**4. The major contributor:** *Pseudomonas aeruginosa*

the lungs of older CF patients [31]. Included in this group of bacteria were the genera *Prevotella, Veillonella, Propionibacterium*, and *Actinomyces* [31]. This indicated that at least part of the lung must become anaerobic during the disease, which added to the notion that the CF lung has a vast array of micro-environments within it, each of which can be colonized by different bacterial species. It also corroborated the evidence of anaerobic niches being produced in the mucus plugs, which to this point had only been associated with bacteria converting to

With the discovery of the aforementioned anaerobic bacteria, it became clear that there was most likely a temporal aspect of CF infections as well. Clinical evidence has shown that depending on the age of the patient, certain bacteria are more likely to be cultured with their sputum (Figure 3). Early in the patient's life, from birth until around ten years of age, it is common for patients to test positive for *S. aureus*; but later in life this infection seems to become less likely [25]. *PA* can also be acquired early in life, however, much effort is often put into trying to eliminate this infection. As a result, a patient may test positive for *PA* several times throughout their life, even if the infections are not chronic. However, often by the patient's teenage years, *PA* has established a persistent infection and may have already converted to its mucoid form [32], leading to 73% of adult CF patients harboring such an infection [33]. In contrast to the "major" bacteria of CF, the newly discovered anaerobic bacteria generally do not infect a patient until later in life. This is associated with the development of more anaerobic niches in the lung, which is indicative of mucus buildup and thickening. This occurs later in life as the detrimental effects of a defective CFTR build and the inflammation worsens. Although treatment would still depend on cultures from the patients, establishing a general timeline is beneficial for physicians who need to create a treatment plan for their patients, and

could allow for proactive treatment as the patient enters different phases of life.

**Figure 3.** Prevalence of respiratory microorganisms by age in 2013.

anaerobic growth.

180 Cystic Fibrosis in the Light of New Research

Perhaps the most problematic and dangerous of the bacteria associated with CF is *PA*. *PA* is a gram-negative opportunistic pathogen that can sometimes be found as part of the normal human skin flora. However, in cases where the immune system is compromised in some way, the bacteria finds a way to infect a host, which can lead to severe complications. It is also perhaps the most deadly bacteria to be associated with CF, as a patients life expectancy decreases by roughly 7 years if the patient cultures positive for *PA* [37]. The reasoning for this is that *PA* produces a large number of virulence factors that stimulate inflammation in the airway, and often converts to a mucoid phenotype that allows it to escape a number of host defenses and the use of many antibiotics.

### **4.1. The role of quorum sensing in PA infections**

To control the expression of its virulence factors, *PA* uses a bacterial "communication" system known as quorum sensing. This is not unique to *PA*, and has been shown to exist in a number of other bacteria, including *E. coli*, *S. pyogenes*, and *S. aureus* (another CF associated bacteria) [39]. However, much research has been conducted into the complex regulatory pathways associated with this system in *PA*, which controls not only the means by which the bacteria move in relation to each other, but also the expression of virulence factors that can affect the patient and the ability to convert into a biofilm [39]. Quorum sensing in *PA* is accomplished by at least two regulatory pathways, the *las* and *rhl* systems [40]. The *las* system is hierarchically first for sensing other molecules. The system is composed of two primary genes, *lasR* and *lasI*, respectively. The transcriptional activator gene *lasR*, responds to environmental cues to produce the transcription factor LasR. LasR on its own, however, cannot properly interact with any genes. It needs a cofactor, specifically the quorum sensing autoinducer 3-oxo-C12-HSL (PAI-1) produced by the lasI gene product. This molecule can be taken up from other nearby bacteria that have released it into the supernatant and especially in biofilms [41]. When PAI-1 enters the bacteria, LasR forms a complex that is then able to act as a transcription factor. It has two major targets in the system, *lasI* and *rhlR*. Importantly, by activating *lasI*, the system begins an autoinduction loop that will maintain it in the cell and allow for the spread of signal to other bacteria. The gene encodes an enzyme, LasI, which is able to produce the 3-oxo-C12- HSL necessary to activate LasR. More active LasR results in the production of more LasI and PAI-1, amplifying the signaling effect.

However, the system looping and increasing would mean nothing if there was not an output somewhere. This output happens to be the second major system for quorum sensing, the *rhl* system. This system behaves very similarly to the *las* system. After activation by the LasR-PAI-1 complex, the RhlR transcription factor is produced. This requires a different signaling molecule, C4-HSL (PAI-2), with which to form a complex. Once the initial signal is received, the complex is formed and activates RhlI, which produces more PAI-2, and this, in turn, perpetuates this signaling process. At the end of this process, the bacteria result in having a buildup of RhlR-PAI-2 complex, which can help upregulate transcription of a number of virulence factors, including *lasA* and *lasB* (elastase related enzymes necessary for invasion) [42, 40], *xcpP* and *xcpR* (components of the type II secretion system), and even the stationary phase sigma factor gene *rpoS*, a gene linked to antibiotic resistance in *PA* [40]. However, one of the most important secreted factors dependent on the *rhl* system is the production of rhamnolipids, for which the system was named. These are important virulence factors that *PA* produces that can lead to inflammation and associated immune responses.

Another important output of the *las* system is the Psedumonas quinolone signal (PQS) regulon. The PQS regulon is responsible for some of the phenotypic changes seen with *PA* once it begins to grow in communities [39, 40]. The regulon itself requires the induction of pqsR, the regulatory transcription factor for the rest of the genes. Once this is active, the transcription factor will homodimerize to activate the rest of the *pqs* genes, as well as the induction of phenazine genes (phnAB). Together, these will allow for the production of HHQ (2-heptyl-4 quinolone, a precursor to PQS) and PQS signals that will not only allow for the autoinduction of the pathway, but also for the eventual production of pyocyanin and pyoverdin, important siderophores that also lead to the green color that is often seen with *PA*-laden CF sputum. In addition, this activates the production of Hydrogen Cyanide (HCN), an important metabolite for *PA* as well as a potential toxic agent for the patient and other bacteria.

### **4.2. Alginate production and the conversion to mucoidy**

Although these quorum-sensing pathways are important for the virulence associated with *PA*, it is also important to note that this is not the only system involved in its infection of a CF patient. Perhaps more important for the chronic infection of patients are the genes and pathways involved in the conversion of *PA* from its normal phenotype to its mucoid form. This mucoid phenotype has been known to be associated with CF infection, especially during chronic infection of patients [43, 31]. It is characterized by the general loss of motility for the bacteria [44], and the overproduction of the viscous exopolysaccharide called alginate. Alginate surrounds the bacteria and creates a material that seems quite like mucus, hence the appropriate coining of the term "mucoid phenotype." Mucoid *PA* has also been associated with the production of biofilms and is involved in general resistance to several types of antibiotics and environmental stresses, such as dessication and nutrient depletion. However, this conversion is a complicated procedure that results in a signficantly altered regulatory network of several genes associated with alginate production.

patient and the ability to convert into a biofilm [39]. Quorum sensing in *PA* is accomplished by at least two regulatory pathways, the *las* and *rhl* systems [40]. The *las* system is hierarchically first for sensing other molecules. The system is composed of two primary genes, *lasR* and *lasI*, respectively. The transcriptional activator gene *lasR*, responds to environmental cues to produce the transcription factor LasR. LasR on its own, however, cannot properly interact with any genes. It needs a cofactor, specifically the quorum sensing autoinducer 3-oxo-C12-HSL (PAI-1) produced by the lasI gene product. This molecule can be taken up from other nearby bacteria that have released it into the supernatant and especially in biofilms [41]. When PAI-1 enters the bacteria, LasR forms a complex that is then able to act as a transcription factor. It has two major targets in the system, *lasI* and *rhlR*. Importantly, by activating *lasI*, the system begins an autoinduction loop that will maintain it in the cell and allow for the spread of signal to other bacteria. The gene encodes an enzyme, LasI, which is able to produce the 3-oxo-C12- HSL necessary to activate LasR. More active LasR results in the production of more LasI and

However, the system looping and increasing would mean nothing if there was not an output somewhere. This output happens to be the second major system for quorum sensing, the *rhl* system. This system behaves very similarly to the *las* system. After activation by the LasR-PAI-1 complex, the RhlR transcription factor is produced. This requires a different signaling molecule, C4-HSL (PAI-2), with which to form a complex. Once the initial signal is received, the complex is formed and activates RhlI, which produces more PAI-2, and this, in turn, perpetuates this signaling process. At the end of this process, the bacteria result in having a buildup of RhlR-PAI-2 complex, which can help upregulate transcription of a number of virulence factors, including *lasA* and *lasB* (elastase related enzymes necessary for invasion) [42, 40], *xcpP* and *xcpR* (components of the type II secretion system), and even the stationary phase sigma factor gene *rpoS*, a gene linked to antibiotic resistance in *PA* [40]. However, one of the most important secreted factors dependent on the *rhl* system is the production of rhamnolipids, for which the system was named. These are important virulence factors that *PA* produces that

Another important output of the *las* system is the Psedumonas quinolone signal (PQS) regulon. The PQS regulon is responsible for some of the phenotypic changes seen with *PA* once it begins to grow in communities [39, 40]. The regulon itself requires the induction of pqsR, the regulatory transcription factor for the rest of the genes. Once this is active, the transcription factor will homodimerize to activate the rest of the *pqs* genes, as well as the induction of phenazine genes (phnAB). Together, these will allow for the production of HHQ (2-heptyl-4 quinolone, a precursor to PQS) and PQS signals that will not only allow for the autoinduction of the pathway, but also for the eventual production of pyocyanin and pyoverdin, important siderophores that also lead to the green color that is often seen with *PA*-laden CF sputum. In addition, this activates the production of Hydrogen Cyanide (HCN), an important metabolite

Although these quorum-sensing pathways are important for the virulence associated with *PA*, it is also important to note that this is not the only system involved in its infection of a CF

PAI-1, amplifying the signaling effect.

182 Cystic Fibrosis in the Light of New Research

can lead to inflammation and associated immune responses.

for *PA* as well as a potential toxic agent for the patient and other bacteria.

**4.2. Alginate production and the conversion to mucoidy**

The most important gene involved in the process of alginate production is *algU* (*algT*), a major component of the 12-gene operon that results in the production of alginate. AlgU is a extracytoplasmic sigma factor that is expressed at a low level in nonmucoid bacteria, and this basal level of protein is sequestered by the membrane spanning anti-sigma factor, MucA (Figure 4). This interaction is very important for the eventual conversion to the mucoid state. When MucA normally sequesters AlgU in its nonmucoid state, the bacteria do not produce any significant amounts of alginate. However, under conditions of stress (oxidative, osmotic, heat shock), the bacteria will eliminate MucA through one of two means [45]. The first is the proteolytic destruction of MucA. In conditions where the bacteria are stressed due to nutrient depletion (iron, carbon, or phosphorus), dessication, or antibiotics, the bacteria can trigger the produc‐ tion of a protease that targets MucA. Upon the degradation of this protein, the bound AlgU is released into the cell to act as a sigma factor (σE,22), in combination with several other proteins, to upregulate the operon responsible for alginate production, including most of the *alg* genes, such as *algD*, *algC*, and many others. Once this is accomplished, the cell will produce and secrete alginate, allowing for expression of the mucoid phenotype.

Under normal growth conditions in *PA* (Panel A), MucA (Blue) interacts with periplasmic MucB (Green) to allow for cytosolic sequestering of AlgU (Red). This prevents AlgU from acting as a transcription factor, effectively keeping mucoid genes inactivated. However, in times of stress, *PA* can either inactivate MucA through proteolysis (Panel B) or hypermutation (Panel C). In these cases, MucA is no longer able to interact with the periplasmic MucB, and the result is the loss of sequestered AlgU, allowing it to now activate genes in the cell, including AlgD and related mucoidy genes. Adapted from Hassett et al., 2009 [46].

However, in cases where constitutive mucoidy is found, such as in CF patients chronically infected with *PA*, the phenotype is most often caused by a mutation within the mucA gene. This is suspected to still be an outcome of environmental stress (as the CF lung is often lacking water and may contain innate immune defenses). However, the result is not a temporary upregulation of alginate related genes, but rather, a hypermutable phenotype that will usually allow for mutations within the *mucA* gene to occur [45]. This hypermutability stems from the mutation of several genes associated with DNA repair, including the DNA mismatch repair system (MMR) and the deoxyguanine repair system (DO) [45], although errors from the DNA polymerase IV could also lead to these mutations. The most common mutation found associ‐

**Figure 4.** MucA interactions with AlgU.

ated with this is in *mucA*. One such mutation, *mucA22* (a deletion of 5 G residues from bases 431–436), is commonly found, although several others have been associated with this pheno‐ type. Because of these base deletions, the protein is truncated to 15.8 kDa, where it has lost its binding domain for AlgU. As a result, AlgU is constitutively active in the bacteria, resulting in a constant mucoid phenotype in vivo. A mucoid phenotype has also been associated with mutations in other *muc* genes, including *mucB*, *mucC*, and *mucD*, however, these are not as frequent.

Once the patient acquires mucoid *PA*, their clinical course usually diminishes dramatically. This conversion is often a result of chronic *PA* infection, which may not cause a drastic change in health immediately, but will cause chronic inflammation and lead to decreased lung function. Currently, much of the effort for treating CF infections is focused on dealing with biofilm associated bacteria, which includes the mucoid *PA*.

### **4.3. PA biofilm formation in CF patients**

ated with this is in *mucA*. One such mutation, *mucA22* (a deletion of 5 G residues from bases 431–436), is commonly found, although several others have been associated with this pheno‐ type. Because of these base deletions, the protein is truncated to 15.8 kDa, where it has lost its binding domain for AlgU. As a result, AlgU is constitutively active in the bacteria, resulting in a constant mucoid phenotype in vivo. A mucoid phenotype has also been associated with mutations in other *muc* genes, including *mucB*, *mucC*, and *mucD*, however, these are not as

Once the patient acquires mucoid *PA*, their clinical course usually diminishes dramatically. This conversion is often a result of chronic *PA* infection, which may not cause a drastic change in health immediately, but will cause chronic inflammation and lead to decreased lung function. Currently, much of the effort for treating CF infections is focused on dealing with

biofilm associated bacteria, which includes the mucoid *PA*.

frequent.

**Figure 4.** MucA interactions with AlgU.

184 Cystic Fibrosis in the Light of New Research

Herein, we will focus on the biofilm formation of *PA* on the biotic or abiotic surface, in vitro. There are five steps for biofilm development on this kind of surface (Figure 5). This process begins when (Step 1) the planktonic or free-living bacteria (Step 2) attach to the surface as show in Figure 5. Several genes and factors are required for this initial step of biofilm formation such as the expression of flagella, type IV pili, Cup fimbria, and the activation of sadB [47]. Next, (Step 3) cells begin to produce the extracellular matrix components that facilitate the bacteria joining together and forming microcolonies. The matrix contains polysaccharides, proteins and eDNA, which plays a role in the up to 1,000-fold increase in antibiotic resistance of the biofilms compared to planktonic bacteria cells [48, 49]. Then, (Step 4) the microcolonies will progress and mature into a thicker biofilm (maturation), thus resulting in the development of a gradient of oxygen in the biofilm. Here, the surface has a higher oxygen concentration than in the deeper part of the biofilm. In this case, the biofilm is composed of aerobic, micro-aerobic, and anaerobic environments. The factors that also play an important role in this maturation step are involved cell motility. For example, the creation of the mushroom cap-like structure (biofilm maturation) required *pilA* (a pilus component). This also involves parts of the quorum sensing system as it has been shown that a lasI mutant will form a flat and thin biofilm while *rhlI* or *pqsA* mutant form microcolonies lacking the mushroom caps [48]. Several genes have been reported to be involved in the biofilm development such as the response regulator of the GacA/GacS twocomponent regulatory system (*gacA*) that is involved with the extracellular matrix compo‐ nents, a sigma factor (*rpoS*) that regulates a number of genes in the stationary phase that overlap with genes regulated by quorum sensing, the alginate biosysthesis regulator (*algR*) that is involved in type IV-mediated twitching motility, and a regulator of exopolysaccharide and Type III secretion (*retS*) [48, 50, 51, 52]. Finally, (Step 5) biofilm dispersion will occur where the biofilm components have been broken down or modified to release the surface proteins resulting in changes of the biofilm structure and also the forming of a new biofilm [47, 53]. Many genes have been studied in regard to biofilm dispersion. One such gene is *rhlA*, one of the genes required for rhamnolipid biosysthesis, which has been reported to be required for the dispersion of the bacteria from the center of the mushroom structure [54]. Another gene involved is the biofilm dispersion locus (*bdlA*), which studies have shown may be a link between sensing environment cues, c-di-GMP levels and detachment of the biofilm [52].

In CF patients, 65%–80% of all microbial infections are biofilm related [48]. The CF lung provides a suitable environment for *PA* to infect and form a biofilm. The normal airway epithelia cells are hydrated by a mucus that contains a mixture of electrolytes, glycoproteins such as mucins, protein, and lipids. The failure to elucidate free-living *PA* by the inflammatory system combined with the bacterial defense mechanisms such as oxidative stress in a CF patient results in a vicious environment [55, 56]. This environment can be oxygen depleted as well, causing it to be considered micro-aerobic or anaerobic [5]. Several studies have supported the ability of *PA* to form a biofilm in the CF lung, such as one that used a microscopic study of CF sputum, showing an increased resistance to antibiotics and phogocytosis because of the higher production of EPS in the biofilm [46]. This is a common reason to initiate biofilm development, as *PA* biofilms resist phagocytosis by immune cells and are also more resistant

**Figure 5.** The five steps of *Pseudomonas* biofilm formation on a biotic or abiotic surface (in vitro).

to antibiotics than a free-living cell [55]. To form a biofilm in the CF lung, *PA* must penetrate the mucus layer and enter into a hypoxic zone, where it can then form a biofilm with a quick transition from aerobic to anaerobic metabolism by using the alternative electron acceptors in the mucus such as nitrate (NO3 - ), nitrite (NO2 - ) or nitrous oxide (N2O) through the respiratory process known as "denitrification" to support anaerobic growth. The ability of *PA* to be able to grow in the anaerobic condition is an important factor for the formation of biofilms in the CF patient. *PA* also can use alginate as an energy source for anaerobic growth [48]. Finally, these macrocolonies of *PA* are formed during a chronic infection of CF [53]. The summary of the biofilm formation in CF patients is shown in Figure 6. Some of the important genes that are involved in the denitrification system are nitrate reductase (*nar*), nitrite reductase (*nir*), nitric oxide reductase (*nor*) and nitrous oxide reductase (*nos*). These genes are under tight regulation in the chromosome. One gene that has been found to regulate this process, *oprF*, is particularly important. It has been found that antibodies against this protein are elevated in CF patients, suggesting that it is upregulated in the bacteria. In addition, an oprF null mutant formed poor anaerobic biofilms due to no NIR activity [53]. Quorum sensing (QS) also regulates the genes in the denitrification system. One study showed that *rhl* was required for an optimum balance of the denitrification pathway [13].

### **4.4. Genetic alterations during** *PA* **infection**

In addition to the changes that are phenotypically eminent such as during mucoid conversion, it is also important to note that additional genetic regulation is occurring in this environment. As mentioned earlier, the CF lung is not a hospitable environment for bacteria, due especially to the dessicated mucus layer, yet it is quite nutrient-rich. If the bacteria can benefit from this, then they will be able to survive and flourish in the lung. In order to do this, the bacteria must undergo several layers of genetic regulation in order to activate specific shunt pathways that will allow them to use the nutrients provided. In the case of *PA*, this has been studied exten‐ sively, but has required significant efforts to unravel, notably the creation of various synthetic media that represents CF lung ASL [57] as close as anything else to date.

An Overview of Infections in Cystic Fibrosis Airways and the Role of Environmental Conditions on… http://dx.doi.org/10.5772/60897 187

to antibiotics than a free-living cell [55]. To form a biofilm in the CF lung, *PA* must penetrate the mucus layer and enter into a hypoxic zone, where it can then form a biofilm with a quick transition from aerobic to anaerobic metabolism by using the alternative electron acceptors in


process known as "denitrification" to support anaerobic growth. The ability of *PA* to be able to grow in the anaerobic condition is an important factor for the formation of biofilms in the CF patient. *PA* also can use alginate as an energy source for anaerobic growth [48]. Finally, these macrocolonies of *PA* are formed during a chronic infection of CF [53]. The summary of the biofilm formation in CF patients is shown in Figure 6. Some of the important genes that are involved in the denitrification system are nitrate reductase (*nar*), nitrite reductase (*nir*), nitric oxide reductase (*nor*) and nitrous oxide reductase (*nos*). These genes are under tight regulation in the chromosome. One gene that has been found to regulate this process, *oprF*, is particularly important. It has been found that antibodies against this protein are elevated in CF patients, suggesting that it is upregulated in the bacteria. In addition, an oprF null mutant formed poor anaerobic biofilms due to no NIR activity [53]. Quorum sensing (QS) also regulates the genes in the denitrification system. One study showed that *rhl* was required for

In addition to the changes that are phenotypically eminent such as during mucoid conversion, it is also important to note that additional genetic regulation is occurring in this environment. As mentioned earlier, the CF lung is not a hospitable environment for bacteria, due especially to the dessicated mucus layer, yet it is quite nutrient-rich. If the bacteria can benefit from this, then they will be able to survive and flourish in the lung. In order to do this, the bacteria must undergo several layers of genetic regulation in order to activate specific shunt pathways that will allow them to use the nutrients provided. In the case of *PA*, this has been studied exten‐ sively, but has required significant efforts to unravel, notably the creation of various synthetic

media that represents CF lung ASL [57] as close as anything else to date.

) or nitrous oxide (N2O) through the respiratory

the mucus such as nitrate (NO3

186 Cystic Fibrosis in the Light of New Research


an optimum balance of the denitrification pathway [13].

**4.4. Genetic alterations during** *PA* **infection**

), nitrite (NO2

**Figure 5.** The five steps of *Pseudomonas* biofilm formation on a biotic or abiotic surface (in vitro).

**Figure 6.** *PA* biofilm formation in the CF lung. A shows the normal epithelia cells. B–F depict CF airway epithelia. B–C show the mucus formation as mentioned previously in the CF lung section of this chapter, resulting in the hypoxic zones in the CF lung. D–F illustrate the free-living cell bacteria moving into the hypoxic area (D). E shows the bacteria adjusting themselves from an aerobic to anaerobic condition and forming macrocolony, biofilm, in this environment. F shows the increasing amount of macrocolonies.

Early gene expression analyses showed several important changes in expression levels, but suffered from lack of application. Some of the first analyses were of general biofilm gene expression, which were important as a seminal work. For example, Whiteley et al. performed a transcriptional profiling analysis in 2001 that examined gene expression by *PA* biofilms grown on granite pebbles [38]. They discovered that only a small subset of genes showed any change in expression (73 genes), roughly splitting 50:50 into up- and downregulated genes. This was quite interesting, as considering the relatively large change in phenotype between normal *PA* and biofilm structure, it would seem that more changes would have had to occur. In addition, several genes promoting the resistance to certain antibiotics were upregulated, and when they treated the biofilms with antibiotics, a different gene expression profile was seen. This all suggested that biofilm bacteria might be able to respond better to antibiotics and environmental stresses than planktonic cells, as the biofilm is not only more inherently resistant, but the bacteria interior to the film experience a lower concentration of the stressor, allowing for genetic regulations and responses [38]. Following this paper, more attempts to analyze biofilm-related genes ensued. These studies involved mostly environmental biofilms, and not those associated with human infection. Eventually, as the prevalence of biofilms in disease became better known, it became important to discover if these changes in biofilm gene regulation were able to be transitioned over to this situation.

In this regard, several attempts to examine human-associated biofilm infections occurred, but it had to be verified that the gene expression difference was reliable, and not an artifact of the harvesting procedure. Some studies did well with this issue, collecting *PA* from patients and analyzing both that sample and in vitro grown cells. These differences did seem quite signif‐ icant, showing changes between the two samples that suggested a decrease in the regulatory machinery for biofilm cells rather than an upregulation of many of the biofilm associated genes (such as *muc, alg, rhl, hcn,* and *plc*) [58]. This was novel, as the biofilm conversion seemed much more complicated than simply via a dysregulatory pathway.

Other genes that were found to be changed in the CF lung associated samples seemed to actually be associated with the acquisition of nutrients in the surrounding area. For example, one such study found that there was an increase in genes associated with arginine metabolism and the glyoxylate shunt (a process found often with the β-oxidation of lipids to acetyl-CoA) [59]. This seems to indicate that the CF lung has a higher available amount of arginine and free lipids for degradation, potentially linked to the death of epithelial cells in the lung. This same study also found the cells generally change to a sessile state, where many of the genes associated with motility and chemotaxis are downregulated. This indicates that the cells initially colonizing the CF lung are able to prosper enough that the organisms can rapidly adapt to such conditions. This same down-regulation of motility genes is often seen with the conversion to a biofilm mode of growth. Once the bacteria have shifted to the mucoid state and are able to survive the thick, dessicated mucus, then they will be able to thrive in the lung. This can help explain why *PA* is often such a major problem for the CF patients.
