In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.

副结核分枝杆菌亚种（Mycobacterium avium ssp. paratuberculosis, MAP）是引发反刍动物慢性肠道疾病的致病菌。当前可用的诊断方法复杂且耗时。体外实验表明，MAP培养物释放的挥发性有机化合物（volatile organic compound, VOC）模式可反映细菌生长状态，并可区分不同菌株。本研究旨在可控动物模型环境中开展体内VOC检测。研究采用针捕集微萃取（needle trap microextraction，用于呼吸样本）与固相微萃取（solid phase microextraction，用于粪便样本），对42只山羊（16只对照组、26只MAP接种动物）的呼吸与粪便样本进行VOC预浓缩，并通过气相色谱-质谱联用法（gas chromatography/ mass spectrometry）完成分析。分别于接种后第18、29、33、41及48周开展检测。从血液样本中检测MAP特异性抗体与MAP特异性γ-干扰素（interferon-γ）应答反应。所有标记性VOC的化学身份均通过纯标准品分析得到确认。基于高pptV级别的检出限与两个数量级的线性范围，可在呼吸样本以及粪便顶空中检出100余种VOC。接种组与非接种组动物间存在28种物质的浓度差异。尽管感染进程中，呋喃类、含氧化合物及烃类等主要物质的VOC模式发生了改变，但在任意检测时间点，粪便样本中仍有16种物质、呼吸样本中仍有3种VOC可区分接种组与对照组动物。粪便顶空的VOC浓度差异可反映MAP细菌的存在情况。呼吸样本的VOC谱型差异则与宿主的γ-干扰素应答反应相关。未来体内VOC分析或可助力克服现有诊断方法的局限性。