| This study has identified and characterized five Borrelia burgdorferi naturally occurring mutations conferring resistance to spectinomycin, kanamycin, gentamicin or streptomycin. These antibiotics target the small subunit of the ribosome, which catalyzes protein synthesis. A naturally occurring coumermycin A1-resistant mutant has also been characterized. Four other B. burgdorferi mutants have been created using site-directed mutagenesis conferring resistance to coumermycin A1. Coumermycin A1 targets the B subunit of DNA gyrase, which catalyzes DNA supercoiling.; 16S rRNA mutations, A1185G and C1186U, homologous to Escherichia coli nucleotides 1191 and 1192, confer 1500-fold and 2500-fold resistance, respectively, to spectinomycin. These mutations appear in a population of B. burgdorferi wild-type parental strain B31-A at a high frequency of 6 x 10-6. After 100 generations in competition with wild-type B31-A, 18% of the cells in a B31-A+ C1186U culture was mutants and 8% of the cells in a B31-A+ A1185G culture were mutants. A 16S rRNA A1402G mutation confers over 100-fold resistance to kanamycin and gentamicin. These mutants appear at a frequency of 1 x 10-8 in a population of B31 and are unable to compete with B31-A after 100 generations. Two streptomycin-resistant mutations were identified in the S12 ribosomal protein. A K88R mutation conferred seven-fold resistance and a K88E mutation conferred 10-fold resistance to streptomycin. Both exhibited only the streptomycin-resistant, SmR, phenotype. The frequency of the streptomycin-resistant mutants was 3 x 10-9 and the K88E mutant was unable to compete with B31-A at 100 generations.; Coumermycin A1-resistant mutants were constructed with amino acid substitutions in gyrase B of T162F, T162L, T162M and T162S. These mutants were from 5 to 6000-fold resistant to coumermycin A1. The naturally occurring T162I mutant was 420-fold resistant to coumermycin A1. |
