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Fungal biofilm

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fungal biofilm

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Fungal biofilm

  1. 1. FUNGAL BIOFILM DR KAMRAN AFZAL MBBS, FCPS, PHD CONSULTANT MICROBIOLOGIST
  2. 2. INTRODUCTION BIOFILM Biofilm is an assemblage of microbial cells that is irreversibly associated with a surface and enclosed in a matrix of a primarily extra-cellular polysaccharide material
  3. 3. • Biofilms are responsible for a broad spectrum of microbial infections in human host • Many medically important fungi produce biofilms • Candida • Aspergillus • Cryptococcus • Trichosporon • Coccidioides • Pneumocystis
  4. 4. HISTORY • Van Leeuwenhoek credited for the discovery of microbial biofilms • Zobell observed number of bacteria on surface was higher than in surrounding medium • Jones used scanning / transmission electron microscopy to examine biofilms • Characklis studied microbial slimes in industrial water systems and showed that they were highly resistant to disinfectants like chlorine
  5. 5. EPIDEMIOLOGY • Transplantation procedures, immunosuppression, use of chronic indwelling devices, and prolonged ICU stays have increased the prevalence of fungal disease • The CDC estimates that over 65% of nosocomial infections are caused by biofilms • Indwelling medical devices (dental implants, catheters, heart valves, vascular bypass grafts, ocular lenses, artificial joints, CNS shunts) act as substrates for biofilm growth • The tenacity with which Candida infects indwelling biomedical devices necessitates their removal to effect a cure • Mortality rate for patients with catheter-related candidemia - 41%
  6. 6. FUNGAL BIOFILM ARCHITECTURE • Biofilms are complex surface-associated sessile cell populations embedded in an ECM that possess distinct phenotypes compared to their planktonic cell counterparts • ECM accumulates as the biofilm matures, and contributes to cohesion • Contributory factors are: • Nutrients • Quorum-sensing molecules • Surface contact
  7. 7. PHASES IN THE FORMATION OF FUNGAL BIOFILM • Fungal biofilm formation progresses in three distinct developmental phases: • Early (0-11 h) • Initially (0 to 2 h), the majority of fungal cells appear as blastospores (yeast forms) adhering to the surface • At 3 to 4 h, distinct microcolonies appear on the surface, by 11 h, fungal colonies appear as tracks of fungal growth along areas of surface irregularities
  8. 8. • Intermediate (12-30 h) • Emergence of predominantly noncellular material (12-14 h), which appear as a haze-like film covering the fungal microcolonies • Basal blastospore communities are covered by the matrix
  9. 9. • Maturation (38-72 h) • Fungal colonies and the extracellular material in which they are embedded constitute the biofilm • Maturation is followed by dispersion
  10. 10. STAGES IN THE FORMATION OF BACTERIAL BIOFILM • Stage 1, initial attachment; stage 2, irreversible attachment; stage 3, maturation I; stage 4, maturation II; stage 5, dispersion (dispersin B)
  11. 11. ENVIRONMENTAL AND CULTURAL FACTORS AFFECTING BIOFILM BIOFILM COMMUNITY STRUCTURE AND EVOLUTION ATTACHMENT EFFICIENCY CYCLIC STAGE ANTI EFFECTIVE HOSTILE FORCES PHYSIOCHEMICAL ENVIRONMENT MECHANICAL FACTORS AND SHEAR FORCES SUBSTRATUM GENOTYPIC FORCES NUTRIENT RESOURCES
  12. 12. DIFFERENTIATING FEATURES • Hyphae formation is not a uniform feature of all fungal biofilms • Hyphal organisation is variable in the species of Aspergillus • C. albicans can exist as yeast cells, or pseudohyphae, or both, its biofilm has a highly heterogeneous structure • The emerging fungal pathogen T. asahii forms biofilms comprised of yeast and hyphal cells embedded in ECM, as do those of Coccidioides immitis • C. neoformans forms biofilms consisting of yeast cells on many abiotic substrates, and shed capsular polysaccharide forming the ECM • Pneumocystis do not produce hyphal structures as part of their biofilms
  13. 13. ROLE OF ECM DURING BIOFILM FORMATION • Biofilm ECM, which is also referred to as "slime“, is a self- produced polymeric jumble of extracellular DNA, dead cells, proteins, capsular polysaccharides and exopolysaccharides • Facilitates attachment • Maintains micro colonies (structural integrity) • Protects the biofilm cells from harsh conditions and predation • Enables the biofilms to capture nutrients (surrounding fluid medium) • Enhances biofilm resistance to environmental stress, fungicidal agents
  14. 14. QUORUM SENSING • Part of physiological structure • QS (Gene expression and regulation) is a phenomenon influencing biofilm attachment and formation • It can occur within a single fungal species as well as between diverse species • It serves as a simple communication network but helps survival of pathogens
  15. 15. SIGNALING IN BIOFILMS • QS fungi release chemical signal molecules 3-oxo-C12 homoserine lactone molecules, used as signals, that increase in concentration as a function of cell density • These biochemicals diffuse through water channels in the matrix
  16. 16. FUNGAL BIOFILM GENETICS • Transcription factors are fundamental in both positive and negative regulation of biofilm formation through regulation of hyphal formation and cell surface proteins for adherence • 124 upregulated transcription factors identified in biofilm culture • Transcription factors responsible for regulation of biofilm formation: • Bcr1 - C. albicans, C. parapsilosis and A. fumigatus • Ace2 - C. albicans and A. fumigatus • Efg1 - C. albicans, C. glabrata and A. fumigatus • LAEA - A. fumigatus
  17. 17. GENE EXPRESSION PORTRAIT OF FUNGAL BIOFILMS • Biofilms of C. albicans and A. fumigatus cells have phenotypes distinct from planktonic cells and have increased expression of genes involved in protein synthesis, encoding: • Ribosomal proteins, protein turnover, and translation factors • Multi-drug resistance transporter genes: • MDR1, MDR2, MDR4 - A. fumigatus • MDR1, CDR1, CDR2 - C. albicans • ERG - C. albicans
  18. 18. • Adherence genes: • ALS1 - C. albicans, A. fumigatus • Amino acid synthesis genes: • GCN4 - C. albicans • LAEA - A. fumigatus • Cell wall biogenesis genes are induced: • FKS1, BGL2, XOG1 - C. albicans • ROD - A. fumigatus • These features may also optimize recycling of cellular constituents
  19. 19. MATING TYPE AND FUNGAL BIOFILMS • Genetic exchange is a feature, mediated by extracellular DNA • Main mechanism of genetic exchange involves mating and cell fusion • Biofilm formation of the mating-capable cell types has revealed a regulatory pathway intimately tied to pheromone signalling • In order to mate, C. albicans must go through a switch from the white to opaque cell type • Upon switching, a pheromone is released that induces a mating response
  20. 20. Human infection s involving biofilms Native valve endocarditi s Periodontiti s Corneal infection Chronic prostatitis Otitis media CLINICAL MANIFESTATIONS
  21. 21. BIOFILM ON MEDICAL DEVICES Indwelling Medical devices Central venous catheter Intrauterine device Artificial hip prosthesis Artificial voice prosthesis Urinary catheter Prosthetic heart valve
  22. 22. MEDICAL DEVICES AND COMMON ORGANISMS WHICH CAUSE BIOFILM MEDICAL DEVICES ORGANISMS Central venous catheter CoNS, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas spp., Candida albicans Prosthetic heart valves Viridans Streptococci, Staphylococcus aureus, Enterococci Urinary catheter Staphylococcus epidermidis, E.coli, Klebsiella, Proteus mirabilis Intrauterine device Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus spp, Enteroccocci spp, Group B Streptococci, Candida albicans Artificial voice prosthesis Candida albicans, Streptococcus spp, Staphylococcus epidermidis.
  23. 23. DETECTION AND MEASUREMENT OF BIOFILMS • Microscopy Techniques • Provides the best direct evidence of biofilm formation by imaging actual cells • Biofilms are examined by fluorescence microscopy using CW dye that binds chitin to highlight fungal cell walls • Most practical microscopy technique is confocal laser scanning microscopy • Scanning electron microscopy, a research tool • Qualitative detection • Quantitative detection • Molecular detection
  24. 24. QUALITATIVE METHOD Decant tubes, wash with phosphate buffer saline Incubate at 37֯C for 24 hours Colony inoculated into 10 ml TSB Dried and stained with 0.1% crystal violet
  25. 25. RESULTS – QUALITATIVE METHODS Positive - visible film on the wall and bottom of the tube Negative – Ring formation at the liquid interface QUANTITATIVE METHODS • Maki’s Roll plate method • Used in case of central venous catheter • Cleri’s Quantitative method A- High B- Non Biofilm Producer C- Moderate High Moderate None
  26. 26. • Calgary biofilm device • Recently discovered device that can detect biofilms as in tissue culture plate along with Antimicrobial Susceptibility testing • Used to detect ability of an organism to form biofilm Peg Microtitre plate
  27. 27. MOLECULAR METHODS • Isolation of nucleic acids (DNA/RNA) and proteins provides evidence of biological materials • FISH, PCR, RT-PCR, RFLP, RAPD MICROARRAYS • Used to assess the genes present in different stages of biofilm formation • One of the best ways to evaluate gene expression • DNA chips are used for a solid support
  28. 28. RECOVERY AND MEASUREMENT OF CLINICALLY RELEVANT BIOFILMS ON MEDICAL DEVICES METHOD BASIC PROTOCOL ADVANTAGE LIMITATION Roll plate Roll the catheter tip over surface of BA Easy to use Examines only outer surface . Inaccurate Vortex then viable count. Catheter section in PBS is vortexed then cultured on different media. Measures intraluminal & extraluminal biofilm. Recovery efficiency unknown. Sonicate , vortex, then viable count. Catheter section in TSB, sonicate, vortex & culture on BA. Measures intraluminal & extraluminal biofilm. Recovery efficiency unknown. Sonicate, vortex, homogenise, Catheter section in PBS/vortex repeatedly then homogenise & Recovery efficiency determined. Measures intraluminal biofilms only.
  29. 29. Acridine orange direct staining. Following roll plate, catheter section is stained with AO Allows direct examination of catheter Method doesn’t allow quantification Endoluminal brush Brush is introduced into the implanted catheter,removed, placed in PBS, sonicated & plated Allows examination of indwelling catheter. Effect of procedure on patient & recovery efficiency unknown
  30. 30. ANTIFUNGAL SUSCEPTIBILITY TESTING • Determination of MICs – Standard method for AST APPARATUS ORGANISM TESTED FLOW DYNAMICS SUBSTRATUM METHOD FOR REMOVING & QUANTIFYING BIOFILM Perfused biofilm fermentor Candida albicans Continuous/op en system Cellulose- acetate filters Shake in sterile water, then viable count
  31. 31. BIOFILMS ARE RESISTANT TO ANTIFUNGALS • Contributing factors include: • Biofilm structural complexity • Presence of ECM causes reduced penetration • Metabolic heterogeneity intrinsic to biofilm • Biofilm-associated up-regulation of efflux pump genes • Microbes impart genetic material to one another to maintain resistance • Highly resistant to both immunological and non-specific defense mechanisms • Colonies communicate with one another through the use of QS molecules
  32. 32. DIFFERENTIATING FEATURES • C. albicans and C. parapsilosis biofilms are relatively resistant to fluconazole, amphotericin B, nystatin and voriconazole • A. fumigatus biofilms are relatively resistant to itraconazole and caspofungin • Cryptococcal biofilms are unaffected by fluconazole and voriconazole • Biofilms of T. asahii display elevated resistance to amphotericin B, caspofungin, voriconazole and fluconazole • Azole and amphotericin B therapies are ineffective against Pneumocystis carinii biofilms
  33. 33. TREATMENT • Treatment is based on MIC and MBC results • Antifungal resistance increases during biofilm development, measurable by increasing MICs • Triazoles, lipid formulations of amphotericin B and echinocandins used for Candida species • Immune modulation • Photodynamic therapy • Ultrasonic wave therapy
  34. 34. CONTROL AND PREVENTION • Antifungal mouth wash • Antifungal impregnated medical devices • Catheters impregnated with antifungals • Catheters coated with a cationic surfactant • Iron chelating compounds • OMP are expressed when iron is restricted • Enzymatic degradation, dispersing biofilms with enzymatic bacteriophage • Antifungal effects of chitosans and chitooligosaccharides
  35. 35. FUTURE RESEARCH PERSPECTIVES • More reliable methods for detection and measurement of biofilms should be developed • Elucidation of the genes specifically expressed by biofilm-associated organisms • Evaluation of various control strategies • Development of improved imaging of biofilms in situ • Development of improved clinically relevant in vitro and in vivo models of biofilms under specific in vivo conditions such as flow rate, nutrient content, and temperature
  36. 36. • Development of better probes (genetic, metabolic, and immunological) for real- time analysis • Elucidation of mechanisms of resistance of biofilms to antimicrobial agents • Studies of host immune responses, both innate and adaptive to biofilms • Studies on the potential of diagnostic procedures such as Broncho-alveloar lavage and bronchoscopy to disturb local biofilm flora and inoculate distant locations • Development of mathematical models and computer simulations of biofilms • Development of the methodology for the prevention and control of biofilms from catheters, water unit lines, and other clinically important solid surfaces
  37. 37. CONCLUSION • With the increasing use of prosthetic devices in the modern practice of medicine, the prevalence of these infections is expected to increase • There are not many answers about how to treat chronic infections caused by biofilm formation!!! • However, this is something that has trickled down into the medical field of research
  38. 38. Thank you

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