TRACING THE CRIMINAL Part Eleven: Criminal in a Organized Group Bacterial biofilm Institute of Microbiology shows L [USEMAP] Survey of topics Clinical cases related to biofilm Characterisation of biofilm Diagnostic and experimental method for biofilm Pictures of biofilm Clinical cases related to biofilm Story one (today a real one) nMale, 58 let, 2001 cardiostimulator, 2002 repeatedly hospitalized on an internal department with fever of unknown origin, elavation of inflamatory markers nIn blood cultures, S. epidermidis, very good susceptibility nSeveral times treated by high doses of antibiotics in combinations (oxacilin, gentamicin, rifampicin, cefazolin, cefalotin, clindamycin) nIn the beginning, a good response, later attacks of fever again nAt transoesofageal examination, vegetation on a chamber electrode sized 1,5 × 1,5 cm. nCardiologists repeatedly refuse cardiostimulator removal. A combination oxacilin + gentamicine + rifampicine, pacient in a good state. nNevertheless, 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 – contiuing Who is guilty? The biofilm!!! nThe therapy could not be successfull, because high resistance of bacteria growing in form of a biofilm was not taken into account. nThe therapy was not strong enough from the beginning and the biofilm was not erradicated. nOnly electrod removal (under antibiotic therapy) enabled pacient status improval. F:\Documents\Biofilm\04 catheter_biofilm.gif webs.wichita.edu Story two nMichal was a 13-year old boy. He hated his parents and he decided to do anything against them. nHe decided that one of methods to fight them could be to resist everything his parents insist on him to do. nSo he decided to stop cleaning his teeth, keeping order in his room and some more activities like that. n But very soon, a toothache started. He had to visit a dentist. The dentist said he has a severe dental caries. She repaired his teeth, but also wanted him to clean his teath again, otherwise he would have problems again – not only with her (and other „come-back-to-your-grave generation), but mostly with his own teeth. The criminal agent was n…an overgrown biofilm again. nIn oral cavity, a biofilm is normal. It is even useful: the normal oral microflora is organised in it, and so it is more resistant to outer influences, including pathogenic agents nNevertheless, too overgrown biofilm (as a result of too many sugars eaten and too little teeth cleaning) makes oral biofilm to be an enemy instead to be a friend of a patient. Biofilm missed by a toothbrash may lead to a caries formation F:\Documents\Biofilm\10 carie_development.gif 3× webs.wichita.edu F:\Documents\Biofilm\08 ging_plaque_diagram.jpg F:\Documents\Biofilm\07 brush_failure.jpg [USEMAP] Characterisation of biofilm Biofilm: what is it? nA biofilm is a complex, organized structure nIt consists of living cells (mostly bacteria), masses produced by them (mostly polysaccarides) and channels nIt 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. Biofilm in a river F:\Documents\Biofilm\17 keevil-biofilm.jpg www.sbs.soton.ac.uk Various pictures of biofilm E:\Binec\02 Fotky\Pracovní\Obrazky na prezentace\Vladane\Foto na web\Tol 01.JPG Photo: Archive of Veronika Holá Biofilm on a cathetre Various pictures of biofilm E:\Binec\02 Fotky\Pracovní\Obrazky na prezentace\Vladane\Foto na web\Tol 01.JPG Photo: Archive of Veronika Holá Biofilm on a catheter Bacteria A channell Catheter Polysaccharides F:\Documents\Biofilm\01 BiofilmWbWithLabels.jpg biology.fullerton.edu Stages of biofilm development nDirect contact of a planctonic bacteria with a surface + nAdhesion to this surface nAggregation of cells into microcolonies nProduction of polymeric matrix nFormation of three-dimensional structure known as biofilm E:\Binec\02 Fotky\Pracovní\Obrazky na prezentace\Vladane\Model.gif F:\Documents\Biofilm\02 biofilm.jpg Development of a biofilm biology.binghamton.edu Biofilm development, another picture F:\Documents\Biofilm\03 biofilm_formation.gif webs.wichita.edu Biofilm development F:\Documents\Biofilm\14 biofilm formation.gif www.ul.ie Biofilm formation, another picture F:\Documents\Biofilm\12 biofilmtutorial.jpg www.uweb.engr.washington.edu Importance of biofilm production in bacteria nBacteria may better regulate their quantity – in the biofilm they inform each other by production of various stuffs (quorum sensing) nBacteria become more resistant to outer influences: ndesinfectants nantibiotics nhost immunity response Biofilm is formed both by common flora bacteria (rather positive for macrorganism and by pathogens F:\Documents\Biofilm\19 biofilm.jpg Biofilm env.snu.ac.kr Mechanisms influencing bacterial resistance nInfluence of surface charge nDecrease of growth rate nPenetration bariere nNon-homogenous matrix nFenotypic differences nIntercelular signalisation nImmunity mechanizms... F:\Documents\Biofilm\15 biofilm.jpg Biofilm www.dms-online.de Biofilm eradication nAntibiotic therapy often only supresses symptomas 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. nTo biofilm eradication we often to use high ATB concentrations (monotherapy or combinations), when treatment is not effective, the biofilm focus should be removed. nIn future we will possibly try to destroy the biofilm, e. g. by enzymotherapy F:\Documents\Biofilm\18 andes%20biofilm%20sem.jpg Yeast Biofilm www.ansci.wisc.edu Prevention nCatheters and bone cements nmade of new generation plastic material (risk of adhesion and biofilm formation lower) nwith coloid silver and similar surface-active compounds nwith antimicrobial substances, e. g. nminocycline nrifampicine nCatheter washing nCorrect asepsis, decontamination methods etc. n [USEMAP] Diagnostic and experimental methods for biofilm Biofilm and microbiologic diagnostics na) Biofilm assessment naa) by phenotypic methods (Christensen‘s method, Congo red agar cultivation) n n n n nab) by genotypic methods nb) Assesment of bacterial susceptibility in biofilm to individual antibiotics or combinations (mostly MBEC) nc) 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 E:\Binec\02 Fotky\Pracovní\Dizertacka obrazky\CTT 01.JPG E:\Binec\02 Fotky\Pracovní\Dizertacka obrazky\CRA 02.JPG Foto: Archiv Veroniky Holé Microscopy of oral biofilm nBesides official methods for biofilm detection there are also other methods how to visualise biofilm. nFor oral biofilm: nGram stain may only visualise cell clusters (both G+ and G- ) and eventually macrooranism cells (epitheliae etc.). Polysacharidic masses remain invisible. E:\Binec\02 Fotky\Pracovní\Obrazky na prezentace\Vladane\Foto na web\Alcian 02.JPG Alciane blue stain enables visualisation of polysaccharicic material, i. e. the acellullar part of biofilm. Cells are visualized by negative staining. Photo: Archive of Veronika Holá Proof of influence of tooth cleaning to oral biofilm E:\Pracovní\Mikrobiální biofilmy\Kapitoly kniha\Zuby\Obrazky\ zubní kámen 2.jpg E:\Binec\02 Fotky\Pracovní\detekce plaku - erytrosin.jpg nA volunteer has a iodine solution or pills with a stain effecting to tooth plaque. n The iodine is let to work in oral cavity during approx. 2 min. Photo: Archive of Veronika Holá Photo: Archive of Veronika Holá Culture of biofilm producing bacteria nIn 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 nFor 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 nClassical 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). nSemiquantitative (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 necesarilly released from the biofilm. nSonification: 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 Proof of influence of saccharides presence to dental plaque formation nThe experiment has a simple principle. One of oral bacteria is cultured on plastic surface (simulating tooth surface) with presence of various concetrations of glucose and for various time value nAfter the incubation, biofilm is visualised using gentiane violet and its density quantified as absorbance using a spectrophotometre To avoid accidental mistake, six adjacent wells have always the same values of both glc concentration and time H:\Documents\Seropanl biofilm.bmp Old and new abbreviations in antibiotic effect measuring nMIC – minimal inhibition concentration is the growth limit of bacteria (the lowest concentration that disables bacterial growth) nMBC – minimal bactericidal concentration is the survival limit of bacteria (the lowest concentration that kills bacteria). In viruses, we would use „minimal virucidal“ etc. nMBIC – minimal biofilm inhibiting concentration nMBEC – minimal biofilm eradication concentration 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) nWhile MIC determinates minimal ninhibitory concentration of atb in planctonic form, nMBEC shows us if eradication of bacterial biofilm is present. nSo it tells us more about effect of antibiotic on normally living bacteria nMBEC corresponts 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 C:\Nika\Pracovní\1.JPG Photo: Archive of Veronika Holá Abbreviations: pen – penicilin, oxa – oxacilin, 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 Diagnostic methods II. nValues of MBEC are often over break point for given antibiotics (bacterie are resistant to them) nValues of MBEC use to be several times higher than MIC nMicrobes in biofilm are usually resistant even to antibiotic combinations, the only possibility is then biofilm focus removal (a catheter, joint implants, tooth implants etc.) The End C:\Uživatel\Ondra\Obrázky a fotky\Rodinné a jiné\2005\2005-12-19 Fotky Ančete Haraščete z kruhovky pětky\A2%20023.jpg [USEMAP] (Student K. C. four years ago forgot to bring her index, so she got the credit in the evening in a pub J) This slideshow was prepared in cooperation of ing. Veronika Holá, MUDr. Lenka Černohorská, PhD., and MUDr. Ondřej Zahradníček Photo: Archive of O. Z.