Pseudomonas aeruginosa is a notorious opportunistic pathogen causing various types of biofilm-related infections. Biofilm formation is a unique microbial strategy that allows P. aeruginosa to survive adverse conditions such as antibiotic treatment and human immune clearance. In this study, we experimentally evolved P. aeruginosa PAO1 biofilms for cyclic treatment in the presence of high dose of imipenem, and enriched hyperbiofilm mutants within six cycles in two independent lineages. The competition assay showed that the evolved hyperbiofilm mutants can outcompete the ancestral strain within biofilms but not in planktonic cultures. Whole-genome sequencing analysis revealed the hyperbiofilm phenotype is caused by point mutations in rpoS gene in all independently evolved mutants and the same mutation was found in P. aeruginosa clinical isolates. We further showed that mutation in rpoS gene increased the intracellular c-di-GMP level by turning on the expression of the diguanylate cyclases. Mutation in rpoS increased pyocyanin production and virulence in hyperbiofilm variants. Here, our study revealed that antibiotic treatment of biofilm-related P. aeruginosa infections might induce a hyperbiofilm phenotype via rpoS mutation, which might partially explain antimicrobial treatment failure of many P. aeruginosa biofilm-related infections.

中文翻译：

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的rpoS -mutation变体在选择的铜绿假单胞菌生物膜亚胺培南压力下

铜绿假单胞菌是一种臭名昭著的机会性病原体，可导致各种类型的生物膜相关感染。生物膜形成是一种独特的微生物策略，使铜绿假单胞菌能够在抗生素治疗和人体免疫清除等不利条件下存活。在这项研究中，我们通过实验进化了铜绿假单胞菌 PAO1 生物膜，用于在高剂量亚胺培南存在的情况下进行循环治疗，并在两个独立谱系的六个循环内富集了超生物膜突变体。竞争试验表明，进化出的超生物膜突变体可以在生物膜中胜过祖先菌株，但在浮游培养物中则不然。全基因组测序分析表明，高生物膜表型是由所有独立进化的突变体中 rpoS 基因的点突变引起的，并且在铜绿假单胞菌临床分离株中发现了相同的突变。我们进一步表明 rpoS 基因的突变通过打开双鸟苷酸环化酶的表达增加了细胞内 c-di-GMP 水平。rpoS 中的突变增加了超生物膜变体中绿脓素的产生和毒力。在这里，我们的研究表明，生物膜相关铜绿假单胞菌感染的抗生素治疗可能会通过 rpoS 突变诱导高生物膜表型，这可能部分解释了许多铜绿假单胞菌生物膜相关感染的抗菌治疗失败。