Functional metagenomic approach to identify overlooked antibiotic resistance mutations in bacterial rRNA

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 Delta 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.