培养条件对克柔念珠菌MALDI-TOF MS鉴定结果的影响分析

中国真菌学杂志　 2020, Vol. 15 　Issue (5): 262-267.

培养条件对克柔念珠菌MALDI-TOF MS鉴定结果的影响分析

黄晶晶^1,2, 范欣^1,2,3, 肖盟^1,2, 徐志鹏¹, 张戈^1,2, 徐英春^1,2

1. 中国医学科学院北京协和医院检验科, 北京 100730;
2. 侵袭性真菌病机制研究与精准诊断北京市重点实验室, 北京 100730;
3. 首都医科大学附属北京朝阳医院感染和临床微生物科, 北京 100020

收稿日期:2019-06-18 出版日期:2020-10-28 发布日期:2020-11-23
通讯作者: 徐英春,E-mail:xycpumch@139.com E-mail:xycpumch@139.com
作者简介:黄晶晶,女(汉族),博士在读.E-mail:hjj_6620@163.com
基金资助:
北京协和医学院研究生创新基金（2019-1002-02）；国家科技重大专项子课题"基于飞行质谱技术的真菌鉴定研究"（2018ZX10712001-008）；北京市科技计划"飞行时间质谱的示范推广研究"（Z181100001618015）

Impact of different culture conditions on the identification of Candida krusei clinical isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system

HUANG Jing-jing^1,2, FAN Xin^1,2,3, XIAO Meng^1,2, XU Zhi-peng¹, ZHANG Ge^1,2, XU Ying-chun^1,2

1. Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China;
2. Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases, Beijing 100730, China;
3. Beijing Chao-Yang hospital, Capital Medical University, Beijing 100020, China

Received:2019-06-18 Online:2020-10-28 Published:2020-11-23

摘要/Abstract

摘要：

目的探究不同的培养时间、培养温度、培养基种类及培养气体环境对克柔念珠菌基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry，MALDI-TOF MS）鉴定结果的影响。方法收集中国侵袭性真菌耐药监测网（CHIF-NET）2012-2013年中17所医院克柔念珠菌25株，每株菌平行接种在沙堡弱氯霉素琼脂平板（SDA-C）、血琼脂平板等6种培养基，28℃和35℃ 2种孵育温度，空气和5% CO₂ 2种气体环境中孵育，以真菌rDNA ITS测序为金标准，孵育24 h和48 h后，使用Vitek MALDI-TOF质谱仪（简称Vitek MS）和Bruker Autoflex Speed型MALDI-TOF质谱仪（简称Bruker MS）分别进行菌种鉴定，并对其鉴定正确率进行分析。结果克柔念珠菌孵育24 h和48 h时Vitek MS鉴定正确率均为100%，Bruker MS鉴定正确率分别是98.86%和87.43%。28℃ SDA-C上生长的克柔念珠菌种水平鉴定准确率较35℃生长的菌株高。比较各种培养基，孵育48 h时SDA-C上生长的克柔念珠菌Bruker MS鉴定正确率（100%）最高。结论克柔念珠菌接种在SDA-C上，28℃空气培养24 h质谱鉴定效果最佳。常规细菌培养基上生长的克柔念珠菌，Bruker MS鉴定分值≥1.700即可认为菌种鉴定可靠。

关键词: 克柔念珠菌, 鉴定, 培养条件, MALDI-TOF MS

Abstract:

Objective To investigate the effects of different culture media, incubation time, culture temperature and gas environment on the identification of Candida krusei by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Twenty-five strains of Candida krusei were collected from 17 hospitals of China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program. Six culture media were tested, including Sabouraud dextrose agar supplemented with chloramphenicol (SDA-C), CHROMagar (CCP), trypticase soy agar supplemented with 5% sheep blood (BAP), chocolate agar supplemented with vancomycin (CAP-VA), China blue lactose rosolic acid agar (CBA/R) and MacConkey agar (MAC). All isolates were identified by sequencing of the rDNA internal transcribed spacer (ITS) region as "gold standard". Isolates were incubated at 28℃ and 35℃ for 24 or 48 hours, with/without 5% CO₂ gas, and then identification was carried out by two MALDI-TOF MS systems. Results All (175/175) isolates incubated for 24 hours and 48 hours were identified correctly by Vitek MS. In comparison, Bruker Biotyper correctly identified 98.86% (173/175) isolates incubated for 24 hours and 87.43% (153/175) isolates incubated for 48 hours. All strains inoculated on SDA-C medium were correctly identified by both MALDI-TOF MS systems, but the accuracy of the species level of identification of which grown on SDA-C at 28℃ is higher than those grown at 35℃. With 48 hours' incubation, the correct identification rates by Bruker Biotyper were higher for strains grown on SDA than other media. Conclusion The correct identification rate of 24-hour incubated strains was higher than the rate of 48-hour incubated strains by Bruker MS. Compared with cultured at 35℃, the identification of Candida krusei strains grown at 28℃ was more accurate. SDA-C was the most suitable media for MALDI-TOF MS identification in our study. The results were reliable for Bruker Biotyper MALDI-TOF MS system with score values of≥1.700.

Key words: Candida krusei, identification, culture conditions, MALDI-TOF MS

中图分类号:

R379.4

黄晶晶, 范欣, 肖盟, 徐志鹏, 张戈, 徐英春. 培养条件对克柔念珠菌MALDI-TOF MS鉴定结果的影响分析[J]. 中国真菌学杂志, 2020, 15(5): 262-267.

HUANG Jing-jing, FAN Xin, XIAO Meng, XU Zhi-peng, ZHANG Ge, XU Ying-chun. Impact of different culture conditions on the identification of Candida krusei clinical isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system[J]. Chinese Journal of Mycology, 2020, 15(5): 262-267.

参考文献 15

[1]	Jamal WY, Ahmad S, Khan ZU, et al. Comparative evaluation of two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems for the identification of clinically significant yeasts[J]. Int J Infect Dis, 2014, 26:167-170.
[2]	Xiao M, Sun ZY, Kang M, et al. Five-year national surveillance of invasive candidiasis:species distribution and azole susceptibility from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Study[J]. J Clin Microbiol, 2018, 56(7):e00577-18.
[3]	Singhal N, Kumar M, Kanaujia PK, et al. MALDI-TOF mass spectrometry:an emerging technology for microbial identification and diagnosis[J]. Front Microbiol, 2015, 6:791.
[4]	Buchan BW, Ledeboer NA. Advances in the identification of clinical yeast isolates using matrix-assisted laser desorption ionization-time of flight mass spectrometry[J]. J Clin Microbiol, 2013, 51(5):1359-1366.
[5]	Santos C, Lima N, Sampaio P, et al. Matrix-assisted laser de-sorption/ionization time-of-flight intact cell mass spectrometry to detect emerging pathogenic Candida species[J]. Diagn Microbiol Infect Dis, 2011, 71(3):304-308.
[6]	王欢,贾天野,鲍春梅,等.质谱仪在真菌鉴定中的应用与评价[J].传染病信息,2015,(4):210-214.
[7]	Emonet S, Shah HN, Cherkaoui A, et al. Application and use of various mass spectrometry methods in clinical microbiology[J]. Clin Microbiol Infect, 2010, 16(11):1604-1613.
[8]	Wang H, Li Y, Fan X, et al. Evaluation of Bruker Biotyper and Vitek MS for the identification of Candida tropicalis on different solid culture media[J]. J Microbiol Immunol Infect, 2017, pii:S1684-1182(17)30239-6.
[9]	Kardos T, Kovács R, Kardos G,et al. Poor in vivo efficacy of caspofungin, micafungin and amphotericin B against wild-type Candida krusei clinical isolates does not correlate with in vitro susceptibility results[J]. J Chemother, 2018, 30(4):233-239.
[10]	Hakki M, Staab JF, Marr KA. Emergence of a Candida krusei isolate with reduced susceptibility to caspofungin during therapy[J]. Antimicrob Agents Chemother, 2006, 50(7):2522-2524.
[11]	Kahn JN, Garcia-Effron G, Hsu MJ, et al. Acquired echinocandin resistance in a Candida krusei isolate due to modification of glucan synthase[J]. Antimicrob Agents Chemother, 2007, 51(5):1876-1878.
[12]	Pappas PG, Kauffman CA, Andes DR, et al. Clinical practice guideline for the management of candidiasis:2016 update by the Infectious Diseases Society of America[J]. Clin Infect Dis, 2016, 62(4):e1-50.
[13]	Balá?ová T, Makovcová J, Šedo O, et al. The influence of culture conditions on the identification of Mycobacterium species by MALDI-TOF MS profiling[J]. FEMS Microbiol Lett, 2014, 353(1):77-84.
[14]	Wunschel DS, Hill EA, McLean JS, et al. Effects of varied pH, growth rate and temperature using controlled fermentation and batch culture on matrix assisted laser desorption/ionization whole cell protein fingerprints[J]. J Microbiol Methods, 2005, 62(3):259-271.
[15]	Buskirk AD, Hettick JM, Chipinda, et al. Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi[J]. Anal Biochem, 2011, 411(1):122-128.