TRACING THE CRIMINAL Part Twelve: Criminal in a Organized Group Bacterial biofilm Institute of Microbiology shows Survey of topics Clinical cases related to biofilm Story one (today a real one) n Male, 58 let, 2001 cardiostimulator, 2002 repeatedly hospitalized on an internal department with fever of unknown origin, elavation of inflamatory markers n In blood cultures, S. epidermidis, very good susceptibility n Several times treated by high doses of antibiotics in combinations (oxacilin, gentamicin, rifampicin, cafazolin, cefalotin, clindamycin) Story – contiuing n In the beginning, a good response, later attacks of fever again n At transoesofageal examination, vegetation on a chamber electrode sized 1,5 × 1,5 cm. n Cardiologists repeatedly refuse cardiostimulator removal. A combination oxacilin + gentamicine + rifampicine, pacient in a good state. n 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. Who is guilty? The biofilm!!! Story two n Michal was a 13-year old boy. He hated his parents and he decided to do anything against them. n He decided that one of methods to fight them could be to resist everything his parents insist on him to do. n So 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. n In 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 n Nevertheless, 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. Characterisation of biofilm Biofilm: what is it? n A biofilm is a complex, organised structure n It consists of living cells (mostly bacteria) and masses produced by them (mostly polysaccarides) n 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. Stages of biofilm development n Direct contact of a planctonic bacteria with a surface + n Attachment to this surface n Adhesion, growth, and aggregation of cells into microcolonies n Production of polymeric matrix n Formation of three-dimensional structure known as biofilm Development of biofilm – timing 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: n desinfectants n antibiotics n host immunity response Mechanisms influencing bacterial resistance n Influence of surface charge n Decrease of growth rate n Penetration bariere n Non-homogenous matrix n Fenotypic differences n Intercelular signalisation n Immunity mechanizms... Biofilm eradication n Antibiotic therapy often only supresses symptomas of infection caused by cells released from biofilm matrix and reacting with immunity systém. Cells fixed in biofilm matrix cannot be destroyed by such therapy. n To biofilm eradication it is possible to use high ATB concentrations (monotherapy or combinations), when treatment is not effective, the biofilm focus should be removed. Prevention n Catheters and bone cements with antimicrobial substances, e. g. n minocycline n rifampicine n Cathether washing n Correct asepsis, decontamination methods etc. Diagnostic and experimental methods for biofilm Diagnostic possibilities n Biogilm detection by fenotypic methods (Christensen‘s method, Congo red agar cultivation) n Biofilm detection by genetic methods n Assesment of bacterial susceptibility in biofilm to individual antibiotics (MBIC, MBEC) 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 macrooranism cells (epitheliae etc.). Polysacharidic masses remain invisible. Culture of biofilm producing bacteria Methods n 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). n Semiquantitative (Maki) method: It enables us to assess catether surface and semiquantitativelly assess the finding, but we have no information about intraluminal bacteria and bacteria are not necesarilly released from the biofilm. n Sonification: destroys biofilm on the catether surface and catether lumen. Inoculation of a defined specimen volume is a quantitative method, that enables as to assess microbial amount. Proof of Influence of saccharides presence to dental plaque formation n The 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 n After 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 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 MIC versus MBEC n 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 n MBEC corresponts the lowest concentration of antibiotic, where biofilm eradication is proven (absence of living cell, no pH medium change, the well remains red) Differences in MIC, MBIC, MBEC Diagnostic methods II. n Values of MBIC and MBEC arre often over break point for given antibiotics (bacterie are resistant to them) n Values of MBIC and MBEC are several times higher than MIC n Microbes in biofilm are usually resistant even to antibiotic combinations, the only possibility is then biofilm focus removal (a catether, joint implants, tooth implants etc.) Pictures of biofilm Pictures of the criminal Various pictures of biofilm Pictures of the criminal Various pictures of biofilm Development of biofilm Biofilm development, another picture Biofilm development Biofilm formation, another picture Biofilm in a river Catheter biofilm Biofilm on ileum Prostetic biofilm Biofilm missed by a toothbrash may lead to a caries formation Biofilm Biofilm Biofilm Yeast Biofilm Yeast Biofilm Biofilm The End