*Burkholderia cepacia* **Complex Infections Among Cystic Fibrosis Patients: Perspectives and Challenges**

Jorge H. Leitão, Joana R. Feliciano, Sílvia A. Sousa,

Tiago Pita and Soraia I. Guerreiro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67712

#### **Abstract**

The *Burkholderia cepacia* complex (Bcc) is a group of closely related bacterial species that emerged in the 1980s as the etiological agents of severe and often lethal respiratory infections among cystic fibrosis (CF) patients. After several outbreaks in CF centers in Europe and North America, segregation measures were introduced to avoid patientto-patient transmission. Presently, the prevalence of Bcc infections among CF patients worldwide is below 5% in the majority of CF centers, although exceptions are registered in some European countries. Infections by these pathogens remain problematic due to the high resistance to antimicrobials, the easy patient-to-patient transmission, and the unpredictable outcome of infections that range from asymptomatic carriage to the cepacia syndrome, a fulminating pneumonia often associated with septicemia that can lead to the decease of patients within a period of time as short as 1 week. In this chapter, we review the evolving epidemiology of Bcc infections in CF patients, the virulence traits and mechanisms used by these bacteria, and the recent developments in vaccine and vaccine components research to prevent Bcc infections.

**Keywords:** *Burkholderia cepacia* complex, emerging species, evolving epidemiology, virulence determinants, immunoreactive proteins, vaccine development

## **1. Introduction**

The *Burkholderia cepacia* complex (hereafter referred to as Bcc) is a group of closely related bacteria that emerged in the 1980s as problematic pathogens to cystic fibrosis (CF) patients [1]. Infections by Bcc are particularly feared due to (1) the easy patient-to-patient transmission of

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specific strains; (2) the ability to resist to multiple antibiotics; and (3) the unpredictable outcome of infections, which ranges from asymptomatic carriage to the so-called cepacia syndrome, an often lethal necrotizingpneumonia accompanied with septicemia [1, 2].Initiallydescribedin the 1950s by Burkholder [3] as the cause of soft rot in onions, the species then named *Pseudomonas cepacia* was moved into the new genus *Burkholderia* after the work of Yabuuchi and colleagues in 1992 [4]. However, the most impressive developments on the taxonomy of this group of bacteria have been achieved after the seminal work of Vandamme and colleagues who proposed the division of the species into distinct genomovars [5]. Presently, the Bcc comprises 20 species (**Table 1**), and the genome sequence of several strains is publicly available in databases such as the Burkholderia Genome DB and the Integrated Microbial Genomes & Microbiomes [6, 7].


**Table 1.** *Burkholderia cepacia* complex species names and genome sequence availability in the databases Burkholderia Genome DB and Integrated Microbial Genomes & Microbiomes [6, 7].

## **2. Evolving epidemiology of Bcc infections**

specific strains; (2) the ability to resist to multiple antibiotics; and (3) the unpredictable outcome of infections, which ranges from asymptomatic carriage to the so-called cepacia syndrome, an often lethal necrotizingpneumonia accompanied with septicemia [1, 2].Initiallydescribedin the 1950s by Burkholder [3] as the cause of soft rot in onions, the species then named *Pseudomonas cepacia* was moved into the new genus *Burkholderia* after the work of Yabuuchi and colleagues in 1992 [4]. However, the most impressive developments on the taxonomy of this group of bacteria have been achieved after the seminal work of Vandamme and colleagues who proposed the division of the species into distinct genomovars [5]. Presently, the Bcc comprises 20 species (**Table 1**), and the genome sequence of several strains is publicly available in databases such as the Burkholderia Genome DB and the Integrated Microbial Genomes & Microbiomes [6, 7].

**Bcc species Genome sequence availability Reference** *B. ambifaria* 4 complete genomes (strains AMMD, MC40-6, MEX-5, IOP-120) [8] *B. anthina* In progress [9] *B. arboris* In progress [10]

PC184, HI2424, DDS 22E-1, DWS 37E-2, ST32, 842, 895, MSMB384

[11]

[4]

*B. cenocepacia* 18 complete genomes (strains J2315, H111, AU1054, B1, MCO-3,

WGS, 6, 7, CEIB, 869T2, TAtl-371)

7H-2, GG4, JBK9, LO6)

Databases were assessed by the end of July 2016.

74 Progress in Understanding Cystic Fibrosis

Genome DB and Integrated Microbial Genomes & Microbiomes [6, 7].

*B. cepacia* 8 complete genomes (strains 383, AMMD, ATCC 25416; Bu72, DDS

*B. contaminans* 1 complete genome (strain MS14) [12] *B. diffusa* In progress [10] *B. dolosa* 1 complete genome (strain AU0158) [13] *B. lata* 1 complete genome (strain 383) [12] *B. latens* In progress [10] *B. metallica* No information [10] *B. multivorans* 3 complete genomes (ATCC17616, ATCC BAA-247, DDS 15A-1) [5] *B. pseudomultivorans* In progress [14] *B. pyrrocinia* 1 complete genome (strain DSM 10685) [9] *B. seminalis* In progress [10] *B. stabilis* No information [15] *B. stagnalis* In progress [16] *B. territorii* In progress [16] *B. ubonensis* 1 complete genome (strain MSMB22) [17] *B. vietnamiensis* 3 complete genomes (strains G4, LMG10929, WPB) [18]

**Table 1.** *Burkholderia cepacia* complex species names and genome sequence availability in the databases Burkholderia

All Bcc species are virtually potential pathogens to CF patients. However, epidemiology studies have shown an uneven geographical and regional distribution of clinical isolates among the Bcc species, with the predominance of *Burkholderia cenocepacia*, followed by *Burkholderia multivorans*. Early studies performed during the 1980s and 1990s have shown that in addition to cases of chronic infection due to specific strains, many outbreaks reported in Europe and North America were due to the spread of particularly virulent strains that easily disseminated within a given CF center [1]. Although the environment is thought to be the natural reservoir of these strains, a definitive proof is still lacking.

A few particularly epidemic strains became notorious for the worst reasons. Perhaps, the best-known strain is the Edinburgh-Toronto lineage also known as the ET12 clone, an intercontinental clone responsible for several infections and fatalities in CF centers in the UK and Canada [19]. The best-known representative strain of this highly transmissible clone is the *B. cenocepacia* J2315 strain, the first Bcc strain with its genome sequence publicly available (**Table 1**) and one of the best studied Bcc strains [20]. Another example of a strain that disseminated within centers and even among centers is the PHDC strain. The strain, responsible for almost 20% prevalence in one CF center in the USA, was later found in another CF center, where an increase in Bcc prevalence was experienced. The dissemination of the strain was associated with the transfer of an infected patient from the initial center to the second one [21]. A later study by Coenye et al. [22] showed that the PHDC strain was also present in European patients (namely in France, Italy, and the UK), concluding that the PHDC strain was the second-identified Bcc transatlantic clone. Interestingly, both intercontinental clones belong to the *B. cenocepacia* species, although the ET12 belongs to subgroup IIIA and the PHDC belongs to subgroup IIIB. The *B. cenocepacia* species includes other clones that spread among CF centers, namely the Midwest American clone and the CZI Czech epidemic clone [23, 24]. Evidence of transmission of particularly epidemic strains of *B. cenocepacia* led to the introduction of segregation measures in CF centers in Europe and America, with a significant reduction of prevalence of infections [1, 25–27]. However, these segregation policies had a devastating impact on patients infected with Bcc due to social isolation and stigma and negative psychological impacts [28]. Although effective in interrupting strain transmission, segregation measures do not prevent new acquisitions. Nevertheless, these measures led to a reduction of prevalence of Bcc infections from more than 20% in several centers to less than 5% both in the USA and the majority of European countries [29, 30]. However, prevalence of chronic Bcc infections is still ranging 5–10% in Denmark, Portugal, Slovak Republic, Russian Federation, and Latvia, reaching values of 15 and 23% in Serbia and Lithuania, respectively [30].

Although the Bcc strains responsible for the vast majority of infections both in Europe and North America belong to the *B. cenocepacia* species, recent evidence indicates a changing epidemiology. *B. multivorans* emerged as the dominant species in France by 2004 and as the second most important species in the USA [31, 32]. Recent reports also indicate *Burkholderia contaminans* as an emerging Bcc species associated with CF infections. Early reports of a high incidence of the species among CF patients came from Portugal and Argentina [33–35].

Interestingly, in the case of the Portuguese CF population, two *B. contaminans* clones infecting CF patients were found as indistinguishable from two *B. contaminans* strains isolated from nonsterile nasal saline solutions of commercial origin during routine surveillance by the Portuguese Medicines and Health Products Authority [36]. A recent work by Medina-Pascual and colleagues on the surveillance of Bcc infections in Spanish CF patients also reported a *B. contaminans* overall incidence of 36.5% in the period 2008–2012, surpassing the previously dominant species *B. cenocepacia* and *B. multivorans* [37]. The emergence of *B. contaminans* among Spanish CF patients was hypothesized to be due to unspecified ecological advantages that enable the species to increase its presence in hospitals or in the environment [37]. In the case of Swiss CF-patients, *B. cenocepacia* was the most frequently isolated species in the period 1998–2013, but *B. multivorans* and *B. contaminans* emerged during the last years of the study period [38]. A 30-year study of Bcc infections among CF patients from British Columbia (Canada) evidenced a major impact of segregation measures in Bcc epidemiology; while *B. cenocepacia* was dominant before the introduction of these measures, *B. multivorans* strains became dominant after implementation of novel infection control measures in 1995 [39]. This study and others highlight the impact of infection control measures on Bcc species recovered from CF patients. It is now apparent that while epidemic *B. cenocepacia* strains dominated in early years, nonclonal *B. multivorans* and *B. contaminans* strains are emerging.

## **3. Bcc virulence factors and traits**

Over the last 20 years, substantial progress has been achieved on the knowledge of Bcc virulence factors and determinants, although the exact contribution of some of them to the success of infection remains to be fully understood. It is currently accepted that Bcc virulence does not rely on a single virulence factor, being multifactorial. Bacterial structures such as flagella, the cable pili, and the 22-kDa adhesin are considered virulence factors since they play important roles in the initial steps of interaction with the host cell, promoting the adherence to the lung surface and the invasion of lung epithelial cells [39–41]. In addition, the majority of *B. cenocepacia* strains are able to survive and replicate intracellularly in airway epithelial cells and macrophages, evading the primary cellular defense mechanisms of the lung and avoiding clearance. The factors involved in this ability, exopolysaccharide (EPS) biosynthesis, biofilm formation, resistance to antibiotics, and oxidative stress resistance, as well as the iron acquisition ability are also among virulence determinants described for Bcc [20, 42, 43]. Some of these virulence factors are further detailed below.

#### **3.1. Alternative sigma factors**

RpoE and RpoN are two alternative sigma factors involved in the regulation of the ability of intracellular *B. cenocepacia* to delay phagolysosomal fusion in murine macrophages [44, 45]. RpoE is the extra-cytoplasmic stress response regulatorrequired by *B. cenocepacia* to grow under conditions of high osmolarity and high temperature [44]. RpoN, or sigma factor σ54, is best known for its involvement in nitrogen-related gene regulation. In *B. cenocepacia,* σ54 is involved in motility and biofilm formation [45]. Results from the mapping of σ54 regulon and the characterization of a *B. cenocepacia* H111-derived σ54 mutant suggest that this alternative sigma factor plays an important role in the control of nitrogen metabolism, in the metabolic adaptation of *B. cenocepacia* H111 to stressful and nutrient-limited environments and in virulence toward the nematode *Caenorhabditis elegans*[46]. In addition, it was also reported that RpoN regulates genes involved in exopolysaccharide production, biofilm formation, motility, and virulence [46]. A *B. cenocepacia* mutant defective in a gene encoding a putative σ54-related transcription regulator (BCAL1536) was found as attenuated in the rat agar bead infection model [47].

## **3.2. Lipopolysaccharides and extracellular polysaccharides**

Interestingly, in the case of the Portuguese CF population, two *B. contaminans* clones infecting CF patients were found as indistinguishable from two *B. contaminans* strains isolated from nonsterile nasal saline solutions of commercial origin during routine surveillance by the Portuguese Medicines and Health Products Authority [36]. A recent work by Medina-Pascual and colleagues on the surveillance of Bcc infections in Spanish CF patients also reported a *B. contaminans* overall incidence of 36.5% in the period 2008–2012, surpassing the previously dominant species *B. cenocepacia* and *B. multivorans* [37]. The emergence of *B. contaminans* among Spanish CF patients was hypothesized to be due to unspecified ecological advantages that enable the species to increase its presence in hospitals or in the environment [37]. In the case of Swiss CF-patients, *B. cenocepacia* was the most frequently isolated species in the period 1998–2013, but *B. multivorans* and *B. contaminans* emerged during the last years of the study period [38]. A 30-year study of Bcc infections among CF patients from British Columbia (Canada) evidenced a major impact of segregation measures in Bcc epidemiology; while *B. cenocepacia* was dominant before the introduction of these measures, *B. multivorans* strains became dominant after implementation of novel infection control measures in 1995 [39]. This study and others highlight the impact of infection control measures on Bcc species recovered from CF patients. It is now apparent that while epidemic *B. cenocepacia* strains dominated in

early years, nonclonal *B. multivorans* and *B. contaminans* strains are emerging.

Over the last 20 years, substantial progress has been achieved on the knowledge of Bcc virulence factors and determinants, although the exact contribution of some of them to the success of infection remains to be fully understood. It is currently accepted that Bcc virulence does not rely on a single virulence factor, being multifactorial. Bacterial structures such as flagella, the cable pili, and the 22-kDa adhesin are considered virulence factors since they play important roles in the initial steps of interaction with the host cell, promoting the adherence to the lung surface and the invasion of lung epithelial cells [39–41]. In addition, the majority of *B. cenocepacia* strains are able to survive and replicate intracellularly in airway epithelial cells and macrophages, evading the primary cellular defense mechanisms of the lung and avoiding clearance. The factors involved in this ability, exopolysaccharide (EPS) biosynthesis, biofilm formation, resistance to antibiotics, and oxidative stress resistance, as well as the iron acquisition ability are also among virulence determinants described for Bcc [20, 42, 43]. Some

RpoE and RpoN are two alternative sigma factors involved in the regulation of the ability of intracellular *B. cenocepacia* to delay phagolysosomal fusion in murine macrophages [44, 45]. RpoE is the extra-cytoplasmic stress response regulatorrequired by *B. cenocepacia* to grow under conditions of high osmolarity and high temperature [44]. RpoN, or sigma factor σ54, is best known for its involvement in nitrogen-related gene regulation. In *B. cenocepacia,* σ54 is involved

**3. Bcc virulence factors and traits**

76 Progress in Understanding Cystic Fibrosis

of these virulence factors are further detailed below.

**3.1. Alternative sigma factors**

One of the central components of the outer membrane in Gram-negative bacteria is the lipopolysaccharide (LPS), a complex molecule composed by the lipid A, the core oligosaccharide, and the O-antigen moieties (reviewed in Ref. [48]). The genes involved in LPS production by *B. cenocepacia* are located in chromosome I, organized in three main clusters, one for each LPS component (lipid A: *BCAL1929* to *BCAL1935*; core: *BCAL2402* to *BCAL2408*; O antigen: *BCAL3110* to *BCAL3125*) together with additional genes encoding sugar modification enzymes [49, 50]. Bcc bacteria LPS differs from other Gram-negative bacteria LPS due to the complete lack of negatively charged residues and the presence of the heterodimeric disaccharide D-glycero-D-talo-oct-2-ulosonic acid-(2–4)-3-deoxy-D-manno-oct-2-ulosonic acid (Ko-(2–4)-Kdo) in the core region; the presence of a 4-amino-4-deoxyarabinose (Ara4N) residue, either in the core or in lipid A; and the structure of O-antigen [50, 51]. This particular composition changes the bacterial surface charge, inhibiting the binding and successful action of antibiotics, contributing to the persistence of bacterial infection [51]. Recently, it was demonstrated that although L-Ara4N modifications do not affect recognition, they are critical for the establishment of infection [52]. Several studies have demonstrated that when neutrophils interact with Bcc LPS, the expression of CD11b on their surface increases, stimulating neutrophil respiratory burst response [53]. In addition, macrophages and human blood cells are also stimulated by Bcc LPS, producing pro-inflammatory cytokines such as TNF-α, IL-6, and IL-8 [54, 55].

*B. cenocepacia* J2315 is unable to produce the O-antigen. In this particular strain, this is due to an interruption in the *wbcE* gene-encoding BCAL 3125 [56]. The expression of O-antigen by Bcc strains was demonstrated to reduce phagocytosis by macrophages without interfering with the intracellular survival of bacteria [56].

The production of exopolysaccharides (EPSs) was described for several *Burkholderia* species. EPS production by Bcc is regarded as playing an important role in the chronicity of Bcc infections [57–62]. Cepacian is the most common EPS produced by Bcc and non-Bcc species, both from clinical and environmental sources [59, 63]. Cepacian interferes with phagocytosis by human neutrophils, facilitating the bacterial persistence in a mouse model of infection [64, 65]. The EPS was shown to inhibit the production of ROS by neutrophils and to scavenge reactive oxygen species (ROS), playing a role in the survival of cepacian-producing strains in different environments [64–67]. As a result of a frameshift mutation in the *bceB* gene (*BCAM0856*) encoding a putative glycosyltransferase, Cepacian is not produced by the *B. cenocepacia* ET12 representative strain J2315 [49, 62].

#### **3.3. Biofilms**

Bcc bacteria were found to persist in biofilms *in vitro*. Biofilm formation and maturation depend on many factors, including EPS production, motility, iron availability, and multiple gene regulatory systems, such as quorum sensing, alternative sigma factors, or global regulators such as the ShvR and AtsR [45, 58, 68–73]. In addition, Bcc can form small colony variants *in vitro*, a colony morphology that is associated with enhanced biofilm formation, antibiotic resistance, and persistence [74].

Several studies have been performed to understand the importance and relevance of biofilm formation in Bcc biology. Bcc bacteria growing in biofilms are usually more tolerant to multiple antibiotics, although similar susceptibilities were reported for plancktonic and biofilm cells to the antibiotics kanamycin, amikacin, and ciprofloxacin [75, 76]. Recently, Bcc biofilms were shown to contain persister cells that are able to survive in the presence of high concentrations of antibiotics by avoiding production ofreactive oxygen species [77]. In addition, using neutrophil-like dHL60 cells, it was shown that the presence of these immune system cells enhanced biofilm formation that protected Bcc bacteria against neutrophils by inducing their necrosis, acting as a barrier to the migration of neutrophils, and masking the bacteria from being recognized by neutrophils [78]. Although some evidence suggests that biofilm formation plays a role in bacterial persistence in the CF airways, this topic needs to be further studied.

#### **3.4. Quorum sensing**

Quorum sensing is a mode of regulation of gene expression that is dependent on the density of the bacterial population. Bcc bacteria have at least four quorum sensing systems. The CepIR quorum sensing system is homologous to the LuxIR system of *Vibrio fischeri* (reviewed in Ref. [79]). The CepIR system positively regulates the virulence of *B. cenocepacia* toward model organisms like *C. elegans*, *Galleria mellonella*, rodents, zebrafish, alfalfa, and onions [80–83]. In addition to the CepIR, *B. cenocepacia* encodes the CciIR, the CepR2, and the BDSF quorum sensing systems [84, 85]. While the CepIR and CciR quorum sensing systems rely on acyl homoserine lactones as signaling molecules, the BDSF system uses cis-2-dodecenoic acid as the signaling molecule, and the CepR2 is an orphan quorum sensing system [85]. An arsenal of genes regulated by quorum sensing in Bcc bacteria was described, including the negatively regulated siderophore synthesis and the positively regulated expression of the genes encoding zinc metalloproteases (Zmps), swarming motility and biofilm formation, all thought to have an impact when the bacterium is infecting the CF patient [71, 80, 86, 87].

#### **3.5. Protein secretion systems**

Both Gram-negative and positive bacteria use protein secretion systems to secrete toxins or other proteins, either directly into the environment or into host cells. These systems are particularly well studied in the CF pathogens Bcc and *Pseudomonas aeruginosa*. For instance, Bcc strains of the ET12 lineage and *Burkholderia vietnamiensis* harbor type I and II secretion systems (T1SS, T2SS) implicated, for instance, in the secretion of hemolytic proteins [88, 89]. The T2SS is also involved in *B. cenocepacia* secretion of two zinc metalloproteases, ZmpA and ZmpB, which play a role in virulence [80, 90]. Two T4SSs are encoded by *B. cenocepacia*; the T4SS-1 encoded in a plasmid, and the T4SS-2 encoded in chromosome 2 [91]. Until now, only the T4SS-1 was identified in *B. cenocepacia* strains as necessary for virulence in onions and intracellular survival in phagocytes [92].

In a mouse agar bead infection model, the T3SS has been shown to be important for bacterial pathogenesis [93]. Although the precise mechanism is still not clear, T3SS seems to play no role in intracellular survival of *B. cenocepacia* [94].

Four type V secretion systems are encoded within the genome of *B. cenocepacia* J2315 [49]. Proteins transported by this type of transporters contain pertactin and hemagglutinin domains and are thought to play a role in bacterial adhesion [49].

*B. cenocepacia* also encodes a T6SS, which was shown to affect the actin cytoskeleton of macrophages and the assembly of the reduced nicotinamide adenine dinucelotide phosphate (NADPH) oxidase complex in *B. cepacia-*containing vacuoles (BcCV's) by inactivation of Rac1 and Cdc42 [73, 95, 96]. *B. cenocepacia* was found to efficiently activate the inflammasome by a yet uncharacterized T6SS effector [97]. Consequently, monocytes and THP-1 cells release IL-1β in a pyrin-, Asc-, and T6SS-dependent manner [97]. The T6SS also enhances caspase-1 activation, negatively regulated by the sensor kinase-response regulatorAtsR [73]. In addition, a recent paper suggests that the T6SS might be important for the secretion of T2SS effectors into the host cytoplasm, such as ZmpA and ZmpB, revealing an unanticipated role for type II secretion systems in intracellular survival and replication of *B. cenocepacia* [96]. Although membrane vesicles cannot be considered a canonical secretion system, they can effectively allow the secretion of several hydrolytic enzymes and toxins [98]. **Table 2** summarizes and compares the most relevant information available about secretion systems of Bcc bacteria and their counterparts in the major CF pathogen *P. aeruginosa*.

#### **3.6. Iron uptake**

glycosyltransferase, Cepacian is not produced by the *B. cenocepacia* ET12 representative strain

Bcc bacteria were found to persist in biofilms *in vitro*. Biofilm formation and maturation depend on many factors, including EPS production, motility, iron availability, and multiple gene regulatory systems, such as quorum sensing, alternative sigma factors, or global regulators such as the ShvR and AtsR [45, 58, 68–73]. In addition, Bcc can form small colony variants *in vitro*, a colony morphology that is associated with enhanced biofilm formation,

Several studies have been performed to understand the importance and relevance of biofilm formation in Bcc biology. Bcc bacteria growing in biofilms are usually more tolerant to multiple antibiotics, although similar susceptibilities were reported for plancktonic and biofilm cells to the antibiotics kanamycin, amikacin, and ciprofloxacin [75, 76]. Recently, Bcc biofilms were shown to contain persister cells that are able to survive in the presence of high concentrations of antibiotics by avoiding production ofreactive oxygen species [77]. In addition, using neutrophil-like dHL60 cells, it was shown that the presence of these immune system cells enhanced biofilm formation that protected Bcc bacteria against neutrophils by inducing their necrosis, acting as a barrier to the migration of neutrophils, and masking the bacteria from being recognized by neutrophils [78]. Although some evidence suggests that biofilm formation plays a

role in bacterial persistence in the CF airways, this topic needs to be further studied.

Quorum sensing is a mode of regulation of gene expression that is dependent on the density of the bacterial population. Bcc bacteria have at least four quorum sensing systems. The CepIR quorum sensing system is homologous to the LuxIR system of *Vibrio fischeri* (reviewed in Ref. [79]). The CepIR system positively regulates the virulence of *B. cenocepacia* toward model organisms like *C. elegans*, *Galleria mellonella*, rodents, zebrafish, alfalfa, and onions [80–83]. In addition to the CepIR, *B. cenocepacia* encodes the CciIR, the CepR2, and the BDSF quorum sensing systems [84, 85]. While the CepIR and CciR quorum sensing systems rely on acyl homoserine lactones as signaling molecules, the BDSF system uses cis-2-dodecenoic acid as the signaling molecule, and the CepR2 is an orphan quorum sensing system [85]. An arsenal of genes regulated by quorum sensing in Bcc bacteria was described, including the negatively regulated siderophore synthesis and the positively regulated expression of the genes encoding zinc metalloproteases (Zmps), swarming motility and biofilm formation, all thought to have an impact when the bacterium is infecting the CF patient [71, 80, 86, 87].

Both Gram-negative and positive bacteria use protein secretion systems to secrete toxins or other proteins, either directly into the environment or into host cells. These systems are

J2315 [49, 62].

78 Progress in Understanding Cystic Fibrosis

**3.3. Biofilms**

**3.4. Quorum sensing**

**3.5. Protein secretion systems**

antibiotic resistance, and persistence [74].

In order to carry out iron chelation and uptake, members of the Bcc can produce up to four distinct siderophores: ornibactin, pyochelin, cepabactin, and cepaciachelin [122]. Ornibactin appears to be the most important and abundant siderophore produced by *B. cenocepacia* strains [123, 124]. The pathways and regulatory mechanisms of ornibactin synthesis and uptake are relatively well known [87, 125–127]. The requirement of this siderophore for *B. cenocepacia* virulence was demonstrated in different infection models, including the rat agar bead, *G. mellonella,* and *C. elegans* [82, 125, 127].

The competition for available iron by Bcc bacteria and other CF lung colonizing organisms such as *P. aeruginosa* was reported to occur in the CF lung, although it is not completely clear how Bcc organisms acquire iron from host proteins [128, 129].


**Table 2.** Summary of secretion systems from Bcc and the respective counterparts from the CF major pathogen *P. aeruginosa*.

#### **3.7. Resistance to antimicrobials**

Difficulties in eradicating Bcc infections mainly result from their intrinsic resistance to multiple antibiotics, including polymyxins, aminoglycosides, and most β-lactams. In addition, these bacteria have the ability to develop *in vivo* resistance tovirtuallyall classesofantibiotics [20, 130, 131]. Antibiotics administration to CF patients was also reported to affect resistance profiles of Bcc bacteria [132]. Various mechanisms involved in the resistance of Bcc to multiple antibiotics have been described and include enzymatic inactivation (β-lactamases, aminoglycoside-inactivating enzymes, dihydrofolate reductase), alteration of drug targets, integrons, cell wall impermeability, and active efflux pumps [88, 133–140]. However, major contributions to intrinsic and acquired multidrug resistance by Bcc seem to be due to efflux pumps of the resistance nodulation cell division (RND) family. In fact, the *B. cenocepacia* J2315 genome encodes at least 16 efflux systems of the RND family [141]. At least six of these RND efflux pumps were implicated in drug resistance—RND-1, RND-3, RND-4, RND-8, RND-9, and RND-10 [138–140, 142, 143]. RND-3 and RND-4 efflux pumps were described as being involved in the resistance to various antimicrobial drugs including tobramycin and ciprofloxacin; the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin; the RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance; and RND-10 efflux pump seems to confer resistance to chloramphenicol, fluoroquinolones, and trimethoprim [140, 143]. It was suggested that mutations in the RND-3 regulator-encoding gene may be responsible for the prevalent overexpression of this efflux pump in clinical Bcc isolates, contributing to their high levels of antibiotics resistance [144].

## **3.8. Motility**

Genes involved in the synthesis and assembly of *B. cenocepacia* flagella are located in chromosome I, distributed within five clusters, with two additional genes found on chromosomes 2 and 3 [49]. These genes were found as being upregulated when the organism was incubated in CF sputum, contributing to its virulence in a murine agar bead infection model [145, 146]. More recently, flagellin expression and flagellar morphology of *B. cenocepacia* grown in a medium mimicking the CF sputum was analyzed [147]. Those nutritional conditions led to increased motility and flagellin expression, by inducing the synthesis of multiple flagella on the cell surface of *B. cenocepacia* K56-2 [147]. A link between the loss of bacterial motility and the development of the cepacia syndrome was recently established based on a transcriptomics analysis comparing the *B. cenocepacia* ST32 CF isolates recovered from bloodstream, at the time of cepacia syndrome, with their sputum counterparts, recovered prior to the development of this syndrome, revealing that flagellar genes were downregulated in isolates recovered from the bloodstream [148].

#### **3.9. Intracellular survival**

**3.7. Resistance to antimicrobials**

Difficulties in eradicating Bcc infections mainly result from their intrinsic resistance to multiple antibiotics, including polymyxins, aminoglycosides, and most β-lactams. In addition, these bacteria have the ability to develop *in vivo* resistance tovirtuallyall classesofantibiotics [20, 130, 131]. Antibiotics administration to CF patients was also reported to affect resistance profiles of Bcc bacteria [132]. Various mechanisms involved in the resistance of Bcc to multiple antibiotics have been described and include enzymatic inactivation (β-lactamases, aminoglycoside-inactivating enzymes, dihydrofolate reductase), alteration of drug targets, integrons, cell wall impermeability, and active efflux pumps [88, 133–140]. However, major contributions to intrinsic and acquired multidrug resistance by Bcc seem to be due to efflux pumps of the resistance nodulation cell division (RND) family. In fact, the *B. cenocepacia* J2315 genome encodes at least 16 efflux systems of the RND family [141]. At least six of these RND efflux pumps were implicated in drug resistance—RND-1, RND-3, RND-4, RND-8, RND-9, and RND-10 [138–140, 142,

**Table 2.** Summary of secretion systems from Bcc and the respective counterparts from the CF major pathogen *P. aeruginosa*.

**Secretion system** *Burkholderia cepacia* **complex** *P. aeruginosa*

T3SS No effector described yet, plays a role

80 Progress in Understanding Cystic Fibrosis

[93, 94]

T4SS T4SS-1: Plant cytotoxic proteins, T4SS-2:

T5SS Four T5SS: two containing pertactin

Membrane vesicles (MV) MV-associated (metallo)proteases,

Plasmid mobilization [91]

T1SS Hemolytic proteins [88, 89] HasAp (heme-binding) [99]; AprA and

T2SS ZmpA and ZmpB [80, 90] LasB (Major extracellular protease) [102],

in evasion of the host immune system

domains involved in adhesion, other two contain haemagglutinin repeats [49]

T6SS Hcp and VgrGs [73, 95, 96] Hcp and VgrGs [119, 120]

(phospho)lipases, peptidoglycandegrading enzymes [98]

AprX (alkaline proteases) [100, 101]

GTPase-activator ExoS and ADPribosyltransferase ExoT [109], adenylate cyclase ExoY [110], phospholipase A2

Integrative and conjugative elements (ICEs): ICE*clc* [112], Pathogenicity islands: pKLC102 (includes the type IV sex piliencoding pil cluster and the *chvB* gene encoding a virulence factor) [113], and PAP-I (includes several virulence factors, such as CupD type fimbriae, and the PvrSR/RcsCB regulatory system) [114]

Autotransporter: EstA (esterase activity) [115]; Two-partner secretion systems LepA/LepB [116] and CupB [117], and the

Multiple virulence factors: Alkaline phosphatase, hemolytic phospholipase C; the Cif toxin that inhibits CFTR-mediated chloride secretion in the airways [121]

ExoU and ExoS [111]

PdtA/PdtB system [118]

Staphylolysin LasA [102], Aminopeptidase PaAP [103], Protease IV [104], Lipases LipA, LipC, phospholipase C, PlcH, and PlcN [105, 106], CbpD Chitin-binding protein CbpD [107]; Exotoxin A [108]

> Infection assays using free-living amoeba demonstrated that *B. cenocepacia* can survive in an acidified intracellular compartment [94, 149]. These bacteria were also demonstrated to have the ability to delay the maturation of phagolysosomes in murine macrophages [94–96, 150]. Although the *B. cenocepacia* containing vacuoles (BcCVs) progress normally to the early phagosomal stage, the fusion of the BcCV's with late endosomes and subsequent maturation is significantly delayed comparing with vacuoles containing heat-killed bacteria [94]. In contrast to heat-killed bacteria that ended up in phagolysosomes with a pH of 4.5, BcCVs did not acidify normally maintaining a luminal pH around 6.4 [94]. This ability of *B. cenocepacia* to alter the acidification of the vacuole seems to be correlated with the delay in recruitment or assembly on the BcCV membrane of both the 16-kDa subunit of the phagosomal vacuolar ATPase (vATPase) and the NADPH phagocyte oxidase [96, 151]. In contrast, Al-Khodor and colleagues demonstrated that *B. cenocepacia* J2315 only transiently interacts with the endocytic pathway, event after which the bacterium is able to rapidly escape to the cytosol [152]. Escaped bacteria are afterward targeted by the host autophagy pathway, through the recruitment to the bacterial vicinity of the ubiquitin conjugation system, the autophagy adaptors p62 and NDP52, and the autophagosome membrane-associated protein LC3B. However, apparently, this host cell control through autophagy ultimately fails in a high proportion of infected cells,

being *B. cenocepacia* able to block the autophagosome completion and replicate in the cytosol of the host cell [152].

To better understand the intracellular behavior of *B. cenocepacia* in CF infected patients, studies have also been performed in Cystic fibrosis transmembrane conductance regulator (CFTR) defective macrophages. Remarkably, the delayed maturation arresting of BcCV's is more exaggerated in CFTR-defective macrophages than in normal macrophages and is specific to live *B. cenocepacia* [153]. Although it is not clear how the CFTR defect enhances the *B. cenocepacia* intracellular survival, there is evidence of a link between the defective CFTR with autophagy deficiency and decreased clearance of protein aggregates and inflammation [154]. The elucidation of these survival details, especially the ability of *B. cenocepacia* to synergize with the CFTR defect and its consequences on the mechanism of autophagy will provide new avenues to explore novel therapeutic approaches for CF patients [155].

## **4. Toward a vaccine to prevent Bcc infections**

No objective guidelines for eradication strategies are available for Bcc infections, as these pathogens are intrinsically resistant to the majority of the clinical available antimicrobials [156]. Currently, no immunotherapeutic strategy to protect CF patients from Bcc infections is available. Several studies on the immune response elicited by Bcc species in CF patients have been performed; however, they are challenging due to the ability of this bacteria to modulate and overcome the host immune responses and the ability to survive intracellularly in phagocytes and epithelial cells [157, 158].

An important aspect to consider during vaccine design is the optimal balance of Th1 and Th2 responses required for effective pathogen clearance. For example, a Th1 bias elicits a cell-mediated response, while Th2 induces a humoral immune response [159]. In the case of CF, their immune phenotype appears to be skewed toward Th2 responses [160]. In the case of Bcc, the type of host response necessary to clear the pathogen is still not fully understood, making it difficult to develop a protective vaccine (**Table 3**). Recently, BALB/c mice immunized intraperitoneally with the proteins Linocin and OmpW showed a significant reduction of *B. cenocepacia* and *B. multivorans* cells in the lung and lower dissemination of bacteria to the spleen [161]. While Linocin led to a robust Th1 response, the OmpW led to a mixed Th1/Th2 response [161]. The protection achieved with these proteins was greater against *B. cenocepacia* infection, and OmpW immunization was more efficient in reducing the lung bacterial load [161].

Nonpurified outer membrane proteins (OMP) from *B. multivorans*, supplemented with the mucosal adjuvant adamantylamide dipeptide (AdDP) that promotes a robust Th2 response, were tested for immunization of BALB/c mice [162]. A statistically significant increase in IgG and in mucosal IgA OMP-specific antibodies was observed, together with a reduction of *B. multivorans* burden and lung pathology, but only a moderate cross protection to *B. cenocepacia* was reported. The specificity of the immune response was found to be against 90, 72, 66, and 60 kDa proteins. Elicitation of specific IgA antibodies by mucosal immunization was also reported to be important to prevent the colonization of the respiratory tract by Bcc bacteria. In another study, the intranasal immunization of CD-1 mice with outer membrane proteins (OMP) from *B. cenocepacia* was described to originate a Th2-biased response with the maintenance of the bacterial burden, while mice immunized with OMP and the noninflammatory mucosal adjuvant nanoemulsion (NE) elicited a Th1/Th2-balanced response that led to a significant reduction of the *B. cenocepacia* cell burden [163]. The serum derived from mice vaccinated with OMP-NE could also inhibit *B. multivorans* growth by 80.1%, showing that induction of cross-reactive antibodies occurred after mice immunization. Additionally, a highly conserved 17-kDa OmpA-like protein was recently identified as a new immunedominant epitope in mucosal immunization [163].

being *B. cenocepacia* able to block the autophagosome completion and replicate in the cytosol

To better understand the intracellular behavior of *B. cenocepacia* in CF infected patients, studies have also been performed in Cystic fibrosis transmembrane conductance regulator (CFTR) defective macrophages. Remarkably, the delayed maturation arresting of BcCV's is more exaggerated in CFTR-defective macrophages than in normal macrophages and is specific to live *B. cenocepacia* [153]. Although it is not clear how the CFTR defect enhances the *B. cenocepacia* intracellular survival, there is evidence of a link between the defective CFTR with autophagy deficiency and decreased clearance of protein aggregates and inflammation [154]. The elucidation of these survival details, especially the ability of *B. cenocepacia* to synergize with the CFTR defect and its consequences on the mechanism of autophagy will provide new

No objective guidelines for eradication strategies are available for Bcc infections, as these pathogens are intrinsically resistant to the majority of the clinical available antimicrobials [156]. Currently, no immunotherapeutic strategy to protect CF patients from Bcc infections is available. Several studies on the immune response elicited by Bcc species in CF patients have been performed; however, they are challenging due to the ability of this bacteria to modulate and overcome the host immune responses and the ability to survive intracellularly

An important aspect to consider during vaccine design is the optimal balance of Th1 and Th2 responses required for effective pathogen clearance. For example, a Th1 bias elicits a cell-mediated response, while Th2 induces a humoral immune response [159]. In the case of CF, their immune phenotype appears to be skewed toward Th2 responses [160]. In the case of Bcc, the type of host response necessary to clear the pathogen is still not fully understood, making it difficult to develop a protective vaccine (**Table 3**). Recently, BALB/c mice immunized intraperitoneally with the proteins Linocin and OmpW showed a significant reduction of *B. cenocepacia* and *B. multivorans* cells in the lung and lower dissemination of bacteria to the spleen [161]. While Linocin led to a robust Th1 response, the OmpW led to a mixed Th1/Th2 response [161]. The protection achieved with these proteins was greater against *B. cenocepacia* infection, and OmpW immunization was more efficient in reducing the

Nonpurified outer membrane proteins (OMP) from *B. multivorans*, supplemented with the mucosal adjuvant adamantylamide dipeptide (AdDP) that promotes a robust Th2 response, were tested for immunization of BALB/c mice [162]. A statistically significant increase in IgG and in mucosal IgA OMP-specific antibodies was observed, together with a reduction of *B. multivorans* burden and lung pathology, but only a moderate cross protection to *B. cenocepacia* was reported. The specificity of the immune response was found to be against

avenues to explore novel therapeutic approaches for CF patients [155].

**4. Toward a vaccine to prevent Bcc infections**

in phagocytes and epithelial cells [157, 158].

lung bacterial load [161].

of the host cell [152].

82 Progress in Understanding Cystic Fibrosis

Metalloproteases are also considered as potential effective candidates for vaccine development [90]. It was demonstrated that immunizations of rats using a conserved zinc metalloprotease peptide 15 (PSCP) decreased the severity of *B. cenocepacia* infection and the lung damage was reduced by 50% upon challenge with a *B. cenocepacia* strain after immunization [90].

In 2012, it was shown that the bacterial surface polysaccharide poly-β-(1-6)-N-acetyl-glucosamine (PNAG) confers protective immunity against Bcc infection in a lethal peritonitis mice model [164]. In this study by Skurnik and colleagues using opsophagocytic assays, it was observed that goat-raised antibodies against PNAG could kill Bcc strains (>80%) of the *B. ceno-*


**Table 3.** Summary of vaccine development against Bcc infections.

*cepacia*, *Burkholderia dolosa* and *B. multivorans* species. Furthermore, bacterial killing was found to depend of the presence of the complement [164].

Other proteins of putative immunogenic activity have been reported as potential vaccine candidates. However, studies in a Bcc infection animal model are still lacking (**Table 3**). One of these promising antigens is the OmpA-like BCAL2958 protein that was shown to be highly conserved in Bcc, to elicit IgG antibodies in CF patients and to elicit an increase of TNFα, elastase, NO, and MPO in neutrophils [166].

Mussonandcolleagueshave shownthatT-cellhybridomas againstthe*Burkholderia pseudomallei* flagellarproteinFliCepitope cross-reactedwithorthologousFliCsequences from*B. multivorans* and *B. cenocepacia* [165]. FliC epitopes were accessible for processing and presentation from live or heat-killed *B. cenocepacia* bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with *B. cenocepacia*.

Studies referred above revealed that subunit vaccines that only produce an antibody response cannot fully prevent an infection caused by Bcc bacteria [157, 161, 164]. Therefore, Bcc vaccines containing multiple antigens that elicit a balanced Th1 and Th2 response are expected to be effective in preventing Bcc infections. With this aim, immunoproteomics approaches have been performed. For instance, Mariappan and colleagues identified 18 immunogenic proteins from culture supernatants of *B. cepacia* that reacted with mice antibodies raised against inactivated *B. cepacia* whole cells [167]. More recently, the analysis of the imunoproteome of two clinical relevant strains of *B. cenocepacia* and *B. multivorans* revealed 15 common immunoreactive proteins that reacted with CF human serum samples [168].

## **5. Concluding remarks**

An overview of Bcc infections in CF from early 1980s until the more recent available data was presented. The prevalence of Bcc species in CF patients worldwide is still evolving, most probably as a result of infection control measures and segregation policies. Many virulence factors have been identified, and the resulting wealth of information prompted the establishment of new research lines envisaging the development of novel protective strategies and products, namely vaccines and vaccine components.

## **Acknowledgements**

Funding received by iBB-Institute for Bioengineering and Biosciences from FCT-Portuguese Foundation for Science and Technology (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317) is acknowledged. This work was also partially funded by FCT through contract PTDC/BIA-MIC/1615/2014 and grants to SAS (SFRH/BPD/102006/2014), JRF (BL184), and TP (BL183).

## **Author details**

*cepacia*, *Burkholderia dolosa* and *B. multivorans* species. Furthermore, bacterial killing was found

Other proteins of putative immunogenic activity have been reported as potential vaccine candidates. However, studies in a Bcc infection animal model are still lacking (**Table 3**). One of these promising antigens is the OmpA-like BCAL2958 protein that was shown to be highly conserved in Bcc, to elicit IgG antibodies in CF patients and to elicit an increase of TNFα,

Mussonandcolleagueshave shownthatT-cellhybridomas againstthe*Burkholderia pseudomallei* flagellarproteinFliCepitope cross-reactedwithorthologousFliCsequences from*B. multivorans* and *B. cenocepacia* [165]. FliC epitopes were accessible for processing and presentation from live or heat-killed *B. cenocepacia* bacteria, demonstrating that flagellin enters the HLA class II

Studies referred above revealed that subunit vaccines that only produce an antibody response cannot fully prevent an infection caused by Bcc bacteria [157, 161, 164]. Therefore, Bcc vaccines containing multiple antigens that elicit a balanced Th1 and Th2 response are expected to be effective in preventing Bcc infections. With this aim, immunoproteomics approaches have been performed. For instance, Mariappan and colleagues identified 18 immunogenic proteins from culture supernatants of *B. cepacia* that reacted with mice antibodies raised against inactivated *B. cepacia* whole cells [167]. More recently, the analysis of the imunoproteome of two clinical relevant strains of *B. cenocepacia* and *B. multivorans* revealed 15 common

An overview of Bcc infections in CF from early 1980s until the more recent available data was presented. The prevalence of Bcc species in CF patients worldwide is still evolving, most probably as a result of infection control measures and segregation policies. Many virulence factors have been identified, and the resulting wealth of information prompted the establishment of new research lines envisaging the development of novel protective strategies and products,

Funding received by iBB-Institute for Bioengineering and Biosciences from FCT-Portuguese Foundation for Science and Technology (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317) is acknowledged. This work was also partially funded by FCT through contract PTDC/BIA-MIC/1615/2014 and grants to SAS

Ag presentation pathway during infection of macrophages with *B. cenocepacia*.

immunoreactive proteins that reacted with CF human serum samples [168].

to depend of the presence of the complement [164].

elastase, NO, and MPO in neutrophils [166].

84 Progress in Understanding Cystic Fibrosis

**5. Concluding remarks**

**Acknowledgements**

namely vaccines and vaccine components.

(SFRH/BPD/102006/2014), JRF (BL184), and TP (BL183).

Jorge H. Leitão\*, Joana R. Feliciano, Sílvia A. Sousa, Tiago Pita and Soraia I. Guerreiro

\*Address all correspondence to: jorgeleitao@tecnico.ulisboa.pt

Departamento de Bioengenharia, Instituto Superior Técnico (IST), Universidade de Lisboa, iBB – Institute for Bioengineering and Biosciences, Lisboa, Portugal

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## *Pseudomonas aeruginosa* **Extracellular Secreted Molecules Have a Dominant Role in Biofilm Development and Bacterial Virulence in Cystic Fibrosis Lung Infections** *Pseudomonas aeruginosa* **Extracellular Secreted Molecules Have a Dominant Role in Biofilm Development and Bacterial Virulence in Cystic Fibrosis Lung Infections**

Theerthankar Das and Jim Manos Theerthankar Das and Jim Manos

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66308

#### **Abstract**

Cystic fibrosis (CF) is a genetic disorder that predominantly affects Caucasian populations. *Pseudomonas aeruginosa* is the most important Gram‐negative pathogen that persists in CF patients' lungs. By evading host defence mechanisms and persisting, it is ultimately responsible for the morbidity and mortality of about 80% of CF patients worldwide. *P. aeruginosa* is also responsible for infections in burns, wounds, eyes, nosocomial patients and HIV patients. Prevalence and progression of infection by *P. aeruginosa* in the host is dependent on secretion of numerous extracellular molecules such as polysaccharides, proteases, eDNA, pyocyanin and pyoverdine. These molecules have multiple roles in facilitating *P. aeruginosa* colonisation and virulence. Pyocyanin is one of the major factors dictating progression of infection and biofilm formation. Pyocyanin is a potent virulence factor causing host cell death in CF patients. In this chapter, we have outlined the roles of various extracellular molecules secreted by *P. aeruginosa* and specifically focused on the role of pyocyanin in inducing eDNA production, binding to eDNA via intercalation and facilitating biofilm promoting factors, whilst inducing oxidative stress to host cells via production of reactive oxygen species. In line with this, we have described the current challenges in treatment of CF infections and the development of new strategies to control *P. aeruginosa* infections.

**Keywords:** *Pseudomonas aeruginosa*, polysaccharides, protease, pyoverdine, pyocyanin, eDNA, glutathione, biofilm

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. © 2017 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.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

## **1. Introduction**

Cystic fibrosis (CF) is a genetic disorder whose effects are felt from birth. It predominantly affects Caucasian populations; however, it is also present in non‐Caucasians [1]. The preva‐ lence of CF varies around the globe; however, extensive evidence suggests that in the USA, Canada, Australia, New Zealand and European countries the ratio of newborns with CF is 1:2000–3000 [2]. CF is induced by mutations (amino acid deletions/substitutions) in the cystic fibrosis transmembrane conductance regulator (CFTR), with a loss of the phenylalanine at position 508 (∆F508) leading to the most severe outcome. The dysfunctional CFTR leads to greatly reduced transport of ions across epithelial cells and membranes, resulting in dehy‐ dration of the mucus in the host respiratory tract/lungs and the digestive pathway, reduced mucus clearance and severe breathing problems [1, 2]. The slow‐moving mucous facilitates the growth of microbes, including potentially life‐threatening bacteria such as *Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae* and *Burkholderia cenocepacia*, as well as fungal species such as *Candida*, *Aspergillus* and *Malassezia* spp. and viruses [3]. Chronic microbial infections and concomitant airway inflammation induced by the bacterial are primarily responsible for respiratory failure in about 95% of CF patients [1]. In spite of intensive antibiotic therapy and other associated therapy (chest physical therapy, pure oxygen therapy) and finally lung transplantation to combat the effects of CF, the mean life expectancy of CF sufferers is still shorter than that of non‐CF people, ranging between 35 and 50 years [2].

*P. aeruginosa* is the most important Gram‐negative pathogen that persists in CF patients' lungs, and this persistence is achieved primarily by evading host defence mechanisms through a shutdown of potential trigger genes. *P. aeruginosa* is ultimately responsible for the morbidity and mortality of about 80% of CF patients worldwide [2]. Clinical research has shown that during a CF patient's infancy and childhood more infections are caused by *S. aureus* and *H. influenzae*, whereas in adulthood, the severity of infection is accelerated by *P. aeruginosa* colonisation [4]. *P. aeruginosa* is the most prevalent Gram‐negative pathogen in CF patients' lungs by adolescence, by which time the strains isolated from patients are usually multidrug resistant. Evidence suggests that *P. aeruginosa* and its associated infections are more persistent and dominant in CF patients aged over 18 years (91%) than in patients less than 18 years (39%) [5]. In addition to CF‐related infections, *P. aeruginosa* is also primarily responsible for airway infections in bronchiectasis, infection of burns and wounds, surgery‐associated infections, eye infections due to contact lens contamination and nosocomial infections such as pneumonia and urinary tract infections in the immunocompromised [6]. In CF and bronchiectasis patients, *P. aeruginosa* infection results in chronic airway inflammation, lung tissue damage, declining lung function, respiratory failure and premature death [1, 6].

Persistence of bacterial infections in the host is due to the bacterium's ability to form biofilms via secretion of numerous extracellular biopolymers, collectively known as extracellular polymeric substances (EPS) and small molecules [7, 8]. Different extracellular biopolymers and small molecules conjugate with each other through physico‐chemical interactions to form a highly complexed and structurally integrated matrix [7]. This matrix represents a critical interface between bacterial cells and the host or its environment. Extracellular biopolymers (EPSs) play a primary role in immobilising planktonic cells (cell adhesion) and cell‐cell communication (aggregation), leading to colonisation and biofilm formation on both biotic and abiotic surfaces. It also provides bacterial cells/biofilms with inherent protection against physical stress, traditional antibiotic therapy and host immune defences, thus making eradication extremely difficult [7, 9]. Potentially all biopolymers (e.g. proteins, polysacchar‐ ides, eDNA) in EPS serve as an excellent source of nutrients and specifically eDNA promotes horizontal gene transfer between cells within the biofilm [7].

**1. Introduction**

102 Progress in Understanding Cystic Fibrosis

Cystic fibrosis (CF) is a genetic disorder whose effects are felt from birth. It predominantly affects Caucasian populations; however, it is also present in non‐Caucasians [1]. The preva‐ lence of CF varies around the globe; however, extensive evidence suggests that in the USA, Canada, Australia, New Zealand and European countries the ratio of newborns with CF is 1:2000–3000 [2]. CF is induced by mutations (amino acid deletions/substitutions) in the cystic fibrosis transmembrane conductance regulator (CFTR), with a loss of the phenylalanine at position 508 (∆F508) leading to the most severe outcome. The dysfunctional CFTR leads to greatly reduced transport of ions across epithelial cells and membranes, resulting in dehy‐ dration of the mucus in the host respiratory tract/lungs and the digestive pathway, reduced mucus clearance and severe breathing problems [1, 2]. The slow‐moving mucous facilitates the growth of microbes, including potentially life‐threatening bacteria such as *Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae* and *Burkholderia cenocepacia*, as well as fungal species such as *Candida*, *Aspergillus* and *Malassezia* spp. and viruses [3]. Chronic microbial infections and concomitant airway inflammation induced by the bacterial are primarily responsible for respiratory failure in about 95% of CF patients [1]. In spite of intensive antibiotic therapy and other associated therapy (chest physical therapy, pure oxygen therapy) and finally lung transplantation to combat the effects of CF, the mean life expectancy of CF sufferers is still shorter than that of non‐CF people, ranging between 35 and 50 years [2].

*P. aeruginosa* is the most important Gram‐negative pathogen that persists in CF patients' lungs, and this persistence is achieved primarily by evading host defence mechanisms through a shutdown of potential trigger genes. *P. aeruginosa* is ultimately responsible for the morbidity and mortality of about 80% of CF patients worldwide [2]. Clinical research has shown that during a CF patient's infancy and childhood more infections are caused by *S. aureus* and *H. influenzae*, whereas in adulthood, the severity of infection is accelerated by *P. aeruginosa* colonisation [4]. *P. aeruginosa* is the most prevalent Gram‐negative pathogen in CF patients' lungs by adolescence, by which time the strains isolated from patients are usually multidrug resistant. Evidence suggests that *P. aeruginosa* and its associated infections are more persistent and dominant in CF patients aged over 18 years (91%) than in patients less than 18 years (39%) [5]. In addition to CF‐related infections, *P. aeruginosa* is also primarily responsible for airway infections in bronchiectasis, infection of burns and wounds, surgery‐associated infections, eye infections due to contact lens contamination and nosocomial infections such as pneumonia and urinary tract infections in the immunocompromised [6]. In CF and bronchiectasis patients, *P. aeruginosa* infection results in chronic airway inflammation, lung tissue damage, declining

Persistence of bacterial infections in the host is due to the bacterium's ability to form biofilms via secretion of numerous extracellular biopolymers, collectively known as extracellular polymeric substances (EPS) and small molecules [7, 8]. Different extracellular biopolymers and small molecules conjugate with each other through physico‐chemical interactions to form a highly complexed and structurally integrated matrix [7]. This matrix represents a critical interface between bacterial cells and the host or its environment. Extracellular biopolymers

lung function, respiratory failure and premature death [1, 6].

*P. aeruginosa* EPS plays multiple roles in bacterial adhesion, colonisation, biofilm formation and pathogenesis of *P. aeruginosa* infections [7]. EPS primarily consists of bio‐polymers such as polysaccharides (alginate, lipopolysaccharides), proteins (protease, elastase), nucleic acids such as extracellular DNA (eDNA) and RNA, and small molecules such as siderophores and metabolites (phenazines/pyocyanin) [8, 9]. Secretion of EPS and metabolites (phenazines) by *P. aeruginosa* is regulated by the quorum sensing (QS) system. With QS, bacterial cells com‐ municate with each other via small molecules comprising N‐acyl homoserine lactones (AHL) and the *Pseudomonas* quinolone signal (PQS). These AHL and PQS promote *P. aeruginosa* biofilm formation through activation of numerous genes expressing extracellular molecules at different stages of the bacterial growth phase [7, 8], with roles in virulence and biofilm development (**Figure 1**).

**Figure 1.** Schematic diagram showing quorum‐sensing‐mediated production of various extracellular molecules (poly‐ saccharides, protease, pyoverdine, eDNA, pyocyanin) by *P. aeruginosa* and their potential roles in biofilm development and virulence.

Of the many extracellular molecules secreted by *P. aeruginosa*, phenazine‐pyocyanin stands out as a molecule that has numerous functions including assistance in growth and multiplication of the cell population, biofilm promotion and virulence. Pyocyanin is a small metabolite with oxidant properties that act as a virulence factor by producing reactive oxygen species (ROS) and generating oxidative stress in the host [10]. Pyocyanin is also a key metabolite in strength‐ ening the e‐DNA backbone of the *P. aeruginosa* biofilm [10]. The major focus of this chapter will be on pyocyanin in its role as a *P. aeruginosa* virulence factor. This will involve a review of the literature in the field as well as our work in understanding pyocyanin's role in strengthening the *P. aeruginosa* biofilm and inducing virulence in the host. In addition, we will briefly review the role of other essential molecules such as polysaccharides, protease, e‐DNA and pyoverdine, secreted by *P. aeruginosa* in establishment of the biofilm and progression of infection. This chapter will also address various developments in therapeutic treatment that involves these extracellular metabolites and biopolymers, and our development of new approach disrupts *P. aeruginosa* biofilms in vivo using a combined antioxidant/DNase‐I/antibiotic approach.

## **2. Role of** *P. aeruginosa* **secreted extracellular molecules in development of biofilm and pathogenesis**

## **2.1. Polysaccharides**

Alginate (capsular polysaccharide) is acknowledged as a virulence factor responsible for mucoidal *P. aeruginosa* infection in CF lung [11]. Transformation from initial non‐mucoid *P. aeruginosa* colonies occurs after a mutation in the negative regulator of mucoidy, *mucA*, leads to expression of the alginate biosynthesis operon [12] and extracellular secretion of alginate, the basis of the robust mucoid phenotype. Alginate is also partly responsible for the pathoge‐ nicity of *P. aeruginosa* infection and has been shown to enhance the resistance of biofilms against antibiotics and the host immune response, by scavenging reactive oxygen species (ROS) released by host immune cells [13, 14]. In line with this, studies have shown that mucoid *P. aeruginosa* biofilms treated with alginate lyase demonstrated enhanced efficacy to antibiotic treatment [15]. However, evidence suggests that alginate is not essential for *P. aeruginosa* biofilm development since *P. aeruginosa* wild‐type alginate‐producing and alginate deficient strains form morphologically and structurally similar biofilms [16].

Other polysaccharides that are essential and partly associated with biofilm formation include Psl and Pel (coded by the *psl* and *pel* gene clusters, respectively) [16]. Interestingly, studies show that *P. aeruginosa* laboratory strains that do not produce detectable amounts of alginate (UCBPP‐PA14 (PA14) and PAO1), still form robust biofilms through expression of Psl, indicat‐ ing that biofilm formation is independent of alginate production [16]. Psl and Pel polysacchar‐ ides are distinct biochemically and play different roles in the establishment of *P. aeruginosa* biofilms. Psl is a mannose and galactose‐rich polysaccharide and is essential for initiation of *P. aeruginosa* cell surface adhesion and aggregation (cell‐cell interactions) and maintenance of the structural integrity of established biofilms [17]. In respect to the host, Psl plays a significant role in initiating *P. aeruginosa* adhesion to mucin‐coated surfaces, airway epithelial cells and biotic surfaces, thus triggering colonisation of CF lung [18]. Pel is a glucose‐rich matrix poly‐ saccharide that is essential for pellicle formation and biofilm structure in *P. aeruginosa* [11]. Studies with Pel‐deficient mutants concluded that Pel only influences morphological changes in *P. aeruginosa* colonies and does not influence biofilm initiation [19].

#### **2.2. Proteases**

*P. aeruginosa* secretes several protease enzymes identified as important virulence factors, such as alkaline protease (AP), elastase (Ela) B, elastase A (LasA protease), toxin A, phospholipase C and protease IV [20, 21]. Through their activity, these proteases contribute to the pulmonary damage seen in CF patients [21]. Interestingly, studies have shown that both environmental (soil and water) and clinical *P. aeruginosa* isolates produce similar concentrations of toxin A, phospholipase C, AP and Ela and have similar levels of elastolytic activity [22]. Protease production in *P. aeruginosa* is triggered through the QS system and numerous genes including *lasA* (elastase A/LasA protease), *lasB* (elastase B), *piv* (protease IV) and the *apr* (alkaline protease) operon are involved [23]. A significant amount of AP, Ela and protease IV has been detected in bronchial secretions from the lungs of CF patients [23]. These bacterial proteases can significantly influence a broad range of biological functions including the infection process, by hydrolysing peptide bonds and degrading proteins essential for basic biological functions in the host. They are also active against the host's humoral immunity system [23]. For example, AP and Ela cleave the major human immunoglobulins IgA and IgG in the respiratory tract [24]. In infected CF lung, protease has been shown to induce a severe inflammatory response, with increased interleukin‐8 (IL‐8) and interleukin‐6 (IL‐6) cytokine levels in the airways [25]. *P. aeruginosa* protease secretions in infected burn and wounds patients have been shown to induce sepsis, leading to an increased mortality rate [26]. However, the effectiveness of proteases is limited, as studies have shown that chronically infected CF patients produce specific antibodies against proteases and that these antibodies provide a defensive mechanism for the host by inhibiting protease‐mediated cleavage of secretory immunoglobulins [27].

*P. aeruginosa‐*secreted elastase B degrades human elastin, and over time, the decreased levels of elastin and increased levels of collagen in lung tissue result in lung fibrosis [25]. Elastase A cleaves glycine‐containing proteins and interestingly influences the activity of several other host elastolytic proteases, including human leukocyte elastase, human neutrophil elastase [28]. *P. aeruginosa* protease IV potentially cleaves IgG and fibrinogen (required for blood clotting). Low levels of fibrinogen lead to haemorrhaging, which is a characteristic of *P. aeruginosa* CF infection [29, 30]. In vitro studies demonstrated that secretion of *P. aeruginosa* proteases is significantly affected by antibiotic (ciprofloxacin) treatment [31]. Biofilms of *P. aeruginosa* PA1159 and PA1230 when treated with 64 μg/ml ciprofloxacin(twice the minimum inhibitory concentration (MIC)) showed up to a 65% decrease in total proteolytic activity [31]. However, the remaining *P. aeruginosa* population displayed increased resistance to ciprofloxacin com‐ pared to their planktonic counterparts when grown in fresh medium [31].

## **2.3. Pyoverdine**

the *P. aeruginosa* biofilm and inducing virulence in the host. In addition, we will briefly review the role of other essential molecules such as polysaccharides, protease, e‐DNA and pyoverdine, secreted by *P. aeruginosa* in establishment of the biofilm and progression of infection. This chapter will also address various developments in therapeutic treatment that involves these extracellular metabolites and biopolymers, and our development of new approach disrupts *P. aeruginosa* biofilms in vivo using a combined antioxidant/DNase‐I/antibiotic approach.

**2. Role of** *P. aeruginosa* **secreted extracellular molecules in development of**

Alginate (capsular polysaccharide) is acknowledged as a virulence factor responsible for mucoidal *P. aeruginosa* infection in CF lung [11]. Transformation from initial non‐mucoid *P. aeruginosa* colonies occurs after a mutation in the negative regulator of mucoidy, *mucA*, leads to expression of the alginate biosynthesis operon [12] and extracellular secretion of alginate, the basis of the robust mucoid phenotype. Alginate is also partly responsible for the pathoge‐ nicity of *P. aeruginosa* infection and has been shown to enhance the resistance of biofilms against antibiotics and the host immune response, by scavenging reactive oxygen species (ROS) released by host immune cells [13, 14]. In line with this, studies have shown that mucoid *P. aeruginosa* biofilms treated with alginate lyase demonstrated enhanced efficacy to antibiotic treatment [15]. However, evidence suggests that alginate is not essential for *P. aeruginosa* biofilm development since *P. aeruginosa* wild‐type alginate‐producing and alginate deficient

Other polysaccharides that are essential and partly associated with biofilm formation include Psl and Pel (coded by the *psl* and *pel* gene clusters, respectively) [16]. Interestingly, studies show that *P. aeruginosa* laboratory strains that do not produce detectable amounts of alginate (UCBPP‐PA14 (PA14) and PAO1), still form robust biofilms through expression of Psl, indicat‐ ing that biofilm formation is independent of alginate production [16]. Psl and Pel polysacchar‐ ides are distinct biochemically and play different roles in the establishment of *P. aeruginosa* biofilms. Psl is a mannose and galactose‐rich polysaccharide and is essential for initiation of *P. aeruginosa* cell surface adhesion and aggregation (cell‐cell interactions) and maintenance of the structural integrity of established biofilms [17]. In respect to the host, Psl plays a significant role in initiating *P. aeruginosa* adhesion to mucin‐coated surfaces, airway epithelial cells and biotic surfaces, thus triggering colonisation of CF lung [18]. Pel is a glucose‐rich matrix poly‐ saccharide that is essential for pellicle formation and biofilm structure in *P. aeruginosa* [11]. Studies with Pel‐deficient mutants concluded that Pel only influences morphological changes

*P. aeruginosa* secretes several protease enzymes identified as important virulence factors, such as alkaline protease (AP), elastase (Ela) B, elastase A (LasA protease), toxin A, phospholipase

strains form morphologically and structurally similar biofilms [16].

in *P. aeruginosa* colonies and does not influence biofilm initiation [19].

**biofilm and pathogenesis**

104 Progress in Understanding Cystic Fibrosis

**2.1. Polysaccharides**

**2.2. Proteases**

Iron is an important cofactor required for bacterial metabolism, growth and survival and also essential for induction of infection in host by various pathogenic bacteria including *P. aerugi‐ nosa* [32]. Various iron‐binding proteins (a class of ferritin) secreted by mammalian systems reduce the bioavailability of free iron essential for progress of infection and growth by pathogens, thus ferritin acts as an innate immunity molecule against bacterial infection [32]. Under iron limitation conditions, bacteria secrete siderophores (iron‐chelating molecules) to acquire iron from the host [32]. *P. aeruginosa* secretes two types of siderophores: pyoverdine (the predominant siderophore) and pyochelin, with high and low affinity for Fe3+ ions, respectively [33, 34]. Pyoverdine production is encoded mainly bythe *pvc* gene cluster and pyochelin production by the *pch* gene cluster [35]. Pyoverdine is more efficient in releasing iron from human ferritin and also has high affinity for Fe2+ ions [33, 34]. Studies have demonstrated that pyoverdine is more important than its counterpart pyochelin for the development of *P. aeruginosa* biofilm and infection, whereas mutants that produce pyochelin but are deficient in pyoverdine production are significantly hampered in their biofilm‐forming ability [34]. In line with this, a study using an animal model (immunosuppressed mice) showed that pyoverdine predominantly contributes to *P. aeruginosa* virulence and infection [36].

Various factors influence the bioavailability of iron for *P. aeruginosa* and other pathogens in the host; in vitro studies show mutations in the CFTR gene trigger increased release of ex‐ tracellular iron from lung epithelial cells in comparison to healthy epithelial cells, while ele‐ vated iron levels in CF patients directly correlated with an increase in the *P. aeruginosa* population [34]. The proteolytic activity of *P. aeruginosa* protease degrades human ferritin so that it cannot bind iron, thus allowing pyoverdine to scavenge iron and triggering *P. aerugi‐ nosa* pathogenicity [34]. Tate et al. showed that iron acquisition by *P. aeruginosa* in CF also occurs through the heme uptake (FeoABC and EfeU) pathways, which are independent of regular siderophore uptake pathways [37]. The presence of an elevated concentration of haem in CF sputum due to haemolysis resulting from pulmonary exacerbations provides bacteria in general with an excellent source of iron. Studies have also demonstrated that un‐ der oxygen‐deficient conditions in *P. aeruginosa* biofilms or in CF airways,iron exists as Fe2+ ions and *P. aeruginosa* takes up Fe2+ via the FeoABC and EfeU pathways [37].

Interestingly, mammalian biological systems have an innate defence strategy against sidero‐ phores, a neutrophil‐gelatinase‐associated lipocalin (NGAL). NGAL functions as a scavenger by directly binding with siderophores, blocking *P. aeruginosa's* ability to sequester iron and thereby inhibiting bacterial growth and infection [32]. However, studies have reported that pyoverdine does not bind to NGAL and consequently is able to assist *P. aeruginosa* growth, as demonstrated by biofilm formation and chronic infection in CF lung in spite of elevated amounts of NGAL in lung secretions and bronchoalveolar lavage fluid [32].

#### **2.4. Role of eDNA**

eDNA is currently recognised as an essential constituent of EPS and plays a pivotal role in the various processes of biofilm formation in numerous medicallyrelevant Gram‐negative and Gram‐positive bacteria [8, 9]. In *P. aeruginosa*, eDNA is recognised as an essential molecule in facilitating biofilm formation, including assisting initial bacterial adhesion to surfaces, cell‐to‐ cell interaction (aggregation), microcolony formation and enhancement of biofilm strength and stability [38–41]. eDNA, which is similar to chromosomal DNA in its primary structure [42], is not only released by many bacterial species, predominantly through cell‐lytic, but also partly through non‐lytic mechanisms [9, 43, 44]. In cell‐lytic release, various cell lysing agents such as prophages, autolysin proteins, enzymes and phenazines lyse bacterial cells and trigger eDNA release [8, 38]. Non‐lytic eDNA release occurs through the lysis of bacterial outer membrane blebs/vesicles that contain large amounts of DNA [44, 45]. In *P. aeruginosa,* both lytic and non‐lytic eDNA releases have been recorded [38, 43, 44]. Studies show that mutants deficient in eDNA production are significantly hampered in biofilm formation. In the same vein, biofilm treatment with DNase I, an enzyme that non‐specifically cleaves DNA via hydrolysis of phosphodiester bonds in DNA, significantly inhibits biofilm formation and dispersal of mature biofilms [39, 40, 43].

pyochelin production by the *pch* gene cluster [35]. Pyoverdine is more efficient in releasing iron from human ferritin and also has high affinity for Fe2+ ions [33, 34]. Studies have demonstrated that pyoverdine is more important than its counterpart pyochelin for the development of *P. aeruginosa* biofilm and infection, whereas mutants that produce pyochelin but are deficient in pyoverdine production are significantly hampered in their biofilm‐forming ability [34]. In line with this, a study using an animal model (immunosuppressed mice) showed that pyoverdine

Various factors influence the bioavailability of iron for *P. aeruginosa* and other pathogens in the host; in vitro studies show mutations in the CFTR gene trigger increased release of ex‐ tracellular iron from lung epithelial cells in comparison to healthy epithelial cells, while ele‐ vated iron levels in CF patients directly correlated with an increase in the *P. aeruginosa* population [34]. The proteolytic activity of *P. aeruginosa* protease degrades human ferritin so that it cannot bind iron, thus allowing pyoverdine to scavenge iron and triggering *P. aerugi‐ nosa* pathogenicity [34]. Tate et al. showed that iron acquisition by *P. aeruginosa* in CF also occurs through the heme uptake (FeoABC and EfeU) pathways, which are independent of regular siderophore uptake pathways [37]. The presence of an elevated concentration of haem in CF sputum due to haemolysis resulting from pulmonary exacerbations provides bacteria in general with an excellent source of iron. Studies have also demonstrated that un‐ der oxygen‐deficient conditions in *P. aeruginosa* biofilms or in CF airways,iron exists as Fe2+

Interestingly, mammalian biological systems have an innate defence strategy against sidero‐ phores, a neutrophil‐gelatinase‐associated lipocalin (NGAL). NGAL functions as a scavenger by directly binding with siderophores, blocking *P. aeruginosa's* ability to sequester iron and thereby inhibiting bacterial growth and infection [32]. However, studies have reported that pyoverdine does not bind to NGAL and consequently is able to assist *P. aeruginosa* growth, as demonstrated by biofilm formation and chronic infection in CF lung in spite of elevated

eDNA is currently recognised as an essential constituent of EPS and plays a pivotal role in the various processes of biofilm formation in numerous medicallyrelevant Gram‐negative and Gram‐positive bacteria [8, 9]. In *P. aeruginosa*, eDNA is recognised as an essential molecule in facilitating biofilm formation, including assisting initial bacterial adhesion to surfaces, cell‐to‐ cell interaction (aggregation), microcolony formation and enhancement of biofilm strength and stability [38–41]. eDNA, which is similar to chromosomal DNA in its primary structure [42], is not only released by many bacterial species, predominantly through cell‐lytic, but also partly through non‐lytic mechanisms [9, 43, 44]. In cell‐lytic release, various cell lysing agents such as prophages, autolysin proteins, enzymes and phenazines lyse bacterial cells and trigger eDNA release [8, 38]. Non‐lytic eDNA release occurs through the lysis of bacterial outer membrane blebs/vesicles that contain large amounts of DNA [44, 45]. In *P. aeruginosa,* both lytic and non‐lytic eDNA releases have been recorded [38, 43, 44]. Studies show that mutants deficient in eDNA production are significantly hampered in biofilm formation. In the same

predominantly contributes to *P. aeruginosa* virulence and infection [36].

ions and *P. aeruginosa* takes up Fe2+ via the FeoABC and EfeU pathways [37].

amounts of NGAL in lung secretions and bronchoalveolar lavage fluid [32].

**2.4. Role of eDNA**

106 Progress in Understanding Cystic Fibrosis

eDNA also serves as a nutrient source (an excellent source of carbon,phosphate and nitrogen), facilitates horizontal gene transfer through Type IV pili and competence stimulating peptides and helps maintain the structural integrity of the biofilm by binding to various extracellular molecules (proteins, polysaccharides, metabolites) in the biofilm matrix [7, 8]. Recent investi‐ gations have revealed that eDNA protects bacterial cells in biofilm from physical challenges such as shear stress by increasing biofilm viscosity, and from chemical challenges by antibiotics and detergents. For example, eDNA binds to various positively charged antibiotics (amino‐ glycosides) thus shielding *P. aeruginosa* in biofilms against their action [46]. eDNA at sub‐MIC concentrations creates a cation‐limited atmosphere by chelating divalent cations such as Ca2+. This results in the induction of genes involved in resistance to cationic antimicrobial peptides [47]. Swartjeset al. demonstrated that continuous exposure of bacterial cells (*P. aeruginosa* and *S. aureus*) to a DNase I‐coated surface inhibits biofilm formation [40]. Treating biofilms with DNase I alters the biofilm architecture leading to penetration by antibiotics, thus promoting the efficacy of antibiotics in killing mature biofilms [48]. It is important to note that *P. aeruginosa‐* infected CF lung secretions and bronchitis sputum contain a significant amount of eDNA (3– 14 mg/ml), compared to none in non‐CF patients [49]. eDNA aids bacterial viability by inducing antibiotic resistance [48] and it also contributes tothe high viscosity of CF sputum [49].

While eDNA is well‐recognised as one of the prime factors in the establishment of *P. aeruginosa* biofilms [39, 43], it has also been demonstrated to have such a role in other biofilm‐forming bacteria [50, 51]. eDNA initiates biofilm formation by binding with bacterial extracellular bio‐ molecules such as polysaccharides, peptides/enzymes/proteins and other bacterial cell surface structures. In *Listeria monocytogenes* (a food‐borne pathogen), Harmsen et al. demonstrated that eDNA binds with peptidoglycan *(N*‐acetyl glucosamine), and this molecular interaction initiates adhesion by *L. monocytogenes* to surfaces [50]. In *Caulobacter crescentus* (environmental freshwater bacterium) biofilms, eDNA binds to polar adhesive structure called 'hold‐fast' that is present on the tip of the stalk cell (a part of the cell wall that is essential for *C. crescentus* adherence to surfaces), while eDNA from lysed cells masking the adhesive properties of hold‐ fast, inhibit swarmer cell adherence to the same surface [52]. Rather than acting as an essential structural element of the biofilm, this unusual role for eDNA means that it functions as a regulatory component assisting in the escape of cells from the biofilm and thus promoting development of new, independent colonies [52]. Peptide‐eDNA interactions have also been found to be an essential factor promoting biofilm growth of *Streptococcus mutans* (an oral pathogen responsible for dental plaque). In *S. mutans*, uptake of eDNA is triggered through a competence‐stimulating peptide, whereas bacterial cell‐to‐cell interaction and biofilm forma‐ tion are initiated through the DNA‐binding protein ComGB [51]. In *P. aeruginosa*, Das et al. were the first to discover that the phenazine metabolite (pyocyanin) binds with DNA to facilitate *P. aeruginosa* biofilm formation [53].

#### **2.5. Role of pyocyanin**

#### *2.5.1. Pyocyanin production in P. aeruginosa*

Pyocyanin, a member of the phenazine class, is a molecule only known to be expressed by *P. aeruginosa*, and thus distinguishes it from other pathogens. Up to 95% of *P. aeruginosa* isolates synthesise pyocyanin [54]. It is a bluish‐green‐coloured extracellular metabolite that is secreted incopiousquantitiesbothinvitroandinvivo.In*P. aeruginosa*,phenazineproductionis regulated through the bacterium's complex QS mechanism. The primary QS molecules, AHL and PQS, triggerthe inductionofthephenazine operon(*phzA‐G*)toproducephenazine‐1‐carboxylic acid (PCA). Seven genes have been identified as having a role in pyocyanin synthesis, namely *phzCDEFGMS*.Amongstthese, *phzM*and*phzS*are centraltothe conversionofPCAtopyocyanin in a two‐step reaction. First, PCA is converted to 5‐methylphenazine‐1‐carboxylic acid betaine (encoded by *phzM*) and then to pyocyanin (encoded by *phzS*) [54, 55]. PCA is also converted in much lower ratios to other types of phenazines, including phenazine‐1‐carboxamide (PCN, encoded by *phzH*) and 1‐hydroxyphenazine (1‐OHPHZ, encoded by *phzS*) [54].

In chronic CF lung infection, up to 85 μM of pyocyanin has been recorded in *P. aeruginosa‐* infected CF lung secretions and up to 130 μM in bronchitis sputum [56]. In vitro measurement of pyocyanin production by *P. aeruginosa* in both clinical CF and laboratory reference strains showed, in most cases, the expression of large amounts of pyocyanin within 24 h of growth in Luria‐Bertani (LB) medium. Amongst CF isolates, the Liverpool Epidemic Strain LESB58 and the Australian Epidemic Strain‐2 (AES‐2) produced close to 100 μM pyocyanin, as did the laboratory reference strain DKN‐370 (a pyocyanin overproducing strain), while the laboratory reference strain PA14 and the Australian epidemic strain‐1 isolate AES‐1R produced 70–80 μM pyocyanin. Conversely, the chronic infection isogen of AES‐1R (AES‐1M) produced less than 5 μM pyocyanin indicating expression is reduced as the strain adapts to the CF lung [11]. Evidence suggests that many factors activate pyocyanin production, including low iron [57] and phosphate depletion [58].

#### *2.5.2. Pyocyanin facilitates eDNA release*

Pyocyanin is a redox molecule and electrochemically active (has potential to accept and donate electrons as a shuttle) with a multitude of biological activities [59]. Recent investigations have demonstrated that pyocyanin facilitates eDNA release in *P. aeruginosa*. Comparison of eDNA release by *P. aeruginosa* PA14 wild‐type and a phenazine/pyocyanin‐deficient PA14 mutant (Δ*phzA‐G*) showed up to 50% increase in eDNA release by the wild‐type under laboratory growth conditions in LB. In line with this, the Δ*phzA‐G* mutant showed a significant increase in eDNA release when grown in the presence of exogenous pyocyanin, with the rate of eDNA release directly correlated to the concentration of pyocyanin [38]. Pyocyanin‐mediated eDNA release is induced through cell lysis due to hydrogen peroxide (H2O2) expression. In PAO1 and PA14 planktonic growth cultures, pyocyanin has been shown to donate electrons to molecular oxygen to form H2O2 and initiate an increase of up to 14% in cell lysis under laboratory growth conditions [38]. Interestingly, the surviving *P. aeruginosa* population is protected from H2O2 by catalase, whose expression is upregulated by *P. aeruginosa* as a self‐defence mechanism against its own and host‐released H2O2 molecules [60]. H2O2‐mediated eDNA release has also been documented in other bacterial species including *Streptococccus sanguinis,* an oral bacterium responsible for dental disease. In this species, pyruvate oxidase activity by *S. sanguinis*induces a ca. 10% increase in cell death in its own population and consequently facilitates eDNA release [61].

#### *2.5.3. Pyocyanin and eDNA intercalate in biofilms*

**2.5. Role of pyocyanin**

108 Progress in Understanding Cystic Fibrosis

and phosphate depletion [58].

*2.5.2. Pyocyanin facilitates eDNA release*

*2.5.1. Pyocyanin production in P. aeruginosa*

Pyocyanin, a member of the phenazine class, is a molecule only known to be expressed by *P. aeruginosa*, and thus distinguishes it from other pathogens. Up to 95% of *P. aeruginosa* isolates synthesise pyocyanin [54]. It is a bluish‐green‐coloured extracellular metabolite that is secreted incopiousquantitiesbothinvitroandinvivo.In*P. aeruginosa*,phenazineproductionis regulated through the bacterium's complex QS mechanism. The primary QS molecules, AHL and PQS, triggerthe inductionofthephenazine operon(*phzA‐G*)toproducephenazine‐1‐carboxylic acid (PCA). Seven genes have been identified as having a role in pyocyanin synthesis, namely *phzCDEFGMS*.Amongstthese, *phzM*and*phzS*are centraltothe conversionofPCAtopyocyanin in a two‐step reaction. First, PCA is converted to 5‐methylphenazine‐1‐carboxylic acid betaine (encoded by *phzM*) and then to pyocyanin (encoded by *phzS*) [54, 55]. PCA is also converted in much lower ratios to other types of phenazines, including phenazine‐1‐carboxamide (PCN,

In chronic CF lung infection, up to 85 μM of pyocyanin has been recorded in *P. aeruginosa‐* infected CF lung secretions and up to 130 μM in bronchitis sputum [56]. In vitro measurement of pyocyanin production by *P. aeruginosa* in both clinical CF and laboratory reference strains showed, in most cases, the expression of large amounts of pyocyanin within 24 h of growth in Luria‐Bertani (LB) medium. Amongst CF isolates, the Liverpool Epidemic Strain LESB58 and the Australian Epidemic Strain‐2 (AES‐2) produced close to 100 μM pyocyanin, as did the laboratory reference strain DKN‐370 (a pyocyanin overproducing strain), while the laboratory reference strain PA14 and the Australian epidemic strain‐1 isolate AES‐1R produced 70–80 μM pyocyanin. Conversely, the chronic infection isogen of AES‐1R (AES‐1M) produced less than 5 μM pyocyanin indicating expression is reduced as the strain adapts to the CF lung [11]. Evidence suggests that many factors activate pyocyanin production, including low iron [57]

Pyocyanin is a redox molecule and electrochemically active (has potential to accept and donate electrons as a shuttle) with a multitude of biological activities [59]. Recent investigations have demonstrated that pyocyanin facilitates eDNA release in *P. aeruginosa*. Comparison of eDNA release by *P. aeruginosa* PA14 wild‐type and a phenazine/pyocyanin‐deficient PA14 mutant (Δ*phzA‐G*) showed up to 50% increase in eDNA release by the wild‐type under laboratory growth conditions in LB. In line with this, the Δ*phzA‐G* mutant showed a significant increase in eDNA release when grown in the presence of exogenous pyocyanin, with the rate of eDNA release directly correlated to the concentration of pyocyanin [38]. Pyocyanin‐mediated eDNA release is induced through cell lysis due to hydrogen peroxide (H2O2) expression. In PAO1 and PA14 planktonic growth cultures, pyocyanin has been shown to donate electrons to molecular oxygen to form H2O2 and initiate an increase of up to 14% in cell lysis under laboratory growth conditions [38]. Interestingly, the surviving *P. aeruginosa* population is protected from H2O2 by

encoded by *phzH*) and 1‐hydroxyphenazine (1‐OHPHZ, encoded by *phzS*) [54].

Pyocyanin's intercalation with DNA has been demonstrated using various bio‐physical techniques (circular dichroism, Fourier transform infrared spectroscopy, fluorescence and UV‐ Vis spectroscopy) [53]. In a preliminary study using fluorescence emission spectroscopy, it was shown that pyocyanin displaces ethidium bromide bound to dsDNA, indicating pyocyanin is an intercalating agent. Fluorescence emission spectroscopy data were further complemented using the UV‐Vis spectra of the DNA‐pyocyanin complex. Results indicated a significant shift (from 259 to 253 nm) and increase in absorbance intensity in the DNA peak. This marked change in the DNA peak from 259 nm indicates effective intercalation of pyocyanin molecules between the nitrogenous base‐pairs of DNA [53]. Meanwhile, the circular dichroism spectra of the DNA‐pyocyanin mixtures confirmed that pyocyanin binds to the sugar‐phosphate backbone of DNA and strongly intercalates with the nitrogenous bases of DNA, consequently creating local perturbations in the DNA double helix structure [53]. This type of interaction is a typical characteristic feature of all intercalating molecules. In the same study, Das et al. also discovered that pyocyanin significantly increased the viscosity of DNA solutions, and that by intercalating with DNA pyocyanin‐facilitated electron transfer [53]. These results are in line with previous studies concluding that in order to remain viable in biofilms, *P. aeruginosa* exploits redox‐active metabolites such as pyocyanin, where direct access to electron acceptors such as oxygen or nitrate is diffusion‐limited [59].

#### *2.5.4. Pyocyanin‐eDNA binding influences biofilm formation via physico‐chemical interactions*

Molecules that bind to both biological and non‐biological surfaces are known to influence hydrophobicity, charge and the physico‐chemical properties that assist or resist interactions. Previous studies have demonstrated that in both bacteria and fungi, the presence of such bio‐ molecules (eDNA or proteins) plays a significant role in dictating cell surface hydrophobicity and physico‐chemical interactions [41]. In *P. aeruginosa*, the presence of eDNA has been shown to increase cell surface hydrophobicity. Water contact angle measurements on DNase I‐treated *P. aeruginosa* PA14 and PAO1 reduced the angle from 50 to 34° and 46 to 31°, respectively. Interestingly, the PA14 phenazine deficient mutant (∆*phzA‐G*) had a water contact angle similar to DNase I‐treated PA14, and DNase I treatment of Δ*phnzA‐G* did not show any further reduction in cell surface hydrophobicity [41], indicating that pyocyanin‐DNA binding is an essential factor influencing *P. aeruginosa* cell surface hydrophobicity. eDNA‐mediated modu‐ lation in cell surface hydrophobicity has also been reported in other pathogenic strains, including *Staphylococcus epidermidis* and *S. aureus* [62].

Analysis of bacteria‐to‐bacteria and bacteria‐to‐substratum physico‐chemical interactions (Lifshitz‐Van der Waals interactions forces, acid‐base interactions forces) has revealed that the presence of pyocyanin and eDNA facilitates attractive physico‐chemical interactions [41]. Removal of eDNA from the *P. aeruginosa* wild‐type cell surface or absence of pyocyanin in the *∆phzA‐G* strain showed significant impact, that is, resulted in non‐attractive interaction, especially on the short‐range acid‐base interactions, which include electron donating and electron accepting parameters. However, the long‐range Lifshitz‐Van der Waals interactions remained unaffected between wild‐type and *∆phz*A‐G regardless of DNase I treatment [42]. Similarly, the effect of eDNA on physico‐chemical forces between *S. epidermidis* cells has been reported, and results suggest that eDNA triggers *S. epidermidis* cell‐to‐cell interactions [62]. Similarly, adhesion force analysis in *S. mutans* using atomic force microscopy and phase‐ contrast microscopy imaging and quantification indicates that in the presence of eDNA, *S. mutans* has a stronger adhesion force and adheres to surfaces in significantly higher cell numbers [63].

It should be noted, however, that physico‐chemical interactions do not explain bacterial interaction in all cases, since bacterial cell structures (pili, fimbriae) and bio‐polymers (poly‐ saccharides, proteins, eDNA) extend up to hundreds of nanometres from the bacterial cell surface and can affect other interaction types [64]. These cell structures and bio‐polymers initiate hydrogen bonding and ionic interactions by colliding with bio‐molecules anchored on the bacterial cell surface to stabilise the biofilm matrix and also to its adjacent cells and thereby help bacterial cells to overcome the physico‐chemical energy barrier and promote bacterial cell‐to‐cell interactions and biofilm formation [7, 64]. Confocal laser scanning microscopy (CLSM) analysis revealed that the intercalation of pyocyanin with eDNA facilitates *P. aerugi‐ nosa* PA14 wild‐type biofilm formation while the absence of pyocyanin significantly inhibits biofilm formation [65]. To investigate this further, Klare et al. grew the CF *P. aeruginosa*AES‐1 isolate R (isolated at the acute stage of infection)in an artificial sputum media (ASMDM+) that mimics CF sputum, and found it formed robust biofilms in comparison to its isogenic coun‐ terpart AES‐1M (isolated at chronic infection). AES‐1M which produces about 15 times less pyocyanin than AES‐1R, and the exogenous addition of pyocyanin to AES‐1M cultures facilitated enhanced biofilm formation [65] (**Figure 2**).

**Figure 2.** Biofilm formation by *P. aeruginosa* CF isogens in ASMDM+ medium (a) AES‐1R, (b) AES‐1M and (c) AES‐1M grown in the presence of exogenous pyocyanin. The biofilm architecture of (c) indicates pyocyanin facilitates/enhances biofilm formation. Images taken with permission from Ref. [65].

#### *2.5.5. Pyocyanin as a virulence factor*

Analysis of bacteria‐to‐bacteria and bacteria‐to‐substratum physico‐chemical interactions (Lifshitz‐Van der Waals interactions forces, acid‐base interactions forces) has revealed that the presence of pyocyanin and eDNA facilitates attractive physico‐chemical interactions [41]. Removal of eDNA from the *P. aeruginosa* wild‐type cell surface or absence of pyocyanin in the *∆phzA‐G* strain showed significant impact, that is, resulted in non‐attractive interaction, especially on the short‐range acid‐base interactions, which include electron donating and electron accepting parameters. However, the long‐range Lifshitz‐Van der Waals interactions remained unaffected between wild‐type and *∆phz*A‐G regardless of DNase I treatment [42]. Similarly, the effect of eDNA on physico‐chemical forces between *S. epidermidis* cells has been reported, and results suggest that eDNA triggers *S. epidermidis* cell‐to‐cell interactions [62]. Similarly, adhesion force analysis in *S. mutans* using atomic force microscopy and phase‐ contrast microscopy imaging and quantification indicates that in the presence of eDNA, *S. mutans* has a stronger adhesion force and adheres to surfaces in significantly higher cell

It should be noted, however, that physico‐chemical interactions do not explain bacterial interaction in all cases, since bacterial cell structures (pili, fimbriae) and bio‐polymers (poly‐ saccharides, proteins, eDNA) extend up to hundreds of nanometres from the bacterial cell surface and can affect other interaction types [64]. These cell structures and bio‐polymers initiate hydrogen bonding and ionic interactions by colliding with bio‐molecules anchored on the bacterial cell surface to stabilise the biofilm matrix and also to its adjacent cells and thereby help bacterial cells to overcome the physico‐chemical energy barrier and promote bacterial cell‐to‐cell interactions and biofilm formation [7, 64]. Confocal laser scanning microscopy (CLSM) analysis revealed that the intercalation of pyocyanin with eDNA facilitates *P. aerugi‐ nosa* PA14 wild‐type biofilm formation while the absence of pyocyanin significantly inhibits biofilm formation [65]. To investigate this further, Klare et al. grew the CF *P. aeruginosa*AES‐1 isolate R (isolated at the acute stage of infection)in an artificial sputum media (ASMDM+) that mimics CF sputum, and found it formed robust biofilms in comparison to its isogenic coun‐ terpart AES‐1M (isolated at chronic infection). AES‐1M which produces about 15 times less pyocyanin than AES‐1R, and the exogenous addition of pyocyanin to AES‐1M cultures

**Figure 2.** Biofilm formation by *P. aeruginosa* CF isogens in ASMDM+ medium (a) AES‐1R, (b) AES‐1M and (c) AES‐1M grown in the presence of exogenous pyocyanin. The biofilm architecture of (c) indicates pyocyanin facilitates/enhances

facilitated enhanced biofilm formation [65] (**Figure 2**).

biofilm formation. Images taken with permission from Ref. [65].

numbers [63].

110 Progress in Understanding Cystic Fibrosis

Pyocyanin was formerly recognised only as a bacterial secondary metabolite, but has recently gained significant attention for its involvement in a variety of crucial roles in microbial ecology, specifically correlated with the severity of *P. aeruginosa* pathogenicity in plants and humans [66]. **Figure 3** is a schematic representation of pyocyanin‐induced H2O2 production and toxicity on bacterial, fungal and human cells. Pyocyanin also has antibacterial and antifungal activity that is toxic to other pathogenic bacteria and fungi. Pyocyanin‐mediated bactericidal activity occurs through production of H2O2, which consequently depletes oxygen supply to cells and disables electron flow and metabolic transport processes [67]. Studies suggest that pyocyanin potentially kills *Staphylococcus* sp. and other species in the CF lung environment; and that it also has anti‐*Escherichia coli* activity [67, 68] (**Figure 3**). The inhibitory effect of pyocyanin on the growth of fungi such as *Aspergillus fumigatus* and *Candida albicans*isolated from the sputum of CF patients has also been reported earlier [69] (**Figure 3**). These results could be interpreted as a pyocyanin‐mediated modulation of the microbial community in the CF lung by *P. aeruginosa*, resulting in its predominance [70].

**Figure 3.** Schematic diagram of pyocyanin induced H2O2 production and toxicity on bacterial, fungal and human cells.

In the host, pyocyanin appears to participate in a reduction mechanism, which is capable of reducing and releasing the iron from transferrin in host cells to stimulate the growth of *P. aeruginosa* [71]. Previous research concluded that a direct correlation exists between pyocyanin concentration in CF sputum and severity of infection [71]. Studies using *P. aeruginosa*‐infected bronchiectasis airways in a mouse model of lung infection demonstrated that pyocyanin rapidly inhibited lung function and caused cell hyperplasia and metaplasia (abnormal changes in cell or tissue morphology), airway fibrosis and alveolar airspace destruction [71]. Harmer et al. analysed the difference between *P. aeruginosa* epidemic and non‐epidemic isogenic strains that were collected 5–8 years apart from five chronically infected adult CF patients, this study suggested that epidemic (FCC) strains are more virulent and more efficient in killing *Caeno‐ rhabditis elegans* than their non‐epidemic counterparts [72]. The isogens collected early in the infection produced more virulence factors including elastase, pyoverdine and pyocyanin. Over the course of chronic infection, the isogens undergo a significant downregulation in virulence factors *lasB*, *rsaL*, *lecB* and *oprG*, with a significant decrease in elastase and pyoverdine production, however, pyocyanin production increased in three out of five strains and so did biofilm production [72]. Fluctuations in pyocyanin concentration observed among different CF strains are probably due to adaptation of a particular strain to the host and time of acquisition of sample, for example, at exacerbation (when the patient is seriously ill and hospitalised). At exacerbation, the pyocyanin levels may be switched on by the *P. aeruginosa* strain as a protective mechanism against host defences, and this leads to the increased lung damage seen at that time [72]. If the sample was taken when the patient was not in exacerbation, the pyocyanin expression may be very low or negligible [72]. Other phenazine‐like PCA molecules secreted by *P. aeruginosa* were also shown to be highly toxic, killed *C. elegans* and caused serious cell damage in a mouse model of lung infection [73].

Pyocyanin has also been extensively studied due to its electrochemical and redox activity. The diffusible nature and small size of pyocyanin means it can easily pass through the host cell membrane and undergo redox reactions with other molecules [74]. For example, it accepts electrons fromNADHandsubsequentlydonates electrons tomolecularoxygentoformreactive oxygen species (ROS) such as H2O2 [74] (**Figure 3**). Pyocyanin‐mediated ROS cause oxidative stress and affect calcium homeostasis while also obstructing cellular respiration and depleting intracellular cAMP and ATP levels [75]. Pyocyanin significantly alters human protease activity, inhibits nitric oxide production and consequently influences blood flow, blood pressure and immune functions. It also modulates the host immune response to support evasion of the host immune system and establish chronic infection [75]. In CF, pyocyanin‐mediated ROS oxidise host intracellular and extracellular reduced glutathione (GSH) to form glutathione disulphide or oxidised glutathione (GSSG) [76]. Depleted GSH levels during the chronic stage of CF infection lead to widespread epithelial cell death and consequent lung damage and leading to respiratory failure and death [75, 76]. Pyocyanin also inhibits catalase activity in airway epithelial cells, thus increasing oxidative stress in these cells and initiating pulmonary tissue damage [77]. In a recent study, Rada et al. showed that pyocyanin promotes neutrophil extracellular trap (NET) formation [78]. NET formation is an important innate immune mechanism initiated by neutrophils to trap and kill pathogens, however, the aberrant NET release triggered by pyocyanin‐mediated intracellular ROS production directly damages host tissues and has been linked to the severity of many diseases including CF [78].

## **3. Treating** *P. aeruginosa* **infections**

Substantial research over many decades has led to a good degree of understanding of the mechanisms *P. aeruginosa* utilises to cause infection and colonisation. In brief, *P. aeruginosa* has been shown to evade the host's innate defence system through production of various extracellular molecules and render antibiotics ineffective through several efflux pump mechanisms [6, 8, 79]. This research has particular implications for CF, burns and wounds patients, particularly as *P. aeruginosa* antibiotic resistance is a serious concern. This in turn has given impetus to the development of new therapeutic methods. Prominent amongst the extracellular molecules available to *P. aeruginosa* are the previously discussed eDNA, pro‐ tease, pyocyanin and pyoverdine.

that were collected 5–8 years apart from five chronically infected adult CF patients, this study suggested that epidemic (FCC) strains are more virulent and more efficient in killing *Caeno‐ rhabditis elegans* than their non‐epidemic counterparts [72]. The isogens collected early in the infection produced more virulence factors including elastase, pyoverdine and pyocyanin. Over the course of chronic infection, the isogens undergo a significant downregulation in virulence factors *lasB*, *rsaL*, *lecB* and *oprG*, with a significant decrease in elastase and pyoverdine production, however, pyocyanin production increased in three out of five strains and so did biofilm production [72]. Fluctuations in pyocyanin concentration observed among different CF strains are probably due to adaptation of a particular strain to the host and time of acquisition of sample, for example, at exacerbation (when the patient is seriously ill and hospitalised). At exacerbation, the pyocyanin levels may be switched on by the *P. aeruginosa* strain as a protective mechanism against host defences, and this leads to the increased lung damage seen at that time [72]. If the sample was taken when the patient was not in exacerbation, the pyocyanin expression may be very low or negligible [72]. Other phenazine‐like PCA molecules secreted by *P. aeruginosa* were also shown to be highly toxic, killed *C. elegans* and

Pyocyanin has also been extensively studied due to its electrochemical and redox activity. The diffusible nature and small size of pyocyanin means it can easily pass through the host cell membrane and undergo redox reactions with other molecules [74]. For example, it accepts electrons fromNADHandsubsequentlydonates electrons tomolecularoxygentoformreactive oxygen species (ROS) such as H2O2 [74] (**Figure 3**). Pyocyanin‐mediated ROS cause oxidative stress and affect calcium homeostasis while also obstructing cellular respiration and depleting intracellular cAMP and ATP levels [75]. Pyocyanin significantly alters human protease activity, inhibits nitric oxide production and consequently influences blood flow, blood pressure and immune functions. It also modulates the host immune response to support evasion of the host immune system and establish chronic infection [75]. In CF, pyocyanin‐mediated ROS oxidise host intracellular and extracellular reduced glutathione (GSH) to form glutathione disulphide or oxidised glutathione (GSSG) [76]. Depleted GSH levels during the chronic stage of CF infection lead to widespread epithelial cell death and consequent lung damage and leading to respiratory failure and death [75, 76]. Pyocyanin also inhibits catalase activity in airway epithelial cells, thus increasing oxidative stress in these cells and initiating pulmonary tissue damage [77]. In a recent study, Rada et al. showed that pyocyanin promotes neutrophil extracellular trap (NET) formation [78]. NET formation is an important innate immune mechanism initiated by neutrophils to trap and kill pathogens, however, the aberrant NET release triggered by pyocyanin‐mediated intracellular ROS production directly damages host

caused serious cell damage in a mouse model of lung infection [73].

112 Progress in Understanding Cystic Fibrosis

tissues and has been linked to the severity of many diseases including CF [78].

Substantial research over many decades has led to a good degree of understanding of the mechanisms *P. aeruginosa* utilises to cause infection and colonisation. In brief, *P. aeruginosa* has been shown to evade the host's innate defence system through production of various

**3. Treating** *P. aeruginosa* **infections**

#### **3.1. Current antibiotic treatment and challenges against** *P. aeruginosa* **infections in CF patients**

Many antibiotics developed in recent decades such as aminoglycosides, ticarcillin, ureidope‐ nicillins, ceftazidime, cefepime, aztreonam, the carbapenems, ciprofloxacin and levofloxacin display anti‐pseudomonal activity. However, the choice of best antibiotic to use in a particular case remains a major challenge as *P. aeruginosa* can readily adapt by mutation or horizontal gene transfer to acquire resistance in a portion of remaining cells, leading to consequent treatment failure.

Antibiotics commonly used to treat *P. aeruginosa* infection in CF patients include tobramycin, colistin, aztreonam, ciprofloxacin and azithromycin. Administration methods include nebul‐ ised, dry powder inhalation, oral or intravenous, or a combination of different strategies [2, 80]. Studies have shown the size of inhaled antibiotic particles is very important in determining whether they will reach deep infection sites. Particles of 1–5 **µ**m diameters are more effective in reaching deep lung tissue efficiently [81]. However, one of the major concerns in inhalation therapy is that most antibiotics are trapped in the thick viscous mucus that covers both the large respiratory zone (respiratory bronchioles, alveolar ducts and alveolar sacs) and the conductive zone (trachea, bronchi and terminal bronchioles) [81, 82]. With intravenous or oral therapy, antibiotics are readily transported through the bloodstream mainly reaching the respiratory zone but not effectively reaching the conductive zone. A combination of both strategies has been shown to enhance the access of antibiotics to infection sites at both the conductive zone and respiratory zones [82].

Other serious challenges with nebuliser treatment (in comparison to dry powder inhalation) strategies are that the antibiotic particles do not reach infection sites at a faster rate, but even with dry powder inhalation does not provide immediate relief to CF patients [83]. For example, studies with CF patients demonstrated that inhaled tobramycin is effective in reducing *P. aeruginosa* density from the lower airways but has no effect in reducing lung inflammation, and consequently certain infection loci and disease symptoms remain [84]. Azithromycin has been shown to improve lung function (lung inflammation, exacerbations and cough) in CF patients compared to other antibiotics and lead to a reduction in *P. aeruginosa* colonisation [85]. However, azithromycin or the macrolide class of antibiotics has significant side effects, including a significant increase in macrolide resistant *S. aureus* and *H. influenzae* strains in CF sputum [85]. In general, many antibiotics are known to cause adverse side‐effects in patients, targeting the central nervous system, gastrointestinal tract and urinary tract leading to kidney failure [86, 87]. With the increase in antibiotic resistance, there is an urgent need to develop novel therapeutic approaches to disrupt bacterial biofilms and eradicate the causative bacteria in the host.

#### **3.2. Current non‐antibiotic strategies against CF lung infection**

Non‐antibiotic treatment strategies that have shown potential to reduce the severity of respiratory symptoms in CF patients and bacterial associated infections have largely centred on the use of aerosolised recombinant human DNase I (rhDNase I (Pulmozyme)) in a nebu‐ liser [88]. Earlier studies showed DNase I reduced the viscosity of CF sputum by cleaving DNA present in sputum and thus leading to increased pulmonary function [49]. As noted above, eDNA is an essential biofilm promoting factor in many pathogenic bacterial species, is the backbone of the *P. aeruginosa* biofilm matrix, which by its impenetrable structure constitutes a defence strategy against antibiotics [46–48]. In line with this studies have shown that DNase I inhalation reduces the prevalence of bacterial strains in CF patients [88].

#### **3.3. New non‐antibiotic treatments**

A new potential treatment strategy involves the use of reduced GSH to bind to pyocyanin and prevent its intercalation with eDNA. Intracellular GSH levels in mammalian cells are in the millimolar (mM) range, and lower concentrations are found in some bacterial cells. However, in CF patients, GSH levels in whole blood, blood neutrophils lymphocytes and epithelial lung fluid are markedly decreased [89]. Replenishment of GSH levels in CF has thus been investi‐ gated in a number of human studies using either inhaled GSH [90, 91] or oral N‐acetylcysteine, a GSH precursor [92]. These studies demonstrated the feasibility of successfully delivering GSH to human lung, with a significant improvement in lung function (FEV1), especially in patients with moderate lung disease. The GSH therapy was well tolerated by CF patients with no noticeable side effects [91].

GSH, being a thiol antioxidant, will donate electrons/protons to pyocyanin directly through the –SH group from cysteine [53, 76], thereby interfering in the pyocyanin oxidation process by inhibiting H2O2 generation [76]. The antioxidant properties of GSH make it a potential inhibitor of pyocyanin toxicity. GSH binding to pyocyanin tends to modulate pyocyanin's structure, and this has been confirmed using nuclear magnetic resonance (NMR) spectrosco‐ pyand mass spectrometry [53, 93]. This structural change consequently inhibits the intercala‐ tion of pyocyanin with DNA, confirmed using circular dichroism [53]. In line with this, Muller and Merrett concluded that GSH forms a cell‐impermanent conjugate with pyocyanin and consequently inhibits pyocyanin entry into host cells, thus preventing pyocyanin‐mediated lung epithelial cell lysis [93].

Recent studies in the Manos laboratory by Klare et al. have demonstrated the excellent utility of GSH in disrupting *P. aeruginosa* biofilms. It was demonstratedusing CLSM that GSH‐medi‐ ated inhibition of pyocyanin‐DNA binding modulates *P. aeruginosa* biofilm architecture, sig‐ nificantly decreases biofilm biomass, surface coverage and leads to a significant increase in the percentage of dead bacterial cells [65]. GSH alone was shown to have a significant effect on disruption of mature 72‐h‐old biofilms of the epidemic isolate AES‐1R grown in ASMDM+, while the combined treatment with GSH and DNase I of biofilms from a range of CF isolates showed greater disruption and significantly increased susceptibility to ciprofloxacin killing. GSH‐treated biofilms were also shown by RNA‐sequencing to display a transcriptomic profile that was distinctly different from those of both mature biofilms and dispersed cells, including those resulting from dispersal agents such as NO [65]. In contrast to dispersed cells, GSH‐dis‐ rupted biofilm cells significantly upregulated cyclic‐di‐GMP synthesis genes (*siaA* and *siaB*), and there was no concomitant induction of flagellar biosynthesis genes. Cyclic‐di‐GMP gates the transition from sessile to motile lifestyle, and its expression prevents this transition [94]. GSH‐disrupted cells also significantly upregulated the pyoverdine biosynthesis operon, in contrast to the downregulation of pyoverdine shown by dispersed cells. The active expression of pyoverdine is essential for biofilm structure formation [95]. CF sputum and ASMDM+ both have low levels of iron, and this may have triggered increased pyoverdine expression to se‐ quester iron for processes required to re‐form the disrupted biofilm.

In comparison to other techniques, GSH treatment has a distinct advantage, being an intrinsic and essential antioxidant for host cells that not only has antibiofilm properties but has also been proven to enhance lung epithelial growth and increase pulmonary function in CF patients [91].

#### **3.4. Development of new antibacterial agents**

novel therapeutic approaches to disrupt bacterial biofilms and eradicate the causative bacteria

Non‐antibiotic treatment strategies that have shown potential to reduce the severity of respiratory symptoms in CF patients and bacterial associated infections have largely centred on the use of aerosolised recombinant human DNase I (rhDNase I (Pulmozyme)) in a nebu‐ liser [88]. Earlier studies showed DNase I reduced the viscosity of CF sputum by cleaving DNA present in sputum and thus leading to increased pulmonary function [49]. As noted above, eDNA is an essential biofilm promoting factor in many pathogenic bacterial species, is the backbone of the *P. aeruginosa* biofilm matrix, which by its impenetrable structure constitutes a defence strategy against antibiotics [46–48]. In line with this studies have shown that DNase I

A new potential treatment strategy involves the use of reduced GSH to bind to pyocyanin and prevent its intercalation with eDNA. Intracellular GSH levels in mammalian cells are in the millimolar (mM) range, and lower concentrations are found in some bacterial cells. However, in CF patients, GSH levels in whole blood, blood neutrophils lymphocytes and epithelial lung fluid are markedly decreased [89]. Replenishment of GSH levels in CF has thus been investi‐ gated in a number of human studies using either inhaled GSH [90, 91] or oral N‐acetylcysteine, a GSH precursor [92]. These studies demonstrated the feasibility of successfully delivering GSH to human lung, with a significant improvement in lung function (FEV1), especially in patients with moderate lung disease. The GSH therapy was well tolerated by CF patients with

GSH, being a thiol antioxidant, will donate electrons/protons to pyocyanin directly through the –SH group from cysteine [53, 76], thereby interfering in the pyocyanin oxidation process by inhibiting H2O2 generation [76]. The antioxidant properties of GSH make it a potential inhibitor of pyocyanin toxicity. GSH binding to pyocyanin tends to modulate pyocyanin's structure, and this has been confirmed using nuclear magnetic resonance (NMR) spectrosco‐ pyand mass spectrometry [53, 93]. This structural change consequently inhibits the intercala‐ tion of pyocyanin with DNA, confirmed using circular dichroism [53]. In line with this, Muller and Merrett concluded that GSH forms a cell‐impermanent conjugate with pyocyanin and consequently inhibits pyocyanin entry into host cells, thus preventing pyocyanin‐mediated

Recent studies in the Manos laboratory by Klare et al. have demonstrated the excellent utility of GSH in disrupting *P. aeruginosa* biofilms. It was demonstratedusing CLSM that GSH‐medi‐ ated inhibition of pyocyanin‐DNA binding modulates *P. aeruginosa* biofilm architecture, sig‐ nificantly decreases biofilm biomass, surface coverage and leads to a significant increase in the percentage of dead bacterial cells [65]. GSH alone was shown to have a significant effect on disruption of mature 72‐h‐old biofilms of the epidemic isolate AES‐1R grown in ASMDM+,

**3.2. Current non‐antibiotic strategies against CF lung infection**

inhalation reduces the prevalence of bacterial strains in CF patients [88].

**3.3. New non‐antibiotic treatments**

no noticeable side effects [91].

lung epithelial cell lysis [93].

in the host.

114 Progress in Understanding Cystic Fibrosis

Several new antibacterial agents are being developed and undergoing stringent testing both in vitro and in vivo (animal models) against *P. aeruginosa* and other CF pathogens. QS‐ inhibiting molecules against *P. aeruginosa* biofilms, such as furanone‐based compounds (naturally secreted by the alga *Delisea Pulchra*) and synthetic furanone compounds have high affinity and compete with the cognate AHL signal for the LuxR receptor site in *P. aeruginosa* [96]. Thus by binding with and controlling the LuxR mechanism, these furanone molecules significantly alter biofilm architecture and enhance the efficiency of the antibiotic tobramycin against planktonic cells and biofilms [96]. Most interestingly, furanones have been shown to repress numerous QS‐regulated virulence genes and production of concomitant virulence factors, including LasA protease and elastase B (encoded by *lasA* and *lasB,* respectively), rhamnolipid (encoded by the *rhlAB* operon) and phenazine biosynthesis (encoded by the *phzA‐ G* operon) [96].

Other antibiofilm agents under investigation include nitric oxide (NO) which has recently been discovered to induce dispersal of *P. aeruginosa* biofilms by mediating an increase in bacterial phosphodiesterase activity and a decrease in intracellular levels of the secondary messenger cyclic di‐GMP, thereby inhibiting signal transduction in bacteria [97]. NO was shown to disperse released cells, and the remaining biofilms displayed enhanced sensitivity towards antibiotics [97]. A recent study by Kimyon et al. showed that prodigiosin (a heterocyclic bacterial secondary metabolite secreted by *Serratia sp.*) induces biofilm disruption and exhibits bactericidal activity against *P. aeruginosa* [98]. Prodigiosin‐mediated *P. aeruginosa* biofilm disruption occurs by the release of H2O2 and generation of hydroxyl radicals in the presence of copper ions that consequently cleaves/damages eDNA and alters *P. aeruginosa* cell surface hydrophobicity. Prodigiosin also induces bacterial cell lysis as a consequence of the oxidative stress generated by H2O2 [98].

## **4. Conclusions**

Extracellular molecules released by bacteria form a scaffold for biofilm formation. In *P. aeruginosa,* polysaccharides, eDNA and pyocyanin are major factors that integrate the biofilm matrix and provide defence against cationic antibiotics by binding to it [8]. On the other hand, molecules such as pyoverdine help promote bacterial growth and prevalence in the host by chelating iron [34]. Increased resistance to antibiotic therapies and the persistence of bacterial colonisation within the CF lung is associated with bacteria‐secreted extracellular molecules. Novel treatment strategies seek to act on molecules that are essential for bacterial persistence such as biofilm constituents. Biofilm matrix disruption is associated with increased antibiotic susceptibility and clearance of bacteria. Current antibiotics strategies target growth inhibition without cleaving the biofilm matrix, whereas other strategies including DNase I and GSH cleave or disrupt biofilm matrix constituents, but have no bactericidal activity. In CF patients, the severity of disease due to *P. aeruginosa* infection is the leading cause of death, so there is an urgent need to develop new strategies that could disrupt bacterial biofilm matrix and facilitate bactericidal activity, ultimately allowing for repair and re‐growth of lung epithelial tissue. The combination of biofilm‐disrupting agents with traditional antibiotics could serve as a new line of therapy for CF patients in the near future.

## **Author details**

Theerthankar Das1,2\* and Jim Manos1

\*Address all correspondence to: theerthankar\_das11@rediffmail.com

1 Department of Infectious Diseases and Immunology, Sydney Medical School, University of Sydney, Sydney, Australia

2 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Syd‐ ney, Australia

## **References**

[1] Jeffrey BL, Carolyn LC, Gerald BP. Lung infections associated with cystic fibrosis. Clinical Microbiology Reviews. 2002;15:194–222. DOI: 10.1128/CMR.15.2.194–222.2002. [2] Hoiby N. Recent advances in the treatment of *Pseudomonas aeruginosa* infections in cystic fibrosis. BioMed Central Medicine. 2011;9:1–7. DOI: 10.1186/1741‐7015‐9‐32

hydrophobicity. Prodigiosin also induces bacterial cell lysis as a consequence of the oxidative

Extracellular molecules released by bacteria form a scaffold for biofilm formation. In *P. aeruginosa,* polysaccharides, eDNA and pyocyanin are major factors that integrate the biofilm matrix and provide defence against cationic antibiotics by binding to it [8]. On the other hand, molecules such as pyoverdine help promote bacterial growth and prevalence in the host by chelating iron [34]. Increased resistance to antibiotic therapies and the persistence of bacterial colonisation within the CF lung is associated with bacteria‐secreted extracellular molecules. Novel treatment strategies seek to act on molecules that are essential for bacterial persistence such as biofilm constituents. Biofilm matrix disruption is associated with increased antibiotic susceptibility and clearance of bacteria. Current antibiotics strategies target growth inhibition without cleaving the biofilm matrix, whereas other strategies including DNase I and GSH cleave or disrupt biofilm matrix constituents, but have no bactericidal activity. In CF patients, the severity of disease due to *P. aeruginosa* infection is the leading cause of death, so there is an urgent need to develop new strategies that could disrupt bacterial biofilm matrix and facilitate bactericidal activity, ultimately allowing for repair and re‐growth of lung epithelial tissue. The combination of biofilm‐disrupting agents with traditional antibiotics could serve as a new line

stress generated by H2O2 [98].

116 Progress in Understanding Cystic Fibrosis

of therapy for CF patients in the near future.

\*Address all correspondence to: theerthankar\_das11@rediffmail.com

1 Department of Infectious Diseases and Immunology, Sydney Medical School, University of

2 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Syd‐

[1] Jeffrey BL, Carolyn LC, Gerald BP. Lung infections associated with cystic fibrosis. Clinical Microbiology Reviews. 2002;15:194–222. DOI: 10.1128/CMR.15.2.194–222.2002.

Theerthankar Das1,2\* and Jim Manos1

Sydney, Sydney, Australia

ney, Australia

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**Author details**


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