Centre for Bacterial Resistance Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, UK.
Faculty of Natural Sciences, Department of Mathematics, Imperial College London, London, UK.
Rapid Commun Mass Spectrom. 2024 Oct 30;38(20):e9888. doi: 10.1002/rcm.9888.
Rapid, accurate, and easy-to-perform diagnostic assays are required to address the current need for the diagnosis of resistant pathogens. That is particularly the case for mycobacteria, such as the human pathogen Mycobacterium tuberculosis, which requires up to 2 weeks for the determination of the drug susceptibility profile using the conventional broth microdilution method. To address this challenge, we investigated the incorporation of deuterium, the stable isotope of hydrogen, into lipids as a read out of the drug susceptibility profile.
Deuterium is incorporated into newly synthesized proteins or lipids in place of hydrogen as bacterial cells grow, increasing the mass of the macromolecules, which can then be observed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). As proof-of-concept, we used the non-pathogenic Mycobacterium smegmatis mc155 strain, which is susceptible to the aminoglycoside antibiotic kanamycin, and M. smegmatis mc155 containing the empty vector pVV16, which is kanamycin-resistant. Bacteria were incubated in a culture medium containing 50% of deuterium oxide (DO) and either 1 or 2 times the minimal inhibitory concentration (MIC) of kanamycin. Lipids were then analyzed using the MBT lipid Xtract matrix combined with routine MALDI mass spectrometry in the positive ion mode to evaluate the changes in the lipid profile.
Using this approach, we were able to distinguish susceptible from resistant bacteria in less than 5 h, a process that would take 72 h using the conventional broth microdilution method.
We therefore propose a solution for the rapid determination of drug susceptibility profiles using a phenotypic assay combining DO stable isotope labelling and lipid analysis by routine MALDI mass spectrometry.
需要快速、准确且易于执行的诊断检测方法来满足当前对抗菌药物耐药性病原体的诊断需求。这在分枝杆菌等微生物中尤其如此，例如人类病原体结核分枝杆菌，使用常规肉汤微量稀释法确定药敏谱需要长达 2 周的时间。为了解决这一挑战，我们研究了将氘（氢的稳定同位素）掺入脂质中作为药物敏感性谱的读出值。
随着细菌细胞的生长，氘会取代氢掺入到新合成的蛋白质或脂质中，从而增加大分子的质量，然后可以通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）观察到。作为概念验证，我们使用了非致病性分枝杆菌 mc155 菌株，该菌株对氨基糖苷类抗生素卡那霉素敏感，以及含有空载体 pVV16 的分枝杆菌 mc155 菌株，该菌株对卡那霉素具有抗性。细菌在含有 50%重水（DO）的培养基中孵育，并分别含有 1 或 2 倍最小抑菌浓度（MIC）的卡那霉素。然后使用 MBT 脂质 Xtract 基质结合常规 MALDI 质谱在正离子模式下分析脂质，以评估脂质谱的变化。
使用这种方法，我们能够在不到 5 小时内区分敏感和耐药细菌，而使用常规肉汤微量稀释法则需要 72 小时。
因此，我们提出了一种使用结合 DO 稳定同位素标记和常规 MALDI 质谱分析脂质的表型测定法快速确定药敏谱的解决方案。
