Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
PLoS One. 2023 May 16;18(5):e0285856. doi: 10.1371/journal.pone.0285856. eCollection 2023.
Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of individual mutations and mutation combinations. Thirty-five mutants with reduced susceptibility to ceftazidime were evolved from two antibiotic-sensitive P. aeruginosa reference strains PAO1 and PA14. Mutations were identified by whole genome sequencing. The evolved mutants tolerated ceftazidime at concentrations between 4 and 1000 times that of the parental bacteria, with most mutants being ceftazidime resistant (minimum inhibitory concentration [MIC] ≥ 32 mg/L). Many mutants were also resistant to meropenem, a carbapenem antibiotic. Twenty-eight genes were mutated in multiple mutants, with dacB and mpl being the most frequently mutated. Mutations in six key genes were engineered into the genome of strain PAO1 individually and in combinations. A dacB mutation by itself increased the ceftazidime MIC by 16-fold although the mutant bacteria remained ceftazidime sensitive (MIC < 32 mg/L). Mutations in ampC, mexR, nalC or nalD increased the MIC by 2- to 4-fold. The MIC of a dacB mutant was increased when combined with a mutation in ampC, rendering the bacteria resistant, whereas other mutation combinations did not increase the MIC above those of single mutants. To determine the clinical relevance of mutations identified through experimental evolution, 173 ceftazidime-resistant and 166 sensitive clinical isolates were analysed for the presence of sequence variants that likely alter function of resistance-associated genes. dacB and ampC sequence variants occur most frequently in both resistant and sensitive clinical isolates. Our findings quantify the individual and combinatorial effects of mutations in different genes on ceftazidime susceptibility and demonstrate that the genetic basis of ceftazidime resistance is complex and multifactorial.
铜绿假单胞菌可引起多种严重感染。头孢他啶是一种头孢菌素，是治疗感染的关键抗生素，但相当一部分分离株对头孢他啶具有耐药性。本研究旨在鉴定导致耐药性的突变，并定量评估单个突变和突变组合的影响。从两种抗生素敏感的铜绿假单胞菌参考株 PAO1 和 PA14 中进化出 35 株对头孢他啶敏感性降低的突变株。通过全基因组测序鉴定突变。进化后的突变体可耐受头孢他啶的浓度为亲本细菌的 4 至 1000 倍，大多数突变体对头孢他啶耐药（最低抑菌浓度 [MIC]≥32mg/L）。许多突变体也对美罗培南（一种碳青霉烯类抗生素）耐药。28 个基因在多个突变体中发生突变，其中 dacB 和 mpl 突变最为频繁。将 6 个关键基因的突变分别和组合导入 PAO1 基因组中。dacB 突变本身使头孢他啶 MIC 增加 16 倍，尽管突变体细菌仍对头孢他啶敏感（MIC<32mg/L）。ampC、mexR、nalC 或 nalD 突变使 MIC 增加 2-4 倍。dacB 突变与 ampC 突变组合可使细菌耐药，而其他突变组合的 MIC 增加不超过单个突变体。为了确定通过实验进化鉴定的突变的临床相关性，分析了 173 株头孢他啶耐药和 166 株敏感的临床分离株，以确定可能改变耐药相关基因功能的序列变异。dacB 和 ampC 序列变异在耐药和敏感的临床分离株中最常见。本研究定量评估了不同基因中的突变对头孢他啶敏感性的个体和组合影响，并证明了头孢他啶耐药的遗传基础是复杂的、多因素的。
