**3. Inhibition of initial attachment**

*Bacterial Biofilms*

other organs also [6].

based infections.

**2. The process of biofilm development**

CLABSI can cause an increased rate of mortality and morbidity, and every year in the

When cells adhere and attach to surfaces biofilm formation begins. Several factors can promote the attachment of microorganisms to biomaterials including increased shear forces, bacterial motility, and electrostatic as well as hydrodynamic interactions between the surface and microbial cells [4]. It has been observed that adherence of biomaterials to bacteria via biomaterial-surface interactions and cell-surface is facilitated by numerous factors, such as protein autolysin, surface, and adhesion proteins and capsular polysaccharides, etc. For example, '*Staphylococcus* species' show cell-surface proteins that are vital for adherence of '*Staphylococcus epidermidis'* to polystyrene which is named as staphylococcal surface protein-1 and -2 (SSP-1 and SSP-2). After attachment to the extracellular surfaces, microbial cells will start aggregate, multiply, and eventually differentiate into the biofilm network [5]. Such microbial cells can then be separated from mature biofilms, can cause chronic infections and can spread to

Another worrying characteristic of infections associated with biofilm formation is increased biofilm cell tolerance to biocides. As biofilms provide an excellent niche for exchange of plasmid, so increased resistance to the drug can affect genes containing plasmids which results in multidrug resistance (MDR) phenotypes. Enhanced drug resistance mechanisms include incomplete or slow infiltration of antimicrobials within the extracellular matrix, the formation of dormant cells during the non-dividing phase, reduced cell's growth rate within the biofilm, hence ultimately decreasing total targets for antimicrobial molecules [7]. Furthermore, it is difficult to treat biofilm formation with the traditional antimicrobial approach and the therapy is further inhibited by increased resistance to the antibiotic because under antibiotic selective pressure microbial cells develop resistance. For instance, it has been observed that almost above 70% of hospital isolate of *'Staphylococcus epidermidis'* show resistance to methicillin and surprisingly there are many strategies to prevent infections associated with biofilm formation other than antibiotic treatment [8]. In this chapter, we have focused on anti-biofilm approaches and some promising efforts for controlling these biofilm-

The production and maturation of biofilm are complex, subsequent and dynamic processes, depending upon several factors i.e. cellular metabolism, intrinsic properties of the cells, genetic control, the substratum, and the medium signaling molecules. Biofilm formation is introduced with a conditioning film of inorganic or organic material on the cell surface; furthermore, this layer modifies the surface feature of substratum which ultimately favors microbes for colonization on the cell surface. The formation of biofilm consist of several different steps: (i) initially the reversible attachment of microbial cells with biotic or abiotic surfaces through weak forces for example van der Waals forces, (ii) irreversible attachment to the cell surface with the help of different attachment structure i.e. lipopolysaccharides, flagella, adhesive proteins or fimbriae by hydrophobic or hydrophilic interactions, (iii) and then eventually biofilm architecture development due to the production and proliferation of extracellular polysaccharide (EPS) matrix which is self-produced and is made up of proteins, extracellular deoxyribonucleic acid (DNA) and polysaccharides [9] (iv) in the

USA almost 250,000 cases of bloodstream infections are reported [3].

**86**

## **3.1 Altering physical properties of biomaterials**

Biofilm development starts with a reversible weak adhesion of microbial cells to the exterior surface of medical equipment, however, if they are not removed from the exterior of devices, they adhered permanently through their adhesion structures i.e. fimbriae, pili and thereby forming biofilm matrix [12]. Surface charge and hydrophobicity of implant constituents play a significant role in controlling the ability of microbes to anchor to cell surfaces. Therefore, alteration in the hydrophobicity and surface charge of polymeric constituents are proved as efficient for controlling biofilm formation by using numerous antimicrobial agents and backbone compounds [13]. Poly N-vinylpyrrolidone and Hydrophilic polymers i.e. hyaluronic acid [14] on silicone shunt and polyurethane catheters have been widely used to decrease the adherence of '*Staphylococcus epidermidis'*. Furthermore, several hydrogel membranes have been introduced particularly for ureteral stents that decrease bacterial adherence because of their hydrophilic characteristics. It has been observed that due to very low wettability superhydrophobic coatings play a significant role to reduce the biofilm matrix formation and adhesion of bacteria [15]. Later, it has been suggested that *S. aureus* and *Pseudomonas aeruginosa* poorly attached on superhydrophobic fluorinated silica coating as well as on titanium coatings. However, it was demonstrated that *Escherichia coli* and *Staphylococcus aureus* poorly adhered on other superhydrophobic surfaces i.e. (AACVD) aerosol assisted chemical vapor deposition-coated [16]. In some cases, it was observed that hairpin coating affects colonization and adhesion of bacteria because it forms vascular catheter negatively charged, so contribute to reducing the catheter-related infections, inhibiting microbial colonization and thrombosis [17]. It has been described that the surface roughness can modulate hydrophobicity, which ultimately influences the bacterial adhesion [18].

## **3.2 Altering the chemical properties of biomaterials**

There are several chemical approaches used to alter the exterior of biomedical equipment to inhibit the biofilm formation comprising ion coatings, biocides and also antibiotics [19]. Catheters that are impregnated with antibiotics, for example, rifampin and minocycline have been revealed to reduce the occurrence of biofilmbased infections by *Staphylococcus aureus*. Furthermore, catheters are coated with several antibiotics that play a significant role in biofilm production during urinary tract infections (UTI) like norfloxacin, nitrofurazone, and gentamicin [20]. Several chemical molecules are identified through screening of chemical libraries, these molecules are used as potential drugs to control infection and biofilm development. Furthermore, such molecules do not provoke antimicrobial action, and hence reduces the development of resistance due to no selective pressure against biofilm matrix formation. In *Staphylococcus aureus* and *Streptococcus pyogenes* a series of

small chemical molecules have an inhibitory effect on the expression of different important virulent factors during infection and biofilm formation [21]. Several aryl rhodamines showed inhibitory effect on early stages of biofilm development in *Enterococcus faecalis, S. epidermidis,* and *S. aureus*. Moreover, it was reported that a mucolytic mediator N-acetylcysteine has inhibited the formation of exopolysaccharides in the biofilm layer in case of *S. epidermidis* [22]. In another microorganism *Vibrio cholerae*, small substances suppressed the initiation of cyclic di-GMP that acts as the second messenger to control switch in-between the aquatic and sessile way of living of microbes [23].

It has been observed that numerous antibacterial peptides also inhibit biofilm formation in several microbes. For instance, it is considered that peptide 1018 has inhibitory effects in different microbes such as in *Acinetobacter baumannii, Burkholderia cenocepacia, Klebsiella pneumoniae, P. aeruginosa, E. coli, Salmonella typhimurium* and *S. aureus* [24]. Furthermore, class of peptide antibiotics called lantibiotics i.e. gallidermin, epidermin, subtilin, and nisin has been reported and control the biofilm production in *S. aureus, S. epidermidis* and also in *Lactococcus lactis*.

Chelators hindering the role of metal ions in the production of biofilm are considered as biofilm inhibitors, for example, silver salts, metallic silver and also silver nanoparticles are commonly employed as antibacterial agents in clinical implants against *P. aeruginosa, Salmonella typhimurium, Klebsiella species, E. coli,* and *S. aureus* [25]. It is observed that antibiotics i.e. amoxicillin, clindamycin, vancomycin, penicillin G and erythromycin show increased antimicrobial activity against *Staphylococcus aureus* in the presence of nanoparticles [26]. Treatment with silver substances prevents DNA replication, expression of cellular as well as ribosomal proteins, and also respiration process that leads to death of the cell [27]. In addition, It is also suggested that silver-coated implants inhibit *Staphylococcus aureus* biofilm production without aggregating silver inside the host tissue [28].

### **4. Quorum quenching**

In the majority of Gram-negative and Gram-positive bacteria, an essential cellular communicating system is presently called as Quorum sensing, which regulates a variety of genes in accordance with the density of signaling molecules furthermore, signaling molecules are called autoinducers [29]. On the bases of signaling molecules QS is classified into three i.e. autoinducing peptide (AIPbased) for Gram-positive bacteria, N-acyl homoserine lactones (AHLs-based) for Gram-negative bacteria and autoinducer-2 (AI-2-based) for both Gram-negative and Gram-positive bacteria [30]. When the biofilm is formed, after the initial attachment, cells secrete QS molecules that alter the expression of the microbial gene, thus changing planktonic form into a sessile form. Furthermore, QS plays a significant role in biofilm development, so It has been observed that QS inhibition i.e. quorum quenching (QQ ) would be a striking approach to control biofilm formation [31]. QS system is thought to be a target for developing new antimicrobial agents, moreover, QS system plays a crucial role in regulating pathogenetic factors and also virulence factors production in several pathogens [32]. The most important benefit of preventing biofilm formation by QQ is that this approach decreases the risk of multidrug resistance (MDR) and thus creating this approach noticeable to prevent biofilm-based infections in clinical settings. The different approaches for the inhibition or removal of biofilms are summarized in **Table 1** and **Figure 1**.

**89**

**Bacteria** *P. aeruginosa*

**Compound** N-Acyl homoserine lactones

Patriniae Hordenine

Quercetin '*Piper betle*' Leaves

(Ethanolic Extract)

Parthenolide

Extracellular polymeric substance and

transcriptional regulators of quorum sensing

related genes

*E. coli* O157:H7

Ginkgolic acids (GAs)

Phloretin Cinnamaldehyde

'*Zingiber officinale*'

(Methanolic fraction)

Leaf extract of '*Bergenia* 

*crassifolia*' (L.)

Quercetin '*Rhodomyrtus tomentosa*'

(Ethanol extract)

Phloretin

Efflux protein genes

*Staphylococcus aureus* and

*Staphylococcus epidermidis*

*S. aureus* strains

(Gtfs)

pH Not mentioned

*S. mutans*

Curli gene expression, prophage genes

Toxin genes, autoinducer-2 importer genes curli

genes, prophage genes

LuxR-DNA-binding

F-ATPase activity, virulence genes, surface

protein antigen (SpaP)

Exopolysaccharides (EPSs), glucosyltransferases

Biofilm related genes

Quorum sensing related genes

Transcriptional regulators of quorum sensing related genes

Pyocyanin

**Mechanism** Transcriptional regulators (LuxR and LasR)

Reduced the production of exopolysaccharide

Blocked QS-controlled phenotypes like biofilm formation

Inhibition of biofilm formation

Inhibited Pyocyanin production and reduced twitching

Inhibition of the expression of QS related genes expression

[54]

and downregulation of extracellular polymeric substance

Biofilm formation was inhibited on the polystyrene, glass

Decreased biofilm formation and production of fimbria

Affected the biofilm formation and virulence

Affected the cell-surface hydrophobicity index, Inhibited

surface protein antigen (SpaP)

Decreased adherence properties of bacterial cells

Disrupted the pH in biofilm

Inhibition of biofilm formation and disruption of mature

biofilm

Anti-biofilm formation at low

[58]

[59]

[60]

[61]

[56]

[57]

[55]

[55]

and nylon membrane

production

[53]

ability

[51] [52, 53]

[50]

**Antibiofilm activity**

Decreased the production of QS signals and virulence factors

**References**

[49]

*Innovative Strategies for the Control of Biofilm Formation in Clinical Settings*

*DOI: http://dx.doi.org/10.5772/intechopen.89310*


### *Innovative Strategies for the Control of Biofilm Formation in Clinical Settings DOI: http://dx.doi.org/10.5772/intechopen.89310*

*Bacterial Biofilms*

living of microbes [23].

also in *Lactococcus lactis*.

host tissue [28].

**4. Quorum quenching**

small chemical molecules have an inhibitory effect on the expression of different important virulent factors during infection and biofilm formation [21]. Several aryl rhodamines showed inhibitory effect on early stages of biofilm development in *Enterococcus faecalis, S. epidermidis,* and *S. aureus*. Moreover, it was reported that a mucolytic mediator N-acetylcysteine has inhibited the formation of exopolysaccharides in the biofilm layer in case of *S. epidermidis* [22]. In another microorganism *Vibrio cholerae*, small substances suppressed the initiation of cyclic di-GMP that acts as the second messenger to control switch in-between the aquatic and sessile way of

It has been observed that numerous antibacterial peptides also inhibit biofilm formation in several microbes. For instance, it is considered that peptide 1018 has inhibitory effects in different microbes such as in *Acinetobacter baumannii, Burkholderia cenocepacia, Klebsiella pneumoniae, P. aeruginosa, E. coli, Salmonella typhimurium* and *S. aureus* [24]. Furthermore, class of peptide antibiotics called lantibiotics i.e. gallidermin, epidermin, subtilin, and nisin has been reported and control the biofilm production in *S. aureus, S. epidermidis* and

Chelators hindering the role of metal ions in the production of biofilm are considered as biofilm inhibitors, for example, silver salts, metallic silver and also silver nanoparticles are commonly employed as antibacterial agents in clinical implants against *P. aeruginosa, Salmonella typhimurium, Klebsiella species, E. coli,* and *S. aureus* [25]. It is observed that antibiotics i.e. amoxicillin, clindamycin, vancomycin, penicillin G and erythromycin show increased antimicrobial activity against *Staphylococcus aureus* in the presence of nanoparticles [26]. Treatment with silver substances prevents DNA replication, expression of cellular as well as ribosomal proteins, and also respiration process that leads to death of the cell [27]. In addition, It is also suggested that silver-coated implants inhibit *Staphylococcus aureus* biofilm production without aggregating silver inside the

In the majority of Gram-negative and Gram-positive bacteria, an essential cellular communicating system is presently called as Quorum sensing, which regulates a variety of genes in accordance with the density of signaling molecules furthermore, signaling molecules are called autoinducers [29]. On the bases of signaling molecules QS is classified into three i.e. autoinducing peptide (AIPbased) for Gram-positive bacteria, N-acyl homoserine lactones (AHLs-based) for Gram-negative bacteria and autoinducer-2 (AI-2-based) for both Gram-negative and Gram-positive bacteria [30]. When the biofilm is formed, after the initial attachment, cells secrete QS molecules that alter the expression of the microbial gene, thus changing planktonic form into a sessile form. Furthermore, QS plays a significant role in biofilm development, so It has been observed that QS inhibition i.e. quorum quenching (QQ ) would be a striking approach to control biofilm formation [31]. QS system is thought to be a target for developing new antimicrobial agents, moreover, QS system plays a crucial role in regulating pathogenetic factors and also virulence factors production in several pathogens [32]. The most important benefit of preventing biofilm formation by QQ is that this approach decreases the risk of multidrug resistance (MDR) and thus creating this approach noticeable to prevent biofilm-based infections in clinical settings.

The different approaches for the inhibition or removal of biofilms are

summarized in **Table 1** and **Figure 1**.

**88**

