[ODP31] 7. Antimicrobial agents and resistance -b. Antimicrobial resistance

[ODP-219] Streptomycin dependent Mycobacterium bovis BCG possessing a 513 cytosine insertion in 16S rRNA gene

Naoko Honda¹, Norito Sato², Masaaki Nakayama², Takayuki Matsumura³, Tsuyoshi Sekizuka⁴, Makoto Kuroda⁴, Manabu Ato⁵, Kazuo Kobayashi², Kouji Ishii¹, Naoya Ohara² (¹Dept. Qual. Assur. Radiol. Protect., Natl. Inst. Infect. Dis., ²Dept. Oral Microbiol., Okayama Uni. Grad. Sch. Med. Dent. & Pharm. Sci., ³Dept. Immunol., Natl. Inst. Infect. Dis., ⁴Path. Genomics Ctr., Natl. Inst. Infect. Dis., ⁵Dept. Mycobacteriology., Natl. Inst. Infect. Dis.)

A streptomycin (SM) dependent mutant M. tuberculosis 18b was originally isolated from the laboratory strain H2 by Hashimoto in 1955. In 18b strain, an additional cytosine residue was inserted between the 513 and 514 nucleotides in the 530 loop of the 16S rRNA gene and Glu insertion after 23Gly was found in initiation factor IF-3. To clarify the 16S rRNA mutation is responsible for SM dependence, we introduced the same nucleotide insertion on the currently used vaccine strain, M. bovis BCG Tokyo. This strain required more than 0.05 mg/ml SM for its optimal growth and stopped growth after SM removal from culture medium. SM dependent phenotype of 18b was reported to be stable and revertant mutants did not appear by long term culture, however, we obtained SM resistant and sensitive BCG strains from SM dependent BCG. Most mutations were mapped on 16S rRNA and the cytosine insertion mutation was retained in SM resistant strains. To analyze the effect of SM on translation accuracy, we purified ribosomes from SM dependent BCG and performed in vitro translation experiments in the presence or absence of SM. The amount of translated Luciferase did not increase by SM, however, translation error rate was smaller in the absence of SM and increased with SM similar to the error rate of WT ribosome without SM.