Division of Virology
Indian Veterinary Research Institute; Mukteshwar, Nainital, UK-263 138
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Introduction:
A biofilm is well organized cooperating community of microorganisms attached to the surfaces. Biofilm associated cell is differentiated from suspended counterparts by reduced growth rate up and down regulation of gene and generation of extracellular polymeric matrix. They require intracellular signaling and transcribe genes different from floating cells. Its formation is a developmental process and also shares some of the features of other bacterial development processes [1]. Biofilms are broadly defined as assemblages of microorganisms and their associated extracellular products at an interface and typically attached to an abiotic or biotic surfaced.
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The bacterial growth and activity is substantially enhanced by the incorporation of surfaces to which microorganisms could attach (bottle effect). With the use of polysaccharide strains Ruthenium red and Osmium tetra-oxide it is proved that extracellular polymeric substance (EPS) is made up of polysaccharides.
According to a report from National Institute of Health (NIH) more than 60% of all infections are caused by biofilm [3]. Diabetic foot ulcers are very prone to infection which may lead to biofilm formation and chronic complications if not treated properly in early stage.
Biofilm formation is a step by step method which includes bacterial adhesion bacterial growth and biofilm expansion. It can exist on all types of surfaces such as plastic metal glass soil particles medical implants and tissue and food products. Bacterial attachment is mediated by fimbriae pilli flagella and EPS that act to form a bridge between bacteria and the conditioning film [1]. A single bacterial species can form a biofilm but in natural environment often biofilms are formed from various species of bacteria fungi algae protozoa and debris along with corrosion products. Adhesion to surfaces provides considerable advantage for the biofilm forming bacteria such as protection from anti-microbial agents exchange of nutrients metabolites or genetic material from close proximity to other micro organisms. Such symbiotic relationships although benefit the participating bacterial growth but the physical presence of biofilm either damages surfaces or causes obstruction so that the efficiency of the surface is reduced. This kind of surface damage is collectively teamed as "biofouling" and is usually observed to cause problems as dental decay metal pipe line corrosion and colonization of various medical implants product contamination equipment failure and decreased productivity. Biofilms can vary in thickness from a mono cell layer to 6-8 cm thick but mostly on an average are of about 100μm thickness.
Role of Biofilm in Microbial Communities: there are many factors to be considered due to which microbes have tendency to form biofilm like-
1) Protection: Bacteria secretes a very important extra-polymeric substance (EPS) which is known as matrix and composed of a mixture of components such as protein nucleic acids carbohydrate and other substances. This matrix adsorbed a variety of toxic substances including metals cations toxins and antibiotics and protects residing bacteria from the external environment such as UV radiation pH shifts osmotic shock and desiccation without affecting nutrient supply.
2) Nutrition: The metabolic activities of bacteria within a biofilm are different from their planktonic counterparts. Within the biofilm itself the central bacteria have limited access to the nutrients and have low oxygen supply. They communicate each other by cellular channels and environment signaling.
3) Genetic variation: Today the emergence of multidrug-resistant bacteria is of great concern due to extensive use of antibiotics genetically engineered microorganisms and so on. Most bacteria in their natural environment reside within biofilms and follow conjugation as a likely mechanism of gene transfer within or between populations.
Biofilm Structure
Biofilm is composed primarily of micro colonies of same and different species of microbial cells (+15%) and of matrix material (85%). EPS may vary in chemical and physical properties but it primarily consists of polysaccharides. The presence of uronic acids confer the anionic properties which help in the association of divalent cations such as Ca+2 and Mg+2 which have been shown to cross-link with the polymer strands and provide greater binding force in a developed biofilm. The amount of EPS produced by different organisms may vary and increases with the age of biofilm. EPS production is affected by nutrient status of the growth medium excess available of carbon; but limitation of N K and P promotes the EPS synthesis.
Electron microscopy has been used for examination and characterization of biofilm on medical devices and in human infection. Fluorescent in-situ hybridization (FISH) and 16-23S rRNA hybridization along with confocal scanning laser microscopes are used to observed microstructures and metabolism of biofilm.
Biofilm Formation:
It usually starts with colonization of bacteria on a surface. Attachment of the bacteria to the surface may be brought by different mechanisms including surface charge gravity and chemo-attraction provided the surface has nutrients.
Production of exo-polysaccharides (also known as glycol-calyx) by bacteria is very important substance to make cell to cell attachment in a developing biofim. Bacteria divide and grow freely within this glycol-calyx to form micro-colonies eventually forming a biofilm.
Factors Favouring Biofilm Formation:
Biofilm may be formed on a variety of surfaces including living tissues indwelling medical devices water pipes etc.
1) Substrate effect: Rough surfaces favor more colonization compare to smooth surfaces. Surfaces with high surface free energies such as stainless steel and glass are more hydrophilic and thus show greater bacterial attachment than hydrophobic surfaces such as Teflon and fluorinated hydrocarbons.
2) Conditioning: Solid surfaces which have been exposed liquid or polymers become easily conditioned compared to dry surfaces. The chemical modification of surfaces affects the rate and extent of microbial attachment. E.g. alkali or strong acid treatment make the surface hydrophilic. A number of host-produced conditioning substrate such as blood saliva tears urine acquired pellicle on tooth enamel intravascular fluid respiratory secretions and etc. influence the attachment of the bacteria to tissues and organs.
3) Hydrodynamics: Flow of liquids alter the biofilm structure making it sticky while air-flow make it patchy.
4) Aqueous media: Physicochemical characteristics of the medium such as pH nutrient levels ionic strength temperature etc. play an important role in the rate of microbial attachment to the surfaces. It is found that an increase in concentration of several cations such as Na+ Ca++ Fe+++ ions affects the attachment by reducing the repulsive force between the cells and glass surface.
5) Genetic diversity: During the evolution bacteria acquire to new genetic traits via horizontal gene transfer (HGT) in new environment rather than gene mutation. Some genes may be expressed in response to a specific surface on which bacterium has chosen to settle.
6) Quorum sensing: Intracellular communication (cell to cell signaling) between bacteria is generally carried out by bacterial products that are able to diffuse away from one cell to another. Production of quorum sensing molecules like acyl-homoserine lactone (acyl-HSL) makes the biofilm sturdy.
Biofilm Infections:
Acute infections can be treated effectively with antibiotics while chronic infection leads to development of biofilm making it more difficult to treat.
Mechanisms of resistance in biofilm bacteria:
1) Phenotypic changes in bacteria due to genetic variation resulting in resistance
2) Inactivation of the antibiotics by extracellular polymers or modifying enzymes and
3) Nutrient limitation resulting in slowed growth rate.
Clinical biofilm infections are marked by symptoms that typically recur even after repeated treatment. Standard antibiotic therapy is only able to eliminate the planktonic cells leaving the sessile forms to propagate within the biofilm and to disseminate when the therapy is terminated. Moreover biofilm infections are rarely resolved by the host�s immune system. Biofilm bacteria release antigens and stimulate the production of antibodies yet bacteria residing in biofilms are resistant to these defense mechanisms.
Biofilm Disassembly:
Biofilm disassembly probably plays an important role in most biofilm-associated infections by dissemination of bacteria to other part of the host body. E.g. the devastating embolic events of endocarditis caused by detachment of the biofilm growing on heart valves severe acute infections such as sepsis etc. The mechanism of biofilm disassembly utilized by S. aureus and S. epidermidis is the production of extracellular enzymes or surfactants that degrade and solubilize adhesive components in the biofilm matrix. One accessory gene regulatory (agr) system has been found to regulate the production of matrix-degrading enzymes such as Proteases DNases Dispersin B etc. [6]. The polysaccharide secreted by the sponge-associated B. licheniformis has been shown to have negative effect on biofilm development by a range of Gram-positive and Gram-negative bacteria. EPSs from E. coli (group II capsular polysaccharide) V. vulnificus (capsular polysaccharide) P. aeruginosa (mainly extracellular polysaccharide) and marine bacterium Vibrio sps. QY101 (exopolysaccharide) display selective or broad spectrum anti-biofilm activity [7]. Biofilms often have a limited lifespan as nutrients become exhausted and waste products accumulate; thus disassembling. Bacillus subtilis produces biofilm disassembly factors like D-amino acids and Norspermidine (interacts directly and specifically with exopolysaccharide). Both act together to break down existing biofilms.
Biofilm Inhibition: Treatment Strategies in the Post-antibiotic Era
Antibiotics are currently the preferred treatment strategy to prevent bacterial infections. Although antibiotics have been used to eliminate bacterial pathogens since long time but their excessive use also damage the host beneficial microbes creating an environment where opportunistic pathogens can prevail and increase the selective pressure toward antibiotic resistance. So antibiotic treatment should be specific rather than broad spectrum. Moreover although prophylactic antibiotic administration preceding surgery is highly successful in reducing infection rate it has little or no protective effects in surgical procedures involving implants or prostheses. In most cases the best treatment for foreign body-associated biofilm infections is to remove the infected device [9]. Enzymes have been used to remove biofilms by destroying the physical integrity of the EPS. Among enzymes tested Savinase and Everlase were the most effective for the degradation EPS of the biofilm produced by P. fluorescens whereas Amylase was less effective due to its activity on carbohydrate while the biofilm EPS was made up of mostly proteins.
Biofilm dissolution treatment:
a) Bactericidal Strategies: Phage therapy Silver nanoparticles Antimicrobial peptides
b) Lectoferrin has antimicrobial activity due to its Fe-chelating nature which participates in various metabolic pathways of the microbes
c) Antiadhesion Agents: Mannosides Pilicides and Curlicides polysaccharides from other bacteria Signal Transduction Interference �Antimatrix� Agents (enzymes Chelating Agents and D-Amino Acids and Norspermidine)
d) Cold atmospheric plasma technique significantly reduced the viability and quantity of biofilms.
Applications:
1) Foreign body infections � These are more commonly associated with the colonization of microbes on indwelling medical devices. E.g. Implant biofilm-mediated diseases: Prosthetic valve- endocarditis (Staphylococcus epidermidis) Contact lenses- Keratitis (Pseudomonas aeruginosa) Intravascular catheters- Septicemia (S. epidermidis S. aureus) Urinary catheters- bacteriuria (E. coli P. aeruginosa E. faecalis Proteus mirabilisi) Endo-tracheal tube- Pneumonia (P. aeruginosa E. coli S. epidermidis S. aureus) etc.
2) Native tissue infections � Some biofilm related infections involve no foreign bodies. E.g. Non-implant biofilm-mediated diseases: Micro-colonies of bacteria in sections of lung of cystic fibrosis patient (P. aeruginosa) chronic ear infection (otitis media) Periodontitis (Porphyromonas gingivalis) etc. Urinary tract infections by uropathogenic Escherichia coli native valve endocarditis by streptococcus viridians etc.
3) Biofilm and Food Industry: Biofilm growth in food processing units leads to increase opportunity for microbial contamination of processed food. As microbes in the biofilms are protected from cleaning and sanitization therefore survival of the microbes and spoilage of the food is increased. It may be avoided by equipment design temperature control and by reduction of nutrients and water contents along with effective cleaning of potential growth sites with alkali compounds and sanitizers.
4) The Bacterial Biofilm Matrix as a Platform for Protein Delivery: The technology can be exploited as a vehicle for concentration of enzymes and antigens on the surfaces of cells and as a delivery system targeting abiotic surfaces. For example Commensal bacterium such as E. coli or a commonly used pro-biotic might be used to deliver lactase or pancreatic enzymes to the intestine of hosts deficient in these enzymes. A nonpathogenic bacterium colonizing the lung of a cystic fibrosis patient might be re- engineered to deliver mucinase or alginase to clear biofilm-associated P. aeruginosa from the lung. Enzymes delivery to contaminated surfaces are often used in bioremediation.
5) Bio-surfactants are reported to be produced by bacteria yeasts and fungi can serve as green surfactants. These are considered to be less toxic and eco-friendly and thus have the potential to be commercially produced for use in pharmaceutical cosmetics and food and agriculture industries. Many rhizosphere and plant associated microbes produce bio-surfactant which play a vital role in motility signaling and biofilm formation indicating that bio-surfactant governs plant�microbe interaction. In agriculture bio-surfactants can be used for plant pathogen elimination and for increasing the bioavailability of nutrient for beneficial plant associated microbes and can widely be applied for improving the agricultural soil quality by soil remediation. These bio-molecules can replace the harsh surfactant presently being used in million dollar pesticide industries.
6) Impact of biofilm on deterioration of water quality: Deterioration of water quality during storage and in distribution system is one of the major problems. As distribution system is one of the vital importance in determining the quality of portable water its proper cleaning and sanitation is must. Various factors like type of piping materials temperature type of disinfectants (chlorine chloramines ozone hydrogen peroxide etc.) resistant of bacteria to disinfectants etc. influence the biofilm formation.
7) Micro-organisms present in biofilm can cause paint degradation and its spoilage [16]. Slow moving fluid near the biofilm poses a barrier to diffusive transport of solutes into and out of the biofilm while fast moving fluid can cause damage to the biofilm by stretching rolling and rippling; and thus its desimation.
8) It has been reported that microbes have ability to convert the energy stored in the chemical bonds of complex organics into an electrical current as a renewable energy source in future. These microbes are used on the anode surface to transfer electrons from the oxidation of the organic compounds in a cell factory known as Microbial fuel cell (MFC). Oxidation of the substrate produces electrons protons and CO2. The electrons flow from the anode to the cathode creating a current and protons migrate to the cathode to complete the electrical circuit.
Summary:
Biofilm is a well assembled microbial community of bacteria in general along with other microbes like fungi algae protozoa. Bacterial biofilm is made up of bacteria and their extracellular polymeric substances by which they attach to biotic or abiotic surface. Biofilm protect the bacteria from the external harsh environment and antimicrobial agents in one way while on the other hand they harm the contact surfaces tissues and fluids. Today development of biofilms in medical devices tissues and organ is very challenging to the available surgeries and antibiotic treatments. Their presence in water pipelines food processing units are also a subject of worry. Moreover their formation leads to development of different species which are multi-drug resistant and is a cause of great havoc to available treatment. Thus it is very essential to stop their formulation at first stage by use of proper and clean material as well as effective sanitization and treatment. A lot of research work has been going on to understand the each step involved in biofilm formation and its disassembly to get rid of it but still its prevention and removal is challenging.
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