器的子类型<em>空肠弯曲菌</em> SSP。<em> doylei</em>分离株使用基于光谱质谱PhyloProteomics（MSPP）
Summary
基于质谱的phyloproteomics（MSPP）用于键入空肠弯曲杆菌过磷酸钙的集合。doylei隔离在相比于多位点序列分型（MLST）的应变水平。
Abstract
MALDI-TOF MS提供不仅在种和亚种水平，但甚至低于，在应变水平区分某些细菌的可能性。可检测生物标志物离子的等位基因亚型产生具体的分离质量的变化。质谱为基础的phyloproteomics（MSPP）是一种新技术，它结合了质谱检测生物标志物的群众一个方案，允许从比较基因组分离特定质量偏移phyloproteomic关系扣除测序参考株。然后将推断的氨基酸序列被用于计算基于MSPP-系统树图。
在这里，我们通过键入空肠弯曲杆菌 SSP描述MSPP的工作流程。doylei的7株分离的集合。这七株人类起源和多位点序列分（MLST）的展示了其遗传多样性。 MSPP打字造成了七种不同的MSPP序列类型，充分反映他们的PHYlogenetic关系。
C.在菌 SSP。doylei MSPP方案包括14个不同的生物标志物离子，主要是核糖体蛋白在2至11 kDa的质量范围。 MSPP可以在原则上，可以适用于其他质谱平台具有延伸的质量范围。因此，这种技术有可能成为应变水平的微生物类型的有用工具的潜力。
Introduction
在过去十年中，时间飞行基质辅助激光解吸电离质谱（MALDI-TOF MS）已经发展到在临床微生物学1,2微生物属和种鉴定高度评价标准方法。物种鉴定是基于完整细胞或细胞裂解物的小蛋白指纹记录。在常规的临床微生物学中使用的质谱仪的典型质量范围是2-20 kDa的。此外，所得的光谱可以被用于鉴别在下面种菌株和低于亚种级别3。创业初期的研究已经确定了具体的生物标志物离子来株的空肠弯曲菌 4特定亚型， 难辨梭状芽孢杆菌 5， 沙门氏菌 SSP。 伤寒 沙门菌 6， 金黄色葡萄球菌 7 – 9，和Escherichia大肠杆菌10 – 12。
对应等位基因亚型几个变量的生物标记物群众相结合，提供了更深层次的子类型的选项。此前，我们成功地实施大规模型材这些变化转换成一个叫做C.质谱法基于phyloproteomics（MSPP）有意义的和可重复的phyloproteomic关系的方法菌 SSP。 杆菌菌株收集13。 MSPP可以使用质谱等同于基于DNA序列分型技术如多位点序列分型（MLST）。
弯曲杆菌是细菌性胃肠炎的全球领先的原因14，15。作为弯曲菌病感染后后遗症的结果，即，格林-巴利综合症，反应性关节炎和炎症性肠病可能出现16。感染的主要来源是从鸡，火鸡，猪，牛，羊，鸭，牛奶及地表水15,17污染畜肉。因此，在食品安全的背景下定期流行病学监测研究是必要的。 MLST是“黄金标准”的分子分型弯曲杆菌种18。因为基于MLST方法桑格测序是劳动密集，耗时且相对昂贵的，MLST打字被限制为相对小的分离同伙。因此，有必要更便宜和更快的子类型的方法。这种需要可以通过质谱方法，如MSPP得到满足。
本文介绍用空肠弯曲菌 SSP的集合MSPP打字了详细的方案。doylei株及其与MLST潜力比较。
Protocol
1.考虑生物安全条件准备一个安全的工作环境熟悉实验室和安全法规的相关性与微生物工作。大多数人类病原微生物必须在生物安全水平进行处理2的条件，但一些，如伤寒沙门菌，需要生物安全3级上处理每一病原体可在www.cdc.gov/biosafety访问级别的信息。 不管具体微生物的生物危害的分类，把中随附传染性废物传染性病原体必须在处置前进行高压灭菌接触的所有材料。尊重有…
Representative Results
此前，我们成功地建立了一个C. MSPP方案菌 SSP。 空肠弯曲菌 13。在这里，我们的目的是同级亚种C.延长方法菌 SSP。doylei。在这个特定的设置，七C.菌 SSP。doylei株微生物微生物学根特大学BCCM / LMG比利时根特/实验室比利时集合收购。用于我们的分析所有7株均来源于人。基因组测序菌株ATCC 49349（LMG 8843），最初是?…
Discussion
在建立MSPP计划的最关键的一步是生物标志物离子身份的明确的遗传决定。如果它是不可能无疑鉴定的生物标记，那么它应该被排除在方案13。
C.在菌 SSP。doylei方案包括14种不同的生物标志物离子。这些都是5少比C.在检测到C之间杆菌 SSP。 杆菌 MSPP方案13。最显著差异菌 SSP。 菌和C.杆菌 SSP。…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
We are grateful to Hannah Kleinschmidt for excellent technical support. This paper was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universität Göttingen.
Materials
| acetonitrile | Sigma-Aldrich, Taufkirchen, Germany | 34967 | |
| Autoflex III TOF/TOF 200 system | Bruker Daltonics, Bremen, Germany | GT02554 G201 | Mass spectrometer |
| bacterial test standard BTS | Bruker Daltonics, Bremen, Germany | 604537 | |
| BioTools 3.2 SR1 | Bruker Daltonics, Bremen, Germany | 263564 | Software Package |
| Bruker IVD Bakterial Test Standard | Bruker Daltonics, Bremen, Germany | 8290190 | 5 tubes |
| Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG8843 | ATCC 49349;IMVS 1141;NCTC 11951;strain 093 |
| Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG9143 | Goossens Z90 |
| Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG7790 | ATCC 49350;CCUG 18265;Kasper 71;LMG 8219;NCTC 11847 |
| Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG9243 | Goossens N130 |
| Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG8871 | NCTC A603/87 |
| Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG9255 | Goossens B538 |
| Campylobacter jejuni subsp. doylei isolate | Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium | LMG8870 | NCTC A613/87 |
| Columbia agar base | Merck, Darmstadt, Germany | 1.10455 .0500 | 500 g |
| Compass for FlexSeries 1.2 SR1 | Bruker Daltonics, Bremen, Germany | 251419 | Software Package |
| defibrinated sheep blood | Oxoid Deutschland GmbH, Wesel, Germany | SR0051 | |
| ethanol | Sigma-Aldrich, Taufkirchen, Germany | 02854 Fluka | |
| formic acid | Sigma-Aldrich, Taufkirchen, Germany | F0507 | |
| HCCA matrix | Bruker Daltonics, Bremen, Germany | 604531 | |
| Kimwipes paper tissue | Kimtech Science via Sigma-Aldrich, Taufkirchen, Germany | Z188956 | |
| MALDI Biotyper 2.0 | Bruker Daltonics, Bremen, Germany | 259935 | Software Package |
| Mast Cryobank vials | Mast Diagnostica, Reinfeld, Germany | CRYO/B | |
| MSP 96 polished steel target | Bruker Daltonics, Bremen, Germany | 224989 | |
| QIAamp DNA Mini Kit | Qiagen, Hilden, Germany | 51304 | |
| recombinant human insulin | Sigma-Aldrich, Taufkirchen, Germany | I2643 | |
| trifluoroacetic acid | Sigma-Aldrich, Taufkirchen, Germany | T6508 | |
| water, molecular biology-grade | Sigma-Aldrich, Taufkirchen, Germany | W4502 |
Referencias
- Seng, P., et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis. 49 (4), 543-551 (2009).
- Bader, O. MALDI-TOF-MS-based species identification and typing approaches in medical mycology. Proteomics. 13 (5), 788-799 (2013).
- Sandrin, T. R., Goldstein, J. E., Schumaker, S. MALDI TOF MS profiling of bacteria at the strain level: a review. Mass Spectrom Rev. 32 (3), 188-217 (2013).
- Zautner, A. E., et al. Discrimination of multilocus sequence typing-based Campylobacter jejuni subgroups by MALDI-TOF mass spectrometry. BMC Microbiol. 13, 247 (2013).
- Reil, M., et al. Recognition of Clostridium difficile PCR-ribotypes 001, 027 and 126/078 using an extended MALDI-TOF MS system. Eur J Clin Microbiol Infect Dis. 30 (11), 1431-1436 (2011).
- Kuhns, M., Zautner, A. E., et al. Rapid discrimination of Salmonella enterica serovar Typhi from other serovars by MALDI-TOF mass spectrometry. PLoS One. 7 (6), e40004 (2012).
- Wolters, M., et al. MALDI-TOF MS fingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages. Int J Med Microbiol. 301 (1), 64-68 (2011).
- Josten, M., et al. Analysis of the matrix-assisted laser desorption ionization-time of flight mass spectrum of Staphylococcus aureus identifies mutations that allow differentiation of the main clonal lineages. J Clin Microbiol. 51 (6), 1809-1817 (2013).
- Lu, J. J., Tsai, F. J., Ho, C. M., Liu, Y. C., Chen, C. J. Peptide biomarker discovery for identification of methicillin-resistant and vancomycin-intermediate Staphylococcus aureus strains by MALDI-TOF. Anal Chem. 84 (13), 5685-5692 (2012).
- Novais, A., et al. MALDI-TOF mass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131) and D (ST69, ST405, ST393). Eur J Clin Microbiol Infect Dis. , (2014).
- Matsumura, Y., et al. Detection of extended-spectrum-beta-lactamase-producing Escherichia coli ST131 and ST405 clonal groups by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 52 (4), 1034-1040 (2014).
- Christner, M., et al. Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli. PLoS One. 9 (7), e101924 (2014).
- Zautner, A. E., Masanta, W. O., Weig, M., Groß, U., Bader, O. Mass Spectrometry-based PhyloProteomics (MSPP): A novel microbial typing Method. Scientific Reports. 5, (2015).
- Dasti, J. I., Tareen, A. M., Lugert, R., Zautner, A. E., Gross, U. Campylobacter jejuni: a brief overview on pathogenicity-associated factors and disease-mediating mechanisms. Int J Med Microbiol. 300 (4), 205-211 (2010).
- Zautner, A. E., et al. Seroprevalence of campylobacteriosis and relevant post-infectious sequelae. Eur J Clin Microbiol Infect Dis. 33 (6), 1019-1027 (2014).
- Zautner, A. E., Herrmann, S., Groß, U. Campylobacter jejuni – The Search for virulence-associated factors. Archiv Fur Lebensmittelhygiene. 61 (3), 91-101 (2010).
- Dingle, K. E., et al. Multilocus sequence typing system for Campylobacter jejuni. J Clin Microbiol. 39 (1), 14-23 (2001).
- Dingle, K. E., et al. Molecular characterization of Campylobacter jejuni clones: a basis for epidemiologic investigation. Emerg Infect Dis. 8 (9), 949-955 (2002).
- Cody, A. J., et al. Real-time genomic epidemiological evaluation of human Campylobacter isolates by use of whole-genome multilocus sequence typing. J Clin Microbiol. 51 (8), 2526-2534 (2013).
- Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 30 (12), 2725-2729 (2013).
- Jolley, K. A., Chan, M. S., Maiden, M. C. mlstdbNet – distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics. 5, 86 (2004).
- Verroken, A., et al. Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nocardia Species. J Clinl Microbiol. 48 (11), 4015-4021 (2010).
- El Khéchine, A., Couderc, C., Flaudrops, C., Raoult, D., Drancourt, M. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Identification of Mycobacteria in Routine Clinical Practice. PLoS ONE. 6 (9), e24720 (2011).
- Goujon, M., et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Research. 38, 695-699 (2010).
- Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 41, 95-98 (1999).
- Jolley, K. A., et al. Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain. Microbiology. 158, 1005-1015 (2012).
- Suarez, S., et al. Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory. J Microbiol Methods. 94 (3), 390-396 (2013).
- Teramoto, K., et al. Phylogenetic classification of Pseudomonas putida strains by MALDI-MS using ribosomal subunit proteins as biomarkers. Anal Chem. 79 (22), 8712-8719 (2007).
- Teramoto, K., Kitagawa, W., Sato, H., Torimura, M., Tamura, T., Tao, H. Phylogenetic analysis of Rhodococcus erythropolis based on the variation of ribosomal proteins as observed by matrix-assisted laser desorption ionization-mass spectrometry without using genome information. J Biosci Bioeng. 108 (4), 348-353 (2009).
- Bernhard, M., Weig, M., Zautner, A. E., Gross, U., Bader, O. Yeast on-target lysis (YOTL), a procedure for making auxiliary mass spectrum data sets for clinical routine identification of yeasts. J Clin Microbiol. 52 (12), 4163-4167 (2014).
- Stark, T., et al. Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay. Anal Bioanal Chem. 405 (1), 191-201 (2012).
Citar este artículo
Zautner, A. E., Lugert, R., Masanta, W. O., Weig, M., Groß, U., Bader, O. Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP). J. Vis. Exp. (116), e54165, doi:10.3791/54165 (2016).