Aunado a lo anterior, obsérvese cómo, uno de los elementos que se introducen en la prestación de los servicios que involucren el uso del espectro es el de asegurar el pluralismo

Of the many human infections associated with biofilms, CRS is one of the most common, resulting in 18 to 22 million visits to general practitioners in the USA per year (Benninger et al., 2003). Furthermore, CRS affects approximately 10% of the adult European population (Hastan et al., 2011). Rhinosinusitis is an inflammation of the mucous membrane that lines the paranasal sinuses. The disease is divided into several categories, largely based on the length of time a patient has experienced symptoms of rhinosinusitis. Acute and recurrent acute rhinosinusitis last up to four weeks, and may consist of many episodes (between disease-free states) in the latter case. The transition to CRS is known as subacute rhinosinusitis and is defined as rhinosinusitis that has lasted four to twelve weeks. Rhinosinusitis is deemed ‘chronic’ if it has lasted twelve

41 Figure 1.5 Functional endoscopic sinus surgery. Chronic rhinosinusitis mucus is aspirated during FESS (A) and placed in a test tube filled with reduced transport fluid (B).The aspirate is a tenacious, thick, bloody mucus, although the appearance varies widely between patients.

42 consecutive weeks or longer. The symptoms patients experience are similar, and as a result the disease can be hard to classify, which is why classification is defined chronologically. In addition, the location of the infection can be based in one or more of the paranasal sinuses. There are several paranasal sinuses, including, maxillary, frontal, ethmoidal, and sphenoidal sinuses.

Treatment of the infection begins with medical therapy and only in severe cases is surgical therapy recommended (Ragab et al., 2004). Wood and Douglas (2010) advocate 3 week medical treatment with a corticosteroid (prednisone) and an antibiotic (e.g. doxycycline). After this period, patients use a sinus douche to administer saline, and also use a corticosteroid spray. Ultimately, if symptoms still persist after this regime, surgery is instructed. Although antibiotics are favoured by clinicians there is little evidence for their efficacy (Lim et al., 2010). Macrolides, such as roxithromycin, may be beneficial because of their anti-inflammatory effect (Wallwork et al., 2006). Furthermore, the use of the antifungal amphotericin B improved symptoms in 75% of patients tested (Ponikau et al., 2002). Nasal lavage with amphotericin B has been shown to have no treatment efficacy (Ebbens et al., 2006). Chronic rhinosinusitis is challenging to treat because it caused by a number of factors (discussed in the next paragraph). Surgical therapy, primarily the non-invasive procedure, functional endoscopic sinus surgery (FESS) (Figure 1.5A), is carried out in hard to treat cases, to open up the airways and relieve some symptoms. However, the efficacy of this treatment is unproven (Khalil and Nunez, 2006). An example of the material removed during FESS is shown in Figure 1.5B.

In all cases CRS is an inflammatory disease. However, the cause of the inflammation can be due to many factors, and it is likely that CRS is an overarching name for a number of different diseases with similar symptoms. For instance, the development of CRS has been linked to smoking (Hastan et al., 2011), allergies (Perez-Novo et al., 2005), viruses (Benninger et al., 2003), fungi (Lanza et al., 2006), bacteria (Benninger

et al., 2003), staphylococcal super antigens (Seiberling et al., 2005), and underlying

systemic diseases (Benninger et al., 2003). Increasingly over the last decade bacterial and fungal biofilms have been recognised as a major cause of CRS, with increasing evidence from several authors (see Table 1.3). It is important to note that microbial biofilms are not the sole cause of CRS, but it appears they are a significant factor in the pathogenesis of CRS. Furthermore, the presence of biofilms is independent of other clinical factors like allergies (Zhang et al., 2011).

43 Table 1.3 Bacterial biofilms on clinical mucosal specimens removed from sufferers and non-sufferers of chronic rhinosinusitis.

Reference Imaging Method CRS Mucosa Control Mucosa

(Bezerra et al., 2011) SEM 24/33 (73%) 13/27 (48%)

(Healy et al., 2008) FISH-epifluorescent microscopy 9/11 (82%) 2/3 (67%) (Hochstim et al., 2010) Hematoxylin-eosin staining 15/24 (63%) 1/10 (10%)

(Foreman et al., 2009) FISH-CLSM 36/50 (72%) 0/10 (0%)

(Psaltis et al., 2007) CLSM 17/38 (45%) 0/9 (0%)

(Ramadan et al., 2005) SEM 5/5 (100%) NDa

(Sanclement et al., 2005) SEM 24/30 (80%) 0/4 (0%)

(Sanderson et al., 2006) FISH-epifluorescent microscopy 14/18 (78%) 2/5(40%)

a

ND, not determined.

44 Evidence of microbial biofilms on the surface of sinus mucosa is produced through microscopic analysis of mucosal biopsies. A range of techniques have been used to visualise micro-organisms in chronically infected sinuses, including, FISH-CLSM (Foreman et al., 2009), hematoxylin-eosin staining (Hochstim et al., 2010), and SEM (Sanclement et al., 2005). They are present on the sinus mucosa of CRS patients in 45- 100% of cases (Table 1.3) (Foreman et al., 2012). The most convincing visual studies of biofilms on CRS patient mucosa is via the use of FISH stains, as these are selective for prokaryotic or eukaryotic organisms. Bacteria appear as punctate dots against large eukaryotic cells (Shields et al., 2013). Further evidence for the biofilm pathogenesis of CRS was revealed through testing isolated micro-organisms for their ability to form biofilms in vitro. Prince et al., (2008) cultured sinonasal aspirates in a Calgary Biofilm Detection Assay (CBDA) and 45 out of 157 were positive for biofilm formation. Again, biofilm formation was linked to surgical interventions. The CBDA has also been used more recently, with biofilm formation shown in 21% of patient samples (Zhang et al., 2011). Lastly, the simple microplate crystal violet assay has also been used to show biofilm formation in bacteria isolated from the paranasal sinuses of CRS patients (Bendouah et al., 2006). It is important to consider that the threshold set for biofilm formation will have a strong influence on the amount of biofilm-forming micro- organisms reported and there is no gold standard approach for this.

Although micro-organisms are implicated in the pathogenesis of CRS, the bacterial flora of healthy and diseased paranasal sinuses are largely similar (Araujo et al., 2007). There is a high prevalence of coagulase-negative staphylococci, Streptococcus spp., and

S. aureus in both groups. More recently, culture-independent techniques have been used

to study the microflora of CRS (Stressmann et al., 2011; Feazel et al., 2012; Boase et

al., 2013). Although molecular methods have greater detection sensitivity, culture-

dependent and culture-independent techniques appear to demonstrate similar micro- organisms in CRS disease (Feazel et al., 2012). It is unclear if specific pathogens are involved in disease manifestation. Pathogenic species like S. aureus, M. catarrhalis, P.

aeruginosa, and S. pneumoniae can be more prevalent in CRS patient sinuses (Boase et al., 2013). However, it seems increasingly likely that a biofilm phenotype contributes to

the symptoms of CRS, and it is unknown how biofilm formation begins. Although it is important to note that biofilms are found on the surface of healthy sinus mucosa (Table 1.3).

There are still a number of aspects of CRS microbiology that are unclear, including the prevalence of anaerobic micro-organisms (Ramadan et al., 2002) and the role of

45 fungi in facilitating disease (Ebbens et al., 2009). Fungi are either cultured in high incidence (Ponikau et al., 1999), or detected in few cases (Araujo et al., 2007). The fungal pathogenesis of CRS is distinct to biofilm-associated CRS, and probably affects a minor proportion of sufferers of the disease. Similarly, anaerobic micro-organism carriage is either found to be in a high number of cases (Brook, 1989) or very low (Doyle and Woodham, 1991). Culture technique will likely bias results because laboratories close to sample collection will facilitate greater survival of anaerobes. The oxygen content of sinuses can drop when blocked with extensive amounts of mucin (Carenfelt and Lundberg, 1977). Therefore, in extreme cases, anaerobic micro- organisms may be more common.

As previously discussed, biofilm-forming micro-organisms exhibit greater levels of antibiotic resistance, which may explain the lack of efficacy in treating CRS with antibiotics in some cases (Lim et al., 2010; Fokkens et al., 2012). Interestingly, biofilms are often observed in patients who have previously undergone FESS, which is suggestive of the difficulty in eliminating biofilm infections (Bendouah et al., 2006; Zhang et al., 2011) Novel ideas for eradicating microbial biofilms are required to improve the management of CRS. Extracellular DNA is a common characteristic of the biofilm matrices of micro-organisms. Therefore, targeting the eDNA molecule and reducing biofilm stability may increase treatment efficacy. Bacteria that are CRS- associated and have been dispersed or inhibited with DNase I include S. pneumoniae (Hall-Stoodley et al., 2008), Neisseria spp. (Lappann et al., 2010), P. aeruginosa (Whitchurch et al., 2002), S. aureus (Kaplan et al., 2012), and E. coli (Tetz and Tetz, 2010). In addition, Bordetella spp. have been dispersed from the upper respiratory tract of mice (Conover et al., 2011), highlighting the efficacy of delivering a DNase solution to a nasal biofilm. However, it is unknown how recently isolated bacteria from a clinical condition will respond to DNase treatment. It is also unknown if eDNA is important in CRS-associated biofilm-forming micro-organisms. At this early stage it would be useful to culture isolates in uncomplicated models to test their sensitivity to DNase enzymes.

No optimal therapy for CRS has yet been discovered. Medical therapy with antibiotics has limited efficacy (Lim et al., 2010), as does FESS (Khalil and Nunez, 2006). Nasal irrigation with saline solutions does have some benefits, although it is less effective than topical steroids (Harvey et al., 2007). Adding a chemical to a solution that can be delivered to the sinus that subsequently dissolves the biofilm is a concept that could improve treatment. One previous attempt of this using sterile water, citric acid and caprylyl sulfobetaine (zwitterionic surfactant) applied at pressure to biofilm

46 forming isolates from CRS was successful at causing biofilm reduction (Desrosiers et

al., 2007). There was a 99.9% reduction of P. aeruginosa biofilm mass. Given this

research was an in vitro study this cannot yet be applied in a clinical environment but it does suggest that this concept may work in vivo. Addition of the novel bacterial nuclease, NucB, to a saline solution could work in much the same way. Administered prior to surgery to soften or even dissolve the biofilm it could allow for less intrusive surgery. Remaining planktonic bacteria may be more susceptible to conventional antibiotics. However, before this application the efficacy of nuclease treatment needs to be researched in clinical isolates from CRS patient sinuses. An improvement in clinical practice would undoubtedly save health services considerable amounts of time and money. Furthermore, it would improve the quality of life for up to 10% of the adult European population.