Department of Life Science and Biotechnology, Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba, 277-8561, Japan.
Sci Rep. 2018 Apr 3;8(1):5179. doi: 10.1038/s41598-018-23474-4.
Our knowledge as to how bacteria acquire antibiotic resistance is still fragmented, especially for the ribosome-targeting drugs. In this study, with the aim of finding novel mechanisms that render bacteria resistant to the ribosome-targeting antibiotics, we developed a general method to systematically screen for antibiotic resistant 16 S ribosomal RNAs (rRNAs), which are the major target for multiple antibiotics (e.g. spectinomycin, tetracycline, and aminoglycosides), and identify point mutations therein. We used Escherichia coli ∆7, a null mutant of the rrn (ribosomal RNA) operons, as a surrogate host organism to construct a metagenomic library of 16 S rRNA genes from the natural (non-clinical) environment. The library was screened for spectinomycin resistance to obtain four resistant 16 S rRNA genes from non-E. coli bacterial species. Bioinformatic analysis and site-directed mutagenesis identified three novel mutations - U1183C (the first mutation discovered in a region other than helix 34), and C1063U and U1189C in helix 34 - as well as three well-described mutations (C1066U, C1192G, and G1193A). These results strongly suggest that uncharacterized antibiotic resistance mutations still exist, even for traditional antibiotics.
我们对于细菌如何获得抗生素耐药性的了解仍然很零散，特别是对于针对核糖体的药物。在这项研究中，我们旨在寻找使细菌对针对核糖体的抗生素产生耐药性的新机制，因此开发了一种系统筛选抗生素耐药性 16S 核糖体 RNA（rRNA）的通用方法，这些 rRNA 是多种抗生素（例如壮观霉素、四环素和氨基糖苷类抗生素）的主要靶标，并确定其中的点突变。我们使用大肠杆菌 ∆7（rrn（核糖体 RNA）操纵子的 null 突变体）作为替代宿主生物，从自然（非临床）环境中构建了 16S rRNA 基因的宏基因组文库。该文库经过壮观霉素耐药性筛选，从非大肠杆菌细菌物种中获得了四个耐药 16S rRNA 基因。生物信息学分析和定点诱变鉴定了三个新突变-U1183C（首次在螺旋 34 以外的区域发现的突变）以及螺旋 34 中的 C1063U 和 U1189C-以及三个描述良好的突变-C1066U、C1192G 和 G1193A。这些结果强烈表明，即使是传统抗生素，仍然存在未被表征的抗生素耐药性突变。
