Department of Microbiology, Cornell University, Ithaca, New York, USA.
Department of Medicine, Division of Infectious Diseases, Weill Cornell Medicine, New York, New York, USA.
mBio. 2023 Apr 25;14(2):e0316822. doi: 10.1128/mbio.03168-22. Epub 2023 Feb 13.
Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance-determining region (RRDR) of that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness. Here, we studied the evolution of resistance in Bacillus subtilis to the synergistic combination of RIF and the β-lactam cefuroxime (CEF). Two independent evolution experiments led to the recovery of a single allele (S487L) that was able to confer resistance to RIF and CEF through a single mutation. Two other common RRDR mutations made the cells 32 times more sensitive to CEF (H482Y) or led to only modest CEF resistance (Q469R). The diverse effects of these three mutations on CEF resistance are correlated with differences in the expression of peptidoglycan (PG) synthesis genes and in the levels of two metabolites crucial in regulating PG synthesis, glucosamine-6-phosphate (GlcN-6-P) and UDP--acetylglucosamine (UDP-GlcNAc). We conclude that RRDR mutations can have widely varying effects on pathways important for cell wall biosynthesis, and this may restrict the spectrum of mutations that arise during combination therapy. Rifampicin (RIF) is one of the most valued drugs in the treatment of tuberculosis. TB treatment relies on a combination therapy and for multidrug-resistant strains may include β-lactams. Mutations in present a common route for emergence of resistance to RIF. In this study, using B. subtilis as a model, we evaluate the emergence of resistance for the synergistic combination of RIF and the β-lactam cefuroxime (CEF). One clinically relevant mutation conferred resistance to both RIF and CEF, whereas one other increased CEF sensitivity. We were able to link these CEF sensitivity phenotypes to accumulation of UDP--acetylglucosamine (UDP-GlcNAc), which feedback regulates GlmS activity and thereby peptidoglycan synthesis. Further, we found that higher CEF concentrations precluded the emergence of high RIF resistance. Collectively, these results suggest that multidrug treatment regimens may limit the available pathways for the evolution of antibiotic resistance.
细菌可以通过影响 RNA 聚合酶核心亚基的突变来适应应激条件，从而导致转录的有益变化。为了对抗 Rifampicin（RIF）的选择，会在导致抗生素结合减少的决定 Rif 耐药性区域（RRDR）中出现突变。这些变化也可以改变转录，从而对细菌适应性产生多效性影响。在这里，我们研究了枯草芽孢杆菌对 RIF 和β-内酰胺头孢呋辛（CEF）协同组合的耐药性进化。两个独立的进化实验导致了单个 等位基因（S487L）的恢复，该基因通过单个突变能够赋予对 RIF 和 CEF 的抗性。另外两个常见的 RRDR 突变使细胞对 CEF 的敏感性提高了 32 倍（H482Y）或仅导致适度的 CEF 抗性（Q469R）。这三个突变对 CEF 抗性的不同影响与肽聚糖（PG）合成基因表达和两种在调节 PG 合成中至关重要的代谢物水平的差异有关，这两种代谢物是葡萄糖胺-6-磷酸（GlcN-6-P）和 UDP--乙酰葡萄糖胺（UDP-GlcNAc）。我们得出结论，RRDR 突变对细胞壁生物合成途径有广泛的影响，这可能限制了组合治疗过程中出现的突变谱。Rifampicin（RIF）是治疗结核病最有价值的药物之一。结核病的治疗依赖于联合疗法，对于耐多药菌株可能包括β-内酰胺类药物。 中的突变是产生 RIF 耐药性的常见途径。在这项研究中，我们使用枯草芽孢杆菌作为模型，评估了 RIF 和β-内酰胺头孢呋辛（CEF）协同组合出现的耐药性。一个临床相关的 突变赋予了对 RIF 和 CEF 的抗性，而另一个突变增加了 CEF 的敏感性。我们能够将这些 CEF 敏感性表型与 UDP--乙酰葡萄糖胺（UDP-GlcNAc）的积累联系起来，UDP-GlcNAc 反馈调节 GlmS 活性，从而调节肽聚糖的合成。此外，我们发现更高的 CEF 浓度阻止了高 RIF 耐药性的出现。总的来说，这些结果表明，多药治疗方案可能会限制抗生素耐药性进化的可用途径。
