2019-09-07

The components of biofilms are very vital as they contribute towards the structural and functional aspects of the biofilms. Microbial biofilms comprise of major classes of macromolecules like nucleic acids, polysaccharides, proteins, enzymes, lipids, humic substances as well as ions.
The presence of these components indeed makes them resilient and enables them to surviveIn nature, microorganisms exist primarily by attaching to and growing upon living and inanimate surfaces. These surfaces may take many forms, including those found in soil and aquatic systems, those on the spectrum of indwelling medical devices, and those of living tissues such as tooth enamel, heart valves, or the lung, and middle ear. The common feature of this attached growth state is that the cells develop a biofilm. Biofilm formation is a process whereby microorganisms irreversibly attach to and grow on a surface and produce extracellular polymers that facilitate attachment and matrix formation, resulting in an alteration in the phenotype of the organisms with respect to growth rate and gene transcription. The biofilms are the communities that are formed by bacteria, and which may subsequently be occupied by many different microorganisms including algae, protozoa, and some fungi.
The biofilms are formed when bacteria adhere to a solid surface and enclose themselves in a sticky polysaccharide. Once this polysaccharide is formed the bacteria can no longer leave the surface, and when new bacteria are produced they stay within the polysaccharide layer. This layer, which is the biofilm, is highly protective for the organisms within it. In fact, it is considered a fact that many bacteria could not survive in the environment outside of biofilms.

Biofilm Growth
The cells that attach irreversibly to surfaces (i.e., those not removed by gentle rinsing) will begin cell division, form microcolonies, and produce the extracellular polymers that define a biofilm. These extracellular polymeric substances (EPSs) consist primarily of polysaccharides and can be detected microscopically and by chemical analysis. EPSs provide the matrix or structure for the biofilm. They are highly hydrated (98% water) and tenaciously bound to the underlying surface. The structure of the biofilm is not a mere homogeneous monolayer of slime but is heterogeneous, both in space and over time, with “water channels” that allow transport of essential nutrients and oxygen to the cells growing within the biofilm [16]. Biofilms have a propensity to act almost as filters to entrap particles of various kinds, including minerals and host components such as fibrin, RBCs, and platelets.
Biofilm-associated organisms grow more slowly than planktonic organisms [17], probably because the cells are limited by nutrient and/or oxygen depletion. Cells detach from the biofilm as a result of either cell growth and division or the removal of biofilm aggregates that contain masses of cells. It is possible for these detached cells to cause a systemic infection, depending on a number of factors, including the response of the host immune system.

Resistance to Antimicrobial Agents
The biofilm mode of growth confers on the associated organisms a measurable decrease in antimicrobial susceptibility. For example, Ceri et al. [18] found that biofilm-associated Escherichia coli required >500 times the MIC of ampicillin to provide a 3-log reduction. Williams et al. [19] found that Staphylococcus aureus biofilms required >10 times the MBC of vancomycin to provide a 3-log reduction.

The effect on susceptibility may be -
• intrinsic (i.e., inherent in the biofilm mode of growth) or
• acquired (i.e., caused by the acquisition of resistance plasmids).
There are at least 3 reasons for the intrinsic antimicrobial resistance of biofilms.
• antimicrobial agents must diffuse through the EPS matrix to contact and inactivate the organisms within the biofilm. EPSs retard diffusion either by chemically reacting with the antimicrobial molecules or by limiting their rate of transport.
• biofilm-associated organisms have reduced growth rates, minimizing the rate that antimicrobial agents are taken into the cell and therefore affecting inactivation kinetics. An increase in growth rate resulted in an increase in susceptibility of Staphylococcus epidermidis biofilms.
• The environment immediately surrounding the cells within a biofilm may provide conditions that further protect the organism. It is found that agar-entrapped E. coli demonstrated a decreased susceptibility to aminoglycoside antibiotics as a result of decreased uptake of the antibiotic by the oxygen-deprived cells.

6. Use of AMPs in Preventing Biofilm
6.1. Biofilm Formation
Extended cultivation of bacterial cells results in adherence to animal tissues and inorganic materials. This, in turn, allows the formation of a biofilm, which is a multilayered community of sessile bacterial cells. Biofilms provide a survival advantage over planktonic or free-floating bacteria by enhancing nutrient trapping and colonization. Currently, biofilms are a widespread problem in hospitals and healthcare facilities. Indeed, the United States National Institutes of Health found that 80% of chronic infections are related to biofilms. Moreover, many studies have found that biofilms are associated with dental plaque, endocarditis, lung infection, and infection through medical devices.
Biofilm-formation by bacteria is achieved via responses to various factors, such as nutritional cues, cellular recognition of attachment sites on the surface, exposure to sublethal concentrations of antibiotics, and environment stresses. The biofilm-formation is generally initiated by the attachment of planktonic cells to a surface through weak van der Waals forces, and the colonists are anchored tightly or irreversibly by pili. To facilitate the arrival and attachment of other planktonic cells, the initial cells construct various adhesion sites and the matrix. Bacterial cells are then embedded within this matrix of extracelluar polymeric substance (EPS), which is composed of extracelluar DNA, proteins, lipids, and polysaccharides with various configurations. These components are very important targets for overcoming both biofilms and drug-resistant bacteria. During colonization, some bacteria can communicate through a quorum sensing (QS) system via small molecules called autoinducers and controls collective behaviors, such as bioluminescence, virulence factor production, and biofilm formation. Autoinducers in Gram-negative and -positive bacteria were known to acyl-homoserine lactone molecules and oligopeptides, respectively. It is currently considered a good target for preventing biofilm infection. Subsequently, the grown or developed biofilm provides increased antibiotic-resistance to bacterial colonies through cell division and recruitment. Later, the developed biofilms are dispersed and the bacteria move to other surfaces, such as organs, tissues, and medical devices, where the biofilm formation process occurs again.

6.2. Applications to Prevent or Remove Biofilms
Two main concepts in the prevention of biofilms are dispersion of the biofilm EPS and eradication of the bacteria embedded in the EPS. Typically, lethal or inhibiting concentrations of antibiotics are significantly increased by up to 1000-fold against biofilm bacteria because they are unable to translocate into EPS and therefore do not reach the bacterial cells. In contrast, antimicrobial peptides are believed to have the potential for use as anti-biofilm agents due to their different mechanisms, which include membrane-disrupting action, functional inhibition of proteins, binding with DNA, and detoxification of polysaccharides (lipopolysaccharide and lipoteichoic acid). The EPSs of biofilms contain considerable amounts of polysaccharides, proteins, nucleic acids and lipids. For example, certain antimicrobial peptides can be transferred in biofilm EPS through holes or pores formed in the lipid component of the EPS, while others can disperse biofilms.

6.2.1. In Vitro Anti-Biofilm Activity of Antimicrobial Peptides against Biofilm of MDR Bacteria
Pseudomonas aeruginosa is the significant pulmonary pathogen affecting patients with cystic fibrosis, and this organism forms a biofilm on medical devices and tissues. LL-37, a human cationic host defense peptide, showed a potent inhibitory activity in biofilm formation at a concentration of 0.5 μg/mL against P. aeruginosa biofilm and reduced pre-grown biofilms. It was also demonstrated that these effects were achieved by decreasing the attachment of bacterial cells onto the surface, stimulating twitching motility mediated by type IV pili, and down-regulating the genes related to the QS system. LL-37 also inhibited both the attachment action and development of biofilms by Staphylococcus epidermidis, being commensal in human skin and mucous membrane. Moreover, LL-37 potently inhibited the growth of planktonic cells and biofilm formation against Francisella novicida, which causes the disease tularemia. Dashper et al. reported that kappacin, nonglycosylated κ-casein (109-169), showed a significant reduction of Streptococcus mutans biofilm in the presence of ZnCl2. In addition, systematic replacement of an N-terminal amino acid with fatty acids or conjugation of fatty acids in N-terminus of synthetic short peptide leads to enhanced antibiofilm activity.

6.2.2. Anti-Biofilm Activity in Medical Devices
Recently, the beneficial effects on the survival and quality of life of patients have led to increased use of medical implants. However, medical device-related infections are often serious because contaminating bacteria on the surface of these devices can form biofilms with dense layers that are very difficult to completely remove. Currently available ailable antibiotics fail to eradicate such infections because they are inactive in the presence of biofilms or MDR bacteria. Therefore, many researchers are suggesting that antimicrobial peptide administered alone or in combination with other molecules may be able to solve this problem

6.2.3. Anti-Biofilm Activity against Oral Plaque
Dental plaque is a complex biofilm community that forms on the teeth and oral tissues of shedding and retentive surfaces. Dental plaque develops under a variety of conditions and environments, and is composed of different bacterial species. Oral biofilms cause dental cavities and periodontal diseases, such as gingivitis and chronic periodontitis. Various therapeutic approaches have been investigated to prevent or remove oral biofilm.
An approach to inhibiting biofilm formation is the use enzymes that can degrade the EPS of biofilm and detach established biofilm colonies. Moreover, biofilm-dispersing enzymes administered in combination treatment with antimicrobial agents will allow them to kill bacteria embedded in EPS . Kaplan et al. suggested that deoxyribonuclease I and glycoside hydrolase dispersin B are useful as anti-biofilm agents due to the dispersing action of EPS layers on medical devices. In addition, therapeutic treatment of combination treatments with antimicrobial peptides may result in significant synergetic-effects against MDR bacteria and the formation of biofilms

 

These extra cellular slime natured cover encloses the microbial cells and protects from various external factors.

The components of biofilms are very vital as they contribute towards the structural and functional aspects of the biofilms. Microbial biofilms comprise of major classes of macromolecules like nucleic acids, polysaccharides, proteins, enzymes, lipids, humic substances as well as ions.
The presence of these components indeed makes them resilient and enables them to surviveIn nature, microorganisms exist primarily by attaching to and growing upon living and inanimate surfaces. These surfaces may take many forms, including those found in soil and aquatic systems, those on the spectrum of indwelling medical devices, and those of living tissues such as tooth enamel, heart valves, or the lung, and middle ear. The common feature of this attached growth state is that the cells develop a biofilm. Biofilm formation is a process whereby microorganisms irreversibly attach to and grow on a surface and produce extracellular polymers that facilitate attachment and matrix formation, resulting in an alteration in the phenotype of the organisms with respect to growth rate and gene transcription. The biofilms are the communities that are formed by bacteria, and which may subsequently be occupied by many different microorganisms including algae, protozoa, and some fungi.
The biofilms are formed when bacteria adhere to a solid surface and enclose themselves in a sticky polysaccharide. Once this polysaccharide is formed the bacteria can no longer leave the surface, and when new bacteria are produced they stay within the polysaccharide layer. This layer, which is the biofilm, is highly protective for the organisms within it. In fact, it is considered a fact that many bacteria could not survive in the environment outside of biofilms.

Biofilm Growth
The cells that attach irreversibly to surfaces (i.e., those not removed by gentle rinsing) will begin cell division, form microcolonies, and produce the extracellular polymers that define a biofilm. These extracellular polymeric substances (EPSs) consist primarily of polysaccharides and can be detected microscopically and by chemical analysis. EPSs provide the matrix or structure for the biofilm. They are highly hydrated (98% water) and tenaciously bound to the underlying surface. The structure of the biofilm is not a mere homogeneous monolayer of slime but is heterogeneous, both in space and over time, with “water channels” that allow transport of essential nutrients and oxygen to the cells growing within the biofilm [16]. Biofilms have a propensity to act almost as filters to entrap particles of various kinds, including minerals and host components such as fibrin, RBCs, and platelets.
Biofilm-associated organisms grow more slowly than planktonic organisms [17], probably because the cells are limited by nutrient and/or oxygen depletion. Cells detach from the biofilm as a result of either cell growth and division or the removal of biofilm aggregates that contain masses of cells. It is possible for these detached cells to cause a systemic infection, depending on a number of factors, including the response of the host immune system.

Resistance to Antimicrobial Agents
The biofilm mode of growth confers on the associated organisms a measurable decrease in antimicrobial susceptibility. For example, Ceri et al. [18] found that biofilm-associated Escherichia coli required >500 times the MIC of ampicillin to provide a 3-log reduction. Williams et al. [19] found that Staphylococcus aureus biofilms required >10 times the MBC of vancomycin to provide a 3-log reduction.

The effect on susceptibility may be -
• intrinsic (i.e., inherent in the biofilm mode of growth) or
• acquired (i.e., caused by the acquisition of resistance plasmids).
There are at least 3 reasons for the intrinsic antimicrobial resistance of biofilms.
• antimicrobial agents must diffuse through the EPS matrix to contact and inactivate the organisms within the biofilm. EPSs retard diffusion either by chemically reacting with the antimicrobial molecules or by limiting their rate of transport.
• biofilm-associated organisms have reduced growth rates, minimizing the rate that antimicrobial agents are taken into the cell and therefore affecting inactivation kinetics. An increase in growth rate resulted in an increase in susceptibility of Staphylococcus epidermidis biofilms.
• The environment immediately surrounding the cells within a biofilm may provide conditions that further protect the organism. It is found that agar-entrapped E. coli demonstrated a decreased susceptibility to aminoglycoside antibiotics as a result of decreased uptake of the antibiotic by the oxygen-deprived cells.

6. Use of AMPs in Preventing Biofilm
6.1. Biofilm Formation
Extended cultivation of bacterial cells results in adherence to animal tissues and inorganic materials. This, in turn, allows the formation of a biofilm, which is a multilayered community of sessile bacterial cells. Biofilms provide a survival advantage over planktonic or free-floating bacteria by enhancing nutrient trapping and colonization. Currently, biofilms are a widespread problem in hospitals and healthcare facilities. Indeed, the United States National Institutes of Health found that 80% of chronic infections are related to biofilms. Moreover, many studies have found that biofilms are associated with dental plaque, endocarditis, lung infection, and infection through medical devices.
Biofilm-formation by bacteria is achieved via responses to various factors, such as nutritional cues, cellular recognition of attachment sites on the surface, exposure to sublethal concentrations of antibiotics, and environment stresses. The biofilm-formation is generally initiated by the attachment of planktonic cells to a surface through weak van der Waals forces, and the colonists are anchored tightly or irreversibly by pili. To facilitate the arrival and attachment of other planktonic cells, the initial cells construct various adhesion sites and the matrix. Bacterial cells are then embedded within this matrix of extracelluar polymeric substance (EPS), which is composed of extracelluar DNA, proteins, lipids, and polysaccharides with various configurations. These components are very important targets for overcoming both biofilms and drug-resistant bacteria. During colonization, some bacteria can communicate through a quorum sensing (QS) system via small molecules called autoinducers and controls collective behaviors, such as bioluminescence, virulence factor production, and biofilm formation. Autoinducers in Gram-negative and -positive bacteria were known to acyl-homoserine lactone molecules and oligopeptides, respectively. It is currently considered a good target for preventing biofilm infection. Subsequently, the grown or developed biofilm provides increased antibiotic-resistance to bacterial colonies through cell division and recruitment. Later, the developed biofilms are dispersed and the bacteria move to other surfaces, such as organs, tissues, and medical devices, where the biofilm formation process occurs again.

6.2. Applications to Prevent or Remove Biofilms
Two main concepts in the prevention of biofilms are dispersion of the biofilm EPS and eradication of the bacteria embedded in the EPS. Typically, lethal or inhibiting concentrations of antibiotics are significantly increased by up to 1000-fold against biofilm bacteria because they are unable to translocate into EPS and therefore do not reach the bacterial cells. In contrast, antimicrobial peptides are believed to have the potential for use as anti-biofilm agents due to their different mechanisms, which include membrane-disrupting action, functional inhibition of proteins, binding with DNA, and detoxification of polysaccharides (lipopolysaccharide and lipoteichoic acid). The EPSs of biofilms contain considerable amounts of polysaccharides, proteins, nucleic acids and lipids. For example, certain antimicrobial peptides can be transferred in biofilm EPS through holes or pores formed in the lipid component of the EPS, while others can disperse biofilms.

6.2.1. In Vitro Anti-Biofilm Activity of Antimicrobial Peptides against Biofilm of MDR Bacteria
Pseudomonas aeruginosa is the significant pulmonary pathogen affecting patients with cystic fibrosis, and this organism forms a biofilm on medical devices and tissues. LL-37, a human cationic host defense peptide, showed a potent inhibitory activity in biofilm formation at a concentration of 0.5 μg/mL against P. aeruginosa biofilm and reduced pre-grown biofilms. It was also demonstrated that these effects were achieved by decreasing the attachment of bacterial cells onto the surface, stimulating twitching motility mediated by type IV pili, and down-regulating the genes related to the QS system. LL-37 also inhibited both the attachment action and development of biofilms by Staphylococcus epidermidis, being commensal in human skin and mucous membrane. Moreover, LL-37 potently inhibited the growth of planktonic cells and biofilm formation against Francisella novicida, which causes the disease tularemia. Dashper et al. reported that kappacin, nonglycosylated κ-casein (109-169), showed a significant reduction of Streptococcus mutans biofilm in the presence of ZnCl2. In addition, systematic replacement of an N-terminal amino acid with fatty acids or conjugation of fatty acids in N-terminus of synthetic short peptide leads to enhanced antibiofilm activity.

6.2.2. Anti-Biofilm Activity in Medical Devices
Recently, the beneficial effects on the survival and quality of life of patients have led to increased use of medical implants. However, medical device-related infections are often serious because contaminating bacteria on the surface of these devices can form biofilms with dense layers that are very difficult to completely remove. Currently available ailable antibiotics fail to eradicate such infections because they are inactive in the presence of biofilms or MDR bacteria. Therefore, many researchers are suggesting that antimicrobial peptide administered alone or in combination with other molecules may be able to solve this problem

6.2.3. Anti-Biofilm Activity against Oral Plaque
Dental plaque is a complex biofilm community that forms on the teeth and oral tissues of shedding and retentive surfaces. Dental plaque develops under a variety of conditions and environments, and is composed of different bacterial species. Oral biofilms cause dental cavities and periodontal diseases, such as gingivitis and chronic periodontitis. Various therapeutic approaches have been investigated to prevent or remove oral biofilm.
An approach to inhibiting biofilm formation is the use enzymes that can degrade the EPS of biofilm and detach established biofilm colonies. Moreover, biofilm-dispersing enzymes administered in combination treatment with antimicrobial agents will allow them to kill bacteria embedded in EPS . Kaplan et al. suggested that deoxyribonuclease I and glycoside hydrolase dispersin B are useful as anti-biofilm agents due to the dispersing action of EPS layers on medical devices. In addition, therapeutic treatment of combination treatments with antimicrobial peptides may result in significant synergetic-effects against MDR bacteria and the formation of biofilms