Abstract
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.
Highlights
Antibiotic resistance is a serious problem for human beings because pathogenic microorganisms that acquire such resistance void antibiotic treatments
The full-length 16 S ribosomal RNAs (rRNAs) genes were PCR-amplified from the metagenomic mixture using a set of universal primers[24] and the amplicon was cloned in the expression vector pMY205mPAG2 by replacing the pre-existing E. coli 16 S rRNA gene in the vector with the amplicon[24]
We introduced reverting point mutations in metagenomically-retrieved 16 S rRNA genes to confirm that the putative resistance mutations we predicted in Table 2 were involved in Spc resistance
Summary
Antibiotic resistance is a serious problem for human beings because pathogenic microorganisms that acquire such resistance void antibiotic treatments. There are a large number of antibiotics that target the ribosome This is because ribosomes play an essential role in protein biosynthesis, translating messenger RNA-encoded genetic information into proteins, which consists of sequential multistep reactions - initiation, elongation, termination, and recycling. H. halobium and M. smegmatis only have one rrn operon in their genome, and can partly solve the underlying problem in the E. coli system, they only show slow growth phenotypes and make it difficult to conduct reliable genetic experiments. It is uncertain whether all possible resistant mutations to an antibiotic have successfully and correctly been listed using these systems. It should be noted that Thermus thermophilus, a thermophilic strain with a single rrn operon, has been used for similar purposes i.e. to generate interesting insights on antibiotic resistance mutations[14,15]
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