The efficacy and safety of chitosan dextran gel in a burr hole neurosurgical sheep model.

BACKGROUND: Achieving and maintaining haemostasis is of paramount importance in neurosurgery. Chitosan has been shown in both animal and human models to be significantly effective in haemostasis as well as in reducing adhesion formation. OBJECTIVES: To evaluate the haemostatic potential and to study histopathological changes caused by novel chitosan dextran gel in a neurosurgical sheep model. METHOD: Ten sheep underwent neurosurgical burr hole procedure. Bleeding control was tested at the level of bone, dura and brain separately with both chitosan gel and Gelfoam paste on separate burr holes. Baseline bleeding was measured at the time of injury using the Boezaart scale, and then every 2 min after the application of each agent until complete haemostasis or 10 min, whichever was earlier. Safety was assessed through MRI scans and histopathological analysis. RESULTS: Mixed modeling showed no statistical difference in time to haemostasis between chitosan gel and Gelfoam paste (means of log-normalized areas under the curve were 1.3688 and 1.3196 respectively) for each burr hole (p = 0.7768). Logistic regression modeling showed that Chitosan significantly decreased the incidence of bleeding beyond the first time point measured after application of the treatment when compared to Gelfoam (OR = 2.7, p = 0.04). Average edema volume (cm(3)) on post-operative MRI was 0.97 for Gelfoam and 1.11 for (p = 0.49) while average histology scores were 2.5 for Gelfoam versus 3.3 for chitosan (p = 0.32). CONCLUSION: Chitosan dextran gel is an effective haemostatic agent to control bleeding in brain tissue. It is safe and nontoxic to neural tissue.

Hypopharyngeal perforation caused by blunt trauma in buffalo attack.

A male in his 50s arrived by ambulance at a regional Australian hospital after being pinned by a buffalo against a fence by the chest and abdomen. Primary and secondary surveys identified an open fibula fracture and superficial abrasions. CT trauma series identified retropharyngeal free gas extending to the right carotid sheath. Flexible nasoendoscopy revealed a normal upper airway and no site of perforation. Oesophagoscopy and gastroscopy were completed to evaluate for a site of free gas leakage. A hypopharyngeal tear was identified 15 cm from the incisors at the cricopharyngeal sphincter. A gastrograffin swallow was completed which showed no leak. The decision was made to manage the patient conservatively with intravenous dexamethasone and intravenous ceftriaxone/metronidazole for antibiotic prophylaxis. The patient had his diet gradually upgraded and was discharged home 4 days later with oral amoxicillin and clavulanic acid.

A single nucleotide polymorphism in an IgA1 protease gene determines Streptococcus pneumoniae adaptation to the middle ear during otitis media.

Factors facilitating the chronicity of otitis media (OM) in children are, to date, not fully understood. An understanding of molecular factors aiding bacterial persistence within the middle ear during OM could reveal pathways required for disease. This study performed a detailed analysis of Streptococcus pneumoniae populations isolated from the nasopharynx and middle ear of one OM case. Isolates were assessed for growth in vitro and infection in a mouse intranasal challenge model. Whole genome sequencing was performed to compare the nasopharyngeal and middle ear isolates. The middle ear isolate displayed a reduced rate of growth and enhanced potential to transit to the middle ear in a murine model. The middle ear population possessed a single nucleotide polymorphism (SNP) in the IgA1 protease gene igA, predicted to render its product non-functional. Allelic exchange mutagenesis of the igA alleles from the genetic variant middle ear and nasopharyngeal isolates was able to reverse the niche-adaptation phenotype in the murine model. These results indicate the potential role of a SNP in the gene encoding the IgA1 protease, in determining S. pneumoniae adaptation to the middle ear during chronic OM. In contrast, a functional IgA1 protease was associated with increased colonisation of the nasopharynx.

Surgical outcomes of endoscopic management of adenocarcinoma of the sinonasal cavity.

OBJECTIVE: To report the surgical outcomes of endoscopic resection of adenocarcinomas of the Sinonasal cavity. METHODS: Retrospective chart review of patients presenting with adenocarcinoma of the anterior skull base between 1998-2008. All patients who underwent wholly endoscopic resection were included in the study. RESULTS: Twelve patients presented with adenocarcinoma involving the sino-nasal cavity. At diagnosis 6 patients were staged as a T2, 5 as a T3 and one as a T4. All of the patients had successful removal of the tumour entirely endoscopically. Three patients recurred: 2 locally and 1 with distant metastases. Overall, 11 patients are alive and free of disease and 1 patient dead of disease. We found an overall disease free survival rate and overall survival rate of 91.6%. The follow-up period ranged from 10 to 96 months with a median of 30 months. CONCLUSION: Endoscopic management of adenocarcinoma of the sino-nasal cavity can be a viable treatment option to craniofacial resection. With the advancement in endoscopic equipment and surgeon skill, larger tumours may be managed wholly endoscopically.

An in vivo safety and efficacy demonstration of a topical liposomal nitric oxide donor treatment for Staphylococcus aureus biofilm-associated rhinosinusitis.

The burden of drug resistance emerges in the wake of chronic and repeated antibiotic use. This underpins the importance of discovering alternatives to current antibiotic regimens. In chronic rhinosinusitis (CRS), topical therapy such as nasal douches and steroid sprays is the mainstay of treatment. However, bacterial sinusitis such as those with Staphylococcus aureus biofilm infection point to more recalcitrant CRS subtypes, focusing research efforts into topical antimicrobial therapies. In the sinuses, both local mucosal and systemic effects must be considered in designing any new topical medication. Nitric oxide (NO), an endogenous antimicrobial agent, is found at extremely low levels in CRS sinuses and high levels in healthy sinuses. As a novel treatment modality, we have designed a liposomal formulation of an NO donor (LFNO) using isosorbide mononitrate, as a topical sinus wash in a sheep model of S. aureus biofilm rhinosinusitis. Heart rate (HR), blood pressure, mean arterial pressure (MAP), and histologic and ciliary analyses were assessed in the safety component. Efficacy was assessed by quantifying biofilm biomass post-treatment. LFNO-treated sheep had lesser inflammation (P = 0.02), and comparable ciliary preservation (P = 0.86) than the control group. A transient increase in HR and decrease in MAP were observed in the LFNO group (P < 0.05), but this was not accompanied by observable side effects. LFNO sheep had significantly lower biofilm biomass vs controls (P = 0.044). Our findings demonstrate the localized and systemic safety of LFNO in an animal model despite using high NO concentrations, thus warranting further investigation for its possible therapeutic role in CRS.

Corticosteroids directly reduce Staphylococcus aureus biofilm growth: an in vitro study.

OBJECTIVES/HYPOTHESIS: Clinical improvement in patients with chronic rhinosinusitis (CRS) treated with steroids alone has previously been ascribed to the steroids' anti-inflammatory properties rather than any direct effect on the bacteria. The aim of this study was to determine if commonly used intranasal steroids directly reduce bacterial biofilm production in vitro. STUDY DESIGN: In vitro comparative controlled trial. METHODS: Staphylococcus aureus biofilms were grown on minimum biofilm eradication concentration device pegs and treated with the commonly prescribed CRS topical steroids fluticasone, mometasone, or budesonide. These were dissolved in vehicle solvents and added to cerebrospinal fluid (CSF) broth. Concentrations (including therapeutic doses) tested for fluticasone and mometasone ranged from 25 µg/200 µL to 400 µg/200 µL, and from 16 µg/200 µL to 2000 µg/200 µL for budesonide. Control pegs were exposed to equivalent volumes of the appropriate solvent/CSF broth. Confocal scanning laser microscopy and COMSTAT software were used to quantify biofilms at 24 hours after treatment. RESULTS: Significant differences from control were found for fluticasone at 400 µg/200 µL (difference = -0.3065 µm(3)/µm(2), P = .007), mometasone at 300 µg/200 µL and 400 µg/200 µL (difference = -0.15 µm(3)/µm(2), P = .006, and difference = -0.9193 µm(3)/µm(2), P = .034, respectively), and budesonide at 750 µg/200 µL, 1000 µg/200 µL and 2000 µg/200 µL (difference = -1.0137 µm(3)/µm(2), P = .038, difference = -0.6164, P = .009, and difference = -0.1906 µm(3)/µm(2), P = .029, respectively). CONCLUSIONS: The concentrations of 400 µg/200 µL of fluticasone, 300 µg and 400 µg/200 µL of mometasone, and 750 µg, 1,000 µg, and 2,000 µg/200 µL of budesonide directly reduce biofilm production in vitro, outside of the inflammatory milieu.

Colloidal silver: a novel treatment for Staphylococcus aureus biofilms?

BACKGROUND: Colloidal silver is an alternative medicine consisting of silver particles suspended in water. After using this solution as a nasal spray, the symptoms of a previously recalcitrant Staphylococcus aureus (S. aureus)-infected chronic rhinosinusitis patient were observed to have improved markedly. The aim of this study was to determine whether colloidal silver has any direct bactericidal effects on these biofilms in vitro. METHODS: S. aureus biofilms were grown from the ATCC 25923 reference strain on Minimum Biofilm Eradication Concentration (MBEC) device pegs, and treated with colloidal silver. Concentrations tested ranged from 10 to 150 µL colloidal silver diluted to 200 µL with sterile water in 50 µL cerebrospinal fluid (CSF) broth. Control pegs were exposed to equivalent volumes of CSF broth and sterile water. The sample size was 4 biomass values per treatment or control group. Confocal scanning laser microscopy and COMSTAT software were used to quantify biofilms 24 hours after treatment. RESULTS: Significant differences from control were found for all concentrations tested bar the lowest of 10 µL colloidal silver in 200 µL. At 20 µL colloidal silver, the reduction in biomass was 98.9% (mean difference between control and treatment = -4.0317 µm(3) /µm(2) , p < 0.0001). A maximum biomass reduction of 99.8% was reached at both 100 and 150 µL colloidal silver (mean differences = -4.0681 and -4.0675µm(3) /µm(2) , respectively, p < 0.0001). CONCLUSION: Colloidal silver directly attenuates in vitro S. aureus biofilms.

Early and late complications of endoscopic hemostatic techniques following different carotid artery injury characteristics.

BACKGROUND: The most dreaded hemorrhagic complication in endoscopic endonasal surgery is injury to the internal carotid artery (ICA). Although a number of treatment protocols are currently used, none have been formally investigated. This study aims to compare the efficacy of the muscle patch, bipolar diathermy, and aneurysm clip on hemostasis, pseudoaneurysm formation, and long-term vessel patency for different injury types in a sheep model of carotid bleeding. METHODS: Twenty-seven sheep underwent ICA dissection/isolation followed by the artery placement within a modified "sinus model otorhino neuro trainer" (SIMONT) model. Standardized linear, punch, and stellate injuries were made. Randomization of sheep to receive 1 of 3 hemostatic techniques was performed (muscle, bipolar, clip). Specific outcome measures included attainment of primary hemostasis, time to hemostasis, blood loss, pseudoaneurysm formation, and carotid patency on follow-up magnetic resonance imaging (MRI). RESULTS: Bipolar achieved primary hemostasis in 7 of 9 cases and 2 cases of secondary hemorrhage. It had no associated pseudoaneurysm formation. Carotid patency was variable on follow-up MRI. Muscle patch achieved 100% primary hemostasis with 2 cases of secondary hemorrhage. There were 2 cases of pseudoaneurysm and 100% patency rate on follow-up MRI. Aneurysm clip achieved 100% primary hemostasis with 1 case of secondary hemorrhage. No pseudoaneurysm formation and a 50% rate of carotid insufficiency on MRI. CONCLUSION: This study shows that the crushed muscle patch and aneurysm clip can be viable options in the management of ICA injury with short-term and long-term benefits. Complications associated with these techniques were comparable if not reduced when compared to the published literature.

Methylglyoxal-augmented manuka honey as a topical anti-Staphylococcus aureus biofilm agent: safety and efficacy in an in vivo model.

BACKGROUND: Bacterial biofilms are thought to contribute to recalcitrance in chronic rhinosinusitis (CRS) patients. Manuka honey (MH) and its active component methylglyoxal (MGO) have demonstrated antibiofilm activity in vitro. This study evaluated the safety and efficacy of these agents in an in vivo model. METHODS: To assess safety, ovine frontal sinuses were flushed twice daily for 14 days. In each sheep, 1 sinus was flushed with a panel of MGO concentrations ranging from 0.5 to 7.2 mg/mL alone and flushed with a panel of with 16.5% wt/vol MH enriched with MGO at the same range of concentrations (0.5-7.2 mg/mL; designated MH/MGO). Contralateral sinuses were flushed with saline control. Tissue morphology was assessed histologically and with scanning electron microscopy. Efficacy was tested by developing Staphylococcus aureus biofilms in sheep sinuses. Twice-daily irrigation for 5 days was commenced with either saline, MGO (0.5-3.6 mg/mL) alone, or MH/MGO (with 0.5-3.6 mg/mL MGO). Biofilm biomass was compared between the groups (n = 4) using LIVE/DEAD BacLight staining and confocal scanning laser microscopy. RESULTS: The results of the safety assessment, for normal sinuses treated with MGO alone or with MH/MGO (≤1.8 mg/mL) showed normal pseudostratified epithelium and cilia structure; however, higher concentrations caused cilia denudation and squamous metaplasia. As for efficacy, when compared to saline flush, treatment with MH/MGO at 0.9 mg/mL (0.608 ± 0.110 vs 0.316 ± 0.197 µm(3) /µm(2) , respectively; p = 0.015) and 1.8 mg/mL (0.676 ± 0.079 vs 0.114 ± 0.033 µm(3) /µm(2) , respectively; p = 0.001) significantly reduced biofilm biomass. CONCLUSION: Sinus irrigation with MH/MGO at MGO concentrations between 0.9 and 1.8 mg/mL is both safe to mucosa and efficacious against S. aureus biofilm. MH/MGO irrigation could represent a viable treatment option for recalcitrant CRS.

Bacteriophage reduces biofilm of Staphylococcus aureus ex vivo isolates from chronic rhinosinusitis patients.

BACKGROUND: Staphylococcus aureus is the most common organism in recalcitrant chronic rhinosinusitis (CRS) and is often resistant to traditional antibiotic therapy. Bacteriophages ("phages") are a potential candidate for a new, effective therapy. For phages to be useful in the setting of CRS, two minimum requirements must be presented: (1) phages must be effective against S. aureus biofilms and (2) phages must have a broad spectrum of activity. This study aimed to assess the in vitro activity of a phage cocktail (CockTail of Staphylococcus aureus specific bacteriophage [CT-SA]) against S. aureus biofilms and a broad panel of strains isolated from patients with CRS. METHODS: The study examined 66 clinical isolates (CIs) of S. aureus. All isolates were tested for the susceptibility to phage lysis by spotting CT-SA onto bacterial lawns. To measure its effect on S. aureus biofilms, a minimum biofilm eradication concentration assay was used, using five S. aureus isolates. Biofilms of these isolates were grown, treated with CT-SA for 48 hours, fluorescently stained, and viewed using confocal scanning laser microscopy. RESULTS: CT-SA lysed 62 of 66 (94%) CIs of S. aureus. CT-SA treatment yielded significant reductions in biofilm mass for 4/5 CIs tested and for ATCC 25923. Challenge of S. aureus with a single phage resulted in the emergence of bacteriophage-insensitive mutants (BIM) with a frequency of 10(-7), and challenge with CT-SA completely prevented their development. CONCLUSION: This study indicates that phage cocktail CT-SA can effectively eliminate S. aureus, in planktonic and biofilm forms, from the great majority of CIs from this hospital setting. In addition, its potential effect in preventing the emergence of BIMs was also established. Thus, CT-SA has the potential to treat S. aureus infection and biofilm in CRS patients.

Liposome-encapsulated ISMN: a novel nitric oxide-based therapeutic agent against Staphylococcus aureus biofilms.

BACKGROUND: Staphylococcus aureus in its biofilm form has been associated with recalcitrant chronic rhinosinusitis with significant resistance to conventional therapies. This study aims to determine if liposomal-encapsulation of a precursor of the naturally occurring antimicrobial nitric oxide (NO) enhances its desired anti-biofilm effects against S. aureus, in the hope that improving its efficacy can provide an effective topical agent for future clinical use. METHODOLOGY: S. aureus ATCC 25923 biofilms were grown in-vitro using the Minimum Biofilm Eradication Concentration (MBEC) device and exposed to 3 and 60 mg/mL of the NO donor isosorbide mononitrate (ISMN) encapsulated into different anionic liposomal formulations based on particle size (unilamellar ULV, multilamellar MLV) and lipid content (5 and 25 mM) at 24 h and 5 min exposure times. Biofilms were viewed using Live-Dead Baclight stain and confocal scanning laser microscopy and quantified using the software COMSTAT2. RESULTS: At 3 and 60 mg/mL, ISMN-ULV liposomes had comparable and significant anti-biofilm effects compared to untreated control at 24 h exposure (p = 0.012 and 0.02 respectively). ULV blanks also had significant anti-biofilm effects at both 24 h and 5 min exposure (p = 0.02 and 0.047 respectively). At 5 min exposure, 60 mg/mL ISMN-MLV liposomes appeared to have greater anti-biofilm effects compared to pure ISMN or ULV particles. Increasing liposomal lipid content improved the anti-biofilm efficacy of both MLV and ULVs at 5 min exposure. CONCLUSION: Liposome-encapsulated "nitric oxide" is highly effective in eradicating S. aureus biofilms in-vitro, giving great promise for use in the clinical setting to treat this burdensome infection. Further studies however are needed to assess its safety and efficacy in-vivo before clinical translation is attempted.

Safety and efficacy of topical bacteriophage and ethylenediaminetetraacetic acid treatment of Staphylococcus aureus infection in a sheep model of sinusitis.

BACKGROUND: Treatment of sinonasal bacterial biofilms continues to be a challenge in modern rhinology. This study's objective was to assess the safety and efficacy of topically applied Cocktail of S. aureus specific phage (CTSA) alone and in combination with ethylenediaminetetraacetic acid (EDTA) for treatment of Staphylococcus aureus biofilms in vivo. METHODS: Using a sheep model of sinusitis, frontal sinuses (n = 6 per treatment) were flushed once daily with a CTSA (2 × 10(6) plaque forming units [PFU]/mL), with or without EDTA (0.075 mg/mL), and compared to a control flush containing saline and heat-inactivated CTSA. Safety was assessed using histology and scanning electron microscopy (SEM) after treatment for 3 days. Efficacy was assessed by quantifying the generation of S. aureus biofilms in the frontal sinuses after 5 days of treatment. Biofilm mass was compared between treatment groups and controls using LIVE/DEAD BacLight staining and confocal scanning laser microscopy to visualize the tissue sections. COMSTAT2 software was used to compute the biofilm mass present on tissue sections. RESULTS: Tissue morphology was conserved, with no significant signs of inflammation, when comparing control and test treatments. Furthermore, SEM analysis indicated test treatments were not toxic or damaging to mucosal cilia. COMSTAT2 quantification of biofilm showed a significant reduction in biofilm levels when comparing the control with CTSA (p = 0.0043), EDTA (p = 0.0095), and CTSA-EDTA (p = 0.0022) treatments. CONCLUSION: Results indicate that CTSA and EDTA are safe and efficacious for short-term topical application against S. aureus infection in a sheep sinusitis model, and have the potential to be translated to a clinical setting.

A human nasal explant model to study Staphylococcus aureus biofilm in vitro.

BACKGROUND: Staphylococcus aureus (S. aureus) biofilm has been associated with severe and recalcitrant cases of chronic rhinosinusitis (CRS). However, its role in the pathophysiology of this condition is not completely understood. This study aims to develop a sinonasal tissue explant model to analyze the interaction of S. aureus biofilm with the mucosa in vitro. METHODS: Sinonasal tissue samples from 5 control patients undergoing pituitary surgery were cultured with and without S. aureus biofilm in vitro. Confocal scanning laser microscopy (CSLM) using the Live/Dead BacLight stain and histology were performed on the tissue explants after 24 hours of biofilm challenge. Measurements of IL-6, at both the messenger RNA (mRNA) level (using quantitative reverse-transcriptase polymerase chain reaction [qRT-PCR]) and the protein level (using enzyme-linked immunosorbent assay [ELISA]), were undertaken to evaluate biofilm-mucosa interaction. RESULTS: Viability of the explants after 24 hours was confirmed by CSLM and histology. Although light microscopy failed to identify S. aureus biofilms, its presence was confirmed in the biofilm-challenged samples by CSLM. IL-6 mRNA transcript levels were 4.9-fold upregulated in biofilm-treated tissue compared to controls (p = 0.0485). A similar trend was observed at the protein level (p = 0.0313). CONCLUSION: The sinonasal tissue explant is a viable and functional model capable of analyzing direct biofilm-mucosal interactions and can advance our understanding of the role played by S. aureus biofilm in sinus inflammation. Our model suggests that S. aureus biofilms in the initial phase of growth are not inert bystanders but elicit an immune response in the sinonasal mucosa.

Intracellular Staphylococcus aureus: the Trojan horse of recalcitrant chronic rhinosinusitis?

BACKGROUND: Despite recent evidence suggesting that Staphylococcus aureus exists within the sinonasal epithelium of chronic rhinosinusitis (CRS) patients, certain questions remain. The intracellular environment may provide a protective niche for pathogenic bacteria to evade host immunity and yet provide a reservoir for reinfection. To date, no studies have examined the impact of this bacterial phenotype; therefore, this study was designed to evaluate the role of intracellular S. aureus on postsurgical outcomes. METHODS: This study included 51 patients undergoing endoscopic sinus surgery (ESS) for medically-recalcitrant CRS. Sinonasal mucosa harvested at the time of surgery was dually stained with fluorescent molecular probes and imaged using confocal scanning laser microscopy for biofilm and intracellular status. Patients were followed in their early and late postoperative course for evidence of ongoing disease and signs of clinical relapse. RESULTS: Intracellular S. aureus was identified in 20 of 51 (39%) patients, and all were associated with surface biofilm. Biofilm alone was found in 16 of 51 (31%) patients and 15 of 51 (29%) patients had no evidence of S. aureus. Intracellular positive patients had a significantly higher risk of late clinical and microbiological relapse (p = 0.014). In this study, biofilm status without coexisting intracellular bacteria did not appear to impact on outcomes. CONCLUSION: Clinical and microbiological relapse of disease following ESS is significantly associated with intracellular S. aureus. Evidence suggests that this disease association is independent to surface biofilm status. Intracellular bacteria should be taken into consideration when designing novel treatment strategies to lessen the chance of reinfection.

Gene expression differences in nitric oxide and reactive oxygen species regulation point to an altered innate immune response in chronic rhinosinusitis.

BACKGROUND: The complex interplay between host, environment, and microbe in the etiopathogenesis of chronic rhinosinusitis (CRS) remains unclear. This study focuses on the host-microbe interaction, specifically the regulation of nitric oxide (NO) and reactive oxygen species (ROS) against the pathogenic organism Staphylococcus aureus (S. aureus). NO and ROS play crucial roles in innate immunity and in the first-line defense against microbial invasion. METHODS: Sinonasal tissue samples were harvested from CRS and control patients during surgery. CRS patients were classified S. aureus biofilm-positive (B+) or biofilm-negative (B-) using fluorescence in situ hybridization and clinically as polyp-positive (P+) or polyp-negative (P-). Samples were assessed using an NO polymerase chain reaction (PCR) array containing 84 genes involved in NO and ROS regulation, and gene expression of all subgroups were compared to each other. RESULTS: Twenty-three samples were analyzed with 31 genes significantly changed, the greatest seen in the B+P+ CRS patients. Four genes consistently displayed differential expression between the groups including the cytoprotective oxidation resistance 1 (OXR1) and peroxiredoxin 6 (PRDX6), neutrophil cytosolic factor 2 (NCF2), and the prion protein (PRNP) genes. CONCLUSION: Alteration in gene expression points to impaired innate immune responses differing among CRS subgroups based on S. aureus biofilm and polyp status. The consistent alteration of 4 genes among distinct groups demonstrates that S. aureus biofilms and polyps are associated with specific changes in gene expression. Further studies are required to validate these findings in a wider cohort of patients and correlate this to protein expression and disease manifestation.

Quantitative analysis of in vivo mucosal bacterial biofilms.

BACKGROUND: Quantitative assays of mucosal biofilms on ex vivo samples are challenging using the currently applied specialized microscopic techniques to identify them. The COMSTAT2 computer program has been applied to in vitro biofilm models for quantifying biofilm structures seen on confocal scanning laser microscopy (CSLM). The aim of this study was to quantify Staphylococcus aureus (S. aureus) biofilms seen via CSLM on ex situ samples of sinonasal mucosa, using the COMSTAT2 program. METHODS: S. aureus biofilms were grown in frontal sinuses of 4 merino sheep as per a previously standardized sheep sinusitis model for biofilms. Two sinonasal mucosal samples, 10 mm × 10 mm in size, from each of the 2 sinuses of the 4 sheep were analyzed for biofilm presence with Baclight stain and CSLM. Two random image stacks of mucosa with S. aureus biofilm were recorded from each sample, and analyzed using COMSTAT2 software that translates image stacks into a simplified 3-dimensional matrix of biofilm mass by eliminating surrounding host tissue. Three independent observers analyzed images using COMSTAT2 and 3 repeated rounds of analyses were done to calculate biofilm biomass. RESULTS: The COMSTAT2 application uses an observer-dependent threshold setting to translate CSLM biofilm images into a simplified 3-dimensional output for quantitative analysis. Intraclass correlation coefficient (ICC) between thresholds set by the 3 observers for each image stacks was 0.59 (p = 0.0003). Threshold values set at different points of time by a single observer also showed significant correlation as seen by ICC of 0.80 (p < 0.001). CONCLUSION: COMSTAT2 can be applied to quantify and study the complex 3-dimensional biofilm structures that are recorded via CSLM on mucosal tissue like the sinonasal mucosa.

Identifying intracellular Staphylococcus aureus in chronic rhinosinusitis: a direct comparison of techniques.

BACKGROUND: The emerging concept of intracellular pathogens such as Staphylococcus aureus playing a role in chronic rhinosinusitis (CRS) has led to the development of numerous imaging techniques for their identification. Traditional methods of bacterial culture are not effective at localizing bacteria to the surface or within tissue samples. The aim of this study was to develop and validate a novel imaging technique using confocal scanning laser microscopy (CSLM) coupled with a fluorescence in situ hybridization (FISH) probe and nucleic acid counterstain (propidium iodide [PI]) that allows for simultaneous analysis of S. aureus intracellular status and surface biofilm within whole mucosal samples. METHODS: A prospective study was performed including 17 patients undergoing endoscopic sinus surgery for CRS. Tissue samples were analyzed with both CSLM-FISH/PI and immunohistochemistry (IHC) for intracellular S. aureus status. RESULTS: Using CSLM-FISH/PI intracellular S. aureus was identified in 9/17 (47%) patients and in 7/17 (39%) using IHC. Surface biofilm can be identified with CSLM-FISH/PI in the same piece of tissue; however, deeper imaging to the submucosa is impossible. IHC showed submucosal bacteria in three patients. CONCLUSION: Both CSLM-FISH/PI and IHC are complementary techniques that can be used to identify intracellular S. aureus. CSLM-FISH/PI allows for the simultaneous detection of intracellular status and surface biofilm within the tissue analyzed. IHC has a role in the identification of intracellular and submucosal S. aureus within these tissues. Additional investigation is required to identify the true pathogenic nature of intracellular organisms as well as any relationship to surface biofilm status.

The multiplicity of Staphylococcus aureus in chronic rhinosinusitis: correlating surface biofilm and intracellular residence.

OBJECTIVES/HYPOTHESIS: The biofilm paradigm of chronic rhinosinusitis (CRS) is increasingly understood to play a key role in the pathophysiology of this disease. The role of intracellular infection of sinonasal epithelial cells has been suggested as a potential reservoir of pathogenic organisms that can lead to recalcitrant disease despite maximal medical and surgical treatment. Could a surface biofilm play a role in allowing intracellular infection to occur, and what are the factors associated with potential intracellular infections? The aim of this study was to investigate these questions. STUDY DESIGN: A prospective study including 36 CRS patients undergoing endoscopic sinus surgery and five control patients undergoing endonasal pituitary surgery. METHODS: Sinonasal mucosa harvested at the time of surgery was examined with a Staphylococcus aureus fluorescence in situ hybridization probe and propodium iodide counterstain using the confocal scanning laser microscope for both biofilm status and evidence of intracellular organisms. RESULTS: Intracellular S aureus was identified in 20/36 (56%) CRS patients compared to 0/8 (0%) control patients. CRS patients with intracellular infection were significantly more likely to harbor surface biofilm (20/20, P = .0014) and have a S aureus-positive culture swab (12/20, P = .0485). CONCLUSIONS: This study gives further evidence supporting a role of intracellular S aureus in CRS. In all cases intracellular infection was associated with surface biofilm, suggesting a potential relationship between the two. Further work is required to delineate the true mechanisms of intracellular persistence and also the role that it plays in the recalcitrant nature of CRS.

The effects of nitric oxide on Staphylococcus aureus biofilm growth and its implications in chronic rhinosinusitis.

BACKGROUND: The relationship between sinonasal nitric oxide (NO) levels and the pathogenic organism Staphylococcus aureus is yet to be established. High NO levels measured in healthy sinuses likely contribute to maintenance of relative sterility. Lower concentrations such as is found in the sinuses of chronic rhinosinusitis (CRS) patients may decrease this effect. S. aureus in biofilm form has recently been implicated in recalcitrant CRS, its isolation predicting a higher risk of posttreatment reinfection. This in vitro study aims to characterize the changes in S. aureus biofilm formation when exposed to different NO levels mimicking the normal and diseased NO sinus concentrations reported in previous literature in an in vitro setting. METHODS: S. aureus ATCC 25923 and 7 clinical isolates were cultured in biofilm form using the MBEC device and the established biofilms exposed to 1 to 1000 µM NO concentrations. Biofilms were visualized using Live/Dead Baclight stain and confocal scanning laser microscopy, and quantified using Comstat2, a biofilm quantification software. RESULTS: Biofilm biomass decreases from an average of 0.105 to 0.057 µm(3) /µm(2) at higher NO concentrations (125-1000 µM), but is increased to 0.470 µm(3) /µm(2) at lower NO concentrations (0.9-2.0 µM). The average biomass at high vs low concentrations are statistically significant (p < 0.001). CONCLUSION: S. aureus biofilm formation varies across exposure to different NO levels, with antibiofilm effects at higher concentrations, and enhanced biofilm formation at lower or subphysiologic concentrations. These results coincide with the often dualistic function of NO, and have implications in its future use in the treatment of CRS.