Effects of oral streptococci and selected probiotic bacteria on the pathogen Streptococcus pyogenes: viability, biofilms, molecular functions, and virulence traits [Elektronische Ressource] / vorgelegt von Catur Riani

Cr169

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• Gesundheit

Informations

• Publié par: universitat_rostock
• Publié le: 01 janvier 2009
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Effects of oral streptococci and selected probiotic bacteria on the pathogen
Streptococcus pyogenes: viability, biofilms, molecular functions, and
virulence traits






Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat)
der Mathematisch-Naturwissenschaftlichen Fakultät
der Univesität Rostock






vorgelegt von
Catur Riani
geb. am 13.08.1976 auf Pulau Sambu
aus Indonesien




Rostock, Januar 2009

urn:nbn:de:gbv:28-diss2009-0087-1








Prof. Johannes Knobloch
(Gutachter / Reviewer)
Universitätsklinikum Schleswig-Holstein
Campus Lübeck Ratzeburger Allee 160
23538 Lübeck

Prof. Dr. Hubert Bahl
(Gutachter / Reviewer)
Uni Rostock
Institut für Biologie
Albert Einstein Str. 3
18059 Rostock

Prof. Dr. Regine Hakenbeck
(Gutachter / Reviewer)
Technische Universität Kaiserslautern
FB Biologie
P.-Ehrlich-Str.
67663 Kaiserslautern


Prof. Dr. Andreas Podbielski
(Gutachter & Betreuer / Reviewer & Supervisor)
Uni Rostock
Medizin
Abt. für Medizinische Mikrobiologie, Virologie und Hygiene
Schillingalle 70
18057 Rostock




Abgabedatum / date of submission: 30 Januar 2009
Verteidigungsdatum / date of defence: 4 Mai 2009






“Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes”Table of content

Table of content

Abbreviations
I. Introduction ........................................................................................................... 1
I.1 Streptococcus pyogenes as a human pathogen ............................................. 1
I.2 The physiological microflora of the upper respiratory tract ........................ 4
I.3 Streptococci and their value as upper airways probiotics ............................ 7
I.4 Aims of the present study ............................................................................. 10
II. Material and Methods ............................................................................................ 12
II.1 Material ........................................................................................................ 12
II.1.1 Bacterial strains ................................................................................ 12
II.1.2 Culture media for bacteria ................................................................ 12
II.1.3 Eukaryotic cells and media for cell culture ...................................... 13
II.1.4 Plasmid ............................................................................................. 14
II.1.5 Antibodies ........................................................................................ 14
II.1.6 Reagents and buffers ........................................................................ 14
II.1.7 Instruments ....................................................................................... 14
II.2 Methods ........................................................................................................ 15
II.2.1 Bacterial culture condition ............................................................... 15
II.2.2 Culture condition and preparation of eukaryotic cell culture ........... 16
II.2.3 DNA/RNA methods and manipulation ............................................ 16
II.2.3.1 S. pyogenes DNA preparation ........................................... 16
II.2.3.2 Plasmid isolation from E. coli 17
II.2.3.3 HEp-2 cells RNA isolation ................................................ 17
II.2.3.4 DNA/RNA concentration measurement ............................ 18
II.2.3.5 Polymerase Chain Reaction (Mullis et al., 1986) ............. 18
II.2.3.6 DNA restriction digest ...................................................... 19
II.2.3.7 DNA ligation reaction ....................................................... 19
II.2.3.8 Agarose electrophoresis for DNA (Sambrook et al., 1989) 19
II.2.3.9 Construction of the S. pyogenes M6 sagA-luc reporter
gene strain ......................................................................... 20
II.2.4 Preparation and transformation of E. coli DH5competent cells ... 21
II.2.5 S. pyogenes competent cells ...... 21
II.2.6 Quantitative co-culture and transwell system .................................. 22
II.2.7 Bacteriocin assay .............................................................................. 23
II.2.8 Growth curve measurement ............................................................. 23
iTable of content
II.2.9 Quantitative assays for sagA-luciferase activity .............................. 23
II.2.10 Hemolysis assay ............................................................................... 24
II.2.11 Coaggregation assay (modified from Cisar et al., 1979) ................. 24
II.2.12 Biofilm quantification with safranin assay ....................................... 25
II.2.13 Microscopic observation and documentation of biofilms
(Fluorescence, SEM, CLSM) ........................................................... 25
II.2.13.1 Fluorescence microscopy ............................................... 25
II.2.13.2 Scanning Electron Microscopy (SEM) .......................... 25
II.2.13.3 Confocal Laser Scanning Microscope (CLSM) ............. 26
II.2.14 Adherence and internalization assay ................................................ 26
II.2.15 Eukaryotic cell viability assay .......................................................... 27
II.2.16 Double-immunofluorescence assay .................................................. 28
II.2.17 HEp-2 cells microarray .................................................................... 29
III. Results ................................................................................................................... 31
III.1 S. pyogenes co-culture experiments: direct and indirect contact ................. 31
III.2 Bacteriocin assay .......................................................................................... 35
III.3 Effect on S. pyogenes sagA transcription ..................................................... 35
III.3.1 Construction of an S. pyogenes serotype M6 sagA-luc reporter
gene strain ........................................................................................ 36
III.3.2 sagA-luc activity measurement in the presence of selected oral
bacteria and E. coli Nissle ................................................................ 39
III.4 Effect of spent medium on S. pyogenes hemolytic activity ......................... 42
III.5 Coaggregation of S. pyogenes with oral bacteria and E. coli Nissle ............ 42
III.6 Effect of oral bacteria and E. coli Nissle on S. pyogenes biofilms ............... 43
III.6.1 Evaluation of growth medium and monospecies biofilm
behaviour .......................................................................................... 44
III.6.2 Investigation of mixed-species biofilms .......................................... 46
III.6.3 The effect of artificial saliva on the species interaction ................... 50
III.7 Effect of oral bacteria and E. coli Nissle on S. pyogenes adherence to and
internalization into host cells ........................................................................ 53
III.7.1 Quantitative assay ............................................................................ 53
III.7.2 Double immunofluorescence ............................................................ 59
III.8 Effect of oral bacteria and E. coli Nissle on S. pyogenes cytotoxicity ......... 61
III.9 Transcriptional response of HEp-2 cells in the presence of S. salivarius
and S. oralis .................................................................................................. 63


iiTable of content
IV. Discussion ............................................................................................................. 68
IV.1 General considerations ................................................................................. 68
IV.2 Changes of S. pyogenes numbers and viability in co-culture experiments 69
IV.3 Co-culture effects on S. pyogenes virulence factor expression .................... 71
IV.4 biofilms .................................................. 73
IV.5 S. pyogenes interactions with eukaryotic cells .......... 75
IV.6 Co-culture effects on the integrity and metabolism of eukaryotic cells ....... 78
V. Conclusion 82
VI. Reference ............................................................................................................... 84
VII. Appendix 95
iiiAbbreviations
Abbreviations

aad9 resistance gene for spectinomycin
ATCC American Type Culture Collection
aqua dest. aqua destillata
bar pressure unit
BHI Brain Heart Infusion
bp base pair
BSA bovine serume albumin
C Coulomb, international unit for electric charge
°C