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 ...........................................................................................................