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To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with defined Mycobacterium smegmatis laboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.

Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. The presence of several rRNA operons (rrn) and a single rpsL gene in most bacterial genomes prohibits the isolation of streptomycin-resistant mutants in which resistance is mediated by mutations in the 16S rRNA gene (rrs). Three strains were constructed in this investigation: Mycobacterium smegmatis rrnB, M. smegmatis rpsL(3+), and M. smegmatis rrnB rpsL(3+). M. smegmatis rrnB carries a single functional rrn operon, i.e., rrnA (comprised of 16S, 23S, and 5S rRNA genes) and a single rpsL+ gene; M. smegmatis rpsL(3+) is characterized by the presence of two rrn operons (rrnA and rrnB) and three rpsL+ genes; and M. smegmatis rrnB rpsL(3+) carries a single functional rrn operon (rrnA) and three rpsL+ genes. By genetically altering the number of rpsL and rrs alleles in the bacterial genome, mutations in rrs conferring streptomycin resistance could be selected, as revealed by analysis of streptomycin-resistant derivatives of M. smegmatis rrnB rpsL(3+). Besides mutations well known to confer streptomycin resistance, novel streptomycin resistance conferring mutations were isolated. Most of the mutations were found to map to a functional pseudoknot structure within the 530 loop region of the 16S rRNA. One of the mutations observed, i.e., 524G-->C, severely distorts the interaction between nucleotides 524G and 507C, a Watson-Crick interaction which has been thought to be essential for ribosome function. The use of the single rRNA allelic M. smegmatis strain should help to elucidate the principles of ribosome-drug interactions.

Clinical resistance to aminoglycosides in general is due to enzymatic drug modification. Mutational alterations of the small ribosomal subunit rRNA have recently been found to mediate acquired resistance in bacterial pathogens in vivo. In this study we investigated the effect of 16S rRNA heterozygosity (wild-type [wt] and mutant [mut] operons at position 1408 [1408wt/1408mut]) on aminoglycoside resistance. Using an integrative vector, we introduced a single copy of a mutated rRNA operon (1408 A-->G) into Mycobacterium smegmatis, which carries two chromosomal wild-type rRNA operons; the resultant transformants exhibited an aminoglycoside-sensitive phenotype. In contrast, introduction of the mutated rRNA operon into an M. smegmatis rrnB knockout strain carrying a single functional chromosomal wild-type rRNA operon resulted in aminoglycoside-resistant transformants. Subsequent analysis by DNA sequencing and RNase protection assays unexpectedly demonstrated a homozygous mutant genotype, rRNAmut/rRNAmut, in the resistant transformants. To investigate whether RecA-mediated gene conversion was responsible for the aminoglycoside-resistant phenotype in the rRNAwt/rRNAmut strains, recA mutant strains were generated by allelic exchange techniques. Transformation of the recA rrnB M. smegmatis mutant strains with an integrative vector expressing a mutated rRNA operon (Escherichia coli position 1408 A-->G) resulted in transformants with an aminoglycoside-sensitive phenotype. Subsequent analysis showed stable heterozygosity at 16S rRNA position 1408 with a single wild-type allele and a single resistant allele. These results demonstrate that rRNA-mediated mutational resistance to aminoglycosides is recessive.

Clarithromycin is a potent macrolide that has been used for treating infections with nontuberculous mycobacteria. Pairs of susceptible and resistant Mycobacterium intracellulare strains were obtained from patients with chronic pulmonary M. intracellulare infections undergoing monotherapy with clarithromycin. Nucleotide sequence comparisons of the peptidyltransferase region in 23S rRNAs from parental and resistant strains revealed that in three of six resistant strains, for which the MIC was > 32 micrograms/ml, a single base was mutated (Escherichia coli equivalent, A-2058-->G, C, or U). As the modification of adenine 2058 by dimethylation is a frequent cause of macrolide resistance in a variety of different bacteria, we suggest that mutation of A-2058 confers acquired resistance to clarithromycin in M. intracellulare.