**Abstract**

Biofilms are structured aggregates of bacterial cells that are embedded in selfproduced extracellular polymeric substances. Various pathogens initiate a disease process by creating organized biofilms that enhance their ability to adhere, replicate to accumulate, and express their virulence potential. Quorum sensing, which refers to the bacterial cell-to-cell communication resulting from production and response to *N*-acyl homoserine lactone signal molecules, also plays an important role in virulence and biofilm formation. Attenuation of microorganisms' virulence such that they fail to adapt to the hosts' environment could be a new strategic fight against pathogens. Thus, agents or products that possess anti-biofilm formation and/or anti-quorum sensing activities could go a long way to manage microbial infections. The incidence of microbial resistance can be reduced by the use of anti-biofilm formation and anti-quorum sensing agents.

**Keywords:** biofilm, quorum sensing, bacteria, acyl homoserine lactone

### **1. Introduction**

Biofilm is a population of cells growing on a surface and enclosed in an exopolysaccharide matrix [1]. The physiology, structure and chemistry of the biofilm vary with the nature of its resident microbes and local environment [2].

Most important feature among biofilms is that their structural integrity critically depends upon the extracellular matrix produced by their constituent cells. They are notoriously difficult to eradicate and are a source of many recalcitrant infections [2]. Biofilms are associated with serious health issues stemming from persistent infections due to the contamination of medical devices (intravenous and urinary catheters), artificial implants and drinking water pollution among others [3].

Intercellular signaling, often referred to as quorum sensing (QS), has been shown to be involved in biofilm development [4]. Quorum sensing relies on small, secreted signaling molecules; much like hormones in higher organisms, to initiate coordinated responses across a population and it contributes to behaviors that enable microbes to resist antimicrobial compounds [5]. Quorum sensing signaling activation can lead to antimicrobial resistance of the pathogens, thus increasing the therapy difficulty of diseases [4].

### *Bacterial Biofilms*

The key concern about biofilms is their contribution to the development of resistance against antimicrobial agents, and with the on-going emergence of antibiotic-resistant pathogens, there is a current need for development of alternative therapeutic strategies [6].

An anti-virulence approach by which quorum sensing is impeded could be a viable means to manipulate bacterial processes, especially pathogenic traits that are harmful to human and animal health and agricultural productivity [7]. Further research into the identification and development of chemical compounds and enzymes that facilitate quorum-sensing inhibition (QSI) by targeting signaling molecules, signal biogenesis, or signal detection are required [7]. Anti-QS agents can abolish the QS signaling and prevent the biofilm formation, therefore reducing bacterial virulence without causing drug-resistant to the pathogens, suggesting that anti-QS agents could be potential alternatives for antibiotics [8]. An effective clinical strategy for treating bacterial diseases in the near future will be to combine anti-QS agents with conventional antibiotics since this can significantly improve the efficacy of therapeutic drugs and decrease the cost of human healthcare [9].

### **2. Microbial biodiversity in biofilm systems**

Biofilms are mixed microbial cultures normally consisting predominantly of prokaryotes with some eukaryotes. Thus, in addition to microbial cells, the surrounding environment contains a range of macromolecular products in which exopolysaccharide secreted by the cells is the dominant macromolecular component, while the water content is probably about 90–97% [10, 11]. Secreted products also include enzymes and other proteins, bacteriocins, and low mass solutes and nucleic acid released through cell lysis. The lysis may occur either naturally with cell aging or through the action of phage and bacteriocins.

Opportunistic pathogens, viruses, parasitic protozoa, toxin releasing algae and fungi and enteric bacteria e.g. *Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter agglomerans, Helicobacter pylori, Shigella spp., Campylobacter spp., Salmonella spp., Clostridium perfringens, Enterococcus faecium, Enterococcus faecalis* and environmental pathogenic bacteria like *Legionella pneumophila, Pseudomonas aeruginosa, Pseudomonas fluorescens, Aeromonas hydrophila, Aeromonas caviae, Mycobacterium avium, Mycobacterium xenopi* etc. are associated with biofilms present in drinking water [12, 13].

Biofilms present complex assemblies of microorganisms attached to surfaces. They are dynamic structures in which various metabolic activities and interactions between the component cells occur [10]. Studies on microorganisms and biofilm formation have revealed diverse complex social behavior including cooperation in foraging, building, reproduction, dispersion and communication among microorganisms [14]. The organisms within a biofilm setup may include a single or diverse species of microorganisms. In the biofilm, bacteria can share nutrients and are sheltered from harmful factors in the environment, such as desiccation, antibiotics, and a host body's immune system.

Bacteria, fungi, viruses, protozoa and cyanobacteria that are common pathogens are all involved in biofilm formation [15].

### **2.1 Bacterial biofilms**

About 99.9% of all bacteria live in biofilm communities [16]. A biofilm usually begins to form when a free-swimming bacterium attaches to a surface. Pathogenic organisms are found on most food items including seafoods and biofilm forming

**115**

*Combating Biofilm and Quorum Sensing: A New Strategy to Fight Infections*

pathogens are found on such seafoods as crabs [17], pacific oysters [18], shrimps [19] etc. Public health and clinical microbiologists recognize that biofilms are present everywhere in nature and are responsible for a number of human infections. Infectious caused by microbial communities include urinary tract infections, middle-ear infections, dental plaque, gingivitis, endocarditis, cystic fibrosis. Biofilms on persistent indwelling devices such as catheter, contact lenses, heart valves and joint prostheses are also responsible for many recurrent infections [20, 21]. Biofilms on indwelling medical devices may be composed of Gram-positive or Gram-negative bacteria. Bacteria commonly isolated from these devices include the Gram-positive *Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis*, and *Streptococcus viridans;* and the Gram-negative *Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis,* and *Pseudomonas aeruginosa* [22]. It has been shown that virtually all indwelling central venous catheters are colonized by microorganisms embedded in a biofilm matrix. Among these *S. epidermidis* and *S. aureus* are commonly present on cardiovascular devices [23], causing about 40–50% of infec-

The organisms that form biofilms on medical devices originate from patient's skin microflora, exogenous microflora from health-care personnel, or contaminated infusates. Biofilms associated with catheters may initially be composed of single species, but with the passage of time they become multi-specie communities. Some urinary tract and bloodstream infections are also caused by biofilm-associated indwelling medical devices with 50–70% of infections related to catheter [12]. Chronic infections, inflammation and tissue damage caused by many strains of

Bacteria that reside in a biofilm community usually will not grow when cultured, a situation normally referred to as "viable, but not culturable". The reason is that to change to the planktonic state from a biofilm-producing phenotype, bacteria require complex and specific environmental and signaling factors that are not available in a culture plate [25]. This therefore suggests that analyzing biofilm samples for bacterial infective agents during infections may show negative results and the real cause of the infections may not be detected if culturing is the only investigative procedure.

Many medically important fungi produce biofilms and they include *Candida*, *Aspergillus*, *Cryptococcus*, *Trichosporon*, *Coccidioides*, and *Pneumocystis*. *Candida albicans* biofilms are primarily made up of yeast-form and hyphal cells, both of which are required for biofilm formation [26]. The formation of *Candida albicans* biofilm follows a sequential process that involves adherence to a substrate (either abiotic or mucosal surface), proliferation of yeast cells over the surface, and induction of hyphal formation [27]. As the biofilm matures extracellular matrix (ECM) accumulates. Many other Candida spp. form ECM-containing biofilms but do not produce true hyphae and they include *Candida tropicalis*, *Candida parapsilosis*, and *Candida glabrata* [28]. Aspergillus biofilms can form both on abiotic and biotic surfaces and the initial colonizing cells that adhere to the substrate are conidia. Mycelia (the hyphal form) develop as the biofilm matures [29]. *Aspergillus fumigatus* produces two forms of biofilm infections: Aspergilloma and Aspergillosis. Aspergilloma infections present an intertwined ball of hyphae while aspergillosis infections pres-

*Trichosporon asahii* forms biofilms comprised of yeast and hyphal cells embedded in matrix, as do those of *Coccidioides immitis*. *Cryptococcus neoformans* forms biofilms consisting of yeast cells on many abiotic substrates [31]. Although *Cryptococcus neoformans* forms hyphae in the course of mating, no hyphae have

single species are often found in polymicrobial communities [24].

*DOI: http://dx.doi.org/10.5772/intechopen.89227*

tions related to heart valve [14].

**2.2 Fungal biofilms**

ent individual separated hyphae [30].

### *Combating Biofilm and Quorum Sensing: A New Strategy to Fight Infections DOI: http://dx.doi.org/10.5772/intechopen.89227*

pathogens are found on such seafoods as crabs [17], pacific oysters [18], shrimps [19] etc. Public health and clinical microbiologists recognize that biofilms are present everywhere in nature and are responsible for a number of human infections. Infectious caused by microbial communities include urinary tract infections, middle-ear infections, dental plaque, gingivitis, endocarditis, cystic fibrosis. Biofilms on persistent indwelling devices such as catheter, contact lenses, heart valves and joint prostheses are also responsible for many recurrent infections [20, 21]. Biofilms on indwelling medical devices may be composed of Gram-positive or Gram-negative bacteria. Bacteria commonly isolated from these devices include the Gram-positive *Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis*, and *Streptococcus viridans;* and the Gram-negative *Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis,* and *Pseudomonas aeruginosa* [22]. It has been shown that virtually all indwelling central venous catheters are colonized by microorganisms embedded in a biofilm matrix. Among these *S. epidermidis* and *S. aureus* are commonly present on cardiovascular devices [23], causing about 40–50% of infections related to heart valve [14].

The organisms that form biofilms on medical devices originate from patient's skin microflora, exogenous microflora from health-care personnel, or contaminated infusates. Biofilms associated with catheters may initially be composed of single species, but with the passage of time they become multi-specie communities. Some urinary tract and bloodstream infections are also caused by biofilm-associated indwelling medical devices with 50–70% of infections related to catheter [12]. Chronic infections, inflammation and tissue damage caused by many strains of single species are often found in polymicrobial communities [24].

Bacteria that reside in a biofilm community usually will not grow when cultured, a situation normally referred to as "viable, but not culturable". The reason is that to change to the planktonic state from a biofilm-producing phenotype, bacteria require complex and specific environmental and signaling factors that are not available in a culture plate [25]. This therefore suggests that analyzing biofilm samples for bacterial infective agents during infections may show negative results and the real cause of the infections may not be detected if culturing is the only investigative procedure.

### **2.2 Fungal biofilms**

*Bacterial Biofilms*

tive therapeutic strategies [6].

The key concern about biofilms is their contribution to the development of resistance against antimicrobial agents, and with the on-going emergence of antibiotic-resistant pathogens, there is a current need for development of alterna-

An anti-virulence approach by which quorum sensing is impeded could be a viable means to manipulate bacterial processes, especially pathogenic traits that are harmful to human and animal health and agricultural productivity [7]. Further research into the identification and development of chemical compounds and enzymes that facilitate quorum-sensing inhibition (QSI) by targeting signaling molecules, signal biogenesis, or signal detection are required [7]. Anti-QS agents can abolish the QS signaling and prevent the biofilm formation, therefore reducing bacterial virulence without causing drug-resistant to the pathogens, suggesting that anti-QS agents could be potential alternatives for antibiotics [8]. An effective clinical strategy for treating bacterial diseases in the near future will be to combine anti-QS agents with conventional antibiotics since this can significantly improve the

efficacy of therapeutic drugs and decrease the cost of human healthcare [9].

Biofilms are mixed microbial cultures normally consisting predominantly of prokaryotes with some eukaryotes. Thus, in addition to microbial cells, the surrounding environment contains a range of macromolecular products in which exopolysaccharide secreted by the cells is the dominant macromolecular component, while the water content is probably about 90–97% [10, 11]. Secreted products also include enzymes and other proteins, bacteriocins, and low mass solutes and nucleic acid released through cell lysis. The lysis may occur either naturally with cell aging

Opportunistic pathogens, viruses, parasitic protozoa, toxin releasing algae and fungi and enteric bacteria e.g. *Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter agglomerans, Helicobacter pylori, Shigella spp., Campylobacter spp., Salmonella spp., Clostridium perfringens, Enterococcus faecium, Enterococcus faecalis* and environmental pathogenic bacteria like *Legionella pneumophila, Pseudomonas aeruginosa, Pseudomonas fluorescens, Aeromonas hydrophila, Aeromonas caviae, Mycobacterium avium, Mycobacterium xenopi* etc. are associated

Biofilms present complex assemblies of microorganisms attached to surfaces. They are dynamic structures in which various metabolic activities and interactions between the component cells occur [10]. Studies on microorganisms and biofilm formation have revealed diverse complex social behavior including cooperation in foraging, building, reproduction, dispersion and communication among microorganisms [14]. The organisms within a biofilm setup may include a single or diverse species of microorganisms. In the biofilm, bacteria can share nutrients and are sheltered from harmful factors in the environment, such as desiccation, antibiotics,

Bacteria, fungi, viruses, protozoa and cyanobacteria that are common pathogens

About 99.9% of all bacteria live in biofilm communities [16]. A biofilm usually begins to form when a free-swimming bacterium attaches to a surface. Pathogenic organisms are found on most food items including seafoods and biofilm forming

**2. Microbial biodiversity in biofilm systems**

or through the action of phage and bacteriocins.

with biofilms present in drinking water [12, 13].

and a host body's immune system.

**2.1 Bacterial biofilms**

are all involved in biofilm formation [15].

**114**

Many medically important fungi produce biofilms and they include *Candida*, *Aspergillus*, *Cryptococcus*, *Trichosporon*, *Coccidioides*, and *Pneumocystis*. *Candida albicans* biofilms are primarily made up of yeast-form and hyphal cells, both of which are required for biofilm formation [26]. The formation of *Candida albicans* biofilm follows a sequential process that involves adherence to a substrate (either abiotic or mucosal surface), proliferation of yeast cells over the surface, and induction of hyphal formation [27]. As the biofilm matures extracellular matrix (ECM) accumulates. Many other Candida spp. form ECM-containing biofilms but do not produce true hyphae and they include *Candida tropicalis*, *Candida parapsilosis*, and *Candida glabrata* [28]. Aspergillus biofilms can form both on abiotic and biotic surfaces and the initial colonizing cells that adhere to the substrate are conidia. Mycelia (the hyphal form) develop as the biofilm matures [29]. *Aspergillus fumigatus* produces two forms of biofilm infections: Aspergilloma and Aspergillosis. Aspergilloma infections present an intertwined ball of hyphae while aspergillosis infections present individual separated hyphae [30].

*Trichosporon asahii* forms biofilms comprised of yeast and hyphal cells embedded in matrix, as do those of *Coccidioides immitis*. *Cryptococcus neoformans* forms biofilms consisting of yeast cells on many abiotic substrates [31]. Although *Cryptococcus neoformans* forms hyphae in the course of mating, no hyphae have

been observed in *Cryptococcus neoformans* biofilms. Similarly, *Pneumocystis species* do not produce hyphal structures as part of their biofilms [32]. Hyphal formation is therefore, not a uniform feature of fungal biofilms.

### **2.3 Protozoan biofilms**

Free-living protozoans are single celled eukaryotic organisms and are divided into amoebae, flagellates and ciliates. All the three protozoan groups have been found in fresh water biofilms. Although many different species are found in association with biofilms, their level of association differs. The protozoans *Cyclospora cayetanensis, Cryptosporidium spp., and Toxoplasma gondii* have all been found in biofilm communities [22].

## **2.4 Virus involvement in biofilms**

Viruses are obligatory intracellular parasites and are found in communities where cells in which they live are found. Viruses are, thus, found in biofilms communities associated with the bacteria, fungi and protozoa they infect.

Many phages may produce polysaccharases or polysaccharide lyases. Some phages are also known to produce enzymes that degrade the poly-Q-glutamic acid capsule of *Bacillus* spp. [33]. Various structures including extracellular polymers and heterologous microbial cells may impede viral access to the bacterial cell surface. Phage may carry on their surfaces enzymes that degrade bacterial polysaccharides including those of biofilm structures. These enzymes are very specific and seldom act on more than a few closely related polysaccharide structures [34]. Numerous phages have been isolated which induce enzymes capable of degrading the exopolysaccharide of various Gram-negative bacterial genera. These include phage for biofilm-forming bacteria. It has been observed that the extracellular matrix of the biofilms does not protect the bacterial cells from infection with phage T4 [35].

Many biofilms possess an open architecture with water-filled channels, which would allow the phage access to the biofilm interior [36]. As biofilms age and cells die and slough off, potential new viral receptor sites may become available. As bacteria excel at adapting to differing nutrient conditions, changes to the host cell surface could be expected with either loss or gain of possible phage receptors. A further factor which might influence phage retention within biofilms lies in the role of hydrophobic and electrostatic interactions. In the interaction of a coliphage with both hydrophobic and hydrophilic membranes, a critical factor in the retention of the phage was its iso-electric point [37].

In complex biofilms in natural environments, eukaryotic algae may also be present [38]. Under these circumstances algal cell lysis through viral action is also possible as many viruses for algal species have now been isolated and identified [39].
