铜绿假单胞菌PAO1 ku基因缺失菌株的构建及其对生物被膜耐药性的影响

Construction of Pseudomonas aeruginosa ku gene deletion mutant and its effects on antibiotic resistance of biofilm

摘要:

【背景】铜绿假单胞菌是常见的条件致病菌，易形成生物被膜，具有基因突变率高、耐药性强的特点。非同源末端连接是DNA双链断裂的主要修复途径之一，修复过程会导致DNA突变产生。【目的】研究非同源末端连接对生物被膜中的铜绿假单胞菌基因突变率和耐药性的影响。【方法】通过基因无痕敲除的方法构建PAO1菌株的ku基因缺失突变株Δku并构建其回补株。对比研究突变株和野生菌株生物被膜形成能力、生物被膜状态下各菌的基因突变率以及对抗生素的耐受性。通过荧光定量PCR检测生物被膜中PAO1菌株ku基因的表达水平。【结果】各突变株生物被膜形成能力无显著差异；与野生菌株相比，突变株Δku在生物被膜中的基因突变率以及对环丙沙星和庆大霉素的最低抑菌浓度(minimum inhibitory concentration，MIC)下降。荧光定量PCR结果表明，ku基因在生物被膜形成早期转录水平有明显上调。【结论】非同源末端连接修复途径对生物被膜中的铜绿假单胞菌基因突变率以及耐药性的提高有一定的作用。本研究将为后续进一步阐释铜绿假单胞菌耐药产生机制提供一定的理论依据。

Abstract:

[Background] Pseudomonas aeruginosa is a common opportunistic pathogen, most of them can form biofilms which show the characteristics of high mutation rate and strong antibiotic resistance. Non-homologous end joining (NHEJ) is one of the major DNA double-strand break repair pathway which leads to DNA mutations. [Objective] To study the effects of NHEJ on biofilm-forming ability, mutation rate and drug resistance within biofilm in P. aeruginosa. [Methods] The ku gene deletion mutant strain Δku was constructed by in-frame deletion and its complementary strain was also obtained. We studied the biofilm-forming ability of each strain as well as the mutation rate and drug resistance within their biofilms. The transcription level of ku gene in P. aeruginosa biofilm was determined by qPCR. [Results] Compared with the wild-type strain, the ku gene deletion strain did not show significant difference in biofilm-forming ability but displayed a slight reduction in mutation rate and minimum inhibitory concentration (MIC) to ciprofloxacin and gentamicin within biofilms. The qPCR results showed that the transcription level of the ku gene was increased when biofilms were formed. [Conclusion] NHEJ affects the mutation rate and antibiotic resistance of P. aeruginosa biofilm. This study will provide a basis for exploiting the mechanisms of antibiotic resistance in P. aeruginosa.