2019-09-02

which has resulted in antimicrobial peptides being considered as an alternative to conventional drugs. Antimicrobial peptides are ancient host defense effector molecules in living organisms. These peptides have been identified in diverse organisms and synthetically developed by using peptidomimetic techniques. This review was conducted to demonstrate the mode of action by which antimicrobial peptides combat multidrug-resistant bacteria and prevent biofilm formation and to introduce clinical uses of these compounds for chronic disease, medical devices, and oral health. techniques. The substance behind this information is to demonstrate the mode of action by which antimicrobial peptides combat multidrug-resistant bacteria and prevent biofilm formation and to introduce clinical uses of these compounds for chronic disease, medical devices, and oral health. In addition, combinations of antimicrobial peptides and conventional drugs were considered due to their synergetic effects and low cost for therapeutic treatment.

1. Introduction 

Since penicillin was first discovered by Fleming in 1928, a large number of antibiotics have been identified, developed and clinically used in antimicrobial pharmacotherapeutics. However, the widespread use of antibiotics was soon followed by the emergence of multidrug-resistant (MDR) microbes due to various reasons including abuse and the increasing use of antibiotics in the biomedical and agricultural fields. In addition to bacterial evolution, a number of patients in hospitals worldwide are currently suffering from superbugs such as vancomycin resistant enterococci (VRE), methicillin resistant Staphylococcus aureus (MRSA) and MDR bacteria. Indeed, from 1999 to 2005, the number of hospitalizations associated with MRSA infections increased by 119%, or ~14% per year [1]. In addition, the Center for Disease Control and Prevention (CDC) reported that 1.7 million people were nosocomially infected in hospitals in 2002 and 99,000 deaths were occurring annually in the United States due to drug-resistant microbes [2]. Moreover, as of early 2005 the number of deaths in the United Kingdom attributed to MRSA was estimated to be 3,000 per year. Biofilms are sessile microbial communities of microbes that are adhered to various surfaces and encaged in a self-produced extracellular matrix, and have given rise to another problem in clinical therapeutics. Specifically, bacterial cells growing in a biofilm are physiologically distinct from planktonic cells of the same bacteria and are embedded within a self-produced matrix of extracelluar polymeric substance (EPS) , which can increase antibiotic resistance by up to 1000 folds.

Currently, many studies are being conducted to address the above problems, multidrug-resistant bacteria and biofilm formation. The results of these studies have led to antimicrobial peptides being considered as an alternative drug for conventional antibiotics. They have weak antimicrobial activity but potent and broad immune modulatory activity when the host organism is invaded by pathogenic microbes or viruses. Indeed many use the generic term “host defense” peptides . They do not activate adaptive immunity, but rather increase the efficiency thereof through adjuvant activity. The mode of action of antibiotic peptides is not fully understood, but it is believed that their major targets are cytoplasmic membrane and intracellular molecules. It is also believed that it is very difficult for bacteria to develop resistance to antimicrobial peptides because most kill bacterial cells quickly through their actions on the entire cytoplasmic membrane or can act through complex mechanism . Although resistance for antimicrobial peptide has been reported, acquirement of resistance by changing the charge on surface molecules or proteolytic cleavage by the release of extracelluar protease, is limited and also takes long periods when compared to conventional drugs. Although antimicrobial peptides are much more expensive than antibiotics, many studies have found that antimicrobial peptides act effectively in synergy with currently used antibiotics against multidrug-resistant bacteria because they function through different mechanisms.

2. Use of AMPs in Preventing Multidrug-Resistant Bacteria 

Major targets of antimicrobial peptides in bacterial cells can be divided into two cellular sites, the cell wall containing outer membrane and inner membrane and cytoplasm. Although the mechanisms inducing antibiotic-resistance are also diverse, the cellular action of antimicrobial peptides is separated from these mechanisms. For that reason, antimicrobial peptides have the potential for use in a unique antibiotic drug for combating or preventing the formation of multidrug-resistant bacteria.

2.1) Modes of Antibacterial Action-

1.) Lipopolysaccharide (LPS) Neutralization or Disaggregation by Antimicrobial Peptides
 

LPSs are major components of the outer leaflet of the outer membrane in Gram-negative bacteria. LPSs consist of an O-specific chain that is highly variable in different bacterial strains, a core oligosaccharide, and lipid A. LPSs are essential for bacterial growth and viability, but macrophages stimulated by LPS induce the release of pro-inflammatory cytokines (TNF-α, IL1 and IL6) into the blood, resulting in septic shock. Accordingly, LPSs are an excellent target for antimicrobial peptides because they have the potential to both directly inhibit the growth of multidrug-resistant bacteria and to neutralize the action of released LPS due to its stimulation of immune cells.

Antimicrobial peptides generally bind to LPS through electrostatic interactions between their cationic amino acids (Lysine and Arginine) and head groups of LPS, and this complex is stabilized through hydrophobic interactions between the hydrophobic amino acids of the peptide and fatty acyl chains of LPS. Since polymyxins, which are pentabasic decapeptide antibiotics, were discovered in Bacillus polymyxa, only two have been produced commercially, polymyxin B and E (colistin). Their action, which occurs via binding to lipid A of LPS and permeabilization of the outer membrane, is restricted to Gram-negative bacteria.

Sushi peptides, which are derived from Factor C (LPS-sensitive serine protease of the horseshoe crab coagulation cascade), disrupt LPS aggregates through detergent-like action and also have LPS-neutralising activity.

2.2. Cell Wall-Lipid II

Cell walls of Gram-positive bacteria are formed by peptidoglycan, which are composed of polymers of sugars and amino acids outside the plasma membrane. Occasionally, inhibition of the production of peptidoglycan leads to resistance against antibiotics such as penicillin, which is inhibited via penicillin-binding proteins or transpeptidases. MRSA is also related to the existence of the penicillin-binding protein 2a (PBP2a), which is not present in susceptible S. aureus. Vancomycin resistance is caused by the production of depsipeptide d-Ala-d-Ala in the peptidoglycan. Although a number of antimicrobial peptides have been shown to be active against MRSA and VRE. Here we focus on antibacterial peptides with unusual amino acids, which are known as lantibiotics, because many of them exert antibacterial action through the interaction with cell wall components.
Lantibiotics are ribosomally-synthesized and post-translationally modified peptides that contain an intramolecular ring structure. These compounds are produced by Gram-positive bacteria and exert potent inhibitory action against a wide-spectrum of bacteria. These compounds are classified as either type-A or type-B, and damage the bacterial membrane and inhibit the production of enzymes, respectively . Type-A lantibiotics include nisin, subtilin, epidermin, and Pep5, while type-B include mersacidin and cinnamycin. The most well-known lantibiotic is nisin, which was isolated from Lactococcus latis and is used as a food preservative worldwide. It was initially shown that nisin forms complexes with lipid I and lipid II, and then inhibits cell wall biosynthesis.

2.3. Alteration of Membrane Potential or Induction of Membrane Permeabilization 

Two major mechanisms of multidrug-resistance are phenotypic alteration of microbes under specific growth conditions, such as biofilms, and reduction of drug accumulation into microbes through limited uptake or pumping out drugs by multidrug-resistant proteins (MDRPs). Mode of action of antimicrobial peptides in the cytoplasmic membrane is considered to be more important than other targets.

Furthermore, antimicrobial peptides must permeate the cell wall and cytoplasmic membrane to reach their intracellular targets, which are nucleic acids and functional proteins. Although the exact mechanisms of antimicrobial peptides are not fully understood, they are known to cause the efflux of intracellular materials by- 

• disrupting the cytoplasmic membrane via either pore formation through a barrel-stave, or 

• a toroidal pore mechanism, or

 • through a nonporous carpet-like mechanism

2.4. Inhibition of Cytoplasmic Proteins Related to Cell Division or Survival Although most antimicrobial peptides primarily contribute to membrane perturbation, some antimicrobial peptides can penetrate the bacterial cytosol through a flip-flop mechanism or outer membrane protein forming channel. Among these, proline-rich antibacterial peptides such as pyrrhocoricin, apidaecin and drosocin have been shown to kill bacterial species by binding to the multi-helical lid region of the bacterial DnaK heat shock protein, which plays an essential role in the initiation of chromosomal DNA replication in an ATP-dependent manner with the other protein, DnaJ. The C-terminus of pyrrhocoricin was allowed to penetrate into cytosol of bacteria and the N-terminus responded to inhibit the ATPase activity of DnaK protein. Microcin B17, which is ribosomally synthesized antimicrobial peptides from Enterobacteriaceae, is also believed to inhibit DNA replication by targeting DNA gyrase.

2.5. Inhibition of Macromolecular Synthesis through Interaction with Nucleic Acids

It has been suggested that inhibition of intracellular processes by certain antimicrobial peptides that penetrate bacterial cells, such as buforin II PR-39 indolicidin, and tPMP , may contribute to inhibition of the growth of bacterial cells or lead to cell death. Chloe et al found that buforin II, a 21-amino acid peptide derived from the Asian toad, Bufo bufo gargarizans, kills bacteria through interaction with nucleic acids without membrane permeabilization, although further investigation is needed to identify other interactions with as yet unidentified intracellular targets. PR-39, which was isolated from the small intestine of the pig, required a lag period of about 8 min to penetrate the outer membrane, after which it rapidly killed growing E. coli cells via a mechanism that stops protein and DNA synthesis. In the case of indolicidin, although it induced permeabilization of the bacterial membrane, it did not lyse the bacterial cells. Its lethal concentration allowed their filamentous morphology by inhibition of DNA synthesis in E. coli cells, and it was also found to bind specifically to DNA rather than RNA.

3. Synergetic Effects between Antimicrobial Peptides and Clinically used Antibiotics 

The combined administration of antibiotics has gained interest because it often results in a synergistic antibacterial effect, which enables the dose of the individual drugs to be reduced. In addition, certain combination therapies have prevented the development of drug-resistance in bacteria. A membranolytic action of antimicrobial peptide is expected to produce synergetic effects when administered in combination with conventional antibiotics, and several studies have reported such findings.

5. Clinical Development of Antimicrobial Peptides

Several antimicrobial peptides are being evaluated in preclinical and clinical trials with limited applications. For example, Omeganan/MX-226, which is an indolicidin analogue, has recently completed phase III trials the prevention of catheter-related local and bloodstream infection, but was dropped for development. Additionally, pexiganan/MSI-78 has completed phase III clinical trials in the prevention of diabetic foot ulcers and plectasin is a fungal defensin peptide that exerts bactericidal action against drug-resistant bacteria and is currently in the preclinical phase. Opebacan, which is a human bactericidal/permeability-increasing protein derivative, has reached the phase II clinical trial for endotoxemia in hematopoetic stem cell transplant recipients. Iseganan/IB-367 from pig protegrin-1 has failed in the prevention of oral mucositis because it did not have a comparative advantage to existing therapeutics. Although several antimicrobial peptides are progressing to commercial development, records of clinical trials for antimicrobial peptides have been restricted to topical applications.

8. Conclusions 

Antimicrobial peptides can be the next generation of antibiotics for combating multi-drug resistant. These peptides have a strong potential for application as nanofilms or other coating materials for surgical devices, including catheters. Even though there are drawbacks to the use of peptides as therapeutics, such as low bioavailability and high cost, these obstacles may be overcome since a great deal of effort is being conducted to circumvent the problems associated with various methods including the use of d- or unnatural amino acid, formulation, recombinant DNA expression of peptides, addition of fatty acyl chains to short peptides. Therefore, it is expected that antimicrobial peptides will become the drugs of choice for emerging bacterial infections in the future.

As a result, there is a continuous search to overcome or control such problems

which has resulted in antimicrobial peptides being considered as an alternative to conventional drugs. Antimicrobial peptides are ancient host defense effector molecules in living organisms. These peptides have been identified in diverse organisms and synthetically developed by using peptidomimetic techniques. This review was conducted to demonstrate the mode of action by which antimicrobial peptides combat multidrug-resistant bacteria and prevent biofilm formation and to introduce clinical uses of these compounds for chronic disease, medical devices, and oral health. techniques. The substance behind this information is to demonstrate the mode of action by which antimicrobial peptides combat multidrug-resistant bacteria and prevent biofilm formation and to introduce clinical uses of these compounds for chronic disease, medical devices, and oral health. In addition, combinations of antimicrobial peptides and conventional drugs were considered due to their synergetic effects and low cost for therapeutic treatment.

1. Introduction 

Since penicillin was first discovered by Fleming in 1928, a large number of antibiotics have been identified, developed and clinically used in antimicrobial pharmacotherapeutics. However, the widespread use of antibiotics was soon followed by the emergence of multidrug-resistant (MDR) microbes due to various reasons including abuse and the increasing use of antibiotics in the biomedical and agricultural fields. In addition to bacterial evolution, a number of patients in hospitals worldwide are currently suffering from superbugs such as vancomycin resistant enterococci (VRE), methicillin resistant Staphylococcus aureus (MRSA) and MDR bacteria. Indeed, from 1999 to 2005, the number of hospitalizations associated with MRSA infections increased by 119%, or ~14% per year [1]. In addition, the Center for Disease Control and Prevention (CDC) reported that 1.7 million people were nosocomially infected in hospitals in 2002 and 99,000 deaths were occurring annually in the United States due to drug-resistant microbes [2]. Moreover, as of early 2005 the number of deaths in the United Kingdom attributed to MRSA was estimated to be 3,000 per year. Biofilms are sessile microbial communities of microbes that are adhered to various surfaces and encaged in a self-produced extracellular matrix, and have given rise to another problem in clinical therapeutics. Specifically, bacterial cells growing in a biofilm are physiologically distinct from planktonic cells of the same bacteria and are embedded within a self-produced matrix of extracelluar polymeric substance (EPS) , which can increase antibiotic resistance by up to 1000 folds.

Currently, many studies are being conducted to address the above problems, multidrug-resistant bacteria and biofilm formation. The results of these studies have led to antimicrobial peptides being considered as an alternative drug for conventional antibiotics. They have weak antimicrobial activity but potent and broad immune modulatory activity when the host organism is invaded by pathogenic microbes or viruses. Indeed many use the generic term “host defense” peptides . They do not activate adaptive immunity, but rather increase the efficiency thereof through adjuvant activity. The mode of action of antibiotic peptides is not fully understood, but it is believed that their major targets are cytoplasmic membrane and intracellular molecules. It is also believed that it is very difficult for bacteria to develop resistance to antimicrobial peptides because most kill bacterial cells quickly through their actions on the entire cytoplasmic membrane or can act through complex mechanism . Although resistance for antimicrobial peptide has been reported, acquirement of resistance by changing the charge on surface molecules or proteolytic cleavage by the release of extracelluar protease, is limited and also takes long periods when compared to conventional drugs. Although antimicrobial peptides are much more expensive than antibiotics, many studies have found that antimicrobial peptides act effectively in synergy with currently used antibiotics against multidrug-resistant bacteria because they function through different mechanisms.

2. Use of AMPs in Preventing Multidrug-Resistant Bacteria 

Major targets of antimicrobial peptides in bacterial cells can be divided into two cellular sites, the cell wall containing outer membrane and inner membrane and cytoplasm. Although the mechanisms inducing antibiotic-resistance are also diverse, the cellular action of antimicrobial peptides is separated from these mechanisms. For that reason, antimicrobial peptides have the potential for use in a unique antibiotic drug for combating or preventing the formation of multidrug-resistant bacteria.

2.1) Modes of Antibacterial Action-

1.) Lipopolysaccharide (LPS) Neutralization or Disaggregation by Antimicrobial Peptides
 

LPSs are major components of the outer leaflet of the outer membrane in Gram-negative bacteria. LPSs consist of an O-specific chain that is highly variable in different bacterial strains, a core oligosaccharide, and lipid A. LPSs are essential for bacterial growth and viability, but macrophages stimulated by LPS induce the release of pro-inflammatory cytokines (TNF-α, IL1 and IL6) into the blood, resulting in septic shock. Accordingly, LPSs are an excellent target for antimicrobial peptides because they have the potential to both directly inhibit the growth of multidrug-resistant bacteria and to neutralize the action of released LPS due to its stimulation of immune cells.

Antimicrobial peptides generally bind to LPS through electrostatic interactions between their cationic amino acids (Lysine and Arginine) and head groups of LPS, and this complex is stabilized through hydrophobic interactions between the hydrophobic amino acids of the peptide and fatty acyl chains of LPS. Since polymyxins, which are pentabasic decapeptide antibiotics, were discovered in Bacillus polymyxa, only two have been produced commercially, polymyxin B and E (colistin). Their action, which occurs via binding to lipid A of LPS and permeabilization of the outer membrane, is restricted to Gram-negative bacteria.

Sushi peptides, which are derived from Factor C (LPS-sensitive serine protease of the horseshoe crab coagulation cascade), disrupt LPS aggregates through detergent-like action and also have LPS-neutralising activity.

2.2. Cell Wall-Lipid II

Cell walls of Gram-positive bacteria are formed by peptidoglycan, which are composed of polymers of sugars and amino acids outside the plasma membrane. Occasionally, inhibition of the production of peptidoglycan leads to resistance against antibiotics such as penicillin, which is inhibited via penicillin-binding proteins or transpeptidases. MRSA is also related to the existence of the penicillin-binding protein 2a (PBP2a), which is not present in susceptible S. aureus. Vancomycin resistance is caused by the production of depsipeptide d-Ala-d-Ala in the peptidoglycan. Although a number of antimicrobial peptides have been shown to be active against MRSA and VRE. Here we focus on antibacterial peptides with unusual amino acids, which are known as lantibiotics, because many of them exert antibacterial action through the interaction with cell wall components.
Lantibiotics are ribosomally-synthesized and post-translationally modified peptides that contain an intramolecular ring structure. These compounds are produced by Gram-positive bacteria and exert potent inhibitory action against a wide-spectrum of bacteria. These compounds are classified as either type-A or type-B, and damage the bacterial membrane and inhibit the production of enzymes, respectively . Type-A lantibiotics include nisin, subtilin, epidermin, and Pep5, while type-B include mersacidin and cinnamycin. The most well-known lantibiotic is nisin, which was isolated from Lactococcus latis and is used as a food preservative worldwide. It was initially shown that nisin forms complexes with lipid I and lipid II, and then inhibits cell wall biosynthesis.

2.3. Alteration of Membrane Potential or Induction of Membrane Permeabilization 

Two major mechanisms of multidrug-resistance are phenotypic alteration of microbes under specific growth conditions, such as biofilms, and reduction of drug accumulation into microbes through limited uptake or pumping out drugs by multidrug-resistant proteins (MDRPs). Mode of action of antimicrobial peptides in the cytoplasmic membrane is considered to be more important than other targets.

Furthermore, antimicrobial peptides must permeate the cell wall and cytoplasmic membrane to reach their intracellular targets, which are nucleic acids and functional proteins. Although the exact mechanisms of antimicrobial peptides are not fully understood, they are known to cause the efflux of intracellular materials by- 

• disrupting the cytoplasmic membrane via either pore formation through a barrel-stave, or 

• a toroidal pore mechanism, or

 • through a nonporous carpet-like mechanism

2.4. Inhibition of Cytoplasmic Proteins Related to Cell Division or Survival Although most antimicrobial peptides primarily contribute to membrane perturbation, some antimicrobial peptides can penetrate the bacterial cytosol through a flip-flop mechanism or outer membrane protein forming channel. Among these, proline-rich antibacterial peptides such as pyrrhocoricin, apidaecin and drosocin have been shown to kill bacterial species by binding to the multi-helical lid region of the bacterial DnaK heat shock protein, which plays an essential role in the initiation of chromosomal DNA replication in an ATP-dependent manner with the other protein, DnaJ. The C-terminus of pyrrhocoricin was allowed to penetrate into cytosol of bacteria and the N-terminus responded to inhibit the ATPase activity of DnaK protein. Microcin B17, which is ribosomally synthesized antimicrobial peptides from Enterobacteriaceae, is also believed to inhibit DNA replication by targeting DNA gyrase.

2.5. Inhibition of Macromolecular Synthesis through Interaction with Nucleic Acids

It has been suggested that inhibition of intracellular processes by certain antimicrobial peptides that penetrate bacterial cells, such as buforin II PR-39 indolicidin, and tPMP , may contribute to inhibition of the growth of bacterial cells or lead to cell death. Chloe et al found that buforin II, a 21-amino acid peptide derived from the Asian toad, Bufo bufo gargarizans, kills bacteria through interaction with nucleic acids without membrane permeabilization, although further investigation is needed to identify other interactions with as yet unidentified intracellular targets. PR-39, which was isolated from the small intestine of the pig, required a lag period of about 8 min to penetrate the outer membrane, after which it rapidly killed growing E. coli cells via a mechanism that stops protein and DNA synthesis. In the case of indolicidin, although it induced permeabilization of the bacterial membrane, it did not lyse the bacterial cells. Its lethal concentration allowed their filamentous morphology by inhibition of DNA synthesis in E. coli cells, and it was also found to bind specifically to DNA rather than RNA.

3. Synergetic Effects between Antimicrobial Peptides and Clinically used Antibiotics 

The combined administration of antibiotics has gained interest because it often results in a synergistic antibacterial effect, which enables the dose of the individual drugs to be reduced. In addition, certain combination therapies have prevented the development of drug-resistance in bacteria. A membranolytic action of antimicrobial peptide is expected to produce synergetic effects when administered in combination with conventional antibiotics, and several studies have reported such findings.

5. Clinical Development of Antimicrobial Peptides

Several antimicrobial peptides are being evaluated in preclinical and clinical trials with limited applications. For example, Omeganan/MX-226, which is an indolicidin analogue, has recently completed phase III trials the prevention of catheter-related local and bloodstream infection, but was dropped for development. Additionally, pexiganan/MSI-78 has completed phase III clinical trials in the prevention of diabetic foot ulcers and plectasin is a fungal defensin peptide that exerts bactericidal action against drug-resistant bacteria and is currently in the preclinical phase. Opebacan, which is a human bactericidal/permeability-increasing protein derivative, has reached the phase II clinical trial for endotoxemia in hematopoetic stem cell transplant recipients. Iseganan/IB-367 from pig protegrin-1 has failed in the prevention of oral mucositis because it did not have a comparative advantage to existing therapeutics. Although several antimicrobial peptides are progressing to commercial development, records of clinical trials for antimicrobial peptides have been restricted to topical applications.

8. Conclusions 

Antimicrobial peptides can be the next generation of antibiotics for combating multi-drug resistant. These peptides have a strong potential for application as nanofilms or other coating materials for surgical devices, including catheters. Even though there are drawbacks to the use of peptides as therapeutics, such as low bioavailability and high cost, these obstacles may be overcome since a great deal of effort is being conducted to circumvent the problems associated with various methods including the use of d- or unnatural amino acid, formulation, recombinant DNA expression of peptides, addition of fatty acyl chains to short peptides. Therefore, it is expected that antimicrobial peptides will become the drugs of choice for emerging bacterial infections in the future.