Environmental Sciences Department, Metropolitan Autononous University (Lerma Unit), Lerma de Villada, Mexico.
Department of Molecular Evolution, Center of Astrobiology (CSIC-INTA), Torrejón de Ardoz, Spain.
Int Microbiol. 2013 Jun;16(2):103-11. doi: 10.2436/20.1501.01.185.
The exploration of novel antibiotic resistance determinants in a particular environment may be limited because of the presence of uncultured microorganisms. In this work, a culture-independent approach based on functional metagenomics was applied to search for chloramphenicol resistance genes in agro-industrial wastewater in Lerma de Villada, Mexico. To this end, a metagenomic library was generated in Escherichia coli DH10B containing DNA isolated from environmental samples of the residual arsenic-enriched (10 mg/ml) effluent. One resistant clone was detected in this library and further analyzed. An open reading frame similar to a multidrug resistance protein from Aeromonas salmonicida and responsible for chloramphenicol resistance was identified, sequenced, and found to encode a member of the major facilitator superfamily (MFS). Our results also showed that the expression of this gene restored streptomycin sensitivity in E. coli DH10B cells. To gain further insight into the phenotype of this MFS family member, we developed a model of the membrane protein multiporter that, in addition, may serve as a template for developing new antibiotics.
在特定环境中探索新型抗生素耐药决定因素可能会受到限制，因为存在未培养的微生物。在这项工作中，应用了一种基于功能宏基因组学的非培养方法，在墨西哥 Lerma de Villada 的农业工业废水中寻找氯霉素抗性基因。为此，在含有从环境样品中分离的 DNA 的大肠杆菌 DH10B 中生成了一个宏基因组文库，这些样品来自于富含砷（10 mg/ml）的剩余流出物。在这个文库中检测到一个抗性克隆，并进一步进行了分析。鉴定并测序了一个与鲑鱼气单胞菌多药耐药蛋白相似的开放阅读框，该蛋白负责氯霉素耐药性，并被发现编码主要易化子超家族（MFS）的成员。我们的结果还表明，该基因的表达恢复了大肠杆菌 DH10B 细胞对链霉素的敏感性。为了更深入地了解这种 MFS 家族成员的表型，我们开发了一种膜蛋白多载体模型，该模型还可以作为开发新抗生素的模板。
