Yakupoğulları Yusuf, Otlu Barış, Çelik Betül, Gözükara Bağ Harika Gözde
İnönü University Faculty of Medicine, Department of Medical Microbiology, Malatya, Turkey.
İnönü University Faculty of Medicine, Department of Biostatistics and Medical Informationt, Malatya, Turkey.
Mikrobiyol Bul. 2019 Jan;53(1):1-11. doi: 10.5578/mb.67523.
Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a new method that is increasingly used in microbiology laboratories due to its ability of reliable and rapid identification (ID) of bacteria and fungi. However, some problems emerge in the routine clinical diagnosis since only one gram-negative selective medium has been suggested up to date. Though EMB agar is one of the traditional gram-negative selective media, there is no data in the scientific literature, about the ID performance of MALDI TOF MS with the gram-negative bacteria grown on this medium. In this study, we tested the ID performance of MALDI-TOF MS for gram-negative isolates on EMB agar and aimed to develop a rapid and easy sample preparation method for improving this performance. A total of 468 clinical isolates of gram-negative bacteria, consisting of 37 different species from 20 genera, were included in this study. The isolates were identified using the Vitek MS MALDI-TOF MS (Bio Mérieux, France) both directly from EMB agar, and also through a two-step colony washing (once with physiologic saline, and three times with 70% ethanol) method. The performances of these two IDs were compared. In the direct reading from EMB medium, 382 (81.6%) of 468 studied isolates were correctly identified at species level; no ID was detected for 80 (17%) isolates, and 6 (1.2%) isolates (four at the genus level) were misidentified Performance of MALDI-TOF MS directly from EMB agar was excellent (100%) for 14 species including Stenotrophomonas maltophilia, Klebsiella oxytoca, Salmonella spp., and Proteus mirabilis; and lowest for Providencia spp. (62.5%), Escherichia coli (70.5%) and Acinetobacter spp. (70.7%). Following the washing procedure which was performed about 20 min with simple laboratory equipment, 434 (92.7%) isolates were correctly identified at species level; 30 (6.4%) strains could not be identified, and four (0.85%) isolates (two at the genus level) were misidentified. Statistical analyses indicated that the washing procedure defined here significantly increased the overall ID performance of MALDI-TOF MS with EMB agar (p= 0.001), particularly with improving the IDs of those markedly dye-absorbing genera, such as E.coli and A.baumannii. In this study, EMB agar which has no data until today on its suitability for mass spectrometric identification has been shown to be useful for bacterial identification with MALDI-TOF MS. In addition, the unidentified gram-negative bacteria in the direct reading from the EMB medium have been shown to be identified after the colony washing method as described here. Determination of the different medium alternatives will contribute to effective usage of MALDI-TOF MS in microbiology laboratories.
基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）是一种新方法，因其能够可靠且快速地鉴定细菌和真菌，在微生物实验室中越来越常用。然而，由于目前仅推荐了一种革兰氏阴性选择性培养基，常规临床诊断中出现了一些问题。尽管伊红美蓝琼脂（EMB琼脂）是传统的革兰氏阴性选择性培养基之一，但科学文献中尚无关于在此培养基上生长的革兰氏阴性菌的MALDI TOF MS鉴定性能的数据。在本研究中，我们测试了MALDI-TOF MS对在EMB琼脂上的革兰氏阴性分离株的鉴定性能，并旨在开发一种快速简便的样品制备方法以提高该性能。本研究共纳入了468株革兰氏阴性菌临床分离株，它们由来自20个属的37个不同物种组成。这些分离株直接从EMB琼脂上，以及通过两步菌落洗涤法（先用生理盐水洗涤一次，再用70%乙醇洗涤三次），使用Vitek MS MALDI-TOF MS（法国生物梅里埃公司）进行鉴定。比较了这两种鉴定方法的性能。在直接从EMB培养基读取结果时，468株研究分离株中有382株（81.6%）在种水平上被正确鉴定；80株（17%）分离株未检测到鉴定结果，6株（1.2%）分离株（4株在属水平）被错误鉴定。对于嗜麦芽窄食单胞菌、产酸克雷伯菌、沙门氏菌属和奇异变形杆菌等14个物种，直接从EMB琼脂上进行MALDI-TOF MS鉴定的性能极佳（100%）；对于普罗威登斯菌属（62.5%）、大肠杆菌（70.5%）和不动杆菌属（70.7%），该性能最低。使用简单实验室设备进行约20分钟的洗涤程序后，434株（92.7%）分离株在种水平上被正确鉴定；30株（6.4%）菌株无法鉴定，4株（0.85%）分离株（2株在属水平）被错误鉴定。统计分析表明，此处定义的洗涤程序显著提高了MALDI-TOF MS对EMB琼脂上细菌的总体鉴定性能（p = 0.001），特别是在提高对那些明显吸收染料的属，如大肠杆菌和鲍曼不动杆菌的鉴定方面。在本研究中，此前尚无关于其是否适合质谱鉴定数据的EMB琼脂已被证明可用于MALDI-TOF MS进行细菌鉴定。此外，从EMB培养基直接读取结果时未鉴定出的革兰氏阴性菌，在此处描述的菌落洗涤法后已被证明可以被鉴定。确定不同的培养基选择将有助于微生物实验室有效使用MALDI-TOF MS。
