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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\u2009S ribosomal RNAs (rRNAs), which are the major target for multiple antibiotics (e.g. spectinomycin, tetracycline, and aminoglycosides), and identify point mutations therein. We used\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    \u22067, a null mutant of the\n                    <jats:italic>rrn<\/jats:italic>\n                    (ribosomal RNA) operons, as a surrogate host organism to construct a metagenomic library of 16\u2009S rRNA genes from the natural (non-clinical) environment. The library was screened for spectinomycin resistance to obtain four resistant 16\u2009S rRNA genes from non-\n                    <jats:italic>E. coli<\/jats:italic>\n                    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.\n                  <\/jats:p>","DOI":"10.1038\/s41598-018-23474-4","type":"journal-article","created":{"date-parts":[[2018,3,28]],"date-time":"2018-03-28T21:40:34Z","timestamp":1522273234000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Functional metagenomic approach to identify overlooked antibiotic resistance mutations in bacterial rRNA"],"prefix":"10.1038","volume":"8","author":[{"given":"Kentaro","family":"Miyazaki","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kei","family":"Kitahara","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2018,4,3]]},"reference":[{"key":"23474_CR1","doi-asserted-by":"publisher","first-page":"1555","DOI":"10.2165\/11317030-000000000-00000","volume":"69","author":"XZ Li","year":"2009","unstructured":"Li, X. 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