Culture-independent characterisation of microbial biofilm communities occluding biliary stents [Elektronische Ressource] / von Britta Kristina Scheithauer

Britschy

Sujets

• Gesundheit

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2008
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 24 Mo

Extrait

Culture-independent characterisation of microbial biofilm
communities occluding biliary stents







Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades einer

Doktorin der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n








von Britta Kristina Scheithauer
aus Alzenau in Unterfranken






























Privatdozent Dr. Dietmar Pieper 1. Referent:
Professor Dr. Michael Steinert 2. Referent:
22.10.2007 eingereicht am:
17.12.2007 mündliche Prüfung (Disputation) am:
Druckjahr 2008

Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen
vorab veröffentlicht:


Tagungsbeiträge


- B Scheithauer, KN Timmis and DF Wenderoth: An experimental system to
monitor microbial biofilm community formation in biliary stents. (Poster) Biofilms
- Prevention of microbial adhesion, Osnabrück (31.03.-02.04.2004).

- B Scheithauer, DH Pieper, B Ferslev, SJ Ott and KN Timmis: Culture-
independent survey of microbial biofilm communities occluding biliary stents.
(Poster) Pseudomonas 2005 - 10th International Congress on Pseudomonas,
Marseille, France (27.08.-31.08.2005).

- B Scheithauer, DH Pieper, B Ferslev, SJ Ott and KN Timmis: Culture-
independent survey of microbial biofilm communities occluding biliary stents.
(Poster) VAAM Jahrestagung 2005, Göttingen (25.09.-28.09.2005).

- B Scheithauer, DH Pieper, B Ferslev, SJ Ott and KN Timmis: Culture-
independent survey of microbial biofilm communities occluding biliary stents.
(Talk) Development and control of functional diversity at micro- and
macroscales, München (05.10.-07.10.2005).

- B Scheithauer, H Lünsdorf, H Jablonowski, DH Pieper and KN Timmis:
Characterisation of mixed microbial biofilms occluding biliary stents. (Poster)
Biofilms II – Attachment and detachment in pure and mixed cultures, Leipzig
(22.03.-24.03.2006).

- B Scheithauer, H Jablonowski and DH Pieper: Characterisation of microbial
biofilm communities persisting in biliary stents. (Poster) ISME 11 - 11th
international symposium on microbial ecology, Vienna, Austria (20.08.-
25.08.2006).

Table of contents I
Table of contents

1 Introduction ______________________________________________________ 1
1.1 Biliary stents__________________________________________________ 1
1.2 Microbiota inherent to the gastrointestinal system_____________________ 6
1.3 Molecular methods for microbial community analysis and use of 16S
ribosomal RNA genes as phylogenetic markers___________________________ 11
1.4 Metabolic activities present in biliary stent biofilms? __________________ 14
1.5 Possible mechanisms contributing to biliary stent occlusion ____________ 17
1.6 Goal of the study _____________________________________________ 19
2 Materials and methods ____________________________________________ 21
2.1 Bacterial strains and plasmids ___________________________________ 21
2.2 Media and growth conditions ____________________________________ 21
2.3 Chemicals __________________________________________________ 25
2.4 Solutions ___________________________________________________ 25
2.5 Biliary stent samples __________________________________________ 26
2.6 Isolation of microorganisms _____________________________________ 27
2.6.1 Aerobic isolation procedures_________________________________ 27
2.6.2 Anaerobic isolation procedures_______________________________ 27
2.6.3 Strain maintenance________________________________________ 28
2.7 Molecular techniques __________________________________________ 28
2.7.1 Extraction of genomic DNA__________________________________ 28
2.7.2 Extraction of RNA _________________________________________ 28
2.7.3 Determination of nucleic acid concentration _____________________ 29
2.7.3.1 Photometric determination_______________________________ 29
2.7.3.2 Determination using fluorescent dyes ______________________ 29
2.7.4 Agarose gel electrophoresis _________________________________ 29
2.7.5 Polymerase chain reaction (PCR)_____________________________ 30
2.7.5.1 Com PCR____________________________________________ 31
2.7.5.2 Reverse Transcriptase Com PCR _________________________ 32
2.7.5.3 16S rDNA PCR _______________________________________ 33
2.7.5.4 Taurine–pyruvate aminotransferase PCR ___________________ 34
2.7.5.5 Bile acid inducible (bai) operon PCR_______________________ 35
2.7.6 M13 PCR _______________________________________________ 36
2.7.7 Purification of nucleic acids__________________________________ 36 Table of contents II
2.7.7.1 Purification of PCR products _____________________________ 36
2.7.7.2 Extraction of DNA from agarose gels ______________________ 36
2.7.8 Single-Strand-Conformation Polymorphism (SSCP) fingerprinting____ 37
2.7.8.1 Enzymatic digestion of double stranded DNA ________________ 37
2.7.8.2 SSCP gel and running conditions _________________________ 37
2.7.8.3 Silver staining and documentation_________________________ 38
2.7.8.4 Excision of SSCP bands ________________________________ 38
2.7.8.5 Analysis of SSCP fingerprint data _________________________ 38
2.7.9 Cloning of PCR products ___________________________________ 40
2.7.9.1 Ligation _____________________________________________ 40
2.7.9.2 Transformation________________________________________ 41
2.7.9.3 Blue-white selection____________________________________ 41
2.7.10 Sequencing of DNA _______________________________________ 41
2.7.10.1 Sequencing reaction ___________________________________ 41
2.7.10.2 Purification and sequencing work _________________________ 42
2.8 Sequence data analysis________________________________________ 42
2.9 Biochemical methods__________________________________________ 44
2.9.1 Screening for bile salt hydrolase (bsh) activity ___________________ 44
2.10 Transmission electron microscopy ______________________________ 44
3 Results ________________________________________________________ 46
3.1 Origin of biliary stents__________________________________________ 46
3.2 Placement and removal of biliary stents by means of endoscopic retrograde
cholangiopancreatography (ERCP) ____________________________________ 47
3.3 Biliary stent microbial community composition analysed by SSCP
fingerprinting ______________________________________________________ 48
3.3.1 SSCP phylotypes detected in biliary stents from Surgery Clinic of
Braunschweig and Medical Clinic of Salzgitter __________________________ 49
3.3.2 Surgery Clinic of Braunschweig ______________________________ 52
3.3.3 Medical Clinic of Salzgitter-Lebenstedt_________________________ 56
3.3.4 Comparison of the bacterial community composition of stents from both
hospitals _______________________________________________________ 60
3.3.4.1 Combined abundance of phylotypes in biliary stents evaluated at the
family level ___________________________________________________ 60
3.3.4.2 Combined abundance of phylotypes in biliary stents evaluated at the
phylum level___________________________________________________ 62 Table of contents III
3.3.4.3 Multivariate statistical analysis of fingerprints from both hospitals 63
3.3.5 Comparing the communities at different positions of the stents ______ 64
3.3.6 Host influence on microbial community structure _________________ 66
3.3.6.1 Consecutively implanted biliary stents of the same patients _____ 66
3.3.6.2 Simultaneously implanted biliary stents of the same patients ____ 69
3.3.6.3 Dispersion analysis ____________________________________ 72
3.3.7 Is the biliary stent bacterial community structure influenced by patient
characteristics? __________________________________________________ 73
3.3.8 RT-PCR SSCP fingerprints of selected samples _________________ 75
3.4 Analysis of 16S rDNA clone libraries of biliary stents _________________ 78
3.4.1 Sequence analysis and phylogenetic classification _______________ 78
3.4.1.1 Bacterial community structure of library I____________________ 79
3.4.1.2 Bacterial community structure of library II ___________________ 81
3.4.1.3 OTUs observed in sequences from both libraries _____________ 83
3.4.1.4 Diversity measures ____________________________________ 84
3.4.2 Comparison of biliary stent microbial community structure as revealed by
random sequencing of 16S rDNA clone libraries and SSCP fingerprinting_____ 88
3.4.3 Isolating and sequencing members from biliary stent communities ___ 92
3.5 Bile acid modifications in biliary stent biofilms? ______________________ 94
3.5.1 Bile salt hydrolase activity of biliary stent isol