Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser315 which are present in approximately 50–90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser315 appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser315 mutations are favoured in the slow growing cultures.优化结核病（TB）治疗的一项重要目标，是在不提升复发率、不诱导抗生素耐药菌株产生的前提下，缩短抗生素治疗疗程。在任何结核分枝杆菌（Mycobacterium tuberculosis, M. tuberculosis）种群中，均存在一定比例的药物耐受细菌；这或许源于预先存在的生长缓慢/不复制细菌种群——这类细菌通过表达可限制药物活性的表型，从而免受抗生素的杀伤作用。本研究借助恒化器（chemostats）中可控且定义明确的培养体系，观察了平均代时分别为69.3h的缓慢生长杆菌与23.1h的快速生长杆菌在异烟肼（isoniazid）暴露后的应答反应，以此解答该科学问题。研究检测到两种生长速率下的菌群存在表型差异，包括外排机制（efflux mechanisms）的表达，以及反义RNA/小RNA（antisense RNA/small RNA）参与调控药物耐受表型——这一调控通路此前尚未在结核分枝杆菌中被探究。基因型分析结果显示，缓慢生长杆菌通过katG基因密码子Ser315（katG codon Ser315）的特异性突变获得异烟肼耐药性；该类突变在所有异烟肼耐药临床分离株中的占比约为50%~90%。快速生长杆菌则以混合种群形式存续，其katG基因突变分布于整个基因区域。katG基因密码子Ser315的突变在体外培养中，尤其是快速生长的培养物内，似乎存在适合度代价（fitness cost）。本研究结果表明，缓慢生长杆菌需要具备功能完整的katG编码的过氧化氢酶-过氧化物酶（catalase-peroxide），而快速生长细菌则无此需求，这或可解释为何在缓慢生长的培养物中更易出现katG基因密码子Ser315的突变。