biofilm: Sudmitted by- SUDESHNA DEY biofilm MICROBIAL GROWTH ON SURFACE Beginning of Microbes: Beginning of Microbes Bacteria first appeared on earth about 3.6 billion years ago, long before the appearance of Homo sapiens around 100,000 years ago.. Van Leeuwenhoek was the first person to visualize, graphically illustrate, and label "animalcules" (bacteria) that he found in plaque scraped from his own teeth. Bio film defination: Bio film defination A biofilm is an aggregate of microorganisms in which cells are stuck to each other and/or to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS, which is also referred to as "slime," is a polymeric jumble of DNA, proteins and polysaccharides. Biofilm is a complex substance: Biofilm is a complex substance A biofilm is a complex aggregation of microorganisms growing on a solid substrate. Biofilms are characterized by structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances. Biofilms found everywhere in nature: Biofilms found everywhere in nature More properly known as biofilm , slime cities thrive wherever there is water - in the kitchen, on contact lenses,coating the inside of water pipes or dangling slippery and green from plumbing ." (Coghlan 1996) Bioflim contribute to new phenotypes: Bioflim contribute to new phenotypes Biofilm cells are distinctly different from planktonic cells by reduce growth rate, changing gene regulation and generation of extracellular polymeric matrix. Biofilm cell require cell signaling and transcribe different set of genes. Why biofilm are formed??: Microorganism form biofilm response to many substance – Cellular recognization of specific and non specific attachment site on surface Nutritional cues Exposure to antibiotics Why biofilm are formed?? Stages of bioflim formation: Stages of bioflim formation Three major components needed for biofilm formation are. Microbes Fluid medium Solid surface PowerPoint Presentation: Step 1 : - Adsorption of inorganic and organic molecules to the solid surface, forming a thin layer termed as conditioning layer. S tep 2 : - Adhesion of microbial cells to this layer. Phase 1 - Transport of microbes to surface. Phase 2 - Initial non specific microbial-substrate adherence phase. Phase 3 - Specific microbial-substrate adherence phase. Step 3 : - Bacterial growth and expansion. Mechanisims of Biofilm formation: Mechanisims of Biofilm formation Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists adhere to the surface initially through weak, reversible van der Waals forces. They can anchor themselves more permanently using cell adhesion structure such as pili Factors Influencing Rate and Extent of Biofilm Formation : Factors Influencing Rate and Extent of Biofilm Formation The rate of cell attachment depends on the number and types of cells in the liquid to which the device is exposed, the flow rate of liquid through the device, and the physicochemical characteristics of the surface Steps in Biofilm Development: Steps in Biofilm Development Biofilm development can be divided into several key steps including attachment micro colony formation biofilm maturation dispersion ; Stages of biofilm development : Stages of biofilm development Steps in Biofilm formation: Steps in Biofilm formation Dispersal: Dispersal Detachment can be caused by external perturbations, such as increased fluid Shear by internal biofilm processes, such as endogenous enzymatic degradation or by the release of EPS . Three distinct biofilm dispersal strategies can be identified: Swarming/seeding dispersal Clumping dispersal Surface dispersal Structure of Biofilm: Biofilm is composed primarily of micro colonies of different microbial cell(15% by volume) and matrix material of(85%). EPS may vary in chemical and physical properties but are primarily consist of polysaccharide are neutral or polyanionic. The presence of uronic acid(glucouronic acid and galactouronic acid and mannuronic acid) or ketal linked pyruvate confer the anionic properties. These properties helps in the association of divalent cations such as calcium and magnesium, which cross link with polymer strands. Backbone of EPS contains 1,3 or 1,4- linked hexose residuse.EPS production is affected by nutrient status of medium. Structure of Biofilm FACTORS AFFECTING BIOFILM FORMATION: SUBSTRATUM EFFECT : The extent of microbial colonization appears to increase the surface roughness, due to diminished surface area. Maximum attachment depends upon high surface free energy or wettability of surfaces such as stainless steel and glass(hydrophilic) CONDITIONING FLIMS : Solid surfaces which have been exposed in an aqueous medium become conditioned or coated with polymer from the medium. The chemical modification of surfaces effects the rate and extend of microbial attachment. The surface is converted to hydrophilic by alkali or acid. Once exposed to air or water which froms cromium oxide layer to which organic soil adheres . FACTORS AFFECTING BIOFILM FORMATION Hydrodynamics: Biofilms grown under laminar flow are found to be patchy and separated by interstitial voids. Biofilm under turbulent flow cells are also patchy but are elongated streamers that oscillate in bulk fluid. Aqueous medium: Physicochemical characteristic of aqueous medium such as pH, neutrient levels…etc may play important role in attachment to the surfaces.It is affected by season.: Hydrodynamics : Biofilms grown under laminar flow are found to be patchy and separated by interstitial voids. Biofilm under turbulent flow cells are also patchy but are elongated streamers that oscillate in bulk fluid. Aqueous medium : Physicochemical characteristic of aqueous medium such as pH, neutrient levels…etc may play important role in attachment to the surfaces.It is affected by season. PowerPoint Presentation: Horizontal gene transfer : It is important for the evolution and genetic diversity of natural microbial communites. The mobile genetics elements mediates gene transfer between bacteria such as conjugative plasmid, transposon or bacteriophages. Quorum sensing: Quorum sensing As their density increases, the organisms secrete low molecular weight molecules that signal when the population has reached a critical threshold. This process, called quorum sensing , is responsible for the expression of virulence factors. Production of quorum sensing molequels such as acyl-homoserine lactone(AHL).This signal may alter distribution of specific bacterial species,alter protein expression in neighbouring cells,introduce new genetic traits in the cells. Biofilms a Great threat to Implants: Biofilms a Great threat to Implants A significant number of people are affected by biofilm infections which develop on medical devices implanted in the body such as catheters ,artificial joints, and mechanical heart valves. When implanted material becomes colonized by microorganisms, a slow developing but persistent infection results. Dental plaque: Dental plaque Dental plaque is a yellowish biofilm that build up on the teeth. If not removed regularly, it can lead to dental caries . Dental plaques: Dental plaques The formation of dental plaque bio films includes a series of steps that begins with the initial colonization of the pellicle and ends with the complex formation of a mature bio film . Biofilms in Cystic fibrosis: Biofilms in Cystic fibrosis Biofilms are involved in numerous diseases. In cystic fibrosis patients have Pseudomonas infections that often result in antibiotic resistant biofilms. Endocarditis and Biofilms: Endocarditis and Biofilms Microorganisms may attach and develop biofilms on components of mechanical heart valves and surrounding tissues of the heart, leading to a condition known as prosthetic valve endocarditis. The primary organisms responsible for this condition are S. epidermidis, S. aureus, Streptococcus spp ., gram-negative bacilli, diphtheroids, enterococci, and Candida spp. These organisms may originate from the skin, other indwelling devices such as central venous catheters, or dental work. Biofilms and Contact lenses: Biofilms and Contact lenses Contact lenses are classified as soft or hard lenses depending upon construction, design and frequency of disposal. The degree of attachment to the lenses depends on the nature of substrate, water content, electrolyte concentration…etc. Organism mainly are E.coli, S. aureus….etc Urinary catheters and Biofilms: Urinary catheters and Biofilms Urinary catheters are tubular latex or silicone devices, which when inserted may readily acquire biofilms on the inner or outer surfaces. The organisms commonly contaminating these devices and developing biofilms are S. epidermidis, Enterococcus faecalis, E. coli, Proteus mirabilis, P. aeruginosa, K. pneumoniae , and other gram-negative organisms. The longer the urinary catheter remains in place, the greater the tendency of these organisms to develop biofilms and result in urinary tract infections. Biofilms and indwelling medical devices : Biofilms and indwelling medical devices Biofilms on indwelling medical devices may be composed of gram-positive or gram-negative bacteria or yeasts. Bacteria commonly isolated from these devices include the gram-positive Enterococcus faecalis , Staphylococcus aureus, Staphylococcus epidermidis , and Streptococcus viridians ; and the gram-negative Escherichia coli, Klebsiella pneumoniae , Proteus mirabilis, and Pseudomonas aeruginosa . Biofilm and Antibiotic resistance: Biofilm and Antibiotic resistance Biofilm cells are covered by EPS which is an encapsulation and provide antibiotic resistant. Bacteria in biofilms resist antibiotics via several mechanisms, including (i)Decreased penetration or diffusion of antimicrobial agents into biofilms (ii)Increased activity of multidrug efflux pumps, turn susceptible planktonic cells into antibiotic-resistant persisters (iii) involvement of quorum sensing systems, (iv) starvation or stress responses, (v) genetic switches that change wildtype to biofilm cells: ( iii) involvement of quorum sensing systems , (iv) starvation or stress responses , (v) genetic switches that change wildtype to biofilm cells Biofilms protects from Immune responses: Biofilms protects from Immune responses Bacteria embedded within biofilms are resistant to both immunological and non- specific defence mechanisms of the body. Contact with a solid surface triggers the expression of a panel of bacterial enzymes which catalyze the formation of sticky polysaccharides that promote colonization and protection . Biofilms – Protects from Phagocytosis: Biofilms – Protects from Phagocytosis Phagocytes are unable to effectively engulf a bacterium growing within a complex polysaccharide matrix attached to a solid surface. This causes the phagocyte to release large amounts of pro-inflammatory enzymes and cytokines, leading to inflammation and destruction of nearby tissues. Biofilms interfere in Antibiotic Therapy: Biofilms interfere in Antibiotic Therapy Bacteria growing in a biofilm are highly resistant to antibiotics, up to 1,000 times more resistant than the same bacteria not growing in a biofilm. Standard antibiotic therapy is often useless and the only recourse may be to remove the contaminated implant . Bacterial resistance and Biofilms: Bacterial resistance and Biofilms Another area of great importance from a public health perspective is the role of biofilms in antimicrobial-drug resistance. Bacteria within biofilms are intrinsically more resistant to antimicrobial agents than plank tonic cells because of the diminished rates of mass transport of antimicrobial molecules to the biofilm associated cells Emerging Methods: Emerging Methods Several researchers are finding solutions for the cure of Biofilms , yet it is experimental, with advances in molecular biology better model treatments can be identified to reduce the problem of Biofilm interference in Antibiotic therapy. Current objectives on Biofilm research: Current objectives on Biofilm research 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 Studies of quorum sensing/signaling molecules Current objectives on Biofilm research: Current objectives on Biofilm research Further characterization of biofilm-specific gene expression Studies of the exchange of genetic material within biofilms Studies of organic contaminants on substrata, and their influence on biofilm structure Animation of biofilm formation: Animation of biofilm formation References: References 1. Yang L, Liu Y, Wu H et al. Current understanding of multi-species biofilms. Int J Oral Sci 2011; 3: 75-81. 2. Myers BA. Wound management principles and practice. 2nd ed. Upper Saddle River, NJ: Pearson; 2008. 3. Simões M, Simões LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT – Food Sci Tech 2010; 43: 573-585. 4. Chen Li and Wen Y-m. The role of bacterial biofilm in persistent infections and control strategies. Int J Oral Sci. 2011; 3:66-73.