Peptides, Enzymes, and Bacteriophages
Suzana Meira Ribeiro
Osmar Nascimento Silva
Bruna de Oliveira Costa
Octávio Luiz Franco
Resistance to anti-infective drugs is considered a worldwide threat.1 The ability to overcome the action of antimicrobial drugs can be achieved through conditions related to an individual bacterial cell (eg, change the antimicrobial bacteria target, antibiotic deactivation, block entrance, and efflux of the antibiotic) and/or may be related to bacterial life in the form of a community, known as biofilm.2,3 Bacteria in biofilm can show resistance to antimicrobial agents, even if individual bacteria within biofilm present susceptibility to antimicrobials. In this context, where there are specific microorganisms that are resistant to antibiotics, such as extended-spectrum β-lactamases (ESBL) and carbapenemases, biofilm formation can further amplify the resistance phenotype.4
Resistant microorganisms can impact the economy in different fields such as public health, agriculture, livestock, food industries, and water distribution systems.5,6 In public health, difficulty in combatting bacterial infections caused by resistant planktonic bacteria and biofilms can prolong the stay of patients in hospitals, leading to reduced quality of life and raising costs due to hospitalization.7 In agriculture, difficulties in bacterial control can increase the yield losses of plant and animal inputs.8,9 In the production chains of food industries, microbial biofilms, for example, can favor the contamination and consequent spoilage of foods.10 Some pathogens that contaminate food can synthetize toxins or cause infections in humans or animals.11 This microbial organization can also develop in systems of water distribution, affecting the water quality and contributing to the corrosion process of pipes.12 In addition, biofilms in water systems can serve as a contamination source of infectious bacteria.12,13
In order to minimize the issues occasioned by microorganisms in many of these scenarios, sterilization and disinfection techniques can be used.14 Sterilization allows the elimination of all forms of microorganism life, including spores.14 Disinfection consists of a process that eliminates most microorganisms, except their spores.15 The term disinfectant is commonly employed for chemical agents used on inanimate surfaces, whereas the term antiseptic is used for disinfectants applied on living tissues and skin.16 Antiseptics and disinfectants can minimize or prevent economic losses related to the presence of undesirable microorganisms in a particular context.
Many marketed disinfectant and antiseptic agents have become inefficient in combatting resistant microorganisms and their biofilms.17 A wide variety of proposals have been presented to overcome these concerns, such as peptides, other proteinaceous compounds (eg, enzymes), and bacteriophages.18,19,20 Peptides have been isolated from different classes of living organisms. They can be promiscuous in their activity, showing, for example, antimicrobial, antibiofilm, and immunomodulatory effects (Figure 26.1).21,22 These activities could be useful to disinfect a contaminated surface, such as skin, and additionally improve the immune response against invader pathogens.21,22 Some enzymes have shown efficiency in disinfecting medical devices and in removing biofilms in food.23,24 Bacteriophages (bacterial viruses) have also shown potential to be used in multiple situations in inanimate and live surfaces. Because their targets are bacterial cells, the advantage of bacteriophages are not only their lytic activity against bacterial cells but also their ability to replicate in bacteria and increase their dose, yet minimally interfering with the normal flora (when narrow-spectrum phages are used) and the absence of cross-resistance with normal flora.25
An interesting use of peptides, enzymes, and bacteriophages involves their application in biomolecular engineering.26,27,28 This approach allows antimicrobial compounds to be manufactured with increased antimicrobial properties and with nonexistent or minimal toxicity and/or potential to cause hazardous waste.26,27,28 Thus, in the era where antibiotics become a limited resource to combat harmful bacteria, the use of innovative approaches could prevent infections or issues caused by microorganisms
in industrial sectors, the environment, and agriculture. Peptides, enzymes, and bacteriophages are interesting strategies to combat bacteria. This chapter sheds light on their potential use for the prevention and eradication of unwanted microorganisms and discusses the biotechnological approaches to improve the use of these agents in different contexts.
in industrial sectors, the environment, and agriculture. Peptides, enzymes, and bacteriophages are interesting strategies to combat bacteria. This chapter sheds light on their potential use for the prevention and eradication of unwanted microorganisms and discusses the biotechnological approaches to improve the use of these agents in different contexts.
 FIGURE 26.1 Action of peptides and enzymes against planktonic bacteria and biofilms. |
PEPTIDES AND ENZYMES FOR DISINFECTION AND ANTISEPSIS OF SURFACESNumerous peptides and proteins, such as enzymes, with antimicrobial properties have been isolated from different classes of organisms.29 Alternatively, they can be used as a template for the development of compounds with improved antimicrobial and antibiofilm action.30,31 In this context, peptides and enzymes could be used as for disinfectant or antiseptic applications.
For disinfection, peptides have been used alone or in combination to eradicate planktonic microorganisms and their biofilms.27,28 Most studies have focused on the elimination of human or veterinary infectious disease. In dentistry, peptides are the potential active components in some products (eg, mouth rinse, root canal sealers, and composite resins) or can be promising candidates for the treatment of oral infections.18 This strategy can be exemplified by the synthetic antibiofilm peptide 1018, which in developed studies showed potent activity against oral multispecies biofilms, thus being a promising candidate as an antibiofilm agent that is nontoxic and effective for antisepsis of bacterial plaques in clinical dentistry.18 This peptide has also shown effectiveness in reducing biofilm on wounds22 and therefore could be used as potential antiseptic agent.
Many peptides have emerged as potential candidates for development as antimicrobial drugs, such as magainin, a potent peptide that reached to phase III clinical trials. Some peptides are already commercially exploited, such as with nisin and aprotinin.32,33 In food applications, nisin
is a peptide produced by the bacterium Lactococcus lactis and has been used as a preservative.28,32 Nisin can combat bacteria associated with food spoilage and human infections.28,32 Aprotinin, an antifibrinolytic molecule, has been primarily explored for use in surgery to reduce bleeding33; however, studies have shown its potential as a direct antimicrobial and as a protease inhibitor (inhibiting surface-related proteases that are important virulence factors in certain bacteria).34,35
is a peptide produced by the bacterium Lactococcus lactis and has been used as a preservative.28,32 Nisin can combat bacteria associated with food spoilage and human infections.28,32 Aprotinin, an antifibrinolytic molecule, has been primarily explored for use in surgery to reduce bleeding33; however, studies have shown its potential as a direct antimicrobial and as a protease inhibitor (inhibiting surface-related proteases that are important virulence factors in certain bacteria).34,35
Another approach in antisepsis is to combine peptides with other agents, such as commercial antiseptics.20 This approach is designed to prevent the emergence of resistant organisms, to expand the spectrum of target microorganisms, and to decrease toxicity, allowing the use of lower doses of both agents.36 For example, the combination of the synthetic peptide 1018 with the commercial antiseptic chlorhexidine increased the antibiofilm activity of each compound compared to when they were used alone to combat infections caused by oral multispecies biofilms.18
Peptide mechanisms of action against planktonic bacteria involves electrostatic interaction between peptides and bacterial membranes, which then leads to increased cell membrane permeability, loss of barrier function, and leakage of cytoplasmic components, culminating in cell death (see Figure 26.1).37 They also can prevent the cell-wall, protein, and nucleic acid synthesis (see Figure 26.1),30 whereas the antibiofilm action has been related to the interference with a universal compound, known as (p)ppGpp (guanosine tetra- and penta-phosphates) (see Figure 26.1). This molecule seems to play an important role in the response to stress in bacteria and consequently in the development of biofilm in multiple bacterial species.38
Enzymes have been commonly used in cleaning formulations, used as prerequisites to disinfection of reusable medical devices or surfaces.39 In particular, proteases have been employed to aid in the removal of bacterial biofilms from the various surfaces of medical devices, such as endoscopes, colonoscopes, and contact lenses. In the food processing industry, enzymatic detergents are also used to aid in the removals of foodborne pathogens40 that contaminate equipment surfaces and the infrastructure, including thawing rooms, conveyor belts, shredders, commercial kitchens, and packaging materials.41
The mechanisms of action of antibiofilm enzymes consist in the degradation of polysaccharides, DNA, and proteins that compose the biofilm matrix.42,43 They can also lead to cell lysis and affect cell signaling, thus interfering mechanisms associated with biofilm formation and maintenance.23 Lysostaphin (an endopeptidase), for example, is able to disrupt biofilms formed by Staphylococcus aureus that grows on plastic and glass surfaces.44 In addition, this enzyme has the ability to kill the bacterial cells present in biofilm.44,45 An advantage of lysostaphin is its antimicrobial activity against nondividing as well as dividing cells.46 The activity against nondividing cells could be explored against dormant cells in biofilms, a bacterial state resistant to antimicrobials.47 But overall, the antimicrobial activity is limited to certain staphylococci, particularly S aureus.
One potential enzyme that could be used as an antiseptic is α-amylase from Bacillus subtilis.48 This enzyme was able to inhibit the biofilm of methicillin-resistant S aureus (MRSA) and Pseudomonas aeruginosa, bacteria commonly associated with wound infections.49 Another strategy in this context would be the combination of enzymes with commercial antiseptic agents for a more effective antisepsis. In this situation, enzymes can degrade the biofilm matrix and thus facilitate the access of the antiseptics to the adhered bacteria in the living tissue or skin.42
A combination of enzymes and peptides also has been proved to be a potential strategy to inhibit pathogens. A combination of the antimicrobial peptide ranalexin with the enzyme lysostaphin reduced MRSA infection on human skin ex vivo to a greater extent than any of the individually tested compounds.20
As presented, there are many approaches to enable the disinfection of surfaces. Examples of antimicrobial and/or antibiofilm peptides and enzymes that have been used to combat pathogens are summarized in Table 26.1. These include various health and food industry applications for the use of these biomolecules.
BACTERIOPHAGES AND ANTIMICROBIAL APPROACHESBacteriophages, viruses that infect bacteria, are considered ubiquitous microorganisms. There are more than 6000 phages described that infect bacteria or archaea.50 These are classified based on their morphology, genetic content (DNA or RNA),51,52 habitats (eg, soil, marine, or human gut bacteriophages),53,54,55 particular hosts (eg, Klebsiella and Pseudomonas phages),56,57 and their life cycle (lytic or lysogenic).58 Because such organisms have the potential to destroy specific pathogenic bacteria and their biofilms,59 and self-replicate, causing minimal or no effect on bacterial microflora of treated organisms (eg, animals infected with pathogenic bacteria),60 they can potentially be used in different scenarios, such as clinical (medical and veterinary), industrial, and environmental (eg, treatment of contaminated water).59,61
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