Understanding the Role of Biofilms and Superantigens in Chronic Rhinosinusitis

Abstract

Purpose of review:

This review explores recent discoveries in our understanding of how biofilms and superantigens contribute to the pathogenesis of chronic rhinosinusitis (CRS). It also examines clinical implications and novel treatment approaches for biofilm associated CRS.

Recent findings:

While the role of biofilms in CRS has been studied for 14 years, research interest has now turned toward elucidating new methods of biofilm detection, microbial diversity, and novel treatment approaches. Recent studies on biofilm superantigens aim to clarify the immunological mechanisms of upper airway inflammation, particularly the type-2 response seen in nasal polyposis.

Summary:

Biofilms are a topic of research interest for their role in the pathogenesis of chronic rhinosinusitis, particularly when they elute superantigens. New studies on this topic focus on the molecular and cellular mechanisms at play.

Introduction

Chronic rhinosinusitis (CRS) is a complex and common syndrome affecting up to 16% of the United States population [1]. CRS invokes a direct treatment cost estimated between $10–13 billion per year in the US and CRS patients suffer from significantly impaired quality of life [2]. Though a broad syndrome, CRS is often classified into two phenootypes based on the presence or absence of nasal polyps on nasal endoscopy. CRS without nasal polyps (CRSsNP) is characterized by a T helper type 1 (Th1) mediated pattern of inflammation and CRS with nasal polyps (CRSwNP) has greater propensity for a T helper type 2 (Th2) mediated immune response [3].

Despite its impact on a societal and individual level, the aetiology of CRS remains unclear. In recent years, research has focused on the multifactorial interactions of host and environmental factors that determine susceptibility to CRS. Examples of host factors include genetic disorders affecting mucociliary clearance like cystic fibrosis and primary ciliary dyskinesia, while environmental factors include pathogens, allergens and tobacco exposure. However, the majority of cases of CRS are idiopathic and multiple hypotheses have been proposed to explain the pathophysiology of CRS, including the potential role of biofilms and superantigens. The purpose of this review is to explore recent advances in our understanding of the biofilm and superantigen hypotheses, examine the clinical implications of biofilms and superantigens associated CRS, summarize emerging interventions based on these hypotheses and suggest future directions for research.

The Biofilm Hypothesis

Biofilm Formation

Bacteria can exist in two distinct forms— planktonic or biofilm— which demonstrate differential growth and gene expression patterns. In planktonic form, bacteria are free floating; in biofilm form, the state in which 99% of bacteria prefer to exist, bacteria form closely organized microcolonies encased in extracellular matrix (ECM) [4]. Biofilm formation occurs when planktonic bacteria irreversibly adhere to a surface via the upregulation of cell adhesion structures and proliferate to form microcolonies which secrete a protective layer formed from polysaccharides, proteins, nucleic acids, and extracellular DNA [4]. Once a critical mass is reached, a phenomenon known as quorum sensing begins which allows bacteria within the biofilm to communicate and coordinate via small signal molecules called autoinducers. This bacterial cross-talk culminates in signal transduction cascades that alter gene expression leading to the final biofilm phenotype. Existing in biofilm form enhances bacterial survival via several mechanisms. First, the protective layer of ECM allows bacteria to evade detection by the host immune system [5]. Second, the exchange of extrachromosomal DNA is more efficient in the biofilm state, enabling the propagation of adaptive mutations [5]. Third, the ECM protection and the coordinated bacterial stress response via quorum sensing confer antibiotic resistance in biofilm form that is up to 1000 fold greater than in planktonic form [6]. Biofilms can then spread by releasing planktonic bacteria to distant sites which can adhere and begin the complex process of biofilm formation again [7].

Biofilm Detection

Biofilms have been implicated in multiple infectious processes significant to the practice of otolaryngology including otitis media [8] and chronic tonsillitis [9]. Recently, research has focused on investigating the prevalence and potential pathophysiological role of biofilms in CRS. Biofilms were first identified in CRS patients in 2004 [10] and several studies have subsequently reported biofilm prevalence ranging from 25% to 100% using a variety of detection methods [11] The earliest studies to attempt biofilm detection in CRS utilized nasal swab cultures which rely on selective growth methods that favor more abundant and metabolically active organisms. Given that bacteria in biofilm have reduced metabolic activity, traditional culture methods often sample planktonic bacteria, misrepresenting the apparent diversity of the microbial community in CRS [8]. Newer and more reliable modalities for biofilm detection include scanning electron microscopy (SEM), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), and confocal scanning laser microscopy (CSLM), which is currently considered the gold standard.

Microbial Diversity

Since the advent of biofilm detection in CRS, multiple bacterial organisms have been implicated including Staphylococus aureus, Pseudomonas aeruginosa, Haemophilus influenza and Moraxella cattarhalis [8]. Of these, S. aureus biofilms have the greatest association with severely recurrent and recalcitrant cases of CRS [12]. Recently, the ability of S. aureus to produce superantigens, which induce a local and at times wide-spread non-specific immune response, has been thought to explain the enhanced pathogenicity of S. aureus compared to other bacterial species.

While research has mostly focused on the bacterial components of biofilms, several studies have established a high correlation between bacterial and fungal biofilms in patients with CRS, suggesting a potentially mutualistic relationship. In a sheep model of CRS, co-inoculation of the frontal sinus with Aspergillus fumigatus and either S. aureus, P. aeruginosa or S. epidermidis promoted fungal biofilm formation, while inoculation with A. fumigatus alone did not [13]. Co-inoculation with bacterial and fungal elements also led to greater degrees of inflammation and ciliary damage than fungal inoculation alone. This synergistic effect of bacterial and fungal organisms is thought to occur at least partially due to bacterial induced mucociliary injury which facilitates opportunistic infections by fungal species [13, 14].

Potential Mechanisms of Pathophysiology

Despite a growing body of evidence implicating biofilms in the pathogenesis of CRS, not all CRS patients demonstrate evidence of biofilm formation during their disease process. To further understand the potential role of biofilms in CRS it is necessary to explore the factors that dictate susceptibility to biofilm formation. Evidence suggests that host defects in innate and adaptive immunity drive susceptibility to biofilms leading to a subgroup of CRS patients with recurrent and recalcitrant disease.

The recent discovery of taste receptors in the upper airway has yielded evidence suggesting a potential role in protecting against biofilm formation. Bitter and sweet taste receptors are G- protein coupled receptors which when stimulated activate the canonical taste signaling cascade [15]. Taste receptors belonging to Taste Receptor Family 1 subtypes 2 and 3 (T1R2/T1R3) detect sweet compounds while those belonging to Taste Receptor Family 2 (T2R) respond to a variety of bitter compounds [16]. In the upper airway, taste receptors are present on a variety of cell types and mediate components of innate immune defense. Ciliated cells express T2Rs and respond to bitter compounds including specific autoinducers involved in quorum sensing called acyl-homoserine lactones released by biofilm forming gram-negative bacteria such as P. aeruginosa. Stimulation of T2R38 results in a signaling cascade with resultant calcium- dependent release of bactericidal nitric oxide and increased ciliary beat frequency [17–19]. Similarly, solitary chemosensory cells (SCC) are rare epithelial cells that express both T1R2/3 and T2Rs. SCC T2R stimulation elicits a calcium response that results in the downstream release of antimicrobial peptides, while stimulation of T1R2/3 acts to antagonize this T2R response. During bacterial infection, glucose levels in mucus decrease thereby reducing stimulation of TIRs which disinhibits the T2R response to bacterial products [15].

Dysfunction of either bitter or sweet taste receptors in the upper airway can impair the detection of and response to quorum-sensing AHLs as evidenced by recent work investigating T2R38, one of the most well studied T2Rs. T2R38 is encoded by the TAS2R38 gene and is particularly sensitive to the bitter compounds propylthiouracil (PROP) and phenylthiocarbamide (PTC) [20]. Genetic polymorphisms dictated by missense mutations in SNPs determine T2R38 functionality and, therefore, sensitivity to PTC and PROP. The T2R38 protein exists in two common haplotypes based on the amino acids encoded by missense mutations in single nucleotide polymorphism. One form, PAV (proline, alanine, valine) is functional and the other, AVI (alanine, valine, isoleucine) is non-functional. These two haplotypes result in three common genotypes and the level of PAV expression correlates with T2R38 receptor sensitivity to bitter compounds [21]. Sensitivity to PTC, as measured by a simple taste test, can serve as a proxy for TAS2R38 genotype and T2R38 functionality. A recent study by Adappa et al. sought to investigate whether T2R38 function is implicated in biofilm formation given its role in detecting quorum sensing molecules. Endoscopic nasal swabs were obtained from CRS patients who were also evaluated for T2R38 functionality with a PTC taste test. An inverse linear relationship was found between in vitro biofilm formation and PTC sensitivity in the entire cohort, but this association was driven specifically by CRSsNP patients. These results suggest that differential T2R38 receptor activity determines the innate immune response in the sinonasal tract. Furthermore, taste tests using appropriate bitter compounds, like quinine hydrochloride and denatonium benzoate, at optimized concentrations have been shown to serve as a cost-effective and accurate proxy for sinonasal immune function [22].

Another component of the innate immune system that demonstrates dysfunction in biofilm associated CRS is the mucociliary apparatus. Through coordinated movement, ciliated cells eliminate mucus produced by goblet cells which traps pathogens traveling through the upper airway. In a rabbit model of sinusitis, Jia et al. traced the natural history of biofilm formation after bacterial inoculation with S. aureus. The earliest changes seen 1 week post-operatively were goblet cell hyperplasia with resultant increase in mucous production, followed by ciliary warping and concomitant reduction in ciliary activity [23]. Other studies have demonstrated varying degrees of epithelial destruction ranging from ciliary disarray to complete absence of cilia [24, 25]. It is unclear whether this ciliary dysfunction precedes or is a consequence of biofilm formation. Biofilm formation has also been associated with the upregulation of mucin related genes, namely MUC5AC in goblet cells and MUC5B in submucous glands collected from mucosa of CRS patients. This increased expression and overproduction of mucin may act to trap more inhaled microbes and this, in concert with stasis from disordered ciliary function, may give bacteria opportunity to adhere and begin the process of biofilm formation [26].

Another potential mechanism for biofilm pathogenicity is intracellular uptake of particular pathogens. Once intracellular, S. aureus undergoes phenotype switching secondary to transcriptional changes that alter its expression of genes related to evasion of host immune response and antibiotic resistance. CRS patients who demonstrate evidence of intracellular s aureus at time of functional endoscopic sinus surgery (FESS) are more likely to have delayed clinical and microbiological relapse post-operatively, suggesting that intracellular residence can serve as a nidus for infection in recurrent disease [27].

Biofilm formation also evokes significant pro-inflammatory changes in several ways. Cantero et al. found that healthy human sinonasal explants exposed to S. aureus biofilms upregulated the expression of genes involved in the Nod2/NF-kB pathway with downstream release of proinflammatory factors IL-6, IL-8, and the neutrophil chemoattractant CXCL2 [28]. S. aureus biofilms have also been associated with changes in the inflammasome pathway. Inflammasomes are cytoplasmic protein complexes with pattern recognition receptors that detect microbial products and activate caspase-1. Caspase-1 then elicits a downstream release of pro- inflammatory cytokines like IL-1B and IL-18. Jardaleza et al. found that CRSwNP mucosal samples positive for S. aureus biofilms demonstrated upregulation of genes involved in the inflammasome signaling pathway, potentially explaining the persistent inflammation characteristic of CRS [29]. This pro-inflammatory state also contributes to the higher degree of osteitis seen in mucosa underlying biofilms in CRS affected mucosa; inflamed bone may in turn act as a “depot” for inflammatory cytokines contributing to the vicious cycle [30]. These changes overall induce an inflammatory infiltrate in the subepithelium and lamina propria featuring lymphocytes, dendritic cells, eosinophils, and plasma cells as early as 2 weeks into the disease. This infiltrate is followed by biofilm adherence to the epithelial surface with resultant damage to mucociliary function hindering clearance of the primary infection. This mucociliary impairment in turn increases susceptibility to other opportunistic pathogens [23, 31, 32].

Clinical Implications of Biofilm Positive CRS

The clinical implications associated with biofilm formation in CRS have been extensively investigated. CRS patients with biofilm have more severe disease prior to intervention as evidenced by worse objective clinical indicators of disease [33, 34] and higher subjective symptom burden [34, 35]. Patients with biofilm positive CRS also report significantly worse outcomes in quality of life at 6 months post-FESS compared to their biofilm negative counterparts [36]. Additionally, biofilm positive CRS patients demonstrate significantly worse objective outcomes with more severe disease on pre- and post-operative nasal endoscopy, more follow up visits and additional courses of antibiotics when compared to biofilm negative CRS patients [14]. The presence of biofilms in CRS is also independently associated with greater risk of recurrence [34] and revision surgery [36].

The clinical outcomes of biofilm associated CRS have been found to depend on the bacterial species involved in the disease process. S. aureus biofilms in particular are associated with more severely recalcitrant disease and patients with S. aureus biofilms demonstrate worse objective markers of inflammation and report poorer quality of life than patients with biofilms formed from other bacterial or fungal species[14, 37]. This phenomenon is potentially due to the severe inflammatory response to S. aureus superantigens.

The Superantigen Hypothesis

Mechanism of Superantigen Stimulation

Bacterial biofilms are an import source for eluting superantigens. Superantigens (SAgs) are exotoxins produced by bacteria in order to evade host immune response. SAgs confer virulence by short-circuiting normal antigen presentation leading to massive release of inflammatory cytokines [38, 39]. In addition, the host experiences massive T cell activation, expansion, and subsequent anergy as well as B cell activation [38, 39]. In effect, host immunity is debilitated, and pathogenic bacteria persist. While multiple bacterial species are known to secrete superantigenic exotoxins, Staphylococcus aureus enterotoxins (SAEs) elicit the most pronounced clinical manifestations, such as toxic shock syndrome, gastroenteritis, atopic dermatitis, asthma, and chronic rhinosinusitis with nasal polyps [40]. SAEs, which include staphylococcal enterotoxins A through U and toxic shock syndrome toxin (TSST-1), are a relatively well-studied family of SAgs that are related to the larger class of exotoxins, called thermal stable proteins [41, 42].

In normal physiology, antigen presenting cells (APCs) internalize and process non-self peptides before presenting them on their MHC II molecules to T cells via TCRs. SAEs, however, can bind directly in an unprocessed form to MHC II molecules outside of peptide-binding groove. Upon presentation to TCRs, SAEs cause a hyper-stimulation of T cells, with nearly 20–30% activation of host T cells, compared to the normal 0.001%−0.001% [43, 44]. Given this robust effect, SAEs are capable of inducing clinical symptoms at concentrations as low as 0.1 pg/ml [45] Interestingly, SAgs are known to specifically interact with only the beta chain in the variable region of TCRs (Vβ), and each SAg interacts with only a subset of Vβ motifs [46]. Therefore, SAg-dependent T cell stimulation leads to characteristic expansion of oligoclonal T cells with specific Vβ signatures [47–49]. While optimal interaction of SAg-MHC II complex with TCRs involves co-stimulatory signals B7 and CD28 on APCs and T cells respectively, some SAgs are also known to stimulate TCRs directly in an MHC ΙΙ-independent manner [50]. However, this mode of stimulation does not yield as strong of a T cell response.

Staphylococcal Superantigens in CRS

Studies have shown a link between superantigen-stimulated immune response and the development of CRS. It is established that superantigens, particularly SAEs, tend to promote a Th2 immune response that favors eosinophilic inflammation found in CRSwNP, rather than a Th1 response found in CRSsNP [51, 52]. Initial data implicating SAEs in CRS demonstrated higher S. aureus culture-positive rates in patients with CRSwNP (45.4–71%) compared to CRSsNP (7.6%−42.8%) and healthy controls (0%−27.3%) [53–57]. Likewise, numerous studies have shown that SAEs were directly detectable by immunoassay in patients with CRSwNP [40, 55, 58–60]. However, other studies have shown conflicting data in which the association of S. aureus and SAEs with CRS was not found [56, 61]. It is well known that even healthy individuals are commonly carriers of S. aureus in the nose and sinuses [62]; moreover, the aforementioned studies showed that not every patient with CRSwNP was colonized. To address conflicting data, a 2014 meta-analysis of 340 cases concluded that four studies collectively showed an overall significantly greater association between CRSwNP and positive S. aureus nasal cultures (OR 4.85, 95 % Cl 1.80–13.05, p = 0.002) [63]. Data from another five studies in the same meta-analysis also demonstrated significant association between CRSwNP and directly measured SAEs (OR 12.07, 95 % Cl 4.57–31.90, p < 0.00001) [63]. While meta-analysis is limited by variation in study design and patient populations, the data do support that S. aureus infection and SAEs are found more often in patients with CRSwNP.

Research interest has since turned toward elucidating the underlying mechanism whereby SAEs drive Th2 inflammation observed in CRSwNP. Patou et al. first demonstrated using multiplex immunoassay that staphylococcal enterotoxin B (SEB) stimulates cultured nasal polyps to secrete a predominately type-2 cytokines (i.e., IL-2, IL-4, IL-5, IL-10, IL-13) [52]. Li et al., recently showed that presence of S. aureus reduced levels of Th1-promoking cytokine, IP-10, thereby contributing to Th2-bias [64]. Superantigens also stimulate local B cell immunoglobulin class switching and production. Numerous studies have demonstrated elevated IgE antibodies specific to SAEs in CRSwNP compared to CRSsNP and healthy controls, independent of atopy [53, 60, 65–67]. In addition, Van Zele et al. reported a rate of SAE-IgEs as high as 80% in a subgroup of CRSwNP patients with AERD and asthma, compared to 28% in CRSwNP lacking such comorbid lower-airway disease, 6% in CRSsNP, and 15% in healthy controls [68]. Levels of IgEs specific to SAEs also correlated with IL-5, eotaxin, eosinophilic cationic protein, which are responsible for driving eosinophilic infiltrate characteristic of the aberrant mucosa found in CRSwNP [69].

Emerging Treatments

The recurrent and recalcitrant nature of biofilm associated CRS and its significant impact on quality of life have recently prompted a focus on the search for effective anti-biofilm therapies. Here we summarize the most recent developments in potential treatment strategies.

The antibiotic resistance conferred by the biofilm state poses a challenge for systemic antibiotic penetration. Recently macrolides have been a focus of potential treatment given their ability to impair biofilm formation by interfering with quorum sensing. In a randomized controlled trial (RCT) by Tatar et al., 32 CRSwNP patients on an 8 week course of oral clarithromycin therapy prior to undergoing FESS were randomized to receive either concomitant topical mometasone furoate or oral macrolide therapy alone. Pre- and post-treatment nasal tissue samples were compared for biofilm density and extension on SEM using a quantitative grading scale (range = 1–3). While overall biofilm prevalence decreased with treatment in both groups (75% vs 43.8%), and post-treatment grade was significantly decreased in both groups (median difference = 1.0, p <0.01), no additional benefit was found for adding intranasal steroid to oral macrolide therapy in CRSwNP [11]. In a similar RCT, Korkmaz et al. compared the efficacy of intranasal steroid as monotherapy versus combined with low-dose oral macrolide in 34 CRSwNP patients [70]. The combined macrolide and intranasal steroid group had significant reduction of biofilm compared to control but the intranasal steroid group alone did not. Despite these studies suggesting a possible role for macrolides in eradicating biofilms, a recent meta-analysis found limited evidence to support macrolide efficacy in CRS. While oral macrolides can interfere with biofilm formation, penetration into an established biofilm may prove difficult and might account for the discrepancies in efficacy.

Topical antibiotic formulations have been a focus of recent research due to their ability to locally deliver higher concentrations of antibiotic therapy with limited systemic absorption. In a prospective trial Ezzat et al. investigated the efficacy of topical ofloxacin in 10 patients with refractory CRS and evidence of biofilm 6 months post-FESS. After 12 weeks of topical ofloxacin treatment there were no reported side effects and 80% of the study group demonstrated complete eradication of biofilm on SEM. The remaining 20% had reduced number of inflammatory cells, restoration of ciliary architecture, and only scattered areas of thin biofilm [71]. Other studies, however, have demonstrated limited efficacy of topical antibiotics like tobramycin and mupirocin and more large scale RCTs are needed before a recommendation for or against topical antibiotics can be reached [72–75].

Sinusitis is one of the most common indications for outpatient antibiotic prescription in adults, accounting for approximately 15–21% of all antibiotic prescriptions in the United States [76]. Finding novel non-antibiotic treatment modalities for CRS is of special importance given the growing concern for antibiotic resistance. Cirkovic et al. performed a prospective study to analyze the effects of intranasal steroids (mometasone furoate and fluticasone proprionate) and intranasal saline irrigation (isotonic and hypertonic) on the biofilm forming capacity of bacterial strains isolated from CRSwNP patients [77]. The effect of treatment was dependent on both the bacterial species and biofilm mass. All four treatment modalities resulted in a decrease in biofilm density but the greatest reduction of biofilm density was observed for S. aureus, P. aeruginosa and S. pneumoniae (p <0.01, p <0.05 and p <0.05 respectively). Treatment effects were also more pronounced with bacterial strains that produced greater biofilm mass. These results demonstrate a need for directed antibiofilm treatment depending on the identity and potency of the microbial strain.

One such potential species specific treatment is a chimeric protein, P128, which combines a cell- wall binding domain specific to Staphylococcal species, and a phage derived hydrolase domain capable of breaking down peptidoglycan based cell walls. Biofilms formed by methicillin sensitive and methicillin resistant S. aureus (MSSA and MSRA) clinical isolates from CRS patients and a reference strain of S. aureus were incubated with varying concentrations of P128 ranging from 1.56–50 μg/mL and assessed for persistence after treatment. Biofilm mass was found to be significantly reduced in all S. aureus isolates at P128 concentrates greater than 25 μg/mL compared to 0.9% saline control (p <0.05). Given its action against virulent and resistant strains of S. aureus P128 has promising implications for the most severely recalcitrant CRS cases and antimicrobial stewardship.

Manuka honey (MH) includes high levels of a phenol compound, methylglyoxal (MGO) which has shown strong bactericidal activity against S. aureus and P. aeruginosa biofilms[78]. In a sheep model of sinusitis, MH augmented with additional MGO was found to significantly reduce biofilm mass at MGO concentrations of 1.8 mg/mL (0.676 ± 0.079 μm³/μm² vs 0.114 ± 0.033 μm³/μm², p = 0.001) and 3.6 mg/mL (0.608 ± 0.110 μm³/μm² vs 0.316 ± 0.197 μm³/μm², p = 0.015) when compared with saline flushes. However, when administered alone, MGO caused severe sinus inflammation and squamous metaplasia of the respiratory epithelium although this effect was not seen when MGO was administered in combination with MH [79]. Clinical trials assessing the safety and efficacy of MH/MGO in patients with biofilm positive CRS are needed.

Another naturally occurring compound with antibiofilm activity is xylitol - a five-carbon sugar alcohol. Jain et al. demonstrated that 5% and 10% solutions of xylitol significantly reduced S. epidermidis biofilm when compared to saline control (Δ 1.1 ± 0.1, P<0.001 and Δ 1.0 ± 0.1, P<0.001, respectively) but no significant difference was seen in biofilm reducing capacity for s aureus or P. aeruginosa biofilms compared to control. This result again demonstrates the complexity of determining the optimal treatment and management of biofilm positive CRS given that clinical behavior and therapeutic response differ based on the biofilm forming species.

Antimicrobial photodynamic therapy (aPDT) harnesses the power of oxygen free radicals inducing the perforation of bacterial cell membranes and allowing the penetration of photoreactive dyes. Once activated by laser light, these dyes induce photodamage to inner organelles and promote apoptosis. In a silicone model of the human maxillary sinus inoculated with MRSA and antibiotic resistant P. aeruginosa, Biel et al. demonstrated a greater than 99.9% reduction of biofilms grown from each species after a single treatment of aPDT [80]. aDPT has also demonstrated efficacy in preventing surgical site infections when used in pre-operative decolonization therapy in conjunction with chlorhexidine body wipes, in the treatment of chronic periodontitis, and in reducing biofilm formation on orthopedic prosthetic devices [81–83]. aPDT does not cause histologic damage to the underlying respiratory mucosa and appears to be a promising and viable option for biofilm eradication, especially in antibiotic resistant cases [84].

Recently, the use of ultrasound to physically disrupt established biofilms has been evaluated in two in vitro studies. Karosi et al. examined the efficacy of biofilm disruption using low- frequency ultrasound (LFU) on nasal polyps isolated from 10 CRSwNP patients. In LFU untreated polyps, the average biofilm thickness was 17.3 pm while polyps in the treated condition demonstrated total absence of bacterial biofilm (p <0.001). The number of inflammatory cells present in the subepithelial layer was also significantly reduced in the LFU treated versus untreated group (p < 0.001). In a double blind RCT, researchers assessed the impact of LFU on subjective symptom severity for 60 CRS patients after 10 treatment sessions. Patients were randomized to LFU using either normal ultrasound transmission gel or erythromycin ointment applied to the superficial facial skin overlying the maxillary and frontal sinuses[85]. Patients in both conditions reported significantly improved post-treatment symptom scores with large effect sizes (LFU d=1.36, LFU + erythromycin d=2.15). The percent improvement in pre- to post-treatment scores was significantly higher in the LFU plus erythromycin condition than the LFU alone condition (67.2 ± 23.7 vs 49.3 ± 37.2,p=0.03). The superior symptom relief in the erythromycin condition is thought to be from increased delivery of erythromycin to target tissues via synergistic ultrasound vibrations and increased blood flow in the skin overlying target tissues.

Future directions

Biofilms

A growing body of evidence implicates the role of biofilms in the pathophysiology of recurrent and recalcitrant CRS. Future studies exploring novel detection measures and antibiofilm treatments are needed. Non-invasive identification methods would allow for earlier and easier detection of biofilms and objective assessment of pre- and post-treatment changes. Poly-A- acetylglucosamine (PNAG) is a polysaccharide produced by S. aureus that is critical to biofilm formation that can serve as target for both S. aureus biofilm detection and disruption. Foremen et al. have developed a non-invasive immunofluorescence protocol that detects S. aureus biofilms in CRS patients using PNAG with similar accuracy to invasive methods [86]. Given that alterations in bitter and sweet taste receptors, like T2R38, can increase susceptibility to biofilm formation, bitter and sweet receptor sensitivity as assessed by a simple in-office taste test can serve to stratify patients most at risk for biofilm formation, and identify patients for whom T2R pathway agonists may be beneficial.

Future studies should also make every effort to stratify patients by CRS endotype given that CRSwNP and CRSsNP are heterogeneous disease processes with significantly different inflammatory profiles. In addition, the majority of treatment related studies have focused on S. aureus given its association with biofilm formation and severe CRS. However, it is clear that other bacterial species capable of forming biofilms demonstrate differential clinical behavior and therapeutic response.

Superantigens

The exact mechanisms by which superantigens perturb the immune system in CRSwNP is still being elucidated. Multiple studies have shown that distinct Vβ signatures are produced when host T cells undergo SAE-mediated stimulation in the setting of CRSwNP [49, 87, 88], and Tripathi et al. reported 58% of CRSwNP had detectable signatures Vβ clonal expansions of T cells [49]. In an era of more granular endotyping of CRS, understanding how these clonal expansions may serve as methods of immunophenotyping will add value.

In addition, further understanding host characteristics is key. Across most studies, data demonstrate that only half of patients with CRSwNP have detectable SAEs; therefore, host factors may be underlying SAE sensitivity [89, 90]. Recent investigation is underway examining variations in host HLAs, Vβ chains, and various forms of immunocompromised states. For example, hosts’ abilities to rid pathogenic bacteria may be related not only to SAE-eluting biofilms as discussed above, but also to intracellular residing S. aureus producing SAgs, which recent studies have identified by immunohistochemistry [91] and PNA-FISH [51].

New evidence also has implicated SAEs in influencing eicosanoids, which are inflammatory molecules generated from the metabolism of arachidonic acid. Involvement of the eicosanoid pathway was first suggested in patients with lower respiratory disease presenting with aspirin intolerance [92]. More recently, eicosanoids such as leukotrienes and pro-inflammatory leukotrienes have been known to be downstream of superantigens in the setting of Th2 upper airway disease [93–95]. Furthermore, Murawat et al. demonstrated that SAEs cause cytokine secretion in nasal polyps via phospholipase A2, the first and key regulatory enzyme in metabolizing eicosanoids [96]. Further investigation will hopefully shed increasing light on how these molecules are contributing.

Conclusions

CRS is a complex and multifactorial disease with a still unclear etiology that includes inflammatory, infectious, host and environmental factors. Two emerging areas of research of recent interest explore the potential role for biofilms and superantigens in the pathophysiology of CRS. A growing body of evidence suggests both entities play potentially intersecting roles in the development of recalcitrant disease. Despite the discovery of several novel, hypothesis driven, antibiofilm therapeutics, the optimal means of management for biofilm and superantigen associated CRS remains unclear. As our understanding of the role that biofilms and superantigens play in CRS deepens, we will be able to target the specific underlying causes of persistent CRS with species and mechanism specific multimodal treatments.