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

Copyright © 2017 Drilling, Ooi, Miljkovic, James, Speck, Vreugde, Clark and Wormald. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.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

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 Structure of the Accretion Disk in the ADC X-Ray Binary 4U 1822-371 at Optical and Ultraviolet Wavelengths

The eclipsing low-mass X-ray binary 4U 1822-371 is the prototypical accretion disk corona (ADC) system. We have obtained new time-resolved UV spectroscopy of 4U 1822-371 with the Advanced Camera for Surveys/Solar Blind Channel (ACS/SBC) on the Hubble Space Telescope (HST) and new V- and J-band photometry with the 1.3-m SMARTS telescope at CTIO. We use the new data to construct its UV/optical spectral energy distribution and its orbital light curve in the UV, V, and J bands. We derive an improved ephemeris for the optical eclipses and confirm that the orbital period is changing rapidly, indicating extremely high rates of mass flow in the system; and we show that the accretion disk in the system has a strong wind with projected velocities up to 4000 km/s. We show that the disk has a vertically-extended, optically-thick component at optical wavelengths.This component extends almost to the edge of the disk and has a height equal to ~0.5 of the disk radius. As it has a low brightness temperature, we identify it as the optically-thick base of a disk wind, not as the optical counterpart of the ADC. Like previous models of 4U 1822-371, ours needs a tall obscuring wall near the edge of the accretion disk, but we interpret the wall as a layer of cooler material at the base of the disk wind, not as a tall, luminous disk rim.Comment: 37 pages, 12 figures, submitted to Ap

Bacteriophage therapy for application against Staphylococcus aureus infection and biofilm in chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is a debilitating condition characterised by critical inflammation of the mucosa of the nose and paranasal sinuses. Effecting up to 14% of the world’s population CRS severely impacts a patient’s quality of life. The aetiology of CRS is complex and relatively undefined encompassing a multitude of contributing factors. Bacterial infection is one factor thought to play a role in the pathogenesis of CRS. More specifically biofilm forms of the bacterial species Staphylococcus aureus have been shown to negatively influence post-operative progression. Current practice treatment strategies often fail to remove biofilms from the mucosa of the nose. It is therefore of import to develop novel anti-biofilm therapeutics. Our understanding of the epidemiology of S. aureus infections and biofilms in CRS is also limited. Increasing our epidemiological knowledge would help in the development of effective treatment strategies against recurrent infections. Investigation into the epidemiology of S. aureus infections was undertaken by collecting S. aureus isolates from mucous and biofilm structures of CRS patients. The clonal type of each isolate was then compared to the other isolates using pulse field gel-electrophoresis. Results of this study indicated that the majority of patients experiencing recurrent infections maintained the same clonal type. Furthermore the study suggested that long-term antibiotic therapy in some patients can lead to the development of bacterial antibiotic resistance. Therefore development of a novel antibacterial therapy outside of antibiotics is required. A potential anti-biofilm therapy both eliminating and preventative in nature is the application of bacteriophage. Bacteriophage (phage) are viruses that specifically target, infect and destroy bacterial cells. Initially in vitro study was undertaken to assess the anti-biofilm activity of a phage cocktail specific for S. aureus (CT-SA) using a minimal biofilm eradication assay plate. S. aureus isolates from CRS patients were grown to mature biofilm form and treated with CT-SA for 48hrs. Following treatment biofilm biomass was determined by staining bacteria with a Live/Dead BacLight stain, imaging the biofilm using confocal scanning laser microscopy and determining biofilm biomass using software COMSTAT2. Results showed CT-SA significantly reduced S. aureus biofilms of susceptible strains. Results also indicated that a cocktail of phage was superior to use of a single phage as it reduced the frequency of bacterial resistant to the phage treatment. Following on from in vitro work, the safety and efficacy of CT-SA was assessed in vivo using a sheep model of frontal sinusitis associated with S. aureus infections. CT-SA was also combined with ethylenediaminetetraaceticacid (EDTA) to observe if these therapies would synergise. Results indicated both CT-SA and EDTA were safe for short term topical application to the sinus regions. Furthermore both CT-SA and EDTA individually significantly reduced S. aureus biofilm levels in the frontal sinus, but were not seen to synergise. Work conducted in this thesis has helped lead towards development of a novel anti-S. aureus biofilm agent. Future translation of CT-SA to a clinical trial setting may not only reduce or remove S. aureus biofilm from CRS patient noses but also improve their symptomatology and quality of life.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2015

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

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

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 (cm3) 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.Sukanya Rajiv, Marguerite Harding, Ahmed Bassiouni, Camille Jardeleza, Amanda Drilling, Craig James, Thanh Ha, Steve Moratti, Simon Robinson, Peter-John Wormal