1. Multi-species Biofilms

2. Biofilms A biofilm is a community of microorganisms, associated with a surface, and encased in an extracellular polymeric matrix.A biofilm is a community of microorganisms, associated with a surface, and encased in an extracellular polymeric matrix.

3. Why is Biofilm Formation Important? Many, if not the majority of infections involve biofilm formation (particularly chronic infections)Many, if not the majority of infections involve biofilm formation (particularly chronic infections) Important in device-related infectionsImportant in device-related infections Biofilm formation can complicate therapyBiofilm formation can complicate therapy

4. Dental Biofilms Very complex community; multi-species, multi-kingdomVery complex community; multi-species, multi-kingdom Develop in stagesDevelop in stages

5. Tooth surface Acquired Pellicle Stage 1: Pellicle formation Stage 2: Initial Adherence Tooth surface Acquired Pellicle Stage 3: Aggregation Tooth surface Acquired Pellicle Stage 4: Maturation Tooth surface Acquired Pellicle Stage 5: Dispersal Tooth surface Acquired Pellicle

6. Stage 1: Pellicle formation

7. Stage 2: Initial Adherence Tooth surface Acquired Pellicle Streptococci Actinomyces Streptococci and Actinomyces are initial colonizers

8. Stage 1: Adherence Why is adherence important?Why is adherence important? –Adherence is a critical first step in pathogenesis. –All pathogenic bacteria produce adhesins What is an adhesin?What is an adhesin? –ADHESIN: Bacterial macromolecule that binds to specific ligands or receptors on host cells and defines the tropism of the microbe for various cells or tissues.

9. Role of adherence 1) To avoid physical removal by host defenses –Mucociliary escalator –Washing action of saliva tears, mucus tears, mucus –Blood and urine flow 2) To make intimate contact with host tissues –Nutrient uptake –Invasion

10. Types of Adhesins 1.Pili / fimbriae 2.Non-pilus surface proteins 3.Polysaccharides

11. Types of Adhesins; 1. Pili Streptococcus pyogenes pilus promotes adhesion to pharyngeal epithelial cells.Streptococcus pyogenes pilus promotes adhesion to pharyngeal epithelial cells. This is necessary for streptococcal pharyngitisThis is necessary for streptococcal pharyngitis

12. Types of adhesins; 2. Surface Proteins S. gordonii: Amylase-binding protein, AbpAS. gordonii: Amylase-binding protein, AbpA S. gordonii

13. Types of adhesins; 3. Polysaccharides e.g. S. mutans produces glucosyltransferases that convert sucrose into polysaccharides which;e.g. S. mutans produces glucosyltransferases that convert sucrose into polysaccharides which; –1. Act as receptors for adhesins on other bacteria –2. Contribute to adherence of S. mutans to the tooth surface

14. Approaches to reducing adherence Physical disruption; oral hygiene.Physical disruption; oral hygiene. Replacing sucrose with non- utilizable sugars.Replacing sucrose with non- utilizable sugars. Anti-adhesin vaccinesAnti-adhesin vaccines

16. Stage 3: Aggregation Tooth surface Acquired Pellicle

19. Stage 4: Maturation Tooth surface Acquired Pellicle Increased species diversity; late colonizersIncreased species diversity; late colonizers

20. Co-aggregation Mediated by interactions between lectins on one bacterial cell and sugar residues on another.

21. Co-aggregation

23. Bacterial Vaginosis (BV) Most common vaginal disorder worldwideMost common vaginal disorder worldwide Mixed species biofilm forms on the vaginal epitheliumMixed species biofilm forms on the vaginal epithelium Normal vaginal secretions BV vaginal secretions

24. BV Associated Risk Factors Number of known risk factors includingNumber of known risk factors including –Sexual activity and number of sexual partners –IUD usage –Douching –Antibiotic treatment –Smoking

25. Diagnosis Wet mount: prepared from vaginal sample and observed for presence of clue cellsWet mount: prepared from vaginal sample and observed for presence of clue cells Whiff test with KOHWhiff test with KOH Vaginal pH greater than 4.5Vaginal pH greater than 4.5 Culture or Gram stainCulture or Gram stain Positive 3 out of 4 is indicative of BV, treatment with oral or topical MetronidazolePositive 3 out of 4 is indicative of BV, treatment with oral or topical Metronidazole

26. Complications of BV –Preterm delivery, low birth weight –Complications during pelvic surgeries can lead to PID –Increased risk of infection with HIV and other STDs –Frequently relapses or recurs after treatment

27. Etiology of BV G. vaginalis is the predominant species in >= 90% of cases, but pure cultures don’t reliably cause infectionG. vaginalis is the predominant species in >= 90% of cases, but pure cultures don’t reliably cause infection Koch’s postulates have not been satisfiedKoch’s postulates have not been satisfied Is G. vaginalis necessary but not sufficient to cause the disease??Is G. vaginalis necessary but not sufficient to cause the disease??

28. Gardnerella vaginalis Facultative anaerobe, Gram positive or variable, rod shapedFacultative anaerobe, Gram positive or variable, rod shaped Non-motile, non-flagellated, non-spore forming, non- encapsulated, may produce piliNon-motile, non-flagellated, non-spore forming, non- encapsulated, may produce pili G. vaginalis forms a biofilm on the vaginal epithelium of women with bacterial vaginosis (BV)G. vaginalis forms a biofilm on the vaginal epithelium of women with bacterial vaginosis (BV) Electron microscopy of G. vaginalis

29. Hypothesis Similar to Streptococci in dental biofilms, G. vaginalis is an initial colonizer and forms a biofilm on the vaginal epithelium. Biofilm formation allows G. vaginalis and other anaerobic species to survive in the face of oxygen, acid, hydrogen peroxide, and immune factors in the vagina. Similar to Streptococci in dental biofilms, G. vaginalis is an initial colonizer and forms a biofilm on the vaginal epithelium. Biofilm formation allows G. vaginalis and other anaerobic species to survive in the face of oxygen, acid, hydrogen peroxide, and immune factors in the vagina.

30. How would a biofilm limit local O 2 concentrations?

33. Forces of diffusion and convection (mixing)

39. Mathematical Modeling of Biofilms Is our hypothesis valid?Is our hypothesis valid? Could biofilm formation by G. vaginalis sufficiently deplete local oxygen concentrations?Could biofilm formation by G. vaginalis sufficiently deplete local oxygen concentrations?