Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to 16S rRNA gene sequencing for species identification of nonfermenting bacteria
Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to 16S rRNA gene sequencing for species identification of nonfermenting bacteria
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DOI：
10.1128/jcm.00157-08
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发表时间：
2008-06-01
影响因子：
9.4
通讯作者：
Harmsen, D.
中科院分区：
文献类型：
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作者：
Mellmann, A.;Cloud, J.;Harmsen, D.
非发酵细菌是无处不在的环境机会主义者，可引起人类感染，尤其是受损患者。由于其生物化学反应活性有限且形态不同，经典表型方法的误认屡见不鲜。因此，我们评估了使用基质辅助激光解吸电离飞行时间质谱法（MALDI-TOF MS）进行物种鉴定。根据制造商推荐的microflex测量方法和MALDI BioTyper软件（Bruker Daltonik GmbH, Leipzig, Germany），即使用2000 - 20,000 Da的质量范围和一种新的模式匹配算法，利用与人类感染最相关的37属248株非发酵培养收集菌株，建立了基于MALDI- tof ms的物种鉴定参考数据库。为了评估该数据库，对80个盲编码的临床非发酵菌株进行了分析。采用部分16S rRNA基因测序作为物种命名的参考方法。16S rRNA基因测序结果显示，80株分离株中有57株获得独特的物种鉴定（>= 99%序列相似度）；另外11个分离株在这个阈值上给出了不明确的结果，仅被认定为属水平。10株分离菌株被鉴定为属水平（>= 97%相似度）；如果两个分离株的相似性值低于该阈值，则视为未鉴定，排除在进一步分析之外。MALDI-TOF MS鉴定出78株菌株中的67株（85.9%），与参考方法结果一致；9人被误认，2人身份不明。在3种不同的质谱仪和4种不同的培养基条件下，随机选择10株菌株的鉴定均100%正确。因此，基于MALDI-TOF质谱的非发酵细菌物种鉴定在10分钟内提供了准确且可重复的结果，而无需大量消耗消耗品。
Nonfermenting bacteria are ubiquitous environmental opportunists that cause infections in humans, especially compromised patients. Due to their limited biochemical reactivity and different morphotypes, misidentification by classical phenotypic means occurs frequently. Therefore, we evaluated the use of matrix-assisted laser desorption ionization -time-of-flight mass spectrometry (MALDI-TOF MS) for species identification. By using 248 nonfermenting culture collection strains composed of 37 genera most relevant to human infections, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurement and MALDI BioTyper software (Bruker Daltonik GmbH, Leipzig, Germany), i.e., by using a mass range of 2,000 to 20,000 Da and a new pattern-matching algorithm. To evaluate the database, 80 blind-coded clinical nonfermenting bacterial strains were analyzed. As a reference method for species designation, partial 16S rRNA gene sequencing was applied. By 16S rRNA gene sequencing, 57 of the 80 isolates produced a unique species identification (>= 99% sequence similarity); 11 further isolates gave ambiguous results at this threshold and were rated as identified to the genus level only. Ten isolates were identified to the genus level (>= 97% similarity); and two isolates had similarity values below this threshold, were counted as not identified, and were excluded from further analysis. MALDI-TOF MS identified 67 of the 78 isolates (85.9%) included, in agreement with the results of the reference method; 9 were misidentified and 2 were unidentified. The identities of 10 randomly selected strains were 100% correct when three different mass spectrometers and four different cultivation media were used. Thus, MALDI-TOF MS-based species identification of nonfermenting bacteria provided accurate and reproducible results within 10 min without any substantial costs for consumables.