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.

Hydrocortisone metabolism by Staphylococcus: Effects on anti-inflammatory and antimicrobial action.

KEY POINTS: Hydrocortisone 21-hemisuccinate (HCHS) influenced the growth and metabolism of Staphylococcus aureus S. aureus metabolic activity was high and antibiotic susceptibility low at 1.4 mg/mL HCHS S. aureus metabolized HCHS to cortisol and reduced poly(I:C)-induced IL-6 secretion.

Corrigendum: Corynebacterium accolens inhibits Staphylococcus aureus induced mucosal barrier disruption.

[This corrects the article DOI: 10.3389/fmicb.2022.984741.].

Staphylococcal protein A modulates inflammation by inducing interferon signaling in human nasal epithelial cells.

OBJECTIVE AND DESIGN: Staphylococcus aureus (S. aureus) is one of the leading causes of human respiratory tract infections. The function of Staphylococcal protein A (SpA), expressed on the S. aureus bacterial membrane and released in the environment, on human nasal epithelial cells (HNECs) have not been fully elucidated. In this study, we tested the SpA expression in S. aureus from chronic rhinosinusitis patients and investigated the effects of SpA on HNECs inflammation through Interferon Gamma Receptor 1(IFNGR1)/phosphorylated Janus Kinase 2 (p-JAK2) pathway. METHODS: RNA profiling was performed to investigate inflammatory activation in a S. aureus chronic rhinosinusitis (CRS) mouse model. SpA release by S. aureus clinical isolates was determined using ELISA. The effect of purified SpA and SpA enriched conditioned media from S. aureus clinical isolates on HNECs cytotoxicity, apoptosis and release of inflammatory cytokines was evaluated using lactate dehydrogenase assays, and flow cytometry. SpA dependent IFNGR1 and p-JAK2 expression were assessed by qPCR, immunofluorescence and western blot in HNECs. RESULTS: 49 genes were significantly induced in S. aureus CRS mice indicative of activation of interferon signaling. SpA release was significantly higher in S. aureus clinical isolates from chronic rhinosinusitis with nasal polyps (CRSwNP) patients. Purified SpA significantly increased IFNGR1 mRNA and protein expression in HNECs. SpA induced cytotoxic effects and induced the release of Interleukin-6 (IL-6) and IL-8 in an IFNGR1 dependent way. CONCLUSION: SpA induces interferon signaling through activation of the IFNGR1-JAK-2 pathway, which provides an understanding of how S. aureus SpA affects the inflammatory process in the upper airways.

Corynebacterium accolens inhibits Staphylococcus aureus induced mucosal barrier disruption.

Background: Corynebacterium accolens (C. accolens) is a common nasal colonizer, whereas Staphylococcus aureus (S. aureus) is typically regarded a pathogenic organism in patients with chronic rhinosinusitis (CRS). This study aims to evaluate the interaction of the two bacteria in vitro. Methods: Clinical isolates of C. accolens and S. aureus from sinonasal swabs, as well as primary human nasal epithelial cells (HNECs) cultured from cellular brushings of both healthy and CRS patients were used for this study. The cell-free culture supernatants of all isolates grown alone and in co-cultures were tested for their effects on transepithelial electrical resistance (TER), FITC-Dextran permeability, lactate dehydrogenase (LDH), and IL-6 and IL-8 secretion of HNECs. Confocal scanning laser microscopy and immunofluorescence were also used to visualize the apical junctional complexes. C. accolens cell-free culture supernatants were also tested for antimicrobial activity and growth on planktonic and biofilm S. aureus growth. Results: The cell-free culture supernatants of 3\C. accolens strains (at 60% for S. aureus reference strain and 30% concentration for S. aureus clinical strains) inhibited the growth of both the planktonic S. aureus reference and clinical strains significantly. The C. accolens cell-free culture supernatants caused no change in the TER or FITC-Dextran permeability of the HNEC-ALI cultures, while the cell-free culture supernatants of S. aureus strains had a detrimental effect. Cell-free culture supernatants of C. accolens co-cultured with both the clinical and reference strains of S. aureus delayed the S. aureus-dependent mucosal barrier damage in a dose-dependent manner. Conclusion: Corynebacterium accolens cell-free culture supernatants appear to inhibit the growth of the S. aureus planktonic bacteria, and may reduce the mucosal barrier damage caused by S. aureus.

APTC-C-SA01: A Novel Bacteriophage Cocktail Targeting Staphylococcus aureus and MRSA Biofilms.

The high infection and mortality rate of methicillin-resistant Staphylococcus aureus (MRSA) necessitates the urgent development of new treatment strategies. Bacteriophages (phages) have several advantages compared to antibiotics for the treatment of multi-drug-resistant bacterial infections, and thus provide a promising alternative to antibiotics. Here, S. aureus phages were isolated from patients and environmental sources. Phages were characterized for stability, morphology and genomic sequence and their bactericidal activity against the biofilm form of methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA was investigated. Four S. aureus phages were isolated and tested against 51 MSSA and MRSA clinical isolates and reference strains. The phages had a broad host range of 82−94% individually and of >98% when combined and could significantly reduce the viability of S. aureus biofilms. The phages had a latent period of ≤20 min and burst size of >11 plaque forming units (PFU)/infected cell. Transmission electron microscopy (TEM) identified phages belonging to the family of Myoviridae. Genomic sequencing indicated the lytic nature of all four phages, with no identified resistance or virulence genes. The 4 phages showed a high complementarity with 49/51 strains (96%) sensitive to at least 2/4 phages tested. Furthermore, the frequency of bacteriophage insensitive mutant (BIM) generation was lower when the phages were combined into the phage cocktail APTC-C-SA01 than for bacteria exposed to each of the phages alone. In conclusion, APTC-C-SA01, containing four lytic S. aureus phages has the potential for further development as a treatment against MSSA and MRSA infections.

Characterization of human nasal organoids from chronic rhinosinusitis patients.

Patient-derived organoids grown in three-dimensional cultures provide an excellent platform for phenotypic high-throughput screening and drug-response research. Organoid technology has been applied to study stem cell biology and various human pathologies. This study investigates the characteristics and cellular morphology of organoids derived from primary human nasal epithelial cells (HNECs) of chronic rhinosinusitis (CRS) patients. Nasal organoids were cultured up to 20 days and morphological, cell composition and functional parameters were measured by immunofluorescence, RT-qPCR, western blot and FACS analysis. The results showed that nasal organoids expressed the stem cell marker leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and markers for apical junction genes, goblet cells and ciliated cells. Moreover, we were able to regrow and expand the nasal organoids well after freezing and thawing. This study provides an effective and feasible method for development of human nasal organoids, suitable for the phenotypic high-throughput screening and drug response research.

APTC-EC-2A: A Lytic Phage Targeting Multidrug Resistant E. coli Planktonic Cells and Biofilms.

Escherichia coli (E. coli) are common bacteria that colonize the human and animal gastrointestinal tract, where they help maintain a balanced microbiome. However, some E. coli strains are pathogenic and can cause serious infectious diseases and life-threatening complications. Due to the overuse of antibiotics and limited development of novel antibiotics, the emergence of antibiotic-resistant strains has threatened modern medicine, whereby common infections can become lethal. Phage therapy has once again attracted interest in recent years as an alternative treatment option to antibiotics for severe infections with antibiotic-resistant strains. The aim of this study was to isolate and characterize phage against multi-drug resistant E. coli isolated from clinical samples and hospital wastewater. For phage isolation, wastewater samples were collected from The Queen Elizabeth Hospital (Adelaide, SA, Australia) followed by phage enrichment as required. Microbiological assays, electron microscopy and genomic sequencing were carried out to characterize the phage. From the 10 isolated E. coli phages, E. coli phage APTC-EC-2A was the most promising and could lyse 6/7 E. coli clinical isolates. APTC-EC-2A was stable at a broad pH range (3-11) and could lyse the host E. coli at temperatures ranging between 30-50 °C. Furthermore, APTC-EC-2A could kill E. coli in planktonic and biofilm form. Electron microscopy and genomic sequencing indicated the phage to be from the Myoviridae family and of lytic nature. In conclusion, the newly isolated phage APTC-EC-2A has the desired properties that support its potential for development as a therapeutic agent against therapy refractory E. coli infections.

Preclinical Development of a Bacteriophage Cocktail for Treating Multidrug Resistant Pseudomonas aeruginosa Infections.

A Pseudomonas aeruginosa (P. aeruginosa) airway infection is one of the predominant causes contributing to the high morbidity and mortality rates in cystic fibrosis (CF) patients. The emergence of antibiotic resistant P. aeruginosa strains has led to an urgent need for new therapeutic approaches. Bacteriophages (phages) are viruses that can infect and lyse specific bacteria, providing a potential alternative approach in targeting antibiotic-resistant strains. We aim to isolate and characterise novel P. aeruginosa phages for combination in a cocktail to kill P. aeruginosa. One particular phage, PA4, could lyse 14/20 clinical isolates as observed through spot assays. This phage could significantly reduce the growth of bacteria in vitro, as determined through planktonic adsorption and inhibition assays as well as crystal violet- and LIVE/DEAD-stained biofilm assays. A morphological and genomic analysis revealed that PA4 belongs to the Myoviridae family and contained 66,450 bp. The broad infectivity profile, good stability in various pH and temperature conditions, lytic ability and the absence of the absences of antibiotic resistance, toxic and lysogenic genes suggest that PA4 is a good candidate for clinical grade use. Overall, phage therapy represents a promising alternative treatment option to antibiotics when treating a P. aeruginosa infection.

Cytokine-Induced Modulation of SARS-CoV2 Receptor Expression in Primary Human Nasal Epithelial Cells.

Background: Viral entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) via the spike protein enables endocytosis into host cells using the ACE2 receptor and TMPRSS2. The frequent upper respiratory tract symptoms of COVID-19 and the localization of the virus to the nasopharynx, the most common site of swabbing, indicate that the sinonasal mucosa may play an important role in SARS-CoV2 infection and viral replication. Methods: This paper investigates the presence of ACE2 receptor and TMPRESS2 expression in the primary human nasal epithelial cells (HNECs) from the following: chronic rhinosinusitis without nasal polyps (CRSsNP), CRS with nasal polyps (CRSwNP) and control (non-CRS) patients, and maps the expression changes when exposed to Th1, Th2, Th17-associated cytokines. Results: We found that ACE2 and TMPRSS2 expression was higher in control HNECs than CRSwNP HNECs, and that both ACE2 and TMPRSS2 were downregulated further by Th2 cytokines in CRSwNP HNECs. Conclusions: This indicates an immune dysregulated state of CRSwNP mucosa, which normally contributes to a chronic inflammatory state, and might support an altered susceptibility to SARS-CoV2 infection and transmission.

Overcoming bacteriophage insensitivity in Staphylococcus aureus using clindamycin and azithromycinat subinhibitory concentrations.

BACKGROUND: Staphylococcus aureus is a pathogen of major concern in both acute infections and chronic conditions such as chronic rhinosinusitis (CRS). Bacteriophage (phage) therapy has recently regained interest for its potential to treat infections caused by antibiotic resistant strains including Methicillin Resistant Staphylococcus aureus (MRSA). However, bacteria can adapt and become resistant to phages. The aim of this study is to determine the potential for antibiotics to overcome phage resistance. METHODS: The susceptibility of S. aureus clinical isolates (CIs) to phages J-Sa36, Sa83 and Sa87 alone or in combination with protein synthesis inhibitor (PSI) antibiotics clindamycin, azithromycin and erythromycin was assessed using plaque spot assays, minimum inhibitory concentration (MIC) assays, double layer spot assays and resazurin assays. The safety and efficacy of subinhibitory PSI antibiotics in combination with phage was tested in a Sprague Dawley rat model of sinusitis infected with a phage resistant S. aureus CI. RESULTS: All three antibiotics at subinhibitory concentrations showed synergy when combined with all 3 phages against S. aureus CIs in planktonic and biofilm form and could sensitize phage-resistant S. aureus to promote phage infection. The combination of topical subinhibitory clindamycin or azithromycin and phage was safe and could eradicate S. aureus sinonasal biofilms in vivo. CONCLUSION: Subinhibitory concentrations of PSI antibiotics could sensitize phage-resistant S. aureus and MRSA strains to phages in vitro and in vivo. This data supports the potential use of phage-PSI antibiotic combination therapies, in particular for difficult-to-treat infections with phage-resistant S. aureus and MRSA strains.