Pseudomonas aeruginosa Biofilm Formation in the CF Lung and Its Implications for Therapy

Numerous microorganisms colonize or are associated with the airways of individuals with Cystic Fibrosis (CF). Impairment of the mucociliary clearance in CF lungs leads to a greater number of microbes present for the simple fact that they are not physically removed (Gibson, Burns et al. 2003; Boucher 2004). Microbes thrive in the large mucus plugs in CF airways, probably due to optimal growth temperatures and the abundance of nutrients. Additionally, CF patients display defective antimicrobial peptide activity in their lungs, which can further enhance microbial colonization (Gibson, Burns et al. 2003; Boucher 2004). As a result of these abnormalities, CF lungs are extraordinarily susceptible to infection with a number of bacteria, fungi, and viruses, including Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Haemophilus influenzae, Aspergillus fumigatus, Candida albicans, Respiratory Syncytial virus, and Influenza Virus (Govan and Deretic 1996; Lyczak, Cannon et al. 2002; Saiman and Siegel 2004; Lipuma 2010). The relative abundance and rate of isolation of these various microorganisms varies over time. For instance, early in life, S. aureus is the most often isolated microbe, but by adolescence to young adulthood, P. aeruginosa becomes the predominate microorganism isolated from the airways (Gibson, Burns et al. 2003; Pressler, Bohmova et al. 2011).


Introduction
Numerous microorganisms colonize or are associated with the airways of individuals with Cystic Fibrosis (CF).Impairment of the mucociliary clearance in CF lungs leads to a greater number of microbes present for the simple fact that they are not physically removed (Gibson, Burns et al. 2003;Boucher 2004).Microbes thrive in the large mucus plugs in CF airways, probably due to optimal growth temperatures and the abundance of nutrients.Additionally, CF patients display defective antimicrobial peptide activity in their lungs, which can further enhance microbial colonization (Gibson, Burns et al. 2003;Boucher 2004).As a result of these abnormalities, CF lungs are extraordinarily susceptible to infection with a number of bacteria, fungi, and viruses, including Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Haemophilus influenzae, Aspergillus fumigatus, Candida albicans, Respiratory Syncytial virus, and Influenza Virus (Govan and Deretic 1996;Lyczak, Cannon et al. 2002;Saiman and Siegel 2004;Lipuma 2010).The relative abundance and rate of isolation of these various microorganisms varies over time.For instance, early in life, S. aureus is the most often isolated microbe, but by adolescence to young adulthood, P. aeruginosa becomes the predominate microorganism isolated from the airways (Gibson, Burns et al. 2003;Pressler, Bohmova et al. 2011).
The unique pathogenic characteristics of P. aeruginosa also promote efficient infection in the CF lung.Studies suggest that 20%-25% of CF infants have had a positive P. aeruginosa culture in the United States, and infection rates steadily increase with increasing age, such that 80% of adults 25 years old and older are chronically infected with P. aeruginosa (Gibson, Burns et al. 2003;Stuart, Lin et al. 2010;Woodward, Brown et al. 2010).Initially, individuals with CF experience intermittent infection, wherein transient colonization is followed by P. aeruginosa-free periods (Hoiby, Frederiksen et al. 2005;Stuart, Lin et al. 2010).Clinically, intermittent infection has been defined as either 1) "at least 1 isolate of [P.aeruginosa] with normal [P.aeruginosa] antibody levels," or 2) "[P.aeruginosa] cultures were positive in 50% or less of the 12 months" (Stuart, Lin et al. 2010).Other definitions are possible.These initial, intermittent strains are thought to originate from the environment, but there have also been cases of epidemic strains, demonstrating the potential for direct or indirect person-to-person spread (Salunkhe, Smart et al. 2005;Lipuma 2010;Mowat, Paterson et al. 2011;Saiman 2011).Following the intermittent colonization period, P. aeruginosa eventually establishes chronic infection (Gibson, Burns et al. 2003;Pressler, Bohmova et al. 2011).One of the hallmark characteristics of chronic P. aeruginosa infection in the CF lung is a switch in the colony morphology of P. aeruginosa isolates from a non-mucoid to a mucoid phenotype (Gibson, Burns et al. 2003;Yoon and Hassett 2004).It has been estimated that this conversion to mucoidy takes approximately 1.8 years to occur (Stuart, Lin et al. 2010).Because of mucoidy and other changes (as described below), the bacteria in the chronic state survive the intense immune reaction that occurs in the CF lung as well as the highdose antibiotic treatment given to CF patients to kill infecting microbes (Lyczak, Cannon et al. 2002;Gomez and Prince 2007).Because of their location within the mucus airway plugs, these mucoid bacteria are further protected from immune clearance (Worlitzsch, Tarran et al. 2002;Bjarnsholt, Jensen et al. 2009).Thus, once the chronic infection is established, P. aeruginosa persists for the life of the individual.This chronic colonization is the cause of much of the morbidity and mortality associated with CF (Gibson, Burns et al. 2003).
Chronic P. aeruginosa infection also contributes significantly to the economic burden associated with treatment and care for individuals with CF.Recent studies have calculated an average of $48,098 (US) in overall medical costs per CF patient per year in the United States, with similar estimates for some European countries (Ouyang, Grosse et al. 2009).Thus, with approximately 30,000 CF individuals in the United States (Gibson, Burns et al. 2003), CF accounts for over $1.4 billion (US) in medical expenditures in the United States alone.This calculation is a gross underestimate because it omits increased costs for transplantation, malnutrition, CF-associated diabetes, and other complications (Ouyang, Grosse et al. 2009).Additional analysis has estimated that the costs of treatment with the anti-Pseudomonal antibiotic tobramycin can reach $22,481 per person per year in the United States, which is nearly half of the aforementioned per person total expenditures ($48,098) (Woodward, Brown et al. 2010).It is evident, then, that development of more effective anti-Pseudomonal therapies might lead to decreased P. aeruginosa infection rates and decreased economic burden.Development of new drugs will come through a better understanding of the mechanisms used by P. aeruginosa to establish chronic infection. www.intechopen.com

Biofilm formation
It is generally well accepted that the chronic nature of P. aeruginosa in the CF lung results from the association of the bacteria into organized structures called biofilms (Gibson, Burns et al. 2003;Gomez and Prince 2007).Biofilms are communities of microorganisms bound to a surface, or to each other.During biofilm formation, bacteria undergo phenotypic, and often genotypic, changes that lead to self-aggregation and transition to a lifestyle distinct from their free-swimming (planktonic) counterparts (Costerton, Lewandowski et al. 1995).Numerous infectious states involve a biofilm component, including P. aeruginosa infection in the CF lung, infectious kidney stones, bacterial endocarditis, otitis media, chronic prostatitis, urinary tract infections, periodontitis, and medical device-related infections (Costerton 2001;Donlan and Costerton 2002;Parsek and Singh 2003).Often, these biofilm infections are chronic and/or recurrent.
While the characteristics of biofilms vary depending upon microbial species and growth conditions, there are several general properties that can be used to describe and define biofilms (Figure 1).Focusing specifically on P. aeruginosa, biofilm formation is initiated as planktonic bacteria bind to a surface via their polar flagella (O'Toole and Kolter 1998;Sauer, Camper et al. 2002), although pili have also been shown to mediate attachment to cells and other surfaces (Woods, Straus et al. 1980;Chiang and Burrows 2003).At this point, the bacterium can spin in place as the flagellum continues to rotate (Sauer, Camper et al. 2002;Hinsa, Espinosa-Urgel et al. 2003;Caiazza and O'Toole 2004).However, this initial attachment is reversible because polarly-bound bacteria can detach and swim away from the site of initial attachment (Sauer, Camper et al. 2002;Hinsa, Espinosa-Urgel et al. 2003;Caiazza and O'Toole 2004;Monds, Newell et al. 2007).This initial reversible attachment stage has been referred to as a "sampling" of the surface before full commitment to biofilm formation has been made (Caiazza and O'Toole 2004).Full commitment to the biofilm mode of growth is signaled as the initially-bound P. aeruginosa rods lay down on the surface along their long axis (Sauer, Camper et al. 2002;Caiazza and O'Toole 2004).The bacteria become irreversibly bound at this point and remain on the surface (Stoodley, Sauer et al. 2002)(Figure 1: Inset).Next, through type IV pilus-mediated twitching motility, the individual bacteria begin to associate into structures called microcolonies (O'Toole, Kaplan et al. 2000;Sauer, Camper et al. 2002).Through the efforts of specific signaling molecules called quorum sensing signals (described below), and the resultant change in gene expression, these microcolonies mature into large structures, which can reach a thickness of 100 μM, depending upon the growth conditions (Sauer, Camper et al. 2002).During the maturation process, the constituent bacteria begin to excrete polysaccharides, such that the bacteria in the mature biofilm are encased in a matrix of exopolysaccharides that they produced (Costerton, Lewandowski et al. 1995;Ryder, Byrd et al. 2007).DNA and protein have also been shown to be components of P. aeruginosa biofilms (Parsek and Singh 2003;Bjarnsholt, Tolker-Nielsen et al. 2010).Finally, as the biofilm ages, a sub-population of the bacteria break away from the biofilm bulk, revert to the planktonic state (become motile), and disperse from the biofilm (O'Toole, Kaplan et al. 2000;Sauer, Camper et al. 2002;Kirov, Webb et al. 2007;Harmsen, Yang et al. 2010).Dispersion events appear to be influenced by the production of virulent bacteriophage from dormant prophage (Rice, Tan et al. 2009).While very few studies investigating P. aeruginosa biofilm formation on living tissue have been performed, it is thought that these steps are conserved during infection (Hoffmann, Rasmussen et al. 2005;Anderson, Moreau-Marquis et al. 2008;Moreau-Marquis, Bomberger et al. 2008;Woodworth, Tamashiro et al. 2008;Moreau-Marquis, Redelman et al. 2010)(Figure 1).

Quorum sensing
As mentioned above, the production of quorum sensing (QS) molecules greatly influences biofilm maturation.QS is a method of self-recognition and cell density-dependent gene regulation (Cooley, Chhabra et al. 2008;Galloway, Hodgkinson et al. 2011).As a population of bacteria grows, small molecules (the QS signal) are produced and secreted.Once the density of the population is sufficiently high, the QS signal binds to an intracellular receptor that activates (or represses) a sub-set of genes (Galloway, Hodgkinson et al. 2011).Recognition of the QS signal is a stochastic process and the probability of a QS molecule binding to a receptor is low until the culture reaches a threshold density (Galloway, Hodgkinson et al. 2011).Hence, QS molecules allow bacteria to "sense the quorum", or the relative density of the population, and in this manner, they can coordinate their behaviors (Cooley, Chhabra et al. 2008).Because of the high bacterial density achieved in biofilms, QS plays a large role in regulating gene expression during biofilm development (Bjarnsholt, Tolker-Nielsen et al. 2010).P. aeruginosa contains 3 overlapping QS systems: Las, Rhl, and PQS (Wagner, Bushnell et al. 2003;Harmsen, Yang et al. 2010;Heeb, Fletcher et al. 2011).These systems regulate expression of virulence factors, exopolysaccharides, and other factors important for biofilm formation (Sauer, Camper et al. 2002;Wagner, Bushnell et al. 2003;Bjarnsholt, Tolker-Nielsen et al. 2010;Heeb, Fletcher et al. 2011).It has been found that QS systems are activated during biofilm maturation, and that mutation of QS genes leads to aberrations in overall biofilm architecture and sensitivity of the biofilm to stresses (Sauer, Camper et al. 2002;Bjarnsholt, Tolker-Nielsen et al. 2010;Harmsen, Yang et al. 2010;Heeb, Fletcher et al. 2011).As detailed below, much research is being devoted to the development of QS inhibitors for the disruption of biofilms.

The acute/chronic infection regulatory switch
There are numerous phenotypic changes that occur as planktonic P. aeruginosa transitions to the biofilm state, including a general decrease in expression of toxins and other tissue damaging virulence factors important for acute infections (Furukawa, Kuchma et al. 2006;Gooderham and Hancock 2009;Bjarnsholt, Tolker-Nielsen et al. 2010;Diaz, King et al. 2011).This suggests that P. aeruginosa displays 2 different infection phenotypes: an acute infection phenotype characterized by production of toxins, and a chronic infection phenotype characterized by biofilm formation and secretion of exopolysaccharides.In fact, recent evidence has revealed an inverse regulation of biofilm formation and virulence attributes associated with acute infections (Goodman, Kulasekara et al. 2004;Furukawa, Kuchma et al. 2006;Harmsen, Yang et al. 2010).For instance, expression of the AlgU alternative sigma factor leads to decreased expression of T3SS and increased production of the biofilm exopolysaccharide alginate (Wu, Badrane et al. 2004;Diaz, King et al. 2011).Similarly, the SadARS (also known as RocARS) three component regulatory system positively regulates biofilm maturation but inhibits the transcription of genes encoding components of the T3SS (Kuchma, Connolly et al. 2005;Kulasekara, Ventre et al. 2005).The LadS and GacS sensor proteins also enhance biofilm formation and exopolysaccharide production, but repress T3SS (Ventre, Goodman et al. 2006;Gooderham and Hancock 2009;Harmsen, Yang et al. 2010;Diaz, King et al. 2011).These sensors activate GacA, which, in addition to modulation of T3SS and exopolysaccharide production, can alter levels of pyocyanin, hydrogen cyanide, elastase, and lipase (Burrowes, Baysse et al. 2006;Gooderham and Hancock 2009).On the other hand, the regulatory protein RetS (also known as RtsM) inhibits the actions of GacA, and expression of RetS negatively influences exopolysaccharide production and biofilm formation but positively regulates T3SS gene expression and production of the toxins ToxA and LipA (Goodman, Kulasekara et al. 2004;Laskowski, Osborn et al. 2004).This alternate regulation of genes involved in acute toxicity and genes involved in biofilm formation suggests that during an infection, local environmental conditions might influence the infection phenotype of P. aeruginosa, producing an acute, toxic infection or a chronic, biofilm infection.Indeed, taking the human lung as an example, P. aeruginosa infection can lead to either acute pneumonia or, in the case of the CF lung, chronic colonization (Chastre and Fagon 2002;Furukawa, Kuchma et al. 2006).Depending upon the activity levels of the various acute and chronic regulators, a variety of intermediate bacterial phenotypes could occur on the acute to chronic spectrum.Investigation into this Acute/Chronic regulatory switch, and how it impacts human infections, is ongoing.

Evidence for biofilm formation in the CF lung
Numerous lines of evidence have confirmed that P. aeruginosa persists in CF lungs as biofilms.Perhaps most importantly, microscopic examinations of sputum samples and lung tissue sections have revealed the presence of microcolonies and large biofilm-like structures in the airways (Lam, Chan et al. 1980;Singh, Schaefer et al. 2000;Worlitzsch, Tarran et al. 2002;Bjarnsholt, Jensen et al. 2009;Hoiby, Ciofu et al. 2010;Hoiby, Ciofu et al. 2011).These biofilms can grow to larger than 100 μM in diameter (Worlitzsch, Tarran et al. 2002), and the bacteria within these biofilms have been identified as P. aeruginosa by fluorescent in situ hybridization (FISH) (Bjarnsholt, Jensen et al. 2009).Studies have suggested that in those individuals without sufficient antimicrobial therapy, these biofilms exist throughout the lungs, whereas in those patients that have had aggressive antibiotic therapy, biofilms are confined to the conductive zone and are absent from the lower airways (Bjarnsholt, Jensen et al. 2009).
As further confirmation of biofilm formation in the CF lung, P. aeruginosa QS signaling molecules have been identified and characterized in CF patient sputum samples (Singh, Schaefer et al. 2000).Importantly, the ratios and relative proportion of the different QS molecules was similar to that of P. aeruginosa biofilms grown on abiotic surfaces.These data suggest that the bacteria within CF airways receive signals that induce them toward a chronic biofilm infection phenotype.Indeed, P. aeruginosa isolates from CF airways display a number of characteristics indicative of biofilm formation.The conversion to mucoidy seen with chronically-infecting strains results from an overproduction of the biofilm exopolysaccharide alginate (Gibson, Burns et al. 2003;Ramsey and Wozniak 2005).Initially, steep hypoxic gradients in the mucus plugs of the CF airways stimulate the production of alginate (Worlitzsch, Tarran et al. 2002;Yoon and Hassett 2004).Over time, mutations in the gene mucA, encoding the membranelocalized anti-sigma factor MucA, result in constitutive expression of the alginate biosynthesis genes, through activation of the alternative sigma factor AlgU (Ohman and Chakrabarty 1981;Hughes and Mathee 1998;Hentzer, Teitzel et al. 2001;Ramsey and Wozniak 2005;Hoiby, Ciofu et al. 2010).It is thought that the constant oxidative stress encountered in the CF lung environment induces these mutations (Yoon and Hassett 2004;Hoiby, Ciofu et al. 2010).
CF lung isolates also accumulate mutations in T3SS genes (Dacheux, Attree et al. 2001;Jain, Ramirez et al. 2004;Smith, Buckley et al. 2006).Studies have shown an increasing number of T3SS defective isolates with increasing length of P. aeruginosa colonization (Jain, Ramirez et al. 2004).However, T3SS-competent bacteria have also been isolated from the lungs of CF patients (Dacheux, Toussaint et al. 2000;Jain, Ramirez et al. 2004;Jain, Bar-Meir et al. 2008), and it is possible that hyperactivation of AlgU might inhibit T3SS in these strains (Wu, Badrane et al. 2004;Diaz, King et al. 2011).Thus, both mutation and regulation appear to inhibit T3SS production in the CF lung during chronic infection, and this decrease in T3SS further supports the hypothesis that P. aeruginosa forms biofilms in the CF lung.
Intriguingly, many other mutations appear in genes involved in acute toxicity, including genes for lipopolysaccharide (LPS) biosynthesis, twitching motility, regulation of exotoxin A, pyoverdine synthesis, and QS factors (Smith, Buckley et al. 2006).A particular subset of chronic CF isolates, called small-colony variants (SCVs) due to their small colony morphology on agar plates, contains mutations in intracellular signaling proteins that lead to altered expression of polysaccharides, flagella, and type VI secretion (Starkey, Hickman et al. 2009).Thus, chronic CF P. aeruginosa isolates generally display a decrease in acute virulence.It is interesting to note that several studies have shown decreased virulence of chronic P. aeruginosa strains in mouse models of acute infection (Smith, Buckley et al. 2006;Bragonzi, Paroni et al. 2009).This adapted virulence of SCVs and other chronic CF isolates is thought to promote bacterial survival in the CF lung environment.T3SS toxins and other secreted factors are highly immunogenic, and mutation might protect the infecting bacteria from immune clearance.Furthermore, decreased bacterial toxicity would potentially inhibit the destruction of the biofilm habitat.Taken together, the presence of biofilm-like microcolonies and QS molecules, the increase in alginate production, and the decrease in T3SS and other acute infection phenotype factors strongly indicate that P. aeruginosa persists in the CF lung as biofilms.When comparing the biofilm formation cascade to the history of P. aeruginosa in an individual with CF, several intriguing parallels become apparent (Figure 2).Thus, the progression of intermittent infection, followed by chronic infection and conversion to mucoidy, is analogous to reversible binding, followed by irreversible binding and maturation of the in vitro biofilms, albeit chronic infection occurs on a much longer time scale than biofilms grown in the laboratory.

Consequences of biofilm formation in the CF lung
The formation of P. aeruginosa biofilms promotes chronic infection in the CF airways.As discussed in the following sections, several unique properties of biofilms contribute to this bacterial persistence, including antibiotic resistance, resistance to the activities of the immune system, and the high-frequency generation of bacterial mutants.Additionally, it has been found that P. aeruginosa contact with airway fluid leads to reduced flagella production (Wolfgang, Jyot et al. 2004), which potentially limits spread of the microorganism.Indeed, bacterial colonizers of the CF lung generally remain localized to the airways, and systemic spread rarely occurs (Govan and Deretic 1996).The biofilms that result from the growth and accumulation of infecting bacteria confer several survival advantages.

Antibiotic resistance
Biofilm bacteria are more resistant to many stresses than their planktonic counterparts (Costerton, Stewart et al. 1999;Stewart and Costerton 2001;Donlan and Costerton 2002;Dunne 2002;Patel 2005).In fact, biofilm bacteria can display up to 1,000 fold greater antibiotic resistance than planktonic bacteria (Mah and O'Toole 2001).This increased antibiotic resistance is due to several factors, including reduced antibiotic diffusion through the biofilm exopolysaccharide matrix, reduced growth rates of biofilm bacteria, the development of dormant persister cells, and the production of specific antibioticresistance factors (Donlan and Costerton 2002;Mah, Pitts et al. 2003;Lewis 2005;del Pozo and Patel 2007;Anderson 2008).All of these factors appear to influence the antibioticresistant nature of P. aeruginosa biofilms.In particular, alginate has been shown to retard the movement of cationic antimicrobial peptides, quaternary ammonium compounds, and aminoglycosides (including tobramycin) through P. aeruginosa biofilms (Nichols, Dorrington et al. 1988;Campanac, Pineau et al. 2002;Chan, Burrows et al. 2005).Additionally, P. aeruginosa produces biofilm-specific antimicrobial inhibitors (Mah, Pitts et al. 2003).Antibiotic treatment of P. aeruginosa biofilms also stimulates increased production of resistance factors, such as β-lactamases and antibiotic efflux pumps (Whiteley, Bangera et al. 2001;Bagge, Schuster et al. 2004).Further compounding the problem of biofilm antibiotic resistance, chronic P. aeruginosa CF isolates can accumulate mutations in antibiotic resistance genes, resulting in increased expression and activity of the resistance factors.These mutations confer increased survival in the presence of particular antibiotics (Smith, Buckley et al. 2006).The combination of these activities enables P. aeruginosa biofilms to survive the intense, often daily, antibiotic treatment regime taken by individuals with CF.

Immune resistance
P. aeruginosa biofilms also persist despite the high level inflammatory reaction that occurs in the CF lung (Gibson, Burns et al. 2003;Boucher 2004).The biofilm matrix acts as a shield preventing opsonophagocytosis of biofilm bacteria (Worlitzsch, Tarran et al. 2002;Gibson, Burns et al. 2003;Parsek and Singh 2003).In sputum and lung samples, neutrophils have been seen surrounding P. aeruginosa biofilms, but they have rarely been observed within the biofilms (Bjarnsholt, Jensen et al. 2009;Hoiby, Ciofu et al. 2010).P. aeruginosa can also counteract the effects of harmful chemical species produced by immune cells.Alginate has been shown to protect biofilm bacteria from reactive oxygen species (Cochran, Suh et al. 2000;Battan, Barnes et al. 2004;Gomez and Prince 2007;Hoiby, Ciofu et al. 2010), and denitrification pathways expressed in P. aeruginosa can metabolize reactive nitrogen intermediates, such as nitric oxide (Davies, Lloyd et al. 1989;Yoon and Hassett 2004).Thus, neutrophils appear to be recruited to CF lung biofilms, but the bacteria are protected from attack.Of greatest concern, lysis of spent neutrophils has been shown to add to biofilm viscosity and volume due to the release of DNA and protein (Walker, Tomlin et al. 2005;Parks, Young et al. 2009).

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Chronic biofilm growth might also inhibit immune recognition of P. aeruginosa.Reduced production of flagella, T3SS, and acute phase virulence factors in biofilms can lead to reduced antibody detection of these antigens (Adamo, Sokol et al. 2004;Jain, Ramirez et al. 2004;Wolfgang, Jyot et al. 2004;Smith, Buckley et al. 2006;Starkey, Hickman et al. 2009).Furthermore, some chronic P. aeruginosa isolates display a modified LPS, which can further contribute to immune evasion (Ernst, Yi et al. 1999).

The insurance hypothesis
Biofilm formation can also result in genetic diversity.In addition to mutations in virulence factors and antibiotic resistance factors, a host of other P. aeruginosa genes are mutated within biofilms (Boles, Thoendel et al. 2004;Smith, Buckley et al. 2006;Starkey, Hickman et al. 2009).It is thought that the constant stress encountered in the CF lung environment leads to DNA damage, and hence mutations.Several studies have found mutations in DNA mismatch repair genes in chronic CF P. aeruginosa isolates, which enhances the mutation rate (Smith, Buckley et al. 2006;Doring, Parameswaran et al. 2011).It has been suggested that this genetic diversification with P. aeruginosa biofilms supports the "Insurance Hypothesis", which states that diversity within a population provides protection for the community as a whole against a wide range of adverse or changing conditions (Boles, Thoendel et al. 2004).In other words, in the CF lung, mutation of individual P. aeruginosa cells within a biofilm will give rise to subpopulations with resistance to a wide range of different stresses and the ability to grow in a variety of different environments.In fact, it has been shown that genetic diversity within P. aeruginosa biofilms confers protection from oxidative stress (Boles, Thoendel et al. 2004).In this manner, genotypic changes, along with antibiotic resistance and immune evasion, promote P. aeruginosa survival and chronic infection in the CF lung.

Seed for recurring exacerbations?
Considering the presence of a large persistent population of P. aeruginosa in the lungs of individuals with CF, it is possible that biofilms serve as a reservoir of bacterial pathogens that emerge during a pulmonary exacerbation (VanDevanter and Van Dalfsen 2005).Indeed, during an exacerbation, lung function decreases while the symptoms of bacterial infection increase (VanDevanter, O'Riordan et al. 2010).Clinically, exacerbations have been defined as a sudden worsening of symptoms requiring physician intervention and the need for altered antibiotic treatment (Rogers, Hoffman et al. 2011), although some clinicians and researchers argue for more objective criteria (Bilton, Canny et al. 2011).This definition implies that bacterial activity is a large part of an exacerbation.However, the role of bacteria during an exacerbation remains a mystery.Some studies have shown that bacterial densities increase during an exacerbation (Mowat, Paterson et al. 2011), while others report similar bacterial levels before and during an exacerbation (Stressmann, Rogers et al. 2011).It has also been suggested that a virulent sub-population of bacteria emerge during an exacerbation, thus leading to symptoms of acute infection (Jaffar-Bandjee, Lazdunski et al. 1995;Stressmann, Rogers et al. 2011).In support of this hypothesis, researchers have found increased levels of P. aeruginosa exoenzyme S, exotoxin A, elastase, and alkaline protease in sputum samples during exacerbations (Grimwood, Semple et al. 1993;Jaffar-Bandjee, Lazdunski et al. 1995).
Moreover, it has been shown that high-dose antibiotic intervention for exacerbations decreases bacterial density and results in improved pulmonary symptoms, indication that bacterial activity plays a large role in initiation and progression of an exacerbation (Jaffar-Bandjee, Lazdunski et al. 1995;VanDevanter, O'Riordan et al. 2010;Tunney, Klem et al. 2011).Thus, during an exacerbation, it is possible that some fraction of the biofilm bacteria disperses from the biofilm and reverts to the acute planktonic phenotype, which will cause more tissue damage and lead to greater immune stimulation (Figure 2).

Treatment of P. aeruginosa infections of the CF lung: Triumphs and challenges
Treatment of P. aeruginosa lung infections remains challenging.The best course of action might be prevention of infection through aggressive infection control procedures.These procedures are meant to prevent person-to-person transmission as well as transmission from contaminated surfaces.It has been found that sputum-encased P. aeruginosa can survive on inanimate surfaces for up to 8 days (Saiman and Siegel 2004).Thus, thorough cleaning and sterilization of clinical rooms, apparatuses, and home respirators is recommended (Saiman and Siegel 2004;Saiman 2011).Furthermore, healthcare workers should practice good hand and respiratory hygiene (Saiman and Siegel 2004;Hoiby, Ciofu et al. 2011;Saiman 2011).Isolation and separation of individuals infected with particular pathogens, such as P. aeruginosa and multi-drug resistant bacteria, has also been suggested to reduce patient-to-patient spread.Many clinics also encourage re-gowning and re-gloving with each new patient contact.However, despite the best infection control protocols, most CF individuals still acquire P. aeruginosa, either from environmental sources or from other CF patients.As discussed below, there are a number of antimicrobial therapies implemented to control lung infection with P. aeruginosa.

Antibiotic treatments
Numerous antibiotics have been used to treat CF lung infection with P. aeruginosa, although the aminoglycoside antibiotic tobramycin has most often been used and has been studied the most (Gibson, Burns et al. 2003;Ryan, Singh et al. 2011).In order to achieve high concentration in the airways, tobramycin and other antibiotics are often inhaled in a nebulized form (Ryan, Singh et al. 2011).Studies have investigated the efficacy of a number of inhaled antibiotics, including tobramycin, colistin, gentamicin, ceftazidime, cephaloridine, aztreonam lysine, taurolidine, and a gentamicin/carbenicillin combination (Ryan, Singh et al. 2011).The use of inhaled antibiotics, can lead to increased lung function and decreased exacerbation frequency over placebo (Ryan, Singh et al. 2011).During stable periods, inhaled antibiotics such as tobramycin or colistin can be given as chronic suppressive therapies to maintain low bacterial levels within the airways (Hoiby, Ciofu et al. 2011).An economics study estimated that increased usage of inhaled tobramycin would lead to increased cost for medication but decreased physician and hospital visits.This would have a net decrease in healthcare costs (Woodward, Brown et al. 2010).It has also been suggested that this maintenance therapy be supplemented with 2-week courses of intravenous (IV) antibiotic combinations every 3 months for added anti-Pseudomonal pressure (Hoiby, Ciofu et al. 2011).In addition to antimicrobial therapies, other medications such as DNase and hypertonic saline are widely used to increase airway clearance (Fuchs, Borowitz et al. 1994;Donaldson, Bennett et al. 2006;Elkins, Robinson et al. 2006;Parks, Young et al. 2009).
However, despite suppressive therapies, pulmonary exacerbations still occur.The types of antibiotics, dosing, and treatment schedules for exacerbation therapy vary greatly countryto-country and site-to-site.Synergy and a reduction in antibiotic resistance have been shown with thrice daily IV infusions of an aminoglycoside antibiotic and a β-lactam antibiotic (Bals, Hubert et al. 2011;Plummer and Wildman 2011).A recent study has found that twice daily treatments of tobramycin and ceftazidime are just as effective as thrice daily infusions, and this reduced treatment regimen can be safer and more convenient than a three times a day schedule.Studies have demonstrated that the bacterial response to antibiotic treatment is completed within 14 days (Adeboyeku, Jones et al. 2011), although in some cases, patients respond better to shorter or longer treatments (VanDevanter, O'Riordan et al. 2010;Plummer and Wildman 2011).Many more antibiotic treatment regimens are used in clinics and hospitals, and optimization of therapy for an individual exacerbation event often relies on symptoms and pulmonary function testing.Home-based IV antibiotic therapy of exacerbation has also been explored as an alternative to inpatient treatment.Athome therapy, while requiring specialized training for family members and friends, can reduce costs to families and hospitals, reduce incidence of hospital-acquired infections, improve disease manifestations, and can be more convenient for the affected individual (Balaguer and Gonzalez de Dios 2008).

Early colonization eradication
The period of intermittent infection, before the establishment of chronic P. aeruginosa biofilms, presents a unique opportunity for therapeutic intervention.Many studies have shown the efficacy of early aggressive antibiotic therapy to eradicate P. aeruginosa during this early colonization period.In the Copenhagen Model, which has been in place for over 20 years in the Copenhagen CF center, infected CF patients are given inhaled colistin and IV ciprofloxacin for 3 months (Hoiby, Frederiksen et al. 2005;Hansen, Pressler et al. 2008).80% of patients treated with this regimen were free of chronic P. aeruginosa infection for up to 15 years, and the bacterial isolates recovered exhibited little resistance to colistin and ciprofloxacin (Hansen, Pressler et al. 2008).In a different study of other European CF centers, treatment with inhaled colistin and IV ciprofloxacin for 3 months was found to be 81% effective (Taccetti, Campana et al. 2005).In this study, treated patients were completely free from P. aeruginosa infection for an average of 18 months, and 73% of subsequent P. aeruginosa infections were found to involve a genotypically distinct strain, suggesting that the original isolate had been eradicated (Taccetti, Campana et al. 2005).This treatment was also associated with reduced overall treatment costs and little development of antibiotic resistance (Taccetti, Campana et al. 2005).
There are numerous variations on eradication therapies, and many studies evaluating the efficacy of these treatments (Stuart, Lin et al. 2010;Hayes, Feola et al. 2011).In an effort to develop standardized treatment guidelines for early eradication therapies, there have been 2 large, multicenter studies: the Early Inhaled Tobramycin for Eradication (ELITE) study in Europe, and the Early Pseudomonas Infection Control (EPIC) program in the United States.The EPIC program, the results of which have yet to be published, is comparing standard culturebased therapy with twice daily inhaled tobramycin (300 mg) for 28 days every yearly quarter (Stuart, Lin et al. 2010;Hayes, Feola et al. 2011).The tobramycin-treatment group is further split into groups that additionally receive 14 days of either oral ciprofloxacin or oral placebo.The ELITE study treated participants for 28 days with twice daily inhaled tobramycin (300mg/5mL), and found that 93% of those treated were P. aeruginosa-free after 1 month (Ratjen, Munck et al. 2010).66% of participants were free of P. aeruginosa infection for 2 years (Ratjen, Munck et al. 2010).Similar results were obtained with individuals treated for 56 days.
The early promise of eradication therapy studies demonstrates that these treatments will likely enhance overall patient health and reduce healthcare costs related to P. aeruginosa infection.Indeed, such early eradication protocols have dramatically increased the age at which chronic P. aeruginosa infection is established (Hoiby, Frederiksen et al. 2005;Hansen, Pressler et al. 2008).Furthermore, eradication can be achieved regardless of the age of the patient, provided there has been no evidence of prior P. aeruginosa infection (Hayes, Feola et al. 2011).However, even with constant monitoring and treatment, intermittent P. aeruginosa infection will eventually give way to chronic biofilm formation.It is well established that once chronic infection is initiated, eradication of P. aeruginosa from the CF lung is essentially impossible (Hoiby, Frederiksen et al. 2005;Hansen, Pressler et al. 2008).

Antibiotic resistance
Perhaps not surprisingly, treatment of CF lung infections with large doses of antibiotics leads to high levels of antibiotic resistance.Resistance of a CF lung isolate to a particular antibiotic is generally associated with treatment with that antibiotic (Emerson, McNamara et al. 2010).Greater resistance leads to longer therapies, which consequently induces more resistance (Plummer and Wildman 2011).The formation of P. aeruginosa biofilms plays a large role in the emergence of antibiotic resistance.In addition to the general resistance of biofilms and other stresses (Costerton, Stewart et al. 1999;Stewart and Costerton 2001;Donlan and Costerton 2002;Dunne 2002;Patel 2005), the high mutation rate and generation of diversity that occurs in P. aeruginosa biofilms results in variant strains that display increased resistance to antibiotics, through mutation of antibiotic targets and increased production and activity of multidrug efflux pumps (Smith, Buckley et al. 2006;Bals, Hubert et al. 2011;Mowat, Paterson et al. 2011).Moreover, the SCVs that develop in biofilms, and appear with increasing prevalence in CF patient sputum samples over time, are more innately resistant to a multitude of antibiotics (Starkey, Hickman et al. 2009;Bals, Hubert et al. 2011).The appearance of multidrug-resistant P. aeruginosa clones, which are associated with more severe lung disease and declining lung function (Plummer and Wildman 2011), has lead to an antibiotic dilemma.Novel antimicrobial strategies must be developed to combat these multidrug-resistant P. aeruginosa infections in the CF lung.

Hope for the future: Novel therapies and model systems
Recent investigations into anti-Pseudomonal treatments, with an eye toward inhibiting P. aeruginosa biofilm formation, have led to new drugs and novel implementation strategies of existing antimicrobials (Table 1).Likewise, advances in CF lung infection model systems are leading to new insights into the nature of chronic P. aeruginosa infection in the CF lung.

Anti-biofilm strategies
Because biofilm formation plays an integral role in the persistence and antibiotic resistance of P. aeruginosa in the CF lung, many researchers have begun searching out ways to specifically destroy biofilms.The most obvious place to start in the development of anti-biofilm therapies is testing the efficacy of our current antibiotics against P. aeruginosa biofilms (Moskowitz, Emerson et al. 2011)(Table 1).In one retrospective analysis, the reported planktonic antibiotic susceptibilities of P. aeruginosa CF isolates were compared to the antibiotic susceptibilities of these strains grown as biofilms (Keays, Ferris et al. 2009).Those patients that were treated with antibiotics that could kill biofilmstate bacteria experienced lower treatment failure, decreased exacerbation risk, and decreased hospital stays (Keays, Ferris et al. 2009).Other studies have also shown that treatment tailored to biofilm susceptibility patterns can be effective (Moskowitz, Emerson et al. 2011).The recent development of the Minimum Biofilm Eradication Concentration (MBEC) Assay (also known as the Calgary Biofilm Device, distributed through Innovotech, Edmonton, CA), has permitted high throughput analysis of biofilm formation and biofilm susceptibilities of P. aeruginosa and other CF pathogens (Ceri, Olson et al. 1999;Tomlin, Malott et al. 2005;Davies, Harrison et al. 2007;Alfa and Howie 2009).Antibiofilm therapies developed using the MBEC or other biofilm assays can thus be of great clinical benefit.
Another promising avenue of anti-biofilm research is the identification of molecules that interrupt QS signaling (Geske, Wezeman et al. 2005;Rasmussen and Givskov 2006;Galloway, Hodgkinson et al. 2011)(Table 1).By interfering with inter-bacterial communication and gene regulation, these compounds can lead to the dispersion of biofilm bacteria as well as alter virulence factor production.Generally, QS inhibitors fall into one of three categories: 1) those that block production of the QS signaling molecule, 2) those that degrade the QS molecule, and 3) those that prevent bacterial recognition of the QS signal (Rasmussen and Givskov 2006).Many large screens of natural compounds have been completed or are taking place to identify novel QS inhibitors, and several active compounds have emerged from these studies.For instance, halogenated furanones from the alga Delisea pulchra and synthetic furanones have been shown to block P. aeruginosa QS and biofilm formation, and they lead to increased P. aeruginosa killing when used in combination with traditional antibiotics (Steinberg, Schneider et al. 1997;Hentzer, Riedel et al. 2002;Bjarnsholt, Tolker-Nielsen et al. 2010).Likewise, garlic extract, patulin and penicillic acid from Penicillium species, cis-2-decanoic acid from P. aeruginosa, salicylic acid, 4-nitro-pyridine-N-oxide (4-NPO), and solenopsin A from fire ant venom have all demonstrated inhibition of P. aeruginosa QS, and some have shown direct biofilm disruption activity (Rasmussen, Skindersoe et al. 2005;Rasmussen and Givskov 2006;Bjarnsholt, Tolker-Nielsen et al. 2010;Harmsen, Yang et al. 2010).Some of these QS inhibitors, such as garlic extract, patulin, 4-NPO, and furanones, have also displayed a therapeutic effect in models of P. aeruginosa infection (Rasmussen and Givskov 2006).Importantly, it is thought that resistance to QS inhibitors will not develop because these compounds do not directly affect bacterial growth, and thus exert little selective pressure (Bjarnsholt, Tolker-Nielsen et al. 2010).
Intriguingly, it has been found that some traditional antibiotics can affect biofilm formation and virulence factor production through QS inhibition or other mechanisms.For instance, azithromycin, ceftazidime, and ciprofloxacin were all shown to affect P. aeruginosa QS and virulence factor production (Skindersoe, Alhede et al. 2008;Bjarnsholt, Tolker-Nielsen et al. 2010).Sub-inhibitory concentrations of clarithromycin can also alter biofilm morphology (Wozniak and Keyser 2004).Thus, treatment with these antibiotics has yielded unexpected consequences for P. aeruginosa infection.

Newer antimicrobial strategies
Development of traditional antibiotics, and novel delivery methods for antibiotics, has also yielded some successes (Table 1).Several new antibiotics have recently hit the markets in various countries, including tigecycline, doripenem, and the 5 th generation cephalosporin ceftibiprole (Parkins and Elborn 2010).Many more antibiotics with potential efficacy against Gram-negative CF pathogens are in development, such as tomopenem, CXA-101, NXL104/ceftazidime, ACHN-490, CB182,804, and BLI-489/pipericillin (Parkins and Elborn 2010;Bals, Hubert et al. 2011).However, because many of these compounds are derivatives of existing antibiotics, resistance and toxicity might hinder further research on these novel therapies (Parkins and Elborn 2010).
On the other hand, the development of inhaled versions of existing antibiotics has been shown to improve delivery times and concentrate the antibiotic at the site of infection (Anderson 2010;Bals, Hubert et al. 2011;Ryan, Singh et al. 2011).Nebulized tobramycin has been used for years as an effective anti-Pseudomonal therapy, and many other inhaled antibiotic formulation have been studied (Ryan, Singh et al. 2011).Recently, Aztreonam Lysine for Inhalation has been approved in many countries for treatment of chronic CF lung infections, and studies have shown that use of this drug can improve quality of life and pulmonary function of CF patients, while decreasing P. aeruginosa burden and lower exacerbation severity (McCoy, Quittner et al. 2008;Retsch-Bogart, Burns et al. 2008;Anderson 2010;Parkins and Elborn 2010;Bals, Hubert et al. 2011).Work continues on other inhaled antibiotics, including aerosolized levofloxacin, fosfomycin/tobramycin, and inhalable dry powers of tobramycin and ciprofloxacin (Anderson 2010;Bals, Hubert et al. 2011).Inhalation of liposomally-encased antibiotics shows great promise for therapy of biofilm infections, as liposome delivery is thought to increase the penetration of biofilms (Smith 2005;Bals, Hubert et al. 2011).Patients treated with liposomally-encased amikacin showed improved lung function and reduction in sputum P. aeruginosa levels (Anderson 2010;Bals, Hubert et al. 2011).Similarly, liposomal encasement of tobramycin and polymyxin B might hold great promise as alternative treatments for chronic CF infections (Bals, Hubert et al. 2011).
Looking toward the future, there is great interest in identifying and developing novel chemical agents that disrupt bacterial metabolism, adhesins, virulence factor production, efflux pump activity, and bacterial intracellular signaling (Parkins and Elborn 2010;Bals, Hubert et al. 2011)(Table 1).Indeed, treatment with iron chelators (desferasirox and desferoxamine) or gallium, which is a non-reducible mimic for iron, can interfere with P. aeruginosa metabolism, resulting in biofilm disruption and protection against P. aeruginosa infection in animal models (Kaneko, Thoendel et al. 2007;DeLeon, Balldin et al. 2009;Moreau-Marquis, O'Toole et al. 2009).Inhibitors of P. aeruginosa T3SS have also been found www.intechopen.com(Aiello, Williams et al. 2010).Further screening of chemical compound libraries will likely reveal many additional molecules with anti-Pseudomonal activity.

Vaccination
Vaccines against P. aeruginosa have also been proposed as a potential therapy for preventing chronic CF infections (Table 1).Researchers have explored vaccines against P. aeruginosa LPS, alginate, flagella, outer membrane proteins, pili, T3SS components, DNA, and killed whole bacteria (Doring and Pier 2008).Many of these vaccines have been tested in clinical trials, with moderate efficacy.It is thought that clearance and prevention of P. aeruginosa infection by aggressive early eradication programs masks the true effectiveness of these vaccines, and none of them have reached the market (Doring and Pier 2008).Passive immunotherapy with monoclonal antibodies or pooled immune serum has also been investigated for anti-Pseudomonal therapy (Doring and Pier 2008).

Animal models
Researchers have tried for decades to develop an animal CF model that can maintain a chronic P. aeruginosa infection.Mice with various CFTR alleles and/or overexpression of βENaC have been tested as infection models, but P. aeruginosa is generally cleared from the lungs of these animals (Grubb and Boucher 1999;Mall, Grubb et al. 2004;Wilke, Buijs-Offerman et al. 2011;Zhou, Duerr et al. 2011).Mice and humans have very different lung physiologies, which most likely account for the inability to establish chronic infection in "CF" mice (Wilke, Buijs-Offerman et al. 2011).The recently developed pig and ferret CF animal models have been reported to develop spontaneous bacterial infections, and it is possible that chronic P. aeruginosa infection could be reproduced in these animals (Fisher, Zhang et al. 2011).However, these CF animals generally require surgery to correct the meconium ileus that develops in the young (Fisher, Zhang et al. 2011).Thus, the cost of these model systems is high.

Artificial sputum
In order to model the CF lung environment in vitro, several groups have created artificial sputum media.These media replicate the experimentally-determined composition of CF sputum samples, and they can support similar P. aeruginosa growth rates, gene expression patterns, nutritional preferences, and QS patterns as found in CF sputum (Sriramulu, Lunsdorf et al. 2005;Palmer, Aye et al. 2007).These media have also been useful for investigations of P. aeruginosa biofilm formation (Sriramulu, Lunsdorf et al. 2005;Garbe, Wesche et al. 2010).These models could lead to a better understanding of metabolic flux in P. aeruginosa biofilms in the context of CF lung infection.

Biofilms co-cultured with airway cells
Recently, several investigators have developed P. aeruginosa biofilms on cultured airway cells in vitro (Figure 3).These co-culture biofilm models were developed to more closely mimic the CF lung environment and potential signals that the bacteria receive from www.intechopen.commammalian cells during infection.In one model, P. aeruginosa bind to a monolayer or airliquid interface differentiated layer of immortalized CF-derived (CFTR ΔF508 homozygous) bronchial epithelial cells (Moreau-Marquis, Bomberger et al. 2008;Moreau-Marquis, Redelman et al. 2010).Fresh culture medium then flows across the system and large bacterial aggregates form on the epithelial cells.
The aggregates appear morphologically similar to biofilms formed on abiotic surfaces (Moreau-Marquis, Bomberger et al. 2008;Moreau-Marquis, Redelman et al. 2010).Furthermore, formation of these co-culture biofilms requires factors necessary for abiotic biofilm formation, and bacteria within co-culture biofilms display a pattern of gene expression consistent with that found in abiotic biofilms.In a different study, similar co-culture biofilms were formed by static incubation of P. aeruginosa and CF airway cells in the presence of arginine (Anderson, Moreau-Marquis et al. 2008;Moreau-Marquis, Redelman et al. 2010).Importantly, in both systems, the antibiotic resistance of the co-culture biofilms was greatly increased compared to both planktonic bacteria and abiotic biofilms (Anderson, Moreau-Marquis et al. 2008;Moreau-Marquis, Bomberger et al. 2008;Moreau-Marquis, O'Toole et al. 2009).It was also discovered that the co-culture biofilms displayed a different genetic response to tobramycin treatment than biofilms formed on plastic (Anderson, Moreau-Marquis et al. 2008).These data suggest that the surface upon which a biofilm forms can affect the properties of that biofilm, and they also offer clues into the high antibiotic resistance of P. aeruginosa biofilms that form in the CF lung.P. aeruginosa biofilms have also been shown to form on cultured mouse nasal septal epithelial cells (Woodworth, Tamashiro et al. 2008).

Conclusion
There have been a number of great advances in recent years in anti-Pseudomonal therapy of CF lung infections.In particular, early eradication treatments appear to show much promise in delaying the onset of chronic P. aeruginosa biofilm formation.The increasing arsenal against P. aeruginosa, including inhaled aztreonam and liposomal amikacin, will likely prove a benefit for P. aeruginosa treatment.Eradication of chronic P. aeruginosa may be possible, but it will take creative thinking.It is clear that new anti-biofilm treatments need to be discovered and implemented.The development of clinically-relevant models will further aid this process by providing appropriate systems for testing novel molecules.With renewed focus on the biofilm nature of the infection, much progress can be made toward eliminating chronic P. aeruginosa from the CF lung.

Fig. 1 .
Fig. 1.Proposed biofilm formation cascade on living tissue.It is thought that many of the steps involved in bacterial biofilm formation on non-living surfaces also occur as bacteria form biofilms on human cells.Inset: Initially P. aeruginosa bind reversibly (Rev) by their flagellum.Irreversible binding (Irr) begins when the bacteria lay down on the surface along their long axis.See text for details.

Fig. 2 .
Fig.2.Similarities between P. aeruginosa colonization of the CF lung and in vitro P. aeruginosa biofilm formation.The transition from reversible to irreversible attachment is mirrored in the transition from intermittent to chronic colonization.Similarly, just as bacteria in mature biofilms secrete exopolysaccharides, the conversion to mucoidy observed with CF isolates signals the overproduction of the exopolysaccharide alginate.Finally, it is possible that bacteria present during an exacerbation could represent bacteria that have dispersed from the biofilm and reverted to the planktonic phenotype expressing acute virulence factors.

Fig. 3 .
Fig. 3. P. aeruginosa biofilm microcolonies on cultured human CF-derived airway cells.Immortalized human bronchial epithelial cells, originally isolated from an individual with CF, were inoculated with P. aeruginosa constitutively expressing green fluorescent protein.Biofilm microcolonies (green) are attached to the surface of the cells.Bar = 50μm.

Table 1 .
Novel and Emerging Therapies for P. aeruginosa Infection of CF Lungs www.intechopen.com