Increased antibiotic resistance of Pseudomonas aeruginosa isolates from chronic rhinosinusitis patients grown in anaerobic conditions.

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. Level of Evidence: NA.

S. aureus biofilm properties correlate with immune B cell subset frequencies and severity of chronic rhinosinusitis.

Staphylococcus aureus mucosal biofilms are associated with recalcitrant chronic rhinosinusitis (CRS). However, S. aureus colonisation of sinus mucosa is frequent in the absence of mucosal inflammation. This questions the relevance of S. aureus biofilms in CRS etiopathogenesis. This study aimed to investigate whether strain-level variation in in vitro-grown S. aureus biofilm properties relates to CRS disease severity, in vitro toxicity, and immune B cell responses in sinonasal tissue from CRS patients and non-CRS controls. S. aureus clinical isolates, tissue samples, and matched clinical datasets were collected from CRS patients with nasal polyps (CRSwNP), CRS without nasal polyps (CRSsNP), and controls. B cell responses in tissue samples were characterised by FACS. S. aureus biofilms were established in vitro, followed by measuring their properties of metabolic activity, biomass, colony-forming units, and exoprotein production. S. aureus virulence was evaluated using whole-genome sequencing, mass spectrometry and application of S. aureus biofilm exoproteins to air-liquid interface cultures of primary human nasal epithelial cells (HNEC-ALI). In vitro S. aureus biofilm properties were correlated with increased CRS severity scores, infiltration of antibody-secreting cells and loss of regulatory B cells in tissue samples. Biofilm exoproteins from S. aureus with high biofilm metabolic activity had enriched virulence genes and proteins, and negatively affected the barrier function of HNEC-ALI cultures. These findings support the notion of strain-level variation in S. aureus biofilms to be critical in the pathophysiology of CRS.

A Novel Model of Staphylococcus aureus-Induced Lymphoplasmacytic Rhinosinusitis in Rats.

Chronic rhinosinusitis (CRS) is characterized by sinonasal mucosal inflammation. Staphylococcus aureus (S. aureus) is associated with severe CRS phenotypes. Different animal models have been proposed to study the association of CRS and S. aureus. However, current animal models are expensive due to the use of large animals, have high barriers to ethics approval, or require invasive surgical intervention, necessitating a need for a model that can overcome these limitations. This study aimed at establishing a reliable and efficient rat lymphoplasmacytic inflammatory model for rhinosinusitis. Sprague Dawley rats received a daily intranasal application of 20 µL of saline, S. aureus CI-182 exoprotein (250 µg/mL), or exoprotein CI-182 in combination with S. aureus clinical isolate (CI-908 or CI-913) 108 colony-forming unit (CFU)/mL. The rats' sinuses were harvested at 1 and 2 weeks post-intervention. The CFU and histopathologic examination of inflammation were evaluated. S. aureus clinical isolates CI-908 or CI-913 in combination with the exoprotein (CI-182) had higher CFUs and caused persistently higher inflammation at both the 1 and 2-week post-intervention compared to the exoprotein and saline group. The observed inflammatory cell type was lymphoplasmacytic. This study provided evidence that the combination of a S. aureus exoprotein with S. aureus induces inflammation that persists for a minimum of two weeks post-intervention. This model is the first known animal model to create the lymphoplasmacytic inflammation subtype seen in CRS patients. This offers a cost-effective, accessible, non-invasive, and easy-to-replicate model to study the causes and treatment of such inflammation.

An In Vitro Study Evaluating the Safety of Mesalazine on Human Nasoepithelial Cells.

Chronic rhinosinusitis (CRS) is a disease characterised by the inflammation of the nasal and paranasal cavities. It is a widespread condition with considerable morbidity for patients. Current treatment for chronic rhinosinusitis consists of appropriate medical therapy followed by surgery in medically resistant patients. Although oral steroids are effective, they are associated with significant morbidity, and disease recurrence is common when discontinued. The development of additional steroid sparing therapies is therefore needed. Mesalazine is a commonly used therapeutic in inflammatory bowel disease, which shares a similar disease profile with chronic rhinosinusitis. This exploratory in vitro study aims to investigate whether mesalazine could be repurposed to a nasal wash, which is safe on human nasoepithelial cells, and retains its anti-inflammatory effects. CRS patients' human nasal epithelial cells (HNECs) were collected. HNECs were grown at an air-liquid interface (ALIs) and in a monolayer and challenged with mesalazine or a non-medicated control. Transepithelial electrical resistance, paracellular permeability, and toxicity were measured to assess epithelial integrity and safety. The anti-inflammatory effects of mesalazine on the release of interleukin (IL)-6 and tumour necrosis factor alpha (TNF-α) were analysed using human leukemia monocytic cell line (THP-1). mesalazine did not impact the barrier function of HNEC-ALIs and was not toxic when applied to HNECs or THP-1 cells at concentrations up to 20 mM. mesalazine at 0.5 and 1 mM concentrations significantly inhibited TNF-α release by THP-1 cells. mesalazine effectively decreases TNF-α secretion from THP-1 cells, indicating the possibility of its anti-inflammatory properties. The safety profile of mesalazine at doses up to 20 mM suggests that it is safe when applied topically on HNECs.

Deferiprone-gallium-protoporphyrin (IX): A promising treatment modality against Mycobacterium abscessus.

Non-Tuberculous Mycobacterial Pulmonary Disease (NTM-PD) caused by Mycobacterium abscessus is a frequent complication in patients with cystic fibrosis (CF) that worsens lung function over time. Currently, there is no cure for NTM-PD, hence new therapies are urgently required. Disrupting bacterial iron uptake pathways using gallium-protoporphyrin (IX) (GaPP), a heme analog, has been proposed as a novel antibacterial approach to tackle multi-drug resistant M. abscessus. However, the antibacterial activity of GaPP has been tested only in iron-deficient media, which cannot accurately mirror the potential activity in vivo. Herein, we investigated the potential synergistic activity between GaPP and the iron-chelating agent deferiprone (Def) in regular media against M. abscessus-infected macrophages. The safety of the treatment was assessed in vitro using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in Nuli-1 and THP-1 cell lines. Def-GaPP had synergistic activity against M. abscessus-infected macrophages where 10 mM-12.5 mg/L of Def-GaPP reduced the viability by up to 0.9 log10. Furthermore, Def-GaPP showed no cytotoxicity to Nuli-1 and THP-1 cell lines at the effective antibacterial concentrations (10 mM-12.5 mg/L) of Def- GaPP. These data encourage future investigation of Def-GaPP as a novel antimicrobial against NTM-PD.

Colloidal silver against macrophage infections and biofilms of atypical mycobacteria.

Skin and soft tissue infection (SSTI) caused by atypical mycobacteria such as Mycobacterium abscessus and Mycobacterium avium intracellulare complex (MAIC) have increased in recent years. Current therapeutic options are limited, and hence new and better therapies are urgently required. Colloidal Silver (CS) has been identified for its widespread antibacterial properties and silver-impregnated dressings have been used for SSTIs caused by various pathogens. The efficacy of Green Synthesized Colloidal Silver (GSCS) was investigated for bacterial growth inhibition (BGI) using a microdilution method and minimum biofilm eradication concentration (MBEC) using resazurin assay and confocal scanning laser microscopy (CSLM) of M. abscessus (n = 5) and MAIC (n = 5). The antibacterial effect of GSCS against M. abscessus infected macrophages was also evaluated. The in vitro cytotoxicity of GSCS on a human keratinocyte cell line (HaCaT) and neonatal foreskin fibroblasts was analyzed by the crystal violet proliferation assay. Average BGI and MBEC of GSCS varied between 0.7 and 22 ppm for M. abscessus and MAIC. The concentration of 3 ppm reduced M. abscessus-infection in macrophages significantly. GSCS was not cytotoxic to HaCaT and neonatal foreskin fibroblast cells at concentrations < 3 ppm up to 2 h exposure time. GSCS therefore, has the potential for topical application against atypical mycobacterial SSTI.

Silver nanoparticles as a bioadjuvant of antibiotics against biofilm-mediated infections with methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa in chronic rhinosinusitis patients.

Infectious diseases caused by antibiotic-resistant bacteria in planktonic and biofilm form are difficult to treat with conventional antibiotics. Silver nanoparticles (Ag NPs) can be used as alternatives to antibiotics and can alter the susceptibility of bacteria to antibiotics. Here, the antibacterial properties of 16 different antibiotics and Ag NPs, alone and in combination, were tested against clinical isolates of Pseudomonas aeruginosa (n=3), Staphylococcus aureus (n=3) and methicillin-resistant Staphylococcus aureus (MRSA) (n=2) isolated from chronic rhinosinusitis (CRS) patients. The microdilution method and resazurin assay were used to determine the minimum inhibitory concentration and minimum biofilm eradication concentration for planktonic and biofilm forms, respectively. Results showed that Ag NPs and gentamicin combinations had synergistic antibacterial activity against P. aeruginosa planktonic and biofilm forms and MRSA biofilms. Furthermore, additive effects against biofilms were seen for combinations of Ag NPs with tobramycin or ciprofloxacin against P. aeruginosa; with mupirocin against MRSA; and with augmentin, doxycycline, azithromycin and clindamycin against S. aureus. Moreover, additive effects against planktonic forms were observed for combinations of Ag NPs with tobramycin, ciprofloxacin, imipenem, ceftazidime and aztreonam against P. aeruginosa; with gentamicin or linezolid against MRSA; and with doxycycline or clindamycin against S. aureus. In conclusion, Ag NP-antibiotic combinations can result in enhanced antimicrobial action against P. aeruginosa, MRSA and S. aureus clinical isolates in planktonic and biofilm forms and can be used in the context of CRS with confirmed infection.

Green synthesized colloidal silver is devoid of toxic effects on primary human nasal epithelial cells in vitro.

Evaluating the safety of previously fabricated and effective green synthetized colloidal silver (GSCS) on the mucosal barrier structure and function is essential prior to conduct human trials. The GSCS was applied to primary human nasal epithelial cells (HNECs) grown in an air-liquid interface (ALI) culture. Epithelial barrier integrity was evaluated by measuring the transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran paracellular permeability. Ciliary beat frequency (CBF) was quantified. Effects of the GSCS on cell viability and inflammation were examined through lactate dehydrogenase, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide viability assay and interleukin 6 (IL-6) enzyme linked immunosorbent assay. The localization and transportation of GSCS within HNECs and their HNEC-ALI cultures was assessed by transmission electron microscopy and inductively coupled plasma-mass-spectrometry, respectively. Application of GSCS to HNECs-ALI cultures for up to 2 h caused a significant reduction in the TEER values, however, it did not drop within the first 10 and 20 min for CRS and non-CRS control HNECs. The paracellular permeability, cell viability, IL-6 secretion and CBF remained unchanged. No GSCS was observed within or transported across HNECs. In conclusion, application of GSCS to HNECs is devoid of toxic effects.

Colloidal silver combating pathogenic Pseudomonas aeruginosa and MRSA in chronic rhinosinusitis.

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.

Antifungal, antibacterial, antibiofilm and colorimetric sensing of toxic metals activities of eco friendly, economical synthesized Ag/AgCl nanoparticles using Malva Sylvestris leaf extracts.

Silver nanoparticles, one of the most popular nanomaterials, are used extensively in medicine and industries. The present study biosynthesized spherical Ag/AgCl nanoparticles with a size range of 10-50 nm in less than 5 min. The synthesis was performed in a single step, in a low-cost and eco-friendly manner, from the aqueous extract of Malva Sylvestris leaves. The aqueous extract had a large number of phenolic compounds and carbohydrates as reducing and capping agents. The nanoparticles also showed significant antibacterial and anti-biofilm activities against some multi drug resistant bacteria. They additionally showed antifungal activities on several Candida species. The highest concentration of Ag/AgCl-NPs (62.5 µg/ml) was required in order to inhibit P. aeruginosa B 52, C. glabrata and C. parapsilosis growth. The lowest concentration of Ag/AgCl-NPs (7.8125 µg/ml) inhibited the growth of C. orthopsilosis, P. aeruginosa ATCC 27853 and B. subtilis ATCC 6633. A total of 125 µg/ml of Ag/AgCl-NPs was used to prevent P. aeruginosa B 52 biofilm growth. The concentration of 62.5 µg/ml Ag/AgCl-NPs also eradicated both P. aeruginosa 48 and P. aeruginosa B 52 biofilms. The results showed that Hg2+ and Pb2+ contaminants in water could be colorimetrically detected by these nanoparticles.