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-hydro-pteroate 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 homologs 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. This work hence 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.

中文翻译：

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染色体二氢叶酸还原酶基因的祖先和新变体的反复动员驱动了对甲氧苄啶的临床耐药性的出现

甲氧苄氨嘧啶是一种合成的抗菌剂，通过与二氢叶酸还原酶（DHFR）竞争性结合而靶向叶酸的生物合成。甲氧苄氨嘧啶通常与磺胺类药物协同给药，磺胺类药物是在同一途径中靶向二氢蝶呤合酶（DHPS）酶的另一种化疗药物。对这两种药物的临床耐药性都很普遍，并由能够执行其生物学功能而不与这些药物结合的酶变异体介导。这些突变酶被认为是在发现这些合成药物后出现的，但最近的研究表明，细菌泛基因组中存在着赋予磺酰胺抗性的基因。在这里，我们应用系统遗传学和比较基因组学方法来研究最大的移动甲氧苄啶抗性基因（dfrA）家族。我们显示，迄今确定的大多数dfrA基因都映射到两个可能来自独立动员事件的大型进化枝。与磺胺抗性（sul）基因相反，我们发现dfrA基因中反复动员的证据。系统发生学证据使我们能够在新兴病原体鲍曼不动杆菌中鉴定新的dfrA基因，并在体外证实了它们的抗性表型。我们还确定了在隐秘质粒和噬菌体基因组中的dfrA同源簇，但我们表明这些酶不赋予对甲氧苄啶的抗性。我们的方法还使我们能够查明以前报道的dfrA基因的染色体起源，并且我们证明了许多这些古老的染色体基因也赋予了对甲氧苄啶的抗性。我们的工作表明，甲氧苄啶耐药性早于该化学治疗剂的临床应用，但是这种新的突变也很可能出现，并随着其在临床内外的广泛使用而动员起来。因此，这项工作证实了细菌全基因组中可能已经存在对新药的抗药性，并强调了快速动员作为抗药性决定因素出现和全球传播的基本要素的重要性。