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Introduction: In chronic rhinosinusitis (CRS), the congestion and blockage of the nose can cause anaerobic conditions within the sinus cavities which may promote the expression of virulence and antibiotic resistance genes in invading pathogens. Pseudomonas aeruginosa is a facultative anaerobic bacteria and causes severe recalcitrant CRS. In this study, we aimed to evaluate the antimicrobial resistance of P. aeruginosa isolates of CRS patients in planktonic and biofilm form grown in aerobic and anaerobic conditions. Methods: P. aeruginosa clinical isolates of CRS patients (n = 25) were grown in planktonic and biofilm form in aerobic and anaerobic conditions. Minimum inhibitory concentrations (MIC) of planktonic forms and minimum biofilm eradication concentrations (MBEC) were determined. Additionally, metabolic activity by fluorescein diacetate assay, biofilm biomass by crystal violet assay and eDNA concentration were assessed in both conditions. Results: P. aeruginosa planktonic cells grown in anaerobic condition exhibited increased gentamicin resistance (p < .01), whereas P. aeruginosa biofilms grown in anaerobic condition displayed significantly increased MBEC values for gentamicin (p < .0001) and levofloxacin (p < .001). The metabolic activity of anaerobic biofilms was significantly higher compared with aerobic biofilms (p < .0001). However, the biofilm biomass of isolates grown in aerobic conditions was higher than anaerobic conditions (p < .5). Conclusion: P. aeruginosa isolates from CRS patients grown in anaerobic conditions showed significantly increased resistance to antibiotics with an increased metabolic activity but decreased biofilm biomass. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is one of the most common pathogens encountered in clinical wound infections. Clinical studies have shown that P. aeruginosa infection results in a larger wound area, inhibiting healing, and a high prevalence of antimicrobial resistance. Hydroxypyridinone-derived iron chelator Deferiprone (Def) and heme analogue Gallium-Protoporphyrin (GaPP) in a chitosan-dextran hydrogel (Chitogel) have previously been demonstrated to be effective against PAO1 and clinical isolates of P. aeruginosa in vitro. Moreover, this combination of these two agents has been shown to improve sinus surgery outcomes by quickly reducing bleeding and preventing adhesions. In this study, the efficacy of Def-GaPP Chitogel was investigated in a P. aeruginosa biofilm-infected wound murine model over 6 days. Two concentrations of Def-GaPP Chitogel were investigated: Def-GaPP high dose (10 mM Def + 500 µg/mL GaPP) and Def-GaPP low dose (5 mM Def + 200 µg/mL GaPP). The high-dose Def-GaPP treatment reduced bacterial burden in vivo from day 2, without delaying wound closure. Additionally, Def-GaPP treatment decreased wound inflammation, as demonstrated by reduced neutrophil infiltration and increased anti-inflammatory M2 macrophage presence within the wound bed to drive wound healing progression. Def-GaPP Chitogel treatment shows promising potential in reducing P. aeruginosa cutaneous infection with positive effects observed in the progression of wound healing. [ABSTRACT FROM AUTHOR]

Chronic rhinosinusitis (CRS) represents chronic inflammation of the sinus mucosa characterised by dysfunction of the sinuses' natural defence mechanisms and induction of different inflammatory pathways ranging from a Th1 to a Th2 predominant polarisation. Recalcitrant CRS is associated with Staphylococcus aureus dominant mucosal biofilms; however, S. aureus colonisation of the sinonasal mucosa has also been observed in healthy individuals challenging the significance of S. aureus in CRS pathogenesis. We aimed to investigate the relationship between CRS key inflammatory markers, S. aureus biofilm properties/virulence genes and the severity of the disease. Tissue samples were collected during endoscopic sinus surgery from the ethmoid sinuses of CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps and controls (n = 59). CD3+ T‐cell subset frequencies and key inflammatory markers of CD4+ helper T cells were determined using FACS analysis. Sinonasal S. aureus clinical isolates were isolated (n = 26), sequenced and grown into biofilm in vitro, followed by determining their properties, including metabolic activity, biomass, colony‐forming units and exoprotein production. Disease severity was assessed using Lund–Mackay radiologic scores, Lund–Kennedy endoscopic scores and SNOT22 quality of life scores. Our results showed that S. aureus biofilm properties and CRS severity scores correlated positively with total CD4+ T‐cell frequencies but looking into CD4+ T‐cell subsets showed an inverse correlation with Th1 and Th17 cell frequencies. CD4+ T‐cell frequencies were higher in patients harbouring lukF.PV‐positive S. aureus while its regulatory and Th17 cell subset frequencies were lower in patients carrying sea− and sarT/U‐positive S. aureus. Recalcitrant CRS is characterised by increased S. aureus biofilm properties in relation to increased total CD4+ helper T‐cell frequencies and reduced frequencies of its Th1, Th17 and regulatory T‐cell subsets. These findings offer insights into the pathophysiology of CRS and could lead to the development of more targeted therapies. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to determine the safety and efficacy of Chitogel, with and without Deferiprone (Def) and Gallium Protoporphyrin (GaPP), as a promoter of wound healing to improve surgical outcomes after endoscopic sinus susgery. Design: A double‐blinded, randomised control human clinical trial was conducted in patients undergoing ESS as a treatment for chronic rhinosinusitis. Participants underwent functional ESS or FESS with drill out as required and were randomised to receive test product Chitogel, Chitogel in combination with Def or Def‐GaPP versus no packing (control). Setting: Ostial stenosis and persistent inflammation are the main reasons for revision endoscopic sinus surgery (ESS). Post‐operative (PO) dressings can improve PO wound healing and patient outcomes after ESS. Participants: Eighty two patients were included in this study with 79 patients completing the study with 40 undergoing full house FESS and 39 FESS plus frontal drillout. Main Outcome Measures: Patients were followed up at 2, 6 and 12 weeks PO, and outcome scores such as SNOT‐22, VAS and LKS, pre and post‐surgery (12 weeks) were compared. Results: Seventy nine patients completed the study, there was a significant reduction in SNOT‐22 score and improvement of VAS at 12 weeks in patients treated with Chitogel compared to control (p <.05). In those patients, the mean ostium area for the Chitogel and the Chitogel + Def + GaPP groups was higher across all three sinuses compared to the no‐treatment control group, without statistical significance. Sphenoid sinus ostium was significantly more patent in patients treated with Chitogel compared to the control at the 12‐week time point (p <.05). Conclusion: Chitogel as a PO dressing after ESS results in the best patient‐reported symptom scores and objective measurements. The combination of Def and GaPP to Chitogel though proving safe, had no effect on the ostium patency or mucosal healing. [ABSTRACT FROM AUTHOR]

I aeruginosa i clinical isolates from recalcitrant CRS patients in their planktonic and biofilm stages and to investigate I in vitro i safety application of acriflavine on human nasal epithelial cells (HNECs). The minimum biofilm eradication concentration (MBEC) was determined (320 µg/ml) using AlamarBlue (Figure S1C,D) and crystal violet assays (Figure S1E,F), which resulted in the significant killing of biofilms and reduction in biofilm biomass of matured biofilm ( I p i < .05, Figure S1). Novel anti-biofilm drugs that could be topically applied to the nasal mucosa safely and without promoting antibiotic resistance could address multiple current unmet needs. [Extracted from the article]

Chronic rhinosinusitis (CRS) is a persistent inflammation of the nasal cavity and paranasal sinuses associated with tissue remodelling, dysfunction of the sinuses' natural defence mechanisms, and induction of different inflammatory clusters. The etiopathogenesis of CRS remains elusive, and both environmental factors, such as bacterial biofilms and the host's general condition, are thought to play a role. Bacterial biofilms have significant clinical relevance due to their potential to cause resistance to antimicrobial therapy and host defenses. Despite substantial medical advances, some CRS patients suffer from recalcitrant disease that is unresponsive to medical and surgical treatments. Those patients often have nasal polyps with tissue eosinophilia, S. aureus-dominant mucosal biofilm, comorbid asthma, and a severely compromised quality of life. This review aims to summarise the contemporary knowledge of inflammatory cells/pathways in CRS, the role of bacterial biofilm, and their impact on the severity of the disease. Here, an emphasis is placed on S. aureus biofilm and its secreted products. A better understanding of these factors might offer important diagnostic and therapeutic perceptions for recalcitrant disease. [ABSTRACT FROM AUTHOR]

We have also shown, for the first time, that kappa carrageenan has antibacterial and anti-biofilm activity against I S. aureus i clinical isolates from the sinuses. I Staphylococcus aureus i has been reported to be the most common biofilm-forming pathogen in Chronic rhinosinusitis (CRS) pathogenesis and is associatedwith more severe disease and poor treatment response.1 There has been a recent increased interest in the use of topical antibiotic agents to treat CRS. The current study aimed to investigate both the muco-adhesiveness of kappa carrageenan on explanted sinonasal mucosa and its antibacterial and antibiofilm properties. [Extracted from the article]

Background: Chronic rhinosinusitis (CRS) is a common chronic respiratory condition, frequently associated with asthma and affecting the majority of cystic fibrosis (CF) patients. Pseudomonas aeruginosa infections and biofilms have been implicated in recalcitrant CRS. One of the mechanisms of action for bacteria in CRS and CF is mucosal barrier disruption by secreted products that contribute to the inflammation. However, the role of biofilm and planktonic forms of P. aeruginosa in this process is not known. The aim is to determine the effect of P. aeruginosa exoproteins isolated from CF and non‐CF CRS patients on the mucosal barrier. Methods: Exoproteins from 40 P. aeruginosa isolates were collected in planktonic and biofilm forms and applied to air‐liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs). Mucosal barrier integrity was evaluated by transepithelial electrical resistance (TEER), passage of FITC‐dextrans and immunofluorescence of tight junction proteins. Cytotoxicity assays were performed to measure cell viability, and IL‐6 ELISA was carried out to evaluate pro‐inflammatory effects. Results: Planktonic exoproteins from 20/40 (50%) clinical isolates had a significant detrimental effect on the barrier and significantly increased IL‐6 production. Barrier disruption was characterized by a reduced TEER, increased permeability of FITC‐dextrans and discontinuous immunolocalization of tight junction proteins and was significantly more prevalent in isolates harvested from patients with comorbid asthma (P <.05). Conclusion: Exoproteins from planktonic P. aeruginosa clinical isolates from asthmatic CRS patients have detrimental effects on the mucosal barrier and induce IL‐6 production potentially contributing to the mucosal inflammation in CRS patients. [ABSTRACT FROM AUTHOR]

Introduction: Adhesions are often considered to be an inevitable consequence of abdominal and pelvic surgery, jeopardizing the medium and long-term success of these procedures. Numerous strategies have been tested to reduce adhesion formation, however, to date, no surgical or medical therapeutic approaches have been successful in its prevention. This study demonstrates the safety and efficacy of Chitogel with Deferiprone and/or antibacterial Gallium Protoporphyrin in different concentrations in preventing adhesion formation after abdominal surgery. Materials and methods: 112 adult (8–10 week old) male Wistar albino rats were subjected to midline laparotomy and caecal abrasion, with 48 rats having an additional enterotomy and suturing. Kaolin (0.005g/ml) was applied to further accelerate adhesion formation. The abrasion model rats were randomized to receive saline, Chitogel, or Chitogel plus Deferiprone (5, 10 or 20 mM), together with Gallium Protoporphyrin (250μg/mL). The abrasion with enterotomy rats were randomised to receive saline, Chitogel or Chitogel with Deferiprone (1 or 5 mM). At day 21, rats were euthanised, and adhesions graded macroscopically and microscopically; the tensile strength of the repaired caecum was determined by an investigator blinded to the treatment groups. Results: Chitogel with Deferiprone 5 mM significantly reduced adhesion formation (p<0.01) when pathologically assessed in a rat abrasion model. Chitogel with Deferiprone 5 mM and 1 mM also significantly reduced adhesions (p<0.05) after abrasion with enterotomy. Def-Chitogel 1mM treatment did not weaken the enterotomy site with treated sites having significantly better tensile strength compared to control saline treated enterotomy rats. Conclusions: Chitogel with Deferiprone 1 mM constitutes an effective preventative anti-adhesion barrier after abdominal surgery in a rat model. Moreover, this therapeutic combination of agents is safe and does not weaken the healing of the sutured enterotomy site. [ABSTRACT FROM AUTHOR]

The sinonasal microbiome remains poorly defined, with our current knowledge based on a few cohort studies whose findings are inconsistent. Furthermore, the variability of the sinus microbiome across geographical divides remains unexplored. We characterize the sinonasal microbiome and its geographical variations in both health and disease using 16S rRNA gene sequencing of 410 individuals from across the world. Although the sinus microbial ecology is highly variable between individuals, we identify a core microbiome comprised of Corynebacterium, Staphylococcus, Streptococcus, Haemophilus and Moraxella species in both healthy and chronic rhinosinusitis (CRS) cohorts. Corynebacterium (mean relative abundance = 44.02%) and Staphylococcus (mean relative abundance = 27.34%) appear particularly dominant in the majority of patients sampled. Amongst patients suffering from CRS with nasal polyps, a statistically significant reduction in relative abundance of Corynebacterium (40.29% vs 50.43%; P =.02) was identified. Despite some measured differences in microbiome composition and diversity between some of the participating centres in our cohort, these differences would not alter the general pattern of core organisms described. Nevertheless, atypical or unusual organisms reported in short‐read amplicon sequencing studies and that are not part of the core microbiome should be interpreted with caution. The delineation of the sinonasal microbiome and standardized methodology described within our study will enable further characterization and translational application of the sinus microbiota. [ABSTRACT FROM AUTHOR]

Background: Chronic rhinosinusitis patients (CRS) suffer from chronic inflammation of the sinus mucosa associated with chronic relapsing infections. Mucosal biofilms, associated with Staphylococcus aureus, have been implicated as a cause. We compared the effect of exoproteins secreted from clinical isolates of S aureus from CRS patients in planktonic and biofilm form on the nasal epithelial barrier. Methods: Clinical S aureus isolates from 39 CRS patients were grown in planktonic and biofilm forms and their exoproteins concentrated. These were applied to primary human nasal epithelial cells grown at the air‐liquid interface. Transepithelial electrical resistance, permeability of flourescein isothiocyanate‐dextrans, and cytotoxicity were measured. Structure and expression of tight junctions zona occludens‐1, and claudin‐1 proteins were assessed by electron microscopy and immunofluorescence. The Wilcoxon signed rank test was used for statistical analyses. Results: S aureus biofilm exoproteins showed dose‐ and time‐dependent reduction of transepithelial electrical resistance, increased cell toxicity, and increased permeability (p < 0.001) compared with equal concentrations of planktonic cultures. Discontinuity in zona occludens‐1 and claudin‐1 immunofluorescence was confirmed as disrupted tight junctions on electron microscopy. Conclusion: S aureus biofilm exoproteins disrupt the mucosal barrier structure in a time‐ and dose‐dependent manner and are toxic. Damage to the mucosal barrier by S aureus biofilm exoproteins may play a major role in CRS etiopathogenesis. [ABSTRACT FROM AUTHOR]

This study offers a novel description of the sinonasal microbiome, through an unsupervised machine learning approach combining dimensionality reduction and clustering. We apply our method to the International Sinonasal Microbiome Study (ISMS) dataset of 410 sinus swab samples. We propose three main sinonasal "microbiotypes" or "states": the first is Corynebacterium -dominated, the second is Staphylococcus -dominated, and the third dominated by the other core genera of the sinonasal microbiome (Streptococcus, Haemophilus, Moraxella , and Pseudomonas). The prevalence of the three microbiotypes studied did not differ between healthy and diseased sinuses, but differences in their distribution were evident based on geography. We also describe a potential reciprocal relationship between Corynebacterium species and Staphylococcus aureus , suggesting that a certain microbial equilibrium between various players is reached in the sinuses. We validate our approach by applying it to a separate 16S rRNA gene sequence dataset of 97 sinus swabs from a different patient cohort. Sinonasal microbiotyping may prove useful in reducing the complexity of describing sinonasal microbiota. It may drive future studies aimed at modeling microbial interactions in the sinuses and in doing so may facilitate the development of a tailored patient-specific approach to the treatment of sinus disease in the future. [ABSTRACT FROM AUTHOR]

The characterization of two distinct agr loci from all group I strains of Clostridium botulinum ATCC 3502 and of their homologues in a close relative, Clostridium sporogenes NCIMB 10696 is reported. Compared to the wild-type strains, C. botulinum strains exhibited remarkably reduced sporulation and also reduced production of neurotoxin for C. botulinum indicated that both phenotypes are controlled by quorum sensing.

Background: Topical budesonide (Pulmicort; AstraZeneca AB, Sodertalje, Sweden) is commonly used in the management of chronic rhinosinusitis (CRS). Although its use is due to its perceived anti‐inflammatory effect, studies have suggested that it may also have antibacterial properties. To make the hydrophobic steroid molecule suitable for topical administration, pharmaceutical excipients are used in commercial steroid formulations. Herein we investigated the antibacterial action of commercial budesonide and its excipients. Methods: Planktonic and biofilm forms of Staphylococcus aureus and methicillin‐resistant Staphylococcus aureus (MRSA) were treated with Pulmicort or its excipients at clinically relevant concentrations. Bacterial growth was determined by optical density, resazurin assays, colony‐forming unit counts, and Giemsa staining. Minimum inhibitory concentration (MIC) studies assessed excipients' potentiation of antibiotics. Experiments were conducted in triplicate and results analyzed using one‐way analysis of variance. Results: There was significant reduction in planktonic and biofilm growth of S aureus and MRSA on exposure to budesonide (p < 0.0001) and its excipients (p < 0.0001). Excipient ethylene diamine‐tetraactic acid (EDTA) demonstrated an antibacterial property even at the low concentrations used in topical preparations (p < 0.0001). With amoxicillin, excipients exhibited a potential additive/synergistic effect on MIC, whereas erythromycin and aminoglycosides showed an antagonistic action. Conclusion: The commercial product Pulmicort has a direct antibacterial effect on the planktonic and biofilm forms of S aureus and MRSA. This effect is at least in part mediated through the excipient EDTA in the product. Excipients also influenced the antimicrobial activity of antibiotics depending on the bacterial strain and antibiotic tested. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas aeruginosa causes severe chronic respiratory diseases and is associated with recalcitrant chronic rhinosinusitis (CRS). P. aeruginosa exoproteins contain virulence factors and play important roles in the pathogenicity of P. aeruginosa , however their role in CRS pathophysiology remains unknown. Methods: We isolated P. aeruginosa clinical isolates (CIs) and obtained clinical information from 21 CRS patients. Elastase activity of the CIs was measured at different phases of growth. Primary human nasal epithelial cells (HNECs) were cultured at air-liquid interface (ALI) and challenged with P. aeruginosa exoproteins or purified elastase, followed by measuring Transepithelial Electrical Resistance (TEER), permeability of FITC-dextrans, western blot, and immunofluorescence. Results: 14/21 CIs had a significant increase in elastase activity in stationary phase of growth. There was a highly significant strong correlation between the in vitro elastase activity of P. aeruginosa CIs with mucosal barrier disruption evidenced by increased permeability of FITC-dextrans (r = 0.95, p = 0.0004) and decreased TEER (r = −0.9333, P < 0.01) after 4 h of challenge. Western blot showed a significant degradation of ZO-1, Occludin and β-actin in relation to the elastase activity of the exoproteins. There was a highly significant correlation between the in vitro elastase activity of P. aeruginosa CIs and CRS disease severity (for log phase, r = 0.5631, p = 0.0097; for stationary phase, r = 0.66, p = 0.0013) assessed by CT imaging of the paranasal sinuses. Conclusion: Our results implicate P. aeruginosa exoproteins as playing a major role in the pathophysiology of P. aeruginosa associated CRS by severely compromising mucosal barrier structure and function. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase (MMP)-9 is thought to be involved in the etiopathogenesis of chronic rhinosinusitis (CRS) with nasal polyps and cleaves collagen IV, causing hyperpermeability of the basement membrane within mucosal tissue. It is known that MMP-9 expression is negatively affected by sirtuin (SIRT)-1 in human monocytotic cells, retinal endothelial cells, and epithelial carcinoma cells. However, it is unknown which factors affect MMP-9 expression and activity in human nasal epithelial cells (HNECs). To examine factors affecting MMP-9 expression and activity in HNECs, HNECs were stimulated with Toll-like receptor (TLR) agonists, followed by quantitative PCR, immunofluorescence, and zymography to examine MMP-9 expression and activity. MMP-9 expression was evaluated in sinonasal tissue of control subjects without CRS, and patients with CRS without nasal polyps and those with CRS with nasal polyps, in relation to the expression of SIRT1 using a tissue microarray. The effect of SIRT1 stimulation/inhibition on MMP-9 expression in HNECs was also tested. TLR3 agonists increased MMP-9 mRNA expression (473 fold, P = 0.0198) and activity (20.4-fold, P < 0.05). SIRT1 activation or inhibition reciprocally affected MMP-9 expression in the presence of TLR3 agonists. MMP-9 and SIRT1 expression within the epithelial layer of sinonasal tissue was inversely correlated only in patients with CRS but not in control subjects. TLR3 agonists increased MMP-9 expression and activity in HNECs, and the effect was abolished in the presence of SIRT1 activation. SIRT1 and MMP-9 expression was inversely correlated in CRS tissue, supporting SIRT1 as a possible therapeutic target for nasal polyp formation. [ABSTRACT FROM AUTHOR]

[Display omitted] Chronic rhinosinusitis (CRS) is a persistent inflammation of the sinus mucosa associated with dysfunction of the sinuses' natural defense mechanism and induction of different inflammatory clusters. Severe recalcitrant CRS is unresponsive to medical and surgical interventions and often has Staphylococcus aureus –dominant mucosal biofilms. This study aimed to characterize in vitro grown S aureus biofilm properties in relation to inflammation and CRS severity. The spatial pattern of inflammatory cells in patients' sinonasal tissue was also examined. S aureus isolated from the nasal swabs of patients with CRS with nasal polyps (CRSwNP), those with CRS without nasal polyps, and controls (n = 72) were grown into biofilm in vitro , and their metabolic activity, biomass, colony-forming units, and exoprotein production were quantified. S aureus virulence genes were evaluated using whole-genome sequencing. Patients' matched sinonasal tissue blocks (n = 57) were analyzed using Opal multiplex immunostaining, and their disease severity was determined using the Lund-Mackay computed tomography score. Correlations among S aureus biofilm properties, the frequency and localization of key immune cells in corresponding sinonasal mucosa, and the disease severity were investigated. Increased infiltration of CD3+, CD68+, CD20+, and CD138+ cells was observed in tissue of patients with CRSwNP compared to tissue from controls. CD3+, CD138+, and MBP+ cells diffused deeper into the tissue in CRSwNP but clustered close to the epithelium in controls. This study also found CRSwNP–derived S aureus biofilms showed thicker biomass, higher colony-forming units, and higher exoprotein production than those from controls did (P <.05). S aureus biofilm properties, inflammatory cell numbers, and CRS severity scores were positively correlated. These findings support the notion of an aberrant immunolocalization of key immune cells in CRSwNP with a critical role of S aureus biofilms in CRS etiopathogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The present study describes and compares functional properties of Nuli-1 cells and primary human nasal epithelial cells (HNEC) including TLR expression and function. Differences in gene expression were identified for non-TLR genes that play a role in TLR response pathways. However, experiments comparing TLR gene expression for both Nuli-1 cells and HNECs indicated conserved expression in both cell types. Stimulation of the two cell types resulted in a conserved response to TLR3 agonists, but in differences in response to agonists for TLR5 and TLR6/2. HNECs were much more susceptible to infection with Staphylococcus aureus than NuLi-1 cells. Furthermore, when cultured at air-liquid interface (ALI), NuLi-1 cells possessed much lower trans-epithelial resistance than primary HNEC and did not exhibit maintenance of cell morphology or mucous production which was observed in HNECs. Nor did they produce the characteristic interconnecting pattern of tight junction complexes at the apicolateral margin of adjacent cells. Caution should therefore be exercised when selecting cell lines for immunological studies and a thorough screen of properties relevant to the study should always be carried out prior to commencement. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus is one of the most common bacteria associated with chronic rhinosinusitis (CRS). Although S. aureus biofilms have been correlated with disease severity in CRS, little is known about the initial immune response that biofilms induce in the sinonasal mucosa. Objective: The aim of this study was to evaluate the innate immune response (in terms of cytokines) of nondiseased human sinonasal tissue to S. aureus biofilms. Methods: Full-thickness sinonasal explant cultures (n = 7 donors) were challenged with established S. aureus biofilms for 24 hours. The expression profiles of 17 cytokines were measured using multiplex analysis, real-time quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Differences in expression were evaluated using Student's t-test. Results: Interleukin (IL)-1β, IL-10, TNF, IL-17A, and interferon (IFN)-γ were up-regulated at the RNA and protein levels in biofilm-treated tissues compared with controls. Elevation of caspase-3 in biofilm-treated samples indicates S. aureus biofilms induce apoptosis on the sinonasal mucosa. Conclusion: S. aureus biofilms induced apoptosis and a predominant proinflammatory immune response on normal sinonasal mucosal explants. This immune response appeared to be triggered by intrinsic bacterial elements but also by components of the biofilm matrix. Live biofilms were present on the mucosa at the end of the challenge, suggesting an inability of the induced immune response to eliminate the S. aureus biofilms. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis Biofilms are associated with clinical relapse following surgery for chronic rhinosinusitis. Encased bacteria are protected from innate immunity and antimicrobial therapy. Surfactants can disperse the biofilm into its planktonic phenotype so that traditional treatments may be effective. The aim of this study was to assess a surfactant for its cytotoxicity profile. Study Design In vitro explant-based cytotoxicity study. Methods Sinonasal mucosa harvested from patients undergoing sinus surgery was tested using an air-liquid interface explant system. Surfactant at 1×, 2×, and 3× manufacturer's recommended concentrations were compared to control (saline) and Zinc Sulphate (ZnSO4), a known cytotoxic agent. Culture supernatant was analyzed for lactate dehydrogenase (LDH) as a marker of cellular toxicity. After 7 days, specimens were imaged using structured histopathology and scanning electron microscopy. Results Application of surfactant at 1× concentration did not elicit an elevation in LDH, whereas ZnSO4 caused a significant rise 1 day after application. Specimens tested with a 2× and 3× surfactant demonstrated LDH rises 4 days and 2 days after application, respectively. Mucosa tested with the 1× surfactant and control demonstrated intact cellular structures on histopathology and preserved cilial ultrastructure on SEM. In ZnSO4-treated specimens, marked cellular degradation and ciliary denudation occurred. Conclusion The surfactant does not appear to elicit cellular toxicity using an in vitro explant model at the manufacturer's recommended concentration. At higher concentrations, there may be dose-related toxicity that requires further investigation. In vivo testing is required to prove its efficacy in the treatment of recalcitrant chronic rhinosinusitis. Level of Evidence N/A. Laryngoscope, 124:369-372, 2014 [ABSTRACT FROM AUTHOR]

Background The presence of Staphylococcus aureus biofilms on sinonasal mucosal surfaces is associated with recalcitrant chronic rhinosinusitis (CRS), but little is known about the innate immune response they trigger. We aimed to study the human pattern recognition receptor (PRR) nucleotide-binding oligomerization domain containing 2 (Nod2) receptor and downstream pathway in response to initial S. aureus biofilm infection. Methods Using a validated protocol, sinonasal mucosae from 4 non-CRS donors were cultured with and without S. aureus biofilms and planktonic cells. After 24 hours, RNA was extracted and gene expression was analyzed using a human antibacterial response polymerase chain reaction (PCR) array. Immunohistochemistry was performed to confirm the presence and determine the immunolocalization of selected proteins. Results C-X-C motif (CXC) chemokine ligands 1 and 2, interleukin-6 (IL-6), and genes related to the Nod2 pathway were significantly upregulated in biofilm-treated tissues compared with control samples. Nod2 pathway-specific gene expression was increased in biofilm-treated tissues compared with planktonic S. aureus-treated explants. Enhanced expression of Nod2 and nuclear factor kappa B1 (NF-κB1) was also detected with immunohistochemistry in control and biofilm-treated tissues. Conclusion S. aureus biofilms exerted a proinflammatory response in the mucosa and activation of the Nod2 pathway, indicating this receptor to be involved in the innate immune response to S. aureus biofilms. Further studies are required to elucidate the role of this pathway in CRS. [ABSTRACT FROM AUTHOR]

Corynebacterium accolens strains are increasingly recognized as beneficial bacteria that can confer a health benefit on the host. In the current study, the probiotic potential of three C. accolens strains, C779, C781 and C787 derived from a healthy human nasal cavity were investigated. These strains were examined for their adhesion to HNECs, competition with Staphylococcus aureus for adhesion, toxicity, induction of IL-6, antibiotic susceptibility and the presence of antibiotic resistance and virulence genes. Furthermore, the safety and efficacy of strains were evaluated in vivo using Caenorhabditis elegans. The adhesion capacity of C. accolens to HNECs was strain-dependent. Highest adhesion was observed for strain C781. None of the C. accolens strains tested caused cell lysis. All strains were able to outcompete S. aureus for cell adhesion and caused a significant decrease of IL-6 production by HNECs co-exposed to S. aureus when compared to the control groups. All strains were sensitive or showed intermediate sensitivity to 10 different antibiotics. Whole Genome Sequence analysis showed C. accolens C781 and C787 did not possess antibiotic resistance genes whereas strain C779 harboured 5 genes associated with resistance to Aminoglycoside, Chloramphenicol and Erythromycin. In addition, no virulence genes were detected in any of the 3 strains. Moreover, the tested strains had no detrimental effect on worm survival and induced protection from S. aureus -mediated infection. Taken all together, C. accolens strains, C781 and C787 displayed probiotic potential and hold promise for use in clinical applications for combating dysbiosis in chronic rhinosinusitis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Production of the signalling molecule (autoinducer-2) synthesized by LuxS has been proposed to be pivotal to a universal mechanism of inter-species bacterial cell-cell communication (quorum sensing); however recently the function of LuxS has been noted to be integral to central metabolism since it contributes to the activated methyl cycle. This paper shows that when Helicobacter pylori LuxS is overproduced in Escherichia coli, it forms cross-linkable multimers. These multimers persist at comparable levels after 24 h of growth if glucose is omitted from the growth medium; however, the levels of extracellular autoinducer-2 decline (Glucose Retention of AI-2 Levels: GRAIL). Glycerol, maltose, galactose, ribose and L-arabinose could substitute for glucose, but lactose, D-arabinose, acetate, citrate and pyruvate could not. Mutations in (i) metabolic pathways (glycolytic enzymes eno, pgk, pgm; galactose epimerase; the Pta-AckA pathway), (ii) sugar transport (pts components, rbs operon, mgl, trg), and (iii) regulators involved in conventional catabolic repression (crp, cya), cAMP-independent catabolite repression (creC, fruR, rpoS,) the stringent response (relA, spoT) and the global carbon storage regulator (csrA) did not prevent GRAIL. Although the basis of GRAIL remains uncertain, it is clear that the mechanism is distinct from conventional catabolite repression. Moreover, GRAIL is not due to inactivation of the enzymic activity of LuxS, since in E. coli, LuxS contained within stationary-phase cells grown in the absence of glucose maintains its activity in vitro. [ABSTRACT FROM AUTHOR]

[Display omitted] • Corymbia maculata mediated colloidal silver synthesis in a 15-minute process. • Highly stable colloidal silver is spherical, 40 nm and negatively charged. • Safety of colloidal silver against Nuli-1 cell line and C. elegans. • Efficacy of colloidal silver against clinical isolates from chronic rhinosinusitis patients. The emergence of antibiotic resistant bacteria requires for the development of new antimicrobial compounds one of which colloidal silver (CS) having strong bactericidal properties and being the most promising inorganic nanoparticles for the treatment of bacterial infectious diseases. However, their production can be slow and cumbersome. Here, we used Corymbia maculata aqueous leaf extract as a reducing agent to synthesize CS in a single 15-minute process. CS was physico-chemically characterized for shape, size, zeta potential and stability. The Minimal Inhibitory Concentration (MIC) and Minimum Biofilm Eradication Concentration (MBEC) of CS against planktonic and biofilm forms of methicillin-resistant Staphylococcus aureus (MRSA, n = 5), Pseudomonas aeruginosa (n = 5), Haemophilus influenzae (n = 5) and Streptococcus pneumoniae (n = 3) chronic rhinosinusitis clinical isolates were investigated using the microdilution method and resazurin assay, respectively. The in vitro cytotoxicity on bronchial epithelial cells (Nuli-1) was analyzed by the crystal violet proliferation assay. The safety and efficacy of CS was evaluated in an in vivo infection model in Caenorhabditis elegans. CS was spherical in shape with a diameter of between 11−16 nm (TEM analysis) in dried form and 40 nm (NanoSight) in colloidal form and was stable at room temperature and 4 °C for one year. Average MIC and MBEC values varied between 11 and 44 ppm for MRSA, H. influenzae and S. pneumoniae and between 0.2 and 3 ppm for P. aeruginosa. CS was not toxic to Nuli-1 cells or C. elegans at concentrations of 44 ppm and reduced the Colony Forming Units counts by 96.9 % and 99.6 % in C. elegans for MRSA and P. aeruginosa , respectively. In conclusion, a novel, green synthesis of stable CS is demonstrated with good safety and efficacy profiles, particularly against P. aeruginosa in planktonic and biofilm forms. These CS have potential applications against clinical infections, including in the context of CRS. [ABSTRACT FROM AUTHOR]

Corynebacterium accolens is the predominant species of the healthy human nasal microbiota, and its relative abundance is decreased in the context of chronic rhinosinusitis (CRS). This study aimed to evaluate the antimicrobial potential of C. accolens isolated from a healthy human nasal cavity against planktonic and biofilm growth of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) clinical isolates (CIs) from CRS patients. Nasal swabs from twenty non-CRS control subjects were screened for the presence of C. accolens using microbiological and molecular techniques. C. accolens CIs and their culture supernatants were tested for their antimicrobial activity against eight S. aureus and eight MRSA 4CIs and S. aureus ATCC25923. The anti-biofilm potential of C. accolens cell-free culture supernatants (CFCSs) on S. aureus biofilms was also assessed. Of the 20 nasal swabs, 10 C. accolens CIs were identified and confirmed with rpoB gene sequencing. All isolates showed variable antimicrobial activity against eight out of 8 S. aureus and seven out of eight MRSA CIs. Culture supernatants from all C. accolens CIs exhibited a significant dose-dependent antibacterial activity (p < 0.05) against five out of five representative S. aureus and MRSA CIs. This inhibition was abolished after proteinase K treatment. C. accolens supernatants induced a significant reduction in metabolic activity and biofilm biomass of S. aureus and MRSA CIs compared to untreated growth control (p < 0.05). C. accolens exhibited antimicrobial activity against S. aureus and MRSA CIs in both planktonic and biofilm forms and holds promise for the development of innovative probiotic therapies to promote sinus health. [ABSTRACT FROM AUTHOR]