that are evolutionarily unrelated to type I DHFRs. The dfrB genes have been found in pathogenic bacteria recovered from many food sources, including fish (3), pigs (4, 5), and cows (6), where they confer TMP resistance. Bacteria carrying dfrB genes have also been identified in wastewater samples (7). Over the past decade, dfrB genes have been tracked indirectly in antibiotic resistance studies through identification of integron-related elements (8-10). Therefore, the importance of dfrB genes in TMP resistance in human pathogens may be underappreciated (11,12).To date, only seven members of the dfrB gene family are known, and they are highly homologous (77% to 94% genetic identity, 77% to 99% amino acid identity) ( Table 1). Among these, the DfrB1 protein (also known as R67 DHFR) is the best-studied type II DHFR (13)(14)(15)(16)(17). It is proposed to be recently evolved, and it confers a significant survival advantage under TMP exposure to microbes that harbor it (18). To date, the family of dfrB genes has consistently been reported to be contained within the following mobile

Structure-Based Design of Dimeric Bisbenzimidazole Inhibitors to an Emergent Trimethoprim-Resistant Type II Dihydrofolate Reductase Guides the Design of Monomeric Analogues

“…We previously identified the DfrB4 coding sequence, flanked by further antibiotic resistance genes, in a TMP-resistant North American clinical sample. 10 We further demonstrated that expression of DfrB4 in E. coli conferred complete resistance to the highest concentration of TMP that could be dissolved in the medium (600 μg/mL). 10 DfrB4 shares 77% amino acid identity with DfrB1; the catalytic core is highly conserved, whereas the loops and termini differ.…”

“…These findings inspired the design of analogues that were confirmed to bind with 1:1 stoichiometry. We further revealed a broader biological impact by demonstrating that a second member of the DfrB family, identified in a North American TMP-resistant clinical sample, 10 is also effectively inhibited by these novel bisbenzimidazole inhibitors.…”

RecA a universal drug target in pathogenic bacteria

“…The DfrB4 protein, encoded by the integronrelated dfrB4 gene, was identified in antibiotic-resistant Escherichia coli clinical isolates. DfrB4 conferred a markedly increased resistance to the antibiotic trimethoprim in vitro, leading to the suggestion that dfrB4 contributes to clinical antibiotic resistance (21).…”

Recurrent mobilization of ancestral and novel variants of the chromosomal di-hydrofolate reductase gene drives the emergence of clinical resistance to trimethoprim

“…These two families encode evolutionarily unrelated proteins of markedly different sizes. Sequence similarity indicates that dfrA genes are homologs of the chromosomally-encoded folA genes, whereas dfrB genes are functional analogs of unknown origin [23,24]. Most dfrA genes follow a standard naming convention consisting of dfrA followed by a numerical value indicating their discovery rank order.…”