*4.1.2 Mechanisms of resistance to bacteriocins*

The appearance of resistance is always a concern that may be developed as a result of changes in the membrane composition/structure. In this regard, resistance to nisin has been reported in specific strains of *Clostridium* and *Listeria* [54–56]. Moreover, exposure of the bacteriocins microcin-24 and nisin to *Salmonella enterica* and *Streptococcus bovis*, respectively, showed that the resistant cells had also resistance to other antibiotics [57, 58].

Resistance mechanisms have been mainly identified with bacteriocins targeting the cell envelope. In this regard, studies have shown that a decrease in the receptor of the bacteriocin targeting the lipid II conferred resistance to *Staphylococcus aureus* [46] and a regulation of the ABC transporter in *Listeria monocytogenes* [59]. However, mutations on genes encoding the RNA polymerase subunit and the gyrase have been observed [60–62].

In conclusion, resistance to bacteriocins has already been reported and potential solutions should be taken into consideration to reduce the appearance of such resistance. These include the derivatization of the original molecule to synthesize new molecules that may bind the receptors to reduce their recognition by the bacteria [63]. Alternatively, the use of a cocktail of bacteriocins in combination with other antibacterial agents should be evaluated as well.

### *4.1.3 Bacteriocin delivery*

One attractive system for the delivery of bacteriocins is the use of *Lactobacillus* strains. For example, growth inhibition of the pathogens *Listeria monocytogenes* and enterohemorrhagic *Escherichia coli* in a mouse model has been reported using *Lactobacillus casei* str. LAFTI L26 [64, 65]. Interestingly, the use of bacteriocins was successful to control buccal pathogens by using an engineered *Streptococcus mutans* strain, which produces the bacteriocin mutacin 1140. This bacteriocin was able to control plaque formation [66] and the engineered strain was retained in the buccal microbiome for 14 years after the application [67].

### **4.2 Antimicrobial peptides**

Small antimicrobial peptides are produced by probably every organism to cope with bacterial invasion. Antimicrobial peptides are short peptides with a molecular mass of 1000–5000 Da. Analysis of their sequences revealed that they interact with the negatively charged of bacterial membranes based on their net positive charge [68]. Further analysis of antibacterial peptides revealed that in their sequences a hydrophobic sequence is required to bind to the bacterial membrane as well as a conformation change to intercalate in the membrane.

Structural analysis of the peptides showed that they may acquire different 3D-conformations such as helices, sheets, or loops [69]. The structure of the peptides is very important because a redesign of the secondary structures of the peptides may increase their antibacterial activities or their stability being more resistant to the activity of proteases [70–73].

## *4.2.1 Mechanism of action of antibacterial peptides*

It appears that the main mechanism of action of antibacterial peptides is permeabilization. Therefore, they depend on the interaction with the cell membrane. This interaction involves an electrostatic interaction when the cationic peptide

binds the negatively charged outer bacterial envelope. The negative charge on the cell membrane is the result of phosphate or lipoteichoic groups present in the lipopolysaccharides or surface of Gram-negative and Gram-positive bacteria, respectively. Once the electrostatic interaction occurs, hydrophobic interactions allow the insertion of them into the outer membrane structure in Gram-negative strains. Then, a translocation may occur led by an unknown mechanism, which can be the formation of a transient channel, dissolution of the membrane, or translocation across the membrane [74].

Antibacterial peptides act in different targets such as the inhibition of nucleic acid and protein syntheses, enzymatic activity, and cell wall synthesis [75]. For example, buforin II (isolated from a frog) crosses the bacterial membrane by penetration, binding both DNA and RNA molecules in the cytoplasm of *Escherichia coli* [76]. Likewise, other peptides inhibit DNA and RNA synthesis without destabilizing the bacterial membrane [77–79] and protein synthesis [78, 79]. Other inhibitions including the enzymatic activity of pyrrhocidin that inhibits the activity of the heat shock protein DnaK (ATPase activity for a correct folding) and the transglycosylation of lipid II for peptidoglycan synthesis have been reported [80–82].

#### *4.2.2 Mechanisms of resistance to antibacterial peptides*

As similar to bacteriocins, the development of resistance against antimicrobial peptides has been shown. This resistance is apparently associated but studies have shown that certain genes can confer increased resistance to antimicrobial peptides, such as the gene *rcp* in *Legionella pneumophila* [83, 84]. Other resistance mechanisms have not been yet elucidated as well whether or not this resistance is transferred between bacteria.

Antimicrobial peptides, such as gallinacins, have been isolated from leukocytes in chicken and showed antimicrobial activity against *Listeria monocytogenes, Escherichia coli*, and the yeast *Candida albicans* [85]. Other antimicrobial peptides were isolated from turkey and showed activity against *Staphylococcus aureus* and *Escherichia coli* [86].

The use of antimicrobial peptides faces stability issues. As a result of their proteinaceous nature, they are subjected to degradation by proteolytic enzymes highly abundant in the body. Although antimicrobial peptides are also produced by the immune system, they do not face any vulnerability as their activity is very close to the production site. Thus, a potential use of these antimicrobials should address the proteolysis issue perhaps designing more resistant peptides, including chemical modification as well as an encapsulation to protect them or to develop a system of slow release. Other alternatives of delivery have been proposed, such as their production in genetic-modified plants, which can be used as an animal feed [87].

#### **4.3 Bacteriophages**

Bacteriophages or phages are viruses that infect and multiply in bacteria. As mentioned earlier, viruses infecting cells can be released into the environment by a process of bacterial cell destruction or lysis.

Phages are attractive for therapy because of their specificity of interaction with only a specific strain of bacteria. The interaction of phages with their hosts is based on the identification of specific binding sites, rendering strains without these receptors unaffected. On the other hand, this host specificity may signify a challenge for phage therapy. For example, lytic phages able to infect all *Salmonella* serovars (same species but with differences in the surface antigens) have not been yet discovered.

**11**

*DOI: http://dx.doi.org/10.5772/intechopen.83691*

isolates in chicken and fresh-cut fruits [88–91].

but not in apple slices with a pH of 4.2 [89].

resistance in bacteria, its potential use has reemerged.

To overcome this problem, a mixture of phages will be necessary to cover the most common infections caused by the same pathogen. For example, studies have reported that the use of a cocktail of lytic phages was effective to control *Salmonella*

Trials using phage therapy were not enthusiastic and the attractiveness for this therapy has decreased in the past. However, with the appearance of antibiotic

Therapeutic phages also face issues related to their interaction with the target bacteria. The introduction of the viral genetic material can cause undesired changes in the bacterial strain. For example, some phages may integrate into the bacterial chromosome, which may introduce new characteristics or modifies the expression of host genetic characteristic. These characteristics may include effects on the secretion of bacterial virulence factors, such as toxins, or antibiotic-resistant genes [92–95]. Taking together, it is desired that phages will enter in a lytic cycle to destroy their bacterial host rather than to be incorporated into the bacterial chromosome. In this way, cell lysis is preferred in phage therapy because of the destruction of the host, reducing the chances for viral interactions into the bacterial

It seems that future phage therapy will focus principally in the digestive and respiratory tracts with little possibilities to be used as a systemic therapy. In blood, phages will be exposed to circulating antibodies, which will clear the phage from the blood circulation. However, in the digestive tract, phages are subjected to adverse factors such as pH changes, which might change their antimicrobial activity. For example, the load of *Salmonella enteritidis* was reduced on contaminated melons,

Safety concerns have been also elevated in the production of phages for phage therapy. For example, phages should be produced in live microorganisms and then their production is limited to their pathogen hosts. In this regard, phages can carry genetic material from the host that, in this case, is the pathogen and transmit it to other bacteria. It seems that this scenario is not a frequent event, but it will be desirable to produce the phages in a nonvirulent pathogen to reduce this likelihood. In some application, the use of the enzyme responsible for the lysis of the host may suffice to control the pathogen [96], but it may be limited to topical applications or mucosal infections to avoid its travel through the digestive tract with little possibili-

Although disadvantages related to phage therapy have been discussed above, it is still considered a natural alternative to control infections in humans [97, 98]. Its use is supported by studies that showed protecting effects in different animal models. For example, intramuscular injection of phages protected mice infected with *Escherichia coli* O18:K1:H7, and a reduction in the enteropathogenic *Escherichia coli* strain was measured in the digestive tract of infected calves, piglets, and lambs treated with phage therapy [99, 100]. Similar studies showed the effectiveness of phage therapy when mice were infected with a vancomycin-resistant *Enterococcus* 

An alternative approach based on a genetically engineered phage to deliver genetic material into the bacteria has been reported. The approach is based on the use of lysogenic (nonlytic) phage to deliver the genetic material, which encodes

The use of nanoparticles (NPs) to control bacterial diseases has shown promising results. Over the last decade, NPs mainly synthesized from Ag, Au, Zn, and Cu

proteins with bactericidal activity, such as toxins [102].

*A New Era without Antibiotics*

chromosome.

ties to survive.

*faecium* infection [101].

**4.4 Nanoparticles**

### *DOI: http://dx.doi.org/10.5772/intechopen.83691 A New Era without Antibiotics*

*Technology, Science and Culture - A Global Vision*

tion across the membrane [74].

transferred between bacteria.

*Escherichia coli* [86].

**4.3 Bacteriophages**

process of bacterial cell destruction or lysis.

binds the negatively charged outer bacterial envelope. The negative charge on the cell membrane is the result of phosphate or lipoteichoic groups present in the lipopolysaccharides or surface of Gram-negative and Gram-positive bacteria, respectively. Once the electrostatic interaction occurs, hydrophobic interactions allow the insertion of them into the outer membrane structure in Gram-negative strains. Then, a translocation may occur led by an unknown mechanism, which can be the formation of a transient channel, dissolution of the membrane, or transloca-

Antibacterial peptides act in different targets such as the inhibition of nucleic acid and protein syntheses, enzymatic activity, and cell wall synthesis [75]. For example, buforin II (isolated from a frog) crosses the bacterial membrane by penetration, binding both DNA and RNA molecules in the cytoplasm of *Escherichia coli* [76]. Likewise, other peptides inhibit DNA and RNA synthesis without destabilizing the bacterial membrane [77–79] and protein synthesis [78, 79]. Other inhibitions including the enzymatic activity of pyrrhocidin that inhibits the activity of the heat shock protein DnaK (ATPase activity for a correct folding) and the transglyco-

As similar to bacteriocins, the development of resistance against antimicrobial

sylation of lipid II for peptidoglycan synthesis have been reported [80–82].

peptides has been shown. This resistance is apparently associated but studies have shown that certain genes can confer increased resistance to antimicrobial peptides, such as the gene *rcp* in *Legionella pneumophila* [83, 84]. Other resistance mechanisms have not been yet elucidated as well whether or not this resistance is

Antimicrobial peptides, such as gallinacins, have been isolated from leukocytes in chicken and showed antimicrobial activity against *Listeria monocytogenes, Escherichia coli*, and the yeast *Candida albicans* [85]. Other antimicrobial peptides were isolated from turkey and showed activity against *Staphylococcus aureus* and

The use of antimicrobial peptides faces stability issues. As a result of their proteinaceous nature, they are subjected to degradation by proteolytic enzymes highly abundant in the body. Although antimicrobial peptides are also produced by the immune system, they do not face any vulnerability as their activity is very close to the production site. Thus, a potential use of these antimicrobials should address the proteolysis issue perhaps designing more resistant peptides, including chemical modification as well as an encapsulation to protect them or to develop a system of slow release. Other alternatives of delivery have been proposed, such as their production in genetic-modified plants, which can be used as an animal feed [87].

Bacteriophages or phages are viruses that infect and multiply in bacteria. As mentioned earlier, viruses infecting cells can be released into the environment by a

Phages are attractive for therapy because of their specificity of interaction with only a specific strain of bacteria. The interaction of phages with their hosts is based on the identification of specific binding sites, rendering strains without these receptors unaffected. On the other hand, this host specificity may signify a challenge for phage therapy. For example, lytic phages able to infect all *Salmonella* serovars (same species but with differences in the surface antigens) have not been

*4.2.2 Mechanisms of resistance to antibacterial peptides*

**10**

yet discovered.

To overcome this problem, a mixture of phages will be necessary to cover the most common infections caused by the same pathogen. For example, studies have reported that the use of a cocktail of lytic phages was effective to control *Salmonella* isolates in chicken and fresh-cut fruits [88–91].

Trials using phage therapy were not enthusiastic and the attractiveness for this therapy has decreased in the past. However, with the appearance of antibiotic resistance in bacteria, its potential use has reemerged.

Therapeutic phages also face issues related to their interaction with the target bacteria. The introduction of the viral genetic material can cause undesired changes in the bacterial strain. For example, some phages may integrate into the bacterial chromosome, which may introduce new characteristics or modifies the expression of host genetic characteristic. These characteristics may include effects on the secretion of bacterial virulence factors, such as toxins, or antibiotic-resistant genes [92–95]. Taking together, it is desired that phages will enter in a lytic cycle to destroy their bacterial host rather than to be incorporated into the bacterial chromosome. In this way, cell lysis is preferred in phage therapy because of the destruction of the host, reducing the chances for viral interactions into the bacterial chromosome.

It seems that future phage therapy will focus principally in the digestive and respiratory tracts with little possibilities to be used as a systemic therapy. In blood, phages will be exposed to circulating antibodies, which will clear the phage from the blood circulation. However, in the digestive tract, phages are subjected to adverse factors such as pH changes, which might change their antimicrobial activity. For example, the load of *Salmonella enteritidis* was reduced on contaminated melons, but not in apple slices with a pH of 4.2 [89].

Safety concerns have been also elevated in the production of phages for phage therapy. For example, phages should be produced in live microorganisms and then their production is limited to their pathogen hosts. In this regard, phages can carry genetic material from the host that, in this case, is the pathogen and transmit it to other bacteria. It seems that this scenario is not a frequent event, but it will be desirable to produce the phages in a nonvirulent pathogen to reduce this likelihood. In some application, the use of the enzyme responsible for the lysis of the host may suffice to control the pathogen [96], but it may be limited to topical applications or mucosal infections to avoid its travel through the digestive tract with little possibilities to survive.

Although disadvantages related to phage therapy have been discussed above, it is still considered a natural alternative to control infections in humans [97, 98]. Its use is supported by studies that showed protecting effects in different animal models. For example, intramuscular injection of phages protected mice infected with *Escherichia coli* O18:K1:H7, and a reduction in the enteropathogenic *Escherichia coli* strain was measured in the digestive tract of infected calves, piglets, and lambs treated with phage therapy [99, 100]. Similar studies showed the effectiveness of phage therapy when mice were infected with a vancomycin-resistant *Enterococcus faecium* infection [101].

An alternative approach based on a genetically engineered phage to deliver genetic material into the bacteria has been reported. The approach is based on the use of lysogenic (nonlytic) phage to deliver the genetic material, which encodes proteins with bactericidal activity, such as toxins [102].

#### **4.4 Nanoparticles**

The use of nanoparticles (NPs) to control bacterial diseases has shown promising results. Over the last decade, NPs mainly synthesized from Ag, Au, Zn, and Cu have been tested as a potential antibacterial agent. AgNPs are the most studied NPs probably because of the long use of Ag in medicine already described in the ancient literature by Hippocrates of Kos (c.460-c.370 BC). As a result of the enormous amount of papers published regarding AgNPs as antibacterial agents, this section will focus only on these NPs.

NPs possess a range between 1 and 100 nm and have different physicochemical characteristics compared to the bulk material. One of their characteristics is the large surface area compared to their volume, making them very reactive.

During the process of AgNP synthesis, Ag ion (Ag<sup>+</sup> ) is reduced to Ag0 by using chemical reductants. However, over the last years, a more friendly technology using plant extracts has been proposed to diminish the toxicity problems linked to classical chemical synthesis [103, 104].

Physical characterization of the AgNPs revealed that the shape and size are important parameters with a profound effect in their antibacterial activity. For example, maximal activity was achieved when the size of the AgNPs is <40 nm and the highest activity was measured when an elongated or spherical shape was attained [2, 105–107].

#### *4.4.1 Activity mechanisms of AgNPs*

The antibacterial activity of AgNPs appears to be based on different mechanisms. It is not completely clear whether AgNPs internalize into the bacterial cell or as a result of their activity the membrane ruptures allowing their internalization [2, 108]. Many studies indicated that the adsorption of the NPs on the extracellular portion of the bacteria is the main mechanism of toxicity [105]. As a result of the adsorption, a depolarization of the cell wall ensues and the cell becomes more permeable, leading to cell death [109, 110]. Other studies have reported that AgNPs aggregate on the bacterial cell wall, causing a cell envelope disruption [105, 111, 112] with interactions with different functional groups, such as carboxyl, amino, and phosphate groups, leading to Ag precipitation [113].

**13**

*DOI: http://dx.doi.org/10.5772/intechopen.83691*

including lipid peroxidation [115].

depicted in **Figure 4**.

**5. Conclusions**

**Figure 4.**

*cysteines in proteins [2].*

the amino acid cysteine has a high affinity for Ag+

society to be aware of the misuse of antibiotics.

Another mechanism of bacterial toxicity is the generation of reactive oxygen species (ROS) by the AgNPs. ROS (free radicals, superoxides, and peroxides) are generated in any cell as a result of metabolic reaction (**Figure 3**); however, cells have different systems to cope with the toxicity of these ROS. The production of ROS, either intracellular or extracellular, may lead to membrane disruption [114],

*A model showing the toxicity of AgNPs in Escherichia coli. (A) Disruption and disintegration of the membrane/cell wall. (B) AgNPs access the periplasmic space gaining entrance to the cytosol where they interact with (C) DNA, and (E) ribosomes (protein synthesis impaired), generating (F) ROS and (G) binding to* 

Other toxicity mechanisms are related to the inhibition of the bacterial respiration [116–118], and protein and thiol binding [109, 114, 119]. It is noteworthy that

the proper folding of proteins and also is involved in the catalytic activity of many enzymes. Then, AgNPs target a diversity of enzymes at once with detrimental effects on the bacterial cell [119, 120]. A model of AgNP toxicity in *Escherichia coli* is

The continued misuse of antibiotics, as well as other factors, has accelerated the appearance of bacteria showing multidrug resistance. The problem is aggravated by a lack of new antibiotics introduced by the pharmaceutical companies. Both situations have incurred in a dangerous position to humanity, which will need to cope with a lack of antibiotics to combat diseases in a short term. To overcome this problem, the development of new antibacterial agents has ensued. It is of great importance that everyone in our society will take responsibility in reducing the burden of diseases; including regulatory agencies by accelerating the process of approvals, governmental agencies to provide incentives to pharmaceutical companies to continue with the development of new antibacterial agents, agricultural extension to educate the farmers for a wise use of antibiotics, and everyone in

and has an important role in

*A New Era without Antibiotics*

**Figure 3.** *Production of ROS and their activity on AgNPs [2].*

#### **Figure 4.**

*Technology, Science and Culture - A Global Vision*

will focus only on these NPs.

cal chemical synthesis [103, 104].

*4.4.1 Activity mechanisms of AgNPs*

attained [2, 105–107].

have been tested as a potential antibacterial agent. AgNPs are the most studied NPs probably because of the long use of Ag in medicine already described in the ancient literature by Hippocrates of Kos (c.460-c.370 BC). As a result of the enormous amount of papers published regarding AgNPs as antibacterial agents, this section

NPs possess a range between 1 and 100 nm and have different physicochemical characteristics compared to the bulk material. One of their characteristics is the

chemical reductants. However, over the last years, a more friendly technology using plant extracts has been proposed to diminish the toxicity problems linked to classi-

Physical characterization of the AgNPs revealed that the shape and size are important parameters with a profound effect in their antibacterial activity. For example, maximal activity was achieved when the size of the AgNPs is <40 nm and the highest activity was measured when an elongated or spherical shape was

The antibacterial activity of AgNPs appears to be based on different mechanisms. It is not completely clear whether AgNPs internalize into the bacterial cell or as a result of their activity the membrane ruptures allowing their internalization [2, 108]. Many studies indicated that the adsorption of the NPs on the extracellular portion of the bacteria is the main mechanism of toxicity [105]. As a result of the adsorption, a depolarization of the cell wall ensues and the cell becomes more permeable, leading to cell death [109, 110]. Other studies have reported that AgNPs aggregate on the bacterial cell wall, causing a cell envelope disruption [105, 111, 112] with interactions with different functional groups, such as carboxyl, amino, and

) is reduced to Ag0

by using

large surface area compared to their volume, making them very reactive.

During the process of AgNP synthesis, Ag ion (Ag<sup>+</sup>

phosphate groups, leading to Ag precipitation [113].

**12**

**Figure 3.**

*Production of ROS and their activity on AgNPs [2].*

*A model showing the toxicity of AgNPs in Escherichia coli. (A) Disruption and disintegration of the membrane/cell wall. (B) AgNPs access the periplasmic space gaining entrance to the cytosol where they interact with (C) DNA, and (E) ribosomes (protein synthesis impaired), generating (F) ROS and (G) binding to cysteines in proteins [2].*

Another mechanism of bacterial toxicity is the generation of reactive oxygen species (ROS) by the AgNPs. ROS (free radicals, superoxides, and peroxides) are generated in any cell as a result of metabolic reaction (**Figure 3**); however, cells have different systems to cope with the toxicity of these ROS. The production of ROS, either intracellular or extracellular, may lead to membrane disruption [114], including lipid peroxidation [115].

Other toxicity mechanisms are related to the inhibition of the bacterial respiration [116–118], and protein and thiol binding [109, 114, 119]. It is noteworthy that the amino acid cysteine has a high affinity for Ag+ and has an important role in the proper folding of proteins and also is involved in the catalytic activity of many enzymes. Then, AgNPs target a diversity of enzymes at once with detrimental effects on the bacterial cell [119, 120]. A model of AgNP toxicity in *Escherichia coli* is depicted in **Figure 4**.

## **5. Conclusions**

The continued misuse of antibiotics, as well as other factors, has accelerated the appearance of bacteria showing multidrug resistance. The problem is aggravated by a lack of new antibiotics introduced by the pharmaceutical companies. Both situations have incurred in a dangerous position to humanity, which will need to cope with a lack of antibiotics to combat diseases in a short term. To overcome this problem, the development of new antibacterial agents has ensued. It is of great importance that everyone in our society will take responsibility in reducing the burden of diseases; including regulatory agencies by accelerating the process of approvals, governmental agencies to provide incentives to pharmaceutical companies to continue with the development of new antibacterial agents, agricultural extension to educate the farmers for a wise use of antibiotics, and everyone in society to be aware of the misuse of antibiotics.

*Technology, Science and Culture - A Global Vision*
