Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide
Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide
复制标题
DOI：
10.1093/jac/dkn217
复制
发表时间：
2008-09-01
影响因子：
5.2
通讯作者：
Amaral, Leonard
中科院分区：
文献类型：
--
作者：
Couto, Isabel;Costa, Sofia Santos;Amaral, Leonard
目的：通过使敏感的金黄色葡萄球菌菌株适应增加浓度的溴化乙锭（外排泵（EP）的已知底物），并通过对所得后代进行表型和基因型分析，我们表征了S.金黄色葡萄球菌对溴化乙锭的适应性。金黄色葡萄球菌ATCC 25923在浓度递增的溴化乙锭中生长。在有或没有六种外排泵抑制剂（EPI）的情况下，测定了八类抗生素、八种杀生物剂和两种染料的代表对ATCC 25923及其溴化乙锭抗性后代ATCC 25923（EtBr）的MIC。在存在/不存在EPI的情况下，通过实时荧光测定法评价外排活性。分别采用PCR和定量RT-PCR（qRT-PCR）检测8种EP基因的存在和表达。结果：ATCC 25923（EtBr）对溴化乙锭的耐药性是亲本菌株的32倍，对杀菌剂和亲水性氟喹诺酮类药物的耐药性也比亲本菌株高。对这些药物的耐药性可通过EPIs氯丙嗪、硫利达嗪和利血平降低。相同的EPI可抑制ATCC 25923（EtBr）引起的溴化乙锭外排增加。qRT-PCR显示，诺拉在ATCC 25923（EtBr）中过表达35倍，而其余EP基因的表达没有显著增加。诺拉A启动子区的测序显示ATCC 25923（EtBr）中有70 bp的缺失。金黄色葡萄球菌对季铵化合物如溴化乙锭的敏感性降低，导致生物体对多种化合物的敏感性降低，所述化合物包括喹诺酮和杀生物剂，通过流出介导的应答，对于菌株ATCC 25923，所述应答主要是NorA介导的。这种改变的表达可能是由于诺拉启动子区的改变。
Objectives: By adapting an antibiotic-susceptible Staphylococcus aureus strain to increasing concentrations of ethidium bromide, a known substrate of efflux pumps (EPs), and by phenotypically and genotypically analysing the resulting progeny, we characterized the molecular mechanisms of S. aureus adaptation to ethidium bromide.Methods: S. aureus ATCC 25923 was grown in increasing concentrations of ethidium bromide. The MICs of representatives of eight classes of antibiotics, eight biocides and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny ATCC 25923(EtBr) were determined with or without six efflux pump inhibitors (EPIs). Efflux activity in the presence/absence of EPIs was evaluated by real-time fluorometry. The presence and expression of eight EP genes were assayed by PCR and quantitative RT-PCR (qRT-PCR), respectively. Mutations in grlA, gyrA and norA promoter regions were screened by DNA sequencing.Results: Compared with its parental strain, ATCC 25923(EtBr) was 32-fold more resistant to ethidium bromide and also more resistant to biocides and hydrophilic fluoroquinolones. Resistance to these could be reduced by the EPIs chlorpromazine, thioridazine and reserpine. Increased efflux of ethidium bromide by ATCC 25923(EtBr) could be inhibited by the same EPIs. qRT-PCR showed that norA was 35-fold over-expressed in ATCC 25923(EtBr), whereas the remaining EP genes showed no significant increase in their expression. Sequencing of the norA promoter region revealed a 70 bp deletion in ATCC 25923(EtBr).Conclusions: Exposure of S. aureus to quaternary compounds such as ethidium bromide results in decreased susceptibility of the organism to a wide variety of compounds, including quinolones and biocides through an efflux-mediated response, which for strain ATCC 25923 is mainly NorA-mediated. This altered expression may result from alterations in the norA promoter region.