Inhibition of Bacterial Biofilm Formation

*Angela Di Somma, Antonio Moretta, Carolina Canè, Arianna Cirillo and Angela Duilio*

### **Abstract**

Biofilm is a complex matrix consisting of extracellular polysaccharides, DNA, and proteins that protect bacteria from a variety of physical, chemical, and biological stresses allowing them to survive in hostile environments. Biofilm formation requires three different stages: cell attachment to a solid substrate, adhesion, and growth. The inhibition of one of these steps by small molecules, such as antimicrobial peptides, or their action on specific targets will leave pathogens armless against classical antibiotics. Any drug impairing crucial processes for bacterial life will inevitably lead to the development of drug-resistant strains, whereas the inhibition of biofilm formation might prevent the onset of bacterial resistance. In this section, we will focus on proteins involved in biofilm formation as useful targets for the development of new drugs that can effectively and specifically impair biofilm formation with slight effects on cell survival, thus avoiding the generation of drugresistant strains.

**Keywords:** bacterial biofilms, biofilm inhibition, antimicrobial peptides, protein target, mechanism of action

### **1. Introduction**

Microorganisms have the extraordinary ability to live in almost all environments and to protect themselves from external agents through sophisticated survival mechanisms. Bacteria can be found in planktonic form or in specific conditions, as sessile aggregates on both biotic and abiotic surfaces originating complex structures known as biofilm.

Biofilms are an ensemble of microbial cells irreversibly associated with a surface and enclosed in an essentially self-produced matrix. The biofilm matrix consists of polysaccharides, proteins, and DNA and constitutes a stubborn source that protects bacteria from a variety of physical, chemical, and biological stresses. One of its characteristics is the capability to impair antimicrobial molecules to spread through the polymer matrix or the ability of the matrix material to inactivate antibacterial molecules. Today, the increase and spread of antibiotic resistance among microorganisms (bacteria, fungi, viruses, and parasites) represent one of the greatest emergencies for human health worldwide [1]. Based on these characteristics, biofilm plays crucial roles in humans and nonhuman infections and represents the most important adaptive mechanism closely related to pathogenicity.

### *Bacterial Biofilms*

An antibiofilm agent must display several specific characteristics to target the biofilm lifestyle. First, due to the temporal biofilm heterogeneity, it must show a rapid killing ability to face a changing entity and to target cells before their entry into the biofilm community; it must be able to act in different environmental niches and to target different growth rate cells. The cells located in the periphery of biofilm are directly in contact with nutrients and oxygen, while those placed deepest in the biofilm layers may undergo lack of nutrients, anoxia, and acidic conditions. In this way, a metabolic and spatial heterogeneity is generated including both rapidly and slowly growing cells. In particular, due to environmental conditions, inside the biofilm, it is possible to find the so-called persister, dormant, quiescent cells characterized by a low rate of cell division that are believed to play an essential role in the biofilm resistance to antibiotics [2]. Other important characteristics for a good antimicrobial candidate are the ability to interfere with the production of the extracellular matrix and the possibility to penetrate the biofilm architecture. This matrix consists for 90% of EPS, whose principal components are proteins, polysaccharides, lipids, and extracellular DNA, and it is involved into the biofilm architecture maintenance. An antibiofilm agent should also be able to interfere with bacterial cell communication machinery.

This chapter aims to investigate and clarify in detail the inhibition of biofilm formation by different approaches.

Other additional aspects to consider the identification of potential antimicrobial agents are the ability to recruit immune cells and/or modulate the host immune response and the synergy with other conventional and unconventional antimicrobial compounds [3, 4].

Biofilms are very dynamic and spatially heterogeneous structures originating gradients of oxygen, nutrients, and pH, and their formation occurs through three phases: adhesion, maturation, and dispersal phase as described earlier.

## **2. Small molecules capable to inhibit biofilm formation**

The inhibition or prevention of biofilm formation has been a subject of study for a long time. The first important action against biofilm formation is to prevent bacterial adhesion to surfaces and host tissues to reduce infection [5]. Preventing bacterial adhesion is an attractive target [6] for hampering bacterial infection, and several different strategies have been proposed including hindering cellular receptors from recognizing adhesion surfaces or inhibiting the process of bacterial adhesion. Blocking the primary colonizers can prevent initial biofilm colonization and the subsequent infection produced by planktonic cells released from the biofilm itself.

The adhesion process consists of various distinct steps. In the first step, bacterial cell establishes reversible adhesion interactions on host surfaces [7], while in the second step, a stronger type of adhesion is carried out, which involves specific molecules that bind in a complementary manner [5]. In particular, in Gram-negative bacteria, adhesion is mediated by special proteins known as adhesins associated with cell surface structures such as fimbriae or pili [8, 9]. Initial adhesion is then followed by a complex colonization process that offers a number of advantages to bacteria, including increased protection against dislocation by hydrokinetic forces from fluid surfaces or better access to nutrients released by the host cells [10]. Finally, in these favorable conditions, the development of the elaborate biofilm structures can take place.

For a long time, the first strategies used to inhibit the adhesion process were focused on the use of adhesin analogues that bind to the receptor and competitively

**59**

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

applicable.

packaging [21].

ment of resistant strains.

block bacterial adhesion [5]. However, this strategy resulted unpractical because adhesin proteins are not readily available, and they become toxic at the relatively high concentrations that had to be used. An attempt to overcome this problem consisted in the design and use of synthetic peptides mimicking the sequence of cell surface adhesins. For example, the small peptide p1025 inhibits *Streptococcus mutans* binding to dental surfaces [11]. Analogously, a fragment of the fimbrillin adhesin was found to inhibit the adhesion of *Porphyromonas gingivalis* to hydroxyapatite [12]. However, this approach showed several drawbacks as different adhesins usually mediate the adhesion process and the expression of carbohydrates or cell surface ligands may vary depending on environmental conditions, originating a large number of variables and making this approach more difficult and not very

A novel and interesting approach to inhibit bacterial adhesion consists in the use of cell coatings with antimicrobial peptides that alter the chemical properties of the surface [13, 14], thus interfering with bacterial adhesion and preventing surface binding. Although "passive," this method is rather attractive and may serve as a novel approach to address the biofilm problem on artificial medical devices. However, limited successes have been achieved so far due to attachment variability among different bacterial strains. Recently, many active polymeric coatings were designed to bind the surface and release a variety of antimicrobial molecules such as antibiotics, bacteriocins, and metal ions [15–18]. A significant reduction in biofilm formation of *Staphylococcus epidermidis* on hydrogel-coated and serum/ hydrogel-coated silicone catheters was observed following the release of bacteriophagic factors from the polymer with and without supplemental divalent cations [19]. Similarly, treatment of piperacillin-tazobactam coated tympanostomy tubes reduces biofilm infection of ciprofloxacin-resistant *Pseudomonas aeruginosa* (CRPA) [20]. The negative aspect of this approach might be the continuous release of high concentration of antimicrobials in a short time by the active polymer often higher than the MIC values without a specific target. However, target release polymer can be foreseen as the new era of biofilm treatments in industrial food safety and

Recently, great attention was paid to a different approach addressed to killing planktonic cells for prevention and treatment of biofilms. The new catheter lock solution C/MB/P (citrate, methylene blue, and parabens) was able to act against planktonic and sessile bacteria within a biofilm preventing bacterial colonization of hemodialysis catheters [22]. Killing planktonic cells might represent a good approach, but this strategy cannot be carried out on long term because any drug targeting crucial processes for bacterial life will unavoidably lead to the develop-

An effective and positive control of biofilm formation might be obtained by interfering with specific cellular process crucial for biofilm formation. Biofilm formation is often associated with the phenomenon of quorum sensing (QS), in which bacterial cells communicate with each other by small diffusible signal molecules [23]. Moreover, bacterial gene expression has to be synchronized to form biofilms, and to achieve this goal, the quorum-sensing (QS) mechanism is used by bacteria, producing and responding to a several intra and intercellular signals called autoinducers [24]. At low-cell densities, the autoinducer is present in the extracellular media in a small amount that is too dilute to be detected. When the cell density increases, the autoinducer concentration reaches a threshold, and the autoinducer-receptor complex (the regulatory protein) acts to induce or repress the expression of target genes. The QS controls some physiological processes such as secretion of virulence factors, biofilm formation, and antibiotic resistance in several bacterial species [25, 26]. Investigation and elucidation of

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

*Bacterial Biofilms*

bacterial cell communication machinery.

formation by different approaches.

bial compounds [3, 4].

biofilm itself.

structures can take place.

An antibiofilm agent must display several specific characteristics to target the biofilm lifestyle. First, due to the temporal biofilm heterogeneity, it must show a rapid killing ability to face a changing entity and to target cells before their entry into the biofilm community; it must be able to act in different environmental niches and to target different growth rate cells. The cells located in the periphery of biofilm are directly in contact with nutrients and oxygen, while those placed deepest in the biofilm layers may undergo lack of nutrients, anoxia, and acidic conditions. In this way, a metabolic and spatial heterogeneity is generated including both rapidly and slowly growing cells. In particular, due to environmental conditions, inside the biofilm, it is possible to find the so-called persister, dormant, quiescent cells characterized by a low rate of cell division that are believed to play an essential role in the biofilm resistance to antibiotics [2]. Other important characteristics for a good antimicrobial candidate are the ability to interfere with the production of the extracellular matrix and the possibility to penetrate the biofilm architecture. This matrix consists for 90% of EPS, whose principal components are proteins, polysaccharides, lipids, and extracellular DNA, and it is involved into the biofilm architecture maintenance. An antibiofilm agent should also be able to interfere with

This chapter aims to investigate and clarify in detail the inhibition of biofilm

Biofilms are very dynamic and spatially heterogeneous structures originating gradients of oxygen, nutrients, and pH, and their formation occurs through three

The inhibition or prevention of biofilm formation has been a subject of study for a long time. The first important action against biofilm formation is to prevent bacterial adhesion to surfaces and host tissues to reduce infection [5]. Preventing bacterial adhesion is an attractive target [6] for hampering bacterial infection, and several different strategies have been proposed including hindering cellular receptors from recognizing adhesion surfaces or inhibiting the process of bacterial adhesion. Blocking the primary colonizers can prevent initial biofilm colonization and the subsequent infection produced by planktonic cells released from the

The adhesion process consists of various distinct steps. In the first step, bacterial cell establishes reversible adhesion interactions on host surfaces [7], while in the second step, a stronger type of adhesion is carried out, which involves specific molecules that bind in a complementary manner [5]. In particular, in Gram-negative bacteria, adhesion is mediated by special proteins known as adhesins associated with cell surface structures such as fimbriae or pili [8, 9]. Initial adhesion is then followed by a complex colonization process that offers a number of advantages to bacteria, including increased protection against dislocation by hydrokinetic forces from fluid surfaces or better access to nutrients released by the host cells [10]. Finally, in these favorable conditions, the development of the elaborate biofilm

For a long time, the first strategies used to inhibit the adhesion process were focused on the use of adhesin analogues that bind to the receptor and competitively

agents are the ability to recruit immune cells and/or modulate the host immune response and the synergy with other conventional and unconventional antimicro-

phases: adhesion, maturation, and dispersal phase as described earlier.

**2. Small molecules capable to inhibit biofilm formation**

Other additional aspects to consider the identification of potential antimicrobial

**58**

block bacterial adhesion [5]. However, this strategy resulted unpractical because adhesin proteins are not readily available, and they become toxic at the relatively high concentrations that had to be used. An attempt to overcome this problem consisted in the design and use of synthetic peptides mimicking the sequence of cell surface adhesins. For example, the small peptide p1025 inhibits *Streptococcus mutans* binding to dental surfaces [11]. Analogously, a fragment of the fimbrillin adhesin was found to inhibit the adhesion of *Porphyromonas gingivalis* to hydroxyapatite [12]. However, this approach showed several drawbacks as different adhesins usually mediate the adhesion process and the expression of carbohydrates or cell surface ligands may vary depending on environmental conditions, originating a large number of variables and making this approach more difficult and not very applicable.

A novel and interesting approach to inhibit bacterial adhesion consists in the use of cell coatings with antimicrobial peptides that alter the chemical properties of the surface [13, 14], thus interfering with bacterial adhesion and preventing surface binding. Although "passive," this method is rather attractive and may serve as a novel approach to address the biofilm problem on artificial medical devices. However, limited successes have been achieved so far due to attachment variability among different bacterial strains. Recently, many active polymeric coatings were designed to bind the surface and release a variety of antimicrobial molecules such as antibiotics, bacteriocins, and metal ions [15–18]. A significant reduction in biofilm formation of *Staphylococcus epidermidis* on hydrogel-coated and serum/ hydrogel-coated silicone catheters was observed following the release of bacteriophagic factors from the polymer with and without supplemental divalent cations [19]. Similarly, treatment of piperacillin-tazobactam coated tympanostomy tubes reduces biofilm infection of ciprofloxacin-resistant *Pseudomonas aeruginosa* (CRPA) [20]. The negative aspect of this approach might be the continuous release of high concentration of antimicrobials in a short time by the active polymer often higher than the MIC values without a specific target. However, target release polymer can be foreseen as the new era of biofilm treatments in industrial food safety and packaging [21].

Recently, great attention was paid to a different approach addressed to killing planktonic cells for prevention and treatment of biofilms. The new catheter lock solution C/MB/P (citrate, methylene blue, and parabens) was able to act against planktonic and sessile bacteria within a biofilm preventing bacterial colonization of hemodialysis catheters [22]. Killing planktonic cells might represent a good approach, but this strategy cannot be carried out on long term because any drug targeting crucial processes for bacterial life will unavoidably lead to the development of resistant strains.

An effective and positive control of biofilm formation might be obtained by interfering with specific cellular process crucial for biofilm formation. Biofilm formation is often associated with the phenomenon of quorum sensing (QS), in which bacterial cells communicate with each other by small diffusible signal molecules [23]. Moreover, bacterial gene expression has to be synchronized to form biofilms, and to achieve this goal, the quorum-sensing (QS) mechanism is used by bacteria, producing and responding to a several intra and intercellular signals called autoinducers [24]. At low-cell densities, the autoinducer is present in the extracellular media in a small amount that is too dilute to be detected. When the cell density increases, the autoinducer concentration reaches a threshold, and the autoinducer-receptor complex (the regulatory protein) acts to induce or repress the expression of target genes. The QS controls some physiological processes such as secretion of virulence factors, biofilm formation, and antibiotic resistance in several bacterial species [25, 26]. Investigation and elucidation of

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].

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 bacterial specificity [33].
