Sequencing mutant library in the E. coli beta-lactamase gene ampC selected on different drugs. AmpC beta-lactamase mutant sequencing

Beta-lactamase inhibitors are increasingly used to counteract microbial resistance mediated by beta-lactamase enzymes. These inhibitors compete with the beta-lactam drug for the same binding site of the beta-lactamase, thereby generating an inherent evolutionary tradeoff: enzyme mutations that increase its activity against the beta-lactam drug also increase its susceptibility towards the inhibitor. It is unclear how common and accessible are mutants that escape this adaptive tradeoff. Here, systematically constructing and phenotyping a deep mutant library of the ampC beta-lactamase gene of Escherichia coli, we discover mutations, which even in the presence of the enzyme inhibitor allow growth at beta-lactam concentrations far exceeding the native inhibitory levels of the wildtype strain. Importantly, while such escape mutations appear for combinations of avibactam with some beta-lactam drugs, for other drug combinations completely prevent such escape phenotypes. Amplicon sequencing of the selected mutant pool identified these escape mutations and showed that they are rare and drug specific. The differential adaptive potential of ampC to combinations of avibactam and different beta-lactam drugs can help guide drug treatments that are more resilient to evolution of resistance.

β-内酰胺酶抑制剂（Beta-lactamase inhibitors）正被越来越广泛地用于对抗由β-内酰胺酶（beta-lactamase）介导的微生物耐药性。此类抑制剂可与β-内酰胺类药物（beta-lactam drug）竞争β-内酰胺酶的同一结合位点，由此催生一种固有的进化权衡：能够提升酶对β-内酰胺类药物活性的酶突变，同时也会增强该酶对抑制剂的敏感性。目前尚不明确能够规避这类适应性权衡的突变体究竟有多普遍、多易获得。本研究通过系统构建并完成表型鉴定的大肠埃希菌（Escherichia coli）ampC型β-内酰胺酶基因深度突变文库，成功鉴定出一类突变体：即便在酶抑制剂存在的条件下，这类突变体仍可在远高于野生型菌株天然抑制水平的β-内酰胺类药物浓度下实现生长增殖。值得注意的是，尽管此类逃逸突变可在阿维巴坦（avibactam）与部分β-内酰胺类药物联用时出现，但在其他药物联用方案中则可完全阻断此类逃逸表型的产生。对筛选得到的突变体池进行扩增子测序后，我们确认了这些逃逸突变的存在，并证实其发生频率极低且具有药物特异性。ampC型β-内酰胺酶对阿维巴坦与不同β-内酰胺类药物联用方案的差异化适应潜力，可为指导制定更能抵御耐药性进化的药物治疗方案提供科学依据。