Our understanding of chronic rhinosinusitis (CRS) show biofilm and osteitis play a role in the disease’s pathogenesis and refractory. Studies point to its role in pathogenesis and poor prognosis. Outside the research laboratory, biofilm detection remains difficult and specific treatment remains elusive. It is believed that osteitis is a nidus of inflammation and occurs more commonly in patients with refractory CRS. However, osteitis may be exacerbated by surgery and a marker of refractory disease, not a causative agent. Surgery remains the mainstay treatment for biofilm and osteitis with mechanical disruption and removal of disease load providing the most effective treatment.
Key points
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Biofilm is a sessile colony of bacteria with unique phenotypic expressions that interacts with the immune system, producing inflammation and contributing to the disease’s refractory nature.
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Osteitis is predominately an inflammation of bone that is associated with refractory chronic rhinosinusitis (CRS).
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Both osteitis and biofilm are poor prognostic markers and are part of the inflammatory load.
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Surgery is currently the cornerstone of treatment for CRS; reducing inflammatory load with maximal ventilation of the affected paranasal sinuses facilitates postoperative medical treatment.
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Adjunctive treatments are currently being investigated with topical antibiotics being shown to be effective against biofilm.
Biofilm in refractory chronic rhinosinusitis
Definition of Biofilm
Chronic rhinosinusitis (CRS) is a multifactorial inflammatory condition of the upper respiratory tract that results from a complex interplay between the host immune response and multiple extrinsic environmental and disease-causing factors. Bacteria and fungi have long been implicated as pathogens in the development of CRS, although the specific mechanism remains unclear. These microbes can exist either as a planktonic “free-floating” state or as a biofilm.
Biofilm is defined as a microbial-derived sessile community characterized by cells irreversibly attached to a surface or to each other, embedded within a matrix and exhibiting an altered phenotype with respect to growth and gene transcription. Within a biofilm, the microbes are surrounded by an extracellular matrix of polysaccharides, nucleic acids, and proteins. The bacteria and the extracellular polymeric substance form microcolonies with water channels conveying fluid and nutrients. This matrix confers characteristics of biofilm that allows it to adhere to surfaces, evade the immune response, increase resistance to antimicrobials, and provide a reservoir for recalcitrant infections. The ability of the biofilm to evade the host immune response and confer antibiotic resistance are some of the key pathologic features of a biofilm. Not only does the extracellular matrix provide a physical barrier to antibiotics, some of the bacteria in biofilm exists in a hypometabolic, slow-growing state making them more resistant to antibiotic treatment. The bacteria within a biofilm communicate via quorum sensing with small signal molecules that allow for rapid coordination of behavior to the environment. The close proximity of the bacteria within the biofilm also allows frequent gene transfer that, combined with recurrent antibiotic exposure, leads to the development of antibiotic resistance.
Biofilm in CRS was first discovered in 2004 using scanning electron microscopy with subsequent studies showing confocal scanning microscopy to be the best method of detection. Since then, numerous studies have demonstrated the presence of biofilm in CRS patients, with the reported prevalence ranging from 44% to 92%.
Detection of Biofilm
In terms of detection of biofilm, current techniques include the use of scanning electron microscopy, transmission electron microscopy, confocal laser scanning microscopy using BacLight staining, fluorescence in situ hybridization, and molecular techniques. Confocal scanning laser microscopy has been shown to have the greatest sensitivity and specificity for biofilm detection ( Fig. 1 ).
Characterization of biofilm
Identification of the microorganism that forms the biofilm has been demonstrated by using fluorescence in situ hybridization probes ( Figs. 2 and 3 ). Staphylococcus aureus biofilms are the most commonly identified bacterial biofilm. Pseudomonas aeruginosa , Haemophilus influenza , and Streptococcus pnuemoniae may also be represented in CRS biofilm formation. These studies have identified a high proportion of polymicrobial biofilm, including the presence of fungal biofilm in up to 50% of cases. Inoculation with S aureus or a cilia toxin–producing mucosal injury may be a prerequisite for formation of fungal biofilm.
Limitation of current techniques
Although the fluorescence in situ hybridization technique is very accurate, only a limited number of probes can be applied; therefore, researchers need to presumptively decide which organisms to target with the probes. Newer molecular-based polymerase chain reaction techniques can identify DNA of all microorganisms present that make up the complex microbiome of the sinonasal cavity. These findings reinforce the interplay between not just the host and the microbes, but also the interplay between the microorganisms as well. Moreover, a practical test for biofilm detection in the clinical setting is not available currently.
Pathophysiology of Biofilm and Its Role in Refractory Chronic Rhinosinusitis
Does biofilm equal disease?
The pathophysiology of biofilm in CRS has not been elucidated fully. Some studies have indicated that biofilms are not uniformly present in patients with CRS, whereas other studies have found biofilm in healthy controls. Limitations in detection and sampling methods may partly explain this discrepancy. For instance, scanning electron microscopy may not reliably differentiate between biofilm and mucous, resulting in false-positive biofilm detection. These findings emphasize that biofilm is just 1 factor in the multifactorial pathogenesis of CRS.
The effect of biofilm on disease seems to be at least partly related to the nature of the comprising organisms. In particular, S aureus biofilm represents a poor prognostic indicator. Multiple studies have demonstrated that S aureus biofilm-positive disease have worse postoperative outcomes in both objective and subjective scores compared with biofilm-negative disease. S aureus biofilm patients have higher burden of disease with higher symptom scores and Lund-Mackay computed tomography (CT) scores. They are also more likely to have persistent disease postoperatively with worse subjective scores and objective assessments. In contrast, patients with an H influenza biofilm behave more like biofilm-negative patients and have a higher success rate with conventional treatment.
How does biofilm contribute to refractory chronic rhinosinusitis?
Biofilm contributes to the refractory nature of some patients with CRS by its highly resistant nature to both antibiotic treatment and the host immune response. Defects within the innate immune response have been found in patients with biofilm-positive CRS. These are characterized by the downregulation of lactoferrin, which prevents bacterial aggregation and MUC7, an antimicrobial glycoprotein. In addition, there is an upregulation of sialic acid, which exposes more adherent sites for bacteria. The presence of the biofilm leads to significant destruction of the cilia in the epithelial layer of the mucosa, resulting in disruption in the mucociliary blanket. Mucous stasis within the sinus cavity then results, which predisposes to further biofilm formation. The presence of biofilm also elicits a local inflammatory response with elevation of T lymphocytes and macrophage numbers. The life cycle of the biofilm provides a local reservoir of bacteria with shedding of bacteria into planktonic form and leading to recurrent infections. In the case of S aureus biofilms, there is continued release of superantigenic molecules. There are also associations between biofilm and local markers of recalcitrant disease, such as intracellular bacterial infections and osteitis. In addition to the changes in the local environment, biofilm alters the host’s adaptive immune function, with skewing toward a T-helper cell 2 profile and elevated levels of interleukin-5, interleukin-6, and eosinophilic cationic proteins.
Treatment of Biofilm
The effective treatment of biofilm in refractory CRS is challenging. At present, there is no consensus of the optimal treatment modality, although local and topical treatment of sinus cavities has an intuitive appeal. Unfortunately, although some treatments have shown promise, none have been able to provide reliable long-term control. In addition, some potential biofilm treatments having been found to be ciliary toxic.
Surgery
Endoscopic sinus surgery may have a role in biofilm-related refractory CRS. Surgically opening the involved sinuses serves the dual purpose of establishing maximal sinus ventilation and providing a conduit for the postoperative application of topical therapies. Limited study has suggested that surgery may reduce significantly the density of the biofilm. Mechanical disruption of biofilm by irrigation during surgery may also be applied. The efficacy of topical therapy may depend on the successful reduction of the inflammatory load contributed by the biofilm. The principles of functional surgery should be observed to avoid unnecessary mucosal stripping, because doing so would lead to dysfunctional postoperative mucociliary clearance.
Topical steroids
Topical treatment with corticosteroids is one of the cornerstones of treatment of CRS, although its role in biofilm-related disease has not been fully established. The benefit may be greatest in the postoperative setting. In vitro studies have found that it reduces S aureus biofilm. Higher than standard doses of topical steroids may be required, such as budesonide 750 to 2000 mg/2 mL, fluticasone 400 mg/200 mL, or mometasone 300 to 400 mg/200 mL.
Topical antibiotics
The postoperative use of topical antibiotics has shown some promise in treating biofilm disease. A study by Ha and colleagues showed that standard antibiotics needed very high doses in a toxic range to achieve biofilm removal. In contrast, nonabsorbed topical antibiotics such as mupirocin were effective in standard concentrations. An in vitro study of topical antibiotics has suggested that mupirocin of 125 mg/mL can reduce biofilm mass by more than 90%. Subsequently, a randomized control trial demonstrated twice-daily mupirocin rinses were effective in short-term eradication of S aureus compared with normal saline rinses. Unfortunately, the relapse with recurrent infection occurred in up to 74% of patients treated with mupirocin over a 12-month follow-up period.
Surfactants
Topical surfactants such as citric acid zwitterionic surfactant (Medtronic ENT, Jacksonville, FL), Johnson’s Baby Shampoo (Johnson & Johnson, New Brunswick, NJ), and SinuSurf (NeilMed Pharmaceuticals, Santa Rosa, CA) have shown promise. Johnson’s Baby Shampoo diluted to 1% solution was found to improve symptoms of mucus thickness and postnasal drip, but had side effects of nasal irritation and increased mucociliary clearance time. SinuSurf (NeilMed Pharmaceuticals) was found to be initially helpful but was subsequently withdrawn owing to the side effect of anosmia. Citric acid zwitterionic surfactant was found to be ciliary toxic and is not recommended for use.
Other experimental treatments
Although there is not yet a definitive antibiofilm agent, there are a number of avenues are under investigation. In a sheep model, irrigation with Maunka honey (active ingredient methylglyoxal) was found to be safe to mucosa and efficacious against S aureus biofilm when used at a concentration of 0.9 to 1.8 mg/mL. However, it was toxic to the cilia at concentrations greater than this level. Similarly, another in vitro study demonstrated the usefulness of colloidal silver for reduction of biofilm compared with control with maximal effect noted at a concentration of 100 to 150 μL.
Nonconventional antimicrobial treatment has also been investigated. N,N-dichloro- 2,2-dimethyltaurine, is a broad-spectrum antimicrobial agent, has been investigated in a sheep model and demonstrated significant biofilm reduction compared with saline.
Bacteriophages are viruses that directly target and destroy bacteria. Early results with the use of S aureus -specific bacteriophages have been shown to be effective against clinical isolates of S aureus with reduction of biofilm mass.
Photodynamic therapy with treatment of in vitro bacterial colonies with methylene blue trihydrate and subsequent exposure to 664 nm of light demonstrated 99% reduction after a single treatment with no histologic evidence of side effect. Low-frequency ultrasound treatment at 40 kHz, 0.5 W/cm 2 sound pressure on in vitro nasal polyp has found significant reduction in biofilm and inflammatory cells.
Osteitis in refractory chronic rhinosinusitis
Definitions of Osteitis
Osteitis is inflammation of bone without marrow space. It is characterized by neo-osteogenesis with new woven bone formation and thickening of the mucosa. Although infectious mechanisms have been implicated in osteitis, it is predominately a disease of inflammation and bone remodeling There is, however, contention regarding the exact role of osteitis in CRS and its role in refractory disease.
Prevalence of Osteitis
Although osteitis has been described since 1992, the exact prevalence is variable with studies citing an incidence of 36% to 53% depending on the radiologic or histologic criteria used in the study. In patients with refractory CRS who have undergone multiple surgeries, the incidence of osteitis has been reported to be as high as 64%.
Pathophysiology of Osteitis
Early experimental studies demonstrated localized periosteal reaction and bone changes as early as 4 days after inoculation of bacteria. These studies demonstrated that infections can infiltrate through the mucosa, inducing an inflammatory reaction. This not only occurs in the underlying bone, but also at distal sites through widening of the bony vascular network and Harversian canal system. The cycle of mucosal and bony infection and inflammation can theoretically act as a persisting nidus of disease. However, more recent human studies have presented conflicting findings with some studies showing an absence of bacteria within the bone itself and other studies showing bacterial microcolonies existing in the bone without an osteitis reaction.
Biofilm association
Biofilm is certainly associated with osteitis, with the volume of biofilm present correlating with the degree of osteitis. Biofilm potentially also acts as a reservoir for bacteria that cause bony inflammation through a release of eosinophilic inflammatory mediators implicated in the formation of osteitis. The inflammation that occurs in osteitis leads to continuous remodeling of bone with histologic findings of new woven bone formation, periosteal thickening, bone resorption, and finally fibrosis in the most severe case of CRS.
Disease Severity and Osteitis
There is a clear correlation between disease severity and osteitis. From a radiologic perspective, the degree of osteitis correlates with the Lund-Mackay score. From a clinical perspective, patients with osteitis exhibited a greater severity of inflammatory CRS findings on endoscopy. These patients are also more resistant to medical treatment as well as surgical treatment with more severe osteitis in patients undergoing revision surgery than primary. One of the perplexing factors regarding osteitis is that stripping of the mucosa or periosteum in multiple surgeries can also lead to underlying bone changes with worsening of CT grades of osteitis. This raises the question of whether the osteitis is causing recalcitrant disease necessitating further surgery, or alternatively that surgery may play a role in osteitis. Ultimately, osteitis may represent an irreversible common endpoint of recalcitrant CRS disease with inflammatory involvement of both the mucosa and underlying bone.
Diagnosis of Osteitis
Computed tomography scan
Although histology is considered the most accurate way of diagnosing osteitis, radiologic studies are the more commonly used investigations. CT scan was first used by Biedlingmaier to diagnose osteitis and correlated well with histologic findings especially in more severe cases ( Fig. 4 ).
