The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, UK.
J Infect. 2011 Mar;62(3):212-7. doi: 10.1016/j.jinf.2011.01.003. Epub 2011 Jan 13.
It has generally been held that the repeated emergence of resistance in Mycobacterium tuberculosis is due to the effects of large population sizes, slow replication, and prolonged colonization and treatment. However, there have been suggestions that its emergence is facilitated by high mutation rates due to a lack of mismatch repair, error-prone polymerases, and a potentially mutagenic host niche. Genome re-sequencing has indicated higher variability in strains with emergent resistance, but these studies have not been performed in serial isolates in which drug resistance has emerged. We have used genome re-sequencing to address the mutational processes that occur during the evolution of drug resistance during a clinical infection.
Serial isolates from a patient obtained over a 12 month period, and spanning the transition of the colonizing population from fully drug sensitive, to isoniazid resistant, to isoniazid and rifampicin (multiply drug) resistant, spanning an estimated minimum of 100 generations within the host, were deep sequenced using Illumina sequencing. The genomes were compared, and all mutations in non-repetitive sequences were identified.
Specific mutations conferring resistance were identified. No additional mutations in non-repetitive regions were present. The mutations observed were kat S315T and rpoB D516Y.
M. tuberculosis is relatively stable genetically within the host, and demonstrates greater stability than is suggested by in vitro studies of emergent drug resistance, or by models of hypermutability. This indicates that it is primarily the nature and duration of the infection that are sufficient to lead to the repeated emergence of drug resistance in this infection if improperly managed, and that the selective pressure of the drugs limits additional diversification. This emphasizes the central importance of maintaining therapeutic concentrations of at least two effective antibiotics for the duration of treatment to prevent the emergence of resistance.
一般认为，结核分枝杆菌的耐药性反复出现是由于其种群数量大、复制缓慢、长期定植和治疗等因素所致。然而，也有人认为，由于缺乏错配修复、易错聚合酶以及潜在的致突变宿主小生境，其耐药性的出现是由高突变率所促进的。基因组重测序表明，具有耐药性的菌株具有更高的变异性，但这些研究尚未在耐药性出现的连续分离株中进行。我们利用基因组重测序来研究在临床感染过程中耐药性进化过程中发生的突变过程。
从一名患者在 12 个月的时间内获得的连续分离株，跨越了定植菌群从完全敏感到异烟肼耐药，再到异烟肼和利福平（多药）耐药的转变，跨越了宿主内估计至少 100 代的进化过程，使用 Illumina 测序进行了深度测序。对基因组进行了比较，并确定了非重复序列中的所有突变。
鉴定出了赋予耐药性的特定突变。非重复区域没有出现其他突变。观察到的突变是 kat S315T 和 rpoB D516Y。
结核分枝杆菌在宿主内的遗传上相对稳定，其稳定性比体外研究耐药性出现或高突变性模型所表明的要高。这表明，如果管理不当，感染本身的性质和持续时间足以导致该感染中耐药性的反复出现，而药物的选择压力限制了进一步的多样化。这强调了在治疗期间维持至少两种有效抗生素的治疗浓度以防止耐药性出现的重要性。
