Genetic Architecture of Intrinsic Antibiotic Susceptibility

BackgroundAntibiotic exposure rapidly selects for more resistant bacterial strains, and both a drug's chemical structure and a bacterium's cellular network affect the types of mutations acquired.Methodology/Principal FindingsTo better characterize the genetic determinants of antibiotic susceptibility, we exposed a transposon-mutagenized library of Escherichia coli to each of 17 antibiotics that encompass a wide range of drug classes and mechanisms of action. Propagating the library for multiple generations with drug concentrations that moderately inhibited the growth of the isogenic parental strain caused the abundance of strains with even minor fitness advantages or disadvantages to change measurably and reproducibly. Using a microarray-based genetic footprinting strategy, we then determined the quantitative contribution of each gene to E. coli's intrinsic antibiotic susceptibility. We found both loci whose removal increased general antibiotic tolerance as well as pathways whose down-regulation increased tolerance to specific drugs and drug classes. The beneficial mutations identified span multiple pathways, and we identified pairs of mutations that individually provide only minor decreases in antibiotic susceptibility but that combine to provide higher tolerance.Conclusions/SignificanceOur results illustrate that a wide-range of mutations can modulate the activity of many cellular resistance processes and demonstrate that E. coli has a large mutational target size for increasing antibiotic tolerance. Furthermore, the work suggests that clinical levels of antibiotic resistance might develop through the sequential accumulation of chromosomal mutations of small individual effect.研究背景：抗生素暴露会快速筛选出耐药性更强的细菌菌株，而药物的化学结构与细菌的细胞网络均会影响其所获得的突变类型。 研究方法与主要结果：为了更好地表征抗生素敏感性的遗传决定因子，我们将转座子诱变构建的大肠杆菌（Escherichia coli）文库分别暴露于17种覆盖多种药物类别与作用机制的抗生素中。以中等抑制同基因亲本菌株生长的药物浓度持续传代培养该文库多代后，即使仅具有微小适应度优势或劣势的菌株丰度也会发生可检测且可重复的变化。随后我们采用基于微阵列（microarray）的遗传足迹法（genetic footprinting）策略，定量测定了每个基因对大肠杆菌固有抗生素敏感性的贡献。我们发现，既有敲除后可提升整体抗生素耐受性的基因位点，也有下调后可增强对特定药物及药物类别耐受性的通路。所鉴定到的有益突变涉及多条通路，同时我们还发现了成对突变：单个突变仅能轻微降低抗生素敏感性，但二者联合则可赋予更高的耐受性。 研究结论与意义：本研究结果表明，广泛的突变可调控多种细胞耐药通路的活性，同时证实大肠杆菌存在可提升抗生素耐受性的大量突变靶点。此外，本研究提示临床水平的抗生素耐药性可能通过逐步积累单个效应微小的染色体突变而形成。