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.

Keywords: antibiotics; chemotherapeutic agent; evolution; phylogenetics; resistance; sulfonamides; trimethoprim.

Fig. 1.

Consensus tree of DHFR protein sequences. Branch support values are provided as Bayesian posterior probabilities estimated after four independent runs of 20 000 000 generations. Support values are only shown for branches with posterior probability values higher than 0.8. For chromosomal DHFR, the species name is displayed. Mobile DHFRs are denoted by their established dfrA name or by their NCBI GenBank accession number. Reported dfrA genes deemed redundant (>90 % identity) are listed next to the corresponding non-redundant taxon included in the analysis. Next to each tip label, coloured boxes designate trimethoprim-resistant (orange) and -sensitive (purple) DHFR. Numbers between brackets indicate the mol% G+C content of the sequence for the gene encoding the DHFR. Tip label colouring denotes previously reported (green) and novel (blue) DHFRs. Bold label text indicates that resistance has been experimentally assessed in this work. DHFR variants marked with an asterisk are encoded in megaplasmids (>400 kbp). The internal ring shows the mol% G+C of the gene encoding the DHFR in a yellow−red colour scale, while the external ring displays the ratio between the mol% G+C content of the genome harbouring the DHFR gene and the mol% G+C content of the gene. Dotted lines from the inner ring to tip labels denote genes discussed in the text. Reconstructed mobile/chromosomal states are displayed on ancestral nodes as pink/black pie charts.

Fig. 2.

Correlation between the mol% G+C content of mobile dfrA (red circles) and sul (green squares) genes and that of their host genome. Large open circles/squares denote representatives of clusters of redundant sequences (identity >90 %), and dfrA genes from clade 1 and clade 2 in Fig. 1 are marked with an additional corona. A 0.75 % jitter to both x- and y-axis values has been applied for visualization purposes. The red line shows the linear regression for representative dfrA gene values. The Pearson R² coefficient is superimposed. Vertical background bars in (a) designate DfrA sequences harboured by mobile genetic elements (MGEs) identified in E. coli and K. pneumoniae isolates, which are heavily overrepresented in the dataset. Sequences from clusters with more than 100 sequences (represented by dfrA12, dfrA5 and dfrA1) are shown with specific markers, and highlighted by horizontal background bars. The number of MGEs identified as harbouring dfrA genes, before and after filtering DfrA sequence identity (>90 %), is shown in (b).

Fig. 3.

Schematic representation of the two proposed evolutionary processes (based on the results presented in Figs 1 and 2, and Table 1) leading to the dissemination of trimethoprim-resistance determinants. Left panel: upon the introduction of trimethoprim, mobilization events involving pre-existing resistant chromosomal folA genes can be favourably selected. Right panel: following the introduction of trimethoprim, mobilization events involving folA genes with novel mutations that confer resistance to this chemotherapeutic agent may be selected for and disseminated among closely related bacteria.