Paired qualitative and quantitative analysis of bacterial microcolonies in the tonsils of patients with tonsillar hyperplasia.

The discovery of bacterial microcolonies in tonsillar tissue of patients with tonsillar hyperplasia has raised the question of their role in provoking the local immune response. Tonsils collected from patients undergoing tonsillectomy were stained for three clinically relevant bacterial taxa and lymphocytes. The bacterial composition and abundance of microcolonies was investigated using a combination of laser-microdissection, amplicon sequencing and Droplet Digital polymerase chain reaction. Microcolonies were detected in most samples (32/35) with a high prevalence of Haemophilus influenzae (78% of samples). B and T cell lymphocytes were significantly higher in the epithelium adjacent to microcolonies compared to epithelium distal to microcolonies. Furthermore, significant positive and negative correlations were identified between bacterial taxa and lymphocytes. Genus Streptococcus, which includes Group A Streptococcus (traditionally described as the main pathogen of tonsillar hyperplasia), was found in low abundance in this study. These results suggest other potential pathogens may be involved in stimulating the local immune response leading to tonsillar hyperplasia.

The Effect of Amoxicillin with Clavulanate on the Microbiota of Tonsillar Tissue in Disease: a Randomized Control Trial.

Despite antibiotics being the primary medical treatment for recurrent tonsillitis, the impact of antibiotics on the tonsillar microbiome is not well understood. This study aimed to determine the effect of amoxicillin with clavulanate on the composition and quantity of bacteria in the tonsils of children with recurrent tonsillitis. A multicenter randomized clinical trial in Auckland, New Zealand was undertaken between August 1, 2017, and June 30, 2018. Sixty children undergoing tonsillectomy for the indication of recurrent tonsillitis were recruited for this study. Following random allocation, 30 participants were prescribed amoxicillin with clavulanate for the week before surgery. The remaining 30 received no antibiotics. Immediately following surgery, the crypts of the right and left tonsils were swabbed. Bacterial 16S rRNA gene-targeted amplicon sequencing and histological techniques were utilized. In the control group, there were significantly higher relative abundances of Haemophilus, Streptococcus, Neisseria, and Porphyromonas. Members from the genera Fusobacterium and Treponema were found to be significantly more abundant in the antibiotic group. There were no significant differences in the absolute quantities of bacteria between the groups. Microscopic examination found fewer bacterial microcolonies present in the tonsillar crypts of participants in the antibiotic group. Streptococcus pyogenes was not present in these bacterial microcolonies. These results suggest that a single course of antibiotics has a significant impact on the tonsil microbiota composition. The duration of this effect and the effect that the altered microbiome has on the course of the condition need to be determined. IMPORTANCE Several studies have identified the presence of multiple pathogenic bacteria in hyperplastic adenoids and palatine tonsils. However, there are currently no studies that utilize this technology to investigate the effect of oral antibiotics in children with recurrent tonsillitis on the tonsillar microbiome. This is the first study to investigate the effect of antibiotics on the microbiome of tonsillar tissue in children with recurrent tonsillitis using molecular techniques. This study has shown that participants who received amoxicillin with clavulanate immediately before tonsillectomy had a significantly reduced number of bacterial taxa commonly associated with recurrent tonsillitis, as well as the number of bacterial microcolonies observed in the tonsillar crypts. This novel finding suggests that either the effect of antibiotics is not sustained or that they are not an effective treatment for recurrent tonsillitis.

The histological and microbiological characteristics of bacterial microcolonies in paediatric tonsillar hyperplasia.

INTRODUCTION: Paediatric tonsillar hyperplasia (TH) is associated with a spectrum of presentations ranging from recurrent tonsillitis (RT) to sleep-disordered breathing (SDB). The underlying pathogenesis of tonsillar hyperplasia remains poorly understood. Previous studies have implicated bacterial microcolonies as targets of host inflammatory cells and as a potential driver of the chronic inflammation seen in TH. The role of atopy in tonsillar hyperplasia is also largely unknown. In this study, we aimed to determine the allergic responses and microbial factors that may influence TH in children. MATERIALS AND METHODS: Paired tonsils and a serum sample were collected from 21 children undergoing tonsillectomy for RT or SDB in the Auckland region. The disposition of immunoglobulin isotypes (IgG, A, M and E) and local inflammatory cells on histological sections of tonsil tissue were determined using immunohistochemistry techniques. Aeroallergen specific IgE (sIgE) and Staphylococcal enterotoxin C specific IgE (SEC-specific IgE) were measured in serum and tonsil tissue using the ImmunoCAP® system. Finally, tonsil bacterial microcolonies were then excised from histological slides using laser microdissection techniques, before undergoing bacterial and fungal amplicon sequencing. RESULTS: There were no significant differences in any of the measured variables between children with RT and SDB symptoms. IgE staining was not associated with increased levels of mast cells, leukocytes or plasma cells. However, sIgE positivity was more frequently found in local tissue than in serum (p = 0.025). A significant association was observed between tissue sIgE levels and tissue SEC-specific IgE levels (r2 = 0.95, p = 0.0001). The most abundant bacterial and fungal genera identified in the microcolonies were Fusobacterium, Sphingomonas, Porphyromonas, Prevotella and Malassezia. DISCUSSION: These results suggest that there is a local IgE response in children with TH. Local IgE production is unrelated to systemic atopy and may play a key role in the pathogenesis of TH. This is the first study to determine the microbial composition of microcolonies in tonsil tissue. These findings enhance current understanding of the microbiology of tonsils in children with TH and have important implications for antibiotic strategies.

Unilateral Intervention in the Sinuses of Rabbits Induces Bilateral Inflammatory and Microbial Changes.

Background: Chronic rhinosinusitis (CRS) is a globally prevalent inflammatory condition of the paranasal sinuses which severely impairs patients' quality of life. An animal model of unilateral sinusitis by transient sinus occlusion has been described previously in rabbits. The aim of this study was to characterise the sinusitis rabbit model by investigating temporal and bilateral changes in the bacterial community and mucosal inflammation. Methods: Development of sinusitis was achieved by endoscopically placing Merocel ® , a sterile nasal packing material, in the left middle meatus of six New Zealand white rabbits for four weeks. After a total period of 14 weeks, rabbits were assessed for sinusitis by endoscopic examination, magnetic resonance imaging (MRI) and histology. Swabs from the left and right middle meatus were obtained for bacterial community analysis at three time points (week 0, week 4, week 14) during the study. Results: Endoscopic evaluation showed unilateral inflammation in all animals examined after the 4-week blocking period and at week 14. Notably, inflammatory changes were also seen in the contralateral sinus of all animals at week 4. MRI images demonstrated unilateral sinus opacification at week 4 in two rabbits, and partial unilateral sinus opacification at week 14 in one rabbit only. Histological analyses revealed substantial spatial heterogeneity of mucosal inflammation with inconsistent findings across all animals. No significant differences in mucosal inflammatory markers (such as goblet cell hyperplasia, epithelial denudation and oedema) could be identified between nostrils at week 14. The bacterial community in the rabbit sinuses was heavily dominated by Helicobacter at week 0 (baseline). At the end of the blocking period (week 4), bacterial alpha and beta diversity were significantly increased in both nostrils. The bacterial community composition at week 14 had primarily returned to baseline, reflecting the endoscopic and radiological results. Conclusion: This study reaffirmed the ability for development of sinusitis without inoculation of any pathogens in a rabbit model. We were able to demonstrate bilateral sinonasal mucosal inflammation, by inducing unilateral sinus blockage, which resulted in significant changes to the sinonasal bacterial community. These findings may explain some of the clinical observations seen in CRS and warrant further research to reveal potential implications for its therapeutic management.

Impact of Superantigen-Producing Bacteria on T Cells from Tonsillar Hyperplasia.

Staphylococcus aureus and Group A Streptococcus (GAS) are common occupants of the tonsils and many strains produce potent exotoxins (mitogens) that directly target T cells, which could be a driver for tonsillar hyperplasia. Tonsil tissues from 41 patients were tested for these bacteria in conjunction with profiling of B and T cells by flow cytometry. S. aureus and GAS were detected in tonsil tissue from 44% and 7%, respectively, of patients by bacteriological culture; immuno-histology showed bacteria in close proximity to both B and T lymphocytes. The presence of tonsillar S. aureus did not alter B or T cell populations, whereas peripheral blood mucosal-associated invariant T (MAIT) cells were significantly increased in S. aureus culture positive individuals (p < 0.006). Alterations of tonsil CD4+ TCR Vß family members relative to peripheral blood were evident in 29 patients. Three patients had strong TCR Vß skewing indicative of recent exposure to superantigens, their tonsils contained mitogenic bacteria, and supernatants from these bacteria were used to partially recapitulate the skewing profile in vitro, supporting the notion that superantigens can target tonsillar T cells in situ. Tonsils are a reservoir for superantigen-producing bacteria with the capacity to alter the composition and function of key immune cells.

Comparison of Subtyping Approaches and the Underlying Drivers of Microbial Signatures for Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is a heterogeneous condition characterized by persistent sinus inflammation and microbial dysbiosis. This study aimed to identify clinically relevant subgroups of CRS patients based on distinct microbial signatures, with a comparison to the commonly used phenotypic subgrouping approach. The underlying drivers of these distinct microbial clusters were also investigated, together with associations with epithelial barrier integrity. Sinus biopsy specimens were collected from CRS patients (n = 23) and disease controls (n = 8). The expression of 42 tight junction genes was evaluated using quantitative PCR together with microbiota analysis and immunohistochemistry for measuring mucosal integrity and inflammation. CRS patients clustered into two distinct microbial subgroups using probabilistic modelling Dirichlet (DC) multinomial mixtures. DC1 exhibited significantly reduced bacterial diversity and increased dispersion and was dominated by Pseudomonas, Haemophilus, and Achromobacter DC2 had significantly elevated B cells and incidences of nasal polyps and higher numbers of Anaerococcus, Megasphaera, Prevotella, Atopobium, and Propionibacterium In addition, each DC exhibited distinct tight junction gene and protein expression profiles compared with those of controls. Stratifying CRS patients based on clinical phenotypic subtypes (absence or presence of nasal polyps [CRSsNP or CRSwNP, respectively] or with cystic fibrosis [CRSwCF]) accounted for a larger proportion of the variation in the microbial data set than with DC groupings. However, no significant differences between CRSsNP and CRSwNP cohorts were observed for inflammatory markers, beta-dispersion, and alpha-diversity measures. In conclusion, both approaches used for stratifying CRS patients had benefits and pitfalls, but DC clustering provided greater resolution when studying tight junction impairment. Future studies in CRS should give careful consideration to the patient subtyping approach used.IMPORTANCE Chronic rhinosinusitis (CRS) is a major human health problem that significantly reduces quality of life. While various microbes have been implicated, there is no clear understanding of the role they play in CRS pathogenesis. Another equally important observation made for CRS patients is that the epithelial barrier in the sinonasal cavity is defective. Finding a robust approach to subtype CRS patients would be the first step toward unravelling the pathogenesis of this heterogeneous condition. Previous work has explored stratification based on the clinical presentation of the disease (with or without polyps), inflammatory markers, pathology, or microbial composition. Comparisons between the different stratification approaches used in these studies have not been possible due to the different cohorts, analytical methods, or sample sites used. In this study, two approaches for subtyping CRS patients were compared, and the underlying drivers of the heterogeneity in CRS were also explored.

Inflammatory Endotypes and Microbial Associations in Chronic Rhinosinusitis.

A complex mix of inflammatory and microbial associations underscores the chronic inflammatory condition chronic rhinosinusitis (CRS), and the etiology remains poorly understood. Recent work has begun to delineate between variants (endotypes) of CRS on the basis of inflammatory biomarkers. This study aimed to assess inflammatory patterns in CRS phenotypes, identify putative endotypes of CRS, and to assess inflammatory associations with the sinonasal microbiota. Ten cytokines and six inflammatory cell types were assessed in mucosal biopsies from 93 CRS subjects and 17 controls via cytometric bead array and immunohistochemical techniques. Putative endotypes were identified via cluster analysis of subjects on the basis of inflammatory markers and comorbidities including polyposis, asthma, and aspirin sensitivity. Finally, previously published bacterial data for this cohort were reanalyzed to evaluate associations with inflammatory markers and CRS subtypes. Inflammatory patterns were highly variable within standard CRS phenotypes. Cluster analysis identified eight subject clusters, with strong delineation on the basis of polyposis and asthma, but also subtle distinctions in inflammatory markers. An association was also identified between depletion of several "health-associated" bacterial taxa, reduced bacterial diversity and increased overall bacterial load, with markers of inflammation and clinical severity. This study contributes to ongoing efforts to define distinct endotypes of CRS on the basis of underlying inflammatory processes, and also offers compelling evidence of a link between bacterial community dysbiosis and inflammation in CRS. Further resolving the heterogeneity of CRS is vital to inform clinical management and personalized treatment approaches.

The bacterial community and local lymphocyte response are markedly different in patients with recurrent tonsillitis compared to obstructive sleep apnoea.

INTRODUCTION: Obstructive sleep apnea (OSA) is now a more common indication for tonsillectomy than recurrent tonsillitis (RT) [1,2]. Few studies have addressed possible differences in pathogenesis between these two conditions. Children with RT and OSA are often being treated in the community with multiple courses of antibiotics before surgery. Current understanding of the role of bacteria in disorders of the tonsils is mainly based on the culture of tonsil swabs. Swab cultures reflect only a very small fraction of the bacteria present on the mucosal surface and may not represent the bacterial communities within the tonsil crypts [3,4]. This study aimed to evaluate the local lymphocyte response and associations with bacterial community composition using molecular techniques of the tonsils removed from children for RT or OSA. METHOD: The palatine tonsils were removed by extracapsular dissection from 24 patients with age range one to ten years, 14 of whom had RT and 10 had OSA. The fixed tonsil tissues were evaluated for bacteria by Gram-staining and presence of connective tissue by safranin staining. B lymphocytes and T lymphocytes were also measured immunohistochemically. Finally, previously published bacterial community data for this cohort were reassessed for associations with RT and OSA, and with the observed lymphocyte patterns. RESULTS: In tonsils from patients with RT, large micro-colonies of bacteria were observed in the tonsil crypts, and a large number of B and T lymphocytes were noted immediately adjacent to the tonsil crypt itself. In marked contrast, the tonsils from patients with OSA had no bacteria identified, and no significant skewing of lymphocytes based on site (such as follicles or crypts). We observed that the majority of lymphocytes surrounding the bacterial micro-colonies were B lymphocytes with a mean ratio of 109:55 (B lymphocytes: T lymphocytes). Bacterial community diversity was not different between the two cohorts; however, there were significant differences in bacterial community composition. Children with RT had a higher relative abundance of members from the genera Parvimonas, Prevotella, and Treponema. While children with OSA had a higher relative abundance of Haemophilus, and Capnocytophaga. CONCLUSION: These results demonstrate significant differences in the local lymphocyte response and bacterial community composition in tonsil tissue between RT and OSA patients. It suggests that the response to antibiotics used in the treatment of these two conditions may be different. Furthermore, the presence of lymphocytes in RT within the tonsil crypt outside the tonsil epithelium is a unique observation of the location of these cells.

Human papillomavirus and oropharyngeal squamous cell carcinoma: a New Zealand cohort study.

BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas (OPSCC) are clinically, epidemiologically and prognostically distinct from other OPSCCs. The incidence of HPV-related OPSCCs has increased significantly worldwide over the past few decades. However, no studies of OPSCC with direct molecular HPV testing has been conducted in New Zealand. AIMS: To estimate the proportion of OPSCCs attributable to HPV infections in a New Zealand population with a validated HPV testing algorithm. METHODS: HPV-status was determined by p16 immunohistochemistry and polymerase chain reaction (PCR) of both L1 and E6/7 genes on 55 OPSCCs diagnosed in 2010 and 2011 in Central and South Auckland. Baseline and survival analyses were performed according to HPV status. RESULTS: Forty-one (75%) of OPSCC tumours had HPV infections. There was 98% concordance between p16 immunohistochemistry and real-time E6/E7 PCR. After a median follow-up period of 2.6 years, patients with OPSCC of HPV aetiology had more favourable outcomes compared to patients with HPV-negative OPSCC (hazard ratio 0.14, P = 0.02) after adjustment for other variables. CONCLUSION: This study highlights the significant role that HPV plays in the aetiology of OPSCC in New Zealand, and confirms the high rate of accuracy of p16 immunostaining.

Differentially Regulated Host Proteins Associated with Chronic Rhinosinusitis Are Correlated with the Sinonasal Microbiome.

The chronic inflammatory nature of chronic rhinosinusitis (CRS) makes it a morbid condition for individuals with the disease and one whose pathogenesis is poorly understood. To date, proteomic approaches have been applied successfully in a handful of CRS studies. In this study we use a multifaceted approach, including proteomics (iTRAQ labeling) and microbiome (bacterial 16S rRNA gene sequencing) analyses of middle meatus swabs, as well as immune cell analysis of the underlying tissue, to investigate the host-microbe interaction in individuals with CRS (n = 10) and healthy controls (n = 9). Of the total 606 proteins identified in this study, seven were significantly (p < 0.05) more abundant and 104 were significantly lower in the CRS cohort compared with healthy controls. The majority of detected proteins (82% of proteins identified) were not significantly correlated with disease status. Elevated levels of blood and immune cell proteins in the CRS cohort, together with significantly higher numbers of B-cells and macrophages in the underlying tissue, confirmed the inflammatory status of CRS individuals. Protein PRRC2C and Ras-related protein (RAB14) (two of the seven elevated proteins) showed the biggest fold difference between the healthy and CRS groups. Validation of the elevated levels of these two proteins in CRS samples was provided by immunohistochemistry. Members of the bacterial community in the two study cohorts were not associated with PRRC2C, however members of the genus Moraxella did correlate with RAB14 (p < 0.0001, rho = -0.95), which is a protein involved in the development of basement membrane. In addition, significant correlations between certain members of the CRS bacterial community and 33 lower abundant proteins in the CRS cohort were identified. Members of the genera Streptococcus, Haemophilus and Veillonella were strongly correlated with CRS and were significantly associated with a number of proteins with varying functions. The results from this study reveal a strong association between the host and microbes in the sinonasal cavity. Proteins identified as associated with CRS could be new targets for drug therapies and biomarkers for assessment of treatment efficacy.

The in vitro mucolytic effect of xylitol and dornase alfa on chronic rhinosinusitis mucus.

BACKGROUND: The overproduction and stagnation of purulent mucus impair mucociliary clearance and exacerbate the symptoms of chronic rhinosinusitis (CRS). There is a clinical need for effective topical mucolytic agents to facilitate removal of mucus and improve postoperative outcomes. METHODS: The effects of xylitol (5%) and dornase alfa (1 mg/mL) on mucus and mucus crusts were investigated. Viscoelasticity and viscosity of wet mucus derived from 30 CRS patients was measured with a plate rheometer. Postoperative dried mucus crust dissolution was measured by examining peripheral transparency, central transparency, and border definition of treated crust samples from 17 CRS patients. RESULTS: Xylitol and dornase alfa reduced wet mucus viscoelasticity at a frequency of 0.1 Hz significantly more than the saline control. Treatments also produced significantly lower viscosities than saline at a shear rate of 10 and 100 seconds-1 . Xylitol and dornase alfa significantly decreased mucus crust border definition relative to saline. CONCLUSION: Xylitol and dornase alfa may be efficacious mucolytics, encouraging the breakdown of postoperative mucus crusts and the reduction of viscoelasticity and viscosity of wet mucus. In vivo study is required to evaluate the potential of these agents in treating recalcitrant CRS.

Differences in the paranasal sinuses between germ-free and pathogen-free mice.

BACKGROUND: The role of bacteria in the etiology of chronic rhinosinusitis (CRS) is not fully understood. Commensal bacteria may have a significant impact on the development of normal paranasal sinus anatomy and mucosal immunity, as they do in the gut. Studying the paranasal sinuses of germ-free (GF) mice may provide some insight into the effect of commensal bacteria on sinus structure and mucosal function. METHODS: The paranasal sinuses of 5 GF mice were compared to 5 pathogen-free normal mice. Mice heads underwent computed tomography and images were compared for pneumatization and geometry of the sinuses. Histologically, slides were examined by light microscopy and compared for mucosal thickness, epithelial thickness, cilia, collagen, goblet cells, and nasal-associated lymphatic tissue (NALT). RESULTS: No radiological differences were seen between groups. Overall, GF mice were found to have thinner mucosa (Δ 15.2 ± 5.2 µm, p = 0.004), thinner epithelium (Δ 5.5 ± 2.6 µm, p = 0.037), more collagen (Δ 5.8% ± 1.6%, p < 0.001), fewer goblet cells (Δ 29.3 ± 5.4, p < 0.001), and less NALT (Δ 14,900 ± 6700 µm(2) , p = 0.04). Subanalysis by region revealed significant differences for GF mice in the middle (thinner mucosa, thinner epithelium, fewer cilia, and more collagen) and posterior (fewer goblet cells) sinus sections. CONCLUSION: The results of this study demonstrate that commensal microbiota significantly contribute to the structure and function of murine paranasal sinuses. Therefore, changes in commensal microbiota associated with CRS may alter the normal microbe host dialogue in humans and be implicated in the pathogenesis of CRS.

The effects of topical agents on paranasal sinus mucosa healing: a rabbit study.

BACKGROUND: Numerous topical agents have been used intraoperatively to enhance postoperative mucosal healing or reduce scar formation. However, the histological effects of many of these treatments have not been well described. This study investigates the impact of topical mometasone furoate, acitretin, lactoferrin, and Silastic sheet (Medtronic) on sinus mucosal healing in a rabbit model. METHODS: Forty-eight New Zealand white rabbits underwent defined, localized stripping of a bilateral region of maxillary sinus mucosa. One of 6 treatments was placed in 1 maxillary sinus, and the treatment carrier was applied contralaterally (0.1% mometasone furoate, 0.25% and 0.5% acitretin, lactoferrin, Silastic, and no treatment; n = 8 each group). Rabbits were euthanized after 2 weeks and histological sections were examined with light microscopy. RESULTS: Treatment with acitretin 0.25% and 0.5% improved cilial recovery by 0.9 ± 0.5 (p = 0.003) and 0.5 ± 0.5 (p < 0.05), respectively. Acitretin 0.25% treatment also significantly reduced collagen in healing mucosa (5.1% ± 4.8%, p = 0.04). Conversely, rabbits treated with mometasone furoate 0.1% were more likely to have reduced cilial and goblet cell recovery. Intergroup comparisons demonstrated a significant improvement in cilial recovery scores with both acitretin doses compared with mometasone furoate (p < 0.05) and less collagen deposition in rabbits treated with placebo gel over Silastic (p < 0.05). Mucosa directly underlying a blood clot had a lower cilia score and impaired epithelial recovery (p < 0.001). CONCLUSION: Intraoperatively applied agents have the potential to significantly affect wound healing. Acitretin improved cilial recovery and reduced collagen deposition.

The characteristics of intramucosal bacteria in chronic rhinosinusitis: a prospective cross-sectional analysis.

BACKGROUND: We have observed subepithelial bacterial microcolonies within the mucosa of patients with chronic rhinosinusitis (CRS). These were predominantly Staphylococcus aureus and did not appear to elicit a local inflammatory response. We hypothesized that these microcolonies had made adaptations allowing them to exist apparently undetected within the mucosa. We sought to determine whether the tissue colonies had genotypic or phenotypic variations from the surface bacteria. METHODS: Mucosal swabs and tissue biopsies were taken from 31 patients with CRS undergoing functional endoscopic sinus surgery, and 9 with normal sinuses having transnasal pituitary surgery. Biopsied tissues were assessed histologically, by routine culture, and by culture techniques facilitating growth of small colony variants (SCVs). Genotypic typing compared isolates of S. aureus that were cultured from both swab and tissue samples. The activity of the accessory gene regulator (agr) gene, a global regulator of S. aureus virulence, was evaluated indirectly by determining the hemolytic activity of the colonies on blood agar. RESULTS: SCVs were grown from 2 samples but these were found not to possess the nuc gene, specific to S. aureus. When S. aureus was recovered from both swab and mucosa, the genetic profiles were indistinguishable in all but 1 patient. All S. aureus cultured from mucosa demonstrated ß-hemolysis, implying normal agr activity. CONCLUSION: Intramucosal S. aureus are genetically closely related and phenotypically similar to surface S. aureus. Further studies are needed to explore the possible mechanisms by which intramucosal colonies become less immunogenic, and the role of the colonies in the pathophysiology of CRS.