Cameron M, Barkema H W, De Buck J, De Vliegher S, Chaffer M, Lewis J, Keefe G P
Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada C1A 4P3.
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada T2N 1N4.
J Dairy Sci. 2017 Mar;100(3):2137-2147. doi: 10.3168/jds.2016-12020. Epub 2016 Dec 29.
This study evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) for the identification of bovine-associated coagulase-negative staphylococci (CNS), a heterogeneous group of different species. Additionally, we aimed to expand the MALDI-ToF MS database with new reference spectra as required to fill the gaps within the existing commercial spectral library. A total of 258 isolates of CNS were used in the study, covering 16 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 219), and the remainder were identified by sequencing of 16S rRNA, hsp60, or both rpoB and hsp60. The genotypic identification was considered the gold standard identification. All MALDI-ToF MS identifications were carried out using the direct transfer method. In a preliminary evaluation (n = 32 isolates; 2 of each species) with the existing commercial database, MALDI-ToF MS showed a typeability of 81% (26/32) and an accuracy of 96% (25/26). In the main evaluation (n = 226 isolates), MALDI-ToF MS with the existing commercial Biotyper (Bruker Daltonics Inc., Billerica, MA) database achieved a typeability of 92.0% (208/226) and an accuracy of 99.5% (207/208). Based on the assessment of the existing commercial database and prior knowledge of the species, a total of 13 custom reference spectra, covering 8 species, were created and added to the commercial database. Using the custom reference spectra expanded database, isolates were identified by MALDI-ToF MS with 100% typeability and 100% accuracy. Whereas the MALDI-ToF MS manufacturer's cutoff for species-level identification is 2.000, the reduction of the species level cutpoint to ≥1.700 improved the species-level identification rates (from 64 to 92% for the existing commercial database) when classifying CNS isolates. Overall, MALDI-ToF MS using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS.
本研究评估了基质辅助激光解吸/电离飞行时间质谱（MALDI-ToF MS）用于鉴定牛源凝固酶阴性葡萄球菌（CNS）的能力，CNS是一组不同种类的异质菌群。此外，我们旨在根据需要用新的参考光谱扩展MALDI-ToF MS数据库，以填补现有商业光谱库中的空白。本研究共使用了258株CNS分离株，涵盖16种不同的CNS菌种。大多数分离株先前通过rpoB基因测序进行鉴定（n = 219），其余分离株通过16S rRNA、hsp60或rpoB和hsp60两者的测序进行鉴定。基因型鉴定被视为金标准鉴定。所有MALDI-ToF MS鉴定均采用直接转移法进行。在使用现有商业数据库的初步评估（n = 32株分离株；每种菌种2株）中，MALDI-ToF MS的分型成功率为81%（26/32），准确率为96%（25/26）。在主要评估（n = 226株分离株）中，使用现有的商业Biotyper（布鲁克道尔顿公司，马萨诸塞州比勒里卡）数据库的MALDI-ToF MS分型成功率为92.0%（208/226），准确率为99.5%（207/208）。基于对现有商业数据库的评估和菌种的先验知识，共创建了13个涵盖8个菌种的定制参考光谱，并添加到商业数据库中。使用定制参考光谱扩展后的数据库，MALDI-ToF MS对分离株的鉴定分型成功率和准确率均达到100%。虽然MALDI-ToF MS制造商的菌种水平鉴定临界值为2.000，但在对CNS分离株进行分类时，将菌种水平临界值降低至≥1.700可提高菌种水平鉴定率（现有商业数据库从64%提高到92%）。总体而言，采用直接转移法的MALDI-ToF MS被证明是鉴定牛源CNS的高度可靠工具。
