Year : 2020 | Volume
: 9 | Issue : 2 | Page : 74--79
Microbial biofilms—Development, behaviour and therapeutic significance in oral health
S Nithya, Susmita Saxena, Jitin Kharbanda
Department of Oral Pathology and Microbiology, ESIC Dental College and Hospital, Rohini, Delhi, India
Dr. S Nithya
Department of Oral Pathology and Microbiology, ESIC Dental College and Hospital, Rohini, Delhi
Background: Biofilms are communities of microorganisms that are found attached to a surface. They develop on both biotic and abiotic surfaces and could act as a source of infection. The formation of biofilm involves the population of bacteria in an extracellular matrix exhibiting a cooperative group behavior. It is a dynamic process that involves adhesion, growth mobility, and extracellular matrix production with both cells and the environment contributing to the formation of this matrix material. The biofilm exhibits unique properties of protecting itself from host defenses and desiccation, persistence in the flowing system, heterogeneity, spatial organization, and resistance to antimicrobial agents through its ability to influence gene expression and phenotype. Quorum sensing, a means of a cell to cell communication is closely interconnected to the development of biofilm formation and inhibition. Dental plaque is the most common and well known oral biofilm. The preponderance of biofilm-associated diseases and its resistance in eradication has potentiated the need for further research in this field.
Purpose: The purpose of this review is to create an awareness of the dentist on biofilms, its mode of formation, and the effect of biofilms on oral health. A comprehensive search of all literature on biofilms pertaining to oral health using literary search engines like PubMed and PubMed central has been taken into account in reviewing the pathogenesis and significance of biofilms in this article.
|How to cite this article:|
Nithya S, Saxena S, Kharbanda J. Microbial biofilms—Development, behaviour and therapeutic significance in oral health.J NTR Univ Health Sci 2020;9:74-79
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Nithya S, Saxena S, Kharbanda J. Microbial biofilms—Development, behaviour and therapeutic significance in oral health. J NTR Univ Health Sci [serial online] 2020 [cited 2021 Jan 18 ];9:74-79
Available from: https://www.jdrntruhs.org/text.asp?2020/9/2/74/289891
An assemblage of surface-associated microbial cells irreversibly associated and enclosed within an extracellular polymeric substance is called a Biofilm. Generally, microorganisms are considered to be planktonic or freely suspended organisms and are characterized based on their growth characteristics in different nutrient-rich media. Our human body contains many bacteria as commensals (organisms exhibiting a symbiotic relationship with each other without being injurious to each other). The realization that all surfaces of plants, animals, humans, and inanimate objects that have water or air interfaces are covered by complex microbial biofilms has turned the focus of investigations onto microbiome or microbial communities.
This term “Microbiome” coined by Joshua Lederberg and embraced by the Human Microbiome Project, refers to the microorganisms found in the human oral cavity and signifies the ecological community of commensals, both symbiotic and pathogenic which share our body space acting as major determinants of health and disease. Any change in the surrounding environment affects these planktonic organisms, this in turn leads to multiple regulatory signals being influenced, resulting in the reorganization of their spatial and temporal forms. All this reprogramming causes the biofilm to become highly complex and dynamic colonization needed for their survival.
Dental plaque is the most common and well known oral biofilm. The preponderance of biofilm-associated diseases and its resistance in eradication has potentiated the need for further research in this field. A comprehensive search of all literature on biofilms pertaining to oral health using literary search engines like PubMed and PubMed central has been taken into account in reviewing the pathogenesis and significance of biofilms in this article. The purpose of this review is to bring about an understanding of the intricate formation, heterogeneity, and adaptation of the microbiome within biofilms and their implications with respect to oral health.
Composition: The formed biofilm is composed of bacterial cells, and noncellular materials cocooned in an extracellular matrix composed primarily of polysaccharide material produced by the bacteria themselves. This contributes to ~ 90% of its biomass. Carbohydrate-binding proteins, pili, flagella, adhesive fibers, and extracellular DNA (e DNA) are some of the other components found within the biofilm.
Stages of development: The development of biofilm occurs gradually, exhibiting a universal growth cycle with common characteristics. These characteristics are not dependent on the phenotype of the organisms.
Stage I or the attachment phase is activated in seconds and are influenced by environmental signals dependent on many factors like pH, oxygen concentration, temperature, nutrients, nutrient concentration, osmolality, and iron. The signals vary depending on the organism. Rough and hydrophobic surfaces like plastic accumulate more biofilm. This stage is reversible with the detachment of some cells.
Stage II or the stage of irreversible binding begins in a matter of minutes. Adherence is followed by proliferation and intercommunication among the bacterial cells using chemical signals. These signals, on crossing the threshold activate the genetic mechanisms responsible for exopolysaccharide production, a major component of the matrix. This matrix then traps nutrients and planktonic bacteria as cell aggregates with lowered motility.
Stage-III or maturation phase - I is when the cells get layered to a thickness above 10 mm and is in the Stage IV or maturation phase -II, when a thickness of 100 mm or above is reached.
Stage V is the phase of cell dispersion where certain bacteria tend to leave the biofilm after developing the planktonic phenotype.
Structure and Evolution: The structure and evolution of biofilm communities depend on the following factors like Nutrient resources, attachment efficiency, genotypic factors, substratum, cyclic stage, antieffective hostile forces, physiochemical environment, mechanical factors, and shear forces. All this is mostly enhanced and made possible through a number of regulatory mechanisms like two-component regulatory system, quorum sensing, cyclic dimeric guanosine monophosphate (di-GMP) signaling and stigmergic factors (different spp. after differentiation into distinct cell types are kept together through intermolecular signals “stimulation affecting performance”) This has been validated in studies done on pseudomonas biofilm formation.
Two-component systems: Sensor kinase and the response regulators are the two components in this system. This system occurs constantly between the organisms and the environment where the organisms evaluate the environmental conditions and induce or modify responses leading to the growth of a biofilm. For example, the organism Pseudomonas aeruginosa contains genes for 127 two components. This makes it likely to succeed as a biofilm organism.,
Quorum sensing:Quorum sensing is a means of a cell to cell communication where gene expression is synchronized in response to the density of the cell population. It is closely interconnected to the development of biofilm formation and its inhibition through the action of stress response genes and cell signaling. The extracellular molecules, pheromones (chemical substances which when liberated triggers a social response in members of the same spp), acylated homoserine lactone (acyl-HSL) liberated via quorum sensing enable communication between the bacteria. These signals are then translated to concerted gene expression which causes cellular reprogramming by altering the expression of surface molecules, nutrient utilization, and virulence. This makes the bacteria more equipped to survive in unfavorable conditions. This is responsible for the viability of the biofilm community and is utilized by both the pathogenic and nonpathogenic bacteria.
The architecture of a mature biofilm appears to have three main layers. They are arranged in such a manner that microcolonies of the bacteria are seen as stalks of mushroom arising from the matrix which is composed of the extra polysaccharides, proteins, and deoxyribonucleic acid (DNA). Water channels are seen between the stalks of microcolonies and are responsible for the availability of nutrients and removal of the toxic metabolite buildup that could be harmful to the bacterial cell.
Pathogenicity: The biofilm protects its component microbes from host defenses like desiccation through its ability to influence its environment and by altering the gene expression and phenotype of the resident organisms. This results in the bacteria exhibiting features of persistence in the flowing system, heterogeneity, attachment to a solid surface, spatial reorganization, and resistance to antimicrobial agents. Biofilms provide a constant supply of nutrients and is well hydrated keeping the organisms viable., Interbacterial communications help in spreading drug resistance and other factors that enable increased virulence.
Formation and behavior: Biofilms could be formed on a variety of both biotic and abiotic surfaces,, ranging from living tissues, dead tissue like sequestra of dead bone, to indwelling medical devices. Aquatic systems, both natural and industrial piping systems show biofilm formation. Biofilm formation could be the result of single species colonization or a mixture of species involvement. Though the antigens liberated by sessile bacterial cells stimulates antibody production, the antibodies are not effective in killing the bacteria within the biofilm and end up causing damage to the surrounding tissues. Recently organisms within the biofilm, have shown varied behavior, having found to exhibit a new character of producing more than one biofilm. Candida albicans, common oral pathogen, for instance, produces two biofilms that are outwardly similar but functionally different, one being pathogenic and resistant to any challenge, while the other being sexually oriented and nonresistant.
Characterization/Matrix Determination: Biofilms could be examined using electron microscopy like scanning and transmission electron microscopy. The use of a dye called Ruthenium red along with osmium tetraoxide has been utilized to demonstrate the polysaccharide nature of the extracellular matrix that surrounds the cells. The structure can be characterized using scanning microscopy/standard microbiologic culture techniques. Ultrastructure of the biofilm can be visualized using a confocal laser scanning microscope. Gene analysis helps in analyzing its properties of adhesion and formation.
Methods to detect biofilms: Recalcitrant infections, known for their resistance to antibiotics owe their properties of restricted penetration to the expression of genes that cause resistance to biofilm-producing bacteria. This validates the need to detect and assess the biofilm and its associated microbiome. The different modes of detection modalities are
Tissue Culture Plate Method (TCP): Culture of the microbial organism is followed by staining and detecting biofilm formation through the reading of the optical density using Elisa reader.Tube Method: A qualitative assessment involving the formation of visible film lines on the walls and floor of the tube after culturing of the organisms in soyositive broth followed by washing and staining procedures.The Congo Red Agar Method (CRA): Microorganisms show black colony formation indicative of positive film formation.Bioluminescent Assay: Attenuated total reflecting spectroscopy (ATR): This method tries to monitor the conditioning films that are precursors to actual biofilm formation.Piezoelectric Sensors: Monitors accumulating mass of the film through its sensors.
Studies have shown the TCP method to have better detection ability when compared to the tube method and the Congo red agar method.,,
Biofilm Eradication: As one particular method cannot target the different components of a biofilm, a combination of several strategies are required to target its signaling and phenotypic attributes, that promote biofilm formation and attachment. Procedures like sonication (causes mechanical disruption), Immune modulation with low dose chemotherapeutics and antimicrobial agents are a few methods that help in biofilm removal. However, a variety of substances play a role in interfering with the control mechanisms of biofilm formation. This is done through targeted interference of the biofilm's cellular mechanics like detachment induction, dysregulation, the introduction of signal blockers, and cell killing methods., Studies using natural substances or their synthetic analogs have been proved to show varying effects in the inhibition and degradation of biofilms. The features that favor the use of these substances are their nontoxicity towards the eukaryotes and their lack of antibiotic activity. This in turn prevents the creation of resistance with a prolonged anti-biofilm effect. The liberation of hydrolytic enzymes, use of DNases that targets the extracellular DNA generation by organisms using quorum sensing, Concentration of iron' furanones (produced from plants and at certain concentrations regulate colonization of bacteria and epibiota settlement), aqueous extracts from fruits like berries, turmeric and methanolic extracts from Cuminum cyminum all have shown an antibiofilm activity. Studies on a type of honey called Manuka have also been shown to act synergistically with conventional antibiotics causing distortion of microbial morphologies and a decrease in their extracellular matrix.
Dental Plaque: Dental plaque is also a biofilm and can be defined as a diverse community of microorganisms found on the tooth surface as a biofilm, embedded in an extracellular matrix of polymers of host and microbial origin. Dental plaque contains organisms like Streptococcus mutans, Porphyromonas gingivalis, Streptococcus gordinii, Streptococcus cristatus, Porphyromonas aeruginosa as commensals. These plaque bacteria are able to use quorum sensing to modulate the expression of certain genes. This can be observed between P. gingivalis and S. cristatus species where the genes encoding fimbrial expression (fim A) in P. gingivalis gets modulated by the presence of S. cristatus and are prevented from getting attached to the biofilm. Other bacteria like P. aeruginosa utilize multiple or dual-cell to cell signaling. Eg. lasR - lasI and rhlR - rhlI signals. These signals are dependent upon the population density of the involved organisms., As sufficient densities are formed the concentration of the signals produced will be enough to activate the genes that cause differentiation and formation of biofilm. This in turn forms a mutant biofilm that is thicker with more adhesive properties Mutants that fail to provide such signals produce a thinner biofilm that lack the normal biofilm architecture and can be easily removed using surfactant treatment. Further investigations have proved that homoserine lactone signals produced by certain gram-negative bacteria result in a thicker biofilm architecture especially in urethral catheters.
Yung-Hua et al. in his study showed that S. mutans become genetically competent through quorum sensing. This kind of transformational frequencies is higher in biofilms than in planktonic cells. The bacteria within the biofilm exhibit a competitive and a predatory behavior and with the best strategies can coexist together and grow through their association among themselves. The plaque accumulation in the oral cavity occurs at sites that are stagnant and are protected from the removal forces applied in the oral cavity. The plaque passes through certain distinct developmental phases that include adsorption (host and bacteria to tooth surface), passive transport (oral bacteria to tooth surface), co-adhesion (late colonizers onto early colonizers), multiplication (attached microorganisms), and active detachment.
General properties of biofilms, microbial communities, dental plaque, and Dentistry: The general properties of biofilm comprises an open architecture with protection from desiccation and other host defenses and shows enhanced resistance to antimicrobials through inhibitor neutralization. Novel expression of genes includes a broad range of habitat and exhibits heterogeneity both spatially and environmentally. This leads to a more efficient metabolism. Dental plaque biofilm also exhibits these features. The dental plaque biofilm shows the presence of channels and voids and produces extracellular polymers to form a functional matrix. Various mechanisms like ß-Lactamase production, synthesis and upregulation of novel proteins, cell to cell signaling, change in oxygen gradient, and pH level are enhanced. This leads to the growth of obligate anaerobes in an aerobic environment showing increased resistance to chlorhexidine and antibiotics.
Dental plaque-host associated biofilms are made of soft deposits and have to be differentiated from materia alba. Mineralization of these plaques leads to calculus formation which could be either supra gingival or subgingival. Marginal plaque leads to gingivitis and supragingival plaque to caries, while subgingival plaque causes periodontitis and soft tissue destruction. A Study by Tanner et al. demonstrates dental plaque composition to be of varied diversity on anaerobic cultivation and isolation using enriched blood and acid agars. This diversity in different oral niche areas like occlusal surfaces and non-stimulated saliva along with macromorphology of tooth occlusal surfaces enables it to become cariogenic biofilm This has been proved using molecular techniques by Carvalho et al. More than 500 distinct microbial species can be seen colonizing within these plaque biofilms Along with nonbacterial microorganisms like yeasts, protozoa, mycoplasma spp. and viruses  Newer biofilm organisms like Streptococcus wiggsiae have been identified and have been implicated in the etiopathogenesis of early childhood caries. Visible occlusal plaque index (VOPI) has been used to assess the occurrence and distribution of occlusal biofilm in relation to caries. Recent studies have tried to elucidate the various patterns of oral biofilm induced responses of the host cells when the surrounding microenvironment gets altered. Peyyala et al. used anin vitro system model that utilizes rigid gas permeable contact lenses (RGPLs) to form bacterial biofilms and correlated the inflammatory responses of the host cells through calibration of IL-8 liberation thus challenging the oral epithelial cells. These studies would help in determining the significance of the microbiome in the pathogenesis of any oral lesion. Similarly among the various tooth associated structures affected by the formation of biofilms, dental implants, orthodontic wires appliances, and denture prosthesis are encompassed with the dental plaque and are the reason behind caries and certain inflammatory conditions like peri-implantitis, stomatitis, and implant failures.
Significance of biofilms and disease: Biofilm associated diseases could be either device-related or soft tissue infection related exhibiting a varied and a wide spectrum of representation. The commonly seen infections associated with biofilms are catheter-associated urinary tract infection (CAUTI), ventilator-associated pneumonia (VAP), and central line-associated bloodstream infections (CLABSIs) (device-related), cystic fibrosis, recurrent, chronic urinary tract and mucosal infections, chronic tonsillitis, laryngitis, and dental plaque (soft tissue related). The treatment of these biofilm-associated infections is further compounded by their recalcitrance to any kind of antibiotic treatment.
Biofilm has far-reaching implications in the treatment and prognosis of various conditions, especially in immunocompromised patients. The significance of mixed-species biofilm formation as dental plaque plays a crucial role in clinical dentistry. This could be targeted through the development of inhibitors and antiplaque agents that will have to affect the organisms through effective target delivery systems, other areas of future research would involve developing high throughput biofilm models which could be used to identify compounds which could kill/inhibit sessile cells, promote detachment of biofilms, Interfere with the communication of the microbes within the biofilm, prevent colonization by interference or replacement therapy, neutralization of species selection parameters. Tapping these areas and identifying the response of normal cells to the microbiome in their natural and altered state can lead to a better understanding of the prognosis and treatment of diseases.
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