Data from: Macrophage adaptation leads to parallel evolution of genetically diverse Escherichia coli small-colony variants with increased fitness in vivo and antibiotic collateral sensitivity

Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP), and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and show increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.小菌落变异体（Small-colony variants, SCVs）是进化实验与临床分离株中常见的表型，与抗生素耐药性及持续性感染密切相关。我们近期观察到，大肠杆菌（Escherichia coli）SCVs在适应与巨噬细胞的互作过程中反复出现。为鉴定该临床相关表型出现的遗传基础，我们对独立进化得到的SCV克隆开展了全基因组测序（whole-genome sequencing）。本研究发现了此前未与SCVs关联的新型突变靶点（如cydA、pepP），并观察到广泛存在的功能平行性现象；所有SCV克隆均携带与电子传递链（electron-transport chain）相关基因的突变。由于SCVs在巨噬细胞适应过程中出现，且通常表现出更高的抗生素耐药性，我们检测了SCVs在巨噬细胞内的适合度及其抗生素耐药谱。结果显示，SCVs在巨噬细胞内具有适合度优势，体外实验中氨基糖苷类（aminoglycoside）耐药性升高，但对四环素（tetracycline）等其他抗生素存在交叉敏感性。重要的是，我们在体内（in vivo）环境中得到了一致结果：SCVs在小鼠肠道定植时具有适合度优势，且该优势可通过抗生素干预进行调控——氨基糖苷类的卡那霉素（kanamycin）可增强SCVs的适合度，而四环素则会显著降低其适合度。本研究结果彰显了以实验进化（experimental evolution）为框架，解析宿主-微生物互作（host-microbe interactions）过程中适应性演化的成因与后果的研究价值。