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Legionella and Non-Tuberculous Mycobacteria Using MALDI TOF MS (Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry)

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Legionella and Non-Tuberculous Mycobacteria Using MALDI TOF MS (Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry) View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by OTHES DIPLOMARBEIT Titel der Diplomarbeit Establishment of a reference database for Acanthamoeba, Legionella and non-tuberculous mycobacteria using MALDI TOF MS (Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry) Verfasserin Dzenita HASANACEVIC angestrebter akademischer Grad Magistra der Naturwissenschaften (Mag.rer.nat.) Wien, 2012 Studienkennzahl lt. A 442 Studienblatt: Studienrichtung lt. Studienblatt: Anthropologie Betreuerin: Ass. Prof. Univ. Doz. Mag. Dr. Julia Walochnik Contents 1 ABBREVIATIONS ..................................................................................................... 5 2 INTRODUCTION ....................................................................................................... 6 2.1 Acanthamoeba .................................................................................................... 6 2.1.1 Classification ................................................................................................ 6 2.1.1.1 Phylogeny of Acanthamoeba ................................................................. 6 2.1.1.2 Methods of classification ....................................................................... 8 2.1.2 Ecology and geographical distribution ........................................................ 11 2.1.2.1 Life cycle ............................................................................................. 11 2.1.2.2 Trophozoites ....................................................................................... 12 2.1.2.3 Cysts ................................................................................................... 13 2.1.3 Medical relevance ...................................................................................... 15 2.1.3.1 Comparative characteristics ................................................................ 15 2.2 Bacterial endocytobionts in free-living amoeba (FLA)........................................ 19 2.3 Legionella ......................................................................................................... 21 2.3.1 History ........................................................................................................ 21 2.3.2 Phylogeny .................................................................................................. 21 2.3.3 Physiology, morphology and distribution of Legionella ............................... 22 2.3.4 Pathomechanism ....................................................................................... 22 2.3.4.1 Cycle of infection in amoebae and macrophages ................................ 22 2.3.5 Epidemiology and medical relevance ......................................................... 24 2.4 Mycobacteria .................................................................................................... 27 2.4.1 History ........................................................................................................ 27 2.4.2 Phylogeny and morphology ........................................................................ 27 2.4.2.1 Phylogeny ........................................................................................... 27 2.4.2.2 Physiology and morphology ................................................................. 28 2.4.3 Distribution of non-tuberculous mycobacteria ............................................. 31 2.4.4 Pathomechanism of non-tuberculous mycobacteria ................................... 31 2.4.4.1 Medical relevance ............................................................................... 31 2.5 Maldi TOF MS ................................................................................................... 35 2.5.1 Introduction ................................................................................................ 35 2.5.2 Principle of the method ............................................................................... 36 2.5.3 Application in the routine clinical analysis ................................................... 39 2.6 Aims .................................................................................................................. 39 3 MATERIAL AND METHODS ................................................................................... 40 3.1 Samples ............................................................................................................ 40 3.2 Cultivation ......................................................................................................... 40 3.2.1 Acanthamoeba spp. ................................................................................... 40 3.2.2 Legionella spp. ........................................................................................... 44 3.2.3 Mycobacterium spp. ................................................................................... 46 3.2.4 Infection of Acanthamoeba spp. with Legionella spp. ................................. 47 3.2.4.1 Preparation of Legionella spp. and Acanthamoeba before infection .... 47 3.3 Molecular biology .............................................................................................. 49 3.3.1 DNA Isolation MagNa Pure Compact ......................................................... 49 3.3.2 Polymerase chain reaction (PCR) .............................................................. 50 3.3.2.1 Acanthamoeba- PCR........................................................................... 50 3.2.2.2 LegMIP PCR ....................................................................................... 50 3.2.2.3 16S PCR ............................................................................................. 51 3 Contents 3.3.3 Protocol for PCR product clean up ............................................................. 51 3.3.4 Sequencing PCR ........................................................................................ 51 3.3.5 Cleanup with the Centri-Sep 8 strips .......................................................... 51 3.3.4 DNA-Sequencing ........................................................................................ 52 3.3.4.1 Data analysis ....................................................................................... 52 3.4 MALDI TOF ....................................................................................................... 53 3.4.1 Sample preparation .................................................................................... 53 3.4.2 Creation of MSPs with Maldi Biotyper 2.0 and the establishment of a new database ............................................................................................................. 55 4 RESULTS ................................................................................................................ 56 4.1 Culture .............................................................................................................. 56 4.1.1 Acanthamoeba ........................................................................................... 56 4.1.1.1 Culture plates and axenic cultures ....................................................... 56 4.1.2 Legionella ................................................................................................... 56 4.1.3 Non-tuberculous mycobacteria ................................................................... 57 4.1.3.1 Löwenstein medium ............................................................................. 57 4.2 Molecular biology .............................................................................................. 59 4.2.1 Sequencing results ..................................................................................... 59 4.2.1.1 Acanthamoeba .................................................................................... 59 4.2.1.2 Legionella ............................................................................................ 60 4.2.1.3 Non-tuberculous mycobacteria ............................................................ 64 4.2.2 MALDI TOF ................................................................................................ 66 4.2.2.1 Acanthamoeba spp. - classification results and the mass spectra........ 66 4.2.2.2 Legionella spp. – classification results and the mass spectra .............. 70 4.2.2.3 Non-tuberculous mycobacteria – classification results and the mass spectra ............................................................................................................ 73 4.3 Mass spectra of acanthamoebae, Legionella spp. and of the infected acanthamoebae ...................................................................................................... 75 5 DISCUSSION .......................................................................................................... 79 5.1 DNA sequencing analysis vs. MALDI TOF ........................................................ 79 5.1.1 Composition proceedings of the used application ....................................... 79 5.1.2 Reproducibility ............................................................................................ 85 5.2 Differentiation of Legionella species by MALDI Biotyper .................................... 86 5.3 The Biotyper as a promising new method for identification of Acanthamoeba . 88 6 LITERATURE .......................................................................................................... 90 7 APPENDIX ...........................................................................................................
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