**2.** *P. aeruginosa* **biofilms and lung infection in cystic fibrosis**

#### **2.1** *P. aeruginosa* **biofilms and antibiotic resistance**

The ubiquitous presence of *P. aeruginosa*, its prevalence and persistence in clinical settings and its intrinsic resistance to therapeutics are underpinned by an extraordinary arsenal of response mechanisms [10]. In particular, bacteria are protected by biofilms from adverse environmental conditions like phagocytosis, oxidative stress, nutrient/oxygen restriction, metabolic waste accumulation and antimicrobial agents [1, 11]. The matrix – which provides a favorable niche for intense cell–cell interaction and communication and a reservoir of metabolic substances, nutrients and energy [12] – accounts for 90% of the dry weight of the biofilm mass. Its main constituents are extracellular polysaccharides, proteins, extracellular DNA (eDNA), lipids, especially rhamnolipids, and other secreted molecules, such as the siderophores pyoverdine and pyocheclin, pyocyanin and phenazines. The production of all these components is highly regulated by *quorum sensing* (QS). *P. aeruginosa* biofilm development is characterized by the production of large amounts of three types of extracellular polysaccharides: Psl, Pel and alginate. Psl and Pel are the main constituents of the extracellular matrix in non-mucoid strains and are involved in the early stages of biofilm formation and in cell–cell interactions, whereas alginate overproduction is associated with the mucoid phenotype, the hallmark of chronic infection, and is indicative of disease progression and long-term persistence.

Biofilm development is held to be a differentiation process – activated in response to a variety of environmental stimuli – that alters pathogen behavior and results in the adoption of a sessile lifestyle [13]. Biofilms are characterized by an intricate regulation network that induces the development of different bacterial subpopulations and the emergence of antibiotic-resistant variants, which are a typical trait of *P. aeruginosa* biofilms [14]. The heterogeneity of the biofilm bacterial population is associated with the presence of niches with distinctive environmental characteristics that modulate gene expression patterns [15].

Biofilm formation is regulated by a number of redundant mechanisms of which QS is the most widely investigated. Four different QS systems, Las, Rhl, Pqs and Iqs, each characterized by a specific signal molecule and a receptor protein, have been described in *P. aeruginosa*. QS systems are involved in the regulation of several metabolic and pathogenic pathways that have a significant role in bacterial fitness in the environment as well as in the host. Their interplay is governed by a complicated hierarchical network, where the Las system directly regulates the Pqs and the Rhl systems [15].

Additional regulator systems, which sense the changes in the extracellular environment and regulate gene expression accordingly, also seem to be key factors in biofilm population dynamics. The best known is the Gac/Rsm system, which is the main factor controlling the switch from the planktonic to the sessile lifestyle in *P. aeruginosa* [13]. It encompasses two proteins, GacS/GacR, which sense and respond to environmental stimuli, promoting the synthesis of two small RNAs, RsmZ and RsmY, which bind and sequester the post-transcriptional regulator RsmA [16]. It induces the expression of virulence factors and of other genes playing roles

**35**

*Pseudomonas aeruginosa* Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters

in colonization and acute infection processes, such as the genes involved in motility (synthesis of pili) and in the type III secretion system; at the same time, it represses some genes implicated in chronic infections, such as those encoding the production of alginate and other exopolysaccharides, which constitute the biofilm matrix. RsmA sequestration seems to be a central mechanism in the shift from the planktonic to the biofilm lifestyle [16]. The second messenger c-di-GMP acts through an alternative regulation pathway and seems to promote biofilm development by a variety of routes: repression of motility-related genes, exopolysaccharide overproduction and expression of the adhesin CdrA [17, 18]. RsmA and c-di-GMP share overlapping targets and indirectly regulate each other with antagonistic effects, supporting the notion of a redundant system [19]. The fact that the c-di-GMP positively regulated efflux pump overexpression through *brlR* induction highlights the importance of the messenger in the development of antibiotic resistance/persistence phenotypes [20]. In sessile cells, the action of antibiotics is contrasted by a variety of mechanisms that make them less susceptible to antimicrobials than planktonic cells [21]. Notably, the biofilm matrix acts as a barrier, limiting the diffusion of toxic compounds [22]; in particular, binding to eDNA prevents positively charged antibiotics such as aminoglycosides from penetrating the bacterial cells [23]. Moreover, in biofilm-growing *P. aeruginosa* a wide range of resistance determinants are expressed or upregulated in a biofilm-specific manner [24]. Indeed, overexpression of the efflux pumps – particularly MexAB-OprM and MexXY-OprM – is the main cause of the multiple antibiotic-resistant phenotype [25] that characterizes chronic *P. aeruginosa* infection and contributes to the failure of its eradication in CF patients [26, 27]. The *mexAB-oprM* operon is upregulated in biofilms resistant to azithromycin [28] and fluoroquinolones [29] and also seems to be involved in colistin tolerance, which has been described in actively growing *P. aeruginosa* cells [30]. MexXY-OprM is the main aminoglycoside resistance determinant. It is a typical example of inducible adaptive resistance [31]; this is also demonstrated by the frequent recovery, from chronic patients, of strains bearing mutations in *mexZ*, a repressor gene of the *mex-XY* operon, which is considered as a mutation hotspot in biofilm-growing *P. aeruginosa* and a typical example of convergent evolution of different CF clonal lineages [32, 33]. Other remarkable examples of antibiotic resistance associated with biofilm growth are endogenous AmpC ß-lactamase overexpression and upregulation of the *ndvB* gene [34]; the latter is involved in biofilm-specific synthesis of cyclic glucans, which are responsible for aminoglycoside binding and trapping [35]. Finally, the biofilm is an ideal environment for HGT events [8], which contribute to the spread of resistance determinants. Conjugation events are favored by close contact between cells of different strains and/or species [36]; moreover, it has recently been suggested that *P. aeruginosa* biofilms can achieve a natural competence to acquire both genomic and plasmid DNA [37]. This is a cause of particular concern for chronic CF patients, whose lungs are often colonized by different antibiotic-resistant strains, a condition that has the potential

*DOI: http://dx.doi.org/10.5772/intechopen.95590*

to give rise to multidrug resistance [38].

Cystic Fibrosis is a genetic autosomic disease due to mutations in the cystic fibrosis transmembrane conductance regulator (*CFTR*) gene, which involve a wide range of dysfunctions that alter the airway environment and increase susceptibility to bacterial respiratory infections. *CFTR* gene dysfunction affects epithelial cells, the pancreas (malabsorption), the liver (biliary cirrhosis), the sweat glands (heat shock) and the vas deferens (infertility). Patients with late disease suffer from bronchiectasis, small airway obstruction and progressive respiratory impairment [39]. CF is

**2.2** *P. aeruginosa* **CF lung infection**

#### *Pseudomonas aeruginosa* Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters *DOI: http://dx.doi.org/10.5772/intechopen.95590*

in colonization and acute infection processes, such as the genes involved in motility (synthesis of pili) and in the type III secretion system; at the same time, it represses some genes implicated in chronic infections, such as those encoding the production of alginate and other exopolysaccharides, which constitute the biofilm matrix. RsmA sequestration seems to be a central mechanism in the shift from the planktonic to the biofilm lifestyle [16]. The second messenger c-di-GMP acts through an alternative regulation pathway and seems to promote biofilm development by a variety of routes: repression of motility-related genes, exopolysaccharide overproduction and expression of the adhesin CdrA [17, 18]. RsmA and c-di-GMP share overlapping targets and indirectly regulate each other with antagonistic effects, supporting the notion of a redundant system [19]. The fact that the c-di-GMP positively regulated efflux pump overexpression through *brlR* induction highlights the importance of the messenger in the development of antibiotic resistance/persistence phenotypes [20].

In sessile cells, the action of antibiotics is contrasted by a variety of mechanisms that make them less susceptible to antimicrobials than planktonic cells [21]. Notably, the biofilm matrix acts as a barrier, limiting the diffusion of toxic compounds [22]; in particular, binding to eDNA prevents positively charged antibiotics such as aminoglycosides from penetrating the bacterial cells [23]. Moreover, in biofilm-growing *P. aeruginosa* a wide range of resistance determinants are expressed or upregulated in a biofilm-specific manner [24]. Indeed, overexpression of the efflux pumps – particularly MexAB-OprM and MexXY-OprM – is the main cause of the multiple antibiotic-resistant phenotype [25] that characterizes chronic *P. aeruginosa* infection and contributes to the failure of its eradication in CF patients [26, 27]. The *mexAB-oprM* operon is upregulated in biofilms resistant to azithromycin [28] and fluoroquinolones [29] and also seems to be involved in colistin tolerance, which has been described in actively growing *P. aeruginosa* cells [30]. MexXY-OprM is the main aminoglycoside resistance determinant. It is a typical example of inducible adaptive resistance [31]; this is also demonstrated by the frequent recovery, from chronic patients, of strains bearing mutations in *mexZ*, a repressor gene of the *mex-XY* operon, which is considered as a mutation hotspot in biofilm-growing *P. aeruginosa* and a typical example of convergent evolution of different CF clonal lineages [32, 33]. Other remarkable examples of antibiotic resistance associated with biofilm growth are endogenous AmpC ß-lactamase overexpression and upregulation of the *ndvB* gene [34]; the latter is involved in biofilm-specific synthesis of cyclic glucans, which are responsible for aminoglycoside binding and trapping [35]. Finally, the biofilm is an ideal environment for HGT events [8], which contribute to the spread of resistance determinants. Conjugation events are favored by close contact between cells of different strains and/or species [36]; moreover, it has recently been suggested that *P. aeruginosa* biofilms can achieve a natural competence to acquire both genomic and plasmid DNA [37]. This is a cause of particular concern for chronic CF patients, whose lungs are often colonized by different antibiotic-resistant strains, a condition that has the potential to give rise to multidrug resistance [38].

#### **2.2** *P. aeruginosa* **CF lung infection**

Cystic Fibrosis is a genetic autosomic disease due to mutations in the cystic fibrosis transmembrane conductance regulator (*CFTR*) gene, which involve a wide range of dysfunctions that alter the airway environment and increase susceptibility to bacterial respiratory infections. *CFTR* gene dysfunction affects epithelial cells, the pancreas (malabsorption), the liver (biliary cirrhosis), the sweat glands (heat shock) and the vas deferens (infertility). Patients with late disease suffer from bronchiectasis, small airway obstruction and progressive respiratory impairment [39]. CF is

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

**2.** *P. aeruginosa* **biofilms and lung infection in cystic fibrosis**

**2.1** *P. aeruginosa* **biofilms and antibiotic resistance**

progression and long-term persistence.

insects and animals. In humans it is an important nosocomial pathogen responsible for a variety of infections that have a strong tendency to recur, particularly in burn patients and in those with lung involvement. Like other opportunistic pathogens, it typically affects immunocompromised individuals [9]. However, the subjects most prone to develop *P. aeruginosa* infection are patients with cystic fibrosis (CF).

The ubiquitous presence of *P. aeruginosa*, its prevalence and persistence in clinical settings and its intrinsic resistance to therapeutics are underpinned by an extraordinary arsenal of response mechanisms [10]. In particular, bacteria are protected by biofilms from adverse environmental conditions like phagocytosis, oxidative stress, nutrient/oxygen restriction, metabolic waste accumulation and antimicrobial agents [1, 11]. The matrix – which provides a favorable niche for intense cell–cell interaction and communication and a reservoir of metabolic substances, nutrients and energy [12] – accounts for 90% of the dry weight of the biofilm mass. Its main constituents are extracellular polysaccharides, proteins, extracellular DNA (eDNA), lipids, especially rhamnolipids, and other secreted molecules, such as the siderophores pyoverdine and pyocheclin, pyocyanin and phenazines. The production of all these components is highly regulated by *quorum sensing* (QS). *P. aeruginosa* biofilm development is characterized by the production of large amounts of three types of extracellular polysaccharides: Psl, Pel and alginate. Psl and Pel are the main constituents of the extracellular matrix in non-mucoid strains and are involved in the early stages of biofilm formation and in cell–cell interactions, whereas alginate overproduction is associated with the mucoid phenotype, the hallmark of chronic infection, and is indicative of disease

Biofilm development is held to be a differentiation process – activated in response to a variety of environmental stimuli – that alters pathogen behavior and results in the adoption of a sessile lifestyle [13]. Biofilms are characterized by an intricate regulation network that induces the development of different bacterial subpopulations and the emergence of antibiotic-resistant variants, which are a typical trait of *P. aeruginosa* biofilms [14]. The heterogeneity of the biofilm bacterial population is associated with the presence of niches with distinctive environmental

Biofilm formation is regulated by a number of redundant mechanisms of which QS is the most widely investigated. Four different QS systems, Las, Rhl, Pqs and Iqs, each characterized by a specific signal molecule and a receptor protein, have been described in *P. aeruginosa*. QS systems are involved in the regulation of several metabolic and pathogenic pathways that have a significant role in bacterial fitness in the environment as well as in the host. Their interplay is governed by a complicated hierarchical network, where the Las system directly regulates the Pqs and the Rhl systems [15]. Additional regulator systems, which sense the changes in the extracellular environment and regulate gene expression accordingly, also seem to be key factors in biofilm population dynamics. The best known is the Gac/Rsm system, which is the main factor controlling the switch from the planktonic to the sessile lifestyle in *P. aeruginosa* [13]. It encompasses two proteins, GacS/GacR, which sense and respond to environmental stimuli, promoting the synthesis of two small RNAs, RsmZ and RsmY, which bind and sequester the post-transcriptional regulator RsmA [16]. It induces the expression of virulence factors and of other genes playing roles

characteristics that modulate gene expression patterns [15].

**34**

characterized by recurrent pulmonary exacerbations. Worsening of the chronic lung infection symptoms (particularly cough and sputum production), increased bacterial load and inflammation and, often, a reduction in FEV1 (forced respiratory volume in 1 second) impair lung function hence quality of life and overall survival.

The identification of effective treatments requires a greater understanding of the factors underpinning the exacerbations. Notably, the lung of CF patients is initially colonized by *Haemophilus influenzae* and *Staphylococcus aureus*; then, patients gradually become susceptible to infection with a variety of environmental Gramnegative bacteria carrying a broad range of constitutive and acquired antibiotic resistance determinants [39]. *P. aeruginosa* is the main pathogen triggering airway inflammation and the leading cause of CF morbidity and mortality [40]. Most CF patients are susceptible to *P. aeruginosa* respiratory infections from infancy. The 30% of them acquire a strain from the environment resulting in acute infections in the first year of life, this rate increases to about 50% before turning 3 years, while mucoid phenotype and chronic infection usually raise from 3 to 16 years [10].

Lung colonization generally involves alternate asymptomatic periods and relapses with progressive tissue deterioration that eventually lead to lung failure and to premature death. Over the years *P. aeruginosa* develops multiple phenotypic variants such as SCVs, mucoid and persistent forms. In particular, SCVs are typically isolated from the lungs of chronic CF patients. They are small (1–3 mm in diameter) usually non-motile and resistant to several classes of antibiotics; produce high amounts of exopolysaccharide and form biofilms that strongly adhere to surfaces [41]. *In vitro* and *in vivo* tests have demonstrated that exposure to sublethal concentrations of antibiotics, such as aminoglycosides, selects for SCVs. In CF patients, prolonged persistent infection, deterioration of pulmonary function and increased antibiotic resistance all correlate with SCVs detection in sputum [41].

*P. aeruginosa* adaptation to the CF lung environment ultimately results in a mucoid phenotype, a conversion first described by Lam and colleagues [42], which may take several months to years. The mucoid material has subsequently been identified as alginate. In mucoid strains, alginate may favor adhesion to lung epithelial cells, thereby inhibiting clearance. Nutrient restriction, dehydration and suboptimal antibiotic concentrations may result in mucoidity [7, 43]. Host inflammation responses are also believed to contribute to mucoid conversion, a hypothesis that is supported by the absence of mucoid variants among environmental isolates [44].
