Garton Natalie J, Waddell Simon J, Sherratt Anna L, Lee Su-Min, Smith Rebecca J, Senner Claire, Hinds Jason, Rajakumar Kumar, Adegbola Richard A, Besra Gurdyal S, Butcher Philip D, Barer Michael R
Department of Infection, Immunity and Inflammation, University of Leicester Medical School, Leicester, United Kingdom.
PLoS Med. 2008 Apr 1;5(4):e75. doi: 10.1371/journal.pmed.0050075.
Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis.
Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body-positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body-positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R(2) = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population.
As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.
结核痰液提供了一份必须通过化疗清除且可能会传播感染的杆菌样本。一项初步观察发现，结核分枝杆菌细胞在痰液中含有三酰甘油脂质体，但在体外生长时则没有，这促使我们研究这种现象的程度及其生理基础。
通过尼罗红和金胺联合染色，对来自英国和冈比亚的显微镜检查呈阳性的痰液样本进行脂质体阳性分枝杆菌含量的研究。所有样本均含有脂质体阳性菌群，其占抗酸杆菌总数的比例在3%至86%之间。最近有研究发现，分枝杆菌在进入体外非复制性持续状态时会表达三酰甘油合酶，这促使我们研究这种状态是否也与脂质体形成有关。我们发现，当置于诱导非复制性持续状态的实验室条件下时，两株结核分枝杆菌菌株的脂质体水平与在痰液中发现的水平相当。我们通过比较生长和非复制性持续培养的结核分枝杆菌转录组与直接从痰液样本中获得的转录组，进一步研究了这些生理发现。尽管传统上认为痰液中含有活跃生长的结核杆菌，但我们的转录分析反驳了这些细胞占主导地位的假设。相反，它们通过揭示可明确归因于缓慢复制或非复制性分枝杆菌的转录特征，加强了脂质体分析的结果。最后，脂质体计数与诊断液体培养中出现阳性的时间高度相关（R² = 0.64，p < 0.03），从而在这一细胞学特征与潜在非复制菌群的大小之间建立了直接联系。
由于非复制性结核杆菌对抗生素的杀菌作用具有耐受性且对多种应激具有抗性，在痰液中鉴定出这种类似持留菌的结核杆菌群体为研究化疗反应和结核病传播提供了令人兴奋且易于处理的新机会。
