SEMINAR REPORT ON

Biofilm

Submitted to:

Submitted By: Tulina Chatterji ID-09BIOTIM060

INTRODUCTION
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Staphylococcus aureus biofilm on an indwelling catheter A biofilm is an aggregate of microorganisms in which cells adhere to each other on 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 (although not everything described as slime is a biofilm), is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings. The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single-cells that may float or swim in a liquid medium. Microbes form a biofilm in response to many factors, which may include cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or in some cases, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics. When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior in which large suites of genes are differentially regulated.

Composition of biofilm
Biofilms consist of microorganisms and their self-produced extracellular polymeric substances (EPS). A fully developed biofilm contains many layers including a matrix of EPS with vertical structures, and a conditioning film. Vertical structures of microorganisms sometimes take the form of towers or mushrooms, and are separated by interstitial spaces. Interstitial spaces allow the bulk of the biofilm to easily and rapidly take in nutrients from the surrounding liquid and move byproducts away from the biofilm.

Formation

1. initial adsorption to surface 2. cell-cell growth population growth / reproduction 3. production of an extracellular polysaccharide substances / irreversible adhesion 4. trapped biofilm bacteria form a community that controls the structural complexity of the biofilm

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 adhesion via van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili.

The first colonists facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build the matrix that holds the biofilm together. Some species are not able to attach to a surface on their own but are often able to anchor themselves to the matrix or directly to earlier colonists. It is during this colonization that the cells are able to communicate via quorum sensing using such products as AHL. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The final stage of biofilm formation is known as development, and is the stage in which the biofilm is established and may only change in shape and size. The development of a biofilm may allow for an aggregate cell colony (or colonies) to be increasingly antibiotic resistant.

Biofilm structure
Observation of the biofilm without disrupting the community. CSLM, which allows the visualization of fully hydrated sample, has revealed the elaborate 3-dimensional structure of biofilm. Biofilm is composed primarily of micro colonies of different species of microbial cells (+15% by volume) and of matrix material (+85%). EPS may vary in chemical and physical properties but it primarily consists of polysaccharides. Some of the polysaccharides are neutral or polyanionic. The presence of uronic acids D-glucuronic, D-galactouronic and mannuronic) or ketal linked pyruvate confers the anionic properties. This property helps in the association of divalent cations such as calcium and magnesium, which have been shown to cross-link with the polymer strands and provide greater binding force in a developed biofilm. Backbone of EPS contains 1, 3- or 1, 4- linked hexose residues. The amount of EPS produced by different organisms may vary and the amount of EPS increases with the age of biofilm. EPS may associate with metal ions, divalent cautions and macro molecules (proteins, DNA, lipids and even humic substances). EPS production is known to be affected by nutrient status of the growth medium, excess available carbon; however, limitation of nitrogen, potassium, phosphate promotes the EPS synthesis.

Role of Biofilm in Microbial Communities

Biofilm formation is prevailing in bacterial life style. Here, we have described why biofilm strategy 2 has been adopted by so many microbes.

Protection from Environment

EPS of biofilm provides certain degree of shelter and homeostasis to the bacteria residing in biofilm. EPS plays various roles in structure and function of different biofilm communities. The EPS matrix also has the potential to physically prevent the access of certain antimicrobial agents into the biofilm by acting as an anion exchanger. It restricts the diffusion of compounds from surroundings into the biofilm. These characteristics largely depend on the nature of both the agent and the EPS matrix. This effect appears to be more pronounced with the antibiotics that are hydrophilic and positively charged such as 7 aminoglycosides 7, 8 EPS has also been reported sequester. Reported 9, 10 metal to ions, provide cations protection and toxins from variety and of environmental stresses such as pH shift, UV radiation, osmotic shock and desiccation.

Nutrient Availability
The water channel provides effective means of exchanging nutrient and metabolites with the bulk aqueous phase, enhancing the nutrient availability as 11 well as removal of potentially toxic metabolites. Micro colonies in biofilm quite often consist of different microbial communities. These multispecies micro consortia can result from association between metabolically co-operative organisms. Their close proximity facilitates interspecies substrate

exchange and removal and distribution of metabolic products. For example, degradation of complex organic matter into methane and carbon dioxide during anaerobic digestion requires interaction of at least three bacteria. Fermentive bacteria initiate the catabolism producing acids and alcohols, which are then utilized as substrate by acetogenic bacteria. Methanogen obtains energy by converting acetate, carbon dioxide and hydrogen to methane. Biofilm provides an ideal environment relationship. for the establishment of syntrophic case of Syntrophism is a special symbiosis in which two metabolically distinct bacteria depend on each other to utilize certain substrates, typically for energy requirements. Syntrophism has been well degradation studied 11, 12. With regard to methanogenic.

Acquisition of New Genetic Trait

Horizontal gene transfer is important for the evolution and genetic diversity of natural microbial community. Acquisition of new genetic trait gives chances to the microbial communities to transcribe the necessary genes to become the active member of the biofilm communities. This is due to transcription of different genes by biofilm forming communities and the phenotypic characters are the expression of a particular genotypic character. Transcription of algC gene involved in the production of alginate is increased approximately fourfold in biofilm associated cells as compared to planktonic cells. Researchers have noted that the pulmonary isolates (Pseudomonas aeruginosa) are mucoid due to the synthesis of large amounts of alginate. In addition, by mutational analysis researchers have shown that alginate synthesis is positively regulated by sigma factors whereas sigma factor negatively regulates the synthesis of flagellum. This reveals that when synthesis of the exopolysaccharides and alginate are increased in the biofilm-associated cells, flagellar synthesis decreases. Thus, to become an effective member of a biofilm community, the bacterium must differentiate into biofilm-associated cells by repressing the synthesis of flagellum that destabilizes the biofilm and producing exopolysaccharides that 13, 14 will reinforce the biofilm structure biofilm structure and 14, 15 the nature of physiological attributes of biofilm organisms confer inherent resistance to the antimicrobial agents. The mechanisms responsible for the resistance to these antimicrobial agents, antibiotics, disinfectants or germicides are described below.

Development

Each stage of development in the diagram is paired with a photomicrograph of a developing P. aeruginosa biofilm. All photomicrographs are shown to same scale.

There are five stages of biofilm development (see illustration at right): 1. 2. 3. 4. 5. Initial attachment: Irreversible attachment: Maturation I: Maturation II: Dispersion:

Dispersal
Dispersal of cells from the biofilm colony is an essential stage of the biofilm life cycle. Dispersal enables biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm extracellular matrix, such as dispersin B and deoxyribonuclease, may play a role in biofilm dispersal. Biofilm matrix degrading enzymes may be useful as anti-biofilm agents. [10] Recent evidence has shown that a fatty acid messenger, cis-2-decenoic acid, is capable of inducing dispersion and inhibiting growth of biofilm colonies. Secreted by Pseudomonas aeruginosa, this compound induces cyclo heteromorphic cells in several species of bacteria and the yeast Candida albicans. Nitric oxide has also been shown to trigger the dispersal of biofilms of several bacteria species at sub-toxic concentrations. Nitric oxide has the potential for the treatment of patients that suffer from chronic infections caused by biofilms.

Properties
Biofilms are usually found on solid substrates submerged in or exposed to an aqueous solution, although they can form as floating mats on liquid surfaces and also on the surface of leaves, particularly in high humidity climates. Given sufficient resources for growth, a biofilm will quickly grow to be macroscopic (visible to the naked eye). Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performs specialized metabolic functions. However, some organisms will form single-species films under certain conditions.

Extracellular matrix
The biofilm is held together and protected by a matrix of excreted polymeric compounds called EPS. EPS is an abbreviation for either extracellular polymeric substance or exopolysaccharide. This matrix protects the cells within it and facilitates communication among them through biochemical signals. Some biofilms have been found to contain water channels that help distribute nutrients and signalling molecules. This matrix is strong enough that under certain conditions, biofilms can become fossilized. Bacteria living in a biofilm usually have significantly different properties from freefloating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased a thousandfold. Lateral gene transfer is greatly facilitated in biofilms and leads to a more stable biofilm structure. However, biofilms are not always less susceptible to antibiotics. For instance, the biofilm form of Pseudomonas aeruginosa has no greater resistance to antimicrobials than do stationary-phase planktonic cells, although when the biofilm is compared to logarithmic phase planktonic cells, the biofilm does have greater resistance to antimicrobials. This resistance to antibiotics in both stationary phase cells and biofilms may be due to the presence of persister cells.

Biofilm adhesion
How a biofilm adheres to the surface of a material

Polymicrobic biofilm epifluorescence

Adsorption: the interphase accumulation of cells from the bulk liquid directly on the substratum (surface of the material) 1. Once a material is exposed to water, organic molecules begin to adsorb to its surface. 2. Called the conditioning film: mainly composed of glycoproteins (subject to high turnover rate (not static) EPS (extracellular polymeric substances) 1. Pre-requisite for biofilm formation 2. Binds the bacteria together to form the biofilm

Sticking Efficiency Equation

= sticking efficiency = number of cells adsorbed onto substratum = number of cells transported to substratum

Attachment: the acquisition of cells from the bulk liquid by an existing biofilm 1. In order to understand the process of attachment one must first examine the properties of both the substratum and the cell surface

Substratum: can be either very hydrophobic (Teflon) or hydrophilic (glass) 1. Rougher and more hydrophobic materials will develop biofilms faster

Cell properties: flagella, pili, fimbriae, or glycocalyx may impact rate of microbial attachment 1. The reason that these are important is because a cell, once drawn to the substratum must combat the repulsive forces common for all materials; these appendages enable the cell to remain attached until better/more capable attachment mechanisms are set

Inhibiting protein adsorption/ biofilm adhesion

Judging from what has been uncovered through the processes of Adsorption and Attachment, in order to prevent bacteria from forming a biofilm the substratum should be incredibly smooth. This will make it difficult for the cells to attach to the surface. Another method could be to chemically coat the substratum in order to prevent the conditioning layer and the EPS from forming. The Methods section will cover more detail in this regard.

Methods
Chemical
Antimicrobial coatings Chemical modifications are the main strategy for biofilm prevention on indwelling medical devices. Antibiotics, biocides, and ion coatings are commonly used chemical methods of biofilm prevention. They prevent biofilm formation by interfering with the attachment and expansion of immature biofilms. Typically, these coatings are effective only for a short time period (about 1 week), after which leaching of the antimicrobial agent reduces the effectiveness of the coating. The medical uses of silver and silver ions have been known for some time; its use can be traced to the Phoenicians, who would store their water, wine, and vinegar in silver bottles to keep them from spoiling. There has been renewed interest in silver coatings for antimicrobial purposes. The antimicrobial property of silver is known as an Oligodynamic effect, a process in which metal ions interfere with the growth and function of bacteria. Several in vitro studies have confirmed the effectiveness of silver at preventing infection, both in coating form and as nanoparticles dispersed in a polymer matrix. However, concerns remain over the use of silver in vivo. Considering the mechanism by which silver interferes with bacterial cell function, some fear that silver may have a similarly toxic effect on human tissue. For this reason, there has been limited use of silver coatings in vivo. Despite this, silver coatings are commonly used on devices such as catheters.

Polymer modification

Dispersive forces of grafted polymer chains can prevent bacterial adhesion to a surface

To avoid the undesirable effects of leaching, antimicrobial agents can be immobilized on device surfaces using long, flexible polymeric chains. These chains are anchored to the device surface by covalent bonds, producing non-leaching, contact-killing surfaces. One in vitro study found that when N-alkylpyridinium bromide, an antimicrobial agent, was attached to a poly (4vinyl-N-hexylpyridine), the polymer was capable of inactivating 99% of S. epidermidis, E. coli, and P. aeruginosa bacteria. Dispersion forces between the polymer chains and the bacterial cells prevent bacteria from binding to the surface and initiating biofilm growth. The concept is similar to that of Steric Stabilization of colloids. Polymer chains are grafting to a surface via covalent bonding or adsorption. The solubility of these polymers stems from the high conformational entropy of polymer chains in solution. The (Chi) parameter is used to determine whether a polymer will be soluble in a given solution. is given by the equation:

where

and

are the cohesive energy densities of the polymer and solvent, respectively,

is

the molar volume of the solution (assuming ), R is the ideal gas constant, and T is temperature in Kelvins. If 0 < < 2, the polymer will be soluble.

Mechanical
Hydrophobicity

Contact angle of a liquid droplet wetted to a rigid solid surface.

The ability of bacteria to adhere to a surface and begin the formation of a biofilm is determined in part by the enthalpy of adhesion of the surface. Adherence is thermodynamically

favored if the free enthalpy of adhesion is negative and decreases with increasing free enthalpy values. The free energy of adhesion can be determined by measuring the contact angles of the substances in question. Young's Equation can be used to determine whether if adhesion is favorable or unfavorable:

Where

and

are the interfacial energies of the solidliquid, the liquidvapor, and the can be determined.

solidvapor interfaces, respectively. Using this equation

Surface roughness

Wenzel model

Surface roughness has canned also affect biofilm adhesion. Rough, high-energy surfaces are more conducive to biofilm formation and maturation, while smooth surfaces are less susceptible to biofilm adhesion. The roughness of a surface can affect the hydrophobicity or hydrophilicity of the contacting substance, which in turn affects its ability to adhere. The Wetzel equation can be used to estimate the observed contact angle:

Where the apparent contact angle and R is the roughness parameter of the surface. R is the ratio of actual surface area over projected surface area. The Wetzel equation predicts that a hydrophilic surface will have a lower , thus making it easier for bacteria to adhere.

It is thus desirable to maintain a smooth surface on any products that may come in contact with bacteria. Studies have shown that there is a threshold value of surface roughness (Ra = 2 microns) below which biofilm adhesion will reduce no further.

Surface charge

Modification of the surface charge of polymers has also proven to be an effective means of biofilm prevention. Based on the principles of electrostatics charged particles will repel other particles of like charge. The hydrophobicity and the charge of polymeric chains can be controlled by using several backbone compounds and antimicrobial agents. Positively charged polycationic chains enable the molecule to stretch out and generate bactericidal activity.

Techniques
Due to high interest in preventing biofilm formation in the recent years a large number of techniques have been studied to find a good solution. The following section summarizes just a few of the paths being examined in the field but many more techniques outside of these are being developed as well.

Ozone cleaves extracellular polysaccharides re-dissolving the bacteria

Low-energy surface acoustic waves

This technique uses low-energy waves produced from a battery powered device. The device delivers periodic rectangular pulses through an actuator holding a thin piezo plate. The waves spread to the surface, in this case a catheter, creating horizontal waves that prevent the adhesion of planktonic bacteria to surfaces. This technique has been tested on white rabbits and guinea pigs. The results showed a lowered biofilm growth.

 Ozonation
Biofilms form as a way of survival for bacteria in aqueous situations. Ozone targets extracellular polysaccharides, a group of bacterial colonies on a surface, and cleaves them. The ozone cuts through the skeleton of the biofilm at a rapid pace thus dissolving it back to harmless microscopic fragments. Ozone is so effective because it is a very strong oxidant and it encounters biofilms in much larger concentrations than most disinfectants like chlorine. This technique has been employed mainly in the spa and pool industry as a way to purify water.

Water purification
When this technique was studied two purification methods were used to treat water. The first was a typical reverse osmosis technique used for pure water. The other was a double reverse osmosis technique with electric deionization which was continuously disinfected with UV light and disinfected weekly with ozone. The tubing it ran through was tested weekly for bacterial colonies. The highly purified water showed a sharp decrease in bacteria colony adherence. Water purification methods are being scrutinized here because it is in this state that contamination is thought to occur and biofilms are formed.

Surface modification
Surface modifications have been a highly studied technique for biofilm prevention. Many methods have been tested and a variety of results have been recorded. These techniques have been the focus of many biomedical studies aiming to reduce harmful biofilm formation on medical devices, especially catheters. The following table is a quick summary of a few surface modification techniques that have been studied.

Technique Silver ions

Method of action Results Eluted from sol-gel Silver ions initially impeded biofilm coating growth. After ten days a biofilm layer was completely established. Polyurethane Polymer surface Changes in surface roughness showed modified through glow no effect on biofilm formation. discharge techniques Surfaces with negative surface charge lessened formation. pDMAEMA (poly[2- Grafted to polyethylene Biofilm formations were reduced. (dimethylamino)ethyl and silicone rubber Silicone rubber had almost no biofilm methacrylate]) present.

Applications of Biofilm
Biofilm and Devices Associated Infection
Biofilm on indwelling medical devices may be composed of Gram-positive or Gramnegative microorganisms. These organisms may originate from the skin of patient, or health-care workers, tap water to which entry ports are exposed or other sources in the environment. Biofilms may be composed of single species or multiple species, depending on the device and its duration of action. Microorganisms commonly associated with biofilm on indwelling devices are shown in Table 2.

Central Venous Catheter Biofilm

All the indwelling central venous catheters are colonized by microorganisms embedded in a biofilm matrix. The organisms, most commonly isolated from Catheters may be inserted for administration of fluid, blood products, medications, nutritional solution, and hemodynamic monitoring. Biofilms have been reported to be universally present on central venous catheters using SEM and TEM and may be associated with either the outside of the catheter or inner lumen. These organisms originate from patient's skin microflora, exogenous microflora from health-care personnel. They gain access to the catheter by migration externally from skin along the exterior catheter surface or internally from the catheter port. Colonization and biofilm formation may occur within 3 d of atherization. Raad and Sherertz also showed that catheters in place for less than 10 d tended to have more extensive biofilm formation on the external surface of the catheter. During long term catheterization there would be more formation of a biofilm on the inner lumen of catheters. Biofilm on central venous catheters have routinely been detected by a semiquantative procedure termed the roll plate catheter biofilm, are S. epidermidis, S. aureus, C.albicans, P. Aeruginosa, K. Pneumoniae, etc technique. In this procedure, the distal tip of catheter is removed aseptically and rolled over the surface of a non-selective medium. The roll plate technique has the limitation such as low diagnostic sensitivity and low predictive value for catheter-related infection. Therefore, researchers have attempted quantification of biofilm using sonication plus vortexing of catheter

Table 1 Common fluorescent probes used in biofilm studies

Name
FITC Acridine orange Fluoroscein diacetate Nile red Propidium iodide Fluoroscein TRITC RITC Fluo-3 NCECF Name

Excitation\emmision
490\520 490\530 495\520 450\530 530\615 490\520 541/572 575/ 595 506/ 526 500/ 530or 620 362/ 470

Applications
Binds to proteins, may be conjugated to antibodies, lectins, dextrans, ficols Stains DNA and RNA Indicates esterase activity Stains neutral lipid and phospholipids RNA, DNA intercalating agent Negative staining, pH indicator Bind to proteins, may be conjugated to antibodies, lectins and dextran Bind to proteins, may be conjugated to antibodies, lectins and dextran Calcium indicator PH indicator Stains DNA

Table 2 Microorganisms associated with biofilm on indwelling devices

Medical devices Urinary catheter, Intra uterine devices, prosthetic heart valves, central venous catheter Central venous catheter, urinary catheter Artificial hip prosthesis, central venous catheter, urinary catheter Artificial voice prosthesis, central venous catheter, intra uterine devices Artificial hip prosthesis, central venous catheter, intra uterine devices, prosthetic heart valve Artificial hip prosthesis, prosthetic heart valve, urinary catheter

Causative organism Coagulase -negative Staphylococci Klebsiella pneumoniae Pseudomonas aeuginosa Candida albicans Staphylococcus aureus Enterococcus spp. Coagulase -negative Staphylococci

Examples
Several studies have examined the effect of various types of antimicrobial treatment in controlling the biofilm. Researchers have found that addition of sodium metabisulfite to dextrose heparin flush of left arterial catheter eliminated microbial colonization of this catheter. Other research group has found that catheter impregnated with minocycline and rifampicin commonly develops on the tissue surrounding the prosthesis. The primary microorganisms responsible for this condition are S. Epidermidis, S. Aureus, Streptococcus spp, Gram-negative bacilli, diptheroids, enterococci and Candida spp. Antimicrobial agents are usually applied during valve replacement and whenever patient has dental work to prevent the initial attachment by killing the microorganisms introduced into the blood stream were less to be colonized than impregnated sulfadiazine. Likely with chlorhexidine impregnated and with Catheter.

Urinary Catheters
Urinary catheters are tubular latex or silicone devices that are inserted through urethra into the bladder to measure the urine output and collect urine during surgery. Catheters may be pen or closed systems. In the open system, the catheter drains into an open collection center. On the other hand, in closed system, the catheter empties into a securely fastened plastic bag. In open system, catheter quickly gets contaminated and develops urinary tract infection (UTI) within 4 d. Patients using closed system are much less susceptible to UTI. The longer the urinary catheter remains in place, greater the tendency of these organisms to develop biofilm and result in UTI. Only 10 to 20% of patients undergoing short-term catheterization (up to 7 d) but essentially all the patients undergoing long-term catheterization (more than 30 d) get infected with UTI . 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 bacteria. Divalent cations (calcium and magnesium), increase the urinary pH and ionic strength, which results in enhancement of bacterial attachment. Some organisms of these biofilms produce urease, which hydrolyzes the urea to ammonium hydroxide. The higher pH responsible for biofilm-urine interface results in precipitation of minerals such as struvite and hydroxyapatite. These mineral containing biofilms, which form encrustations, may completely block the inner lumen. Several strategies have been attempted to control the urinary catheter biofilm such as antimicrobials, bladder irrigation, and antimicrobial agents in collection bags, and impregnation of Catheter with antimicrobial agents such as silver oxide or systemic antibiotics.

Contact Lenses
Contact lenses have been classified as soft contact lenses and hard contact lenses according to materials those silver cationic Surfactant, which is used to bond cephalosporin, will less likely to develop biofilm than untreated catheter.

Prosthetic Heart Valves

Mechanical valves and bioprostheses are being currently used as prosthetic heart valves. The surgical implantation of the prosthetic valve results in tissue damage, leading to the accumulation of platelets and of construction, design, wear schedule, and frequency of disposal. Microorganisms readily adhere to the surface of both types of lenses. The degree of attachment to the lenses depends on the nature of substrate, water content, electrolyte concentration, polymer composition, type of bacterial strain, etc. Organisms mainly adhering to the contact lenses are P. aeruginosa, E. coli, S. aureus, S. epidermidis and species of Proteus. Serratia, Candida, etc. Biofilms have been observed on the lenses removed from a patient with keratitis caused by P. Aeruginosa using SEM. Biofilms have also been found to develop on contact lenses kept in storage cases. In fact, the lens case has been implicated as the primary source for contamination.

Intrauterine Devices
The intrauterine devices (IUDs) have a tail that facilitates locating the device for removal and it is composed of a plastic monofilament surrounded by a nylon sheath. The tail portion of the IUDs may be a primary source of contamination. Organisms which contaminate the IUDs are actobacillus plantarum, S. Epidermidis, Candida albicans, and S. aureus and species of Corynebacterium, Enterococcus, etc. IUDs removed from women with pelvic inflammatory disease may also contain streptococci, S. Aureus, E. coli and some anaerobic bacteria. Fibrin at the Microorganisms suture also site have and on the device. Greater tendency to colonize these locations.

Biofilm in Yellowstone National Park. Longest raised mat area is about half a meter long.

Thermophilic bacteria in the outflow of Mickey Hot Springs, Oregon, approximately 20 mm thick.

Biofilms are ubiquitous. Nearly every species of microorganism, not only bacteria and archaea, have mechanisms by which they can adhere to surfaces and to each other. Biofilms will form on virtually every non-shedding surface in a non-sterile aqueous (or very humid) environment.

Biofilms can be found on rocks and pebbles at the bottom of most streams or rivers and often form on the surface of stagnant pools of water. In fact, biofilms are important components of food chains in rivers and streams and are grazed by the aquatic invertebrates upon which many fish feed. Biofilms can grow in the most extreme environments: from, for example, the extremely hot, briny waters of hot springs ranging from very acidic to very alkaline, to frozen glaciers. In the human environment, biofilms can grow in showers very easily since they provide a moist and warm environment for the biofilm to thrive. Biofilms can form inside water and sewage pipes and cause clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food preparation areas. Biofilms in cooling- or heating-water systems are known to reduce heat transfer.[18] Biofilms in marine engineering systems, such as pipelines of the offshore oil and gas industry, can lead to substantial corrosion problems. Corrosion is mainly due to abiotic factors; however, at least 20% of corrosion is caused by microorganisms that are attached to the metal subsurface (i.e., microbially-influenced corrosion). Bacterial adhesion to boat hulls serves as the foundation for biofouling of seagoing vessels. Once a film of bacteria forms, it is easier for other marine organisms such as barnacles to attach. Such fouling can reduce maximum vessel speed by up to 20%, prolonging voyages and consuming fuel. Time in dry dock for refitting and repainting reduces the productivity of shipping assets, and the useful life of ships is also reduced due to corrosion and mechanical removal (scraping) of marine organisms from ships' hulls. Biofilms can also be harnessed for constructive purposes. For example, many sewage treatment plants include a treatment stage in which waste water passes over biofilms grown on filters, which extract and digest organic compounds. In such biofilms, bacteria are mainly responsible for removal of organic matter (BOD), while protozoa and rotifers are mainly responsible for removal of suspended solids (SS), including pathogens and other microorganisms. Slow sand filters rely on biofilm development in the same way to filter surface water from lake, spring or river sources for drinking purposes. What we regard as clean water is a waste material to these microcellular organisms since they are unable to extract any further nutrition from the purified water. Biofilms can help eliminate petroleum oil from contaminated oceans or marine systems. The oil is eliminated by the hydrocarbon-degrading activities of microbial communities, in particular by a remarkable recently-discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCB). Stromatolites are layered accretionary structures formed in shallow water by the trapping, binding and cementation of sedimentary grains by microbial biofilms, especially of cyanobacteria. Stromatolites include some of the most ancient records of life on Earth, and are still forming today. Biofilms are present on the teeth of most animals as dental plaque, where they may cause tooth decay and gum disease.

Biofilms are found on the surface of and inside plants. They can either contribute to crop disease or, as in the case of nitrogen-fixing Rhizobium on roots, exist symbiotically with the plant.[21] Examples of crop diseases related to biofilms include Citrus Canker, Pierce's Disease of grapes, and Bacterial Spot of plants such as peppers and tomatoes. Biofilms are used in microbial fuel cells (MFCs) to generate electricity from a variety of starting materials, including complex organic waste and renewable biomass.

Biofilms and infectious diseases

Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections.[23] Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque,[24] gingivitis,[24] coating contact lenses, and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and valves. More recently it has been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds. It has recently been shown that biofilms are present on the removed tissue of 80% of patients undergoing surgery for chronic sinusitis. The patients with biofilms were shown to have been denuded of cilia and goblet cells, unlike the controls without biofilms who had normal cilia and goblet cell morphology.[29] Biofilms were also found on samples from two of 10 healthy controls mentioned. The species of bacteria from interoperative cultures did not correspond to the bacteria species in the biofilm on the respective patient's tissue. In other words, the cultures were negative though the bacteria were present. Biofilms can also be formed on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices.New staining techniques are being developed to differentiate bacterial cells growing in living animals, e.g. from tissues with allergyinflammations.

Pseudomonas aeruginosa biofilms

The achievements of medical care in industrialized societies are markedly impaired due to chronic opportunistic infections that have become increasingly apparent in immunocompromised patients and the aging population. Chronic infections remain a major challenge for the medical profession and are of great economic relevance because traditional antibiotic therapy is usually not sufficient to eradicate these infections. One major reason for persistence seems to be the capability of the bacteria to grow within biofilms that protects them from adverse environmental factors. Pseudomonas aeruginosa is not only an important opportunistic pathogen and causative agent of emerging nosocomial infections but can also be considered a model organism for the study of diverse bacterial mechanisms that contribute to bacterial persistence. In this context the elucidation of the molecular mechanisms responsible for the switch from planktonic growth to a biofilm phenotype and the role of inter-bacterial communication in persistent disease should provide new insights in P. aeruginosa pathogenicity, contribute to a better clinical management of chronically infected patients and should lead to the

identification of new drug targets for the development of alternative anti-infective treatment strategies.[33]

Dental plaque
Dental plaque is the material that adheres to the teeth and consists of bacterial cells (mainly Streptococcus mutans and Streptococcus sanguinis), salivary polymers and bacterial extracellular products. Plaque is a biofilm on the surfaces of the teeth. This accumulation of microorganisms subjects the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease. The bacterial strains identification of the biofilms isolated from the dental plaque or from the biofilms attached to the surfaces of some dental alloys, impression materials, dental implants, restorative and cement materials play an essential role concerning the biofilms establishment dynamics towards the physical-chemical properties of the materials which biofilms are attached to.

Legionellosis
Legionella bacteria are known to grow under certain conditions in biofilms, in which they are protected against disinfectants. Workers in cooling towers, persons working in air conditioned rooms and people taking a shower are exposed to Legionella by inhalation when the systems are not well designed, constructed, or maintained.

Neisseria gonorrhoeae biofilms

Neisseria gonorrhoeae is an exclusive human pathogen. Recent studies have demonstrated that it utilizes two distinct mechanisms for entry into human urethral and cervical epithelial cells involving different bacterial surface ligands and host receptors. In addition it has been demonstrated that the gonococcus can form biofilms on glass surfaces and over human cells. There is evidence for formation of gonococcal biofilms on human cervical epithelial cells during natural disease and that outer membrane blebbing by the gonococcus is crucial in biofilm formation over human cervical epithelial cells.[36]

Molecular genetics
Technological progress in microscopy, molecular genetics and genome analysis has significantly advanced our understanding of the structural and molecular aspects of biofilms, especially of extensively studied model organisms such as Pseudomonas aeruginosa. Biofilm development can be divided into several key steps including attachment, micro colony formation, biofilm maturation and dispersion; and in each step bacteria may recruit different components and molecules including flagellae, type IV pili, DNA and exopolysaccharides.The rapid progress in biofilm research has also unveiled several genetic regulation mechanisms implicated in biofilm regulation such as quorum sensing and the novel secondary messenger cyclic-di-GMP. Understanding the molecular mechanisms of biofilm formation has facilitated the exploration of novel strategies to control bacterial biofilms.

Conclusions
Residual throughout the distribution the importance from a public health perspective is the role of biofilm in antimicrobial drug resistance. The resistance of microbes residing in the biofilms towards various types of antimicrobial agents poses a serious threat the pharmaceutical industries. Therefore, it is recommended to prevent their formation rather than treatment. Further study on the biofilms include effective control strategies to prevent the formation of biofilms, effective treatment strategies for complete eradication of biofilms and complete understanding of make the two biofilm phenotype different Counterparts. Microorganisms wet so on which from planktonic have been surfaces observed to aggregate and grow into micro colonies form 3-dimensional structures, resulting in a complex biofilm. Biofilms are difficult to remove from blood processing surfaces and evironments due to the production associated equipment of EPS materials and the with and cleaning processing Complex difficulties processing environments. Model systems should be developed and used to study biofilm processes on various indwelling medical devices. These systems should closely simulate the in vivo or in situ conditions for each device. This system design could be used to investigate and compare various biofilm control treatments, device design modifications or different media formulations.

REFERENCE

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