Outcome and Prevention of Pseudomonas aeruginosa-Staphylococcus aureus Interactions During Pulmonary Infections in Cystic Fibrosis

Several microorganisms take advantages of the most common single gene disorder afflicting Caucasians and colonize the airways of cystic fibrosis (CF) patients. Although CF is a multisystem disorder, the associated mortality is mostly due to respiratory problems subsequent to chronic bacterial infections. CF is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) which is a cAMP-regulated chloride channel and a regulator of the activity of other channels. Consequently, there is a significant dehydration of the airway mucus when CFTR is dysfunctional. However, the exact reason why CF predisposes the lungs to microbial infections is not completely understood. It is thought that the obstruction of the airways by mucus and the proinflammatory status associated with this disease may be part of the explanation (Lyczak et al., 2002; Riordan, 2008).


Introduction
Several microorganisms take advantages of the most common single gene disorder afflicting Caucasians and colonize the airways of cystic fibrosis (CF) patients.Although CF is a multisystem disorder, the associated mortality is mostly due to respiratory problems subsequent to chronic bacterial infections.CF is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) which is a cAMP-regulated chloride channel and a regulator of the activity of other channels.Consequently, there is a significant dehydration of the airway mucus when CFTR is dysfunctional.However, the exact reason why CF predisposes the lungs to microbial infections is not completely understood.It is thought that the obstruction of the airways by mucus and the proinflammatory status associated with this disease may be part of the explanation (Lyczak et al., 2002;Riordan, 2008).
The recalcitrance of CF pathogens to antibiotic therapies is a major problem and is responsible for most of the morbidity and the mortality associated with CF (Chmiel & Davis, 2003;Lyczak et al., 2002).The ever-growing and overwhelming problems caused by antibiotic-resistant bacteria in human medicine have not spared CF patients.Antibioticresistant bacteria are frequently recovered from CF samples (George et al., 2009;Parkins & Elborn, 2010).The evolution of drug resistance has been accelerated by the extensive use of an antibiotic arsenal which has become limited due to insufficient innovation in the development of antimicrobials (Shah, 2005;Talbot et al., 2006).Indeed, the development of new classes of antibiotics has been almost abandoned for the last four decades and antibiotics that were introduced during this period usually consisted of new-generation molecules derived from existing antibiotics (Wenzel et al., 2005).Furthermore, long-term infections of the CF airways not only allow pathogens to adapt and circumvent the host immune system, but also allow them time to adapt to antibiotic therapies (Goerke & Wolz, 2010;Hogardt & Heesemann, 2010).Several mechanisms that decrease the susceptibility to antimicrobials are known to be at play in the CF airways.Conventional resistance mechanisms include the upregulation of bacterial efflux pumps and mutations of antibiotic target molecules (Høiby et al., 2010).In addition, the formation of persister cells (Mulcahy et al., 2010) and of small-colony variants (Goerke & Wolz, 2010;Proctor et al., 2006;Schneider et al., 2008) as well as bacterial growth in biofilms (Høiby et al., 2010;Mitchell et al., 2010aMitchell et al., , 2010b;;Wagner & Iglewski, 2008) are mechanisms that are well known for their involvement in difficult-to-treat infections (Galli et al., 2007;Stewart, 2002).
The formation of bacterial biofilms may explain, at least in part, the failure of many antimicrobial therapies.Biofilm-growing bacteria are highly persistent in CF because they appear to be physically protected from the host immune system and are inherently resistant to antimicrobials (Costerton et al., 1999;Davies & Bilton, 2009;Høiby et al., 2010;Stewart, 2002).These bacteria are thought to be as much as 1000 times more resistant to antimicrobials than their planktonic counterparts (George et al., 2009).Biofilms also represent complex integrated polymicrobial communities that are strongly influenced by cell-to-cell intraspecies and interspecies communications (Costerton et al., 1999;Hall-Stoodley et al., 2004;Stoodley et al., 2002).The different species of microbes found in polymicrobial infections such as in CF most probably respond to each other's chemical signals in order to survive and this is likely to influence the course of any particular infection (Brogden et al., 2005;Ryan & Dow, 2008).
In the surge to develop new therapies against CF pathogens, multiple aspects should be considered.Indeed, several diverse phenotypic adaptations confer a selective advantage in the host environment or toward antibiotic therapies.Also to consider are the various bacterial signals used for cell-to-cell communication that are involved in the establishment and the development of an infection.These mechanisms enable pathogens from polymicrobial communities to adjust their behavior in response to other neighboring bacteria and they deserve particular attention.This chapter aims to describe some of the mechanisms used by Pseudomonas aeruginosa and Staphylococcus aureus for their mutual coexistence and persistence in the host environment, and to emphasize those that are potential targets for the development of anti-pathogenesis therapies.

Microbiology of CF
Colonization of the CF airways by bacteria usually occurs in infancy and results in the establishment of chronic infections which eventually lead to respiratory failure and death (Harrison, 2007;Lyczak et al., 2002).Recent investigations have shown that the CF airways are colonized by complex polymicrobial communities constituted by numerous bacterial species and not only by the relatively few predominant species originally described that include P. aeruginosa, S. aureus, Heamophilus influenzae, bacteria from the Burkholderia cepacia complex and Stenotrophomonas maltophilia (Sibley & Surette, 2011).Notwithstanding this polymicrobial mixture, P. aeruginosa and S. aureus are still among the most prevalent and dominant bacterial species encountered in CF (Canadian Cystic Fibrosis Foundation, 2009;Cystic Fibrosis Foundation, 2009;European Cystic Fibrosis Society, 2007).It is also important to note that the prevalence of the different pathogens varies as a function of the age of patients, with S. aureus and H. influenzae being more frequent in early childhood and P. aeruginosa being more important as patients become older.Several other microorganisms such as Mycobacterium spp., pathogenic viruses, fungal pathogens (e.g.Aspergillus fulmigatus) and yeasts (e.g.Candida albicans) have also been recovered from the CF airways and may also contribute to disease (Harrison, 2007;Lyczak et al., 2002;Moskowitz et al., 2005).It should be kept in mind that the chronically infected CF airways represent a complex and diverse ecosystem and that the precise contribution of the different microbes to the morbidity of the disease remains undetermined, even for the less frequently encountered microorganisms (Harrison, 2007).QS in P. aeruginosa is controlled by the las and rhl N-acylhomoserine lactone (AHL) regulatory circuits and by the 2-alkyl-4-quinolone (AQ) system (Dubern & Diggle, 2008).In the las and rhl system, the lasI and rhlI gene products direct the synthesis of the homoserine lactones (HSL) 3-oxo-C12-HSL and C4-HSL, which interact with the transcription regulators LasR and RhlR, respectively, and activate target promoters.The las and rhl systems are hierarchically connected and interact together to regulate the production of several virulence determinants such as pyocyanin biosynthesis and biofilm formation (Dubern & Diggle, 2008;R. S. Smith & Iglewski, 2003).The AQ system is also interconnected with las and rhl and leads to the production of 2-heptyl-4-quinolone (HHQ) and the Pseudomonas quinolone signal (PQS).Both HHQ and PQS also have a role in cell-to-cell communication.However, PQS has a number of other biologically important functions (Dubern & Diggle, 2008).P. aeruginosa produces several other quinolone compounds, some of which, such as 4-hydroxy-2-heptylquinoline-N-oxide (HQNO), have antibiotic activity (Leisinger & Margraff, 1979;Lépine et al., 2004;Machan et al., 1992).It should also be kept in mind that the control of virulence factors by these systems is growth and environment dependent.This complex mixture of factors allows P. aeruginosa to modulate its behavior only when necessary (Williams & Camara, 2009).Interestingly, P. aeruginosa QS signals not only serve for intraspecies communications, but are also known to affect other microorganisms and the host (Williams & Camara, 2009).The association between the environment, P. aeruginosa QS systems and virulence is schematized in Fig. 1.Substantial adaptive and genetic changes occur in the genome of P. aeruginosa during chronic infections of the CF airways (Foweraker et al., 2005;E. E. Smith et al., 2006;Sriramulu et al., 2005).These changes cause bacteria to diversify and to exhibit characteristics differing from isolates found in the environment outside the body.The adaptation of P. aeruginosa during persistent infection of CF lungs can often lead to antimicrobial resistance, alginate overproduction and improved metabolic fitness.Overall, P. aeruginosa seems able to adopt a less aggressive profile by repressing its virulence factors and its immunostimulatory products, by growing in biofilms and by metabolically adapting to the microaerobic environment created by airway mucus plugs (Hogardt & Heesemann, 2010).Of these characteristics it appears that the formation of drug-resistant biofilms is strongly associated with the persistence of this bacterium in the CF airways (Høiby et al., 2010;Wagner & Iglewski, 2008).Also, non-mucoid strains that colonize CF patients can, with time, switch to the mucoid phenotype, show resistance to various antibiotics and become more difficult to eradicate from the airways (George et al., 2009).Other phenotypic variants associated with chronic infections and antibiotic tolerance have also been recovered from the CF airways such as persister cells (Mulcahy et al., 2010) and small-colony variants (Häußler et al., 1999;Schneider et al., 2008).The hypermutability of P. aeruginosa CF strains is thought to accelerate the development of antibiotic resistances and the adaptation required for longterm persistence in the host (Maciá et al., 2005).
Fig. 1.The virulence of P. aeruginosa is controlled by interconnected quorum-sensing (QS) systems that integrate environmental cues and influence virulence gene expression.The two N-acylhomoserine lactone systems las and rhl and the 2-alkyl-4-quinolone (AQ) system are hierarchically interconnected.The las system is usually activated first.These QS systems regulate the production of several virulence factors as a function of growth and in response to the environment not only to influence P. aeruginosa behavior but also other bacteria of the microbial community as well as host-pathogen interactions.S. aureus can live as a human commensal but also can be an opportunistic Gram-positive pathogen associated with significant mortality in hospitals (Talbot et al., 2006).This bacterium demonstrates an impressive versatility being able to infect several hosts, organs and body sites and cause both life-threatening and chronic infections (Archer, 1998;Goerke & Wolz, 2010).The treatment of S. aureus is seriously impeded by antibiotic resistance that has spread among staphylococci and is now being considered as a serious threat to the general population (Witte et al., 2008).
The presence of numerous virulence genes in the genome of S. aureus is thought to explain the ability of this bacterium to cause a broad spectrum of diseases (Archer, 1998).The genes involved in pathogenesis are tightly controlled by complex regulatory networks that allow the bacteria to express its virulence factors as a function of the bacterial population density and of its environment (Novick, 2003).One of the most characterized regulatory systems influencing the virulence of S. aureus is agr, the quorum-sensing accessory gene regulator, that upregulates the production of several extracellular proteins while downregulating many cell-surface proteins (Novick & Geisinger, 2008).Other regulatory networks that govern the expression of accessory genes in S. aureus include several two-component regulatory systems and transcription factors such as the alternative sigma factor SigB (Bronner et al., 2004;Novick, 2003).The activity of particular virulence regulators is thought to allow the expression of different sets of factors likely to be required at specific steps during infection or needed for different types of infections.
The contribution of S. aureus to the progression of disease in CF is less obvious than that of P. aeruginosa.Although the presence of this bacterium in the lower respiratory tract is considered as representative of a pathologic situation, there are still questions concerning the impact of S. aureus on the progression of the disease (Lyczak et al., 2002).However, recent data indicate that while the prevalence of P. aeruginosa has declined among CF patients over the past few years, the incidence of methicillin-suceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively) has increased in the USA (Razvi et al., 2009).Importantly, detection of persistent MRSA in the respiratory tract of CF patients has been associated with a decrease in survival and with a more rapid decline in lung function (Dasenbrook et al., 2008;Dasenbrook et al., 2010).Furthermore, MRSA bacteria often present a phenotype of mutiresistance to antibiotics (Chambers & Deleo, 2009;Pruneau et al., 2011) and are proficient in biofilm production (Molina et al., 2008).
Notwithstanding these findings, S. aureus often persists in the CF lungs for many months or even years and is the cause of recurrent and relapsing infections despite antibiotic treatments (Goerke & Wolz, 2010;B. C. Kahl, 2010).Long-term adaptation of S. aureus to the CF environment may occur through mutations (such as those causing the small-colony variant [SCV] phenotype) or through regulatory mechanisms that are still not well understood and that may result in the repression of the agr system and establishment of biofilms (Goerke & Wolz, 2010).SCVs of S. aureus are often isolated from chronic infections such as those of the CF airways (Kahl et al., 1998;Moisan et al., 2006;Proctor et al., 2006).The SCV phenotype is frequently caused by either mutations in genes required for electron transport (e.g.genes involved in the biosynthesis of hemin or menadione) or by mutations in genes enabling thymidine biosynthesis.Almost all the phenotypic characteristics of SCVs such as the slow growth (i.e., the formation of pin-point colonies when grown on solid media), the altered susceptibility to aminoglycoside antibiotics and the decreased production of exotoxins can be explained by their dysfunctional electron transport (Proctor et al., 2006).Several studies have reported that SCVs are less virulent than prototypical strains in vivo yet they can persist as well as the normal strains (Proctor et al., 2006).Our group and others have demonstrated that SCVs are relatively more persistent than their normal counterparts under antibiotic pressure (Bates et al., 2003;Brouillette et al., 2004).A recent study supports the theory that bacterial switching between wild-type and SCV phenotypes is required to sustain chronic infections (Tuchscherr et al., 2011).
The hypothesis that SCVs play a role in the development of chronic infections is well supported by in vitro experiments demonstrating that these variants have an increased ability to adhere to host tissue components (Mitchell et al., 2008;Vaudaux et al., 2002), to form biofilms (Mitchell et al., 2010a(Mitchell et al., , 2010b;;Singh et al., 2009Singh et al., , 2010) ) and to infect and persist within non-professional phagocytes (Mitchell et al., 2011b;Sendi & Proctor, 2009).These in vitro characteristics can be explained by the impact of the defective electron transport chain on the expression of virulence factors which seem to be mostly controlled by the activity of SigB rather than by the agr system in SCVs (Moisan et al., 2006;Senn et al., 2005).This altered activation of virulence regulators triggers a sustained expression of several genes encoding cell-surface proteins (e.g. the fibronectin-binding protein A fnbA gene) and the down-regulation of several exoprotein genes (e.g. the hemolysinhla gene) (Mitchell et al., 2008;Moisan et al., 2006).The sustained expression of fnbA has been associated with efficient binding of SCVs to fibronectin (Mitchell et al., 2008).In turn, the formation of a fibronectin bridge between S. aureus fibronectin-binding proteins and the 5 1 -integrin of eukaryotic cells (Sinha et al., 1999) probably explains the proficient cellular internalization of SCVs (Sendi & Proctor, 2009).Additionally, the low production of exoproteins and toxins by SCVs is likely to account for their increased persistence within host cells (Sendi & Proctor, 2009).Moreover, the increased production of biofilm by SCVs in comparison with wild-type strains may possibly be explained by the relative influence and the interconnection of the agr system and SigB that manipulate the maturation of protein-dependent biofilms (Lauderdale et al., 2009).Furthermore, SCVs have been shown to activate the innate immune response to a lesser extent than that observed with wild-type strains (Tuchscherr et al., 2010).This reduced ability of SCVs to induce an inflammatory response may also be attributable to the repression of the agr system (Grundmeier et al., 2010) and supports the idea that the SCV phenotype confers on S. aureus the ability to remain hidden from the host immune system inside nonprofessional phagocytes.However, to date, the in vitro observations that suggest that the formation of biofilms and the infection of host cells by SCVs allow S. aureus to establish long-term infections in CF patients have not been supported by in vivo experiments.Further work is required, especially in CF models of pulmonary infections, in order to fully understand the role of SCVs in the pathogenesis of S. aureus.Some of the characteristics of the normal and SCV strains are compared in Fig. 2. Fig. 2. The ability to switch from the normal to the small-colony variant (SCV) phenotype may have an impact on the virulence of S. aureus.(A) S. aureus can switch from a normal to a SCV phenotype.The normal and SCV phenotypes are associated with the dissemination of bacteria and acute infections, and with host tissue colonization and persistent infections, respectively.In comparison to normal strains, SCVs form pinpoint colonies, are less or nonhemolytic on blood agar plates and have an increased ability to infect non-professional phagocytes and to form biofilms (B).The host-cell infection pictures were prepared as previously described (Mitchell et al., 2011a(Mitchell et al., , 2011b) and show Calu-3 cells with actin colored in green and internalized S. aureus bacteria in red.Scale bar is 50 μm.Biofilm formation was evaluated by crystal violet staining as previously decribed (Mitchell et al., 2010a(Mitchell et al., , 2010b)).

Interspecies interactions between CF pathogens
It is now accepted that bacteria can sense signal molecules across species' boundaries and that this signaling influences the development of microbial communities and the virulence and persistence of pathogens during infections (Ryan & Dow, 2008).The clinical impact of polymicrobial infections is receiving more and more recognition from the medical community.There are indeed several examples of polymicrobial infections where at least two different microorganisms influence the course of the disease by synergistic, additive or antagonistic effects.Also, biofilms are thought to be of major importance for the pathogenesis of bacteria in the context of CF lung infections (Davies & Bilton, 2009) and can be considered as integrated and complex polymicrobial communities whose development is controlled by interspecies communications (Stoodley et al., 2002).Accordingly, and as previously underlined, infections of the CF airways is highly polymicrobic and there is growing evidence that many of these microorganisms interact (Sibley & Surette, 2011) as observed between P. aeruginosa and S. aureus (Biswas et al., 2009;Hoffman et al., 2006;Mashburn et al., 2005;Mitchell et al., 2010b;Qazi et al., 2006;Yang et al., 2011), P. aeruginosa and Burkholderia spp.(Bakkal et al., 2010;Chattoraj et al., 2010;Riedel et al., 2001;Weaver & Kolter, 2004), P. aeruginosa and S. maltophilia (Ryan et al., 2008), P. aeruginosa and C. albicans (McAlester et al., 2008), and more generally with a large proportion of the organisms found in the CF airways (Duan et al., 2003;Sibley et al., 2008).

Interactions between P. aeruginosa and S. aureus
Given that P. aeruginosa and S. aureus are highly prevalent and commonly co-isolated from the CF airways (Harrison, 2007;Hoffman et al., 2006), a great deal of effort has been directed toward the characterization of the interaction between them.Historically, synergistic interactions between these two species have been proposed and it was thought that S. aureus could sensitize the lungs for subsequent infections by P. aeruginosa (Lyczak et al., 2002).However, it is now clear that antagonistic interactions also exist between these bacteria as P. aeruginosa has the ability to provoke lysis of S. aureus cells (Mashburn et al., 2005;Palmer et al., 2005).The structures of some P. aeruginosa exoproducts that are known to impact on S. aureus are shown in Fig. 3.The antistaphylococcal activities of pseudomonal HQNO (see Fig. 3) and other 4-hydroxy-2alkylquinolines (HAQs) have been known for many years and are molecules that can generally suppress the growth of many Gram-positive bacteria (Lightbown & Jackson, 1954;Machan et al., 1992).Mashburn et al. (2005) suggested that P. aeruginosa could exploit this property to lyse S. aureus cells in order to use the released iron for growth in low-iron environments.Interestingly, HQNO also allows some Gram-positive to grow slowly in presence of aminoglycoside antibiotics (Lightbown, 1954) and the reason for this has remained a mystery for several years.The protection provided by HQNO against the inhibitory activity of aminoglycosides was finally found to be related to the ability of this molecule to inhibit the Gram-positive electron transport chain (Hoffman et al., 2006), which is required for aminoglycoside uptake (Bryan & Van Den Elzen, 1977).It was further shown that prolonged exposure of S. aureus to HQNO (or to P. aeruginosa) selects for SCVs (Hoffman et al., 2006).We subsequently demonstrated that HQNO produced by P. aeruginosa not only stimulates the formation of S. aureus biofilms but also modulates the activity of virulence regulators.More particularly, while increasing the activity of SigB and the expression of sarA, HQNO downregulates the expression of the effector of the agr system (RNAIII).This modulation of regulator activities is likely to influence the expression of several virulence factors as was shown for fnbA and hla (Mitchell et al., 2010b).The interaction of PQS with the cell envelope of P. aeruginosa is also known to trigger the release of membrane vesicles (MVs) which contain toxins, DNA, antimicrobials as well as HHQ, PQS and HQNO.It is thought that MVs are

Outcome and Prevention of Pseudomonas aeruginosa-Staphylococcus aureus Interactions During Pulmonary Infections in Cystic Fibrosis
189 important for the trafficking of PQS within the P aeruginosa population as PQS is poorly soluble in water.However, since MVs contain AQs including HQNO, they could also inhibit staphylococcal growth (Mashburn & Whiteley, 2005).Furthermore, P. aeruginosa produces other small-molecule respiratory inhibitors such as pyocyanin (see Fig. 3) and hydrogen cyanide which can affect the respiration of S. aureus (Voggu et al., 2006) and potentially select for the SCV phenotype (Biswas et al., 2009).Whether and how these other respiratory inhibitors influence the virulence of S. aureus remains to be determined.Fig. 4 shows the effect of P. aeruginosa on the growth of S. aureus, the HQNO-mediated emergence of SCVs and the stimulation of S. aureus biofilm production by HQNO.Bacteria were treated or not with 10 μg/ml of HQNO for 18 h and plated on agar containing gentamicin at 4 μg/ml to reveal the presence of SCVs.(D) HQNO and P. aeruginosa culture supernatants enhance biofilm production by a normal S. aureus strain.S. aureus biofilms were produced in the presence of 10 μg/ml of HQNO or in the presence of culture supernatants from P. aeruginosa or Escherichia coli (acting as a negative control) and were revealed by crystal violet staining as previously described (Mitchell et al., 2010a(Mitchell et al., , 2010b)).
It also appears that factors other than pseudomonal respiratory inhibitors are involved in the microbial interactions occurring between P. aeruginosa and S. aureus.Importantly, most of the antistaphylococcal activity found in P. aeruginosa culture supernatant is attributed to the staphylolytic endopeptidase LasA (Kessler et al., 1993).Qazi et al. (2006) demonstrated that some long-chain 3-oxo-substituted N-acylhomoserine lactones (AHL) such as 3-oxo-C12-homoserine lactone (see Fig. 3) can inhibit the growth of S. aureus as a function of their concentrations.The 3-oxo-C12-HSL also modulates the production of S. aureus exotoxins and cell-surface proteins through the repression of sarA and agr by interaction with a specific and saturable receptor(s) at the cytoplasmic membrane.Furthermore, the 3-oxo-C12-HSL is capable of undergoing internal re-arrangement to form the 3-oxo-C12-tetrameric acid, which also has an inhibitory activity against Grampositive bacteria (Kaufmann et al., 2005).A recent study investigated the formation of biofilms by P. aeruginosa-S.aureus co-cultures using a flow chamber system and confocal microscopy (Yang et al., 2011).This study demonstrated that wild-type P. aeruginosa facilitates S. aureus microcolony formation, but that mucA and rpoN mutants do not have this property and tend to outcompete S. aureus.A role for type IV pili in this phenomenon was proposed to occur through binding of extracellular DNA, and it was demonstrated that P. aeruginosa protects S. aureus against Dictyostelium discoideum phagocytosis when in co-culture biofilms.Some studies have been carried out to substantiate the interactions between P. aeruginosa and Staphylococcus epidermidis, a bacterium closely related to S. aureus.It was shown that some P. aeruginosa extracellular products (possibly polysaccharides) provide a competitive advantage over S. epidermidis (Qin et al., 2009).Furthermore, Pihl et al. (Pihl et al., 2010a(Pihl et al., , 2010b) ) demonstrated that some S. epidermidis strains were better fitted than others to coexist in biofilms with P. aeruginosa whereas the ability of P. aeruginosa to inhibit S. epidermidis biofilms varied between clinical isolates.These authors suggested that specific P. aeruginosa strains might be selected during infections to counteract chronic colonization by S. epidermidis in order to allow the persistence and dominance of P. aeruginosa.Whether the genetic background of each strain also influences interspecies interactions between S. aureus and P. aeruginosa and whether the outcome of these interactions varies in each CF patient remains to be determined.Recent studies, which demontrate that P. aeruginosa lasR mutants are frequently found in CF, indicate that some of the abilities of this bacterium to influence the virulence of S. aureus may indeed be lost during CF lung infections (D'Argenio et al., 2007;Hoffman et al., 2009).
Other questions remain open.For example, the real impact of S. aureus-P.aeruginosa coinfections in CF is not known and convincing clinical data as well as co-infection in adequate experimental models are clearly missing.Whether S. aureus and P. aeruginosa have a synergistic or antagonistic effect on the progression of the disease is not known and may potentially be influenced by the nature of each clinical isolate.Furthermore, whereas the effect of P. aeruginosa on S. aureus has been studied widely, almost no investigations address the potential effect of S. aureus on the virulence of P. aeruginosa.Interestingly, Korgaonkar and Whiteley (2011) proposed a model in which P. aeruginosa senses surrounding bacteria by monitoring the presence of exogenous peptidoglycan and responds to it by increasing the production of the virulence factor pyocyanin.

New therapeutic approaches in CF
Humanity is now facing a post-antibiotic era defined by its limited capability to combat microbial infections caused by antibiotic resistant pathogens.The increased number of nosocomial and community-acquired infections caused by microorganisms resistant to at least two classes of conventional antibiotics is becoming an important public health problem.The global rise of antimicrobial resistance combined with the slow discovery and approval processes for classical antibiotics has resulted in the present urgent need for new and innovative therapeutic approaches.One of the reasons for multidrug resistance is that current antibiotics were designed around a limited number of chemical scaffolds with few major modifications since the 1980s.This has left plenty of opportunities for antibiotic resistance mechanisms to develop and spread worldwide (Shah, 2005;Talbot et al., 2006).The identification of new antimicrobial targets and the development of novel therapeutic approaches may be facilitated by a better understanding of bacterial pathogenesis both at the single species and bacterial community levels.Antibiotic resistant bacteria are often recovered from CF patients but recalcitrance to antibiotic therapies is not only caused by bacterial genes that encode conventional mechanisms of antibiotic resistance.Indeed, the ever changing epidemiology of the CF airways (Razvi et al., 2009) and the acquisition of bacterial phenotypes inherently resistant to antimicrobials contribute to treatment failures.Although some antibiotics have recently been approved or are close to being approved for clinical use and are promising in the context of CF (Parkins & Elborn, 2010), this section will talk about some novel therapeutic approaches that take into account the pathogenesis and the adaptation of CF pathogens in the context of polymicrobial infections.However, in order to appreciate the entirety of the efforts directed toward the cure of CF, it should be kept in mind that a number of strategies other than those using antibiotherapies are also being considered (George et al., 2009).

Modulation of biofilm-forming microbial communities
The formation of biofilms by CF pathogens is thought to be a major virulence asset that promotes resistance both to antimicrobials and the host immune system (Costerton et al., 1999;Davies & Bilton, 2009;Høiby et al., 2010;Stewart, 2002).This fully justifies current research aimed at the development of therapeutic strategies that target biofilm formation and dispersal (Simões, 2011).Current management practices for P. aeruginosa infections include hygienic measures (Høiby & Pedersen, 1989), early aggressive eradication by antimicrobial therapy (Döring & Høiby, 2004), the use of nebulized DNAse (Frederiksen et al., 2006) and chronic suppressive antibiotic therapy (Bjarnsholt et al., 2009;Döring et al., 2000).However, even if these methods are undeniably successful to a certain extent, chronic infection ultimately occurs and a gradual increase in the level of resistance is observed (Ciofu et al., 1994).New therapeutic approaches specifically targeting biofilms should thus be useful in the context of CF lung infections and should decrease the occurrence or development of antibiotic resistance.
Most natural biofilms are polymicrobial (Stoodley et al., 2002) and the polymicrobial nature of CF lung infections needs to be considered in novel therapeutic approaches (Sibley et al., 2009;Sibley & Surette, 2011).Whereas some microbes may predispose the tissue toward the colonization by others, there may also be competition among bacterial populations and the removal of one pathogen could create an opportunity for another to expand (Harrison, 2007).On the other hand, tampering with the CF airway microbial community may lead to more effective treatment of chronic infections (Moore et al., 2005).Some data even suggest that a healthy gut microflora protects against some respiratory pathogens (Alvarez et al., 2001;Villena et al., 2005) and that care should be taken not to deplete the gut microflora when oral or intravenous routes of antibiotic administration are used.It is also conceivable that monitoring the population dynamics of polymicrobial infections can be used to predict the efficacy of antimicrobial therapy and to optimize treatments (Rogers et al., 2010).As such, the impact of antimicrobial chemotherapies on microbial communities should be assessed to detect unwanted effects.As an example, aminoglycosides, which are indicated for the management of acute exacerbations, the control of chronic infections and the eradication of recently acquired P. aeruginosa, have also been shown to induce bacterial biofilms in both P. aeruginosa (Hoffman et al., 2005) and S. aureus (Mitchell et al., 2010a).
The next sections provide examples of methods by which biofilm infections may be potentially overcome using different strategies that include targeting specific microbial phenotypes, influencing the pathogenesis of bacteria through the manipulation of cell-to-cell signaling and the enhancement of preexisting antimicrobial therapies against persistent forms of bacteria.

Targeting the persistent microbial phenotype
Bacteria often encounter unfavorable conditions during infection that limit bacterial growth and oblige the microorganisms to enter a quiescent state in order to persist within the host (Kolter et al., 1993;Nataro et al., 2000).Dormant bacteria are well-known for their tolerance to antibiotics normally active against rapidly dividing cells and often require prolonged periods of treatment (Coates et al., 2002;Neu, 1992).The highly refractory nature of biofilms to eradication by chemotherapy is thought to be at least partly attributable to the presence of metabolically inactive cells (Fux et al., 2005).The inefficacy of antibiotics against nonmultiplying bacteria thus results in slow or partial death, prolonges the duration of therapy and increases the emergence of genotypic resistances.Accordingly, targeting slow-growing or non-dividing bacteria should provide substancial therapeutic benefits.
Membrane-acting agents are usually active against bacteria in all their phases of growth and are thus good candidates for the development of antimicrobials that target slow-growing and non-dividing bacteria.As an example, the novel porphyrin antibacterial agents XF-70 and XF-73 were shown to remain highly active against this type of bacteria (Ooi et al., 2010).Hu et al. (2010) found that the small quinolone-derived compound HT61 was active against non-multiplying MSSA and MRSA by causing depolarization of the cell membrane and destruction of the cell wall.Antimicrobial peptides also interact and permeabilize the bacterial membrane and there is a good probability that they act on slow-growing bacteria that form biofilms (Batoni et al., 2011).Bioactive peptides may even have additional benefits for CF therapeutic applications due to their anti-inflammatory and immunomodulating activities (Scott et al., 2007;Zhang et al., 2005).
The resistance of biofilms to killing by most antimicrobial agents is thought to be more specifically attributable to the presence of non-dividing persister cells (Lewis, 2007).Persisters are dormant bacteria that present a global slowdown in metabolic processes, do not divide and are tolerant to antibiotics.In other words, they have the ability to survive the www.intechopen.com

Outcome and Prevention of Pseudomonas aeruginosa-Staphylococcus aureus Interactions During Pulmonary Infections in Cystic Fibrosis
193 effects of antibiotics without the use of drug-specific resistance mechanisms.Persisters have been described for S. aureus (Allison et al., 2011;Singh et al., 2009) and P. aeruginosa, with a recent study that shows the emergence of strains producing high levels of persister cells in CF patients (Mulcahy et al., 2010).Currently, there are only a few therapeutic strategies that are considered for targeting persister cells.One such is the combination of conventional antibiotics with an inhibitor of an essential persister protein (Lewis, 2007).Also, repeated-or pulse-dosing of antibiotics could allow persister cells to resuscitate in order to be killed by subsequent antibiotic administration.The development of specific pro-antibiotics which could irreversibly bind to bacterial targets is also being considered (Lewis, 2007).An outstanding recent study shows that the use of specific metabolic stimuli enables the killing of persister cells with aminoglycoside antibiotics by modulating the proton-motive force required for the uptake of these drugs.The proof of concept for the latter approach has been demonstrated against biofilms and also in a model of chronic infection (Allison et al., 2011).
As we have previously mentioned, SCVs are often associated with relapsing and persistent infections.In addition to the increased ability of these variants to form biofilms (Al Laham et al., 2007;Häußler et al., 2003;Mitchell et al., 2010aMitchell et al., , 2010b;;Singh et al., 2009Singh et al., , 2010;;von Götz et al., 2004), SCVs are well-known for their ability to infect and persist within non-professional phagocytes (Sendi & Proctor, 2009) and there is a limited choice of antibiotics able to act against intracellular bacteria.Nguyen et al. (Nguyen et al., 2009a) reported a considerable decrease in the efficacy of most antibiotics against intracellular SCVs in comparison to that seen against extracellular bacteria, but, most importantly, in comparison to their efficacy against the normal-phenotype bacteria.Nevertheless, the authors noted that four antibiotics (quinupristin-dalfopristin, moxifloxacin, oritavancin and rifampicin) were more effective in killing intracellular SCVs.In addition, we recently described the first known molecule to specifically target the SCV phenotype of S. aureus (Mitchell et al., 2011a).Tomatidine (TO) is the aglycone form of the plant secondary metabolite tomatine.The structure and the main biological activities of TO against S. aureus are presented in Fig. 5A.We found that TO has a bacteriostatic activity against SCVs, but not against normal strains.More importantly, we showed that TO has the ability to inhibit the replication of SCVs internalized in CF-like human airway epithelial cells (Mitchell et al., 2011a).The specificity of the action of TO against SCVs was linked to the dysfunctional electron transport chain of these variants.Acordingly, HQNO sensitized normal S. aureus strains to TO (see Fig. 5B), which suggests that TO may be especially effective in the context where P. aeruginosa and S. aureus co-infect a CF patient.Although TO causes a marked inhibition of protein synthesis in bacteria showing a dysfunctional electron transport chain, the exact mechanism of action of TO on SCVs remains to be elucidated.Other biological activities for TO are discussed below.

Targeting virulence
Another emerging concept in the development of novel therapeutic approaches is the possibility to modulate the expression of virulence factors that are thought to be of major importance in the establishment of a particular infection.Modulators or blockers of pathogenesis are particularly interesting because it is speculated that, since they do not inhibit growth or kill bacteria, their use will not yield a strong selective pressure for resistance development.In this context, most attention has been directed toward the interference of bacterial QS and cell-to-cell signaling to inhibit virulence or biofilm Bacteria were inoculated at 10 5 -10 6 CFU/ml and incubated 24 h at 35 o C with shaking.(C) TO (8μg/ml) inhibits the hemolytic ability of a normal-growing S. aureus strain.(D) TO may be used in combination with aminoglycosides (e.g., gentamicin [GEN]) to eradicate a population of S. aureus composed of normal and SCV bacteria.Bacteria were inoculated at 10 5 -10 6 CFU/ml and incubated 24 h at 35 o C with shaking in presence of 4 μg/ml of GEN and/or 0.12 μg/ml of TO.The clear test tubes show no bacterial growth.formation (Njoroge & Sperandio, 2009;Rasko & Sperandio, 2010).However, although targeting the QS systems of P. aeruginosa in CF infections may increase the susceptibility of biofilms to clearance by antibiotics (Hentzer et al., 2003), interference with the QS agr system of S. aureus may not be a good strategy since it could increase the formation of biofilms and increase bacterial persistence (Novick & Geisinger, 2008;Otto, 2004).The discovery of compounds that attenuate or abolish the cross talk between the QS systems of different bacterial species may be more promising.It remains to be determined whether interference with interspecies communications has the potential to decrease the overall virulence or the cohesion of polymicrobial communities and more particularly of those found in CF.
Several plant products have been shown to act as "virulence attenuators" of human pathogens (González-Lamothe et al., 2009).Virulence attenuators modulate the virulence or the ability of the bacterium to adapt to the host environment.This gives a competitive advantage to the host immune system.As an example, a garlic extract was shown to interfere with the QS of P. aeruginosa, to sensitize its biofilm to tobramycin treatments and to improve clearance of bacteria in a pulmonary mouse model (Bjarnsholt et al., 2005;Rasmussen et al., 2005).Ginseng extract was also shown to alter the virulence of P. aeruginosa by interfering with QS, destroying biofilms, promoting phagocytosis by airway phagocytes and by protecting animals from the development of chronic lung infections (Song et al., 1997a(Song et al., , 1997b(Song et al., , 2010;;Wu et al., 2011).Some of our own transcriptional analyses of bacteria exposed to plant products have shed light on the effect of TO on the expression of virulence factors by normal S. aureus strains (Bouarab et al., 2007).We demonstrated that TO causes a repression in the expression of many extracellular toxins and of RNAIII, the effector molecule of the agr system, and thus it inhibits the hemolytic activity of S. aureus.We further showed that TO inhibits biofilm formation by S. aureus SCVs, probably through the induction of bacteriostasis (Mitchell et al., 2009).We suggest that the overall negative effects of TO on the virulence and the growth of both normal and SCV S. aureus strains could be used in the management of both acute and chronic lung infections in CF patients.Fig. 5C shows the inhibitory effect of TO on the hemolytic ability of a normal S. aureus strain.
Other studies have also promoted the use of virulence factor-based therapies against S. aureus.For example, QS autoinducing peptide variants were shown to inhibit heterologous agr activation and were proposed as therapeutic agents (Novick & Geisinger, 2008).Also, a blocker of the synthesis of staphyloxanthin, the golden-carotenoid pigment of S. aureus that promotes resistance to reactive oxygen species and host neutrophil-based killing, increased the susceptibility of S. aureus to killing by human blood and the innate immune clearance in a mouse infection model (Liu et al., 2008).Other researchers have attempted to achieve virulence attenuation by manipulation of bacterial metabolism (Lan et al., 2010;Zhu et al., 2009).Another possible approach is to target the bacterial pathways for programmed cell death which have been identified in several species (Engelberg-Kulka et al., 2004).

Enhancing preexisting antimicrobial therapies
The use of synergistic combinations of antimicrobial compounds is an old strategy that continues to be tantalizing especially against the polymicrobial populations found in the CF airways.Accordingly, Høiby (2011) suggests that the effectiveness of combination therapies should be tested in the context of CF lung infections.Several combinations of old antibiotics indeed showed promising synergistic effects in vitro and/or in vivo such as fosfomycintobramycin, tobramycin-colistin and ciprofloxacin-colistin combinations, with the ciprofloxacin-colistin combination thought to be efficient even against P. aeruginosa biofilms (Høiby, 2011).A large screen of double and triple antibiotic combinations was tested on biofilm-grown B. cepacia and P. aeruginosa in order to identify effective antibiotic combinations for treatment of CF patients (Dales et al., 2009).Combinations of antibiotics may also be useful in order to improve the efficiency of antimicrobial treatments against intracellular SCVs.As an example, some drug combinations that included rifampicin were most effective against intracellular S. aureus of both normal and SCV phenotypes.(Baltch et al., 2008).Nguyen et al. (2009b) also showed additive or synergistic effects between oritavancin, moxifloxacin and rifampicin against intracellular SCVs.
The combination of antimicrobial agents with non-antibiotic compounds is also an attractive approach (George et al., 2009).Several plant products are "antibiotic potentiators" that can act as bacterial efflux pump inhibitors, cell wall-acting agents or membrane destabilizing agents to provide synergy with conventional antibiotics (González-Lamothe et al., 2009).Interestingly, we have recently demonstrated that TO potentiates the bactericidal activity of aminoglycoside antibiotics against normal S. aureus strains of diverse clinical origins and antibiotic susceptibility patterns (unpublished results).Although the mechanism(s) by which this effect occurs is yet unknown, TO may prove useful in combination therapy with aminoglycoside antibiotics in the treatment of CF lung infections as exemplified in Fig. 5D.According to Mohtar et al. (Mohtar et al., 2009), a vast number of other plant products with antimicrobial activity await discovery.

Conclusion
Complex polymicrobial communities colonize the CF airways and interspecies interactions are likely to play a role in the course of respiratory infections.P. aeruginosa and S. aureus are prevalent pathogens often simultaneously found in CF patients and for which microbial interactions that modulate virulence are already well documented.In communities and by using intraspecies and interspecies cell-to-cell communication, these pathogens have the ability to form biofilms and to adopt persistent phenotypes such as persister cells and SCVs.These phenotypes confer non-specific resistance to antimicrobials and to the host immune system.The development of novel therapeutic approaches that take into account polymicrobial communities and the various strategies that bacteria have elaborated to adapt and persist within the CF airways should help to eradicate chronic and life-threatening infections in CF.

Acknowledgment
François Malouin (FM) is supported by Cystic Fibrosis Canada and by a team grant from the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT) with Kamal Bouarab and Éric Marsault.Gabriel Mitchell was the recipient of an Alexander-Graham-Bell The authors would like to thank Brian Talbot for critical review of this chapter, Gilles Grondin for technical assistance as well as Alexandre Fugère, David Lalonde Séguin, Éric Brouillette, Isabelle Guay, Karine Pépin Gaudreau and Simon Boulanger for their involvement in the CF research program taking place in the FM laboratory.We thank Josée Lessard and André Cantin from the CF clinic and Eric Frost and the personnel from the clinical microbiology laboratory of the Centre Hospitalier Universitaire de Sherbrooke, and of course, we gratefully thank all CF patients for their willingness and enthusiasm in participating to this research.
www.intechopen.comOutcome and Prevention of Pseudomonas aeruginosa-Staphylococcus aureus Interactions During Pulmonary Infections in Cystic Fibrosis 185 2.2 Pathogenesis of S. aureus in CF

Fig. 4 .
Fig. 4. P. aeruginosa influences the phenotypic switching of S. aureus and may have an impact on its virulence.(A) P. aeruginosa promotes the emergence of SCVs from normal S. aureus strains through the production of respiratory inhibitors such as HQNO and pyocyanin.Dotted arrows indicate other potential interactions between these bacterial species.(B) P. aeruginosa inhibits the growth of S. aureus.(C) HQNO stimulates the emergence of SCVs in S. aureus.Bacteria were treated or not with 10 μg/ml of HQNO for 18 h and plated on agar containing gentamicin at 4 μg/ml to reveal the presence of SCVs.(D) HQNO and P. aeruginosa culture supernatants enhance biofilm production by a normal S. aureus strain.S. aureus biofilms were produced in the presence of 10 μg/ml of HQNO or in the presence of culture supernatants from P. aeruginosa or Escherichia coli (acting as a negative control) and were revealed by crystal violet staining as previously described(Mitchell et al., 2010a(Mitchell et al., , 2010b)).

Fig. 5 .
Fig. 5. Biological activities of tomatidine against S. aureus.(A) Tomatidine (TO) is a steroidal alkaloid molecule that inhibits both the extracellular and intracellular replication of SCVs, represses the expression of several agr-regulated exoproducts and potentiates the bactericidal activity of aminoglycoside antibiotics against prototypical S. aureus.(B) TO (8 μg/ml) inhibits the growth of a normal S. aureus strain in presence of HQNO (20 μg/ml).
Outcome and Prevention of Pseudomonas aeruginosa-Staphylococcus aureus Interactions During Pulmonary Infections in Cystic Fibrosis 197 Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada and received a research scholarship from FQRNT during the course of his doctoral program.