The influence of incubation time, sample preparation and exposure to oxygen on the quality of the MALDI-TOF MS spectrum of anaerobic bacteria
The influence of incubation time, sample preparation and exposure to oxygen on the quality of the MALDI-TOF MS spectrum of anaerobic bacteria
复制标题
DOI：
10.1111/1469-0691.12644
复制
发表时间：
2014-12-01
影响因子：
14.2
通讯作者：
van Winkelhoff, A. J.
中科院分区：
文献类型：
--
作者：
Veloo, A. C. M.;Elgersma, P. E.;van Winkelhoff, A. J.
利用基质辅助激光解吸/电离飞行时间质谱 (MALDI-TOF MS)，可以快速可靠地识别细菌。这尤其适用于厌氧细菌。由于厌氧细菌的生长速率和氧敏感性不同，我们旨在研究孵育时间、氧气暴露和样品制备对使用布鲁克系统的光谱质量的影响。此外，还确定了再现性和检查者间的变异性。根据革兰氏染色特征、生长速率和菌落形态，选择了代表 17 个属的 26 个厌氧菌种。检查者之间的差异表明，制定目标的经验可能是一个重要的变量。在孵育 24 至 96 小时之间确定孵育时间的影响。革兰氏阴性厌氧菌孵育 48 小时后和革兰氏阳性厌氧菌孵育 72 小时后获得可靠的物种鉴定。培养物暴露于氧气并不影响所有测试的革兰氏阳性菌种的 MALDI-TOF MS 鉴定结果。暴露于氧气 >24 小时和 48 小时后，无法分别鉴定坏死梭杆菌和中间普雷沃氏菌。其他测试的革兰氏阴性细菌可在暴露于氧气 48 小时后被识别。大多数测试物种可以使用直接点样方法进行识别。长双歧杆菌和 Finegoldia magna 需要使用 70% 甲酸进行目标提取，以获得可靠的菌种鉴定和象牙胃杆菌的完全提取。光谱质量受到目标上发现的细菌数量、涂片的均匀性和检查者经验的影响。
With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), bacteria can be identified quickly and reliably. This accounts especially for anaerobic bacteria. Because growth rate and oxygen sensitivity differ among anaerobic bacteria, we aimed to study the influence of incubation time, exposure to oxygen and sample preparation on the quality of the spectrum using the Bruker system. Also, reproducibility and inter-examiner variability were determined. Twenty-six anaerobic species, representing 17 genera, were selected based on gram-stain characteristics, growth rate and colony morphology. Inter-examiner variation showed that experience in the preparation of the targets can be a significant variable. The influence of incubation time was determined between 24 and 96 h of incubation. Reliable species identification was obtained after 48h of incubation for gram-negative anaerobes and after 72h for gram-positive anaerobes. Exposure of the cultures to oxygen did not influence the results of the MALDI-TOF MS identifications of all tested gram-positive species. Fusobacterium necrophorum and Prevotella intermedia could not be identified after >24h and 48h of exposure to oxygen, respectively. Other tested gram-negative bacteria could be identified after 48h of exposure to oxygen. Most of the tested species could be identified using the direct spotting method. Bifidobacterium longum and Finegoldia magna needed on-target extraction with 70% formic acid in order to obtain reliable species identification and Peptoniphilus ivorii a full extraction. Spectrum quality was influenced by the amount of bacteria spotted on the target, the homogeneity of the smear and the experience of the examiner.