Opgedragen aan Lotte, Yenthe en Seppe This work was supported by grants from the Bijzonder Onderzoeks Fonds (BOF), Ghent University, Belgium; the Fund for Scientific Research (FWO) Flanders, Belgium and the Marie Marguérite Delacroix Fund, Belgium. Faculty of Medicine and Health Sciences Department Clinical Chemistry, Microbiology and Immunology Laboratory Bacteriology Research Characterization of the vaginal microflora Rita Verhelst Promotors: Prof. Dr. Mario Vaneechoutte Prof. Dr. Marleen Temmerman Dissertation submitted in fulfillment of the requirements for the degree of Doctor in Biomedical Sciences, Faculty of Medicine, University of Ghent April 2006 PROMOTORS LEDEN VAN DE EXAMENCOMMISSIE Prof. Dr. Mario Vaneechoutte VOORZITTER Prof. Dr. Geert Leroux-Roels Vakgroep Klinische biologie, microbiologie en immunologie Vakgroep Klinische biologie, microbiologie en immunologie Universiteit Gent Universiteit Gent Prof. Dr. Marleen Temmerman Prof. Dr. Geert Claeys Vakgroep Uro-gynaecologie Vakgroep Klinische biologie, microbiologie en immunologie Universiteit Gent Universiteit Gent Prof. Dr. Denis Pierard Departement Microbiologie Academisch Ziekenhuis Vrije Universiteit Brussel Prof. Dr. Koenraad Smets Vakgroep Pediatrie en genetica Universiteit Gent Prof. Dr. Guido Van Ham Departement Microbiolgie Instituut voor Tropische Geneeskunde, Antwerpen Prof. Dr. Gerda Verschraegen Vakgroep Klinische biologie, microbiologie en immunologie Universiteit Gent Dr. Nico Boon Vakgroep Biochemische en microbiële technologie Universiteit Gent Table of contents List of abbreviations ........................................................................................................................................... 11 List of bacterial species....................................................................................................................................... 12 I. General introduction....................................................................................................................................... 13 II. Objectives ....................................................................................................................................................... 15 III. Overview of the literature............................................................................................................................ 17 III.1. The vaginal ecosystem............................................................................................................................. 17 III.2. The normal vaginal microflora ............................................................................................................... 19 III.2.1. Vaginal lactobacilli.......................................................................................................................... 19 III.2.2. Taxonomy of vaginal lactobacilli: major taxonomic changes ......................................................... 19 III.3. Bacterial vaginosis.................................................................................................................................. 21 III.3.1. Definition......................................................................................................................................... 22 III.3.2. Epidemiology .................................................................................................................................. 22 III.3.3. Pathophysiology .............................................................................................................................. 22 III.3.3.1. Hydrogen peroxide production ................................................................................................ 23 III.3.3.2. Change in pH ........................................................................................................................... 24 III.3.3.3. Overgrowth of bacterial vaginosis associated organisms ........................................................ 25 III.3.3.4. Reduction in lactobacilli .......................................................................................................... 25 III.3.4. Clinical features............................................................................................................................... 26 III.3.5. Complications.................................................................................................................................. 26 III.3.6. Diagnosis ......................................................................................................................................... 26 III.3.6.1. Clinical diagnosis by Amsel criteria ........................................................................................ 27 III.3.6.2. Commercial point-of-care tests................................................................................................ 28 III.3.6.2.1. pH measurement .............................................................................................................. 28 III.3.6.2.2. Presence of trimethylamine.............................................................................................. 28 III.3.6.2.3. pH measurement and trimethylamine presence................................................................ 28 III.3.6.2.4. Sialidase activity .............................................................................................................. 28 III.3.6.2.5. Proline aminopeptidase activity ....................................................................................... 29 III.3.6.2.6. DNA probe for G. vaginalis rRNA .................................................................................. 29 III.3.6.3. Microscopy .............................................................................................................................. 29 III.3.6.3.1. Scoring of Gram stained vaginal smears according to Nugent ........................................ 29 III.3.6.3.2. Scoring of Gram stained vaginal smears according to Hay/Ison...................................... 31 III.3.6.3.3. Wet smear criteria ............................................................................................................ 31 III.3.6.4. Conclusion ............................................................................................................................... 32 III.3.7. Treatment......................................................................................................................................... 32 III.4.1. Culture............................................................................................................................................. 35 III.4.1.1. Non-selective culture methods................................................................................................. 35 III.4.1.2. Semi-selective culture methods ............................................................................................... 36 III.4.1.3. Phenotypic identification of cultured microorganisms ............................................................ 37 III.4.1.4. DNA-based identification of cultured microorganisms. .......................................................... 38 III.4.1.4.1. PCR-independent DNA-based identification of cultured organisms................................ 38 III.4.1.4.2. PCR-based identification methods of cultured organisms ............................................... 39 III.4.1.4.3. Principle of tRNA intergenic polymorphism length analysis (tDNA-PCR)..................... 40 III.4.2. DNA-based culture-independent methods....................................................................................... 41 III.4.2.1. Advantages of the molecular microbiologic approach............................................................. 41 III.4.2.2. PCR-independent and culture-independent techniques............................................................ 44 III.4.2.3. PCR-based culture-independent techniques............................................................................. 45 III.4.2.3.1. Principle of cloning.......................................................................................................... 46 III.4.2.3.2. Principle of T-RFLP......................................................................................................... 46 III.4.2.3.3. Species specific PCR........................................................................................................ 49 IV. Experimental work....................................................................................................................................... 51 IV.1. PCR-based identification method of cultured organisms ........................................................................ 53 Baele M, Vaneechoutte M, Verhelst R, Vancanneyt M, Devriese LA, Haesebrouck F. Identification of Lactobacillus species using tDNA-PCR. J Microbiol Methods. 2002 Aug;50(3): 263-71. IV.2. Cloning of 16S rRNA genes of normal and disturbed vaginal microflora............................................... 65 Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis. BMC Microbiol. 2004 Apr 21;4: 16. IV.3. Characterization of vaginal microflora at three time points in pregnancy by Gram stain, culture