**3. Biofilm inhibition by antimicrobial peptides**

Antimicrobial peptides (AMPs) are small molecules (10–100 amino acids) widespread in nature that play an essential role in the innate immunity. Recently, much attention has been paid to AMPs as they exert a broad spectrum of action, exploiting different activities as antibacterial, antifungal, antiparasites, anticancer, and antibiofilm factors [34]. This paragraph will focus on the ability of some antimicrobial peptides to inhibit biofilm formation.

The use of antimicrobial peptides to impair biofilm formation is attracting great interest, and many peptides have already been tested on different bacterial biofilms. In particular, the molecular mechanism of biofilm inhibition by AMPs is very much under investigation. The AMPs tested on biofilms so far derive from different natural sources, such as humans, mammals, bacteria, plants, and amphibians, but many synthetic peptides have also been studied. For example, it was demonstrated that the human cathelicidin LL-37 and indolicidin peptides could prevent biofilm formation of *P. aeruginosa* by downregulating the transcription of Las and Rhl, two quorum-sensing systems [35]. Moreover, AMPs could inhibit biofilm formation by increasing twitching motility in *P. aeruginosa* through the stimulation of the expression of genes needed for type IV pilli biosynthesis and function. Type IV pilli has the main function to increase bacteria movement on surfaces, which could facilitate cell removal [35]. The synthetic antimicrobial peptide meta-phenylene ethynylene (mPE), based on magainin, was active against biofilms of *Streptococcus mutans*, both as an intracellular antibiotic by binding to DNA and as a membrane-active molecule inhibiting lipopolysaccharides (LPSs), similar to magainin action [36].

In addition, the LL-37 peptide can also inhibit initial biofilm attachment. In *Pseudomonas aeruginosa*, this peptide downregulates the expression of genes associated with the assembly of flagella involved in the process of initial adherence [37]. Antiadhesion could be one of the major AMPs antibiofilm properties leading

**61**

(SasG) in *S. aureus*.

*Inhibition of Bacterial Biofilm Formation DOI: http://dx.doi.org/10.5772/intechopen.90614*

its N-terminus [39, 40].

to their potential use as an effective pretreatment strategy. For example, the nisin peptide, which interferes with cell wall synthesis and is capable to form membrane pores, delays biofilm formation, but it does not inhibit the *Staphylococcus aureus*

AMPs can also cause biofilm matrix disruption. The human liver-derived hepcidin 20 peptide can reduce the mass of extracellular matrix and can alter the *S. epidermidis* biofilm architecture by targeting polysaccharide intercellular adhesin (PIA). Being endowed with nucleosidase activity, the fish-derived piscidin-3 peptide can degrade *P. aeruginosa* extracellular DNA by coordinating with Cu2+ through

Although several antimicrobial peptides have nowadays been studied for the inhibition of biofilm formation, a further aspect needs to be considered. Several biofilms have developed defense mechanisms to protect themselves from antimicrobial agents. The interaction with EPS is thought to be the principal reason of biofilm resistance to AMPs even if the exact mechanism is not well understood. Gramnegative bacteria, such as *P. aeruginosa*, can secrete alginate, an anionic extracellular polysaccharide consisting of uronic acid D-mannuronate and C-5 epimer-L guluronate. Alginate can interact with cationic AMPs and protect *P. aeruginosa* biofilm from the effect of the antimicrobial peptides [41]. Moreover, the peptide sensing system known as aps, first recognized in *S. epidermidis*, can protect Grampositive bacteria from AMP action. This system upregulates the D-alanylation of teichoic acid and increases the expression of putative AMP efflux pumps. It was demonstrated that *Enterococcus faecalis* D-alanine deficient mutant is more resistant

growth when it is immobilized in multiwalled carbon nanotubes [38].

to AMPs than the wild type even if they produce less biofilm [42].

Planktonic bacteria can adhere to different cells or tissues starting biofilm formation via production of a multitude of proteins, which act at different stages of biofilm formation. Some proteins contribute to biofilm accumulation, while others are involved into the mediation of primary attachment to surfaces [43, 44]. For this reason, the formation and the development of bacterial biofilm can be associated with the production of specific proteins, which play essential roles in the bacterial biofilm formation and development. Strategies leading to the identification of these proteins are fundamental as they could represent interesting targets to inhibit biofilm formation, allowing the development of new antibiofilm agents and procedures [45]. In this paragraph, we will focus on some target proteins involved in the production of biofilms in different bacteria: the N-acetylneuraminate lyase (NanA) in *Escherichia coli*, the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate acetyltransferase (GlmU) in *Mycobacterium smegmatis*, and the surface protein G

The NanA protein of *E. coli* is an enzyme able to recognize the sialic acid, a molecule essential to a number of critical biological processes, such as cell recognition, adhesion, and immune system evasion. NanA catalyzes the transformation of sialic acid into pyruvate and N-acetyl-D-mannosammine [46, 47], favoring cell-cell adhesion. Therefore, NanA plays a fundamental role in the adhesion development of host cells a process of great importance in the formation of biofilm. This enzyme is then considered an important target for developing molecules able to reduce biofilm accumulation. Recently, a relationship between methylation stress in *E. coli* and the reduction of bacterial adhesion properties thus decreasing its ability to form biofilm was reported. This phenomenon was associated with a drastic reduction in the expression levels of the NanA protein, suggesting a possible role of NanA in

**4. Biofilm inhibition by protein targets**

### *Inhibition of Bacterial Biofilm Formation DOI: http://dx.doi.org/10.5772/intechopen.90614*

*Bacterial Biofilms*

bacterial specificity [33].

the molecular mechanisms underlying the QS effects on biofilms including the production of virulence factors may help to control bacterial infection. More than 70 species of Gram-negative bacteria communicate and control their population density and mobility via N-acyl homoserine lactones (AHLs) mediated QS and represented one of the primary scaffolds studied for the design of potential biofilm inhibitors [27]. N-butanoylhomoserine lactone 1 (C4-AHL, for the rhl system) and 3-oxo-C12-AHL 5 (for the las system) are among the most important AHLs involved in QS (REF Small molecule control of bacterial biofilms). In *P. aeruginosa*, one of the most important bacteria involved in human infections, different antibiofilm molecules focused on AHL analogues were designed to develop new strategies to impair biofilm formation. The Blackwell et al. identified, designed, and synthesized several different AHLs capable to significantly reduce biofilm

formation and virulence factor production in *P. aeruginosa* [28, 29].

**3. Biofilm inhibition by antimicrobial peptides**

antimicrobial peptides to inhibit biofilm formation.

A different approach consisted in the use of the synthetic halogenated furanone produced by secondary metabolism of the Australian macroalga *Delisea pulchra*, which is able to penetrate the biofilm matrix and to alter its architecture in flow chambers [30, 31]. Furthermore, T315, an integrin-linked kinase inhibitor previously identified as a potential therapeutic agent against chronic lymphocytic leukemia [32], was shown to selectively inhibit biofilm formation in both *Salmonella typhi* and *Salmonella Typhimurium* at early stages of biofilm development without affecting bacterial viability. T315 was also demonstrated to reduce biofilm formation in *Acinetobacter baumannii* but had no effect on *P. aeruginosa* suggesting a

Antimicrobial peptides (AMPs) are small molecules (10–100 amino acids) widespread in nature that play an essential role in the innate immunity. Recently, much attention has been paid to AMPs as they exert a broad spectrum of action, exploiting different activities as antibacterial, antifungal, antiparasites, anticancer, and antibiofilm factors [34]. This paragraph will focus on the ability of some

The use of antimicrobial peptides to impair biofilm formation is attracting great interest, and many peptides have already been tested on different bacterial biofilms. In particular, the molecular mechanism of biofilm inhibition by AMPs is very much under investigation. The AMPs tested on biofilms so far derive from different natural sources, such as humans, mammals, bacteria, plants, and amphibians, but many synthetic peptides have also been studied. For example, it was demonstrated that the human cathelicidin LL-37 and indolicidin peptides could prevent biofilm formation of *P. aeruginosa* by downregulating the transcription of Las and Rhl, two quorum-sensing systems [35]. Moreover, AMPs could inhibit biofilm formation by increasing twitching motility in *P. aeruginosa* through the stimulation of the expression of genes needed for type IV pilli biosynthesis and function. Type IV pilli has the main function to increase bacteria movement on surfaces, which could facilitate cell removal [35]. The synthetic antimicrobial peptide meta-phenylene ethynylene (mPE), based on magainin, was active against biofilms of *Streptococcus mutans*, both as an intracellular antibiotic by binding to DNA and as a membrane-active molecule

inhibiting lipopolysaccharides (LPSs), similar to magainin action [36].

In addition, the LL-37 peptide can also inhibit initial biofilm attachment. In *Pseudomonas aeruginosa*, this peptide downregulates the expression of genes associated with the assembly of flagella involved in the process of initial adherence [37]. Antiadhesion could be one of the major AMPs antibiofilm properties leading

**60**

to their potential use as an effective pretreatment strategy. For example, the nisin peptide, which interferes with cell wall synthesis and is capable to form membrane pores, delays biofilm formation, but it does not inhibit the *Staphylococcus aureus* growth when it is immobilized in multiwalled carbon nanotubes [38].

AMPs can also cause biofilm matrix disruption. The human liver-derived hepcidin 20 peptide can reduce the mass of extracellular matrix and can alter the *S. epidermidis* biofilm architecture by targeting polysaccharide intercellular adhesin (PIA). Being endowed with nucleosidase activity, the fish-derived piscidin-3 peptide can degrade *P. aeruginosa* extracellular DNA by coordinating with Cu2+ through its N-terminus [39, 40].

Although several antimicrobial peptides have nowadays been studied for the inhibition of biofilm formation, a further aspect needs to be considered. Several biofilms have developed defense mechanisms to protect themselves from antimicrobial agents. The interaction with EPS is thought to be the principal reason of biofilm resistance to AMPs even if the exact mechanism is not well understood. Gramnegative bacteria, such as *P. aeruginosa*, can secrete alginate, an anionic extracellular polysaccharide consisting of uronic acid D-mannuronate and C-5 epimer-L guluronate. Alginate can interact with cationic AMPs and protect *P. aeruginosa* biofilm from the effect of the antimicrobial peptides [41]. Moreover, the peptide sensing system known as aps, first recognized in *S. epidermidis*, can protect Grampositive bacteria from AMP action. This system upregulates the D-alanylation of teichoic acid and increases the expression of putative AMP efflux pumps. It was demonstrated that *Enterococcus faecalis* D-alanine deficient mutant is more resistant to AMPs than the wild type even if they produce less biofilm [42].

### **4. Biofilm inhibition by protein targets**

Planktonic bacteria can adhere to different cells or tissues starting biofilm formation via production of a multitude of proteins, which act at different stages of biofilm formation. Some proteins contribute to biofilm accumulation, while others are involved into the mediation of primary attachment to surfaces [43, 44]. For this reason, the formation and the development of bacterial biofilm can be associated with the production of specific proteins, which play essential roles in the bacterial biofilm formation and development. Strategies leading to the identification of these proteins are fundamental as they could represent interesting targets to inhibit biofilm formation, allowing the development of new antibiofilm agents and procedures [45]. In this paragraph, we will focus on some target proteins involved in the production of biofilms in different bacteria: the N-acetylneuraminate lyase (NanA) in *Escherichia coli*, the bifunctional enzyme N-acetyl-D-glucosamine-1-phosphate acetyltransferase (GlmU) in *Mycobacterium smegmatis*, and the surface protein G (SasG) in *S. aureus*.

The NanA protein of *E. coli* is an enzyme able to recognize the sialic acid, a molecule essential to a number of critical biological processes, such as cell recognition, adhesion, and immune system evasion. NanA catalyzes the transformation of sialic acid into pyruvate and N-acetyl-D-mannosammine [46, 47], favoring cell-cell adhesion. Therefore, NanA plays a fundamental role in the adhesion development of host cells a process of great importance in the formation of biofilm. This enzyme is then considered an important target for developing molecules able to reduce biofilm accumulation. Recently, a relationship between methylation stress in *E. coli* and the reduction of bacterial adhesion properties thus decreasing its ability to form biofilm was reported. This phenomenon was associated with a drastic reduction in the expression levels of the NanA protein, suggesting a possible role of NanA in

biofilm formation and bacteria host interactions. Using a null NanA mutant and DANA, a substrate analog acting as competitive inhibitor, it was demonstrated that the downregulation of NanA or inhibition of its enzymatic activity affects biofilm formation and adhesion properties of *E. coli* [48, 49].

*Inhibition of Bacterial Biofilm Formation DOI: http://dx.doi.org/10.5772/intechopen.90614*

**Acknowledgements**

**Conflict of interest**

**Author details**

Angela Di Somma1

and Angela Duilio1

approach from in silico to *in vivo*."

The authors declare no conflict of interest.

\*, Antonio Moretta<sup>2</sup>

1 Department of Chemical Sciences, Federico II University, Naples, Italy

2 Department of Science, University of Basilicata, Potenza, Italy

\*Address all correspondence to: angela.disomma@unina.it

The administration of a single antibiotic is often not enough to eradicate bacterial invasions, and a high concentration of the antibiotic can be extremely toxic. A possible solution might be the coadministration of antibiotics with antibiofilm peptides that allow the use of low antibiotic concentrations. This strategy can be tuned to affect biofilms without killing bacteria, thus avoiding the emergence of

drug-resistant populations through synergy with existing antibiotics.

This work was supported in part by MIUR grants ARS01\_00597 Project "NAOCON" and PRIN 2017 "Identification and characterization of novel antitumoral/antimicrobial insect-derived peptides: a multidisciplinary, integrated

**63**

provided the original work is properly cited.

3 CEINGE Biotecnologie Avanzate, Naples, Italy

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Carolina Canè3

, Arianna Cirillo3

Another important protein target is GlmU, a bifunctional enzyme with acetyltransferase activity involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a key precursor of β-1,6-N-acetyl-Dglucosamine polysaccharide adhesin required for biofilm formation [50, 51]. GlmU is a possible factor involved in biofilm production in *M. smegmatis*, a nonpathogenic bacterium homologous to the pathogenic *M. tuberculosis*. The response of *M. smegmatis* to alkylating stress is different from *E. coli*, resulting in an increase in biofilm formation possibly due to a very strong defense mechanism. In this contest, GlmU has an important role in the process of biofilm production in *M. smegmatis*, being its expression highly upregulated when the bacterium needs to activate defense mechanisms [52]. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that the downregulation of GlmU decreased *M. smegmatis* capabilities to produce biofilm, whereas the overexpression of enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the *M. smegmatis* defense mechanisms. Focusing on the inhibition of GlmU might then be an efficient method to disable the bacterium defense mechanism.

*S. aureus* is a common pathogen responsible for nosocomial and community infections being able to colonize the squamous epithelium of the anterior nares. One of the adhesins likely to be responsible for this ability is the *S. aureus* surface protein G (SasG), which promotes cellular aggregation leading to biofilm formation [53, 54]. SasG comprises an N-terminal A domain and repeated B domains with only the B domain required for the accumulation of biofilm. Expression of SasG does not increase the adherence of bacteria, and it is not involved in primary attachment but plays a role in the accumulation phase of biofilm formation [55]. For different aspects and playing different roles, NanA, GlmU, and SasG may all represent interesting targets to address the inhibition of biofilm production.

### **5. Conclusions**

Currently, biofilm infections constitute a serious medical problem, and their treatment is far from being satisfactory. Biofilm formation inhibitors have several potential therapeutic applications as coatings in medical devices or in the prophylaxis of implanted surgery. In this respect, the identification of new strategies to counteract biofilm formation is a broad subject of study. The antibiofilm activity of many molecules such as proteins, peptides, and small organic molecules is currently under investigation. Each of these molecules is endowed with specific characteristics and can exert its ability to inhibit bacterial biofilm formation with different mechanisms. Antibiofilm agents are able to act both at the initial stages of biofilm formation, such as bacterial adhesion to the host surface, and on preformed biofilm, leading to the disruption of the EPS architecture. Many small organic molecules are able to interfere with the bacterial QS system, but their lack of activity in *in vivo* models and the high toxicity make these molecules of limited use in clinical applications.

As antimicrobial peptides show a broad spectrum of action, exploiting different activities including antibiofilm capabilities, these molecules might be considered as new promising factors to impair biofilm formation that exploit different mechanisms to hamper biofilms at different stages.

*Inhibition of Bacterial Biofilm Formation DOI: http://dx.doi.org/10.5772/intechopen.90614*

The administration of a single antibiotic is often not enough to eradicate bacterial invasions, and a high concentration of the antibiotic can be extremely toxic. A possible solution might be the coadministration of antibiotics with antibiofilm peptides that allow the use of low antibiotic concentrations. This strategy can be tuned to affect biofilms without killing bacteria, thus avoiding the emergence of drug-resistant populations through synergy with existing antibiotics.
