Differentiation of Streptococcus pneumoniae from other Streptococcus mitis group streptococci (SMGS) remains challenging despite the introduction of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). While the bile solubility test (BST) provides most reliable discrimination of pneumococci, its practical implementation is limited by subjective visual interpretation and frequent inconclusive results. We aimed to develop a rapid confirmation BST based on direct-on-target MALDI-TOF MS assay. After establishment of optimal test conditions, test performance was evaluated on 36 consecutive clinical SMGS isolates. Colony material was suspended and pipetted onto a MALDI target. After drying, sodium deoxycholate in different concentrations (2%, 5%, and 10 %) was added. Incubation for 30 min (at room temperature or 35 °C) was followed by liquid removal and spot washing. After adding 70 % formic acid, spots were overlaid with matrix and measured (MALDI Biotyper smart, Bruker). The absence of microbial spectra (Biotyper score <1.7) in samples with sodium deoxycholate indicated efficient removal of bacterial biomass due to bile solubility, thus, identifying pneumococci. In contrast, scores ≥1.7 were interpreted as lack of bile solubility and confirmation as viridans streptococci other than S. pneumoniae. Highest test accuracy was achieved applying 5% sodium deoxycholate at 35 °C and 10 % sodium deoxycholate at room temperature. These test conditions provided 100 % sensitivity and 100 % specificity for discrimination of S. pneumoniae. The developed MALDI-TOF MS-based BST is an easy-to-perform assay with minimum hands-on time and objective readout. The promising results of this proof-of-principle study warrant confirmation with large collections of epidemiologically diverse strains.

分化肺炎链球菌从其它缓症链球菌尽管引入了基质辅助激光解吸/电离飞行时间质谱仪（MALDI-TOF MS），但是链球菌组（SMGS）仍然具有挑战性。虽然胆汁溶解度测试（BST）提供了对肺炎球菌的最可靠区分，但其实际实施受到主观视觉解释和频繁不确定的结果的限制。我们旨在开发基于直接靶向MALDI-TOF MS分析的快速确认BST。建立最佳测试条件后，对36个连续的临床SMGS分离株进行测试性能评估。将菌落材料悬浮并吸移到MALDI靶上。干燥后，加入不同浓度（2％，5％和10％）的脱氧胆酸钠。温育30分钟（在室温或35℃），然后除去液体并点洗。加入70％的甲酸后，将斑点覆盖在基质上并进行测量（MALDI Biotyper smart，Bruker）。含脱氧胆酸钠的样品中没有微生物谱（Biotyper得分<1.7），表明由于胆汁溶解性有效去除了细菌生物质，因此鉴定出肺炎球菌。相反，得分≥1.7则被解释为缺乏胆汁溶解性，并被确认为绿色链霉菌链球菌，而不是肺炎链球菌。在35°C下使用5％脱氧胆酸钠和在室温下使用10％脱氧胆酸钠可以达到最高的测试精度。这些测试条件为肺炎链球菌的鉴别提供了100％的灵敏度和100％的特异性。研发的基于MALDI-TOF MS的BST是一种易于操作的测定方法，具有最少的动手时间和客观的读数。这项原理验证研究的有希望的结果值得大量流行病学多样菌株的证实。