*3.4.1 Biofilms formed by Pseudomonas aeruginosa*

*Pseudomonas aeruginosa* is a recognized common pathogen in respiratory tract infections although other members of the genus *Pseudomonas* are able to form biofilms [7]. Respiratory infections caused by *P. aeruginosa* are a major globally clinical issue, especially for patients with chronic pulmonary disorders, such as those with cystic fibrosis (CF), non-CF bronchiectasis, severe chronic obstructive pulmonary disease (COPD) and ventilator-associated pneumonia [60]. This bacterium is a difficult opportunistic pathogen that readily forms biofilms on most surfaces [5]. The intricate steps of biofilm formation by *P. aeruginosa* are considered to be a developmental process. The stages of *P. aeruginosa* biofilm formation can be seen by several strategies. One easy technique is the scanning electron microscope (SEM) of *P. aeruginosa* grown on glass surfaces or tracheal explants. Biofilms form when planktonic *P. aeruginosa* bacteria get attached to a surface using adhesins such as type IV fimbriae and flagella, and begin to colonize. In this regard type IV fimbriae and flagella *P. aeruginosa* mutants are severely compromised in initiation of biofilm formation [58, 61]. Additionally, the process of surface translocation mediated by type IV fimbriae (twitching motility) is essential for initiation of biofilm formation by *P. aeruginosa* [58]. Most probable, twitching motility confers synchronized cell movement along the surface as well as cell–cell communications that lead to the formation of micro-colonies. The coordination of events for the initiation and formation of biofilms requires cell– cell interactions that are mediated by quorum sensing [62]. Following this, the micro-colonies mature into distinctive three-dimensional structures that pose the most severe scenario for clinical treatment. This structure is typically trapped in a matrix material that may be composed of protein, polysaccharide, or nucleic acid. Nonetheless, it has been proposed guluronic and mannuronic acids [63] are the major constituents of the biofilm matrix [64]. Recent data also suggest that DNA also contributes to this matrix [60].

### *3.4.2 Biofilms formed by Staphylococcus species*

The adherence of *Staphylococcus* directly to an implanted device (intravascular catheters, prosthetic devices, and other indwelling medical devices) or indirectly via host proteins is the first step in the development of a biofilm. This is followed by a buildup of multilayered cell clusters on the polymer surface [65]. When *Staphylococcus* bacteria get within 50 nm of a surface, they adhere through hydrophobic interactions, van der Waal's forces, and when present, fimbriae and pili also contribute to its adhesion [66]. A biofilm-associated protein (Bap) is reported to contribute to *S. aureus* biofilm formation. The second phase of *Staphylococcus* biofilm formation is the accumulation of complex cell clusters mediated by intercellular adhesion. A 140 kDa extracellular protein, known as the accumulation associated protein (AAP), appears responsible for accumulative growth on polymer substances [67]. It has been hypothesized that AAP is involved in anchoring the polysaccharide adhesion PIA (polysaccharide intercellular adhesion) to the cell surface [63]. The extracellular polysaccharide adhesion antigen PIA is a well-described polysaccharide antigen that is linked to cellular aggregation or clustering. Lastly, the generation of a slime glycocalyx is believed to be the climaxing event in the staphylococcal biofilm developmental process. This slime layer is not essential for surface colonization and appears variable between strains. However, when present, the slime layer protects the bacteria from host defenses and some antibiotics. As in *P. aeruginosa*, organization of complex communities within *Staphylococcus* biofilms is a coordinated effort and requires cell–cell communication [68].

### *3.4.3 Biofilms formed by Haemophilus influenzae*

Non-typeable *H. influenzae* (NTHI) strains are members of the normal human nasopharyngeal flora, as well as frequent opportunistic pathogens of both the upper and lower respiratory tracts. It is an important cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Recently, it has been shown that NTHI can form biofilms both *in vitro* and *in vivo* [69]. Considerable diversity in the ability of NTHi isolates to form biofilms has also been reported. A NTHi pilus defective strain was reduced three-to four fold in biofilm formation compared with its isogenic parental NTHi isolate, signifying a role of the pilus in biofilm development. Although this is the case for other gram-negative bacteria [70], nonetheless, it is quite clear that NTHi strains have the ability to form biofilms both *in vitro* and *in vivo* [69]. Earlier studies of cell envelopes during growth of *H. influenzae* as a biofilm established an increased abundance of a ~30 kDa protein [58], peroxiredoxin-glutaredoxin (PGdx) [71], that is expressed by *H. influenzae* during biofilm growth and this probably contributes to its persistence in the upper respiratory tract infections.

### *3.4.4 Biofilms formed by other microorganisms*

*Streptococcus pneumoniae*: *Streptococcus pneumoniae* is a frequent colonizer of the human nasopharynx and a significant human respiratory pathogen that causes a variety of diseases such as community-acquired pneumonia and otitis media in children [72]. Colonizing pneumococci form well-ordered biofilm communities in the nasopharyngeal environment, but the exact role of biofilms and their interaction with the host during colonization and disease is not yet explicit [73]. However, investigators have speculated that pneumococci form biofilms in the nasopharynx *in vivo* [74]. Recently, pneumococci have been reported for the first time to form

**121**

**4. Quorum sensing**

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

highly structured biofilms during colonization of the murine nasopharynx [75]. Mice were also inoculated intranasally with the pneumococcal strain EF3030, a clinical isolate known to be non-invasive and an efficient colonizer in murine

*Bordetella* species: *Bordetellae* are respiratory pathogens that infect both humans and animals. *Bordetella bronchiseptica* causes asymptomatic and long-term to lifelong infections in animal nasopharynges while the human pathogen, *B. pertussis* is the etiological agent of the acute disease whooping cough in infants and young children. One proposed hypothesis to explain the survival and continued persistence of *Bordetella spp*. in the mammalian nasopharynx is that these organisms produce surface-adherent communities known as biofilms [77]. Researchers have recently established the ability of the three classical *Bordetella species* (*Bordetella pertussis, Bordetella bronchiseptica,* and *Bordetella parapertussis*) to form biofilms on abiotic surfaces [78]. It is assumed that *Bordetella* biofilm formation may play a role in the pathogenic cycle, precisely in persistence within the nasopharynx [79]. The capacity to form biofilms in mice suggests a role for *Bordetella* mode of existence during human infections. Clusters and tangles (reminiscent of biofilms) of *Bordetella pertussis* adherent to ciliated cells in explant cultures and tissue biopsies of pertussis patients have been documented [79]. As reported for other biofilm-forming organisms, extracellular DNA and exopolysaccharide are vital for biofilm formation by *Bordetella bronchiseptica*. The observation of biofilm-like structures *in vivo* in the nasal epithelium of *Bordetella bronchiseptica* infected mice showed that these communities expressed a polysaccharide essential for *in vivo* biofilm development [75, 76]. In *Bordetella*, BvgAS-regulated factors, including the filamentous hemagglutinin and adenylate cyclase, may also contribute to biofilm formation [79]. *Mycobacterium* species: Mycobacterial infections have been shown to form biofilms, most notably *Mycobacterium tuberculosis*, which under the conducive environments, can self-assemble. Among the non-tuberculous mycobacteria, *Mycobacterium avium* complex (MAC) and the rapidly growing mycobacteria, including *Mycobacterium abscessus* complex, have been reported to produce biofilms either *in vitro* or in environmental reservoirs [80], but *in vivo* conditions have not been investigated. *Mycobacterium abscessus* complex is an evolving threat to patients with cystic fibrosis [81], that become infected at an early stage and worsens clinically as the persistent infection results in inflammation and tissue damage.

In the control of microbial infections, two strategies are normally envisaged; killing the organisms or attenuation of the organisms' virulence such that they fail to adapt to the host environment. The former approach is what is generally favored; the latter lacks specific targets for rational drug design. It has, however, been realized that Gram-negative bacteria use small molecules known as acyl homoserine lactones to regulate the production of secondary metabolites and virulence factors, and this could offer a novel target to address the strategy of attenuating the organisms' virulence thereby impairing their adaptation to the host system. Recent research has highlighted the importance of cell-to-cell interactions or communications, referred to as Quorum Sensing (QS), in microorganisms. Many bacterial species employ a complex mechanistic communication system to transmit information among themselves. Bacteria can act in response to a variety of chemical signals produced by the same species along with others produced by other species, and this provides a way for intraspecies and interspecies cross-communication

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

models, and found to form biofilms [76].

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

*Bacterial Biofilms*

*3.4.2 Biofilms formed by Staphylococcus species*

The adherence of *Staphylococcus* directly to an implanted device (intravascular catheters, prosthetic devices, and other indwelling medical devices) or indirectly via host proteins is the first step in the development of a biofilm. This is followed by a buildup of multilayered cell clusters on the polymer surface [65]. When *Staphylococcus* bacteria get within 50 nm of a surface, they adhere through hydrophobic interactions, van der Waal's forces, and when present, fimbriae and pili also contribute to its adhesion [66]. A biofilm-associated protein (Bap) is reported to contribute to *S. aureus* biofilm formation. The second phase of *Staphylococcus* biofilm formation is the accumulation of complex cell clusters mediated by intercellular adhesion. A 140 kDa extracellular protein, known as the accumulation associated protein (AAP), appears responsible for accumulative growth on polymer substances [67]. It has been hypothesized that AAP is involved in anchoring the polysaccharide adhesion PIA (polysaccharide intercellular adhesion) to the cell surface [63]. The extracellular polysaccharide adhesion antigen PIA is a well-described polysaccharide antigen that is linked to cellular aggregation or clustering. Lastly, the generation of a slime glycocalyx is believed to be the climaxing event in the staphylococcal biofilm developmental process. This slime layer is not essential for surface colonization and appears variable between strains. However, when present, the slime layer protects the bacteria from host defenses and some antibiotics. As in *P. aeruginosa*, organization of complex communities within *Staphylococcus* biofilms

is a coordinated effort and requires cell–cell communication [68].

Non-typeable *H. influenzae* (NTHI) strains are members of the normal human nasopharyngeal flora, as well as frequent opportunistic pathogens of both the upper and lower respiratory tracts. It is an important cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Recently, it has been shown that NTHI can form biofilms both *in vitro* and *in vivo* [69]. Considerable diversity in the ability of NTHi isolates to form biofilms has also been reported. A NTHi pilus defective strain was reduced three-to four fold in biofilm formation compared with its isogenic parental NTHi isolate, signifying a role of the pilus in biofilm development. Although this is the case for other gram-negative bacteria [70], nonetheless, it is quite clear that NTHi strains have the ability to form biofilms both *in vitro* and *in vivo* [69]. Earlier studies of cell envelopes during growth of *H. influenzae* as a biofilm established an increased abundance of a ~30 kDa protein [58], peroxiredoxin-glutaredoxin (PGdx) [71], that is expressed by *H. influenzae* during biofilm growth and this probably contributes to

*Streptococcus pneumoniae*: *Streptococcus pneumoniae* is a frequent colonizer of the human nasopharynx and a significant human respiratory pathogen that causes a variety of diseases such as community-acquired pneumonia and otitis media in children [72]. Colonizing pneumococci form well-ordered biofilm communities in the nasopharyngeal environment, but the exact role of biofilms and their interaction with the host during colonization and disease is not yet explicit [73]. However, investigators have speculated that pneumococci form biofilms in the nasopharynx *in vivo* [74]. Recently, pneumococci have been reported for the first time to form

*3.4.3 Biofilms formed by Haemophilus influenzae*

its persistence in the upper respiratory tract infections.

*3.4.4 Biofilms formed by other microorganisms*

**120**

highly structured biofilms during colonization of the murine nasopharynx [75]. Mice were also inoculated intranasally with the pneumococcal strain EF3030, a clinical isolate known to be non-invasive and an efficient colonizer in murine models, and found to form biofilms [76].

*Bordetella* species: *Bordetellae* are respiratory pathogens that infect both humans and animals. *Bordetella bronchiseptica* causes asymptomatic and long-term to lifelong infections in animal nasopharynges while the human pathogen, *B. pertussis* is the etiological agent of the acute disease whooping cough in infants and young children. One proposed hypothesis to explain the survival and continued persistence of *Bordetella spp*. in the mammalian nasopharynx is that these organisms produce surface-adherent communities known as biofilms [77]. Researchers have recently established the ability of the three classical *Bordetella species* (*Bordetella pertussis, Bordetella bronchiseptica,* and *Bordetella parapertussis*) to form biofilms on abiotic surfaces [78]. It is assumed that *Bordetella* biofilm formation may play a role in the pathogenic cycle, precisely in persistence within the nasopharynx [79]. The capacity to form biofilms in mice suggests a role for *Bordetella* mode of existence during human infections. Clusters and tangles (reminiscent of biofilms) of *Bordetella pertussis* adherent to ciliated cells in explant cultures and tissue biopsies of pertussis patients have been documented [79]. As reported for other biofilm-forming organisms, extracellular DNA and exopolysaccharide are vital for biofilm formation by *Bordetella bronchiseptica*. The observation of biofilm-like structures *in vivo* in the nasal epithelium of *Bordetella bronchiseptica* infected mice showed that these communities expressed a polysaccharide essential for *in vivo* biofilm development [75, 76]. In *Bordetella*, BvgAS-regulated factors, including the filamentous hemagglutinin and adenylate cyclase, may also contribute to biofilm formation [79].

*Mycobacterium* species: Mycobacterial infections have been shown to form biofilms, most notably *Mycobacterium tuberculosis*, which under the conducive environments, can self-assemble. Among the non-tuberculous mycobacteria, *Mycobacterium avium* complex (MAC) and the rapidly growing mycobacteria, including *Mycobacterium abscessus* complex, have been reported to produce biofilms either *in vitro* or in environmental reservoirs [80], but *in vivo* conditions have not been investigated. *Mycobacterium abscessus* complex is an evolving threat to patients with cystic fibrosis [81], that become infected at an early stage and worsens clinically as the persistent infection results in inflammation and tissue damage.

### **4. Quorum sensing**

In the control of microbial infections, two strategies are normally envisaged; killing the organisms or attenuation of the organisms' virulence such that they fail to adapt to the host environment. The former approach is what is generally favored; the latter lacks specific targets for rational drug design. It has, however, been realized that Gram-negative bacteria use small molecules known as acyl homoserine lactones to regulate the production of secondary metabolites and virulence factors, and this could offer a novel target to address the strategy of attenuating the organisms' virulence thereby impairing their adaptation to the host system. Recent research has highlighted the importance of cell-to-cell interactions or communications, referred to as Quorum Sensing (QS), in microorganisms. Many bacterial species employ a complex mechanistic communication system to transmit information among themselves. Bacteria can act in response to a variety of chemical signals produced by the same species along with others produced by other species, and this provides a way for intraspecies and interspecies cross-communication

### *Bacterial Biofilms*

and interruption of signals. The ability of bacteria to dispatch, pull together, and process information allow them to act as "multicellular" organisms and enhance their survival in complex environments [82].

Any mechanism capable of disrupting QS signals can be used to reduce survival of the microorganism thereby preventing or reducing virulence in the host environment. Such methods of interruption of the QS include:


Agents capable of inhibiting the growth of microorganisms or disrupting the quorum sensing mechanisms of the microorganisms or interrupting the biofilm formation may be useful in the fight against microbial pathogenicity.
