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Experimental Confirmation that an Uncommon rrs Gene Mutation (g878a) of Mycobacterium tuberculosis Confers Resistance to Streptomycin

The effective treatment of patients diagnosed with drug-resistant tuberculosis is highly dependent on the ability to rapidly and accurately determine the antibiotic susceptibility profile of the Mycobacterium tuberculosis isolate(s) involved. Thus, as more clinical microbiology laboratories advance toward the use of DNA sequence-based diagnostics, it is imperative that their predictive functions extend beyond the well-known resistance mutations in order to also encompass as many of the lower-frequency mutations as possible. ABSTRACT The effective treatment of patients diagnosed with drug-resistant tuberculosis is highly dependent on the ability to rapidly and accurately determine the antibiotic susceptibility profile of the Mycobacterium tuberculosis isolate(s) involved. Thus, as more clinical microbiology laboratories advance toward the use of DNA sequence-based diagnostics, it is imperative that their predictive functions extend beyond the well-known resistance mutations in order to also encompass as many of the lower-frequency mutations as possible. However, in most cases, fundamental experimental proof that links these uncommon mutations with phenotypic resistance is lacking. One such example is the g878a polymorphism within the rrs 16S rRNA gene. We, and others, have identified this mutation within a small number of drug-resistant isolates, although a consensus regarding exactly which aminoglycoside antibiotic(s) it confers resistance to has not previously been reached. Here, we have employed oligonucleotide-mediated recombineering to introduce the g878a polymorphism into the rrs gene of Mycobacterium bovis BCG, a close relative of M. tuberculosis, and demonstrate that it confers low-level resistance to streptomycin alone. It does not confer cross-resistance to amikacin, capreomycin, or kanamycin. We also demonstrate that the rrsg878a mutation exerts a substantial fitness defect in vitro that may at least in part explain why clinical isolates bearing this mutation appear to be quite rare. Overall, this study provides clarity to the phenotype attributable to the rrsg878a mutation and is relevant to the future implementation of genomics-based diagnostics as well as the clinical management of patients in whom this particular polymorphism is encountered.