**3. Bacterial infections**

can be devastating to the patient. In addition to the effects that decreased HCO3

that cannot be cleared from the airway, causing a buildup or mucus plug.

antibiotics and other disease treatments.

176 Cystic Fibrosis in the Light of New Research

**2.3. Pathology of the CF lung**

*PA*. This will be covered in more depth later in this chapter.

the expected for a healthy individual of the same age [16].

on the density of the mucus, the general inability to transport anions across the apical mem‐ brane also affects the airway mucus layer. The PCL that lines the cilia of the epithelial cells is very sensitive to changes in water concentration. When the cells are not exporting significant amounts of ions, the PCL will then lose water as a sequela, resulting in it becoming denser and reducing the effectiveness of ciliary beating. This leads to an overall larger amount of material

In addition to the buildup of mucus as the disease progresses, the patient will experience several other symptoms as well. Commonly, the bronchi become inflamed, caused by an overreactive response from the immune system due to both mucus buildup (containing a plethora of bacterial components such as virulence factors, DNA, and cell debris from lysed bacteria or airway epithelial cells) and a potential infection. While the infections will be covered in more detail later in this chapter, bacteria such as *Pseudomonas aeruginosa* (*PA), H. influen‐ zae,* and *S. aureus* have been found to infect CF patients early in life (roughly 1 year of age) [1], and even if eradicated once, will often arise from a re-infection later in life. *PA* is the organism most commonly associated with a decline in the clinical course of CF patients, as its ability to form biofilms and convert to a mucoid phenotype often provides a large level of resistance to

Interestingly, the CF lung will also develop an oxygen gradient in its luminal mucus [12, 13]. As mentioned earlier, the increased density of the mucus makes it more difficult for oxygen to diffuse across it freely and into the blood. While this has consequences for the overall health of the individual, it also has implications for growth of bacteria enmeshed within it. The oxygen gradient is severe enough that the basal layer of the mucus could be termed microaerobic, or in more severe cases, anaerobic. This leads to the growth and development of bacteria that would normally not be found in the lung, and eventually to the growth of the mucoid form of

As might be expected, the buildup of thick mucus in the CF lungs often has severe clinical implications The significant reduction or loss of mucus clearance often results in infection and leads to inflammation due to the dramatic ~1,500-fold increase in airway neutrophils [14]. Coupling the inflammation and buildup of mucus, it is not surprising that the overall lung capacity of CF patients decreases dramatically throughout life. This can be tested using a series of pulmonary function tests (PFTs) [15]. These often involve a spirometry test, which is a measure of the forced efflux volume in one second (FEV1), or how much air the patient can forcefully exhale in one second. This is a hallmark test for overall lung volume and strength. Clinically it has been shown that the FEV1 of a CF patient will be approximately 10% below

In older patients, the prolonged effects of the disease often lead to chronic infections. These invading organisms can then be cultured and analyzed to determine the best treatment strategy. While we will be covering the type of infections further in this chapter, it is important to note that clinically, this also affects the patient in other ways, primarily leading to inflam‐


levels have

While CF airway disease is based on the genetic mutation of the CFTR gene, this is not usually what leads to the morbidity and mortality associated with the disease. Rather, an infectious agent that grows under the physiological conditions created by this mutation will lead to detrimental symptoms and eventual death. In the lungs, the buildup of thick mucus leads to decreased clearance of bacteria and provides a nutrient rich medium with which they can grow and even thrive. This mucus becomes colonized relatively easily with potentially several different species of bacteria at once [19, 16, 20]. Considering that infection is the major source of morbidity and mortality for CF patients, much research has focused on this aspect of the disease and different means by which to eradicate it. However, time has shown that this is not quite as easy as hoped, but the advancements of alternate treatments are helping this issue.

Bacterial colonization of the lungs of CF patients has been known for many decades. However, our understanding of what bacteria colonize the lungs has evolved dramatically. Early research identified that there were several species of bacteria that could colonize the lungs easily, and were often found associated with CF patients. By far, the most common (and most linked to severe progression of the disease) was *PA*. This gram-negative bacterium is often considered an opportunistic pathogen, existing naturally in soil, water, and in some cases, as part of the flora of human skin. However, in cases where the bacteria come into contact with immune-deficient or severely compromised tissues, they can grow quite prolifically. A common example is found with burn wounds, where *PA* can colonize the exposed flesh and lead to the need for aggressive antibiotic treatment. This type of infection is often associated with a distinct "smell of grapes" and the potential for a green-blue color to develop, indicative of several of the toxins that *PA* produces such as the blue phenazine antibiotic, pyocyanin. There are additional phenotypes that are seen with lung colonizing *PA*, the most dramatic of which was found in 1964 by Doggett et al. [21]. In that work, the authors noted that the multiple strains of *PA* that they could isolate on agar from CF patients all had a similar, mucoid phenotype. Although, at the time, the significance of this was not known, this mucoid phenotype is tightly linked to the overall progression of CF lung disease. It is indicative of the organism's ability to create a biofilm, a highly resistant matrix of exopolysaccharide and bacteria that allows survival in harsh environments. We will cover the details of biofilms further into this chapter, but it is important to note the significance they play in CF disease progression.

In addition to *PA*, three other major strains of bacteria were found associated with the CF lung. The first of these is *Staphylococcus aureus*, a gram-positive organism that is part of the normal skin flora. Often, *S. aureus* is found colonizing the nares of hosts, where it is hypothesized that it can travel to the lungs. It is often the first bacterium identified in CF patients, potentially due to a lack of proper immune defense within the newborns diagnosed with CF [22, 23]. It often resides in the lungs for a prolonged period, although it can be eradicated with proper antibiotic treatment. Similar to *PA* acquiring a mucoid variant, *S. aureus* can also acquire a different phenotype. Small colony variants have been seen associated with lung cultures, which have shown decreased expression of many of the virulence factors associated with *S. aureus* [24]. This could indicate that the bacteria are attempting to avoid the host immune system, allowing survival in the lungs for a longer duration. What potentially worsens the overall clinical course is the acquisition of MRSA in the lungs [23]. Although it is more rare than its antibiotic-sensitive counterpart, it is possible to acquire this strain nosocomially, or even develop the strain independently through the constant use of β-lactam antibiotics to treat existing,methicillinsensitive *S. aureus* (MSSA) infections. Interestingly, it has been found that in situations where a patient is infected with both *S. aureus* and *PA*, treatment of one can lead to the prominence of the other [25]. When patients were given anti-staphylococcal antibiotics, it was found that *PA* infection could develop and fill the niche that the eradicated *S. aureus* no longer occupies. This is another unfortunate side effect of a *S. aureus* infection, that not only can its treatment lead to a potential development of MRSA but it can also cause an exacerbation of any existing *PA* infection. This is of great clinical importance to note for younger patients who have not yet been colonized with *PA.*

The second major strain historically found in addition to *PA* is *Haemophilus influenzae*. This bacterium is similar to *S. aureus* in that it is a normal commensal organism of the nasopharynx region, but can actually be found in normal lungs. As an individual progresses through life, the chances of being colonized with *H. influenzae* increases, rising from 20% in infants, to 50% of children, to more than 75% of adults [26]. This has led some to believe that *H. influenzae* is one of the earliest CF pathogens, and that it modifies the lung in a manner that allows for subsequent *PA* infection [27]. The forms of *H. influenzae* that can colonize an individual vary, depending on the presence or absence of a capsule around the bacterium. When a capsule is absent, the bacterium is referred to as non-typeable *H. influenzae* (NTHi), and this form is most associated with respiratory infections [26]. Within the CF lungs, the bacteria attempt to attach to the epithelial cells underlying the PCL. Once attached, the cells will trigger innate immune responses, which can lead to initial inflammation processes [27]. After this, NTHi will try to evade the immune response, using a series of enzymes to destroy antibodies, and its ability to create microcolonies, and to invade the epithelial cells [26]. For this reason especially, NTHi is an important pathogen to be aware of for treatment strategies.

The last of the historically major three bacteria associated with CF, *Burkholderia cepacia* is often considered the most dangerous lung pathogen requiring rapid and rigorous medical attention. Although first identified as an individual bacterial species related to *Pseudomonas*, it has since been reclassified as a complex of genomovars (*Burkholderia cepacia Complex*, BCC), which have similar characteristics [28]. BCC is generally considered one of the main causes of pneumonia in immunocompromised individuals, which includes CF patients. BCC has been found to coinfect individuals with other CF associated bacteria, such as *PA* [29]. The complex is also known to produce virulent factors, such as elastases and gelatinases, which can weaken cell-to-cell interactions [29]. In addition, BCC fills a unique niche in the CF lung by being able to invade macrophages that have made it to the lung. These tactics caused the bacterium to be of great concern when it was first identified in CF patients in 1984, but since then, epidemiological and clinical studies have greatly advanced treatment [28].

further into this chapter, but it is important to note the significance they play in CF disease

In addition to *PA*, three other major strains of bacteria were found associated with the CF lung. The first of these is *Staphylococcus aureus*, a gram-positive organism that is part of the normal skin flora. Often, *S. aureus* is found colonizing the nares of hosts, where it is hypothesized that it can travel to the lungs. It is often the first bacterium identified in CF patients, potentially due to a lack of proper immune defense within the newborns diagnosed with CF [22, 23]. It often resides in the lungs for a prolonged period, although it can be eradicated with proper antibiotic treatment. Similar to *PA* acquiring a mucoid variant, *S. aureus* can also acquire a different phenotype. Small colony variants have been seen associated with lung cultures, which have shown decreased expression of many of the virulence factors associated with *S. aureus* [24]. This could indicate that the bacteria are attempting to avoid the host immune system, allowing survival in the lungs for a longer duration. What potentially worsens the overall clinical course is the acquisition of MRSA in the lungs [23]. Although it is more rare than its antibiotic-sensitive counterpart, it is possible to acquire this strain nosocomially, or even develop the strain independently through the constant use of β-lactam antibiotics to treat existing,methicillinsensitive *S. aureus* (MSSA) infections. Interestingly, it has been found that in situations where a patient is infected with both *S. aureus* and *PA*, treatment of one can lead to the prominence of the other [25]. When patients were given anti-staphylococcal antibiotics, it was found that *PA* infection could develop and fill the niche that the eradicated *S. aureus* no longer occupies. This is another unfortunate side effect of a *S. aureus* infection, that not only can its treatment lead to a potential development of MRSA but it can also cause an exacerbation of any existing *PA* infection. This is of great clinical importance to note for younger patients who have not yet

The second major strain historically found in addition to *PA* is *Haemophilus influenzae*. This bacterium is similar to *S. aureus* in that it is a normal commensal organism of the nasopharynx region, but can actually be found in normal lungs. As an individual progresses through life, the chances of being colonized with *H. influenzae* increases, rising from 20% in infants, to 50% of children, to more than 75% of adults [26]. This has led some to believe that *H. influenzae* is one of the earliest CF pathogens, and that it modifies the lung in a manner that allows for subsequent *PA* infection [27]. The forms of *H. influenzae* that can colonize an individual vary, depending on the presence or absence of a capsule around the bacterium. When a capsule is absent, the bacterium is referred to as non-typeable *H. influenzae* (NTHi), and this form is most associated with respiratory infections [26]. Within the CF lungs, the bacteria attempt to attach to the epithelial cells underlying the PCL. Once attached, the cells will trigger innate immune responses, which can lead to initial inflammation processes [27]. After this, NTHi will try to evade the immune response, using a series of enzymes to destroy antibodies, and its ability to create microcolonies, and to invade the epithelial cells [26]. For this reason especially, NTHi is

The last of the historically major three bacteria associated with CF, *Burkholderia cepacia* is often considered the most dangerous lung pathogen requiring rapid and rigorous medical attention. Although first identified as an individual bacterial species related to *Pseudomonas*, it has since

an important pathogen to be aware of for treatment strategies.

progression.

178 Cystic Fibrosis in the Light of New Research

been colonized with *PA.*

Several other genera of microbes, including *Achromobacter*, *Pandorea*, *Alcaligenes*, *Stenotropho‐ monas*, and *Ralstonia,* have also begun to be found in CF sputum in the past 20 years. These bacteria are quite often associated with more advanced stages of the disease, indicating that they may need the fertile ground of the CF lung to which they become accustomed before they can thrive [19, 16]. *Alcaligenes* and *Stenotrophomonas* are increasingly found with a *PA* or BCC infection [19]. In addition, several fungal pathogens have been associated with CF as well, such as *Aspergillus fumigatus* and *Candida albicans*. While these fungi have not been the primary focus of CF microbial treatment, recent evidence suggests that they can, in fact, become a problem for CF patients. They have been associated with an increase in inflammation, as well as a possibly more severe disease progression. Both host and fungal factors are equally important in determining if the fungus will become pathogenic [30].

Although each of these pathogens has been studied as a single organism in the CF pathology, limited efforts have been made into looking at the interaction between the bacteria. Consid‐ ering that more often than not multiple genera of bacteria are found in the CF lung, this is an important aspect to consider. Research that has been studying this topic has focused generally on the interaction between *PA* and another species of bacteria, as this is the most common pathogen associated with CF. For instance, work has been conducted to elucidate the rela‐ tionship and interactions between *PA* and BCC. In these cases, initial reports showed that BCC and *PA* formed communal biofilms, where both microbes could be detected in a "biofilm-like" structure. They share similar quorum sensing molecules, and together could be promoting the growth of one another. However, further investigation began to see that these bacteria did not necessarily occupy the same biofilm structures [29]. In some cases, it could occur, and could be recapitulated in vitro. However, in other situations, the bacteria did not cooperate. It seemed that *PA* was producing a secreted chemical that prevented the growth of BCC nearby, which was suspected to be pyocyanin [29]. This suggests that all of the relationships in CF may not be symbiotic or even mutually beneficial. Perhaps it depends on the timeline of when the infections are established. However, more work is needed to determine the actual relationship between these bacteria. The entire web of bacterial networks in the CF lung is not trivial, but deciphering how it works could potentially allow for more effective treatment of lung infections.

While this paradigm of "the big three" bacteria for CF persisted for several decades, along with the recent knowledge that several other species could infect the CF lung, a paradigm shift occurred around 2008, when it was discovered that there were obligate anaerobes present in 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 anaerobic growth.

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.

As a CF patient progresses through life, the likelihood of culturing positive for a particular microorganism changes. This is due mostly to the changing environment in the CF lungs. This graph depicts the percentage of patients registered in the Cystic Fibrosis Foundation patient registry who tested positive for a particular bacterium, separated by age [4].

Given the presence of anaerobic bacteria, this indicates that current antibiotic regimens for CF patients may have to be revisited. Normal antibiotic treatments include tobramycin, kanamy‐ cin, and several other antibiotics of the aminoglycoside class. Those in this class are generally ineffective against anaerobic bacteria due to their mechanism of entry. Aminoglycosides rely on the ability of an organism to respire, using either nitrate or oxygen as the terminal oxygen acceptor [34]. For fermenting bacteria, such as *Actinomyces* [35] and *Prevotella* [36], that means that this class of antibiotics is ineffective. In the case of *PA*, the organism exists as a facultative anaerobe that does use nitrate as a terminal oxygen acceptor. Although that should allow for these antibiotics to work, reports are mixed on the efficacy of the treatment alone, noting the ability for the bacteria to resist the action through a phenomenon termed "impermeability" [37]. It has been observed in multiple studies that CF-related *PA* gains this ability, characterized by a lack of uptake [37]. This may be related to the innate ability of *PA* to produce protective biofilms in its mucoid state, a mechanism already shown to be protective against antibiotic treatment. It may also be due to the action of certain genes involved in the biofilm formation, as one study found that biofilms upregulated *tolA*, whose gene product can alter LPS structure in such a way as to make it more resistant to aminoglycoside recognition, and bacteria which did not produce as much of this protein were far more susceptible to aminoglycosides [38].
