García-Gómez Elizabeth, Jaso-Vera Marcos E, Juárez-Verdayes Marco A, Alcántar-Curiel María D, Zenteno Juan C, Betanzos-Cabrera Gabriel, Peralta Humberto, Rodríguez-Martínez Sandra, Cancino-Díaz Mario E, Jan-Roblero Janet, Cancino-Diaz Juan C
Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas-Instituto Politécnico Nacional, Ciudad de México, Mexico.
Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.
Microb Pathog. 2017 Feb;103:139-148. doi: 10.1016/j.micpath.2016.12.021. Epub 2016 Dec 23.
In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 μg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR G mutation. Isolates carrying the G mutation had higher levels of norA expression compared with those that did not. To corroborate that the G mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro. This strain also presented the G mutation and had a high level of norA expression and EF, indicating that the G mutation is important for the HEA phenotype. The G mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5'UTR G mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene.
在金黄色葡萄球菌ATCC25923菌株中，norA基因5'非翻译区（5'UTR）的flqB突变导致norA mRNA表达增加和高外排活性（HEA）。表皮葡萄球菌中norA基因5'UTR与HEA的关系尚未得到研究；因此，我们研究了该区域在表皮葡萄球菌临床分离株中的功能。基于溴化乙锭（EtBr）的最低抑菌浓度（MIC）值和使用半自动荧光法测定的外排效率（EF）来选择具有HEA的分离株。通过定量norA表达水平（RT-qPCR）和通过序列分析鉴定5'UTR突变来研究5'UTR的功能。在总共165株分离株中，只有10株（6.1%）具有HEA（EtBr MIC = 300 μg/ml，EF范围为48.4%至97.2%）。10株具有HEA的分离株中有8株存在5'UTR G突变。携带G突变的分离株与未携带该突变的分离株相比，norA表达水平更高。为了证实G突变与HEA有关，体外获得了一株适应EtBr的菌株。该菌株也存在G突变，并且具有高水平的norA表达和EF，表明G突变对于HEA表型很重要。G突变在Shine-Dalgarno区域产生了不同的结构，这可能促进norA mRNA的更好翻译。据我们所知，这是第一份证明norA基因5'UTR G突变参与表皮葡萄球菌分离株HEA表型的报告。在此，我们提出EtBr的外排是由norA基因转录和/或翻译的增加引起的。
