**4. Control strategies to** *S. epidermidis* **biofilm formation**

Because the expression of toxins and other virulence factors is less in *S. epidermidis*, the biofilm forming capacity is its major virulence factor. Biofilm growth is characterized by high resistance to antimicrobial agents and host immune responses, making biofilm eradication tremendously difficult. The increasing prevalence of multidrug-resistant *S. epidermidis* strains additionally hampers antimicrobial therapy. Therefore, targeting factors expressed at different phases in biofilm formation might offer new tools to combat *S. epidermidis* infections.

### **4.1 Inhibition of initial attachment**

The first step of biofilm formation is bacterial adherence to the host cell surface. Direct binding to host cell surface is mediated by electrostatic and hydrophobic interactions and van der Waals forces and affected by physicochemical variables [29].

Found in our research, after investigating the antibiofilm activities of spent media from 185 Actinomycete strains using two *S. epidermidis* strains (ATCC 35984 and a clinical strain 5-121-2) as target bacteria, three strains of tested Actinomycete (TRM 46200, TRM 41337, and TRM 46814) showed a significant inhibition against *S. epidermidis* biofilm formation without affecting the growth of planktonic cells. Effect of Actinomycete supernatants on cell surface hydrophobicity (CSH) of *S. epidermidis* was measured by Microbial Adhesion to Hydrocarbon (MATH) assay. The adhesion of staphylococci to n-hexadecane was used to measure the hydrophobicity of *S. epidermidis*. All the crude proteins from spent media showed a reduction in the CSH against *S. epidermidis* ATCC 35984 and 5-121-2, which explain at least in part the inhibitory effect of Actinomycete supernatants on biofilm reduction [19].

Moreover, apart from physico-chemical determinants, it was demonstrated that the major autolysine AtlE is involved in attachment to polystyrene surfaces. Therefore, AtlE may be indirectly involved in cell adhesion via releasing DNA. Treatment of *S. epidermidis* cells with DNaseI was found to inhibit biofilm formation at an early time point, suggesting that release of DNA also contributes to the attachment of *S. epidermidis* to artificial surfaces [30]. In our research, we performed the degradation of the crude proteins from spent media against *S. epidermidis* DNA. The crude protein from spent media of TRM 46200 showed a significant DNA-degradation activity. Importantly, the crude protein from spent medium of TRM 41337 possessed the highest DNA-degradation activity as that of the positive control, 10 μg/ml of DNaseI [19].

*S. epidermidis* foreign-associated infections occurring early are thought to involve direct interactions of the bacterial surface with host extracellular matrix (ECM). Specific binding to surface ECM proteins involves cell wall-associated adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) [31, 32]. The recent studies have shown that antibodies against cell surface components of *S. epidermidis* can affect the rate of biofilm formation or adherence of these bacteria to medical devices in vitro. Using polyclonal antibodies against a fibrinogen-binding protein from *S. epidermidis* (Fbe) could block adherence of *S. epidermidis* to fibrinogen-coated catheters in vitro [33, 34]. Consequently, all these surface-located components are good candidates for vaccine development aiming at the inhibition of the initial attachment step of biofilm formation.

### **4.2 Inhibition of bacterial accumulate**

After adherence to the host cell surface, biofilms develop through intercellular aggregation. The major factor involved in intercellular adhesion is polysaccharide intercellular adhesin (PIA). The de-acetylation of PIA is not only essential for biofilm formation but also crucial for *S. epidermidis* virulence [29]. Hence, PIA was one of the first targets evaluated in view of biofilm-inhibiting *S. epidermidis* vaccine development. Pier and coworkers have significantly contributed to the evaluation of PIA as vaccine target. Following the evidence, high-molecular-weight PIA could elicit an antibody response accompanied by opsonophagocytic killing of the PIA-dependent biofilm-forming *S. epidermidis* M187 and three *S. aureus* strains. The PIA-specific antibodies can prevent biofilm formation or retard already initiated biofilm development [35].

PIA biosynthesis depends on the expression of the icaADBC operon, which is controlled by a complex regulatory network. Gomes et al. studied the effect of rifampicin+gentamicin and rifampicin+clindamycin combinations on the expression of icaA and rsbU genes, responsible for poly-N-acetylglucosamine/polysaccharide intercellular adhesin (PNAG/PIA) production. The results demonstrated that this combinatorial therapy can cause a lower genetic expression of the two specific genes tested and consequently can reduce biofilm formation recidivism [36, 37].

Nevertheless, *S. epidermidis* strains lacking icaADBC but still producing biofilm were isolated, indicating the existence of an ica-independent mechanism of cell accumulation. A proteinaceous intercellular adhesin involved in cell accumulation during biofilm formation was discovered. The accumulation-associated protein (Aap) can functionally substitute PIA as an intercellular adhesin, and there is good evidence that additional proteinaceous intercellular adhesins must exist. They showed that monoclonal antibodies against Aap can significantly reduce the accumulation but not initiation phase of *S. epidermidis* biofilm formation in vitro [38].

**107**

by degrading PIA [41].

*Formation, Antibiotic Resistance, and Control Strategies of* Staphylococcus epidermidis *Biofilm*

Biofilm formation is a result of bacterial interactions and group behavior. Quorum sensing (QS) is one of the regulatory mechanisms suggested to be involved in coordinating biofilm formation. The QS system is a cell-to-cell communication system used by many bacteria to assess the cell density. Quorum sensing inhibitors (QSI) could be a novel way to fight biofilm-associated infections. The study has identified furanones and thiophenones as inhibitors of quorum sensing and biofilm formation. In this study, the effect of both the furanone and the thiophenone could be abolished by the synthetic Autoinducer-2 (AI-2) molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD), indicating that furanone and thiophenone affect biofilm

For the biofilm that has been formed on the surface of the host, if the biofilm can be separated by antibacterial oranti-biofilm substances, the bacteria in the biofilm can be released, and the planktonic bacteria are more easily to be killed if the biofilm is exposed to antibiotics. Biofilms are composed primarily of microbial cells and extracellular polymeric substance (EPS). EPS may account for 50–90% of the total organic carbon of biofilms and can be considered the primary matrix material of the biofilm. The components of EPS include polysaccharides, nucleic

We initially determined the dependent type of biofilm formation by *S. epidermidis* ATCC 35984 and 5-121-2. The biofilm formation by *S. epidermidis* ATCC 35984 mainly depends on EPS consisting of reducing polysaccharides in which dihydroxyl groups are unsubstituted. Thus, sodium-meta-periodate, which specifically destroys sugars containing unsubstituted dihydroxyl groups, significantly decreased biofilm formation in *S. epidermidis* ATCC 35984. However, not only EPS but also proteins, eDNA, are responsible for the biofilm formation of *S. epidermidis* 5-121-2. Moreover, EPS in *S. epidermidis* 5-121-2, which mainly consists of nonreducing polysaccharides, is distinct with those in *S. epidermidis* ATCC 35984. Thus, three enzymes specific to nonreducing glycosides, amylase, β-glucanase, and β-glucosidase, worked effectively in the degradation of EPS, resulting in biofilm

Since extracellular polysaccharides are the main compounds in biofilm matrices, namely in *S. epidermidis*, antimicrobial substances able to disrupt or inhibit EPS are of major interest. N-acetylcysteine (NAC) is an amino acid with strong antioxidant, antimucolytic, and antibacterial properties. As observed by researchers, NAC decreased biofilm formation and reduced the formation of extracellular polysaccharide matrix while promoting the disruption of mature biofilm. NAC has demonstrated not only to reduce adhesion but also to detach bacterial cells adhered to surfaces and to inhibit bacterial growth in vitro. The possible action of NAC in the biofilm matrix can result in the release of cells either individually or in cell clusters, becoming the biofilm and loose cells more exposed and susceptible to the host immune system and to other antimicrobial agents [40]. Kaplan et al. found an enzyme called dispersin B, which can promote biofilm detachment from *Actinobacillus actinomycetemconitans*, which rapidly and effectively removes biofilms formed by *S. epidermidis* on the host surface. Dispersin B is a β-1,6-N-Acetylglucosaminidase that causes *S. epidermidis* to detach from the biofilm matrix

Our results showed that EPS in *S. epidermidis* ATCC 35984 and 5-121-2 was degraded by crude proteins from three Actinomycete strains (TRM 41337, TRM 46200, and TRM 46814) supernatants. Specifically, for the strain ATCC 35984

formation through interference with bacterial communication [39].

*DOI: http://dx.doi.org/10.5772/intechopen.89800*

**4.3 Promotion of biofilm detachment**

acids, lipids, and proteins [36].

reduction in *S. epidermidis* 5-121-2 [19].

*Formation, Antibiotic Resistance, and Control Strategies of* Staphylococcus epidermidis *Biofilm DOI: http://dx.doi.org/10.5772/intechopen.89800*

Biofilm formation is a result of bacterial interactions and group behavior. Quorum sensing (QS) is one of the regulatory mechanisms suggested to be involved in coordinating biofilm formation. The QS system is a cell-to-cell communication system used by many bacteria to assess the cell density. Quorum sensing inhibitors (QSI) could be a novel way to fight biofilm-associated infections. The study has identified furanones and thiophenones as inhibitors of quorum sensing and biofilm formation. In this study, the effect of both the furanone and the thiophenone could be abolished by the synthetic Autoinducer-2 (AI-2) molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD), indicating that furanone and thiophenone affect biofilm formation through interference with bacterial communication [39].

### **4.3 Promotion of biofilm detachment**

*Bacterial Biofilms*

formation.

control, 10 μg/ml of DNaseI [19].

**4.2 Inhibition of bacterial accumulate**

biofilm development [35].

Moreover, apart from physico-chemical determinants, it was demonstrated that the major autolysine AtlE is involved in attachment to polystyrene surfaces. Therefore, AtlE may be indirectly involved in cell adhesion via releasing DNA. Treatment of *S. epidermidis* cells with DNaseI was found to inhibit biofilm formation at an early time point, suggesting that release of DNA also contributes to the attachment of *S. epidermidis* to artificial surfaces [30]. In our research, we performed the degradation of the crude proteins from spent media against *S. epidermidis* DNA. The crude protein from spent media of TRM 46200 showed a significant DNA-degradation activity. Importantly, the crude protein from spent medium of TRM 41337 possessed the highest DNA-degradation activity as that of the positive

*S. epidermidis* foreign-associated infections occurring early are thought to involve direct interactions of the bacterial surface with host extracellular matrix (ECM). Specific binding to surface ECM proteins involves cell wall-associated adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) [31, 32]. The recent studies have shown that antibodies against cell surface components of *S. epidermidis* can affect the rate of biofilm formation or adherence of these bacteria to medical devices in vitro. Using polyclonal antibodies against a fibrinogen-binding protein from *S. epidermidis* (Fbe) could block adherence of *S. epidermidis* to fibrinogen-coated catheters in vitro [33, 34]. Consequently, all these surface-located components are good candidates for vaccine development aiming at the inhibition of the initial attachment step of biofilm

After adherence to the host cell surface, biofilms develop through intercellular aggregation. The major factor involved in intercellular adhesion is polysaccharide intercellular adhesin (PIA). The de-acetylation of PIA is not only essential for biofilm formation but also crucial for *S. epidermidis* virulence [29]. Hence, PIA was one of the first targets evaluated in view of biofilm-inhibiting *S. epidermidis* vaccine development. Pier and coworkers have significantly contributed to the evaluation of PIA as vaccine target. Following the evidence, high-molecular-weight PIA could elicit an antibody response accompanied by opsonophagocytic killing of the PIA-dependent biofilm-forming *S. epidermidis* M187 and three *S. aureus* strains. The PIA-specific antibodies can prevent biofilm formation or retard already initiated

PIA biosynthesis depends on the expression of the icaADBC operon, which is controlled by a complex regulatory network. Gomes et al. studied the effect of rifampicin+gentamicin and rifampicin+clindamycin combinations on the expression of icaA and rsbU genes, responsible for poly-N-acetylglucosamine/polysaccharide intercellular adhesin (PNAG/PIA) production. The results demonstrated that this combinatorial therapy can cause a lower genetic expression of the two specific genes tested and consequently can reduce biofilm formation recidivism [36, 37]. Nevertheless, *S. epidermidis* strains lacking icaADBC but still producing biofilm were isolated, indicating the existence of an ica-independent mechanism of cell accumulation. A proteinaceous intercellular adhesin involved in cell accumulation during biofilm formation was discovered. The accumulation-associated protein (Aap) can functionally substitute PIA as an intercellular adhesin, and there is good evidence that additional proteinaceous intercellular adhesins must exist. They showed that monoclonal antibodies against Aap can significantly reduce the accumulation but not initiation phase of *S. epidermidis* biofilm formation in vitro [38].

**106**

For the biofilm that has been formed on the surface of the host, if the biofilm can be separated by antibacterial oranti-biofilm substances, the bacteria in the biofilm can be released, and the planktonic bacteria are more easily to be killed if the biofilm is exposed to antibiotics. Biofilms are composed primarily of microbial cells and extracellular polymeric substance (EPS). EPS may account for 50–90% of the total organic carbon of biofilms and can be considered the primary matrix material of the biofilm. The components of EPS include polysaccharides, nucleic acids, lipids, and proteins [36].

We initially determined the dependent type of biofilm formation by *S. epidermidis* ATCC 35984 and 5-121-2. The biofilm formation by *S. epidermidis* ATCC 35984 mainly depends on EPS consisting of reducing polysaccharides in which dihydroxyl groups are unsubstituted. Thus, sodium-meta-periodate, which specifically destroys sugars containing unsubstituted dihydroxyl groups, significantly decreased biofilm formation in *S. epidermidis* ATCC 35984. However, not only EPS but also proteins, eDNA, are responsible for the biofilm formation of *S. epidermidis* 5-121-2. Moreover, EPS in *S. epidermidis* 5-121-2, which mainly consists of nonreducing polysaccharides, is distinct with those in *S. epidermidis* ATCC 35984. Thus, three enzymes specific to nonreducing glycosides, amylase, β-glucanase, and β-glucosidase, worked effectively in the degradation of EPS, resulting in biofilm reduction in *S. epidermidis* 5-121-2 [19].

Since extracellular polysaccharides are the main compounds in biofilm matrices, namely in *S. epidermidis*, antimicrobial substances able to disrupt or inhibit EPS are of major interest. N-acetylcysteine (NAC) is an amino acid with strong antioxidant, antimucolytic, and antibacterial properties. As observed by researchers, NAC decreased biofilm formation and reduced the formation of extracellular polysaccharide matrix while promoting the disruption of mature biofilm. NAC has demonstrated not only to reduce adhesion but also to detach bacterial cells adhered to surfaces and to inhibit bacterial growth in vitro. The possible action of NAC in the biofilm matrix can result in the release of cells either individually or in cell clusters, becoming the biofilm and loose cells more exposed and susceptible to the host immune system and to other antimicrobial agents [40]. Kaplan et al. found an enzyme called dispersin B, which can promote biofilm detachment from *Actinobacillus actinomycetemconitans*, which rapidly and effectively removes biofilms formed by *S. epidermidis* on the host surface. Dispersin B is a β-1,6-N-Acetylglucosaminidase that causes *S. epidermidis* to detach from the biofilm matrix by degrading PIA [41].

Our results showed that EPS in *S. epidermidis* ATCC 35984 and 5-121-2 was degraded by crude proteins from three Actinomycete strains (TRM 41337, TRM 46200, and TRM 46814) supernatants. Specifically, for the strain ATCC 35984

### *Bacterial Biofilms*

when treated with crude proteins from spent medium of the strain TRM 41337, arabinose (Ara) was absent in the monosaccharide composition compared with the control. Furthermore, the proportion of mannose (Man) was decreased, while the proportions of glucosamine (GluN), galactosamine (GalN), and galactose (Gal) were increased. When treated with crude proteins from spent medium of the strain TRM 46814, three new monosaccharides, rhamnose (Rha), glucuronic acid (GluA), and galacturonic acid (GalA), appeared. Additionally, the proportions of Man and glucose (Glu) decreased obviously. For the strain 5-121-2, when treated with crude proteins from spent media of TRM 41337 and TRM 46814, a new monosaccharide, Rha, was present [19].
