Discovery of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases in Diverse Environmental Settings Suggests an Evolutionary Advantage Unrelated to Antibiotic Resistance

Abstract

Type B dihydrofolate reductases (DfrB) are intrinsically highly resistant to the widely used antibiotic trimethoprim, posing a threat to global public health. The ten known DfrB family members have been strongly associated with genetic material related to the application of antibiotics. Several dfrB genes were associated with multidrug resistance contexts and mobile genetic elements, integrated both in chromosomes and plasmids. However, little is known regarding their presence in other environments. Here, we investigated the presence of dfrB beyond the traditional areas of enquiry by conducting metagenomic database searches from environmental settings where antibiotics are not prevalent. Thirty putative DfrB homologues that share 62 to 95% identity with characterized DfrB were identified. Expression of ten representative homologues verified trimethoprim resistance in all and dihydrofolate reductase activity in most. Contrary to samples associated with the use of antibiotics, the newly identified dfrB were rarely associated with mobile genetic elements or antibiotic resistance genes. Instead, association with metabolic enzymes was observed, suggesting an evolutionary advantage unrelated to antibiotic resistance. Our results are consistent with the hypothesis that multiple dfrB exist in diverse environments from which dfrB were mobilized into the clinically relevant resistome. Our observations reinforce the need to closely monitor their progression.

Keywords: antibiotic resistance; metagenomic database search; mobile genetic elements; multidrug resistance; type B dihydrofolate reductase.

Figure 1

Structure of DfrB1 and sequence alignment of the known DfrB family members (75–95% sequence identity). (a) The functional, homotetrameric DfrB1 (PDB 1VIE) is constituted of four identical SH3-like protomers (one shown in green) that form the single, central active-site tunnel. The VQIY catalytic tetrad (V66, Q67, I68, and Y69; dark blue) and key residues K32 (red), W38 (magenta) and W45 (orange), are identified. (b) The DfrB1 protomer adopts an SH3-like fold. (c) Multiple sequence alignment of DfrB1–DfrB11 (there is no DfrB8) shows amino acid conservation, using standard annotation beneath the alignment. Conserved residues are highlighted in cyan. The poorly conserved N-terminal domain and the highly conserved SH3-like domain are identified. Functionally and structurally important residues are framed in red.

Figure 2

Newly identified DfrB homologues confer TMP resistance and possess Dfr activity. (a) Minimal inhibitory concentrations were determined on solid media with [TMP] ranging between 0–600 µg/mL. The reported MIC was the lowest TMP concentration where no bacterial growth was observed. The initial rates of reaction were calculated from panel B (n = 3, mean ± SD). The rate of the most active variants is underestimated, since the initial rate was not captured. (b) Dfr activity was determined in E. coli lysate, monitoring substrate consumption as a function of time (n = 3, mean ± SD). The negative control (Neg. Ctrl) is E. coli expressing the cTEM-19m β-lactamase instead of a DfrB.

Figure 3

Phylogenetic tree of dfrB1–dfrB21 and associated putative dfrB genes. Sequences are classified according to their source environment, predicted mobility, and taxonomy. Sequences were aligned using MAFFT; the tree was obtained using IQ-tree and visualized with iTOL. Bootstrap confidence levels are indicated by the size of the circle before each node. Information pertaining to source environment, taxonomy, and predicted mobility is reported in Table 2 and Table S3. The newly identified dfrB12–dfrB21 are in bold. Sequences associated with MDR are marked with an asterisk (*). Taxonomy: “β” for β-Proteobacteria, “γ” for γ-Proteobacteria, “PB” for Proteobacteria; “?” indicates undetermined, as taxonomic information was not available in some cases.