Table_3_Mechanisms for Development of Ciprofloxacin Resistance in a Clinical Isolate of Pseudomonas aeruginosa.XLS

Treatment of infections by Pseudomonas aeruginosa is difficult due to its high intrinsic and acquired antibiotic resistance. Upon colonization in the human hosts, P. aeruginosa accumulates genetic mutations that confer the bacterium antibiotic resistance and ability to better live in the host environment. Characterizing the evolutionary traits would provide important insights into the development of effective combinatory antibiotic therapies to cure P. aeruginosa infections. In this work, we performed a detailed analysis of the molecular mechanisms by which a clinical isolate (CSP18) yields a ciprofloxacin-resistant derivative (CRP42). Genomic DNA re-sequencing and RNAseq were carried out to compare the genomic mutational signature and transcriptional profiles between the two isolates. The results indicated that D87G mutation in GyrA, together with MexEF-OprN hyper-expression caused by F7S mutation in MexS, was responsible for the increased resistance to ciprofloxacin in the isolate CRP42. Further simulation of CRP42 by gene editing in CSP18 demonstrated that D87G mutation in GyrA rendered CSP18 a fourfold increase in minimum inhibitory concentration against ciprofloxacin, while F7S mutation in MexS conferred an additional eightfold increase. Our experimental results demonstrate for the first time that the clinically relevant F7S point mutation in MexS results in hyper-expression of the mexEF-oprN and thus confers P. aeruginosa resistance to ciprofloxacin.

由于铜绿假单胞菌（Pseudomonas aeruginosa）兼具极高的固有耐药性与获得性耐药性，其引发的感染治疗难度极大。当该菌定植于人体宿主后，会积累各类基因突变，使其获得抗生素耐药性，并具备更适配宿主环境的生存能力。解析其进化特征，可为开发治疗铜绿假单胞菌感染的高效联合抗生素疗法提供关键思路。本研究针对一株临床分离株（CSP18）产生环丙沙星（ciprofloxacin）耐药衍生株（CRP42）的分子机制展开了详细分析。通过基因组DNA重测序与RNA测序（RNAseq）技术，本研究对两株分离株的基因组突变特征与转录组表达谱进行了对比分析。研究结果显示，GyrA的D87G突变，联合由MexS基因F7S突变介导的MexEF-OprN高表达，是导致衍生株CRP42对环丙沙星耐药性升高的核心原因。后续通过在CSP18菌株中进行基因编辑以模拟CRP42的遗传特征的实验表明，GyrA的D87G突变可使CSP18对环丙沙星的最低抑菌浓度（minimum inhibitory concentration, MIC）提升4倍，而MexS的F7S突变可进一步使其最低抑菌浓度提升8倍。本实验结果首次证实，MexS基因中与临床相关的F7S点突变可介导mexEF-oprN操纵子高表达，进而使铜绿假单胞菌获得对环丙沙星的耐药性。