The Bacterial Genomic Context of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases Highlights an Emerging Threat to Public Health

Abstract

Type B dihydrofolate reductase (dfrb) genes were identified following the introduction of trimethoprim in the 1960s. Although they intrinsically confer resistance to trimethoprim (TMP) that is orders of magnitude greater than through other mechanisms, the distribution and prevalence of these short (237 bp) genes is unknown. Indeed, this knowledge has been hampered by systematic biases in search methodologies. Here, we investigate the genomic context of dfrbs to gain information on their current distribution in bacterial genomes. Upon searching publicly available databases, we identified 61 sequences containing dfrbs within an analyzable genomic context. The majority (70%) of those sequences also harbor virulence genes and 97% of the dfrbs are found near a mobile genetic element, representing a potential risk for antibiotic resistance genes. We further identified and confirmed the TMP-resistant phenotype of two new members of the family, dfrb10 and dfrb11. Dfrbs are found both in Betaproteobacteria and Gammaproteobacteria, a majority (59%) being in Pseudomonas aeruginosa. Previously labelled as strictly plasmid-borne, we found 69% of dfrbs in the chromosome of pathogenic bacteria. Our results demonstrate that the intrinsically TMP-resistant dfrbs are a potential emerging threat to public health and justify closer surveillance of these genes.

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

“…At the outset of this work, there were ten known DfrB family members (DfrB1–11; there is no DfrB8, [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ]). All procure high TMP resistance in E. coli (MIC > 600 µg/mL; K i ~0.38 to 1.3 mM), and most were originally identified in clinical samples [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ].…”

“…At the outset of this work, there were ten known DfrB family members (DfrB1–11; there is no DfrB8, [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ]). All procure high TMP resistance in E. coli (MIC > 600 µg/mL; K i ~0.38 to 1.3 mM), and most were originally identified in clinical samples [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. The turnover rates of DfrB enzymes for dihydrofolate reduction ( k cat = 0.20–0.41 s −1 ) are at least 100-fold lower than bacterial FolA (e.g., k cat = 230 s −1 for E. coli FolA); nonetheless, a low level of DfrB expression suffices to confer TMP resistance [ 13 , 14 ].…”

“…We recently uncovered the mobility of dfrB genes found in pathogenic bacteria isolated from samples associated with human activity, such as clinical samples [ 11 , 12 ]. However, little is known about the presence or mobility of dfrB genes in environmental settings [ 19 , 20 ].…”

Antibiotic Resistance Genes in River Biofilms: A Metagenomic Approach toward the Identification of Sources and Candidate Hosts

“…On the one hand, this could point toward an environmental origin of the dfr B genes. On the other hand, genes of the dfr B family have already been detected in genomes of various clinically relevant strains 51 with clear evidence for mobility. 52 Hence, the detection of dfr B in environmental samples could be a result of resistance dissemination predominantly from nonpoint sources.…”

Epistasis between promoter activity and coding mutations shapes gene evolvability

“…The DfrB enzymes are obligate homotetramers that form a single, central active site requiring the distinct contribution from each of the four identical protomers ( 12 , 14 ). DfrB enzymes are not homologous to the ubiquitous FolA-type DHFR enzymes that ecDHFR is part of ( 14 – 16 ). While many antibiotic resistance proteins degrade or inactivate molecules, or expel them from the cell, DfrB1 is a metabolic enzyme that circumvents the inhibition of ecDHFR by duplicating the same essential enzymatic reaction: the reduction of dihydrofolate to tetrahydrofolate.…”