Wardell Samuel J T, Rehman Attika, Martin Lois W, Winstanley Craig, Patrick Wayne M, Lamont Iain L
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
Antimicrob Agents Chemother. 2019 Sep 9;63(12). doi: 10.1128/AAC.01619-19. Epub 2019 Sep 30.
is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations we experimentally evolved the antibiotic sensitive strain PAO1 to become resistant to three widely used anti-pseudomonal antibiotics, ciprofloxacin, meropenem and tobramycin. Mutants could tolerate up to 2048-fold higher concentrations of antibiotic than strain PAO1. Genome sequences were determined for thirteen mutants for each antibiotic. Each mutant had between 2 and 8 mutations. For each antibiotic at least 8 genes were mutated in multiple mutants, demonstrating the genetic complexity of resistance. For all three antibiotics mutations arose in genes known to be associated with resistance, but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in this study we analysed the corresponding genes in 558 isolates of from patients with chronic lung disease and in 172 isolates from the general environment. Many genes identified through experimental evolution had predicted function-altering changes in clinical isolates but not in environmental isolates, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. Additionally, large deletions of up to 479kb arose in experimentally evolved meropenem resistant mutants and large deletions were present in 87 of the clinical isolates. These findings significantly advance understanding of antibiotic resistance in and demonstrate the validity of experimental evolution in identifying clinically-relevant resistance-associated mutations.
是一种机会性病原体，可引起多种急性和慢性感染。越来越多的分离株发生了使其产生抗生素耐药性的突变，导致治疗困难。为了鉴定与耐药性相关的突变，我们通过实验使抗生素敏感菌株PAO1进化，使其对三种广泛使用的抗假单胞菌抗生素环丙沙星、美罗培南和妥布霉素产生耐药性。突变体能够耐受比PAO1菌株高2048倍的抗生素浓度。测定了每种抗生素的13个突变体的基因组序列。每个突变体有2至8个突变。对于每种抗生素，多个突变体中至少有8个基因发生了突变，这表明耐药性的遗传复杂性。对于所有三种抗生素，突变不仅出现在已知与耐药性相关的基因中，也出现在以前与耐药性无关的基因中。为了确定本研究中发现的突变的临床相关性，我们分析了来自慢性肺病患者的558株分离株和来自一般环境的172株分离株中的相应基因。通过实验进化鉴定的许多基因在临床分离株中具有预测的功能改变变化，但在环境分离株中没有，这表明实验进化细菌中的突变基因可以预测感染期间发生突变的基因。此外，在实验进化的美罗培南耐药突变体中出现了高达479kb的大片段缺失，并且在87株临床分离株中存在大片段缺失。这些发现显著推进了对其抗生素耐药性的理解，并证明了实验进化在鉴定临床相关耐药性相关突变方面的有效性。
