**Staphylococcal Biofilms: Pathogenicity, Mechanism and Regulation of Biofilm Formation by Quorum-Sensing System and Antibiotic Resistance Mechanisms of Biofilm-Embedded Microorganisms**

### Sahra Kırmusaoğlu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62943

#### **Abstract**

Staphylococcal infections are reported to cause very important problems in hospital‐ ized and immunocompressed patients worldwide due to their tough and irresponsive treatment by antibiotics. Biofilm-embedded bacteria that gain resistance to immune defense and antibiotics by antibiotic degrading enzymes, efflux pumps, and certain gene products of which expression are changed by the quorum sensing cause chronic and recurrent infections such as indwelling device–associated infections. Biofilm-embed‐ ded sessile community has heterogeneous cells that have wide range of different responds to each antimicrobials. *Staphylococcus epidermidis* (*S. epidermidis*) and *Staphylococcus aureus* (*S. aureus*) that are mostly known pathogenic strains can induce gene expression of biofilm that has an important role in the pathogenesis of staphylo‐ coccal infections and causes bacterial attachment and colonization on biotic such as tissues or abiotic surfaces such as prosthetic surfaces that may act as a substrate for microbial adhesion when microorganisms exposed to stress conditions. This ex‐ pressed and matured biofilm causes bacterial spread to whole body, consequently, spread of infection in to whole body. It is hard to treat biofilm infections, and new agents are being researched to prevent formation and dissemination of biofilm. Defining the virulence and the role of biofilm of *S. epidermidis* and *S. aureus* in chronic and recur‐ rent infections such as indwelling device–associated infections, the mechanism and the global regulation of biofilm production by quorum-sensing system, inactivation of biofilm formation, and the resistance patterns of biofilm-embedded microorganism against antimicrobials are important.

© 2016 The Author(s). Licensee InTech. 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, provided the original work is properly cited.

**Keywords:** staphylococcal biofilm, mechanism and regulation of biofilm formation, quorum-sensing system, antimicrobial resistance of biofilm, *Staphylococcus aureus*, *Sta‐ phylococcus epidermidis*, pathogenicity

bic conditions, acid exposure, salinity, pH gradients, desiccation, bacteriophages, and amoebae and to resist antibiotics, antimicrobials, and host immune defense [5–8]. The main pathogen of implant infections is staphylococci that cause 80% of all prosthetic infections [9]. The biofilm of bacteria causes chronic infections such as indwelling device–related infections, chronic wound infections, chronic urinary tract infections (UTI), cystic fibrosis pneumonia, chronic otitis media (OM), chronic rhinosinusitis, periodontitis, and recurrent tonsillitis [10]. The biofilm infections are the main important problems in hospitalized and immunocompressed patients in worldwide due to their tough and irresponsive treatment by antibiotics. In biofilm, bacteria are not distrupted completely by antibiotics even high doses of antibiotics used *in vivo* [3, 11, 12]. Infected device can expose the patient to a higher risk of mortality. Orthopedic surgery and trauma indwelling device-related infections that make treatment difficult by antibiotics [13] cause removal of implant out of the body to eradicate biofilm and overcome biofilm-related infections [14] and may cause functional loss of the infected limb [15, 16].

Staphylococcal Biofilms: Pathogenicity, Mechanism and Regulation of Biofilm Formation by Quorum-Sensing System

and Antibiotic Resistance Mechanisms of Biofilm-Embedded Microorganisms

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191

The biofilm that anchored to abiotic or biotic surfaces is a slime-like glycocalyx in which sessile community of microorganisms embedded. This extracellular polymeric substance that is constituted by matrix of polysaccharide, teichoic acids, extracellular DNA (eDNA), and staphylococcal proteins is produced by biofilm producing microorganisms [4, 17, 18]. Polysaccharide intracellular adhesin (PIA) is a specific polysaccharide in glycocalyx com‐ posed of β-1,6–linked N-acetylglucosamine residues (80–85%) and non-N-acetylated Dglucosaminyl residues that are an anionic fraction and contain phosphate and ester-linked succinate (15–20%) [18]. Although PIA is a main mechanism of biofilm formation in *S. aureus* and *S. epidermidis*, surface proteins are the other alternative mechanism of biofilm formation. Extracellular matrix has large water-filled channels, accumulates antibiotic-degrading enzymes such as β-lactamases [19], and plays a role in the adaptive resistance mechanisms

Bacterial biofilm formation is a complex and multifactorial process. The biofilm formation process consists of adherence/adhesion/attachment, aggregation/maturation/accumulation, and detachment/dispersal phase. The last step is the dispersal of mature biofilm-embedded

When conditions favor biofilm formation, biofilm formation that begins with the adherence of the bacteria to a surface that act as a substrate for microbial adhesion continues with the

Staphylococcal adherence to an abiotic surface of indwelling prosthetic device depends on physico-chemical structure of medical device and surface components of Staphylococci such as wall teichoic acid (WTA) [23], lipoteichoic acid (LTA) [23], accumulation-associated

**2.2. Staphylococcal biofilms as a virulence factor**

due to eDNA constituent [20] (**Figure 3**).

**2.3. Mechanisms of biofilm formation**

bacteria out of the biofilm [21] (**Figure 1**).

*2.3.1. Attachment (adhesion or adherence) phase*

aggregation formed by cell–cell adhesion [22] (**Figure 1**).

### **1. Introduction**

*Staphylococcus epidermidis* (S. epidermidis) and *Staphylococcus aureus* (S. aureus) are the most common causes of indwelling device–associated infections, and nosocomial and community acquired infections can produce biofilm as a virulence factor [1]. The biofilm infections such as *S. epidermidis* and *S. aureus* infections are important problems in hospitalized and immu‐ nocompressed patients worldwide due to their tough and irresponsive treatment by antibi‐ otics. Biofilm-producing bacteria resist to immune defense, antibiotics, and many antimicrobial agents. Biofilm-embedded bacteria gain antibiotic resistance by antibioticdegrading enzymes, efflux pumps, and certain gene products of which expression are changed by the quorum sensing [2, 3]. Biofilm-embedded sessile community has heterogeneous cells that have wide range of different responds to each antimicrobials [2]. So, every antibiotic has a different effect against different metabolically active cells that are present in the different layers of biofilm and persister cells that are evolved to survive in biofilm. It is hard to treat biofilm infections that are generally recurrent infections and of which treatments are tough and irresponsive [3].

Staphylococci that construct the human skin flora can contaminate indwelling devices. By this way, they are inserted to human by contaminated indwelling devices. When microorgan‐ isms exposed to stress conditions, gene expression of biofilm is induced as a stress response. The biofilm that is a slime-like glycocalyx causes bacteria to survive in the stress conditions. Staphylococci adhere, colonize, and infect biotic surfaces such as tissue or abiotic surfaces such as prosthetic surfaces that may act as a substrate for microbial adhesion and causes bacterial spread to whole body by forming biofilm that is a slime-like glycocalyx [1, 4, 5]. The viru‐ lence and the role of biofilm of *S. epidermidis* and *S. aureus* in chronic and recurrent infec‐ tions such as indwelling device–associated infections, the mechanism, and the global regulation of biofilm production by quorum-sensing system, especially *agr*-quorum-sensing system, inactivation of biofilm formation, and the resistance patterns of biofilm-embedded microorganism against antimicrobials are discussed in this chapter.

### **2. The biofilm, virulence, and Staphylococcus**

#### **2.1. The pathogenesis of Staphylococcus biofilm**

The biofilm has an important role in the pathogenesis of staphylococcal infections. The biofilm causes bacteria to survive in the stress conditions such as UV damage, metal toxicity, anaero‐

#### Staphylococcal Biofilms: Pathogenicity, Mechanism and Regulation of Biofilm Formation by Quorum-Sensing System and Antibiotic Resistance Mechanisms of Biofilm-Embedded Microorganisms http://dx.doi.org/10.5772/62943 191

bic conditions, acid exposure, salinity, pH gradients, desiccation, bacteriophages, and amoebae and to resist antibiotics, antimicrobials, and host immune defense [5–8]. The main pathogen of implant infections is staphylococci that cause 80% of all prosthetic infections [9]. The biofilm of bacteria causes chronic infections such as indwelling device–related infections, chronic wound infections, chronic urinary tract infections (UTI), cystic fibrosis pneumonia, chronic otitis media (OM), chronic rhinosinusitis, periodontitis, and recurrent tonsillitis [10]. The biofilm infections are the main important problems in hospitalized and immunocompressed patients in worldwide due to their tough and irresponsive treatment by antibiotics. In biofilm, bacteria are not distrupted completely by antibiotics even high doses of antibiotics used *in vivo* [3, 11, 12]. Infected device can expose the patient to a higher risk of mortality. Orthopedic surgery and trauma indwelling device-related infections that make treatment difficult by antibiotics [13] cause removal of implant out of the body to eradicate biofilm and overcome biofilm-related infections [14] and may cause functional loss of the infected limb [15, 16]. bic acidexposure, pH andamoebaeand to resist and host immune The main of implant infections is staphylococci that of all prosthetic The of bacteria causes chronic infections such as indwelling device–related wound chronic urinary tract cystic fibrosis otitis chronic and recurrent patients in worldwide due to their tough and irresponsive treatment by In bacteria are not distrupted completely by antibiotics even high doses of antibiotics used *in* Infected device can expose the patient to a higher risk of surgery and trauma indwelling device-related infections that make treatment difficult cause removal of implant out of the body to eradicate biofilm and as wall teichoic lipoteichoic acid staphylococcal mechanism and regulation of biofilm degrading enzymes, efflux pumps, and certain gene products of which expression are Staphylococci that construct the human skin flora can contaminate indwelling By they are inserted to human by contaminated indwelling When Staphylococci and infect biotic surfaces such as tissue or abiotic surfaces spread to whole body by forming biofilm that is <sup>a</sup> slime-like The lence and the role of biofilm of and in chronic and recurrent tions such as indwelling device–associated the and the regulation of biofilm production by quorum-sensing especially inactivation of biofilm and the resistance patterns of The biofilm has an importantrole in the pathogenesis of staphylococcal The

### **2.2. Staphylococcal biofilms as a virulence factor**

**Keywords:** staphylococcal biofilm, mechanism and regulation of biofilm formation, quorum-sensing system, antimicrobial resistance of biofilm, *Staphylococcus aureus*, *Sta‐*

*Staphylococcus epidermidis* (S. epidermidis) and *Staphylococcus aureus* (S. aureus) are the most common causes of indwelling device–associated infections, and nosocomial and community acquired infections can produce biofilm as a virulence factor [1]. The biofilm infections such as *S. epidermidis* and *S. aureus* infections are important problems in hospitalized and immu‐ nocompressed patients worldwide due to their tough and irresponsive treatment by antibi‐ otics. Biofilm-producing bacteria resist to immune defense, antibiotics, and many antimicrobial agents. Biofilm-embedded bacteria gain antibiotic resistance by antibioticdegrading enzymes, efflux pumps, and certain gene products of which expression are changed by the quorum sensing [2, 3]. Biofilm-embedded sessile community has heterogeneous cells that have wide range of different responds to each antimicrobials [2]. So, every antibiotic has a different effect against different metabolically active cells that are present in the different layers of biofilm and persister cells that are evolved to survive in biofilm. It is hard to treat biofilm infections that are generally recurrent infections and of which treatments are tough

Staphylococci that construct the human skin flora can contaminate indwelling devices. By this way, they are inserted to human by contaminated indwelling devices. When microorgan‐ isms exposed to stress conditions, gene expression of biofilm is induced as a stress response. The biofilm that is a slime-like glycocalyx causes bacteria to survive in the stress conditions. Staphylococci adhere, colonize, and infect biotic surfaces such as tissue or abiotic surfaces such as prosthetic surfaces that may act as a substrate for microbial adhesion and causes bacterial spread to whole body by forming biofilm that is a slime-like glycocalyx [1, 4, 5]. The viru‐ lence and the role of biofilm of *S. epidermidis* and *S. aureus* in chronic and recurrent infec‐ tions such as indwelling device–associated infections, the mechanism, and the global regulation of biofilm production by quorum-sensing system, especially *agr*-quorum-sensing system, inactivation of biofilm formation, and the resistance patterns of biofilm-embedded

The biofilm has an important role in the pathogenesis of staphylococcal infections. The biofilm causes bacteria to survive in the stress conditions such as UV damage, metal toxicity, anaero‐

microorganism against antimicrobials are discussed in this chapter.

**2. The biofilm, virulence, and Staphylococcus**

**2.1. The pathogenesis of Staphylococcus biofilm**

*phylococcus epidermidis*, pathogenicity

190 Microbial Biofilms - Importance and Applications

**1. Introduction**

and irresponsive [3].

The biofilm that anchored to abiotic or biotic surfaces is a slime-like glycocalyx in which sessile community of microorganisms embedded. This extracellular polymeric substance that is constituted by matrix of polysaccharide, teichoic acids, extracellular DNA (eDNA), and staphylococcal proteins is produced by biofilm producing microorganisms [4, 17, 18]. Polysaccharide intracellular adhesin (PIA) is a specific polysaccharide in glycocalyx com‐ posed of β-1,6–linked N-acetylglucosamine residues (80–85%) and non-N-acetylated Dglucosaminyl residues that are an anionic fraction and contain phosphate and ester-linked succinate (15–20%) [18]. Although PIA is a main mechanism of biofilm formation in *S. aureus* and *S. epidermidis*, surface proteins are the other alternative mechanism of biofilm formation. Extracellular matrix has large water-filled channels, accumulates antibiotic-degrading enzymes such as β-lactamases [19], and plays a role in the adaptive resistance mechanisms due to eDNA constituent [20] (**Figure 3**). The biofilm that anchored to abiotic or biotic surfaces is a slime-like glycocalyx in which community of microorganisms This extracellular polymeric substance that constituted by matrix of teichoic extracellular staphylococcal proteins is produced by biofilm producing Polysaccharide intracellular is a specific polysaccharide in glycocalyx posed linked N-acetylglucosamine and non-N-acetylated glucosaminyl residues that are an anionic fraction and contain phosphate and Extracellular matrix has large water-filled accumulates enzymes such and plays a role in the adaptive resistance

#### **2.3. Mechanisms of biofilm formation**

Bacterial biofilm formation is a complex and multifactorial process. The biofilm formation process consists of adherence/adhesion/attachment, aggregation/maturation/accumulation, and detachment/dispersal phase. The last step is the dispersal of mature biofilm-embedded bacteria out of the biofilm [21] (**Figure 1**). Bacterial biofilm formation is a complex and multifactorial The biofilm process consists of and detachment/dispersal The last step is the dispersal of mature

#### *2.3.1. Attachment (adhesion or adherence) phase*

When conditions favor biofilm formation, biofilm formation that begins with the adherence of the bacteria to a surface that act as a substrate for microbial adhesion continues with the aggregation formed by cell–cell adhesion [22] (**Figure 1**). When conditions favor biofilm biofilm formation that begins with the of the bacteria to a surface that act as a substrate for microbial adhesion continues with

Staphylococcal adherence to an abiotic surface of indwelling prosthetic device depends on physico-chemical structure of medical device and surface components of Staphylococci such as wall teichoic acid (WTA) [23], lipoteichoic acid (LTA) [23], accumulation-associated Staphylococcal adherence to an abiotic surface of indwelling prosthetic device depends

protein (Aap) [24], autolysins AtlA [25] and AtlE [26]. The staphylococcal adherence to a biotic surfaces such as host cells and plasma protein-coated prosthetic surface is mediated by cell wall-anchored (CWA) proteins such as the fibrinogen-binding protein SdrG/Fbe of *S. epidermidis* and fibrinogen-/fibronectin-binding proteins FnBPA and FnBPB and clumping factors A and B of *S. aureus* [27].

*2.3.2. Accumulation (aggregation or maturation) phase*

tion rather than PIA [33] (**Figure 1**).

*2.3.3. The detachment (or dispersal) phase*

**2.4. Types of biofilm formation**

*2.4.1. PIA-dependent biofilm formation*

After adherence of staphylococcus to biotic and abiotic surfaces, exopolysaccharide (EPS) such as PIA or PNAG that are produced by *ica* operon (*ica*-dependent form) starts to be produced, extracellular matrix (ECM) is constructed by PIA/PNAG, extracellular DNA (eDNA), and surface proteins [cell wall-anchored (CWA) proteins] in *ica*-independent form, and bacterial colonies become mature [2, 27]. The cell wall-anchored (CWA) proteins not only provide bacterial adherence but also provide intercellular adhesion, biofilm accumulation, and maturation [27]. Aggregation that is mediated by the synthesis of either polysaccharide intercellular adhesion/poly-N-acetylglucosamine (PIA/PNAG) [30, 32] is formed in cell clusters till multi-layer-structured biofilms formed. Several staphylococcal surface proteins that mediate primary attachment of bacteria such as clumping factors A and B, fibrinogen-/ fibronectin-binding proteins FnbA and FnbB of *S. aureus* or the fibrinogen-binding protein SdrG/Fbe of *S. epidermidis* that are cell wall-anchored proteins (CWA) also promote intercel‐ lular adhesion and construct the aggregation of bacteria in *ica*-independent biofilm forma‐

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and Antibiotic Resistance Mechanisms of Biofilm-Embedded Microorganisms

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193

In the initial cell-surface interaction of motile bacteria, adherence of motile cell to surface is facilitated by flagella of motile cell. After adherence motile species that undergo cellular differentiation in biofilm lose their motility by paralyzing their flagella and become nonmo‐ tile [34]. Klausen et al. [35] revealed that wild-type strain and isogenic flagellar mutant of

In the detachment stage, sessile cells turn into planktonic state that can spread and colonize other surfaces and form biofilm on these infected regions [2] (**Figure 1**). Detachment of microorganisms from biofilm can be caused by bacteria themselves, such as enzymatic degradation of the biofilm matrix such as dissolution of adhesins by proteases, nucleases, and a group of small amphiphilic α-helical peptides, known as phenol-soluble modulins (PSMs) functioning as surfactants [27], and quorum sensing or by external forces such as fluid shear forces, corrosion, and human intervention [36] (**Figure 2**). During detachment of motile microorganism rather than staphylococcus, cells express genes that are for motility such as transcription of pilus and ribosomal proteins and are almost seen in planktonic cells [37].

Positively charged PIA provides intercellular attachment via binding to bacteria of which surface is negatively charged [27]. All *S. aureus* strains contain *icaADBC* gene of which product is PIA constructs biofilm formation [31]. *Ica* locus have been identified in many staphylococ‐ cus species like *S. aureus* and *S. epidermidis* but except *S. haemolyticus* and *S. saphrophyticus* [9]. *ica* is regulated by stress conditions, such as anaerobic conditions, extreme temperature, osmolarity, ethanol, and antibiotics. icaA, icaD, icaC, and icaB are the genes of *ica*ADBC locus.

*Pseudomonas aeroginosa* both forms biofilms which have structural differences.

Several microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that are able to bind to human matrix proteins such as fibronectin and fibrinogen and colonize are expressed in *S. epidermidis* and *S. aureus* at the first step [28]. Adherence of bacteria to an extracellular matrix component, fibronectin, fibrinogen, and plasma clot is mediated by expressed surface adhesins such as Bap coded by bap gene [29], surface protein G (SasG) [22], fibronectin-binding proteins (FnbA and FnbB) of *S. aureus* [30], and the fibrinogen-binding protein SdrG/Fbe of *S. epidermidis* [27]. Adherence of *S. aureus* to collagenous tissues and cartilage is mediated by collagen-binding protein, Cna. Some antibodies can block bacterial attachment to these tissues by blocking Cna. Adherence of *S. aureus* to fibrinogen in the presence of fibronectin is mediated by clumping factor A and B (ClfA, ClfB) that are effec‐ tive in foreign body and wound infections. Also, plasma-sensitive surface protein (Pls) participates in the attachment to fibrinogen and fibronectin. Protein A that is present in cell wall and encoded by *spa* gene in *S. aureus* impair opsonization and phagocytosis by binding to Fc domain of immunoglobulin G (IgG) in the wrong orientation. Endovascular diseases are emerged by *S. aureus* as a result ofthe binding of proteinAto vonWillebrand factorin damaged endothelium [31].

**Figure 1.** The stages of biofilm formation.

Staphylococcal Biofilms: Pathogenicity, Mechanism and Regulation of Biofilm Formation by Quorum-Sensing System and Antibiotic Resistance Mechanisms of Biofilm-Embedded Microorganisms http://dx.doi.org/10.5772/62943 193

#### *2.3.2. Accumulation (aggregation or maturation) phase*

protein (Aap) [24], autolysins AtlA [25] and AtlE [26]. The staphylococcal adherence to a biotic surfaces such as host cells and plasma protein-coated prosthetic surface is mediated by cell wall-anchored (CWA) proteins such as the fibrinogen-binding protein SdrG/Fbe of *S. epidermidis* and fibrinogen-/fibronectin-binding proteins FnBPA and FnBPB and clumping

Several microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that are able to bind to human matrix proteins such as fibronectin and fibrinogen and colonize are expressed in *S. epidermidis* and *S. aureus* at the first step [28]. Adherence of bacteria to an extracellular matrix component, fibronectin, fibrinogen, and plasma clot is mediated by expressed surface adhesins such as Bap coded by bap gene [29], surface protein G (SasG) [22], fibronectin-binding proteins (FnbA and FnbB) of *S. aureus* [30], and the fibrinogen-binding protein SdrG/Fbe of *S. epidermidis* [27]. Adherence of *S. aureus* to collagenous tissues and cartilage is mediated by collagen-binding protein, Cna. Some antibodies can block bacterial attachment to these tissues by blocking Cna. Adherence of *S. aureus* to fibrinogen in the presence of fibronectin is mediated by clumping factor A and B (ClfA, ClfB) that are effec‐ tive in foreign body and wound infections. Also, plasma-sensitive surface protein (Pls) participates in the attachment to fibrinogen and fibronectin. Protein A that is present in cell wall and encoded by *spa* gene in *S. aureus* impair opsonization and phagocytosis by binding to Fc domain of immunoglobulin G (IgG) in the wrong orientation. Endovascular diseases are emerged by *S. aureus* as a result ofthe binding of proteinAto vonWillebrand factorin damaged

factors A and B of *S. aureus* [27].

192 Microbial Biofilms - Importance and Applications

endothelium [31].

**Figure 1.** The stages of biofilm formation.

After adherence of staphylococcus to biotic and abiotic surfaces, exopolysaccharide (EPS) such as PIA or PNAG that are produced by *ica* operon (*ica*-dependent form) starts to be produced, extracellular matrix (ECM) is constructed by PIA/PNAG, extracellular DNA (eDNA), and surface proteins [cell wall-anchored (CWA) proteins] in *ica*-independent form, and bacterial colonies become mature [2, 27]. The cell wall-anchored (CWA) proteins not only provide bacterial adherence but also provide intercellular adhesion, biofilm accumulation, and maturation [27]. Aggregation that is mediated by the synthesis of either polysaccharide intercellular adhesion/poly-N-acetylglucosamine (PIA/PNAG) [30, 32] is formed in cell clusters till multi-layer-structured biofilms formed. Several staphylococcal surface proteins that mediate primary attachment of bacteria such as clumping factors A and B, fibrinogen-/ fibronectin-binding proteins FnbA and FnbB of *S. aureus* or the fibrinogen-binding protein SdrG/Fbe of *S. epidermidis* that are cell wall-anchored proteins (CWA) also promote intercel‐ lular adhesion and construct the aggregation of bacteria in *ica*-independent biofilm forma‐ tion rather than PIA [33] (**Figure 1**).

In the initial cell-surface interaction of motile bacteria, adherence of motile cell to surface is facilitated by flagella of motile cell. After adherence motile species that undergo cellular differentiation in biofilm lose their motility by paralyzing their flagella and become nonmo‐ tile [34]. Klausen et al. [35] revealed that wild-type strain and isogenic flagellar mutant of *Pseudomonas aeroginosa* both forms biofilms which have structural differences.

#### *2.3.3. The detachment (or dispersal) phase*

In the detachment stage, sessile cells turn into planktonic state that can spread and colonize other surfaces and form biofilm on these infected regions [2] (**Figure 1**). Detachment of microorganisms from biofilm can be caused by bacteria themselves, such as enzymatic degradation of the biofilm matrix such as dissolution of adhesins by proteases, nucleases, and a group of small amphiphilic α-helical peptides, known as phenol-soluble modulins (PSMs) functioning as surfactants [27], and quorum sensing or by external forces such as fluid shear forces, corrosion, and human intervention [36] (**Figure 2**). During detachment of motile microorganism rather than staphylococcus, cells express genes that are for motility such as transcription of pilus and ribosomal proteins and are almost seen in planktonic cells [37].

### **2.4. Types of biofilm formation**

### *2.4.1. PIA-dependent biofilm formation*

Positively charged PIA provides intercellular attachment via binding to bacteria of which surface is negatively charged [27]. All *S. aureus* strains contain *icaADBC* gene of which product is PIA constructs biofilm formation [31]. *Ica* locus have been identified in many staphylococ‐ cus species like *S. aureus* and *S. epidermidis* but except *S. haemolyticus* and *S. saphrophyticus* [9]. *ica* is regulated by stress conditions, such as anaerobic conditions, extreme temperature, osmolarity, ethanol, and antibiotics. icaA, icaD, icaC, and icaB are the genes of *ica*ADBC locus.

icaA and icaD contribute to exopolysaccharide synthesis and encode N-acetylglucosaminyl transferase as a transmembrane enzyme to synthesize poly-N-acetylglucosamine polymer. While poly-N-acetylglucosamine polymer is translocated to cell surface of bacteria by *ica*D gene, the polymer is fixed to the outer surface of bacteria by deacylation of poly-N-acetylglu‐ cosamine polymer by the product of *ica*B gene [9]. Regulator gene *icaR* that is located up‐ stream of the *icaADBC* operon encodes a transcriptional repressor in both *S. epidermidis* and *S. aureus* and *icaADBC* genes are upregulated in response to anaerobic growth such as inside of biofilm. Under anaerobic conditions, PIA is induced by SrrAB (the staphylococcal respirato‐ ry response regulator) that binds to upstream of the *icaADBC* operon. Insertion sequence (IS256) can regulate *ica* by reversible inactivation in *S. epidermidis* and some strains of *S. aureus*. TcaR (transcriptional regulator of the teicoplanin-associated locus) and IcaR are repressors of ica operon transcription and repress PIA expression. While deletion of *icaR* gene increases *ica* gene expression, PIA production, deletion of *tcaR* gene had no effect against *ica* gene, PIA production. Transcription of IcaR is repressed by Rbf that is a protein regulator of biofilm formation and leads expression of *ica* gene, PIA production, whereas transcription of IcaR is induced by Spx that is a global regulator of stress response genes and regulates biofilm formation negatively [18].

Autolysin Atl that is a wall-anchored protein of *S. aureus* and causes initial attachment of *S. aureus*to surfaces can be cleaved into amidase and glucosaminidase that cause cell lysis, eDNA release, and cell accumulation. Then, biofilm maturation of FnBP-dependent biofilm pheno‐

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In biofilm production of *S. aureus*, cell-cell interactions are facilitated by α-toxin that is a haemolytic toxin. Nevertheless, the mechanism of integral role of α-toxin has not been known clearly. β-toxin that is a sphingomyelinase and causes hemolysis and lyse lymphocytes plays a stimulative role in the biofilm production of *S. aureus* by covalently cross-linking to itself in

*S. aureus* biofilms can be stabilized by amyloid fibrils that are formed by aggregated PSM on

Biofilm production is provided by the equilibrium between the productions of amyloid fibrils and phenol soluble modulins (PSMs) that are extracellular polymeric substances and their catabolism by enzymes such as nucleases and proteases that are expressed by agr-QS regulator system that use two-component system signal transduction system (TCS). The control of planktonic and sessile bacteria and the biofilm expression is regulated by coordinated

the occurrence of DNA in matrix of staphylococcal biofilms [40].

the surface of bacteria and aggregated signal peptide AgrD [41].

**Figure 2.** The regulation of biofilm formation by agr-quorum-sensing system.

*2.5.1. The regulation of Staphylococcal biofilm by agr-quorum-sensing system*

**2.5. The global regulation of biofilm formation**

type is constructed by FnBPs [25].

mechanisms [41] (**Figure 2**).

#### *2.4.2. PIA-independent biofilm formation*

Biofilms not only can be constructed by *ica* gene of which product is PIA, but also construct‐ ed by *ica*-independent (PIA-independent) form. Biofilm is generated not only by PIA that is a main component of biofilm production but also by a number of proteins. When *icaADBC* is deleted, PIA is not produced but the biofilm formation so, virulence is not affected. In this case, biofilm formation can be constructed rather than PIA. In the catheter infection, biofilm formation of clinical isolates of *S. aureus* of which *ica* cluster is mutated is not reduced [18]. Fitzpatrick et al. revealed that biofilm formation of the *icaADBC* operon-deleted MRSA mutants was not affected, whereas biofilm formation of the *icaADBC* operon-deleted MSSA mutants was impaired. This study showed that ica-independent biofilm formation is strain specific [38].

PIA-independent biofilms were constructed by accumulation-associated proteins (Aap) of *S. epidermidis*, biofilm-associated protein (Bap) that is a surface protein of *S. epidermidis* and *S. aureus* and Bap-related proteins of *S. aureus*[18]. Other surface proteins that involve in the PIAindependent biofilm formation are SasG, SasC, protein A, fibronectin-binding proteins FnBPA and FnBPB, cell wall-anchored (CWA) proteins including clumping factors A and B, autoly‐ sins AtlA and AtlE or wall teichoic acid (WTA), the fibrinogen-binding protein SdrG/Fbe, lipoteichoic acids (LTA) of *S. aureus* and the fibrinogen-binding protein SdrG/Fbe of *S. epidermidis* [27].

Scientists determined that medical MRSA isolates produce protein-dependent biofilm such as FnBP- and Aap-dependent biofilms in animal models that have indwelling device–associat‐ ed infection. O'Neill et al. [30] and McCourt et al. [39] revealed that biofilms of certain isolates of HA-MRSA from CC8 and CC22 and CA-MRSA from USA300 lineage (CC8) were FnBPsdependent.

Autolysin Atl that is a wall-anchored protein of *S. aureus* and causes initial attachment of *S. aureus*to surfaces can be cleaved into amidase and glucosaminidase that cause cell lysis, eDNA release, and cell accumulation. Then, biofilm maturation of FnBP-dependent biofilm pheno‐ type is constructed by FnBPs [25].

In biofilm production of *S. aureus*, cell-cell interactions are facilitated by α-toxin that is a haemolytic toxin. Nevertheless, the mechanism of integral role of α-toxin has not been known clearly. β-toxin that is a sphingomyelinase and causes hemolysis and lyse lymphocytes plays a stimulative role in the biofilm production of *S. aureus* by covalently cross-linking to itself in the occurrence of DNA in matrix of staphylococcal biofilms [40].

*S. aureus* biofilms can be stabilized by amyloid fibrils that are formed by aggregated PSM on the surface of bacteria and aggregated signal peptide AgrD [41].

### **2.5. The global regulation of biofilm formation**

icaA and icaD contribute to exopolysaccharide synthesis and encode N-acetylglucosaminyl transferase as a transmembrane enzyme to synthesize poly-N-acetylglucosamine polymer. While poly-N-acetylglucosamine polymer is translocated to cell surface of bacteria by *ica*D gene, the polymer is fixed to the outer surface of bacteria by deacylation of poly-N-acetylglu‐ cosamine polymer by the product of *ica*B gene [9]. Regulator gene *icaR* that is located up‐ stream of the *icaADBC* operon encodes a transcriptional repressor in both *S. epidermidis* and *S. aureus* and *icaADBC* genes are upregulated in response to anaerobic growth such as inside of biofilm. Under anaerobic conditions, PIA is induced by SrrAB (the staphylococcal respirato‐ ry response regulator) that binds to upstream of the *icaADBC* operon. Insertion sequence (IS256) can regulate *ica* by reversible inactivation in *S. epidermidis* and some strains of *S. aureus*. TcaR (transcriptional regulator of the teicoplanin-associated locus) and IcaR are repressors of ica operon transcription and repress PIA expression. While deletion of *icaR* gene increases *ica* gene expression, PIA production, deletion of *tcaR* gene had no effect against *ica* gene, PIA production. Transcription of IcaR is repressed by Rbf that is a protein regulator of biofilm formation and leads expression of *ica* gene, PIA production, whereas transcription of IcaR is induced by Spx that is a global regulator of stress response genes and regulates biofilm

Biofilms not only can be constructed by *ica* gene of which product is PIA, but also construct‐ ed by *ica*-independent (PIA-independent) form. Biofilm is generated not only by PIA that is a main component of biofilm production but also by a number of proteins. When *icaADBC* is deleted, PIA is not produced but the biofilm formation so, virulence is not affected. In this case, biofilm formation can be constructed rather than PIA. In the catheter infection, biofilm formation of clinical isolates of *S. aureus* of which *ica* cluster is mutated is not reduced [18]. Fitzpatrick et al. revealed that biofilm formation of the *icaADBC* operon-deleted MRSA mutants was not affected, whereas biofilm formation of the *icaADBC* operon-deleted MSSA mutants was impaired. This study showed that ica-independent biofilm formation is strain

PIA-independent biofilms were constructed by accumulation-associated proteins (Aap) of *S. epidermidis*, biofilm-associated protein (Bap) that is a surface protein of *S. epidermidis* and *S. aureus* and Bap-related proteins of *S. aureus*[18]. Other surface proteins that involve in the PIAindependent biofilm formation are SasG, SasC, protein A, fibronectin-binding proteins FnBPA and FnBPB, cell wall-anchored (CWA) proteins including clumping factors A and B, autoly‐ sins AtlA and AtlE or wall teichoic acid (WTA), the fibrinogen-binding protein SdrG/Fbe, lipoteichoic acids (LTA) of *S. aureus* and the fibrinogen-binding protein SdrG/Fbe of *S.*

Scientists determined that medical MRSA isolates produce protein-dependent biofilm such as FnBP- and Aap-dependent biofilms in animal models that have indwelling device–associat‐ ed infection. O'Neill et al. [30] and McCourt et al. [39] revealed that biofilms of certain isolates of HA-MRSA from CC8 and CC22 and CA-MRSA from USA300 lineage (CC8) were FnBPs-

formation negatively [18].

194 Microbial Biofilms - Importance and Applications

specific [38].

*epidermidis* [27].

dependent.

*2.4.2. PIA-independent biofilm formation*

#### *2.5.1. The regulation of Staphylococcal biofilm by agr-quorum-sensing system*

Biofilm production is provided by the equilibrium between the productions of amyloid fibrils and phenol soluble modulins (PSMs) that are extracellular polymeric substances and their catabolism by enzymes such as nucleases and proteases that are expressed by agr-QS regulator system that use two-component system signal transduction system (TCS). The control of planktonic and sessile bacteria and the biofilm expression is regulated by coordinated mechanisms [41] (**Figure 2**).

**Figure 2.** The regulation of biofilm formation by agr-quorum-sensing system.

The biofilm formation of staphylococci is fully expressed *in vivo*, whereas the biofilm forma‐ tion of staphylococci is not fully expressed all the time *in vitro* unless nutrient supplementa‐ tions are added to growth media and is provided. Increased amount of biofilm formation due to fully expression occurs in stress conditions such as starvation, thermal stress, heat shock, salt, certain antibiotics, iron limitation, subinhibitory concentrations of ethanol, accumula‐ tions of metabolites, oxidative stress, low pH, and changes in osmolarity *in vitro*. Bacteria sense stimuli from the environment and bacterial density and then respond to stimuli by upregu‐ lating expression of biofilm formation, virulence factors production such as toxins, etc. [9].

The regulation mechanisms of RNAIII for target genes can be at transcriptional and transla‐ tional level, andits regulation can bedirect orindirect. Fourteen stem-loop andtwo long helices construct structure of RNAIII. Each domain regulates the expression of each target gene. Translation of α-hemolysin (*hla*) upregulated by hairpin loop H2 and H3. In contrast to this, the repression of early expressed virulence genes of *S. aureus* such as coagulase, protein A, and the repressor of toxins (Rot) is comprised by hairpin H13, H14, and H7 of RNAIII. Hairpins such as H7, H13, and H14 that are complementary to Shine-Dalgarno sequences (SD) of target mRNA act as an antisense RNA and inhibit initiation of translation and cause RNAaseIII-

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Staphylococcal virulence factors are expressed with accessory gene regulator (agr) system in response to cell density [9]. During the beginning of the biofilm-related staphylococcal infection, adhesion factors (surface proteins) such as MSCRAMMs are upregulated. After initial attachment and colonization had been happened, during early stationary growth phase

mediated degradation of target mRNA [45] (**Figure 4**).

**Figure 4.** The structure of RNAIII [44]

Staphylococcus use quorum-sensing systems (QS) forintercellular communication and biofilm formation. Accessory gene regulator (Agr) system regulates cell density-dependent gene expression using two-component signal transduction system [42]. Agr and LuxS systems that are required for autoinducer peptide (AIP) production as a pheromone are quorum-sensing systems in staphylococci [43]. Bacteria sense pheromones as stimuli that are released by the density of bacteria belonging to the same group and express biofilm formation [9]. AIP production starts in exponential phase of bacterial growth [44]. There are four proteins that are sensor histidine protein kinase AgrC, DNA-binding response regulator AgrA, AgrD that is a prepheromone, and AgrB that exports and modifies AgrD, present in this system. The signal is transported to bacteria by binding of AIP to AgrC. When AIP binds to AgrC, DNAbinding regulator AgrA is activated by His-dependent phosphorylation of AgrC [42]. By the binding of activated DNA-binding regulator AgrA to P2 and P3 promoters in *agr* operon (*agr*BDCA), RNAII and RNAIII are transcripted, respectively [44]. The *agr*BDCA operon codes RNAII transcript that encodes AgrB, D, C, A from *agrB*, *D*, C, *A* genes as a components of agr system, and RNAIII transcript that include *hld* gene encodes the δ-hemolysin (termed δ-toxin or δ-PSM) [42]. RNAIII regulates the expression of agr-governed virulence factors such as CWA proteins as a surface proteins and exotoxins at transcriptional and translational level. Independently of RNAIII (RNAIII independent control), AgrA also directly regulates the expression of α-PSMs and β-PSMs by binding to their promoters in *psm* operon in *S. aureus* and involves in the downregulation of genes contribute carbohydrate and amino acid metabolism [44] (**Figure 2**).

**Figure 3.** The biofilm-embedded bacteria. (a) The heterogeneous sessile community of biofilm. (b) Antibiotic resistance mechanisms of biofilm-embedded bacteria.

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The regulation mechanisms of RNAIII for target genes can be at transcriptional and transla‐ tional level, andits regulation can bedirect orindirect. Fourteen stem-loop andtwo long helices construct structure of RNAIII. Each domain regulates the expression of each target gene. Translation of α-hemolysin (*hla*) upregulated by hairpin loop H2 and H3. In contrast to this, the repression of early expressed virulence genes of *S. aureus* such as coagulase, protein A, and the repressor of toxins (Rot) is comprised by hairpin H13, H14, and H7 of RNAIII. Hairpins such as H7, H13, and H14 that are complementary to Shine-Dalgarno sequences (SD) of target mRNA act as an antisense RNA and inhibit initiation of translation and cause RNAaseIIImediated degradation of target mRNA [45] (**Figure 4**).

**Figure 4.** The structure of RNAIII [44]

The biofilm formation of staphylococci is fully expressed *in vivo*, whereas the biofilm forma‐ tion of staphylococci is not fully expressed all the time *in vitro* unless nutrient supplementa‐ tions are added to growth media and is provided. Increased amount of biofilm formation due to fully expression occurs in stress conditions such as starvation, thermal stress, heat shock, salt, certain antibiotics, iron limitation, subinhibitory concentrations of ethanol, accumula‐ tions of metabolites, oxidative stress, low pH, and changes in osmolarity *in vitro*. Bacteria sense stimuli from the environment and bacterial density and then respond to stimuli by upregu‐ lating expression of biofilm formation, virulence factors production such as toxins, etc. [9].

Staphylococcus use quorum-sensing systems (QS) forintercellular communication and biofilm formation. Accessory gene regulator (Agr) system regulates cell density-dependent gene expression using two-component signal transduction system [42]. Agr and LuxS systems that are required for autoinducer peptide (AIP) production as a pheromone are quorum-sensing systems in staphylococci [43]. Bacteria sense pheromones as stimuli that are released by the density of bacteria belonging to the same group and express biofilm formation [9]. AIP production starts in exponential phase of bacterial growth [44]. There are four proteins that are sensor histidine protein kinase AgrC, DNA-binding response regulator AgrA, AgrD that is a prepheromone, and AgrB that exports and modifies AgrD, present in this system. The signal is transported to bacteria by binding of AIP to AgrC. When AIP binds to AgrC, DNAbinding regulator AgrA is activated by His-dependent phosphorylation of AgrC [42]. By the binding of activated DNA-binding regulator AgrA to P2 and P3 promoters in *agr* operon (*agr*BDCA), RNAII and RNAIII are transcripted, respectively [44]. The *agr*BDCA operon codes RNAII transcript that encodes AgrB, D, C, A from *agrB*, *D*, C, *A* genes as a components of agr system, and RNAIII transcript that include *hld* gene encodes the δ-hemolysin (termed δ-toxin or δ-PSM) [42]. RNAIII regulates the expression of agr-governed virulence factors such as CWA proteins as a surface proteins and exotoxins at transcriptional and translational level. Independently of RNAIII (RNAIII independent control), AgrA also directly regulates the expression of α-PSMs and β-PSMs by binding to their promoters in *psm* operon in *S. aureus* and involves in the downregulation of genes contribute carbohydrate and amino acid

**Figure 3.** The biofilm-embedded bacteria. (a) The heterogeneous sessile community of biofilm. (b) Antibiotic resistance

metabolism [44] (**Figure 2**).

196 Microbial Biofilms - Importance and Applications

mechanisms of biofilm-embedded bacteria.

Staphylococcal virulence factors are expressed with accessory gene regulator (agr) system in response to cell density [9]. During the beginning of the biofilm-related staphylococcal infection, adhesion factors (surface proteins) such as MSCRAMMs are upregulated. After initial attachment and colonization had been happened, during early stationary growth phase

of bacteria, toxins and other acute virulence factors such as degradative exoenzymes (such as δ-hemolysin, lipases and proteases that disperse bacteria) are upregulated and nonaggressive colonization surface proteins such as MSCRAMMs are downregulated by *agr*-QS regulator system [1, 46]. Adherence is reducedbydownregulatedgenes of CWA, due to surface proteins are no longer needed after colonization, by the way initial biofilm formation is decreased indirectly [5]. Expression of staphylococcal toxins such as enterotoxin B, toxic shock syndrome toxin-1, exfoliative toxins, fibrinolysin, α, β, γ, and δ hemolysins, other phenolsoluble modulins (PSMs), leucocidin, capsular polysaccharide (type 5 and 8), serine protease, and DNase is increased (upregulated), and expression of surface proteins and biofilm formation is decreased (downregulated) by *agr* of *S. aureus* and *S. epidermidis* [9, 44]. Infec‐ tion is dispersed to other surfaces by the detachment of biofilm that is caused by the upregu‐ lation of the expression of PSMs that have an important role in acute infection [1]. In chronic biofilm-associated infection of *S. aureus* high amount of QS or *psm* gene mutants are present, by the way, mutants favor compact biofilm development and biofilm/infection cannot be dispersed to other surfaces [46, 47].

To control biofilm-associated staphylococcal infections, production of virulence factors and antibiotic resistance, QS can be disrupted by inhibition of signal production, degrading signals,

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Two-component regulator gene locus encoded by arlRS is regulated by *agr* and *sarA* loci. *sar*A and *agr* have opposite functions in staphylococcal global regulation. When enough quorum population is present, at the beginning of attachment phase *sar*A is upregulated. During the initial stages, SarA enhances expression of PIA, adhesions, and EPS, by the way, induces attachment and early biofilm formation. SarA also represses nuclease and protease synthesis. After attachment of bacteria, agr system works and virulence factors that cause dispersal,

The *sigB* operon of which product is σ<sup>B</sup> in *S. aureus* upregulates ica transcription, and the factors for early stages of biofilm formation including FnbpA, clumbing factor, and coagulase and downregulates factors that are efficient in dispersal and in passing to planktonic state such as β-hemolysin, enterotoxin B, serine protease (SplA), cysteine protease (SplB), the metallo‐

The biofilm formation of *S. epidermidis* [57] and *S. aureus* [58] can be also regulated by ArIRS that uses TCS. The biofilm formation of *S. epidermidis* is regulated by ArIRS in ica-dependent manner, whereas in *S. aureus*, this is ica-independent manner [59]. ArlRS also plays a role in the modulation of bacterial autolysis, as a result of eDNA release that participates in biofilm

*LytSR* operon that is the other TCS of *S. aureus* plays a role in the activity of murein hydro‐ lase that is an autolysin and distrupt structural components of the bacterial cell wall, conse‐ quently, autolysis. *Lrg/cid* operon that is a target of this system regulates lysis of cell during biofilm formation [60]. The regulator LytR that is effected by stimuli bound LytS sensor histidine kinase protein activates transcription of genes under its control. The regulator LytR upregulates the expression of *lrgA* and *lrgB* genes [61]. Encoded LrgA by *lrgA* is an antiholin and inhibits the extracellular activity of murein hydrolases, whereas *cidA* gene encodes holin protein that effects the activity of murein hydrolase, consequently, cell lysis and release of

and suppressing synthase and receptors [9].

*2.5.2.1. sarA*

*2.5.2.2. sigB*

*2.5.2.3. ArIRS*

matrix [9].

*2.5.2.4. lytSR*

*2.5.2. The regulation of Staphylococcal biofilm by other than Agr*

nucleases and proteases and PSMs are produced [18].

protease Aur, staphopain, and leukotoxin D [18].

eDNA that participate in biofilm matrix [9].

The production of PIA/PNAG, PIA/PNAG-degrading enzymes, and matrix components of staphylococcal biofilm is not regulated by QS [44, 46].

Phenol-soluble modulins (PSMs) are surfactant-like staphylococcal peptides and are control‐ led by *agr* locus function in biofilm maturation, biofilm structuring/destructuring, dispersal, and dissemination by distruption of non-covalent interactions between biofilm matrix molecules. PSMs have a role in the pathogenesis of *S. aureus* and *S. epidermidis* biofilmassociated infections [9, 21, 46]. In contrast to soluble PSMs, PSMs that are aggregated form amyloid fibrils that contribute to stability of the biofilm [27, 41]. *S. aureus* and *S. epidermidis* catheter-related infections can be controlled by PSM surfactant-mediated QS control of biofilms for biofilm maturation and dissemination [48, 49]. The biofilm maturation is not only caused by PSM surfactants but also enzymatic degradation of biofilm matrix components by proteases and nucleases [46]. But Beenken et al. [50] revealed that nuclease did not disperse *S. aureus in vitro*. Hochbaum et al. [51] revealed that D-amino acids trigger biofilm dispersal of *S. aureus*.

Agr (AIPs) of each strain belongs to different *agr* classes of which biofilm-forming capacities and syndromes are different. Four main classes of AIPs (Agr) are present in *S. aureus* and *S. epidermidis*. *S. aureus* strains of which *agr* classes are *agr* II and *agr* III are high and medium biofilm formers due to having defective and inactive *agr*, respectively. Non-defective and active *agr* is present in *agr* I and *agr* IV strains that are weak biofilm producers [52]. *agr* IV *S. aureus* strains are more associated with exfoliative syndromes. *agr*I *S. aureus* strains are isolated from endocarditis and superficial infections. *agr*II and *agr*III *S. aureus* strains are isolated from endocarditis and nasal colonization, respectively [53]. Mortality due to *agr* II–caused infec‐ tions is higher than *agr* I–caused infections [54]. The prevalences of *agr* I type among the *S. epidermidis* clinical isolates and *S. epidermidis* localized in skin flora are approximately 89% and 52%, respectively [55]. The sequences of AIPs that belong to *agr* I, II, III, and IV classes in *S. aureus* and *S. epidermidis* are YSTCDFTM, GVNACSSLF, YINCDFLL, YSTCYFTM, YNPCA‐ SYL, DSVCASYF, YNPCSNYL, YNPCANYL, respectively [55, 56].

To control biofilm-associated staphylococcal infections, production of virulence factors and antibiotic resistance, QS can be disrupted by inhibition of signal production, degrading signals, and suppressing synthase and receptors [9].

### *2.5.2. The regulation of Staphylococcal biofilm by other than Agr*

### *2.5.2.1. sarA*

of bacteria, toxins and other acute virulence factors such as degradative exoenzymes (such as δ-hemolysin, lipases and proteases that disperse bacteria) are upregulated and nonaggressive colonization surface proteins such as MSCRAMMs are downregulated by *agr*-QS regulator system [1, 46]. Adherence is reducedbydownregulatedgenes of CWA, due to surface proteins are no longer needed after colonization, by the way initial biofilm formation is decreased indirectly [5]. Expression of staphylococcal toxins such as enterotoxin B, toxic shock syndrome toxin-1, exfoliative toxins, fibrinolysin, α, β, γ, and δ hemolysins, other phenolsoluble modulins (PSMs), leucocidin, capsular polysaccharide (type 5 and 8), serine protease, and DNase is increased (upregulated), and expression of surface proteins and biofilm formation is decreased (downregulated) by *agr* of *S. aureus* and *S. epidermidis* [9, 44]. Infec‐ tion is dispersed to other surfaces by the detachment of biofilm that is caused by the upregu‐ lation of the expression of PSMs that have an important role in acute infection [1]. In chronic biofilm-associated infection of *S. aureus* high amount of QS or *psm* gene mutants are present, by the way, mutants favor compact biofilm development and biofilm/infection cannot be

The production of PIA/PNAG, PIA/PNAG-degrading enzymes, and matrix components of

Phenol-soluble modulins (PSMs) are surfactant-like staphylococcal peptides and are control‐ led by *agr* locus function in biofilm maturation, biofilm structuring/destructuring, dispersal, and dissemination by distruption of non-covalent interactions between biofilm matrix molecules. PSMs have a role in the pathogenesis of *S. aureus* and *S. epidermidis* biofilmassociated infections [9, 21, 46]. In contrast to soluble PSMs, PSMs that are aggregated form amyloid fibrils that contribute to stability of the biofilm [27, 41]. *S. aureus* and *S. epidermidis* catheter-related infections can be controlled by PSM surfactant-mediated QS control of biofilms for biofilm maturation and dissemination [48, 49]. The biofilm maturation is not only caused by PSM surfactants but also enzymatic degradation of biofilm matrix components by proteases and nucleases [46]. But Beenken et al. [50] revealed that nuclease did not disperse *S. aureus in vitro*. Hochbaum et al. [51] revealed that D-amino acids trigger biofilm dispersal of

Agr (AIPs) of each strain belongs to different *agr* classes of which biofilm-forming capacities and syndromes are different. Four main classes of AIPs (Agr) are present in *S. aureus* and *S. epidermidis*. *S. aureus* strains of which *agr* classes are *agr* II and *agr* III are high and medium biofilm formers due to having defective and inactive *agr*, respectively. Non-defective and active *agr* is present in *agr* I and *agr* IV strains that are weak biofilm producers [52]. *agr* IV *S. aureus* strains are more associated with exfoliative syndromes. *agr*I *S. aureus* strains are isolated from endocarditis and superficial infections. *agr*II and *agr*III *S. aureus* strains are isolated from endocarditis and nasal colonization, respectively [53]. Mortality due to *agr* II–caused infec‐ tions is higher than *agr* I–caused infections [54]. The prevalences of *agr* I type among the *S. epidermidis* clinical isolates and *S. epidermidis* localized in skin flora are approximately 89% and 52%, respectively [55]. The sequences of AIPs that belong to *agr* I, II, III, and IV classes in *S. aureus* and *S. epidermidis* are YSTCDFTM, GVNACSSLF, YINCDFLL, YSTCYFTM, YNPCA‐

SYL, DSVCASYF, YNPCSNYL, YNPCANYL, respectively [55, 56].

dispersed to other surfaces [46, 47].

198 Microbial Biofilms - Importance and Applications

*S. aureus*.

staphylococcal biofilm is not regulated by QS [44, 46].

Two-component regulator gene locus encoded by arlRS is regulated by *agr* and *sarA* loci. *sar*A and *agr* have opposite functions in staphylococcal global regulation. When enough quorum population is present, at the beginning of attachment phase *sar*A is upregulated. During the initial stages, SarA enhances expression of PIA, adhesions, and EPS, by the way, induces attachment and early biofilm formation. SarA also represses nuclease and protease synthesis. After attachment of bacteria, agr system works and virulence factors that cause dispersal, nucleases and proteases and PSMs are produced [18].

#### *2.5.2.2. sigB*

The *sigB* operon of which product is σ<sup>B</sup> in *S. aureus* upregulates ica transcription, and the factors for early stages of biofilm formation including FnbpA, clumbing factor, and coagulase and downregulates factors that are efficient in dispersal and in passing to planktonic state such as β-hemolysin, enterotoxin B, serine protease (SplA), cysteine protease (SplB), the metallo‐ protease Aur, staphopain, and leukotoxin D [18].

#### *2.5.2.3. ArIRS*

The biofilm formation of *S. epidermidis* [57] and *S. aureus* [58] can be also regulated by ArIRS that uses TCS. The biofilm formation of *S. epidermidis* is regulated by ArIRS in ica-dependent manner, whereas in *S. aureus*, this is ica-independent manner [59]. ArlRS also plays a role in the modulation of bacterial autolysis, as a result of eDNA release that participates in biofilm matrix [9].

### *2.5.2.4. lytSR*

*LytSR* operon that is the other TCS of *S. aureus* plays a role in the activity of murein hydro‐ lase that is an autolysin and distrupt structural components of the bacterial cell wall, conse‐ quently, autolysis. *Lrg/cid* operon that is a target of this system regulates lysis of cell during biofilm formation [60]. The regulator LytR that is effected by stimuli bound LytS sensor histidine kinase protein activates transcription of genes under its control. The regulator LytR upregulates the expression of *lrgA* and *lrgB* genes [61]. Encoded LrgA by *lrgA* is an antiholin and inhibits the extracellular activity of murein hydrolases, whereas *cidA* gene encodes holin protein that effects the activity of murein hydrolase, consequently, cell lysis and release of eDNA that participate in biofilm matrix [9].

### *2.5.3. Inactivation of ica by sequences*

### *2.5.3.1. IS256*

Although *S. epidermidis* strains are *ica* positive, they cannot produce biofilm due to IS256 insertion sequence that is inserted within the *ica* operon. Ziebuhr et al.[62] revealed that if bacterial genomicDNAcontainedIS256, IS256 was not seen within *ica* locus. They also revealed that although *S. epidermidis* strains that caused indwelling device–associated infection was *ica* positive and the insertion of IS256 is not seen within *ica* locus, strains did not produce biofilm ("off switch") [62]. These results showed that IS256 is not a natural occurring global regulator mechanism of biofilm production. The similarresults were gained for *S. aureus*. IS256 that was inserted within *ica*C gene of *S. aureus* strain prevented biofilm formation by inacti‐ vating *ica*C gene [63].

enzymes) and disinfectants such as triclosan with antibiotics that are used in the treatment of

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Biofilm-embedded bacteria are more resistant to antimicrobial agents than planktonic bacteria.

Antibiotic resistance (tolerance) that is caused by biofilm and permit bacteria to survive is a physiological state by which mutational changes not caused [73]. Impermeability of peptido‐ glycan by efflux pumps, antibiotic-degrading enzymes, the charge of polymers [73], and certain gene products that are produced in biofilms [3] are the other antibiotic resistance mechanisms of bacteria rather than the biofilm [3]. Biofilm can gain higher antibiotic toler‐ ance by antibiotic degrading enzymes such as beta-lactamases, efflux pumps, and certain gene products of which expression are changed by the quorum sensing as a stress response [3, 74]. Biofilms resist to beta-lactam antibiotics by beta-lactamases. Beta-lactamases that are pro‐ duced by bacteria play a key factor in the biofilm caused resistance to beta-lactam antibiotics

Biofilm-embedded sessile community has heterogeneous cells that are in the different growth states. Bacterial growth rate is reduced by stress conditions such as nutrient and oxygen limitation atthe lower parts ofthe biofilm, and low metabolic activity. Low metabolically active cells (slow growing cells) are seen at the deeper parts of the biofilm, whereas high metabol‐ ically active cells (rapid growing cells) are seen at the surfaces of the biofilm. These heteroge‐ neous cells that consist of low and high metabolically active cells have wide range of different responds to each antimicrobial. Antibiotic penetration through the biofilm is reduced by reduced bacterial growth rate. The biofilm-related resistance mechanisms such as oxygen limitation and low metabolic activity, reduced antibiotic penetration through the biofilm, and gaining genetic adaptations such as increased changes in the genes of the DNA repair systems play a key factor in the biofilm tolerance to antibiotics [3]. But some antibiotics such as colistin are just effective against slow-growing cells seen at the deeper parts of the biofilm not against rapid growing cells that acquired adaptive resistance by upregulation of the LPS-modifica‐ tion (arn) operon [75]. Persister cell population that is present in the biofilms of *S. epidermidis*

Some researchers demonstrated that nutrient limitation-related antibiotic resistance is not due to the reduced growth rate of microorganism, but rather to the activation of regulated stress responses. Nutrient limitation-related antibiotic resistance is controlled by complex regulato‐

wound and skin infections provides synergistic removal of biofilms [71].

**microorganism**

[3].

**3.2. Nutrient limitation**

**3. The mechanisms of antibiotic resistance in biofilm-embedded**

It is difficult to eradicate biofilm, and this causes serious clinical problem [72].

**3.1. The heterogeneous sessile community and the physiology of biofilm**

can withstand to inhibitory concentrations of antibiotics [76] (**Figure 3**).

#### *2.5.3.2. Tetranucleotide tandem repeat*

*ica*C inactivation caused by the expansion or contraction of tetranucleotide tandem repeat inhibits PIA/PNAG formation in *S. aureus* [64]. The reading frame of *ica*C is shifted by tetranucleotide tandem repeat ("ttta"), and this contributes premature stop of IcaC protein, consequently, inhibited PIA/PNAG production ("off switch"). Mutated *ica*C is preferred for the indwelling device-associated infections due to off switching of PIA/PNAG production.

### **2.6. Treatment of biofilm**

To provide protection against *S. aureus* and *S. epidermidis* biofilm-associated infections vaccine that causes production of antibodies against PNAG and PSM peptides can be used. Research‐ ers had revealed that mutant *S. aureus* of which icaB is over-expressed and produces high amount of surface associated PNAG was more opsonized by antibodies and undergoes to phagocytosis. But immune response is ineffective antibodies produced against PIA/PNAG of vaccine bind secreted PIA/PNAG of bacteria rather than surface-associated PIA/PNAG of bacteria [65]. Conjugate vaccine that contains *S. aureus* PNAG and clumping factor A can accelerate immune response [66]. Bacterial dispersal from indwelling medical devices can be prevented by antibodies against PSM peptides [48]. Brady et al. [67] had treated chronic osteomyelitis with a combination of antibiotic and quadrivalent vaccine that contains four antigens, which are glucosaminidase, an ABC transporter lipoprotein, a conserved hypothet‐ ical protein, and a conserved lipoprotein. By this way, Brady et al. [67] had reduced biofilm formation of *S. aureus* on infected tibias.

Kaplan et al. [68] and Whitchurch et al. [69] concluded that DNase I in human serum can degrade eDNA in biofilm matrix, by the way bacterial biofilms are degreased.

Nitric oxide (NO) that is a product of anaerobic respiration can cause dispersal of microor‐ ganism from mature biofilm by stimulation of c-di-GMP phosphodiesterases activity [70]. cdi-GMP biosynthesis inhibitors can be an alternative treatment for preventing biofilm formation and mature biofilm dispersal. The combinations of dispersin B (EPS-degrading enzymes) and disinfectants such as triclosan with antibiotics that are used in the treatment of wound and skin infections provides synergistic removal of biofilms [71].

### **3. The mechanisms of antibiotic resistance in biofilm-embedded microorganism**

Biofilm-embedded bacteria are more resistant to antimicrobial agents than planktonic bacteria. It is difficult to eradicate biofilm, and this causes serious clinical problem [72].

Antibiotic resistance (tolerance) that is caused by biofilm and permit bacteria to survive is a physiological state by which mutational changes not caused [73]. Impermeability of peptido‐ glycan by efflux pumps, antibiotic-degrading enzymes, the charge of polymers [73], and certain gene products that are produced in biofilms [3] are the other antibiotic resistance mechanisms of bacteria rather than the biofilm [3]. Biofilm can gain higher antibiotic toler‐ ance by antibiotic degrading enzymes such as beta-lactamases, efflux pumps, and certain gene products of which expression are changed by the quorum sensing as a stress response [3, 74]. Biofilms resist to beta-lactam antibiotics by beta-lactamases. Beta-lactamases that are pro‐ duced by bacteria play a key factor in the biofilm caused resistance to beta-lactam antibiotics [3].

#### **3.1. The heterogeneous sessile community and the physiology of biofilm**

Biofilm-embedded sessile community has heterogeneous cells that are in the different growth states. Bacterial growth rate is reduced by stress conditions such as nutrient and oxygen limitation atthe lower parts ofthe biofilm, and low metabolic activity. Low metabolically active cells (slow growing cells) are seen at the deeper parts of the biofilm, whereas high metabol‐ ically active cells (rapid growing cells) are seen at the surfaces of the biofilm. These heteroge‐ neous cells that consist of low and high metabolically active cells have wide range of different responds to each antimicrobial. Antibiotic penetration through the biofilm is reduced by reduced bacterial growth rate. The biofilm-related resistance mechanisms such as oxygen limitation and low metabolic activity, reduced antibiotic penetration through the biofilm, and gaining genetic adaptations such as increased changes in the genes of the DNA repair systems play a key factor in the biofilm tolerance to antibiotics [3]. But some antibiotics such as colistin are just effective against slow-growing cells seen at the deeper parts of the biofilm not against rapid growing cells that acquired adaptive resistance by upregulation of the LPS-modifica‐ tion (arn) operon [75]. Persister cell population that is present in the biofilms of *S. epidermidis* can withstand to inhibitory concentrations of antibiotics [76] (**Figure 3**).

#### **3.2. Nutrient limitation**

*2.5.3. Inactivation of ica by sequences*

200 Microbial Biofilms - Importance and Applications

Although *S. epidermidis* strains are *ica* positive, they cannot produce biofilm due to IS256 insertion sequence that is inserted within the *ica* operon. Ziebuhr et al.[62] revealed that if bacterial genomicDNAcontainedIS256, IS256 was not seen within *ica* locus. They also revealed that although *S. epidermidis* strains that caused indwelling device–associated infection was *ica* positive and the insertion of IS256 is not seen within *ica* locus, strains did not produce biofilm ("off switch") [62]. These results showed that IS256 is not a natural occurring global regulator mechanism of biofilm production. The similarresults were gained for *S. aureus*. IS256 that was inserted within *ica*C gene of *S. aureus* strain prevented biofilm formation by inacti‐

*ica*C inactivation caused by the expansion or contraction of tetranucleotide tandem repeat inhibits PIA/PNAG formation in *S. aureus* [64]. The reading frame of *ica*C is shifted by tetranucleotide tandem repeat ("ttta"), and this contributes premature stop of IcaC protein, consequently, inhibited PIA/PNAG production ("off switch"). Mutated *ica*C is preferred for the indwelling device-associated infections due to off switching of PIA/PNAG production.

To provide protection against *S. aureus* and *S. epidermidis* biofilm-associated infections vaccine that causes production of antibodies against PNAG and PSM peptides can be used. Research‐ ers had revealed that mutant *S. aureus* of which icaB is over-expressed and produces high amount of surface associated PNAG was more opsonized by antibodies and undergoes to phagocytosis. But immune response is ineffective antibodies produced against PIA/PNAG of vaccine bind secreted PIA/PNAG of bacteria rather than surface-associated PIA/PNAG of bacteria [65]. Conjugate vaccine that contains *S. aureus* PNAG and clumping factor A can accelerate immune response [66]. Bacterial dispersal from indwelling medical devices can be prevented by antibodies against PSM peptides [48]. Brady et al. [67] had treated chronic osteomyelitis with a combination of antibiotic and quadrivalent vaccine that contains four antigens, which are glucosaminidase, an ABC transporter lipoprotein, a conserved hypothet‐ ical protein, and a conserved lipoprotein. By this way, Brady et al. [67] had reduced biofilm

Kaplan et al. [68] and Whitchurch et al. [69] concluded that DNase I in human serum can

Nitric oxide (NO) that is a product of anaerobic respiration can cause dispersal of microor‐ ganism from mature biofilm by stimulation of c-di-GMP phosphodiesterases activity [70]. cdi-GMP biosynthesis inhibitors can be an alternative treatment for preventing biofilm formation and mature biofilm dispersal. The combinations of dispersin B (EPS-degrading

degrade eDNA in biofilm matrix, by the way bacterial biofilms are degreased.

*2.5.3.1. IS256*

vating *ica*C gene [63].

**2.6. Treatment of biofilm**

formation of *S. aureus* on infected tibias.

*2.5.3.2. Tetranucleotide tandem repeat*

Some researchers demonstrated that nutrient limitation-related antibiotic resistance is not due to the reduced growth rate of microorganism, but rather to the activation of regulated stress responses. Nutrient limitation-related antibiotic resistance is controlled by complex regulato‐

ry pathways [77]. During starvation, the activation of the stringent response participates in antibiotic resistance such as fluoroquinolone resistance in *E. coli* biofilms [23]. Also, some researchers demonstrated that certain efflux pumps in *P. aeruginosa* are upregulated in the lowoxygen conditions [78] (**Figure 3**).

**References**

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[2] Stoodley P, Sauer K, Davies DG and Costerton JW. Biofilms as complex differentiat‐

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### **3.3. Biofilm matrix**

Usually, the decreased antibiotic penetration through the biofilm is caused by antibiotics that may bind to the structural contents of biofilm matrix [3] rather than reduced diffusion of antibiotics through the biofilm matrix [10] (**Figure 3**).

### **3.4. Agr expression**

Antibiotic susceptibility of biofilm-embedded bacteria decreases according to the planktonic state. The virulence of *agr* defective strains is lesser than the wild type. Expression of *agr* that imposes a fitness cost on *S*. *aureus* effects drug resistance of staphylococcal biofilm. It has been revealed that RNAIII production (provides fitness cost of bacteria) of *agr*-positive bacteria is induced by subletal doses of ciprofloxacin, mupirocin, and rifampin [79]. The adaptability of *S*. *aureus* to antibiotics involves the *agr* locus. *S*. *aureus* resists to drugs by adapting to antibi‐ otics with *agr* locus. Ciprofloxacin, mupirocin, and rifampin are more effective against *agr*defective bacteria. These antibiotics just must be used in *agr*-deficient mutants or *agr*-negative *S*. *aureus* when designing antimicrobial chemotherapy. *agr*-defective strains are isolated frequently in hospital-acquired *S*. *aureus* (HA-*S. aureus*) infections. Due to broad antibiotic usage in hospitals, the prevalence of agr-defective strains among hospital-acquired *S. aureus* infections is high and ranges between 15% and 60% [80].

Agr expression of biofilm producer staphylococcus has also been associated with the drug resistance of some antibiotics. It has been also revealed that the effect of rifampin against *agr*defective *S. aureus* mutants was increased, whereas the effect of oxacilline unchanged [79]. *agr* negative or *agr* dysfunction strains have a fitness advantage over *agr* positive strains in the presence of some antibiotics such as vancomycin. Vancomycin susceptibility is reduced in VISA (vancomycin-intermediate *S*. *aureus*) due to the thickening of cell wall that is the result of the combination of cell wall biosynthesis activation and decreased autolytic activity. *agr* mutations have been correlated with the rise of VISA. *agr* defects thatreduce autolysis decrease susceptibility of vancomycin of VISA [81].

### **Author details**

#### Sahra Kırmusaoğlu

Address all correspondence to: kirmusaoglu\_sahra@hotmail.com

Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, T.C. Haliç University, Istanbul, Turkey

### **References**

ry pathways [77]. During starvation, the activation of the stringent response participates in antibiotic resistance such as fluoroquinolone resistance in *E. coli* biofilms [23]. Also, some researchers demonstrated that certain efflux pumps in *P. aeruginosa* are upregulated in the low-

Usually, the decreased antibiotic penetration through the biofilm is caused by antibiotics that may bind to the structural contents of biofilm matrix [3] rather than reduced diffusion of

Antibiotic susceptibility of biofilm-embedded bacteria decreases according to the planktonic state. The virulence of *agr* defective strains is lesser than the wild type. Expression of *agr* that imposes a fitness cost on *S*. *aureus* effects drug resistance of staphylococcal biofilm. It has been revealed that RNAIII production (provides fitness cost of bacteria) of *agr*-positive bacteria is induced by subletal doses of ciprofloxacin, mupirocin, and rifampin [79]. The adaptability of *S*. *aureus* to antibiotics involves the *agr* locus. *S*. *aureus* resists to drugs by adapting to antibi‐ otics with *agr* locus. Ciprofloxacin, mupirocin, and rifampin are more effective against *agr*defective bacteria. These antibiotics just must be used in *agr*-deficient mutants or *agr*-negative *S*. *aureus* when designing antimicrobial chemotherapy. *agr*-defective strains are isolated frequently in hospital-acquired *S*. *aureus* (HA-*S. aureus*) infections. Due to broad antibiotic usage in hospitals, the prevalence of agr-defective strains among hospital-acquired *S. aureus*

Agr expression of biofilm producer staphylococcus has also been associated with the drug resistance of some antibiotics. It has been also revealed that the effect of rifampin against *agr*defective *S. aureus* mutants was increased, whereas the effect of oxacilline unchanged [79]. *agr* negative or *agr* dysfunction strains have a fitness advantage over *agr* positive strains in the presence of some antibiotics such as vancomycin. Vancomycin susceptibility is reduced in VISA (vancomycin-intermediate *S*. *aureus*) due to the thickening of cell wall that is the result of the combination of cell wall biosynthesis activation and decreased autolytic activity. *agr* mutations have been correlated with the rise of VISA. *agr* defects thatreduce autolysis decrease

oxygen conditions [78] (**Figure 3**).

202 Microbial Biofilms - Importance and Applications

antibiotics through the biofilm matrix [10] (**Figure 3**).

infections is high and ranges between 15% and 60% [80].

Address all correspondence to: kirmusaoglu\_sahra@hotmail.com

Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, T.C. Haliç

susceptibility of vancomycin of VISA [81].

**Author details**

Sahra Kırmusaoğlu

University, Istanbul, Turkey

**3.3. Biofilm matrix**

**3.4. Agr expression**


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208 Microbial Biofilms - Importance and Applications

2009;191:7333–7342.

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Drug Res. 1991;37:91–105.

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2011;334(6058):982–986.


**Chapter 11**

*Staphylococcus* **Biofilms**

http://dx.doi.org/10.5772/62910

**Abstract**

microorganisms.

anti-biofilm

**I.** *Staphylococcus* **biofilms**

**Introduction**

Janet Jan-Roblero, Sandra Rodríguez-Martínez, Mario E. Cancino-Diaz and Juan C. Cancino-Diaz

Additional information is available at the end of the chapter

The majority of staphylococci produce biofilm on medical devices, which is the main mechanism to infect humans. Staphylococcal biofilms attach to abiotic or biotic surfaces, forming aggregates and protecting themselves against the immune system and the antimicrobial compounds of the host. Few studies on biofilm formation mechanism in *Staphylococcus epidermidis* and other coagulase-negative staphylococci (CNS) have been performed;however,thereisagreatinterestinstudyingandcontrollingbiofilmformation ofthisgenus.This chapter exhibits the stateofthe artonbiofilmformationin*S.epidermidis* and other staphylococcal species. The main goal of this chapter is to recognize the importance of biofilm formation in *Staphylococcus*. The participating molecules in staphylococcal biofilm formation are described. Currently, biofilm producer strains of *Staphylococcus* and mainly CNS have been frequently isolated at hospitals, causing significant economic losses.This chapterincludespromisingsolutions inordertoprevent medical device-associated infections, as the development of medical devices possessing anti-biofilm materials or surfaces that act against the adhesion or viability of the

**Keywords:** Biofilm, *Staphylococcus epidermidis*, *Staphylococcus aureus*, medical devices,

During the last years, the study of biofilms has become relevant due to their significance on many microbiology areas. In the health field, biofilms have been of great relevance because many pathogenic and non-pathogenic bacteria can produce biofilm as a part of its virulence

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. 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,

### **Chapter 11**

## *Staphylococcus* **Biofilms**

Janet Jan-Roblero, Sandra Rodríguez-Martínez, Mario E. Cancino-Diaz and Juan C. Cancino-Diaz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62910

#### **Abstract**

The majority of staphylococci produce biofilm on medical devices, which is the main mechanism to infect humans. Staphylococcal biofilms attach to abiotic or biotic surfaces, forming aggregates and protecting themselves against the immune system and the antimicrobial compounds of the host. Few studies on biofilm formation mechanism in *Staphylococcus epidermidis* and other coagulase-negative staphylococci (CNS) have been performed;however,thereisagreatinterestinstudyingandcontrollingbiofilmformation ofthisgenus.This chapter exhibits the stateofthe artonbiofilmformationin*S.epidermidis* and other staphylococcal species. The main goal of this chapter is to recognize the importance of biofilm formation in *Staphylococcus*. The participating molecules in staphylococcal biofilm formation are described. Currently, biofilm producer strains of *Staphylococcus* and mainly CNS have been frequently isolated at hospitals, causing significant economic losses.This chapterincludespromisingsolutions inordertoprevent medical device-associated infections, as the development of medical devices possessing anti-biofilm materials or surfaces that act against the adhesion or viability of the microorganisms. performed; however, there is a great interest in studying and controlling biofilm formationof this genus. This chapter exhibits the state of the art on biofilm formation in *S. epidermidis*significant economic losses. This chapter includes promising solutions in order to prevent

**Keywords:** Biofilm, *Staphylococcus epidermidis*, *Staphylococcus aureus*, medical devices, anti-biofilm *Staphylococcus Staphylococcus* medical

### **Introduction**

#### **I.** *Staphylococcus* **biofilms**

During the last years, the study of biofilms has become relevant due to their significance on many microbiology areas. In the health field, biofilms have been of great relevance because many pathogenic and non-pathogenic bacteria can produce biofilm as a part of its virulence

© 2016 The Author(s). Licensee InTech. 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, provided the original work is properly cited.

mechanism and protection against the host. A biofilm is considered a complex microbial community (or communities) attached to a defined surface and embedded within a cell matrix. Regarding the surface, biofilms may be formed on a wide variety of chemical or biological surfaces. Regarding bacteria of the *Staphylococcus* genus, biofilm is the main virulence mecha‐ nism of the coagulase-negative staphylococci (CNS) species. Biofilm formation in staphylococ‐ ci is carried out in at least three stages: i) bacterial attachment to a defined surface, a process termed primary attachment; ii) assembly of these originating bacteria into a small cluster, also known as microcolony or cellular accumulation; and iii) biofilm growth and disassembly (also known as detachment or dispersal) mediated by a mechanical process or by active metabo‐ lites produced by the biofilm-embedded bacteria.

*epidermidis* isolates with both positive- and negative-biofilm phenotypes failed to show evidence that could demonstrate that biofilm-forming isolates are more virulent in compar‐ ing with those possessing a biofilm-negative phenotype. Nevertheless, compelling results were obtained on subsequent studies using genetically defined strains and comparing the wild-type strain with its respective isogenic mutant strain. Using a mouse model of subcuta‐ neous catheter infection and a rat model of venous catheter infection, the polysaccharide intercellular adhesion (PIA)-producing *S. epidermidis* 1457 strain was more virulent than its isogenic counterpart, the biofilm-negative 1457-M10 strain [3]. In a different model of CVC infection, the *icaRADBC*-expressing *S. epidermidis* strain and its *icaRADBC*-negative isogenic mutant displayed the same result [4]. An infection model of *Caenorhabditis elegans* was used in order to study the biofilm-positive phenotype of the *S. epidermidis* 9142 strain, in comparison to the *icaA* mutant, resulting in a higher virulence of the wild type than the mutant [5]. Recently, using a catheter infection model, *icaADBC* inactivation apparently had no effect on coloniza‐ tion, whereas *aap* inactivation completely abolished *S. epidermidis* ability to establish the infection [6]. One explanation for the null pathogenicity of the mutant strain regarding biofilm formation is the lack of protection against the innate immune system. Experiments conduct‐ ed with cell culture showed that the biofilm-positive 1457 strain was less susceptible to antimicrobial peptides (AMPs) and to phagocytosis performed by polymorphonuclear granulocytes (PMNs) compared to the biofilm-negative 1457-M10 isogenic strain [7]. Physiological status is also important, when *S. epidermidis* 1457 grown on biofilm conditions

*Staphylococcus* Biofilms http://dx.doi.org/10.5772/62910 213

was less susceptible to phagocytes than it was grown on planktonic conditions [8].

contribute to the chronic persistence of *S. epidermidis* in inflammatory conditions.

**II. Mechanisms and molecules participating in staphylococci biofilm formation**

grown either in biofilm conditions or in planktonic conditions [9].

PIA-dependent biofilm formation also interferes with the host's complement activation. Biofilm-positive wild-type bacteria pre-opsonized with normal human serum are more resistant to complement-mediated elimination than the corresponding biofilm-negative isogenic bacteria [8]. It has been also shown that *S. epidermidis* biofilm formation interferes with the phagocytosis process and macrophage activation. This biofilm-forming phenotype may

Conversely, *S. epidermidis* produces a set of pro-inflammatory peptides termed phenol-soluble modulins (PSMs), which are produced in a tightly regulated manner by the accessory gene regulator (*agr*) system. It has been demonstrated that PSM δ is able to lyse neutrophils, supporting the concept that these peptides are relevant for *S. epidermidis* pathogenesis. However, PSM δ is expressed at low levels by the biofilm-producing *S. epidermidis* 1457 strain,

In this chapter, we will divide the study of the biofilm formation process in three phases. During primary attachment, bacteria adhere to the biotic or abiotic surface in order to colonize it, whereas on the accumulation phase, bacteria build a tridimensional multi-cell and multilayer array. Then, staphylococci are able to disassemble biofilm structure in order to release those cells capable to colonize other sites on the surface. *S. epidermidis* and *S. aureus* biofilm models have been the most studied among staphylococci and the overall biofilm formation process is very similar. In this chapter, we will address *S. epidermidis* biofilm as the base model.

#### *I.1. Medical and epidemiological relevance of staphylococci biofilms*

Staphylococci are commensal bacteria inhabiting the human skin and mucus. However, they have been identified as infection-causing agents associated to biofilms. Animal models of biofilm-associated infections using staphylococci have allowed to determine the importance of their biofilms as a virulence mechanism. Therefore, staphylococci, particularly *Staphylococ‐ cus epidermidis*, are currently the most studied microorganisms regarding their biofilm formation capacity. Nosocomial Infections Surveillance System recognizes that *Staphylococ‐ cus aureus* and CNS (e.g., *S. epidermidis* and most of the remaining staphylococci species) are the most frequent nosocomial pathogens isolated from patients at the intensive care unit. Epidemiological data show that CNS are the third most common infective agent causing native valve infective endocarditis (NVIE), and they occupy the first place in prosthetic valve infective endocarditis (PVIE), demonstrating their importance for these two clinical entities.

Regarding *S. epidermidis*, it is an inhabitant of the human skin microbiota. *S. epidermidis* is an opportunist pathogen that causes disease only in patients subjected to predisposing factors. This includes patients with particular features such as premature newborns, inborn immuno‐ logical impairments, or concomitant medical conditions, for example, human immunodefi‐ ciency virus (HIV) infection, immunosuppression after solid organ or bone marrow transplants, and chemotherapy-related neutropenia. Epidemiological data point out *S. epidermidis* as the most commonly isolated microorganism from foreign materials-related infections such as infected prosthetic joints, central venous catheters (CVC), cerebrospinal fluid shunts, intracardiac devices, artificial heart valves, and vascular grafts. Regarding prosthetic joints infections, *S. epidermidis* is the main infective agent of prosthetic joint implants. In UK, CNS and *S. epidermidis* are isolated from a 36% of total hip and 49% of total knee arthroplas‐ ty infections [1]. In an additional study on infected total hip and knee arthroplasties, it is pointed out that nearly 70% of the CNS isolates were identified as *S. epidermidis* [2].

### *I.2. Experimental models to study biofilm formation*

The clinical relevance of biofilm formation on foreign materials has been demonstrated using cell culture models, a *Caenorhabditis elegans* infection model, and animal models of device infections, for example, CVC or prosthetic device infection models. The first study on the importance of biofilm formation in vivo using animal models and genetically distinct *S.*

*epidermidis* isolates with both positive- and negative-biofilm phenotypes failed to show evidence that could demonstrate that biofilm-forming isolates are more virulent in compar‐ ing with those possessing a biofilm-negative phenotype. Nevertheless, compelling results were obtained on subsequent studies using genetically defined strains and comparing the wild-type strain with its respective isogenic mutant strain. Using a mouse model of subcuta‐ neous catheter infection and a rat model of venous catheter infection, the polysaccharide intercellular adhesion (PIA)-producing *S. epidermidis* 1457 strain was more virulent than its isogenic counterpart, the biofilm-negative 1457-M10 strain [3]. In a different model of CVC infection, the *icaRADBC*-expressing *S. epidermidis* strain and its *icaRADBC*-negative isogenic mutant displayed the same result [4]. An infection model of *Caenorhabditis elegans* was used in order to study the biofilm-positive phenotype of the *S. epidermidis* 9142 strain, in comparison to the *icaA* mutant, resulting in a higher virulence of the wild type than the mutant [5]. Recently, using a catheter infection model, *icaADBC* inactivation apparently had no effect on coloniza‐ tion, whereas *aap* inactivation completely abolished *S. epidermidis* ability to establish the infection [6]. One explanation for the null pathogenicity of the mutant strain regarding biofilm formation is the lack of protection against the innate immune system. Experiments conduct‐ ed with cell culture showed that the biofilm-positive 1457 strain was less susceptible to antimicrobial peptides (AMPs) and to phagocytosis performed by polymorphonuclear granulocytes (PMNs) compared to the biofilm-negative 1457-M10 isogenic strain [7]. Physiological status is also important, when *S. epidermidis* 1457 grown on biofilm conditions was less susceptible to phagocytes than it was grown on planktonic conditions [8].

mechanism and protection against the host. A biofilm is considered a complex microbial community (or communities) attached to a defined surface and embedded within a cell matrix. Regarding the surface, biofilms may be formed on a wide variety of chemical or biological surfaces. Regarding bacteria of the *Staphylococcus* genus, biofilm is the main virulence mecha‐ nism of the coagulase-negative staphylococci (CNS) species. Biofilm formation in staphylococ‐ ci is carried out in at least three stages: i) bacterial attachment to a defined surface, a process termed primary attachment; ii) assembly of these originating bacteria into a small cluster, also known as microcolony or cellular accumulation; and iii) biofilm growth and disassembly (also known as detachment or dispersal) mediated by a mechanical process or by active metabo‐

Staphylococci are commensal bacteria inhabiting the human skin and mucus. However, they have been identified as infection-causing agents associated to biofilms. Animal models of biofilm-associated infections using staphylococci have allowed to determine the importance of their biofilms as a virulence mechanism. Therefore, staphylococci, particularly *Staphylococ‐ cus epidermidis*, are currently the most studied microorganisms regarding their biofilm formation capacity. Nosocomial Infections Surveillance System recognizes that *Staphylococ‐ cus aureus* and CNS (e.g., *S. epidermidis* and most of the remaining staphylococci species) are the most frequent nosocomial pathogens isolated from patients at the intensive care unit. Epidemiological data show that CNS are the third most common infective agent causing native valve infective endocarditis (NVIE), and they occupy the first place in prosthetic valve infective

endocarditis (PVIE), demonstrating their importance for these two clinical entities.

pointed out that nearly 70% of the CNS isolates were identified as *S. epidermidis* [2].

The clinical relevance of biofilm formation on foreign materials has been demonstrated using cell culture models, a *Caenorhabditis elegans* infection model, and animal models of device infections, for example, CVC or prosthetic device infection models. The first study on the importance of biofilm formation in vivo using animal models and genetically distinct *S.*

Regarding *S. epidermidis*, it is an inhabitant of the human skin microbiota. *S. epidermidis* is an opportunist pathogen that causes disease only in patients subjected to predisposing factors. This includes patients with particular features such as premature newborns, inborn immuno‐ logical impairments, or concomitant medical conditions, for example, human immunodefi‐ ciency virus (HIV) infection, immunosuppression after solid organ or bone marrow transplants, and chemotherapy-related neutropenia. Epidemiological data point out *S. epidermidis* as the most commonly isolated microorganism from foreign materials-related infections such as infected prosthetic joints, central venous catheters (CVC), cerebrospinal fluid shunts, intracardiac devices, artificial heart valves, and vascular grafts. Regarding prosthetic joints infections, *S. epidermidis* is the main infective agent of prosthetic joint implants. In UK, CNS and *S. epidermidis* are isolated from a 36% of total hip and 49% of total knee arthroplas‐ ty infections [1]. In an additional study on infected total hip and knee arthroplasties, it is

lites produced by the biofilm-embedded bacteria.

212 Microbial Biofilms - Importance and Applications

*I.2. Experimental models to study biofilm formation*

*I.1. Medical and epidemiological relevance of staphylococci biofilms*

PIA-dependent biofilm formation also interferes with the host's complement activation. Biofilm-positive wild-type bacteria pre-opsonized with normal human serum are more resistant to complement-mediated elimination than the corresponding biofilm-negative isogenic bacteria [8]. It has been also shown that *S. epidermidis* biofilm formation interferes with the phagocytosis process and macrophage activation. This biofilm-forming phenotype may contribute to the chronic persistence of *S. epidermidis* in inflammatory conditions.

Conversely, *S. epidermidis* produces a set of pro-inflammatory peptides termed phenol-soluble modulins (PSMs), which are produced in a tightly regulated manner by the accessory gene regulator (*agr*) system. It has been demonstrated that PSM δ is able to lyse neutrophils, supporting the concept that these peptides are relevant for *S. epidermidis* pathogenesis. However, PSM δ is expressed at low levels by the biofilm-producing *S. epidermidis* 1457 strain, grown either in biofilm conditions or in planktonic conditions [9].

### **II. Mechanisms and molecules participating in staphylococci biofilm formation**

In this chapter, we will divide the study of the biofilm formation process in three phases. During primary attachment, bacteria adhere to the biotic or abiotic surface in order to colonize it, whereas on the accumulation phase, bacteria build a tridimensional multi-cell and multilayer array. Then, staphylococci are able to disassemble biofilm structure in order to release those cells capable to colonize other sites on the surface. *S. epidermidis* and *S. aureus* biofilm models have been the most studied among staphylococci and the overall biofilm formation process is very similar. In this chapter, we will address *S. epidermidis* biofilm as the base model.

#### *II.1. Participating molecules on the biofilm primary attachment phase*

An essential step performed during the primary attachment stage is the tight binding of bacteria to the foreign material (medical device). This bacterial tight binding leads to a successful establishment of a medical device-associated infection. Regarding *S. epidermidis*, it has been found that cell wall proteins are the main elements of such interactions and this is similar for *S. aureus*. Genetic evidence has allowed establishing that bacterial binding to unmodified polystyrene (non-biotic surface) is conveyed by the *S. epidermidis* AtlE autolysin protein [10]. AtlE is a 115 KDa protein that belongs to the bacterial peptidoglycan (PGN) hydrolases group that plays an important role in bacterial cell wall degradation. The protein is composed by an N-terminal signal peptide, a propeptide, a catalytic domain possessing Nacetylmuramyl-L-alanine amidase activity, three repeated sequences (R1-3), and one Cterminal catalytic domain, possessing N-acetylglucosaminidase activity. In addition to its role during cell wall turnover, AtlE is also important for unmodified polystyrene binding. This function was demonstrated by the *S. epidermidis* O-47 strain harboring a mutation caused by the *atlE*::Tn*917* transposon, which has an impaired ability to adhere to the polystyrene surface [10]. The binding mechanism of AtlE to the polystyrene surface is unclear; however, it is thought that the first event is AtlE recruitment on the bacterial cell wall through the R1-3 domain and those domains possessing enzyme activity. Based on AtlE expression and functional activation studies, it has been suggested that this protein leads to significant changes in cell wall hydrophobicity contributing to the primary attachment process [11]. Another assigned function for AtlE is its autolysin activity in order to cleave the cells wall and thus releasing extracellular DNA (eDNA), which is a common component in staphylococci biofilm [12]. *S. aureus* autolysin also shares this function at this biofilm formation phase.

region containing the serine-aspartate repeat sequence. SdrG specifically binds to a 14-amino acid-long peptide sequence on the N-terminal of fibrinogen's beta chain. SdrF, display a similar organization to SdrG and it specifically binds to collagen I [16]. So far, a specific function has not been assigned to the A region of SdrF. However, it has been demonstrated that its B region is sufficient to interact with collagen I and apparently, this binding occurs through the alpha1 and alpha2 chains of type I collagen [16]. Using a *Lactococcus lactis* heterologous expression system and a murine infection model, it has been established that SdrF may contribute to cardiac assist device driveline infections [17]. SdrF also participates in binding to unmodi‐ fied Dacron surfaces covering drivelines. Anti-SdrF antibodies inhibited 50% of *S. epidermi‐ dis* 9491 binding to collagen using an in vivo model [17], indicating that additional collagen-

*Staphylococcus* Biofilms http://dx.doi.org/10.5772/62910 215

eDNA function during *S. epidermidis* and *S. aureus* biofilm formation has been established as another crucial component for cell attachment to a surface. Some studies confirm that eDNA is a structural component of the biofilm's matrix in both species. Independent studies have demonstrated that eDNA is released through increased cell lysis. In *S. epidermidis*, autolysis is carried out mainly by the autolysin activity of AtlE. A role for eDNA in *S. epidermidis* 1457 was evidenced during primary attachment through the addition of DNase I that results in inhibition of bacterial binding to a glass surface. In spite of the fact that eDNA participates in the primary attachment phase, a function during intercellular attachment phase (accumulation phase) has been ascribed [18]. During the surface colonization phase by *S. aureus* under flow conditions, eDNA is crucial during the transition between primary attachment and accumulation phases [19]. This points out that eDNA plays an important role in early stages of staphylococ‐

The main component during the accumulation phase is the expression of molecules possess‐ ing intercellular (cell-cell) adhesion properties leading to cell aggregation and to subsequent biofilm development having a multi-cell and multi-layer tridimensional structure. Based on the early electron microscopy studies, it has been shown that *S. epidermidis* are embedded within an amorphous matrix. Afterwards, the studies were focused on the biochemical analysis of the matrix components. These efforts resulted in the discovery of the PIA polymer, a

The PIA structure was first described in biofilm-forming *S. epidermidis* 1457 and RP62A strains. Through biochemical analysis, the existence of the structurally related polysaccharide I (>80%) and polysaccharide II (<20%) was determined and separated based on their different ionic properties. Using chemical analysis and NMR spectrometry, it has been demonstrated that polysaccharide I is a lineal homoglycan consisting of beta-1,6-linked 2-amino-2-deoxy-Dglucopyranosil residues. Approximately 80–85% of them are N-acetylated (GlcNAc) and the rest are not N-acetylated, this polymer has an overall negative charge. PIA's polysaccharide II has a low proportion of N-acetylated 2-amino-2-deoxy-D-glucopyranosyl residues and it is modified with succinate residues linked by ester bonds, which confers it with anionic features [20]. The synthesis of an actively functional PIA molecule requires the expression of

binding factors may participate.

ci biofilm formation.

*II.2. Participating molecules during the biofilm accumulation phase*

component participating in great proportion on intercellular adhesion.

The interaction between *S. epidermidis* and an artificial unmodified surface (polystyrene) is mediated by non-specific interactions without the participation of a receptor-specific ligand. On surfaces coated with the host's extracellular matrix (ECM), both *S. epidermidis* and *S. aureus* express cell surface proteins leading to a specific interaction with the components of this ECM of the host. Proteins exhibiting ECM-binding activity are important in order to initiate the infection of medical devices because once foreign materials are introduced inside the body, they are covered by ECM materials (e.g., fibronectin; fibrinogen; vitronectin; collagen). It has been described that *S. epidermidis* AtlE can adhere to vitronectin-covered surfaces, whereas the GehD lipase is involved in interactions with collagen [13]. In addition to these proteins, both *S. epidermidis* and *S. aureus* express proteins possessing a specific function for their interac‐ tion with ECM. These belong to the serine-aspartate repeat (Sdr) protein group and they are members of the microbial surface components family that recognize adhesive matrix mole‐ cules (MSCRAMM) [14]. In *S. epidermidis*, three Sdr proteins referred to as SdrF, SdrG, and SdrH have been identified [14]. SdrG (also known as Fbe) is a protein containing the LPXTG motif that is covalently bound to the bacterial cell wall surface and specifically recognizes fibrinogen and thus *S. epidermidis* cells expressing SdrG adhere to fibrinogen-covered surfaces [15]. The gene coding for SdG/Fbe is found on *S. epidermidis* clinical isolates. The SdrG protein contains four distinct regions: an N-terminal export motif sequence, the A region containing the fibrinogen-binding activity, the B region with unknown function, and the R

region containing the serine-aspartate repeat sequence. SdrG specifically binds to a 14-amino acid-long peptide sequence on the N-terminal of fibrinogen's beta chain. SdrF, display a similar organization to SdrG and it specifically binds to collagen I [16]. So far, a specific function has not been assigned to the A region of SdrF. However, it has been demonstrated that its B region is sufficient to interact with collagen I and apparently, this binding occurs through the alpha1 and alpha2 chains of type I collagen [16]. Using a *Lactococcus lactis* heterologous expression system and a murine infection model, it has been established that SdrF may contribute to cardiac assist device driveline infections [17]. SdrF also participates in binding to unmodi‐ fied Dacron surfaces covering drivelines. Anti-SdrF antibodies inhibited 50% of *S. epidermi‐ dis* 9491 binding to collagen using an in vivo model [17], indicating that additional collagenbinding factors may participate.

eDNA function during *S. epidermidis* and *S. aureus* biofilm formation has been established as another crucial component for cell attachment to a surface. Some studies confirm that eDNA is a structural component of the biofilm's matrix in both species. Independent studies have demonstrated that eDNA is released through increased cell lysis. In *S. epidermidis*, autolysis is carried out mainly by the autolysin activity of AtlE. A role for eDNA in *S. epidermidis* 1457 was evidenced during primary attachment through the addition of DNase I that results in inhibition of bacterial binding to a glass surface. In spite of the fact that eDNA participates in the primary attachment phase, a function during intercellular attachment phase (accumulation phase) has been ascribed [18]. During the surface colonization phase by *S. aureus* under flow conditions, eDNA is crucial during the transition between primary attachment and accumulation phases [19]. This points out that eDNA plays an important role in early stages of staphylococ‐ ci biofilm formation.

#### *II.2. Participating molecules during the biofilm accumulation phase*

*II.1. Participating molecules on the biofilm primary attachment phase*

214 Microbial Biofilms - Importance and Applications

An essential step performed during the primary attachment stage is the tight binding of bacteria to the foreign material (medical device). This bacterial tight binding leads to a successful establishment of a medical device-associated infection. Regarding *S. epidermidis*, it has been found that cell wall proteins are the main elements of such interactions and this is similar for *S. aureus*. Genetic evidence has allowed establishing that bacterial binding to unmodified polystyrene (non-biotic surface) is conveyed by the *S. epidermidis* AtlE autolysin protein [10]. AtlE is a 115 KDa protein that belongs to the bacterial peptidoglycan (PGN) hydrolases group that plays an important role in bacterial cell wall degradation. The protein is composed by an N-terminal signal peptide, a propeptide, a catalytic domain possessing Nacetylmuramyl-L-alanine amidase activity, three repeated sequences (R1-3), and one Cterminal catalytic domain, possessing N-acetylglucosaminidase activity. In addition to its role during cell wall turnover, AtlE is also important for unmodified polystyrene binding. This function was demonstrated by the *S. epidermidis* O-47 strain harboring a mutation caused by the *atlE*::Tn*917* transposon, which has an impaired ability to adhere to the polystyrene surface [10]. The binding mechanism of AtlE to the polystyrene surface is unclear; however, it is thought that the first event is AtlE recruitment on the bacterial cell wall through the R1-3 domain and those domains possessing enzyme activity. Based on AtlE expression and functional activation studies, it has been suggested that this protein leads to significant changes in cell wall hydrophobicity contributing to the primary attachment process [11]. Another assigned function for AtlE is its autolysin activity in order to cleave the cells wall and thus releasing extracellular DNA (eDNA), which is a common component in staphylococci biofilm [12]. *S. aureus* autolysin also shares this function at this biofilm formation phase.

The interaction between *S. epidermidis* and an artificial unmodified surface (polystyrene) is mediated by non-specific interactions without the participation of a receptor-specific ligand. On surfaces coated with the host's extracellular matrix (ECM), both *S. epidermidis* and *S. aureus* express cell surface proteins leading to a specific interaction with the components of this ECM of the host. Proteins exhibiting ECM-binding activity are important in order to initiate the infection of medical devices because once foreign materials are introduced inside the body, they are covered by ECM materials (e.g., fibronectin; fibrinogen; vitronectin; collagen). It has been described that *S. epidermidis* AtlE can adhere to vitronectin-covered surfaces, whereas the GehD lipase is involved in interactions with collagen [13]. In addition to these proteins, both *S. epidermidis* and *S. aureus* express proteins possessing a specific function for their interac‐ tion with ECM. These belong to the serine-aspartate repeat (Sdr) protein group and they are members of the microbial surface components family that recognize adhesive matrix mole‐ cules (MSCRAMM) [14]. In *S. epidermidis*, three Sdr proteins referred to as SdrF, SdrG, and SdrH have been identified [14]. SdrG (also known as Fbe) is a protein containing the LPXTG motif that is covalently bound to the bacterial cell wall surface and specifically recognizes fibrinogen and thus *S. epidermidis* cells expressing SdrG adhere to fibrinogen-covered surfaces [15]. The gene coding for SdG/Fbe is found on *S. epidermidis* clinical isolates. The SdrG protein contains four distinct regions: an N-terminal export motif sequence, the A region containing the fibrinogen-binding activity, the B region with unknown function, and the R

The main component during the accumulation phase is the expression of molecules possess‐ ing intercellular (cell-cell) adhesion properties leading to cell aggregation and to subsequent biofilm development having a multi-cell and multi-layer tridimensional structure. Based on the early electron microscopy studies, it has been shown that *S. epidermidis* are embedded within an amorphous matrix. Afterwards, the studies were focused on the biochemical analysis of the matrix components. These efforts resulted in the discovery of the PIA polymer, a component participating in great proportion on intercellular adhesion.

The PIA structure was first described in biofilm-forming *S. epidermidis* 1457 and RP62A strains. Through biochemical analysis, the existence of the structurally related polysaccharide I (>80%) and polysaccharide II (<20%) was determined and separated based on their different ionic properties. Using chemical analysis and NMR spectrometry, it has been demonstrated that polysaccharide I is a lineal homoglycan consisting of beta-1,6-linked 2-amino-2-deoxy-Dglucopyranosil residues. Approximately 80–85% of them are N-acetylated (GlcNAc) and the rest are not N-acetylated, this polymer has an overall negative charge. PIA's polysaccharide II has a low proportion of N-acetylated 2-amino-2-deoxy-D-glucopyranosyl residues and it is modified with succinate residues linked by ester bonds, which confers it with anionic features [20]. The synthesis of an actively functional PIA molecule requires the expression of all four *icaADBC* genes [21] constituting the *ica* operon. The *icaR* gene confines the repres‐ sion of the *ica* operon expression. The synthesis process has been studied in detail using *S. carnosus* recombinant strains expressing different combinations of the *icaADBC* genes and using UDP-GlcNAc as the sugar donor [21]. The IcaA protein belongs to the glycosyltransfer‐ ase family 2. It is an integral membrane protein consisting of 412 amino acids and four transmembrane domains. This protein performs the synthesis of beta-1,6-linked GlcNAc oligosaccharide composed by up to 20 GlcNAc units. The IcaD protein is required for IcaA full activity in vitro. IcaD is a membrane protein of 101 amino acids possessing two putative membrane space domains and it is thought that it may be a chaperone guiding IcaA protein folding and its membrane insertion and it also may act as a link between the IcaA and IcaC proteins [21]. Essential to PIA synthesis is the presence of the IcaB, an integral membrane protein of 355 amino acids possessing 10 predicted transmembrane domains, which may be involved in the externalization and elongation of the nascent polysaccharide [21]. IcaB is a member of the polysaccharide deacetylase family that includes chitin deacetylases or the chitooligosaccharide deacetylase NodB of *Rhizobium melioti*. IcaB in its mature form is a secreted protein consisting of 259 amino acids with a predicted signal sequence, which is responsible for PIA N-deacetylation and it is crucial for PIA activity during biofilm forma‐ tion and for *S. epidermidis* virulence [22]. A strain harboring an *icaB* deletion mutation, in which the *icaB* gene has been eliminated, produces a weakly retained PIA at the cell's surface, as it does not contain N-deacetylated GlcNAc [22].

terminal end, the A domain, and within the C-terminal, the LPXTG-anchoring domain separated by tandem repeats, which are involved in binding to fibronectin. Both, FnBPA and FnBPB are involved in the biofilm's accumulation phase in isolates from hospitals and under flow conditions. This concept of multifunctional proteins with important roles during some of the biofilm formation and surface colonization phases is also applied to *S. epidermidis* with its respective proteins participating in the accumulation phase: the accumulation-associated

*Staphylococcus* Biofilms http://dx.doi.org/10.5772/62910 217

The Embp protein and its *S. aureus* orthologue, designated as Ebh, were simultaneously pinpointed during identification studies of protein factors [27]. In a clinically relevant *S. epidermidis* isolates collection, the *embp* gene was detected in 90% of all strains [2]. Further‐ more, studies conducted in vivo indicated the presence of anti-Embp antibodies in patients with prosthetic joint infections by *S. epidermidis*, suggesting that the Embp protein is ex‐ pressed and it has a role during the infection. Surprisingly, when using a bacterial cell model in flow conditions, it was observed that addition of anti-Embp antibodies to the system inhibited biofilm formation by the *S. epidermidis* 1457 strain [28]. This result leads to propose the Embp protein as a potential candidate for preventive strategies against biofilm forma‐ tion. Experimental evidences suggest that Embp has a defined role during the primary attachment phase. This proposal is supported by the fact that Embp overexpression had no effect on bacterial attachment on an unmodified polystyrene surface, although it had a negative effect on bacterial binding to fibronectin-covered surfaces [29]. Additionally, it was observed that the Embp-fibronectin interaction is necessary for the biofilm's accumulation phase on

The Aap protein is covalently bound to the cell wall and consists of an A domain and a B domain. The A domain has 584 amino acids and includes an export signal at its N-terminal, 16 amino acid repeats and a globular region of 212 amino acids with alpha-helical and betasheet contents. This 212-amino acid-long region is highly conserved between Aap and its *S. aureus* orthologue, SasG. Through bioinformatics analysis, it has been shown that this domain possess lectin-type activity [6]. The B domain consists in a variable number of repeats of 128 amino acids, the G5 subdomains [2]. The number of G5 subdomains in the B domain is different among the *S. epidermidis* strains, for example, the RP62A reference strain possesses 13 G5 subdomains, whereas the *S. epidermidis* 1457 strain has only seven [6]. This fact has been also observed in clinic isolates with clonal genotypes subsequently recovered from ongoing infections on devices in patients [2]. This observation leads to the hypothesis stating that the number of G5 subdomains of the Aap B domain may represent a mechanism contributing to the immune evasion of *S. epidermidis* mediated by the modification of the major epitopes on the cell's surface [2]. Aap is detected on the bacterial cell wall and its retention mechanism is through anchoring of its C-terminal by a covalent bond with the cell wall PGN [30]. A more detailed analysis by confocal microscopy showed that Aap is strictly localized at the bacteri‐ al cell surface, whereas minimal amounts of Aap are released within the biofilm matrix [30]. This result is supported by electron microscopy observations in which Aap appears as elongated fibers of 120 nm projecting outwards from the cell wall and in form of tufts [31].

protein (Aap) and the extracellular matrix-binding protein (Embp).

plastic surfaces [29].

Conversely, the first observation made through biochemical analysis on biofilm matrix extracts indicated the presence of oligosaccharide, proteins, and nucleic acids. The specific proteins that comprise a biofilm have been identified and characterized; one of them is the biofilmassociated protein Bap [23]. The Bap is rarely found in invasive *S. epidermidis* biofilms from human infections [23] and it similarly occurs for *S. aureus*. Another protein found in biofilms is SesC, which has been proposed as playing an important role during biofilm formation [24]. SesC is a surface protein of 68 KDa containing the *S. epidermidis* LPXTG motif and it is related to the *S. aureus* clumping factor A (ClfA). SesC protein is strongly expressed in biofilm conditions in contrast to planktonic conditions by the *S. epidermidis* 1457 strain [24]. An anti-SesC antibody inhibited biofilm formation in vitro in several *S. epidermidis* isolates. All 105 *S. epidermidis* isolates collected from nose swabs of infection possessed the *sesC* gene in their genomes [24]. Actively or passively immunized animals using SesC as antigen displayed a decreased biofilm formation using the in vivo CVC infection model [25]. Nevertheless, a specific role of SesC during the intercellular adhesion of the accumulation phase of the biofilm remains to be demonstrated.

#### *II.3. Multifunction proteins during the biofilm accumulation phase*

Protein factors contributing to the accumulation phase of staphylococci biofilm have fea‐ tures of multifunctional proteins. In *S. aureus*, it has been found that fibronectin-binding proteins (FnBPs) (FnBPA and FnBPB), ClfA protein and *S. aureus* surface protein (SasG), may be considered multifunctional proteins as they do not have an exclusive role in either of the biofilm phases: primary attachment or accumulation [26]. FnBPs are constituted by an N-

terminal end, the A domain, and within the C-terminal, the LPXTG-anchoring domain separated by tandem repeats, which are involved in binding to fibronectin. Both, FnBPA and FnBPB are involved in the biofilm's accumulation phase in isolates from hospitals and under flow conditions. This concept of multifunctional proteins with important roles during some of the biofilm formation and surface colonization phases is also applied to *S. epidermidis* with its respective proteins participating in the accumulation phase: the accumulation-associated protein (Aap) and the extracellular matrix-binding protein (Embp).

all four *icaADBC* genes [21] constituting the *ica* operon. The *icaR* gene confines the repres‐ sion of the *ica* operon expression. The synthesis process has been studied in detail using *S. carnosus* recombinant strains expressing different combinations of the *icaADBC* genes and using UDP-GlcNAc as the sugar donor [21]. The IcaA protein belongs to the glycosyltransfer‐ ase family 2. It is an integral membrane protein consisting of 412 amino acids and four transmembrane domains. This protein performs the synthesis of beta-1,6-linked GlcNAc oligosaccharide composed by up to 20 GlcNAc units. The IcaD protein is required for IcaA full activity in vitro. IcaD is a membrane protein of 101 amino acids possessing two putative membrane space domains and it is thought that it may be a chaperone guiding IcaA protein folding and its membrane insertion and it also may act as a link between the IcaA and IcaC proteins [21]. Essential to PIA synthesis is the presence of the IcaB, an integral membrane protein of 355 amino acids possessing 10 predicted transmembrane domains, which may be involved in the externalization and elongation of the nascent polysaccharide [21]. IcaB is a member of the polysaccharide deacetylase family that includes chitin deacetylases or the chitooligosaccharide deacetylase NodB of *Rhizobium melioti*. IcaB in its mature form is a secreted protein consisting of 259 amino acids with a predicted signal sequence, which is responsible for PIA N-deacetylation and it is crucial for PIA activity during biofilm forma‐ tion and for *S. epidermidis* virulence [22]. A strain harboring an *icaB* deletion mutation, in which the *icaB* gene has been eliminated, produces a weakly retained PIA at the cell's surface, as it

Conversely, the first observation made through biochemical analysis on biofilm matrix extracts indicated the presence of oligosaccharide, proteins, and nucleic acids. The specific proteins that comprise a biofilm have been identified and characterized; one of them is the biofilmassociated protein Bap [23]. The Bap is rarely found in invasive *S. epidermidis* biofilms from human infections [23] and it similarly occurs for *S. aureus*. Another protein found in biofilms is SesC, which has been proposed as playing an important role during biofilm formation [24]. SesC is a surface protein of 68 KDa containing the *S. epidermidis* LPXTG motif and it is related to the *S. aureus* clumping factor A (ClfA). SesC protein is strongly expressed in biofilm conditions in contrast to planktonic conditions by the *S. epidermidis* 1457 strain [24]. An anti-SesC antibody inhibited biofilm formation in vitro in several *S. epidermidis* isolates. All 105 *S. epidermidis* isolates collected from nose swabs of infection possessed the *sesC* gene in their genomes [24]. Actively or passively immunized animals using SesC as antigen displayed a decreased biofilm formation using the in vivo CVC infection model [25]. Nevertheless, a specific role of SesC during the intercellular adhesion of the accumulation phase of the biofilm

Protein factors contributing to the accumulation phase of staphylococci biofilm have fea‐ tures of multifunctional proteins. In *S. aureus*, it has been found that fibronectin-binding proteins (FnBPs) (FnBPA and FnBPB), ClfA protein and *S. aureus* surface protein (SasG), may be considered multifunctional proteins as they do not have an exclusive role in either of the biofilm phases: primary attachment or accumulation [26]. FnBPs are constituted by an N-

does not contain N-deacetylated GlcNAc [22].

216 Microbial Biofilms - Importance and Applications

remains to be demonstrated.

*II.3. Multifunction proteins during the biofilm accumulation phase*

The Embp protein and its *S. aureus* orthologue, designated as Ebh, were simultaneously pinpointed during identification studies of protein factors [27]. In a clinically relevant *S. epidermidis* isolates collection, the *embp* gene was detected in 90% of all strains [2]. Further‐ more, studies conducted in vivo indicated the presence of anti-Embp antibodies in patients with prosthetic joint infections by *S. epidermidis*, suggesting that the Embp protein is ex‐ pressed and it has a role during the infection. Surprisingly, when using a bacterial cell model in flow conditions, it was observed that addition of anti-Embp antibodies to the system inhibited biofilm formation by the *S. epidermidis* 1457 strain [28]. This result leads to propose the Embp protein as a potential candidate for preventive strategies against biofilm forma‐ tion. Experimental evidences suggest that Embp has a defined role during the primary attachment phase. This proposal is supported by the fact that Embp overexpression had no effect on bacterial attachment on an unmodified polystyrene surface, although it had a negative effect on bacterial binding to fibronectin-covered surfaces [29]. Additionally, it was observed that the Embp-fibronectin interaction is necessary for the biofilm's accumulation phase on plastic surfaces [29].

The Aap protein is covalently bound to the cell wall and consists of an A domain and a B domain. The A domain has 584 amino acids and includes an export signal at its N-terminal, 16 amino acid repeats and a globular region of 212 amino acids with alpha-helical and betasheet contents. This 212-amino acid-long region is highly conserved between Aap and its *S. aureus* orthologue, SasG. Through bioinformatics analysis, it has been shown that this domain possess lectin-type activity [6]. The B domain consists in a variable number of repeats of 128 amino acids, the G5 subdomains [2]. The number of G5 subdomains in the B domain is different among the *S. epidermidis* strains, for example, the RP62A reference strain possesses 13 G5 subdomains, whereas the *S. epidermidis* 1457 strain has only seven [6]. This fact has been also observed in clinic isolates with clonal genotypes subsequently recovered from ongoing infections on devices in patients [2]. This observation leads to the hypothesis stating that the number of G5 subdomains of the Aap B domain may represent a mechanism contributing to the immune evasion of *S. epidermidis* mediated by the modification of the major epitopes on the cell's surface [2]. Aap is detected on the bacterial cell wall and its retention mechanism is through anchoring of its C-terminal by a covalent bond with the cell wall PGN [30]. A more detailed analysis by confocal microscopy showed that Aap is strictly localized at the bacteri‐ al cell surface, whereas minimal amounts of Aap are released within the biofilm matrix [30]. This result is supported by electron microscopy observations in which Aap appears as elongated fibers of 120 nm projecting outwards from the cell wall and in form of tufts [31].

The importance of Aap for *S. epidermidis* biofilm formation was recognized during studies in which the expression of the B domain does not modify the primary adherence properties, although it is very important to cellular aggregation, indicating that Aap is a protein that participates in the intercellular adhesion [32]. Similarly, the importance of the B domain for intercellular adhesion was also described for SasG in *S. aureus* [33]. Another fact that eviden‐ ces the properties of Aap in intercellular adhesion is the ability of the B domain to undergo homodimerization in the presence of Zn [34]. The proposed mechanism of intercellular adhesion through Aap is that the protein must undergo proteolytic processing in order to remove the A domain [32–33]. Thus, Aap proteolytic processing does not normally occur under in vitro growth conditions [32].

is inactivated [38]. Additionally, protease inhibitors have a promoter role during biofilm formation in *S. aureus* under conditions that normally accelerate its disassembly [38]. Similarly, mutations leading to extracellular protease overexpression, such as those on the *sarA* and *sigB* genes, enhance a planktonic growth in *S. aureus* [40]. This leads to a concept of an inverse

*Staphylococcus* Biofilms http://dx.doi.org/10.5772/62910 219

*S. aureus* secretes a potent DNAse, also known as thermonuclease or micrococcal nuclease, which has been implicated on cell detachment from the biofilm. *S. aureus* mature biofilm is disintegrated by the exogenous addition of DNAse I or restriction enzymes [41]. It has been shown that a nuclease-mutant *S. aureus* strain exhibited an increase of biofilm formation under flow conditions regarding to the wild-type strain [41]. These findings suggest that nucleases

PSMs are peptides possessing surfactant features, which are produced by both *S. aureus* and *S. epidermidis*, and they are capable of contributing for mature biofilm disassembly. PSMs are regulated by the *agr* system and their amphiphilic alpha-helix structure confers it with a surfactant-type property. PSMs promote both disassembly of the mature biofilm of *S. epidermidis* in vitro and dissemination from colonized catheters on a mouse model of devicerelated infection [9]. Additionally, antibodies against PSMs inhibit bacterial dispersal from the implanted catheter, indicating that the disassembly manipulation strategy may prevent the

Biofilms are a lifestyle adopted by a wide variety of microorganisms that requires the consumption of an enormous amount of energy. Thus, it is expected that biofilm growth may be controlled by more regulatory mechanisms regarding planktonic growth. Some of the

The *agr* is a quorum-sensing system in staphylococci that regulates the expression of adhe‐ sion molecules, thus it participates in the primary attachment phase. These adhesion mole‐ cules, such as the MSCRAMMs, display an increased expression when cell density is low, a situation favoring the onset of the infection by staphylococci. Once the colonization has concluded, the increased activity of the *agr* quorum-sensing system represses the expression of unnecessary colonization factors. Among these, the MSCRAMMs are included, which are negatively regulated by *agr* in *S. aureus* [42]. In *S. epidermidis*, the knowledge regarding colonization factors and their regulation is more limited. Results obtained by transcriptional profiling and the assessment of MSCRAMM expression [43], suggest that some of the latter do

The regulation of PIA expression is probably the best-studied system among those regula‐ tion systems involved in staphylococci biofilm formation. Anaerobiosis significantly increas‐

factors that impact on biofilm formation are mentioned in the following sections.

*III.1. Regulation of the factors participating on the primary attachment phase*

not follow the classic notion of regulation mediated by the *agr* system.

may function as endogenous mediators for biofilm disassembly in this species.

correlation between protease expression and biofilm formation.

**III. Regulation of biofilm formation in staphylococci**

spreading of the infection.

*III.2. Regulation of PIA synthesis*

Although the intercellular adhesion property of Aap was recognized, currently there is evidence supporting its significant role in the primary attachment phase as well. The bind‐ ing of *S. epidermidis* NCTC 11047 strain expressing Aap to squamous epithelial cells was partially inhibited by the addition of the recombinant A domain of Aap [35]. In a clinical isolate, the binding of *S. epidermidis* CSF41498 strain expressing a non-processed Aap (thus contain‐ ing the A domain) to polystyrene was completely impaired by the addition of an anti-A domain antiserum, whereas an anti-B domain antiserum did not affect its adhesion ability [36]. Thus, a new bifunctional role of Aap during the biofilm formation is suggested: its participation on the primary attachment phase through its A domain and also its participation on the accu‐ mulation phase through its B domain [32, 35–36].

#### *II.4. Molecular mechanisms for mature biofilm disassembly*

A primary biofilm disassembly mechanism used by *S. aureus* and *S. epidermidis* is the produc‐ tion of extracellular enzymes or surfactants that degrade or solubilize the adhesive compo‐ nents of the biofilm matrix. Because this matrix cover bacterial cells within the biofilm colony, its degradation results in cell detachment from the colony and its release toward the environ‐ ment. The products of the matrix-degrading genes, which are implicated on the dispersion of the staphylococcal biofilm, include proteases, DNases, and surfactants.

The *agr* system is a putative regulator controlling the production of the enzymes degrading the biofilm matrix. The *agr* is controlled by a cyclic autoinducing peptide (AIP) that is synthesized and secreted within the environment. When the AIP concentration reaches a critical threshold level, it activates a two-component signal transduction cascade leading to the production of secretory virulence factors [37]. The extracellular proteins induced by the *agr* system are multiple proteases and pore-forming toxins termed PSMs. *S. aureus* does not form a biofilm when the *agr* system is highly active and its reactivation within the mature biofilm results in its disassembly [38]. Furthermore, the *agr* system of *S. aureus* is more active in cells detached from the biofilm [39]; the same phenomenon was observed in *S. epidermidis* [9], contributing evidence showing that induction of the *agr* system results in biofilm disas‐ sembly.

Extracellular protease production has been implicated on the disassembly of the mature biofilm. In *S. aureus*, the mutation of the protease-coding genes results in a significant increase of biofilm formation under flow conditions and the disassembly decrease when the *agr* system

is inactivated [38]. Additionally, protease inhibitors have a promoter role during biofilm formation in *S. aureus* under conditions that normally accelerate its disassembly [38]. Similarly, mutations leading to extracellular protease overexpression, such as those on the *sarA* and *sigB* genes, enhance a planktonic growth in *S. aureus* [40]. This leads to a concept of an inverse correlation between protease expression and biofilm formation.

*S. aureus* secretes a potent DNAse, also known as thermonuclease or micrococcal nuclease, which has been implicated on cell detachment from the biofilm. *S. aureus* mature biofilm is disintegrated by the exogenous addition of DNAse I or restriction enzymes [41]. It has been shown that a nuclease-mutant *S. aureus* strain exhibited an increase of biofilm formation under flow conditions regarding to the wild-type strain [41]. These findings suggest that nucleases may function as endogenous mediators for biofilm disassembly in this species.

PSMs are peptides possessing surfactant features, which are produced by both *S. aureus* and *S. epidermidis*, and they are capable of contributing for mature biofilm disassembly. PSMs are regulated by the *agr* system and their amphiphilic alpha-helix structure confers it with a surfactant-type property. PSMs promote both disassembly of the mature biofilm of *S. epidermidis* in vitro and dissemination from colonized catheters on a mouse model of devicerelated infection [9]. Additionally, antibodies against PSMs inhibit bacterial dispersal from the implanted catheter, indicating that the disassembly manipulation strategy may prevent the spreading of the infection.

### **III. Regulation of biofilm formation in staphylococci**

Biofilms are a lifestyle adopted by a wide variety of microorganisms that requires the consumption of an enormous amount of energy. Thus, it is expected that biofilm growth may be controlled by more regulatory mechanisms regarding planktonic growth. Some of the factors that impact on biofilm formation are mentioned in the following sections.

### *III.1. Regulation of the factors participating on the primary attachment phase*

The *agr* is a quorum-sensing system in staphylococci that regulates the expression of adhe‐ sion molecules, thus it participates in the primary attachment phase. These adhesion mole‐ cules, such as the MSCRAMMs, display an increased expression when cell density is low, a situation favoring the onset of the infection by staphylococci. Once the colonization has concluded, the increased activity of the *agr* quorum-sensing system represses the expression of unnecessary colonization factors. Among these, the MSCRAMMs are included, which are negatively regulated by *agr* in *S. aureus* [42]. In *S. epidermidis*, the knowledge regarding colonization factors and their regulation is more limited. Results obtained by transcriptional profiling and the assessment of MSCRAMM expression [43], suggest that some of the latter do not follow the classic notion of regulation mediated by the *agr* system.

#### *III.2. Regulation of PIA synthesis*

The importance of Aap for *S. epidermidis* biofilm formation was recognized during studies in which the expression of the B domain does not modify the primary adherence properties, although it is very important to cellular aggregation, indicating that Aap is a protein that participates in the intercellular adhesion [32]. Similarly, the importance of the B domain for intercellular adhesion was also described for SasG in *S. aureus* [33]. Another fact that eviden‐ ces the properties of Aap in intercellular adhesion is the ability of the B domain to undergo homodimerization in the presence of Zn [34]. The proposed mechanism of intercellular adhesion through Aap is that the protein must undergo proteolytic processing in order to remove the A domain [32–33]. Thus, Aap proteolytic processing does not normally occur under

Although the intercellular adhesion property of Aap was recognized, currently there is evidence supporting its significant role in the primary attachment phase as well. The bind‐ ing of *S. epidermidis* NCTC 11047 strain expressing Aap to squamous epithelial cells was partially inhibited by the addition of the recombinant A domain of Aap [35]. In a clinical isolate, the binding of *S. epidermidis* CSF41498 strain expressing a non-processed Aap (thus contain‐ ing the A domain) to polystyrene was completely impaired by the addition of an anti-A domain antiserum, whereas an anti-B domain antiserum did not affect its adhesion ability [36]. Thus, a new bifunctional role of Aap during the biofilm formation is suggested: its participation on the primary attachment phase through its A domain and also its participation on the accu‐

A primary biofilm disassembly mechanism used by *S. aureus* and *S. epidermidis* is the produc‐ tion of extracellular enzymes or surfactants that degrade or solubilize the adhesive compo‐ nents of the biofilm matrix. Because this matrix cover bacterial cells within the biofilm colony, its degradation results in cell detachment from the colony and its release toward the environ‐ ment. The products of the matrix-degrading genes, which are implicated on the dispersion of

The *agr* system is a putative regulator controlling the production of the enzymes degrading the biofilm matrix. The *agr* is controlled by a cyclic autoinducing peptide (AIP) that is synthesized and secreted within the environment. When the AIP concentration reaches a critical threshold level, it activates a two-component signal transduction cascade leading to the production of secretory virulence factors [37]. The extracellular proteins induced by the *agr* system are multiple proteases and pore-forming toxins termed PSMs. *S. aureus* does not form a biofilm when the *agr* system is highly active and its reactivation within the mature biofilm results in its disassembly [38]. Furthermore, the *agr* system of *S. aureus* is more active in cells detached from the biofilm [39]; the same phenomenon was observed in *S. epidermidis* [9], contributing evidence showing that induction of the *agr* system results in biofilm disas‐

Extracellular protease production has been implicated on the disassembly of the mature biofilm. In *S. aureus*, the mutation of the protease-coding genes results in a significant increase of biofilm formation under flow conditions and the disassembly decrease when the *agr* system

in vitro growth conditions [32].

218 Microbial Biofilms - Importance and Applications

sembly.

mulation phase through its B domain [32, 35–36].

*II.4. Molecular mechanisms for mature biofilm disassembly*

the staphylococcal biofilm, include proteases, DNases, and surfactants.

The regulation of PIA expression is probably the best-studied system among those regula‐ tion systems involved in staphylococci biofilm formation. Anaerobiosis significantly increas‐

es PIA expression [44]. This constitutes an important finding for biofilm physiology, as the oxygen concentration would restrict biofilm formation at the oxygen-loaded arterial blood‐ stream. In an already established biofilm, PIA expression would be higher at the most deep biofilm sections because oxygen concentration significantly decreases. Conversely, it has been found that sub-inhibitory concentrations of specific antibiotics increase the transcription of the *ica* operon in *S. epidermidis* [45].

Phagocytosis, mainly performed by neutrophils, is a major mechanism by which the innate immune system eliminates microorganisms invading the human body. Staphylococci in a biofilm are not readily subjected to phagocytosis by neutrophils. The responsible elements for this constraint are the PIA exopolysaccharide and the PGA exopolymer, and therefore they

*Staphylococcus* Biofilms http://dx.doi.org/10.5772/62910 221

Medical devices are widely used for diagnostic and therapeutic treatment in most medical specialties. Infection risk is a frequent complication linked to the permanent use of medical devices such as orthopedic or heart prostheses, vascular catheters, urinary catheters, and endotracheal tubes. A promising solution in order to prevent medical device-associated infections is to develop devices possessing materials or surfaces that act against microorgan‐ ism adhesion or their viability. The first strategy was the use of biocides in coatings. A number of clinical assays have been conducted producing conflicting results. Some authors suggest that the extended use of biocide on the coating may lead to an increase of microbial resist‐ ance toward the microbiocide agent. The other strategy consists in the development of

The chemical diversity of the biofilm matrix, including protein material, eDNA, and polysac‐ charides, is susceptible to degradation by a wide variety of exogenously added enzymes. Some research groups have observed that proteinase K and trypsin may disperse the mature biofilm of *S. aureus* and *S. epidermidis* [53]. Bovine DNase addition has also been successful for dispersing the mature biofilm of *S. aureus* [54]. Similarly, the enzymes able to degrade PNAG cleave biofilms containing this polysaccharide as their primary component. An enzyme called dispersin B (DspB) inhibits biofilm formation and promotes its disassembly in several *S. epidermidis* and *S. aureus* strains having PNAG as the main component of their respective biofilm matrices [54]. Finally, the treatment with lysostaphin was effective in a catheter mouse model of *S. aureus* biofilm [55], suggesting that it may be a general therapy against staphylo‐

A current topic is the development of an antimicrobial coating interfering with quorumsensing mechanisms. This has been observed for halogenated furanones synthesized by the red algae *Delisea pulchra* possessing anti-adhesive properties against a wide range of bacteria

Bacterial adhesion depends on hydrophobocity of the cell and material constituting the surface. The self-autoassembled monolayers (SAMs) can modulate the exposure of their different residues on a surface and they are used in bacterial adhesion studies as models of surfaces

contribute to biofilm resistance toward the host's innate defense mechanisms.

**IV. Therapeutic strategies against biofilm formation in medical devices**

materials impeding bacterial adhesion.

coccal biofilm infections.

*IV.2. Anti-adhesive chemical strategies*

*IV.2.1. Hydrophobicity and surface charge*

[56].

*IV.1. Biological strategies for biofilm treatment*

Some overall regulators of *S. aureus* or *S. epidermidis* participate in the *ica* operon transcrip‐ tion regulation or PIA expression, such as the SarA DNA-binding protein and the alterna‐ tive sigma factor SigB that increase the expression of the *ica* operon, whereas the luxS quorumsensing system represses the expression of this operon [46]. Contrastingly, the *agr* system does not regulate PIA. The exact mechanism explaining the influence of SarA and SigB on the *ica* operon transcription is complex. Briefly, the SigB regulator represses the *icaR* gene transcrip‐ tion, as its protein product, IcaR, in turn, represses the transcription of the *icaADBC* operon [47]. Besides, SarA regulates positively the *icaA* gene, independently from IcaR [48].

### *III.3. Regulation of the PSMs expression*

It has been discussed that the *agr* quorum-sensing system represses the expression of surface proteins after the primary attachment. The major *agr* control relies on the expression of the PSMs. The expression of the *agr* system within a biofilm is limited to its periphery, in which the *agr* regulator controls cell detachment from the biofilm by regulating the increased expression of the PSM effector molecules [49]. The staphylococcal PSM δ is a major effector molecule for cell detachment from the biofilm and it is tightly controlled by the *agr* system of *S. aureus* [50]. In *S. epidermidis*, the PSM β is the most important.

#### *III.4. Biofilm regulation against host's defenses and antibiotics*

One of the advantages possessed by bacteria in the biofilm state is high resistance toward antibiotics and the host innate defense, such as AMPs and the phagocytosis performed by neutrophils. However, the molecular basis of this phenomenon has been recently investigat‐ ed. Two of the main mechanisms contributing to biofilm resistance are: (1) keeping antibacte‐ rial substances from reaching their target, for example, by limited diffusion or repulsion and (2) biofilm's specific physiology that limits the efficiency of antibiotics, mainly those target‐ ing active cells, and it may include specific subpopulations of resistant cells ("persistent").

Limited antibiotic diffusion provided by the biofilm is mainly due to the nature of the biofilm matrix. However, this limited diffusion is the resistance mechanism toward some antibiotics, such as ciprofloxacin in *P. aeruginosa* [51], whereas some others (e.g., rifampicin and vanco‐ mycin) are able to cleave the exopolysaccharide envelope of *S. epidermidis* [52]. Interestingly, PIA has the ability to protect the cells within the biofilm from both cationic or anionic AMPs, as it possess an overall positive or negative charge, and thus PIA interacts or repels mole‐ cules depending on its charge. Similarly, the poly-gamma-glutamic acid (PGA) exopolymer, of *S. epidermidis* and a number of CNS species, contributes to the resistance toward AMPs of either charge.

Phagocytosis, mainly performed by neutrophils, is a major mechanism by which the innate immune system eliminates microorganisms invading the human body. Staphylococci in a biofilm are not readily subjected to phagocytosis by neutrophils. The responsible elements for this constraint are the PIA exopolysaccharide and the PGA exopolymer, and therefore they contribute to biofilm resistance toward the host's innate defense mechanisms.

### **IV. Therapeutic strategies against biofilm formation in medical devices**

Medical devices are widely used for diagnostic and therapeutic treatment in most medical specialties. Infection risk is a frequent complication linked to the permanent use of medical devices such as orthopedic or heart prostheses, vascular catheters, urinary catheters, and endotracheal tubes. A promising solution in order to prevent medical device-associated infections is to develop devices possessing materials or surfaces that act against microorgan‐ ism adhesion or their viability. The first strategy was the use of biocides in coatings. A number of clinical assays have been conducted producing conflicting results. Some authors suggest that the extended use of biocide on the coating may lead to an increase of microbial resist‐ ance toward the microbiocide agent. The other strategy consists in the development of materials impeding bacterial adhesion. 

### *IV.1. Biological strategies for biofilm treatment*

es PIA expression [44]. This constitutes an important finding for biofilm physiology, as the oxygen concentration would restrict biofilm formation at the oxygen-loaded arterial blood‐ stream. In an already established biofilm, PIA expression would be higher at the most deep biofilm sections because oxygen concentration significantly decreases. Conversely, it has been found that sub-inhibitory concentrations of specific antibiotics increase the transcription of the

Some overall regulators of *S. aureus* or *S. epidermidis* participate in the *ica* operon transcrip‐ tion regulation or PIA expression, such as the SarA DNA-binding protein and the alterna‐ tive sigma factor SigB that increase the expression of the *ica* operon, whereas the luxS quorumsensing system represses the expression of this operon [46]. Contrastingly, the *agr* system does not regulate PIA. The exact mechanism explaining the influence of SarA and SigB on the *ica* operon transcription is complex. Briefly, the SigB regulator represses the *icaR* gene transcrip‐ tion, as its protein product, IcaR, in turn, represses the transcription of the *icaADBC* operon

It has been discussed that the *agr* quorum-sensing system represses the expression of surface proteins after the primary attachment. The major *agr* control relies on the expression of the PSMs. The expression of the *agr* system within a biofilm is limited to its periphery, in which the *agr* regulator controls cell detachment from the biofilm by regulating the increased expression of the PSM effector molecules [49]. The staphylococcal PSM δ is a major effector molecule for cell detachment from the biofilm and it is tightly controlled by the *agr* system of

One of the advantages possessed by bacteria in the biofilm state is high resistance toward antibiotics and the host innate defense, such as AMPs and the phagocytosis performed by neutrophils. However, the molecular basis of this phenomenon has been recently investigat‐ ed. Two of the main mechanisms contributing to biofilm resistance are: (1) keeping antibacte‐ rial substances from reaching their target, for example, by limited diffusion or repulsion and (2) biofilm's specific physiology that limits the efficiency of antibiotics, mainly those target‐ ing active cells, and it may include specific subpopulations of resistant cells ("persistent").

Limited antibiotic diffusion provided by the biofilm is mainly due to the nature of the biofilm matrix. However, this limited diffusion is the resistance mechanism toward some antibiotics, such as ciprofloxacin in *P. aeruginosa* [51], whereas some others (e.g., rifampicin and vanco‐ mycin) are able to cleave the exopolysaccharide envelope of *S. epidermidis* [52]. Interestingly, PIA has the ability to protect the cells within the biofilm from both cationic or anionic AMPs, as it possess an overall positive or negative charge, and thus PIA interacts or repels mole‐ cules depending on its charge. Similarly, the poly-gamma-glutamic acid (PGA) exopolymer, of *S. epidermidis* and a number of CNS species, contributes to the resistance toward AMPs of

[47]. Besides, SarA regulates positively the *icaA* gene, independently from IcaR [48].

*S. aureus* [50]. In *S. epidermidis*, the PSM β is the most important.

*III.4. Biofilm regulation against host's defenses and antibiotics*

*ica* operon in *S. epidermidis* [45].

220 Microbial Biofilms - Importance and Applications

*III.3. Regulation of the PSMs expression*

either charge.

The chemical diversity of the biofilm matrix, including protein material, eDNA, and polysac‐ charides, is susceptible to degradation by a wide variety of exogenously added enzymes. Some research groups have observed that proteinase K and trypsin may disperse the mature biofilm of *S. aureus* and *S. epidermidis* [53]. Bovine DNase addition has also been successful for dispersing the mature biofilm of *S. aureus* [54]. Similarly, the enzymes able to degrade PNAG cleave biofilms containing this polysaccharide as their primary component. An enzyme called dispersin B (DspB) inhibits biofilm formation and promotes its disassembly in several *S. epidermidis* and *S. aureus* strains having PNAG as the main component of their respective biofilm matrices [54]. Finally, the treatment with lysostaphin was effective in a catheter mouse model of *S. aureus* biofilm [55], suggesting that it may be a general therapy against staphylo‐ coccal biofilm infections. strategy able

A current topic is the development of an antimicrobial coating interfering with quorumsensing mechanisms. This has been observed for halogenated furanones synthesized by the red algae *Delisea pulchra* possessing anti-adhesive properties against a wide range of bacteria [56].

#### *IV.2. Anti-adhesive chemical strategies*

#### *IV.2.1. Hydrophobicity and surface charge*

Bacterial adhesion depends on hydrophobocity of the cell and material constituting the surface. The self-autoassembled monolayers (SAMs) can modulate the exposure of their different residues on a surface and they are used in bacterial adhesion studies as models of surfaces

with chemically controlled properties. SAMs with hydrophilic residues (OH, NH2) tend to decrease bacterial adhesion when compared to those with hydrophobic surfaces containing methyl groups (CH3) [57]. Some hydrophilic linings, such as hydrogels or medical devices with chemically modified surfaces have been developed in order to restrict biofilm development. Some clinical studies have reported that urinary catheters lined with heparin can reduce *Proteus mirabilis* biofilm [58]. However, it has been observed that heparin may stimulate biofilm formation by *S. aureus* [59]. This demonstrates that initial adhesion is not always sufficient to avoid biofilm development. Treatment with plasma can also create hydrophilic residues at the surface of medical devices producing antimicrobial activity.

diameter (30, 70, and 120 nm) [65]. The nanofeature's array is also important for bacterial adhesion and it may form large patterns having several effects on microbial adhesion. For instance, the crest-shaped array (2 μm wide, 3 μm spacing, and different lengths) in polydi‐ methyl siloxane elastomer was bioinspired from the shark's skin. This elastomer structure exhibited no signs of *S. aureus* biofilm formation after 14 days, unlike the smooth surface, which

As mentioned above, the nanoscale level, size, and bacterial shape regarding nanofeature dimensions play a significant role. Bacterial features (adhesion, surface charge) are also important to the adhesion process. The surface of a nanostructure must be tested with several bacterial strains, as they exhibit different adhesion behaviors. For instance, a titanium surface nanostructured by femtosecond laser ablation and mimicking the superhydrophobic surface of the lotus leafs was not colonized after 18 hours by *P. aeruginosa*, whereas *S. aureus* adhe‐ sion was stronger when compared to the smooth surface [67]. This result suggests that some nanostructure surfaces may not be appropriate for medical applications in which the adhe‐

Conversely, silver nanoparticles (AgNPs) are gaining interest for biomedical applications because of their features having a higher surface/mass ratio and a potent antibacterial activity. These AgNPs may be applied as monolayers at the surface of biomaterials. A study on glass surfaces modified with AgNPs was carried out and it was found that they possess a great

anti-biofilm activity against *S. epidermidis* RP62A [68]. These AgNP-coated surfaces could be applied on a great variety of biomaterials. Nevertheless, it is important to conduct more studies to verify the anti-biofilm capacity with clinical isolates of different staphylococci species.

*Staphylococcus* biofilms is a virulence factor widely distributed in this genus, currently there are many studies about this subject; however, there are still questions to be answered about the process of biofilm formation. Some molecules involved in biofilm formation are recog‐ nized; nevertheless, the interaction between them is unknown, such as the formation of the structural network of the biofilm, the assembly and disassembly process, and the mecha‐ nisms of intrinsic and extrinsic regulation during these events. Many molecular pieces remain to be resolved, which allow us to fully understand the construction of a biofilm. Further‐ more, it is evident that a strain of *Staphylococcus* can form different types of biolfilm (PIAdependent or dependent protein), suggesting that staphylococcal biofilm is dynamic and adaptable to growth conditions. In fact, biofilm dynamics can be interpreted as a mechanism of resistance to environmental variations. The use of medical devices covered with anti-biofilm materials represents an alternative strategy but it is not decisive. The picture is complicated by the biological and physical characteristics of the different types of biofilm, the high genetic diversity into the genus, and the lack of comprehensive knowledge on biofilm formation properties, which leads to a complex and complicated scenario that prevents a successful anti-

release without AgNPs detachment and a strong

*Staphylococcus* Biofilms http://dx.doi.org/10.5772/62910 223

allowed the formation of a mature biofilm [66].

sion properties of the microorganisms are unknown.

stability in aqueous media, an extended Ag+

**5. Conclusions**

#### *IV.2.2. Steric barriers*

The chemical modifications of the surface may also consist on grafting long-chain polymers in order to form brush-type structures on it. The density of the chains provides a steric barrier that repels bacterial adhesion. The most widely studied polymers are derivatives of polyethy‐ lene oxides. In fact, residues of SAMs with ethylene glycol (4EG and 3EG) have lower bacterial adhesion in comparison to hydrophilic surfaces [57]. Polymers with ester residues (CHO2-) or cyclic hydrocarbons (C4H-, C6H-) exhibit less bacterial attachment strength than materials containing ethylglycol or hydroxyl group fragments [60].

#### *IV.2.3. Anti-adhesive strategies based on topographic modifications of the surface*

In the theories of bacterial adhesion, the appearance of the surface of the material was not considered. The relief of a surface depends on the scale, that is, for bacterial adhesion, the submicron scale is used. The reliefs are divided into: i) areas with irregular or random traits defined as rough; ii) areas with organized features, often made by an engineering process, defined by the term surface topography.

One study showed that adherence of *S. epidermidis* was similar on titanium surfaces, both rough and smooth [61]. SEM observations showed that this strain tends to adhere to grooves and depressions possessing dimensions similar to that of bacteria [62]. Regarding surface topog‐ raphy, it has been found that a surface constituted by titanium nanotubes is more hydrophil‐ ic than a conventional titanium surface. These properties have a biomedical application in orthopedics by decreasing bacterial adhesion [63]. Additionally, nanotubes could be filled with biocides in order to enhance its activity against biofilm. Superhydrophobic surfaces are being developed with nano or micro-features in order to create bacteria-free medical devices.

#### *IV.2.4. The influence of nanofeature physical structure on bacterial response*

Nanofeatures may adopt different shapes: nanotubes, notches, channels or grooves, holes or pillars. There are few studies regarding the relationship between nanofeatures and bacterial adhesion. Ercan et al. compared *S. aureus* and *S. epidermidis* adhesion on commercial titani‐ um surfaces with nanotubes of 20–80 nm of diameter. A decreased bacterial adhesion was observed for larger diameters (60 and 80 nm) [64]. However, the study conducted by Yu et al. produced opposite results: staphylococci adhesion increased proportionally to nanotube diameter (30, 70, and 120 nm) [65]. The nanofeature's array is also important for bacterial adhesion and it may form large patterns having several effects on microbial adhesion. For instance, the crest-shaped array (2 μm wide, 3 μm spacing, and different lengths) in polydi‐ methyl siloxane elastomer was bioinspired from the shark's skin. This elastomer structure exhibited no signs of *S. aureus* biofilm formation after 14 days, unlike the smooth surface, which allowed the formation of a mature biofilm [66].

As mentioned above, the nanoscale level, size, and bacterial shape regarding nanofeature dimensions play a significant role. Bacterial features (adhesion, surface charge) are also important to the adhesion process. The surface of a nanostructure must be tested with several bacterial strains, as they exhibit different adhesion behaviors. For instance, a titanium surface nanostructured by femtosecond laser ablation and mimicking the superhydrophobic surface of the lotus leafs was not colonized after 18 hours by *P. aeruginosa*, whereas *S. aureus* adhe‐ sion was stronger when compared to the smooth surface [67]. This result suggests that some nanostructure surfaces may not be appropriate for medical applications in which the adhe‐ sion properties of the microorganisms are unknown.

Conversely, silver nanoparticles (AgNPs) are gaining interest for biomedical applications because of their features having a higher surface/mass ratio and a potent antibacterial activity. These AgNPs may be applied as monolayers at the surface of biomaterials. A study on glass surfaces modified with AgNPs was carried out and it was found that they possess a great stability in aqueous media, an extended Ag+ release without AgNPs detachment and a strong anti-biofilm activity against *S. epidermidis* RP62A [68]. These AgNP-coated surfaces could be applied on a great variety of biomaterials. Nevertheless, it is important to conduct more studies to verify the anti-biofilm capacity with clinical isolates of different staphylococci species.

### **5. Conclusions**

with chemically controlled properties. SAMs with hydrophilic residues (OH, NH2) tend to decrease bacterial adhesion when compared to those with hydrophobic surfaces containing methyl groups (CH3) [57]. Some hydrophilic linings, such as hydrogels or medical devices with chemically modified surfaces have been developed in order to restrict biofilm development. Some clinical studies have reported that urinary catheters lined with heparin can reduce *Proteus mirabilis* biofilm [58]. However, it has been observed that heparin may stimulate biofilm formation by *S. aureus* [59]. This demonstrates that initial adhesion is not always sufficient to avoid biofilm development. Treatment with plasma can also create hydrophilic residues at the

The chemical modifications of the surface may also consist on grafting long-chain polymers in order to form brush-type structures on it. The density of the chains provides a steric barrier that repels bacterial adhesion. The most widely studied polymers are derivatives of polyethy‐ lene oxides. In fact, residues of SAMs with ethylene glycol (4EG and 3EG) have lower bacterial adhesion in comparison to hydrophilic surfaces [57]. Polymers with ester residues (CHO2-) or cyclic hydrocarbons (C4H-, C6H-) exhibit less bacterial attachment strength than materials

In the theories of bacterial adhesion, the appearance of the surface of the material was not considered. The relief of a surface depends on the scale, that is, for bacterial adhesion, the submicron scale is used. The reliefs are divided into: i) areas with irregular or random traits defined as rough; ii) areas with organized features, often made by an engineering process,

One study showed that adherence of *S. epidermidis* was similar on titanium surfaces, both rough and smooth [61]. SEM observations showed that this strain tends to adhere to grooves and depressions possessing dimensions similar to that of bacteria [62]. Regarding surface topog‐ raphy, it has been found that a surface constituted by titanium nanotubes is more hydrophil‐ ic than a conventional titanium surface. These properties have a biomedical application in orthopedics by decreasing bacterial adhesion [63]. Additionally, nanotubes could be filled with biocides in order to enhance its activity against biofilm. Superhydrophobic surfaces are being developed with nano or micro-features in order to create bacteria-free medical devices.

Nanofeatures may adopt different shapes: nanotubes, notches, channels or grooves, holes or pillars. There are few studies regarding the relationship between nanofeatures and bacterial adhesion. Ercan et al. compared *S. aureus* and *S. epidermidis* adhesion on commercial titani‐ um surfaces with nanotubes of 20–80 nm of diameter. A decreased bacterial adhesion was observed for larger diameters (60 and 80 nm) [64]. However, the study conducted by Yu et al. produced opposite results: staphylococci adhesion increased proportionally to nanotube

surface of medical devices producing antimicrobial activity.

containing ethylglycol or hydroxyl group fragments [60].

defined by the term surface topography.

*IV.2.3. Anti-adhesive strategies based on topographic modifications of the surface*

*IV.2.4. The influence of nanofeature physical structure on bacterial response*

*IV.2.2. Steric barriers*

222 Microbial Biofilms - Importance and Applications

*Staphylococcus* biofilms is a virulence factor widely distributed in this genus, currently there are many studies about this subject; however, there are still questions to be answered about the process of biofilm formation. Some molecules involved in biofilm formation are recog‐ nized; nevertheless, the interaction between them is unknown, such as the formation of the structural network of the biofilm, the assembly and disassembly process, and the mecha‐ nisms of intrinsic and extrinsic regulation during these events. Many molecular pieces remain to be resolved, which allow us to fully understand the construction of a biofilm. Further‐ more, it is evident that a strain of *Staphylococcus* can form different types of biolfilm (PIAdependent or dependent protein), suggesting that staphylococcal biofilm is dynamic and adaptable to growth conditions. In fact, biofilm dynamics can be interpreted as a mechanism of resistance to environmental variations. The use of medical devices covered with anti-biofilm materials represents an alternative strategy but it is not decisive. The picture is complicated by the biological and physical characteristics of the different types of biofilm, the high genetic diversity into the genus, and the lack of comprehensive knowledge on biofilm formation properties, which leads to a complex and complicated scenario that prevents a successful antibiofilm treatment. By understanding the processes of biofilm, it could control its formation and have biofilm-free medical devices.

[5] Begun J, Gaiani JM, Rohde H, Mack D, Calderwood SB, Ausubel FM, Sifri CD (2007) Staphylococcal biofilm exopolysaccharide protects against *Caenorhabditis elegans*

*Staphylococcus* Biofilms http://dx.doi.org/10.5772/62910 225

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### **Acknowledgements**

This work was supported by a grant from the "CONACyT México" (No. 153268). JJR, SRM, MECD, and JCCD appreciate the COFAA and EDI-IPN fellowships; also that provided by SNI-CONACyT.

### **Author details**

Janet Jan-Roblero1 , Sandra Rodríguez-Martínez2 , Mario E. Cancino-Diaz2 and Juan C. Cancino-Diaz1\*

\*Address all correspondence to: jccancinodiaz@hotmail.com

1 Microbiology department, Escuela Nacional de Ciencias Biológicas-IPN, Mexico City, Mexico.

2 Immunology department, Escuela Nacional de Ciencias Biológicas-IPN, Mexico City, Mexico.

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biofilm treatment. By understanding the processes of biofilm, it could control its formation

This work was supported by a grant from the "CONACyT México" (No. 153268). JJR, SRM, MECD, and JCCD appreciate the COFAA and EDI-IPN fellowships; also that provided by SNI-

1 Microbiology department, Escuela Nacional de Ciencias Biológicas-IPN, Mexico City,

2 Immunology department, Escuela Nacional de Ciencias Biológicas-IPN, Mexico City,

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[56] Baveja JK, Willcox MDP, Hume EBH, Kumar N, Odell R, Poole-Warren LA (2004) Furanones as potential anti-bacterial coatings on biomaterials. Biomaterials.

[57] Tegoulia VA, Cooper SL (2002) *Staphylococcus aureus* adhesion to selfassembled monolayers: effect of surface chemistry and fibrinogen presence. Colloids Surf B.

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6.


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**Chapter 12**

**Biofilm Formation of** *Salmonella*

Additional information is available at the end of the chapter

*Salmonella* spp. may form biofilm, and bacteria in biofilm are more resistant to drug, chemical, physical and mechanical stresses, and host immune system. The progress on biofilm research will be helpful for the development of new tools and strategies to prevent biofilm-related disease and decontaminate biofilm-derived *Salmonella* in food produc‐ tion. In this review, we present a comprehensive overview of biofilm formation in *Salmonella*, included that (1) the component of *Salmonella* biofilm, (2) the detection methods for biofilm, (3) the identification of biofilm-formation-associated genes, (4) the regula‐ tion mechanism of biofilm formation, and (5) virulence or resistance of *Salmonella* in biofilm.

**Keywords:** *Salmonella*, biofilm, component, mechanism, gene, pathogenicity, drug re‐

*Salmonella enteric* is an intracellular gram-negative pathogen that infects various hosts, which is classified into more than 2500 serovars [1]. Many serovars, such as those most commonly associated with human infections, including *Salmonella enteritidis, Salmonella typhimurium*, have a broad host range [2]. In contrast, other serovars, such as *Salmonella typhi, Salmonella paraty‐ phi, Salmonella gallinarum, Salmonella choleraesuis, Salmonella abortusovis*, and *Salmonella dublin*, have restricted host ranges and are associated primarily with one or a few hosts [3]. *Salmonella* can cause disease in domestic animals, ranging in severity of asymptom, diarrhea and enteri‐ tis to systemic syndrome, and result in a huge economic loss in pig and poultry industry. Salmonellosis is also a growing public health concern in both the developed and developing countries, since nontyphoidal *Salmonella* disease, a major cause of diarrheal disease globally, is estimated to cause 93 million enteric infections and 155,000 diarrheal deaths each year [4]. The

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. 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,

Daxin Peng

http://dx.doi.org/10.5772/62905

**Abstract**

sistance

**1. Introduction**


### **Chapter 12**

## **Biofilm Formation of** *Salmonella*

### Daxin Peng

[65] Yu W, Jiang X, Xu L, Zhao Y, Zhang F, Cao X (2011) Osteogenic gene expression of canine bone marrow stromal cell and bacterial adhesion on titanium with different

[66] Chung KK, Schumacher JF, Sampson EM, Burne RA, Antonelli PJ, Brennan AB (2007) Impact of engineered surface microtopography on biofilm formation of *Staphylococ‐*

[67] Fadeeva E, Truong VK, Stiesch M, Chichkov BN, Crawford RJ, Wang J, Ivanova EP (2011) Bacterial retention on superhydrophobic titanium surfaces fabricated by

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nanotubes. J. Biomed. Mater. Res. Part B. 99B:207e216.

femtosecond laser ablation. Langmuir. 27:3012e3019.

*cus aureus*. Biointerphases. 2:89.

230 Microbial Biofilms - Importance and Applications

als. 35:1779–88.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62905

#### **Abstract**

*Salmonella* spp. may form biofilm, and bacteria in biofilm are more resistant to drug, chemical, physical and mechanical stresses, and host immune system. The progress on biofilm research will be helpful for the development of new tools and strategies to prevent biofilm-related disease and decontaminate biofilm-derived *Salmonella* in food produc‐ tion. In this review, we present a comprehensive overview of biofilm formation in *Salmonella*, included that (1) the component of *Salmonella* biofilm, (2) the detection methods for biofilm, (3) the identification of biofilm-formation-associated genes, (4) the regula‐ tion mechanism of biofilm formation, and (5) virulence or resistance of *Salmonella* in biofilm.

**Keywords:** *Salmonella*, biofilm, component, mechanism, gene, pathogenicity, drug re‐ sistance

#### **1. Introduction**

*Salmonella enteric* is an intracellular gram-negative pathogen that infects various hosts, which is classified into more than 2500 serovars [1]. Many serovars, such as those most commonly associated with human infections, including *Salmonella enteritidis, Salmonella typhimurium*, have a broad host range [2]. In contrast, other serovars, such as *Salmonella typhi, Salmonella paraty‐ phi, Salmonella gallinarum, Salmonella choleraesuis, Salmonella abortusovis*, and *Salmonella dublin*, have restricted host ranges and are associated primarily with one or a few hosts [3]. *Salmonella* can cause disease in domestic animals, ranging in severity of asymptom, diarrhea and enteri‐ tis to systemic syndrome, and result in a huge economic loss in pig and poultry industry. Salmonellosis is also a growing public health concern in both the developed and developing countries, since nontyphoidal *Salmonella* disease, a major cause of diarrheal disease globally, is estimated to cause 93 million enteric infections and 155,000 diarrheal deaths each year [4]. The and [4]. AB (2007)

© 2016 The Author(s). Licensee InTech. 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, provided the original work is properly cited.

illnesses and outbreaks are most commonly attributed to exposure to contaminated food, and the eggs, broiler chickens, and pigs are among the top sources [5]. *Salmonella* often exist not only as planktonic cells but also as sessile, multicellular forms such as biofilms attached to surfa‐ ces. Biofilm formation is important for the spread of *Salmonella* because bacteria in the biofilm are resistant to disinfectants and chemical, physical, and mechanical stresses [6–8]. The biofilm formation is also contributed to *Salmonella* virulence, since bacteria in the biofilm are more resistant to antibiotics and host immune system, resulting in a chronic infection and the development of *Salmonella* carrier state [9, 10]. In our review, we present a comprehensive overview of biofilm formation in *Salmonella*.

solved in *Salmonella*. Cellulose is a polysaccharide composed of β(1 → 4)-linked D-glucose units [35], which is an important exopolysaccharide normally synthesized in the *Salmonella* biofilms. The production of cellulose and curli by *Salmonella* leads to a matrix of tightly packed cells covered in a hydrophobic network. The operons, *bcsABZD* and *bcsEFG*, are required for cellulose biosynthesis [36]. Cellulose biosynthesis is positively regulated by CsgD, which stimulates the transcription of AdrA that harbours a cytoplasmic GGDEF domain. AdrA activates cellulose production on the post-transcriptional level either by direct interaction with *bcs* operons or indirect interaction with bis-3′-5′-cyclic dimeric guanosine monophosphate (cdi-GMP) [25, 37, 38]. BapA, a large cell-surface protein required for biofilm formation, is encoded by *bapA* gene and secreted through a type-I protein secretion system (*bapBCD* operons) situated downstream of the *bapA* gene. The expression of *bapA* is coordinated with that of genes encoding curli fimbriae and cellulose, through the action of *csgD* [26, 39]. The *bapA* gene is also highly conserved in *Salmonella* [40]. *Salmonella* produces an O-antigen capsule coregulated with the fimbria- and cellulose-associated extracellular matrix. The operons yihUyshA and yihVW are responsible for capsule assembly and translocation [41] and regulated by CsgD. Although the O-antigen capsule do not appear to be important for multicellular behavior, they play an important role in attachment and environmental persistence [14]. However, the O-antigen capsule is required for biofilm formation of *Salmonella typhimurium* and *Salmonella Typhi* on cholesterol gallstones, and the operons are regulated in a *csgD*independent manner [42]. Extracellular DNA is shown to be a matrix component of *Salmonel‐ la* biofilms cultivated in flow chambers and on glass surfaces [28]. However, the presence of extracellular DNA plays an inhibitive and destabilizing effect during biofilm development of

Biofilm Formation of *Salmonella* http://dx.doi.org/10.5772/62905 233

Biofilm formation of *Salmonella* can be quantitated by microplate-based crystal violet stain‐ ing [43]. Briefly, the overnight broth cultures of bacterium are diluted 1:100 in the diluted tryptic soy broth (TSB). One hundred μl of bacterial suspension is added into 96-well Ubottomed polystyrene microtiter plates. Plates are incubated at 28°C for 24 h under static conditions. Then, non-adherent bacteria are removed and the wells are washed gently three times with 200 μl of distilled water. One hundred μl of 0.4% crystal violet (v/v) is added into each well and stained for 20 min. After discard of staining liquid, all loosely adhering bacteria and dye are gently washed off with distilled water for three times. The dye bound to the adherent cells is solubilized with 100 μl of anhydrous ethanol per well. The optical density (OD) is measured at 590 nm, and OD value of biofilm-formation strain is significantly higher than that of negative control. It provides more reproducible results with an addition of a fixtion step (80°C for 30 min) prior crystal violet staining [19]. Combined with resazurin assay, the number of metabolically active cells is able to be evaluated [44]. With wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in

*Salmonella* on abiotic surfaces [29].

**3. The detection methods for biofilm**

**3.1. Quantification of biofilm formation**

### **2. The component of** *Salmonella* **biofilm**

The biofilm formation is a multistep developmental process that always has several distin‐ guishable steps: (a) attachment to the carrier surface, reversible, (b) irreversible attachment, binding to the surface with the participation of adhesions or exopolysaccharides, (c) the development of microcolonies, a distinct mushroom shape, (d) the maturation of biofilm architecture [11, 12], (e) under favorable conditions, the synthesis of martrix compounds decreases and the matrix is enzymatically cleaved, leading to biofilm dispersion [13]. In natural environments, *Salmonella* forms biofilms on plant [14], abiotic surfaces, including plastics, metal and glass [15–17], meat and meat-processing environments [18, 19]. In addition, *Salmonella* can colonize gallstones under laboratory conditions [20], and the *Salmonella* biofilm can be directly visualized by confocal micrographs of extracellular matrix on the surface of human cholesterol gallstones [21]. They can also form biofilms on chicken intestinal epitheli‐ um [22] or HEp-2 cells that are suspended in once-flow-through continuous culture condi‐ tions [23].

The extracellular matrix of *Salmonella* biofilm is majorly composed of curli (amyloid fim‐ briae), cellulose [24, 25], biofilm-associated protein (Bap) [26], O-antigen capsule [14, 27], extracellular DNA [28, 29]. The expression pattern of the biofilm is serovar specific and correlates with contact surface [30]. Curli were first discovered in the late 1980s on *Escheri‐ chia coli* strains that caused bovine mastitis, and they are mainly involved in adhesion to surfaces, cell aggregation and biofilm formation. Curli also mediate host cell adhesion and invasion, and they are potent inducers of the host inflammatory response [12]. The curli protein is encoded by the divergently transcribed *csgBAC* (*agfBAC*) and *csgDEFG* (*agfDEFG*) operons [31, 32]. The *csgBAC* operon encodes the major structural subunit, CsgA, and the surfaceexposed nucleator protein CsgB. A third gene, *csgC*, is in the *csgBAC* operon, but no tran‐ script for *csgC* has been detected in curli biogenesis [32]. The other study shows that both CsgC and CsgE facilitate extracellular thin aggregative fimbriae synthesis in *Salmonella enteritidis* [33]. The *csgDEFG* operon encodes accessory proteins required for curli assembly. The *csgD* gene encodes a transcriptional regulator belonging to the LuxR family, CsgD, for active transcription of *csgBAC* promoter [24]. Although Giaouris et al. [34] found that CsgF was expressed in biofilm growth when compared with planktonic and biofilm cells of *Salmonella enteritidis* on stainless steel surface, the function of *csgF* and *csgG* genes has not been re‐

solved in *Salmonella*. Cellulose is a polysaccharide composed of β(1 → 4)-linked D-glucose units [35], which is an important exopolysaccharide normally synthesized in the *Salmonella* biofilms. The production of cellulose and curli by *Salmonella* leads to a matrix of tightly packed cells covered in a hydrophobic network. The operons, *bcsABZD* and *bcsEFG*, are required for cellulose biosynthesis [36]. Cellulose biosynthesis is positively regulated by CsgD, which stimulates the transcription of AdrA that harbours a cytoplasmic GGDEF domain. AdrA activates cellulose production on the post-transcriptional level either by direct interaction with *bcs* operons or indirect interaction with bis-3′-5′-cyclic dimeric guanosine monophosphate (cdi-GMP) [25, 37, 38]. BapA, a large cell-surface protein required for biofilm formation, is encoded by *bapA* gene and secreted through a type-I protein secretion system (*bapBCD* operons) situated downstream of the *bapA* gene. The expression of *bapA* is coordinated with that of genes encoding curli fimbriae and cellulose, through the action of *csgD* [26, 39]. The *bapA* gene is also highly conserved in *Salmonella* [40]. *Salmonella* produces an O-antigen capsule coregulated with the fimbria- and cellulose-associated extracellular matrix. The operons yihUyshA and yihVW are responsible for capsule assembly and translocation [41] and regulated by CsgD. Although the O-antigen capsule do not appear to be important for multicellular behavior, they play an important role in attachment and environmental persistence [14]. However, the O-antigen capsule is required for biofilm formation of *Salmonella typhimurium* and *Salmonella Typhi* on cholesterol gallstones, and the operons are regulated in a *csgD*independent manner [42]. Extracellular DNA is shown to be a matrix component of *Salmonel‐ la* biofilms cultivated in flow chambers and on glass surfaces [28]. However, the presence of extracellular DNA plays an inhibitive and destabilizing effect during biofilm development of *Salmonella* on abiotic surfaces [29].

### **3. The detection methods for biofilm**

### **3.1. Quantification of biofilm formation**

illnesses and outbreaks are most commonly attributed to exposure to contaminated food, and the eggs, broiler chickens, and pigs are among the top sources [5]. *Salmonella* often exist not only as planktonic cells but also as sessile, multicellular forms such as biofilms attached to surfa‐ ces. Biofilm formation is important for the spread of *Salmonella* because bacteria in the biofilm are resistant to disinfectants and chemical, physical, and mechanical stresses [6–8]. The biofilm formation is also contributed to *Salmonella* virulence, since bacteria in the biofilm are more resistant to antibiotics and host immune system, resulting in a chronic infection and the development of *Salmonella* carrier state [9, 10]. In our review, we present a comprehensive

The biofilm formation is a multistep developmental process that always has several distin‐ guishable steps: (a) attachment to the carrier surface, reversible, (b) irreversible attachment, binding to the surface with the participation of adhesions or exopolysaccharides, (c) the development of microcolonies, a distinct mushroom shape, (d) the maturation of biofilm architecture [11, 12], (e) under favorable conditions, the synthesis of martrix compounds decreases and the matrix is enzymatically cleaved, leading to biofilm dispersion [13]. In natural environments, *Salmonella* forms biofilms on plant [14], abiotic surfaces, including plastics, metal and glass [15–17], meat and meat-processing environments [18, 19]. In addition, *Salmonella* can colonize gallstones under laboratory conditions [20], and the *Salmonella* biofilm can be directly visualized by confocal micrographs of extracellular matrix on the surface of human cholesterol gallstones [21]. They can also form biofilms on chicken intestinal epitheli‐ um [22] or HEp-2 cells that are suspended in once-flow-through continuous culture condi‐

The extracellular matrix of *Salmonella* biofilm is majorly composed of curli (amyloid fim‐ briae), cellulose [24, 25], biofilm-associated protein (Bap) [26], O-antigen capsule [14, 27], extracellular DNA [28, 29]. The expression pattern of the biofilm is serovar specific and correlates with contact surface [30]. Curli were first discovered in the late 1980s on *Escheri‐ chia coli* strains that caused bovine mastitis, and they are mainly involved in adhesion to surfaces, cell aggregation and biofilm formation. Curli also mediate host cell adhesion and invasion, and they are potent inducers of the host inflammatory response [12]. The curli protein is encoded by the divergently transcribed *csgBAC* (*agfBAC*) and *csgDEFG* (*agfDEFG*) operons [31, 32]. The *csgBAC* operon encodes the major structural subunit, CsgA, and the surfaceexposed nucleator protein CsgB. A third gene, *csgC*, is in the *csgBAC* operon, but no tran‐ script for *csgC* has been detected in curli biogenesis [32]. The other study shows that both CsgC and CsgE facilitate extracellular thin aggregative fimbriae synthesis in *Salmonella enteritidis* [33]. The *csgDEFG* operon encodes accessory proteins required for curli assembly. The *csgD* gene encodes a transcriptional regulator belonging to the LuxR family, CsgD, for active transcription of *csgBAC* promoter [24]. Although Giaouris et al. [34] found that CsgF was expressed in biofilm growth when compared with planktonic and biofilm cells of *Salmonella enteritidis* on stainless steel surface, the function of *csgF* and *csgG* genes has not been re‐

19].

overview of biofilm formation in *Salmonella*.

232 Microbial Biofilms - Importance and Applications

tions [23].

**2. The component of** *Salmonella* **biofilm**

Biofilm formation of *Salmonella* can be quantitated by microplate-based crystal violet stain‐ ing [43]. Briefly, the overnight broth cultures of bacterium are diluted 1:100 in the diluted tryptic soy broth (TSB). One hundred μl of bacterial suspension is added into 96-well Ubottomed polystyrene microtiter plates. Plates are incubated at 28°C for 24 h under static conditions. Then, non-adherent bacteria are removed and the wells are washed gently three times with 200 μl of distilled water. One hundred μl of 0.4% crystal violet (v/v) is added into each well and stained for 20 min. After discard of staining liquid, all loosely adhering bacteria and dye are gently washed off with distilled water for three times. The dye bound to the adherent cells is solubilized with 100 μl of anhydrous ethanol per well. The optical density (OD) is measured at 590 nm, and OD value of biofilm-formation strain is significantly higher than that of negative control. It provides more reproducible results with an addition of a fixtion step (80°C for 30 min) prior crystal violet staining [19]. Combined with resazurin assay, the number of metabolically active cells is able to be evaluated [44]. With wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in

biofilms, the spectrofluorometric assay provides a more sensitive method for quantification and characterization of bacterial biofilms [45].

smooth, brown, and mucoid, indicating a lack of cellulose synthesis but overproduced capsular polysaccharide (SBAM), and (e) smooth and white, indicating a lack of both curli and cellulose

Biofilm Formation of *Salmonella* http://dx.doi.org/10.5772/62905 235

Bacteria cultured on coverslipes, dish, or microplate are stained by 0.1 M phosphate-buf‐ fered saline (pH 7.2) containing SYTO 9 and propidium iodide. After 10 min incubation in the dark at room temperature, stained samples are examined using a confocal scanning laser microscopy. Fluorochromes are excited using an argon laser source at 488 nm. Images are collected in two channels, 490–515 and 620–640 nm, corresponding to the emission maxima for SYTO 9 and propidium iodide, respectively. Optical sections approximately 1 μm in height are collected starting from below the focal plane to upward through the entire biofilm. The

The most common biofilm-formation–associated genes are the genes encode adhesins. The best characterized of the *Salmonella* fimbriae is type-1 fimbriae. This fimbrial type is encoded by the fim gene cluster and is assembled by the chaperone–usher system [52]. The fimA gene encodes the major structural subunit, while the fimH gene encodes the adhesin protein that is located at the tip of the assembled fimbrial structure and mediates binding to the receptor. The FimH adhesin is involved in biofilm formation on HEp-2 tissue culture cells, murine intesti‐ nal epithelium, and chicken intestinal epithelium [22, 23]. The long polar fimbriae (Lpf) are encoded by the *lpfABCDE* genes and have been implicated in the colonization of the murine intestinal mucosa [53, 54]. Plasmid-encoded fimbriae (Pef) are encoded on the 90-kb *Salmonel‐ la* virulence plasmid and are majorly encoded by *pefBCD*, orf5, and orf6 genes. Both Lpf and Pef contribute to the early steps of biofilm formation [55]. *Salmonella enteritidis* produce a variety of potentially adherent fimbrial types including SEF14 (SefA), SEF17 (CsgA), SEF18 (SefD), and SEF21 (type I, FimA), the role of each fimbrial in biofilm formation is different. The SEF17 encoded by *csgA* gene stabilize cell–cell interactions during biofilm formation, while SEF21 fimbriae may involve cell surface adherence [56]. SadA is trimeric autotransporter adhesin of *Salmonella typhimurium*, the expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells [57]. *Salmonella* may persist on post-harvest lettuce during cold storage, the genes *stfC, bcsA, misL*, and *yidR*, encoding a fimbrial outer membrane usher, a cellulose synthase catalytic subunit, an adhe‐ sin of the autotransporter family expressed from the *Salmonella* pathogenicity island-3, and a putative ATP-/GTP-binding protein, respectively, have a role in persistence of the pathogen. The *bcsA, misL*, and *yidR* knockout mutants are impaired in attachment and biofilm forma‐

biofilm cells are clearly observed in a multilayer community [20, 51].

**4. Identification of biofilm-formation–associated genes**

tion, suggesting that these functions are required for biofilm formation [58].

*Salmonella* flagella are not required for the formation of the multicellular morphotype on plates. However, the global behavior of the bacterial community on air–liquid, surface–liquid, or cell–

production (SAW) [19, 31, 50].

**3.6. Confocal laser microscopy**

### **3.2. Biofilm formation in glass tube**

The overnight cultures of bacteria are diluted 1:100 in the diluted TSB. Two milliliters of each bacterial suspension are added into borosilicate glass tubes and incubated at 28°C for 48 h. Then, the liquid is decanted and the tubes are washed gently three times with distilled water. Two ml of 0.4% crystal violet (v/v) are added into each tube and stained at room tempera‐ ture for 20 min. The stained biofilm is observed at the liquid–air interface on the glass test tube walls or at the bottom of the tube [46]. The glass tubes may also be incubated at 37°C at 200 rpm by using an orbital shaker, and biofilm is observed at interphase without staining [47].

### **3.3. Congo red/carbol fuchsin staining**

The overnight culture (1:100 diluted in TSB) is inoculated into 3 ml of fresh TSB in a 6-well plate containing sterile polystyrene coverslip (20 × 20 mm). After incubation at 28°C for 24 or 48 h without agitation, the coverslips are removed carefully, treated with cetylpyridinium chloride (10 mM) for 30 s, rinsed with distilled water and air dried for 20–30 min. After fixation by gentle heating, the coverslips are stained with a mixture of saturated aqueous Congo red solution and 10% Tween-80 (2:1, V/V) for 30 min and rinsed with distilled water. After staining with 10% (v/v) Ziehl carbol fuchsin for 6 min and rinsing in distilled water, the coverslips are air dried and mounted on slides [48]. Under a light microscope, bacterial cells on slides show purple staining, while the exopolysaccharides of biofilm show pink staining [46].

### **3.4. Field emission scanning electron microscopy**

The coverslips with cultured bacteria are fixed in 3% glutaraldehyde in 0.1 M phosphatebuffered saline at 4°C for 2 h. The samples are then dehydrated with increasing concentra‐ tions of ethanol (50, 70, 80, 90, and 100%) followed by isoamyl acetate (100%), each for 15 min. The samples are critical point dried for 5 h, coated with gold palladium alloy, and observed under a field emission scanning electron microscope [49]. The biofilm-formation strain exhibits increased clusters of bacteria cells with curli fimbriae and has meshwork-like structures surrounding the cell surfaces.

### **3.5. Congo red and calcofluor plates**

LB agar plate without salt supplemented with 40 mg/L Congo red and 20 mg/L brilliant blue is used to determine the Congo red-binding property of the colonies. LB agar plate supple‐ mented with 200 mg/L calcofluor (fluorescent brightener) is used to determine the cellulose production by comparing the fluorescence of the test strains under UV light [49]. Biofilm of *Salmonella* is mainly composed of curli and cellulose, and *Salmonella* strains were grouped into distinct morphotypes according to Congo red binding: (a) red, dry, and rough indicating curli and cellulose production (RDAR), (b) brown, dry, and rough, indicating a lack of cellulose synthesis (BDAR), (c) pink, dry, and rough, indicating a defect in curli expression (PDAR), (d)

smooth, brown, and mucoid, indicating a lack of cellulose synthesis but overproduced capsular polysaccharide (SBAM), and (e) smooth and white, indicating a lack of both curli and cellulose production (SAW) [19, 31, 50].

#### **3.6. Confocal laser microscopy**

biofilms, the spectrofluorometric assay provides a more sensitive method for quantification

The overnight cultures of bacteria are diluted 1:100 in the diluted TSB. Two milliliters of each bacterial suspension are added into borosilicate glass tubes and incubated at 28°C for 48 h. Then, the liquid is decanted and the tubes are washed gently three times with distilled water. Two ml of 0.4% crystal violet (v/v) are added into each tube and stained at room tempera‐ ture for 20 min. The stained biofilm is observed at the liquid–air interface on the glass test tube walls or at the bottom of the tube [46]. The glass tubes may also be incubated at 37°C at 200 rpm by using an orbital shaker, and biofilm is observed at interphase without staining [47].

stained

The overnight culture (1:100 diluted in TSB) is inoculated into 3 ml of fresh TSB in a 6-well plate containing sterile polystyrene coverslip (20 × 20 mm). After incubation at 28°C for 24 or 48 h without agitation, the coverslips are removed carefully, treated with cetylpyridinium chloride (10 mM) for 30 s, rinsed with distilled water and air dried for 20–30 min. After fixation by gentle heating, the coverslips are stained with a mixture of saturated aqueous Congo red solution and 10% Tween-80 (2:1, V/V) for 30 min and rinsed with distilled water. After staining with 10% (v/v) Ziehl carbol fuchsin for 6 min and rinsing in distilled water, the coverslips are air dried and mounted on slides [48]. Under a light microscope, bacterial cells on slides show

The coverslips with cultured bacteria are fixed in 3% glutaraldehyde in 0.1 M phosphatebuffered saline at 4°C for 2 h. The samples are then dehydrated with increasing concentra‐ tions of ethanol (50, 70, 80, 90, and 100%) followed by isoamyl acetate (100%), each for 15 min. The samples are critical point dried for 5 h, coated with gold palladium alloy, and observed under a field emission scanning electron microscope [49]. The biofilm-formation strain exhibits increased clusters of bacteria cells with curli fimbriae and has meshwork-like structures

LB agar plate without salt supplemented with 40 mg/L Congo red and 20 mg/L brilliant blue is used to determine the Congo red-binding property of the colonies. LB agar plate supple‐ mented with 200 mg/L calcofluor (fluorescent brightener) is used to determine the cellulose production by comparing the fluorescence of the test strains under UV light [49]. Biofilm of *Salmonella* is mainly composed of curli and cellulose, and *Salmonella* strains were grouped into distinct morphotypes according to Congo red binding: (a) red, dry, and rough indicating curli and cellulose production (RDAR), (b) brown, dry, and rough, indicating a lack of cellulose synthesis (BDAR), (c) pink, dry, and rough, indicating a defect in curli expression (PDAR), (d)

purple staining, while the exopolysaccharides of biofilm show pink staining [46].

and characterization of bacterial biofilms [45].

**3.2. Biofilm formation in glass tube**

234 Microbial Biofilms - Importance and Applications

**3.3. Congo red/carbol fuchsin staining**

**3.4. Field emission scanning electron microscopy**

surrounding the cell surfaces.

**3.5. Congo red and calcofluor plates**

Bacteria cultured on coverslipes, dish, or microplate are stained by 0.1 M phosphate-buf‐ fered saline (pH 7.2) containing SYTO 9 and propidium iodide. After 10 min incubation in the dark at room temperature, stained samples are examined using a confocal scanning laser microscopy. Fluorochromes are excited using an argon laser source at 488 nm. Images are collected in two channels, 490–515 and 620–640 nm, corresponding to the emission maxima for SYTO 9 and propidium iodide, respectively. Optical sections approximately 1 μm in height are collected starting from below the focal plane to upward through the entire biofilm. The biofilm cells are clearly observed in a multilayer community [20, 51].

### **4. Identification of biofilm-formation–associated genes**

The most common biofilm-formation–associated genes are the genes encode adhesins. The best characterized of the *Salmonella* fimbriae is type-1 fimbriae. This fimbrial type is encoded by the fim gene cluster and is assembled by the chaperone–usher system [52]. The fimA gene encodes the major structural subunit, while the fimH gene encodes the adhesin protein that is located at the tip of the assembled fimbrial structure and mediates binding to the receptor. The FimH adhesin is involved in biofilm formation on HEp-2 tissue culture cells, murine intesti‐ nal epithelium, and chicken intestinal epithelium [22, 23]. The long polar fimbriae (Lpf) are encoded by the *lpfABCDE* genes and have been implicated in the colonization of the murine intestinal mucosa [53, 54]. Plasmid-encoded fimbriae (Pef) are encoded on the 90-kb *Salmonel‐ la* virulence plasmid and are majorly encoded by *pefBCD*, orf5, and orf6 genes. Both Lpf and Pef contribute to the early steps of biofilm formation [55]. *Salmonella enteritidis* produce a variety of potentially adherent fimbrial types including SEF14 (SefA), SEF17 (CsgA), SEF18 (SefD), and SEF21 (type I, FimA), the role of each fimbrial in biofilm formation is different. The SEF17 encoded by *csgA* gene stabilize cell–cell interactions during biofilm formation, while SEF21 fimbriae may involve cell surface adherence [56]. SadA is trimeric autotransporter adhesin of *Salmonella typhimurium*, the expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells [57]. *Salmonella* may persist on post-harvest lettuce during cold storage, the genes *stfC, bcsA, misL*, and *yidR*, encoding a fimbrial outer membrane usher, a cellulose synthase catalytic subunit, an adhe‐ sin of the autotransporter family expressed from the *Salmonella* pathogenicity island-3, and a putative ATP-/GTP-binding protein, respectively, have a role in persistence of the pathogen. The *bcsA, misL*, and *yidR* knockout mutants are impaired in attachment and biofilm forma‐ tion, suggesting that these functions are required for biofilm formation [58].

*Salmonella* flagella are not required for the formation of the multicellular morphotype on plates. However, the global behavior of the bacterial community on air–liquid, surface–liquid, or cell–

liquid interfaces is changed in the absence of flagella. In a mutant lacking flagella and thin aggregative fimbriae, the contribution of the latter to the multicellular morphotype is dominant [59]. Biofilm formation of an flgK mutant in meat and poultry broths and their attachment on surfaces of stainless steel and glass are significantly reduced compared with that of the wild-type strain, suggest that expression of flagella could be involved in biofilm formation and attachment of *Salmonella* on contact surfaces [60]. The presence of the flagellar filament enhances binding and biofilm formation in the presence of bile, while flagellar motility and expression of type-1 fimbriae were unimportant in biofilm formation on choles‐ terol gallstones [61].

*ompR* mutant showed a complete loss of production of curli and biofilm formation. The other mutants showed a modified production of curli and cellulose with less effect related to biofilm formation [68]. Therefore, an integral LPS, at both the O-antigen and core polysaccharide levels, are important in the modulation of curli protein and cellulose production, as well as in

Biofilm Formation of *Salmonella* http://dx.doi.org/10.5772/62905 237

Biofilm formation is majorly regulated by CsgD protein, a regulator belonging to the LuxR family [69]. CsgD has an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif. The unphosphorylated CsgD directly binds the *csgBA* and *adrA* promoter regions to activate transcription [70]. Multiple factors bind to the promoter sequence of *csgD* and regulate its transcription, such as OmpR, RpoS, RpoE, integration host factor (IHF), histone-like nucleoid structuring protein (H-NS), and MlrA. OmpR is one of first discovered to be required for *csgD* transcription [71]. Six binding sites (D1–D6) for OmpR are identified in *csgD* promoter regions. Binding of OmpR-P to D2 centered immediately upstream of D1 is proposed to repress promoter activity. IHF competes with OmpR-P for binding at its up‐ stream site IHF1, which overlaps with D3–D6 and thereby activate the transcription of *csgD* [72]. The mutant of *ompR* in *Salmonella enteritidis* and *Salmonella pullorum* has inability to produce cellulose, curli, and biofilm [68, 73]. RpoS, encodes an alternative sigma factor of RNA polymerase, is critical for bacterial endurance under the most-stressful conditions, including stationary-phase entrance and host adaptation. RpoS is required for transcriptional activa‐ tion of the *csgD* promoter in *Salmonella typhimurium* strains that rdar morphotype are normal‐ ly expressed at low temperature [31]. However, in two *Salmonella typhimurium* strains, spontaneous mutants are found forming rdar colonies independent of temperature, the regulation of *csgD* is independent of *rpoS* [71]. Partially independent of *rpoS* for regulation of *csgD* is observed in *Salmonella enteritidis*. The *rpoS* mutant in *Salmonella pullorum* also shows similar biofilm forming ability as the wild-type strain [68], suggests that another sigma factor may recognize the *csgD* promoter. RpoE is an another regulator in the expression of thin aggregative fimbriae in *Salmonella* [74], since the *rpoE* deletion mutant shows significantly reduced amounts of *csgD* expression and modulated biofilm formation. Compared the expression of six different Sigma factors during biofilm formation in a *rpoS*-independent biofilm-formation strain, the expression of *rpoE* gene was the highest, and the *rpoE* mutant could not produce biofilm [75]. Therefore, RpoE acts as a regulator for *csgD* expression. IHF is a histone-like heterodimeric protein composed of two homologous subunits. IHF interacts with a define DNA sequence that has a supportive A-tract upstream of the consensus sequence by binding to the minor groove of the DNA. The *ihf* mutants show altered and reduced biofilm morphotypes on Congo Red agar plates [72]. H-NS prefers to bind AT-rich sites in the intergenic *csgBAC* and *csgDEFG* regions and causes moderate activation of *csgD* promoter. The inactivation of *hns* gene result in reduced expression of the rdar morphotype on agar plate [72]. MlrA (MerR-like regulator) acts directly or indirectly on the *csgD* promoter, the *mlrA* mu‐

**5. Regulation mechanism of biofilm formation**

biofilm formation.

Pathogenicity islands accommodate large clusters of genes that contribute to a particular virulence phenotype. *Salmonella* possess at least seven *salmonella* pathogenicity islands (SPIs). Among these, SPI1 is primarily required for bacterial motility and invasion of host cells. *Salmonella typhimurium* cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type-III secretion functions. Surprisingly, mutations in genes essential for known bacterial biofilm pathways *(bcsA, csgBA, bapA*) did not affect the biofilms formation, indicating that this phenomenon is independent of established biofilm mechanisms [62]. *Salmonella* biofilm cells exposed to superheated steam show decreased transcription of flagella and SPI-1 genes, respectively, whereas increased transcription of SPI-2 genes, important for bacterial survival and replication inside host cells, is detected [63]. In contrast, when compared biofilms of *Salmonella typhimurium* with planktonic cells, the most highly downregulated genes in the biofilm are located on SPI-2 and that a functional SPI2 secretion system regulator (*ssrA*) is required for *Salmonella typhimurium* biofilm formation. Genes involved in tryptophan (*trp*) biosynthesis and transport are upregulated in the biofilm. Deletion of *trpE* results in de‐ creased bacterial attachment and biofilm formation, indicating that aromatic amino acids make an important contribution to biofilm formation [64]. The *aro* mutants of *Salmonella* are frequently used as live vaccines for the oral vaccination of domestic animals, and they are unable to synthesize chorismate, which is a key intermediate in the synthesis of aromatic amino acids. The *aro* mutants exhibit a decreased production of cellulose, N-acetyl-D-glucosamine, or N-acetylneuraminic acid-containing capsular polysaccharide and fimbriae, which ex‐ plains their inability to form biofilms [65].

Lipopolysaccharide (LPS) synthesis also involves the biofilm formation of *Salmonella*. Two Tn5 insertion mutations in genes that are involved in *ddhC* and *waaG* result in diminished expression of colony rugosity. Both mutants have impaired biofilm formation when grown in rich medium with low osmolarity, they constitutively form larger amounts of biofilms when the growth medium was supplemented with either glucose or a combination of glucose and NaCl [49]. The *rfbA* gene also involve in lipopolysaccharide biosynthesis. Biofilm formation by the *rfbA* mutant in meat and poultry broths and their attachment on surfaces of stainless steel and glass is significantly reduced [60]. Using transposon mutagenesis, the genes *metE, ompR, rpoS, rfaG, rfaJ, rfaK, rfaP, rfbH, rhlE, spiA*, and *steB* are found to be associated with biofilm formation of *Salmonella enteritidis* [66, 67]. When eight mutants with knockout of genes *ompR, rpoS, rfaG, rfbH, rhlE, metE, spiA*, or *steB* from the *Salmonella pullorum* are constructed. Only the

*ompR* mutant showed a complete loss of production of curli and biofilm formation. The other mutants showed a modified production of curli and cellulose with less effect related to biofilm formation [68]. Therefore, an integral LPS, at both the O-antigen and core polysaccharide levels, are important in the modulation of curli protein and cellulose production, as well as in biofilm formation.

### **5. Regulation mechanism of biofilm formation**

liquid interfaces is changed in the absence of flagella. In a mutant lacking flagella and thin aggregative fimbriae, the contribution of the latter to the multicellular morphotype is dominant [59]. Biofilm formation of an flgK mutant in meat and poultry broths and their attachment on surfaces of stainless steel and glass are significantly reduced compared with that of the wild-type strain, suggest that expression of flagella could be involved in biofilm formation and attachment of *Salmonella* on contact surfaces [60]. The presence of the flagellar filament enhances binding and biofilm formation in the presence of bile, while flagellar motility and expression of type-1 fimbriae were unimportant in biofilm formation on choles‐

Pathogenicity islands accommodate large clusters of genes that contribute to a particular virulence phenotype. *Salmonella* possess at least seven *salmonella* pathogenicity islands (SPIs). Among these, SPI1 is primarily required for bacterial motility and invasion of host cells. *Salmonella typhimurium* cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type-III secretion functions. Surprisingly, mutations in genes essential for known bacterial biofilm pathways *(bcsA, csgBA, bapA*) did not affect the biofilms formation, indicating that this phenomenon is independent of established biofilm mechanisms [62]. *Salmonella* biofilm cells exposed to superheated steam show decreased transcription of flagella and SPI-1 genes, respectively, whereas increased transcription of SPI-2 genes, important for bacterial survival and replication inside host cells, is detected [63]. In contrast, when compared biofilms of *Salmonella typhimurium* with planktonic cells, the most highly downregulated genes in the biofilm are located on SPI-2 and that a functional SPI2 secretion system regulator (*ssrA*) is required for *Salmonella typhimurium* biofilm formation. Genes involved in tryptophan (*trp*) biosynthesis and transport are upregulated in the biofilm. Deletion of *trpE* results in de‐ creased bacterial attachment and biofilm formation, indicating that aromatic amino acids make an important contribution to biofilm formation [64]. The *aro* mutants of *Salmonella* are frequently used as live vaccines for the oral vaccination of domestic animals, and they are unable to synthesize chorismate, which is a key intermediate in the synthesis of aromatic amino acids. The *aro* mutants exhibit a decreased production of cellulose, N-acetyl-D-glucosamine, or N-acetylneuraminic acid-containing capsular polysaccharide and fimbriae, which ex‐

Lipopolysaccharide (LPS) synthesis also involves the biofilm formation of *Salmonella*. Two Tn5 insertion mutations in genes that are involved in *ddhC* and *waaG* result in diminished expression of colony rugosity. Both mutants have impaired biofilm formation when grown in rich medium with low osmolarity, they constitutively form larger amounts of biofilms when the growth medium was supplemented with either glucose or a combination of glucose and NaCl [49]. The *rfbA* gene also involve in lipopolysaccharide biosynthesis. Biofilm formation by the *rfbA* mutant in meat and poultry broths and their attachment on surfaces of stainless steel and glass is significantly reduced [60]. Using transposon mutagenesis, the genes *metE, ompR, rpoS, rfaG, rfaJ, rfaK, rfaP, rfbH, rhlE, spiA*, and *steB* are found to be associated with biofilm formation of *Salmonella enteritidis* [66, 67]. When eight mutants with knockout of genes *ompR, rpoS, rfaG, rfbH, rhlE, metE, spiA*, or *steB* from the *Salmonella pullorum* are constructed. Only the

terol gallstones [61].

236 Microbial Biofilms - Importance and Applications

plains their inability to form biofilms [65].

Biofilm formation is majorly regulated by CsgD protein, a regulator belonging to the LuxR family [69]. CsgD has an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif. The unphosphorylated CsgD directly binds the *csgBA* and *adrA* promoter regions to activate transcription [70]. Multiple factors bind to the promoter sequence of *csgD* and regulate its transcription, such as OmpR, RpoS, RpoE, integration host factor (IHF), histone-like nucleoid structuring protein (H-NS), and MlrA. OmpR is one of first discovered to be required for *csgD* transcription [71]. Six binding sites (D1–D6) for OmpR are identified in *csgD* promoter regions. Binding of OmpR-P to D2 centered immediately upstream of D1 is proposed to repress promoter activity. IHF competes with OmpR-P for binding at its up‐ stream site IHF1, which overlaps with D3–D6 and thereby activate the transcription of *csgD* [72]. The mutant of *ompR* in *Salmonella enteritidis* and *Salmonella pullorum* has inability to produce cellulose, curli, and biofilm [68, 73]. RpoS, encodes an alternative sigma factor of RNA polymerase, is critical for bacterial endurance under the most-stressful conditions, including stationary-phase entrance and host adaptation. RpoS is required for transcriptional activa‐ tion of the *csgD* promoter in *Salmonella typhimurium* strains that rdar morphotype are normal‐ ly expressed at low temperature [31]. However, in two *Salmonella typhimurium* strains, spontaneous mutants are found forming rdar colonies independent of temperature, the regulation of *csgD* is independent of *rpoS* [71]. Partially independent of *rpoS* for regulation of *csgD* is observed in *Salmonella enteritidis*. The *rpoS* mutant in *Salmonella pullorum* also shows similar biofilm forming ability as the wild-type strain [68], suggests that another sigma factor may recognize the *csgD* promoter. RpoE is an another regulator in the expression of thin aggregative fimbriae in *Salmonella* [74], since the *rpoE* deletion mutant shows significantly reduced amounts of *csgD* expression and modulated biofilm formation. Compared the expression of six different Sigma factors during biofilm formation in a *rpoS*-independent biofilm-formation strain, the expression of *rpoE* gene was the highest, and the *rpoE* mutant could not produce biofilm [75]. Therefore, RpoE acts as a regulator for *csgD* expression. IHF is a histone-like heterodimeric protein composed of two homologous subunits. IHF interacts with a define DNA sequence that has a supportive A-tract upstream of the consensus sequence by binding to the minor groove of the DNA. The *ihf* mutants show altered and reduced biofilm morphotypes on Congo Red agar plates [72]. H-NS prefers to bind AT-rich sites in the intergenic *csgBAC* and *csgDEFG* regions and causes moderate activation of *csgD* promoter. The inactivation of *hns* gene result in reduced expression of the rdar morphotype on agar plate [72]. MlrA (MerR-like regulator) acts directly or indirectly on the *csgD* promoter, the *mlrA* mu‐ mutant

tants of *Salmonella typhimurium* no longer produce curli or rugose colony morphology. However, inactivation of *mlrA* did not affect curli production and aggregative morphology in an upregulated curli producing *Salmonella typhimurium* derivative containing a temperatureand RpoS-independent *csgD* promoter region. Therefore, MlrA acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by *Salmonella typhimurium* [76].

due to the repression of *CsgD* expression, through a mechanism dependent on the accumula‐

Biofilm Formation of *Salmonella* http://dx.doi.org/10.5772/62905 239

Many gram-negative bacteria utilize N-acyl-L-homoserine lactones (AHLs) to bind to transcriptional regulators leading to activation or repression of target genes. *Salmonella* do not synthesize AHLs but do contain the AHL receptor, SdiA. The *Salmonella sdiA* gene regulates the *rck* gene, which mediates its adhesion and invasion of epithelial cells and the resistance of the organism to complement [87]. The *rck* gene is located on the virulence plasmid of pRST98, AHLs increase *rck* expression in pRST98-carrying strains, thereby enhancing bacterial

Biofilm formation may involve in the virulence of *Salmonella. Salmonella enteritidis* stains isolated from either the environment, dairy products, or infected patients are divided into two groups on the basis of their virulence (50% lethal dose) in chickens infected intraperitoneally. Only the virulent strains produce aggregates and formed visible filaments attached to the glass tube [47]. Further study confirms that the virulence of the biofilm-producing strain in infected chickens increases proportionally to the amount of stored glycogen, suggesting a possible role of the glycogen depot in the virulence of *Salmonella enteritidis* [89]. When tested for infection in Caco-2 cells and HEp-2 cells, the more virulent strains of *Salmonella enteritidis*, which are biofilm producers in adherence test medium, are able to disrupt monolayers. In contrast, the low-virulence strains of *Salmonella enteritidis*, which do not produce biofilms in adherence test medium, have no effect on the same cells. The high-virulence *Salmonella enteritidis* strains incubated under optimum biofilm-forming conditions may release a soluble factor, which enables the disruption of the integrity of Caco-2 monolayers [90]. The relationship between biofilm-forming ability and the pathogenicity is also evaluated in *Salmonella pullorum*. Although the virulence of *Salmonella pullorum* strains is independent of their ability of biofilm formation, prior growth as a biofilm for a biofilm producer of *Salmonella pullorum* leads to enhanced virulence in chickens, suggested that biofilm formation may be one of important

The *csgBAC* operon is required for curli biosynthesis in *Salmonella*. The *csgA* mutation is not reduced in ability to attach or colonize alfalfa sprouts, whereas the *csgB* mutation is reduced. Thus, *csgB* alone can play a role in attachment of *Salmonella* to plant tissue [91]. Competitive infection experiments in mice shows that *csgA* mutant cells outcompeted rdar-positive wildtype cells, indicating that aggregation via the rdar morphotype is not a virulence adaptation in *Salmonella typhimurium*. Furthermore, in vivo imaging experiments show that thin aggre‐ gative fimbriae genes are not expressed during infection but are expressed once *Salmonella* was passed out of the mice into the feces [92]. However, *Salmonella typhimurium* strains isolated from water buffalo calves affected by lethal gastroenteritis are tested in vivo in a mouse model of mixed infection. The most pathogenic strain is characterized by a high number of viru‐ lence factors and the presence of the locus *csgA*, coding for a thin aggregative fimbria [93].

adherence, serum resistance, and bacterial biofilm formation [88].

**6. Virulence or resistance for biofilm**

virulence factor for *Salmonella pullorum* infection [46].

tion of the sRNA RprA [86].

c-di-GMP is recognized as a ubiquitous bacterial second messenger and a key regulator in bacterial transition from a motile and planktonic to a sessile and biofilm lifestyle. High intracellular c-di-GMP levels promote extracellular matrix production and subsequent biofilm formation and repress motility, whereas low intracellular c-di-GMP levels suppress matrix production and promote single-cell motility [77]. The synthesis/degradation of c-di-GMP depends on diguanylate cyclase/phosphodiesterase enzymatic activities. The cyclase activity, which converts two molecules of GTP to c-di-GMP, is encoded in the GGDEF protein domain, while phosphodiesterase activity, which hydrolyzes c-di-GMP to linear 5′-pGpG or two GMP molecules, is encoded in the EAL and HD-GYP domains. For example, Adar, containing a GGDEF domain, encodes diguanylate cyclase synthesizing c-di-GMP, is required for cellu‐ lose production and biofilm formation. In another seven GGDEF family (GcpA-G), only GcpA and GcpE are critical for biofilm formation [37]. The EAL domain protein STM4264, STM3611, and the GGDEF-EAL domain protein STM1703 play a determinative role in the expression level of multicellular behavior of *Salmonella typhimurium* [78, 79]. In contradiction, the EALlike protein STM1697, neither degrade nor bind c-di-GMP, promotes biofilm formation and *CsgD* expression through interaction with proteins that regulate flagella function [80]. High intracellular amounts of c-di-GMP in *Salmonella typhimurium* inhibited invasion and abolish‐ ed induction of a pro-inflammatory immune response in the colonic epithelial cell line HT-29. Inhibition of the invasion and IL-8 induction phenotype by c-di-GMP requires the major biofilm activator CsgD and/or BcsA. Therefore, c-di-GMP signaling is at least equally impor‐ tant in the regulation of *Salmonella*–host interaction as in the regulation of biofilm formation at ambient temperature [81].

CsgD synthesis is also regulated at the post-transcriptional level by sRNA. sRNAs have emerged as a diverse group of trans- or cis-encoded regulatory molecules of approximately 50–250 nt in size. The RNA chaperone Hfq protects sRNAs form degradation and facilitates their binding to the target mRNAs. All these sRNA may negatively regulate *csgD* gene expression by binding to the overlapping 5′-region of the transcript, masking the ribosome binding site, resulting in the inhibition of translation or the degradation of mRNA [82]. In *Escherichia coli*, sRNAs, OmrA/B, McaS, RprA, and GcvB are identified, which downregulate *CsgD* translation [83]. In *E. coli* and *Salmonella*, RydC's 5′-domain interacts with *csgD* mRNA translation initiation signals to prevent initiation, stimulation of RydC expression reduces biofilm formation by impairing curli synthesis [84]. Surprisingly, two Hfq-dependent sRNAs (ArcZ and SdsR) are responsible for positively regulation of rdar morphotype expression in *Salmonella typhimurium* [85]. *Salmonella* biofilm development depends on the phosphoryla‐ tion status of RcsB. The unphosphorylated RcsB is essential to activate the expression of the biofilm matrix compounds. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of *CsgD* expression, through a mechanism dependent on the accumula‐ tion of the sRNA RprA [86].

Many gram-negative bacteria utilize N-acyl-L-homoserine lactones (AHLs) to bind to transcriptional regulators leading to activation or repression of target genes. *Salmonella* do not synthesize AHLs but do contain the AHL receptor, SdiA. The *Salmonella sdiA* gene regulates the *rck* gene, which mediates its adhesion and invasion of epithelial cells and the resistance of the organism to complement [87]. The *rck* gene is located on the virulence plasmid of pRST98, AHLs increase *rck* expression in pRST98-carrying strains, thereby enhancing bacterial adherence, serum resistance, and bacterial biofilm formation [88].

### **6. Virulence or resistance for biofilm**

tants of *Salmonella typhimurium* no longer produce curli or rugose colony morphology. However, inactivation of *mlrA* did not affect curli production and aggregative morphology in an upregulated curli producing *Salmonella typhimurium* derivative containing a temperatureand RpoS-independent *csgD* promoter region. Therefore, MlrA acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by *Salmonella typhimurium* [76].

c-di-GMP is recognized as a ubiquitous bacterial second messenger and a key regulator in bacterial transition from a motile and planktonic to a sessile and biofilm lifestyle. High intracellular c-di-GMP levels promote extracellular matrix production and subsequent biofilm formation and repress motility, whereas low intracellular c-di-GMP levels suppress matrix production and promote single-cell motility [77]. The synthesis/degradation of c-di-GMP depends on diguanylate cyclase/phosphodiesterase enzymatic activities. The cyclase activity, which converts two molecules of GTP to c-di-GMP, is encoded in the GGDEF protein domain, while phosphodiesterase activity, which hydrolyzes c-di-GMP to linear 5′-pGpG or two GMP molecules, is encoded in the EAL and HD-GYP domains. For example, Adar, containing a GGDEF domain, encodes diguanylate cyclase synthesizing c-di-GMP, is required for cellu‐ lose production and biofilm formation. In another seven GGDEF family (GcpA-G), only GcpA and GcpE are critical for biofilm formation [37]. The EAL domain protein STM4264, STM3611, and the GGDEF-EAL domain protein STM1703 play a determinative role in the expression level of multicellular behavior of *Salmonella typhimurium* [78, 79]. In contradiction, the EALlike protein STM1697, neither degrade nor bind c-di-GMP, promotes biofilm formation and *CsgD* expression through interaction with proteins that regulate flagella function [80]. High intracellular amounts of c-di-GMP in *Salmonella typhimurium* inhibited invasion and abolish‐ ed induction of a pro-inflammatory immune response in the colonic epithelial cell line HT-29. Inhibition of the invasion and IL-8 induction phenotype by c-di-GMP requires the major biofilm activator CsgD and/or BcsA. Therefore, c-di-GMP signaling is at least equally impor‐ tant in the regulation of *Salmonella*–host interaction as in the regulation of biofilm formation

CsgD synthesis is also regulated at the post-transcriptional level by sRNA. sRNAs have emerged as a diverse group of trans- or cis-encoded regulatory molecules of approximately 50–250 nt in size. The RNA chaperone Hfq protects sRNAs form degradation and facilitates their binding to the target mRNAs. All these sRNA may negatively regulate *csgD* gene expression by binding to the overlapping 5′-region of the transcript, masking the ribosome binding site, resulting in the inhibition of translation or the degradation of mRNA [82]. In *Escherichia coli*, sRNAs, OmrA/B, McaS, RprA, and GcvB are identified, which downregulate *CsgD* translation [83]. In *E. coli* and *Salmonella*, RydC's 5′-domain interacts with *csgD* mRNA translation initiation signals to prevent initiation, stimulation of RydC expression reduces biofilm formation by impairing curli synthesis [84]. Surprisingly, two Hfq-dependent sRNAs (ArcZ and SdsR) are responsible for positively regulation of rdar morphotype expression in *Salmonella typhimurium* [85]. *Salmonella* biofilm development depends on the phosphoryla‐ tion status of RcsB. The unphosphorylated RcsB is essential to activate the expression of the biofilm matrix compounds. The inhibition of biofilm development by phosphorylated RcsB is

at ambient temperature [81].

238 Microbial Biofilms - Importance and Applications

Biofilm formation may involve in the virulence of *Salmonella. Salmonella enteritidis* stains isolated from either the environment, dairy products, or infected patients are divided into two groups on the basis of their virulence (50% lethal dose) in chickens infected intraperitoneally. Only the virulent strains produce aggregates and formed visible filaments attached to the glass tube [47]. Further study confirms that the virulence of the biofilm-producing strain in infected chickens increases proportionally to the amount of stored glycogen, suggesting a possible role of the glycogen depot in the virulence of *Salmonella enteritidis* [89]. When tested for infection in Caco-2 cells and HEp-2 cells, the more virulent strains of *Salmonella enteritidis*, which are biofilm producers in adherence test medium, are able to disrupt monolayers. In contrast, the low-virulence strains of *Salmonella enteritidis*, which do not produce biofilms in adherence test medium, have no effect on the same cells. The high-virulence *Salmonella enteritidis* strains incubated under optimum biofilm-forming conditions may release a soluble factor, which enables the disruption of the integrity of Caco-2 monolayers [90]. The relationship between biofilm-forming ability and the pathogenicity is also evaluated in *Salmonella pullorum*. Although the virulence of *Salmonella pullorum* strains is independent of their ability of biofilm formation, prior growth as a biofilm for a biofilm producer of *Salmonella pullorum* leads to enhanced virulence in chickens, suggested that biofilm formation may be one of important virulence factor for *Salmonella pullorum* infection [46].

The *csgBAC* operon is required for curli biosynthesis in *Salmonella*. The *csgA* mutation is not reduced in ability to attach or colonize alfalfa sprouts, whereas the *csgB* mutation is reduced. Thus, *csgB* alone can play a role in attachment of *Salmonella* to plant tissue [91]. Competitive infection experiments in mice shows that *csgA* mutant cells outcompeted rdar-positive wildtype cells, indicating that aggregation via the rdar morphotype is not a virulence adaptation in *Salmonella typhimurium*. Furthermore, in vivo imaging experiments show that thin aggre‐ gative fimbriae genes are not expressed during infection but are expressed once *Salmonella* was passed out of the mice into the feces [92]. However, *Salmonella typhimurium* strains isolated from water buffalo calves affected by lethal gastroenteritis are tested in vivo in a mouse model of mixed infection. The most pathogenic strain is characterized by a high number of viru‐ lence factors and the presence of the locus *csgA*, coding for a thin aggregative fimbria [93].

The *bcsABZC* and *bcsEFG* operons are required for cellulose biosynthesis in *Salmonella*. Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose-deficient mutants of *bcsC* and *bcsE* genes indicate that the production of cellulose is not involved in the virulence of *Salmonella enteritidis*. However, cellulose-deficient mutants are more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of *S. enteritidis* on surface environ‐ ments [36]. *Salmonella typhimurium* makes cellulose when inside macrophages. An attenuat‐ ed mutant lacking the *mgtC* gene exhibits increased cellulose levels due to increased expression of the cellulose synthase gene *bcsA* and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of *bcsA* restore wild-type virulence to the *Salmonella mgtC* mutant, indicating that *Salmonella* promotes virulence by repressing cellulose production [94].

*emrAB, mdfA, mdtK*, and *macAB* are compromised in their ability to form biofilms. The mutants expressed significantly less *csgB* or *csgD* than wild type, indicating that loss of all multidrug resistance efflux pumps of *Salmonella typhimurium* results in impaired ability to form a biofilm [97]. Further study confirms that mutants of *Salmonella typhimurium* that lack TolC or AcrB, but surprisingly not AcrA, are compromised in their ability to form biofilms. The biofilm defect results from transcriptional repression of curli biosynthesis genes and consequent inhibition of production of curli. Therefore, the inhibition of efflux is a promising antibiofilm strategy [98]. However, recent studies offer contradictory findings about the role of multi‐ drug efflux pumps in bacterial biofilm development. When no selective pressure is applied, *Salmonella typhimurium* is able to produce biofilms even when the AcrAB efflux pumps are inactivated. Upon exposure to chloramphenicol, the formation of biofilms on solid surfaces as well as the production of curli are either reduced or delayed more significantly in both AcrA and AcrAB mutants, implying that the use of efflux pump inhibitors to prevent biofilm formation is not a general solution and that combined treatments might be more efficient [99]. Triclosan is a potent biocide that is included in a diverse range of products. *Salmonella* biofilmderived cells are more resistant to Triclosan. Within biofilms, triclosan upregulate the transcription of *acrAB, marA, bcsA*, and *bcsE* genes. Thus, *Salmonella* within biofilms could experience reduced influx, increased efflux and enhanced exopolysaccharides production. The data suggest that tolerance of *Salmonella* towards triclosan in the biofilm is attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide

Biofilm Formation of *Salmonella* http://dx.doi.org/10.5772/62905 241

In summary, *Salmonella* biofilm formation is major controlled by CsgD regulatory network and regulated by multiple transcriptional factors, c-di-GMP, and sRNAs. More and more genes are found to be associated with both biofilm formation and virulence. Dissection of their func‐ tion and relationship will helpful for development of new tools and strategies to prevent biofilm-related disease and decontaminate biofilm-derived *Salmonella* in food production.

College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, PR China

[1] Popoff MY, Bockemuhl J, Gheesling LL. Supplement 2002 (no. 46) to the Kauffmann-

further resistance to triclosan and to other antimicrobials [100].

Address all correspondence to: daxinpeng@yahoo.com

White scheme. *Res Microbiol* 2004,155:568–570.

**Author details**

Daxin Peng

**References**

BapA, a large cell-surface protein, is required for biofilm formation by *Salmonella*. Studies on the contribution of BapA to *Salmonella enteritidis* pathogenesis reveal that orally inoculated animals with a *bapA*-deficient strain survived longer than those inoculated with the wild-type strain. Also, a *bapA* mutant strain showed a significantly lower colonization rate at the intestinal cell barrier and consequently a decreased efficiency for organ invasion compared with the wild-type strain [26]. Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram-negative bacteria. An *opgGH* mutant strain in *Salmonella typhimurium*, which is defective in OPG biosynthesis, severely impaires biofilm formation. The opgGH mutant strain poorly colonizes mouse organs when introduced orally along with the wild-type strain [95].

Besides, the constitutional components of biofilm, there are many regulation proteins involved in both biofilm formation and virulence. An *ompR* mutant of *Salmonella enteritidis* has no ability to produce cellulose, curli, and biofilm and shows similar adherence percentage to and invasion percentage of epithelial cells as wild-type strain. Intraperitoneal challenge of bacteria in BALB/c mice reveals that the *ompR* mutant strain is significant attenuated [73]. A *spiA* gene mutant shows reduced biofilm formation and significantly decreased curli production, and reduced intracellular proliferation of macrophages during the biofilm phase. In addition, the *spiA* mutant was attenuated in a mouse model in both the exponential growth and biofilm phases [67]. Deletion of genes *ompR* and *spiA* in *Salmonell pullorum* strains contribute to attenuation of virulence in 1-day-old chickens [68]. DksA is a conserved gram-negative regulator that binds directly to the RNA polymerase secondary channel. In *Salmonella typhimurium*, expression of the *dksA* gene is induced during the logarithmic phase and DksA plays an important role in motility and biofilm formation. DksA positively regulates the *Salmonella* pathogenicity island 1 and motility-chemotaxis genes and is necessary for *Salmonella typhimurium* invasion of human epithelial cells and uptake by macrophages. The *dksA* gene is induced at the midcecum during the early stage of the infection and required for gastrointes‐ tinal colonization and systemic infection in a colitis mouse model [96].

*Salmonella* in biofilm is resistant to antibiotic. One of key mechanisms of antibiotic resistance is efflux. There are five families of multidrug resistance (MDR) efflux pumps, in which the AcrAB–TolC efflux system is the best characterized MDR system. Ten mutants of *Salmonella typhimurium* lacking MDR efflux systems, such as *tolC, acrB, acrD, acrEF, mdtABC, mdsABC,* *emrAB, mdfA, mdtK*, and *macAB* are compromised in their ability to form biofilms. The mutants expressed significantly less *csgB* or *csgD* than wild type, indicating that loss of all multidrug resistance efflux pumps of *Salmonella typhimurium* results in impaired ability to form a biofilm [97]. Further study confirms that mutants of *Salmonella typhimurium* that lack TolC or AcrB, but surprisingly not AcrA, are compromised in their ability to form biofilms. The biofilm defect results from transcriptional repression of curli biosynthesis genes and consequent inhibition of production of curli. Therefore, the inhibition of efflux is a promising antibiofilm strategy [98]. However, recent studies offer contradictory findings about the role of multi‐ drug efflux pumps in bacterial biofilm development. When no selective pressure is applied, *Salmonella typhimurium* is able to produce biofilms even when the AcrAB efflux pumps are inactivated. Upon exposure to chloramphenicol, the formation of biofilms on solid surfaces as well as the production of curli are either reduced or delayed more significantly in both AcrA and AcrAB mutants, implying that the use of efflux pump inhibitors to prevent biofilm formation is not a general solution and that combined treatments might be more efficient [99]. Triclosan is a potent biocide that is included in a diverse range of products. *Salmonella* biofilmderived cells are more resistant to Triclosan. Within biofilms, triclosan upregulate the transcription of *acrAB, marA, bcsA*, and *bcsE* genes. Thus, *Salmonella* within biofilms could experience reduced influx, increased efflux and enhanced exopolysaccharides production. The data suggest that tolerance of *Salmonella* towards triclosan in the biofilm is attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance to triclosan and to other antimicrobials [100]. *bcsA*

In summary, *Salmonella* biofilm formation is major controlled by CsgD regulatory network and regulated by multiple transcriptional factors, c-di-GMP, and sRNAs. More and more genes are found to be associated with both biofilm formation and virulence. Dissection of their func‐ tion and relationship will helpful for development of new tools and strategies to prevent biofilm-related disease and decontaminate biofilm-derived *Salmonella* in food production.

### **Author details**

#### Daxin Peng

The *bcsABZC* and *bcsEFG* operons are required for cellulose biosynthesis in *Salmonella*. Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose-deficient mutants of *bcsC* and *bcsE* genes indicate that the production of cellulose is not involved in the virulence of *Salmonella enteritidis*. However, cellulose-deficient mutants are more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of *S. enteritidis* on surface environ‐ ments [36]. *Salmonella typhimurium* makes cellulose when inside macrophages. An attenuat‐ ed mutant lacking the *mgtC* gene exhibits increased cellulose levels due to increased expression of the cellulose synthase gene *bcsA* and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of *bcsA* restore wild-type virulence to the *Salmonella mgtC* mutant,

indicating that *Salmonella* promotes virulence by repressing cellulose production [94].

wild-type strain [95].

240 Microbial Biofilms - Importance and Applications

BapA, a large cell-surface protein, is required for biofilm formation by *Salmonella*. Studies on the contribution of BapA to *Salmonella enteritidis* pathogenesis reveal that orally inoculated animals with a *bapA*-deficient strain survived longer than those inoculated with the wild-type strain. Also, a *bapA* mutant strain showed a significantly lower colonization rate at the intestinal cell barrier and consequently a decreased efficiency for organ invasion compared with the wild-type strain [26]. Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram-negative bacteria. An *opgGH* mutant strain in *Salmonella typhimurium*, which is defective in OPG biosynthesis, severely impaires biofilm formation. The opgGH mutant strain poorly colonizes mouse organs when introduced orally along with the

Besides, the constitutional components of biofilm, there are many regulation proteins involved in both biofilm formation and virulence. An *ompR* mutant of *Salmonella enteritidis* has no ability to produce cellulose, curli, and biofilm and shows similar adherence percentage to and invasion percentage of epithelial cells as wild-type strain. Intraperitoneal challenge of bacteria in BALB/c mice reveals that the *ompR* mutant strain is significant attenuated [73]. A *spiA* gene mutant shows reduced biofilm formation and significantly decreased curli production, and reduced intracellular proliferation of macrophages during the biofilm phase. In addition, the *spiA* mutant was attenuated in a mouse model in both the exponential growth and biofilm phases [67]. Deletion of genes *ompR* and *spiA* in *Salmonell pullorum* strains contribute to attenuation of virulence in 1-day-old chickens [68]. DksA is a conserved gram-negative regulator that binds directly to the RNA polymerase secondary channel. In *Salmonella typhimurium*, expression of the *dksA* gene is induced during the logarithmic phase and DksA plays an important role in motility and biofilm formation. DksA positively regulates the *Salmonella* pathogenicity island 1 and motility-chemotaxis genes and is necessary for *Salmonella typhimurium* invasion of human epithelial cells and uptake by macrophages. The *dksA* gene is induced at the midcecum during the early stage of the infection and required for gastrointes‐

*Salmonella* in biofilm is resistant to antibiotic. One of key mechanisms of antibiotic resistance is efflux. There are five families of multidrug resistance (MDR) efflux pumps, in which the AcrAB–TolC efflux system is the best characterized MDR system. Ten mutants of *Salmonella typhimurium* lacking MDR efflux systems, such as *tolC, acrB, acrD, acrEF, mdtABC, mdsABC,*

tinal colonization and systemic infection in a colitis mouse model [96].

Address all correspondence to: daxinpeng@yahoo.com

College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, PR China

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**Chapter 13**

**Bacterial Biofilms in Diabetic Foot Ulcers: Potential**

Diabetes *mellitus* is a major health problem that affects approximately 171 million people globally. One of its most severe complications is the development of diabetic foot ulcers (DFU). Ischemic and neurophatic lesions are of major importance for DFU onset; however, it is the infection by multidrug-resistant and biofilm-producing microorganisms, along with local microenvironmental conditions unfavorable to antibiotics action that ultimately cause infection chronicity and lower limbs amputa‐ tion. Novel therapeutic protocols for DFU management are extremely urgent. Bacteriophages, probiotics and antimicrobial peptides (AMP) have recently been proposed as alternatives to currently available antibiotics. Bacteriophages are viruses that specifically infect and multiply within bacterial cells. Their ability to diffuse through polymeric matrixes makes them particularly efficient to eradicate biofilmbased bacteria. Promising results were also observed with probiotic therapy. Probiotics are well-characterized strains with the ability to compete with pathogen‐ ic microorganisms and modulate the host immune response. AMP are molecules produced by living organisms as part of their innate immune response. Unlike conventional antibiotics, AMP also act as immunomodulators and resistance to AMP was rarely observed, supporting their potential as therapeutic agents. These innovative therapeutic strategies may in the future substitute or complement antibiotherapy, ultimately contributing for the decrease in multidrug-resistant

**Keywords:** antimicrobial peptides, antimicrobial resistance, bacteriophages, biofilm,

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. 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,

**Alternative Therapeutics**

http://dx.doi.org/10.5772/63085

bacteria dissemination.

diabetic foot ulcer, probiotics

**Abstract**

Raquel Santos, Ana Salomé Veiga, Luis Tavares,

Miguel Castanho and Manuela Oliveira

Additional information is available at the end of the chapter
