Staphylococcus aureus from patients with chronic rhinosinusitis show minimal genetic association between polyp and non-polyp phenotypes.

BACKGROUND: Staphylococcus aureus has a high prevalence in chronic rhinosinusitis (CRS) patients and is suggested to play a more etiopathogenic role in CRS patients with nasal polyps (CRSwNP), a severe form of the CRS spectrum with poorer surgical outcomes. We performed a microbial genome-wide association study (mGWAS) to investigate whether S. aureus isolates from CRS patients have particular genetic markers associated with CRS with nasal polyps (CRSwNP) or CRS without nasal polyps (CRSsNP). METHODS: Whole genome sequencing was performed on S. aureus isolates collected from 28 CRSsNP and 30 CRSwNP patients. A mGWAS approach was employed using large-scale comparative genomics to identify genetic variation within our dataset. RESULTS: Considerable genetic variation was observed, with > 90,000 single nucleotide polymorphisms (SNPs) sites identified. There was little correlation with CRS subtype based on SNPs and Insertion/Delection (Indels). One indel was found to significantly correlate with CRSwNP and occurred in the promoter region of a bacitracin transport system ATP-binding protein. Additionally, two variants of the highly variable superantigen-like (SSL) proteins were found to significantly correlate with each CRS phenotype. No significant association with other virulence or antibiotic resistance genes were observed, consistent with previous studies. CONCLUSION: To our knowledge this study is the first to use mGWAS to investigate the contribution of microbial genetic variation to CRS presentations. Utilising the most comprehensive genome-wide analysis methods available, our results suggest that CRS phenotype may be influenced by genetic factors other than specific virulence mechanisms within the S. aureus genome.

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.

In vitro characteristics of an airway barrier-disrupting factor secreted by Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a major contributor to the pathophysiology of chronic rhinosinusitis (CRS). Previous research has shown that S. aureus-secreted products disrupt the airway barrier. METHODS: S. aureus ATCC 13565 and 25923 strains were grown at exponential, postexponential, and stationary phases. Microbial conditioned media (CM) was collected from the cultures and ultrafiltered (UF). Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was performed on the UF-CM. UF-CM was subjected to heat and protease treatment, size fractionation, and ultracentrifugation (UC) separation. Human nasal epithelial cells grown at air-liquid interface (HNEC-ALI) cultures were exposed to purified alpha hemolysin (Hla), staphylococcal enterotoxin A (SEA), lipoteichoic acid (LTA), and UF-CM. Barrier function outcomes were measured by transepithelial electrical resistance (TEER) and apparent permeability (Papp). UC fraction exposed cultures were subjected to immunofluorescence microscopy for tight junction (TJ) protein zonula occludens-1 (ZO-1). RESULTS: LC-ESI-MS/MS identified 107 proteins, with Hla being most abundant. Hla, SEA, and LTA did not alter the HNEC-ALI barrier as measured by TEER or Papp. Barrier disruption caused by UF-CM peaked in the postexponential phase, was sensitive to heat and protease treatment, >30-kDa in size, and enriched in the UC fraction. HNEC-ALI exposed to UF-CM and UC demonstrated loss of ZO-1 localization. CONCLUSION: These results suggest that the S. aureus factor responsible for TJ disruption in HNEC-ALI cultures is either a protein-macromolecule or a combination of secreted factors. The product is enriched in the UC fraction, suggesting it is associated with large structures such as membrane components or vesicles.

Safety and efficacy of a bacteriophage cocktail in an in vivo model of Pseudomonas aeruginosa sinusitis.

Pseudomonas aeruginosa (PA) is a bacterial pathogen that frequently displays antibiotic resistance. Its presence within the sinuses of chronic rhinosinusitis sufferers is associated with poorer quality of life. Obligately lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death. The aims of this study were to assess the safety and efficacy of a PA phage cocktail (CT-PA) in a sheep model of rhinosinusitis. The sheep rhinosinusitis model was adapted to simulate PA infection in sheep frontal sinuses. To assess efficacy, after a 7-day biofilm formation period, sheep received twice-daily frontal trephine flushes of CT-PA or saline for 1 week. Biofilm quantitation on frontal sinus mucosa was performed using LIVE/DEAD BacLight staining. To assess safety, sheep received twice-daily frontal trephine flushes of CT-PA or vehicle control for 3 weeks. Blood and fecal samples were collected throughout treatment. Histopathology of frontal sinus, lung, heart, liver, spleen, and kidney tissue was performed. Sinus cilia were visualized using scanning electron microscopy (SEM). The Efficacy arm showed a statistically significant reduction in biofilm biomass with all concentrations of CT-PA tested (P < 0.05). Phage presence in sinuses was maintained for at least 16hours after the final flush. All Safety arm sheep completed 3 weeks of treatment. Phage was detected consistently in feces and sporadically in blood and organ samples. Histology and SEM of tissues revealed no treatment-related damage. In conclusion, CT-PA was able to decrease sinus PA biofilm at concentrations of 108-1010 PFU/mL. No safety concerns were noted.

Safety and Tolerability of Bacteriophage Therapy for Chronic Rhinosinusitis Due to Staphylococcus aureus.

IMPORTANCE: Staphylococcus aureus infections are associated with recalcitrant chronic rhinosinusitis (CRS). The emerging threat of multidrug-resistant S aureus infections has revived interest in bacteriophage (phage) therapy. OBJECTIVE: To investigate the safety, tolerability, and preliminary efficacy of ascending multiple intranasal doses of investigational phage cocktail AB-SA01 in patients with recalcitrant CRS due to S aureus. DESIGN, SETTING, AND PARTICIPANTS: This phase 1, first-in-humans, open-label clinical trial of multiple ascending doses was conducted at a single tertiary referral center from December 1, 2015, through September 30, 2016, with follow-up completed on December 31, 2016. Patients with recalcitrant CRS (aged 18-70 years) in whom surgical and medical treatment had failed and who had positive S aureus cultures sensitive to AB-SA01 were recruited. Findings were analyzed from February 2 through August 31, 2017. INTERVENTIONS: Three patient cohorts (3 patients/cohort) received serial doses of twice-daily intranasal irrigations with AB-SA01 at a concentration of 3 × 108 plaque-forming units (PFU) for 7 days (cohort 1), 3 × 108 PFU for 14 days (cohort 2), and 3 × 109 PFU for 14 days (cohort 3). MAIN OUTCOMES AND MEASURES: The primary study outcome was the safety and tolerability of intranasal AB-SA01. Safety observations included vital signs, physical examinations, clinical laboratory test results, and adverse events. The secondary outcome was preliminary efficacy assessed by comparing pretreatment and posttreatment microbiology results, disease-relevant endoscopic Lund-Kennedy Scores, and symptom scores using a visual analog scale and Sino-Nasal Outcome Test-22. RESULTS: All 9 participants (4 men and 5 women; median age, 45 years [interquartile range, 41.0-71.5 years]) completed the trial. Intranasal phage treatment was well tolerated, with no serious adverse events or deaths reported in any of the 3 cohorts. No change in vital signs occurred before and 0.5 and 2.0 hours after administration of AB-SA01 and at the exit visit. No changes in biochemistry were found except for 1 participant in cohort 3 who showed a decrease in blood bicarbonate levels on exit visit, with normal results of physical examination and vital signs. All biochemistry values were normalized 8 days later. No changes in temperature were recorded before, during, or after treatment. Six adverse effects were reported in 6 participants; all were classified as mild treatment-emergent adverse effects and resolved by the end of the study. Preliminary efficacy results indicated favorable outcomes across all cohorts, with 2 of 9 patients showing clinical and microbiological evidence of eradication of infection. CONCLUSIONS AND RELEVANCE: Intranasal irrigation with AB-SA01 of doses to 3 × 109 PFU for 14 days was safe and well tolerated, with promising preliminary efficacy observations. Phage therapy could be an alternative to antibiotics for patients with CRS. TRIAL REGISTRATION: http://anzctr.org.au identifier: ACTRN12616000002482.

Safety and Efficacy of Topical Chitogel- Deferiprone-Gallium Protoporphyrin in Sheep Model.

Objectives: Increasing antimicrobial resistance has presented new challenges to the treatment of recalcitrant chronic rhinosinusitis fuelling a continuous search for novel antibiofilm agents. This study aimed to assess the safety and efficacy of Chitogel (Chitogel®, Wellington New Zealand) combined with novel antibiofilm agents Deferiprone and Gallium Protoporphyrin (CG-DG) as a topical treatment against S. aureus biofilms in vivo. Methods: To assess safety, 8 sheep were divided into two groups of 7 day treatments (n = 8 sinuses per treatment); (1) Chitogel (CG) with twice daily saline flush, and (2) CG-DG gel with twice daily saline flush. Tissue morphology was analyzed using histology and scanning electron microscopy (SEM). To assess efficacy we used a S. aureus sheep sinusitis model. Fifteen sheep were divided into three groups of 7 day treatments (n = 10 sinuses per treatment); (1) twice daily saline flush (NT), (2) Chitogel (CG) with twice daily saline flush, and (3) CG-DG gel with twice daily saline flush. Biofilm biomass across all groups was compared using LIVE/DEAD BacLight stain and confocal scanning laser microscopy. Results: Safety study showed no cilia denudation on scanning electron microscopy and no change in sinus mucosa histopathology when comparing CG-DG to CG treated sheep. COMSTAT2 assessment of biofilm biomass showed a significant reduction in CG-DG treated sheep compared to NT controls. Conclusion: Results indicate that CG-DG is safe and effective against S. aureus biofilms in a sheep sinusitis model and could represent a viable treatment option in the clinical setting.

Chitosan Dextran gel as an anti adhesion agent in a postlaminectomy spinal sheep model.

INTRODUCTION: Post-operative peridural adhesions increase morbidity after neurosurgical procedures. Aim of this study is to assess safety and efficacy of Chitosan-Dextran (CD) gel as an anti-adhesion agent in a spinal laminectomy sheep model. METHODS: Eighteen sheep were used in this study with 6 animals in each treatment arm (namely, CD gel, Gelfoam paste and normal saline control). Posterior lumbar laminectomy was performed in all animals and the dura was exposed intact. Test agents were applied over the exposed dura and the wound was closed in layers. Sheep were euthanized at the end of three months. MRI spine was performed after euthanasia to assess epidural fibrosis. Adhesion in the spinal specimen was assessed by Peel test and histopathology was used to assess safety of the agents. RESULTS: Average scores for the Peel test for CD gel, Gelfoam and normal saline control groups were 1.16 (95% CI, 0.5-1.7), 1.5 (95% CI, 0.6-2.3) and 3 (95% CI, 2.1-3.8) respectively. There was significant reduction in adhesions between treatment and normal saline treated groups (p=0.0292), with no difference between Gelfoam and CD gel groups (p=0.56). Average scores on MRI for CD gel, Gelfoam and normal saline groups were 1.4 (95% CI, 0.9-1.8), 1.5 (95% CI, 1.2-1.8) and 1.6 (95% CI, 1.3-1.8) respectively, with no significant difference in fibrosis amongst (p=0.2992). Histopathology did not show any adverse effects. CONCLUSION: CD gel is an effective agent to reduce epidural adhesions with a good safety profile in neural tissue.

Activity of Bacteriophages in Removing Biofilms of Pseudomonas aeruginosa Isolates from Chronic Rhinosinusitis Patients.

Introduction:Pseudomonas aeruginosa infections are prevalent amongst chronic rhinosinusitis (CRS) sufferers. Many P. aeruginosa strains form biofilms, leading to treatment failure. Lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death. Aim: To assess the activity of a phage cocktail in eradicating biofilms of ex vivo P.aeruginosa isolates from CRS patients. Methods: P. aeruginosa isolates from CRS patients with and without cystic fibrosis (CF) across three continents were multi-locus sequence typed and tested for antibiotic resistance. Biofilms grown in vitro were treated with a cocktail of four phages (CT-PA). Biofilm biomass was measured after 24 and 48 h, using a crystal violet assay. Phage titrations were performed to confirm replication of the phages. A linear mixed effects model was applied to assess the effects of treatment, time, CF status, and multidrug resistance on the biomass of the biofilm. Results: The isolates included 44 strain types. CT-PA treatment significantly reduced biofilm biomass at both 24 and 48 h post-treatment (p < 0.0001), regardless of CF status or antibiotic resistance. Biomass was decreased by a median of 76% at 48 h. Decrease in biofilm was accompanied by a rise in phage titres for all except one strain. Conclusion: A single dose of phages is able to significantly reduce biofilms formed in vitro by a range of P.aeruginosa isolates from CRS patients. This represents an exciting potential and novel targeted treatment for P. aeruginosa biofilm infections and multidrug resistant bacteria.

Long-Term Safety of Topical Bacteriophage Application to the Frontal Sinus Region.

Background:Staphylococcus aureus biofilms contribute negatively to a number of chronic conditions, including chronic rhinosinusitis (CRS). With the inherent tolerance of biofilm-bound bacteria to antibiotics and the global problem of bacterial antibiotic resistance, the need to develop novel therapeutics is paramount. Phage therapy has previously shown promise in treating sinonasal S. aureus biofilms. Methods: This study investigates the long term (20 days) safety of topical sinonasal flushes with bacteriophage suspensions. The bacteriophage cocktail NOV012 against S. aureus selected for this work contains two highly characterized and different phages, P68 and K710. Host range was assessed against S. aureus strains isolated from CRS patients using agar spot tests. NOV012 was applied topically to the frontal sinus region of sheep, twice daily for 20 days. General sheep wellbeing, mucosal structural changes and inflammatory load were assessed to determine safety of NOV012 application. Results: NOV012 could lyse 52/61 (85%) of a panel of locally derived CRS clinical isolates. Application of NOV012 to the frontal sinuses of sheep for 20 days was found to be safe, with no observed inflammatory infiltration or tissue damage within the sinus mucosa. Conclusion: NOV012 cocktail appears safe to apply for extended periods to sheep sinuses and it could infect and lyse a wide range of S. aureus CRS clinical isolates. This indicates that phage therapy has strong potential as a treatment for chronic bacterial rhinosinusitis.

Fighting sinus-derived Staphylococcus aureus biofilms in vitro with a bacteriophage-derived muralytic enzyme.

BACKGROUND: Staphylococcus aureus biofilms are a nidus for exacerbation of infectious conditions including chronic rhinosinusitis (CRS). Resistance of biofilms to current therapeutics stresses the need for the development of novel anti-biofilm strategies. The chimeric muralytic enzyme P128 was specifically engineered to target Staphylococcal sp. by combining the cell wall binding domain of lysostaphin and the peptidoglycan-degrading murein hydrolase derived from phage K. This study assessed the anti-biofilm activity of P128 against sinus-derived S. aureus. METHODS: Biofilms from S. aureus ATCC 25923 and 3 sinus-derived methicillin-sensitive and methicillin-resistant CRS clinical isolates were grown for 48 hours and treated with various concentrations of P128 (0 to 100 µg/mL) for 2 and 24 hours, using the minimum biofilm eradication concentration (MBEC) assay and Alamar Blue (AB) assay. Biofilm present on the MBEC pegs was stained with LIVE/DEAD BacLight stain, imaged using confocal scanning laser microscopy and biomass determined by COMSTAT2 computation. In the AB assay, biofilm was measured by assessing the cell viability. Results were assessed using a Kruskal-Wallis test, with a Wilcoxon post hoc test and Bonferroni correction. RESULTS: Both the MBEC and AB assay indicated that P128 was effective against in vitro S. aureus biofilms with significant reductions in biofilm of up to 95.5% at concentrations ≥12.5 µg/mL for all tested strains. CONCLUSION: The engineered chimeric endolysin P128 was observed to be an effective anti-biofilm agent against S. aureus. Further study will proceed into the appropriate application of P128 to ensure both an economically and clinically feasible preparation.

Association of intracellular Staphylococcus aureus with prognosis in chronic rhinosinusitis.

BACKGROUND: Staphylococcus aureus (S. aureus) has been shown to exist within nasal epithelial cells in chronic rhinosinusitis (CRS) patients. This study investigates the localization of intracellular S. aureus (ICSA) in CRS patients, the associated histopathology changes, and their effect on long-term postoperative outcomes. METHODS: A prospective study of patients with CRS with and without polyps and control patients (n = 25, 15, and 8, respectively) undergoing endoscopic sinus surgery was performed. Validated patient reported symptom scores and objective endoscopic scores were collected preoperatively and 12 months postoperatively. Mucosal tissue samples were collected and examined for the presence of ICSA using immunohistochemical analysis. Tissue also underwent routine hematoxylin and eosin and Sirius Red staining to evaluate the inflammatory cell load and extent of fibrosis. RESULTS: ICSA appeared to localize to the perinuclear region of the pseudostratified columnar respiratory epithelium. ICSA was more prevalent in CRS without nasal polyps (CRSsNP) than in CRS with nasal polyps (CRSwNP) or controls (80% vs 56% vs 38%, respectively). ICSA positive status did not appear to influence symptom or endoscopic scores at the time of surgery nor 12 months postoperatively. Lymphocytes and total inflammatory cells were significantly increased in ICSA(+) group than ICSA(-) groups (36.4 vs 22.4 cells/area and 53.8 vs 29.1 cells/area, respectively). There was no difference found in fibrosis. CONCLUSION: This study indicated that ICSA was most prevalent in CRSsNP patients and was associated with increased lymphocytia and total inflammatory cells but not with worse symptomatology, endoscopy results, or basement membrane (BM) thickening.

Reduced Innate Immune Response to a Staphylococcus aureus Small Colony Variant Compared to Its Wild-Type Parent Strain.

Background:Staphylococcus aureus (S. aureus) small colony variants (SCVs) can survive within the host intracellular milieu and are associated with chronic relapsing infections. However, it is unknown whether host invasion rates and immune responses differ between SCVs and their wild-type counterparts. This study used a stable S. aureus SCV (WCH-SK2SCV) developed from a clinical isolate (WCH-SK2WT) in inflammation-relevant conditions. Intracellular infection rates as well as host immune responses to WCH-SK2WT and WCH-SK2SCV infections were investigated. Method: NuLi-1 cells were infected with either WCH-SK2WT or WCH-SK2SCV, and the intracellular infection rate was determined over time. mRNA expression of cells infected with each strain intra- and extra-cellularly was analyzed using a microfluidic qPCR array to generate an expression profile of thirty-nine genes involved in the host immune response. Results: No difference was found in the intracellular infection rate between WCH-SK2WT and WCH-SK2SCV. Whereas, extracellular infection induced a robust pro-inflammatory response, intracellular infection elicited a modest response. Intracellular WCH-SK2WT infection induced mRNA expression of TLR2, pro-inflammatory cytokines (IL1B, IL6, and IL12) and tissue remodeling factors (MMP9). In contrast, intracellular WCH-SK2SCV infection induced up regulation of only TLR2. Conclusions: Whereas, host intracellular infection rates of WCH-SK2SCV and WCH-SK2WT were similar, WCH-SK2SCV intracellular infection induced a less widespread up regulation of pro-inflammatory and tissue remodeling factors in comparison to intracellular WCH-SK2WT infection. These findings support the current view that SCVs are able to evade host immune detection to allow their own survival.

Topical colloidal silver as an anti-biofilm agent in a Staphylococcus aureus chronic rhinosinusitis sheep model.

BACKGROUND: Treatment of recalcitrant chronic rhinosinusitis (CRS) is a challenge with increasing antibiotic resistance, leading to re-emergence of topical therapies. The aim of this study was to assess safety and efficacy of topical colloidal silver solution for the treatment of Staphylococcus aureus biofilms in a sheep model. METHODS: In the safety study, normal saline (control) and 30-ppm colloidal silver solution (test) was used to flush the frontal sinuses for 14 days in 8 sheep (4 sheep each). In the efficacy study, following frontal sinus infection with Staphylococcus aureus, sheep were treated with either control saline or topical silver solution of varying concentrations (30 ppm/20 ppm/10 ppm/5 ppm) for 5 days, with 4 sheep in each group. Blood silver level, full blood counts, and biochemical parameters were analyzed in both safety and efficacy studies. Sinus tissue was harvested for histological examination and ciliary structure analysis in safety and for biofilm biomass quantification by fluorescence in situ hybridization (FISH) technique and COMSTAT 2 software in the efficacy study. Results were analyzed using appropriate statistical tests. RESULTS: Sheep treated with silver showed a significant decrease in biofilm biomass (0.004, 0.004, 0.004, and 0.007, in the 4 silver-treated groups, respectively) compared to saline control (0.175), p < 0.001. Although average blood silver levels were higher in the treated groups compared to controls (p < 0.05), blood counts and biochemical parameters were normal. Histology and ciliary structure analysis did not show any difference between control and treatment groups. CONCLUSION: Topical colloidal silver solution has effective antibiofilm activity in Staphylococcus aureus CRS in a sheep model and appears safe.

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.

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.

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.

Probiotic manipulation of the chronic rhinosinusitis microbiome.

BACKGROUND: Staphylococcus aureus (SA) is a key pathogenic component of the chronic rhinosinusitis (CRS) microbiome and is associated with increased disease severity and poor postoperative outcomes. Probiotic treatments potentially offer a novel approach to the management of pathogenic bacteria in these recalcitrant patients through supporting a healthy community of commensal species. This study aims to investigate the probiotic properties of Staphylococcus epidermidis (SE) against SA in a mouse model of sinusitis. METHODS: Twenty C57/BL6 mice received intranasal inoculations of phosphate buffered saline (PBS), SE, SA, or a combination of SE and SA (SE+SA) for 3 days. Following euthanasia, the mouse snouts were harvested and prepared for histological analysis. Counts of periodic acid-Schiff (PAS)-positive goblet cells were the primary outcome measure. RESULTS: Goblet cell counts were significantly higher in both the SA and SE+SA groups compared to those receiving PBS or SE alone (p < 0.05). However, the SE+SA group demonstrated significantly lower goblet cell counts compared to the SA group (p < 0.05). Mice receiving SE alone did not show a significant difference to those receiving PBS (p > 0.05). The presence of SA postinoculation was confirmed by culture in both the SA and SE+SA groups. CONCLUSION: This study confirms the probiotic potential of SE against SA in a mouse model of sinusitis. Although the interactions that occur between many probiotic species and pathogens are yet to be fully understood, studies such as this support further exploration of ecologically-based treatment paradigms for the management of CRS.

Cousins, siblings, or copies: the genomics of recurrent Staphylococcus aureus infections in chronic rhinosinusitis.

BACKGROUND: Staphylococcus aureus infection is known to play a role in recalcitrant chronic rhinosinusitis (CRS). However, it is unknown if recurrent S. aureus infections are caused by the same strain or are due to independent acquisitions of different strains. METHODS: Samples were collected from patients with CRS from July 2011 to August 2012. S. aureus was isolated from mucosal swabs and tissue specimens from patients who underwent surgery during the study period, or from swabs of areas of purulence taken in the postoperative period under endoscopic guidance. Pulsed-field gel electrophoresis was used to characterize S. aureus isolates. RESULTS: Thirty-four patients were included in the study; 79% showed persistence of the same S. aureus strain in their paranasal sinuses (p = 0.001; H1 ≠ 50%). Furthermore, a significantly high frequency of patients with known biofilm status were positive for S. aureus biofilm (p = 0.002; H1 ≠ 50%). When patients were stratified according to disease evolution postsurgery, certain strains appeared to be more commonly associated with symptom persistence. CONCLUSION: The same S. aureus strain appears to persist in the paranasal sinuses of CRS patients despite multiple courses of culture-directed antibiotics. This suggests that conventional antimicrobial therapies in patients with CRS may not eliminate the organism. This may be partly explained by the formation of biofilms in the paranasal sinus region.

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.