1. Searching for microbes Part XIV. The Biofilm And The Oral Microbes Ondřej Zahradníček To practical of ZLLM0421c zahradnicek@fnusa.cz

2. Survey of topics Clinical cases related to biofilm Characterisation of biofilm Diagnostic and experimental method for biofilm Diagnostic methods for oral microbes Clinical cases related to oral microbes

3. A clinical case related to biofilm

4. Story one (today a real one) Male, 58 let, 2001 cardiostimulator, 2002 repeatedly hospitalized on an internal department with fever of unknown origin, elevation of inflammatory markers In blood cultures, S. epidermidis, very good susceptibility Several times treated by high doses of antibiotics in combinations (oxacilin, gentamicin, rifampicin, cefazolin, cefalotin, clindamycin)

5. In the beginning, a good response, later attacks of fever again At transoesofageal examination, vegetation on a chamber electrode sized 1,5 × 1,5 cm. Cardiologists repeatedly refuse cardiostimulator removal. A combination oxacillin + gentamicine + rifampicine, patient in a good state. Nevertheless, again temperature and CRP rises. Vancomycin and rifampicin starts to be used, after improval, patient‘s trombus is removed and the electrode changed (under antibiotics), so the patient starts to be better. Story – continuing

6. Who is guilty? The biofilm!!! The therapy could not be successful, because high resistance of bacteria growing in form of a biofilm was not taken into account. The therapy was not strong enough from the beginning and the biofilm was not eradicated. Only electrode removal (under antibiotic therapy) enabled patient status improval.

7. Catheter biofilm webs.wichita.edu

8. Clinical cases connected with oral microbes

9. Story two Mr. Badtooth had dental caries. He visited his stomatologist. The caries was treated with a filling, and Mr. Badteeth was told to improve his dental hygiene. As we know today, microbes play an important role in the dental caries, especially by making acids; acid pH takes a part in the erosion of the enamel Most important bacteria are Streptococcus mutans, and also oral lactobacilli Important is also buffer capacity of saliva

10. Story three Mr. Parodont was not like Mr. Badtooth, he cleaned his teeth, but he did not clean sufficiently the s pace between teeth and gingiva. In his gingival sulcus risky bacteria overmultiplied, especially Porphyromonas gingivalis, Tanerella forsythia and Treponema denticola. Elevated pH and further multiplication of porphyromonads caused that Mr. Parodont had now parodontal pocket instead of a normal gingival sulcus And so Mr. Parodont started to have gingivitis and finally parodontitis

11. Oral microbes Oral microbes are a very complex system. Dental plaque equals to a considerably complex biofilm. We also have to distinguish between supragingival and subgingival plaque, as their composition is different, and influences development of various pathological processes In case of pathogenic processes (dental caries, gingivitis, parodontitis) we are usually not able to name one causative agent. The complete process should be seen as an error in equilibrium between them

12. Other oral microbes The fact that sometimes the dysbalance between normal oral microbes occurs does not mean that other microbes cannot participate as pathogens, too. Especially yeasts may participate on plaque on dental prostheses and may cause oral candidosis called thrush. From outside also bacteria may invade into the oral cavity, but more frequently we find them in adjacent pharyngeal cavity. Important might be „golden“ staphylococci, pyogene streptococci and some haemophlili.

13. Characterisation of biofilm

14. Biofilm: what is it? A biofilm is a complex, organized structure It consists of living cells (mostly bacteria), masses produced by them (mostly polysaccharides) and channels It is present not only inside living body, but also in the environment. For example stones in ponds and rivers are often covered by a biofilm that makes them smooth.

15. Biofilm in a river www.sbs.soton.ac.uk

16. Various pictures of biofilm Photo: Archive of Veronika Holá Biofilm on a cathetre

17. Various pictures of biofilm Photo: Archive of Veronika Holá Biofilm on a catheter Bacteria A channel Catheter Polysaccharides

18. biology.fullerton.edu

19. Stages of biofilm development Direct contact of a planctonic bacteria with a surface + Adhesion to this surface Aggregation of cells into microcolonies Production of polymeric matrix Formation of three-dimensional structure known as biofilm

20. Development of biofilm – timing

21. Development of a biofilm biology.binghamton.edu

22. Biofilm development, another picture webs.wichita.edu

23. Biofilm development www.ul.ie

24. Biofilm formation, another picture www.uweb.engr.washington.edu

25. Importance of biofilm production in bacteria Bacteria may better regulate their quantity – in the biofilm they inform each other by production of various stuffs (quorum sensing) Bacteria become more resistant to outer influences: –disinfectants –antibiotics –host immunity response Biofilm is formed both by common flora bacteria (rather positive for macrorganism and by pathogens

26. Biofilm env.snu.ac.kr

27. Mechanisms influencing bacterial resistance Influence of surface charge Decrease of growth rate Penetration barrier Non-homogenous matrix Phenotypic differences Intercellular signalisation Immunity mechanisms...

28. Biofilm www.dms-online.de

29. Biofilm eradication Antibiotic therapy often only suppresses symptoms of infection caused by cells released from biofilm matrix and reacting with immunity system. Cells fixed in biofilm matrix cannot be destroyed by such therapy. To biofilm eradication we often to use high ATB concentrations (monotherapy or combinations), when treatment is not effective, the biofilm focus should be removed. In future we will possibly try to destroy the biofilm, e. g. by enzymotherapy

30. Yeast Biofilm www.ansci.wisc.edu

31. Prevention Catheters and bone cements –made of new generation plastic material (risk of adhesion and biofilm formation lower) –with coloid silver and similar surface-active compounds –with antimicrobial substances, e. g. minocycline rifampicine Catheter washing Correct asepsis, decontamination methods etc.

32. Diagnostic and experimental methods for biofilm

33. Biofilm and microbiologic diagnostics a) Biofilm assessment aa) by phenotypic methods (Christensen‘s method, Congo red agar cultivation) ab) by genotypic methods b) Assesment of bacterial susceptibility in biofilm to individual antibiotics or combinations (mostly MBEC) c) Regarding to biofilm formation at common bacteriological diagnostics, e. g. at venous catheter cultivation we choose specific methods (see later) instead of classic multiplication in broth Foto: Archiv Veroniky Holé

34. Microscopy of oral biofilm Besides official methods for biofilm detection there are also other methods how to visualise biofilm. For oral biofilm: Gram stain may only visualise cell clusters (both G+ and G- ) and eventually macroorganism cells (epitheliae etc.). Polysaccharidic masses remain invisible. Alciane blue stain enables visualisation of polysaccharidic material, i. e. the acellular part of biofilm. Cells are visualized by negative staining. Photo: Archive of Veronika Holá

35. Proof of influence of tooth cleaning to oral biofilm A volunteer has a iodine solution or pills with a stain effecting to tooth plaque. The iodine is let to work in oral cavity during approx. 2 min. Photo: Archive of Veronika Holá

36. Culture of biofilm producing bacteria In case of likelihood of biofilm formation, it is usually necessary to perform special methods for pre-processing the biological material, that precede the proper culture For central venous catheter culture, there exist two methods. Both of them are better than classical culture in broth without any pre- processing, sonification still remaining better than the Maki method

37. Classical broth culture: Bacteria in planctonic form are released. Bacteria in form of a biofilm are released. Bacteria in biofilm form are released less, or not at all. As broth is used as multiplying medium, we know nothing about its quantity (contamination × infection). Semiquantitative (Maki) method: It enables us to assess catheter surface and semiquantitativelly assess the finding, but we have no information about intraluminal bacteria and bacteria are not necessarily released from the biofilm. Sonification: destroys biofilm on the catheter surface and catheter lumen. Inoculation of a defined specimen volume is a quantitative method, that enables as to assess microbial amount. Methods

38. Proof of influence of saccharides presence to dental plaque formation The experiment has a simple principle. One of oral bacteria is cultured on plastic surface (simulating tooth surface) with presence of various concentrations of glucose and for various time value After the incubation, biofilm is visualised using gentiane violet and its density quantified as absorbance using a spectrophotometre

39. To avoid accidental mistake, six adjacent wells have always the same values of both glc concentration and time

40. Old and new abbreviations in antibiotic effect measuring MIC – minimal inhibition concentration is the growth limit of bacteria (the lowest concentration that disables bacterial growth) MBC – minimal bactericidal concentration is the survival limit of bacteria (the lowest concentration that kills bacteria). In viruses, we would use „minimal virucidal“ etc. MBIC – minimal biofilm inhibiting concentration MBEC – minimal biofilm eradication concentration

41. PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN Kontrola růstu Diagnostic methods MBEC assessment Photo: Archive of Veronika Holá MBEC … minimal biofilm eradicating concentration (Another value exists: MBIC … minimal biofilm inhibitory concentration – a value not approved by all scientists)

42. While MIC determinates minimal inhibitory concentration of atb in planctonic form, MBEC shows us if eradication of bacterial biofilm is present. So it tells us more about effect of antibiotic on normally living bacteria MBEC corresponds the lowest concentration of antibiotic, where biofilm eradication is proven (absence of living cell, no pH medium change, the well remains red) MIC versus MBEC Photo: Archive of Veronika Holá

43. Abbreviations: pen – penicilin, oxa – oxacillin, ams – ampicilin/sulbactam, cmp - chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine, cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine, van – vankomycine Differences in MIC, MBIC, MBEC

44. Diagnostic methods II. Values of MBEC are often over break point for given antibiotics (bacteria are resistant to them) Values of MBEC use to be several times higher than MIC Microbes in biofilm are usually resistant even to antibiotic combinations, the only possibility is then biofilm focus removal (a catheter, joint implants, tooth implants etc.)

45. Diagnostic methods for oral microbes

46. Streptococcus mutans detection in dental caries (1) We are going to use the test Dentocult SM Strip Mutans, that is used to detection of the presence of Streptococcus mutans. Presence and quantity of microbes is detected in the stimulated saliva and plaque. Really we are only going to read already made tests, but we are also going to follow the diagnostic procedure: 1. Preparation of the culture medium. Some five minutes before the sampling the bacitracine disc is placed into the vessel with the cultivation medium and we stir carefully.

47. Streptococcus mutans detection in dental caries(2) 2. Sampling a) Stimulated saliva. Patient is chewing a paraffin globule during one minute, then spits remaining saliva to the collection vessel. Testing strip (with round end) is placed to the tongue dorsum for 10 s. b) Dental plaque. Plaque is taken from all 4 quandrants of a chosen interdental space. A probe, an interdental thread or a microbrush stick is used for sampling. The specimen is stirred on the testing strip with angular end.

48. Streptococcus mutans detection in dental caries(3) 3. Cultivation. Both testing strips are placed into the lid and inserted into the vessel with the selective solution. Specimens are cultivated for 48 hours at 37 °C. 4. Evalutation. Streptococcus mutans forms dark blue colonies elevated from the surface of the testing strip. Number of microorganisms is read according to the pattern on a model table. Values 10 5 and more bacteria for 1 ml of saliva are considered risky.

49. Reading of a Dentocult kit Text and pictures taken from : USE OF MICROBIAL TESTS FOR DENTAL KARIES PREVENTION Practical papar Hana Hecová, Vlasta Merglová, Jaroslava Stehlíková Stomatology clinic, Medical faculty in Pislen, Charles University in Prague and Faculty hospital in Pilsen

50. More approaches in oral microbiology Besides given methods also other tests are used, e. g. PCR, specifically oriented to oral microbes Big problem of all diagnostics is a fact that there are normal components of microflora, only in pathologic numbers. So, the mere finding of a microbe means nothing important.

51. The End (Student K. C. several years ago forgot to bring her index, so she got the credit in the evening in a pub ) This slideshow was prepared in cooperation of ing. Veronika Holá, MUDr. Lenka Černohorská, PhD., and MUDr. Ondřej Zahradníček Photo: Archive of O. Z.