METHODS

Subjects

Patients undergoing nasal endoscopic examination in Rhinological clinics and those undergoing endoscopic sinus surgery procedures by the senior authors (HASJ, PGH and RJS) at University Hospital Southampton between 8/12/2020-7/2/2022 were invited to participate. Subjects which met the EPOS 2020 criteria18 for diagnosis of chronic rhinosinusitis were stratified into CRSsNP and CRSwNP, and those who with other diagnoses including allergic rhinitis, nasal masses and anatomical nasal obstruction were placed in the control group. Exclusion criteria included patients under 18 years of age, cystic fibrosis, primary ciliary dyskinesia, immune deficiency syndromes, inability to provide informed consent and those with blood borne viruses which could put the research team at risk including hepatitis B, C and human immunodeficiency virus. Demographic data including age, sex, atopic status, antibiotic and steroid use in the past month, medical history, history of asthma, and smoking habits was collected. Nasal swabs were taken either intraoperatively or under topical local anaesthetic in clinic from the middle meatal region.

Specimen testing and genomic sequencing

Swabs were spread onto S. aureus 24-hour brilliance agar plates (Oxoid, Basingstoke, UK). Blue coagulase positive colonies were selected and tested for the presence of catalase and DNAse. Coagulase, catalase and DNAse positive colonies were subjected to matrix assisted laser desorption/ionisation time of flight spectrometry to confirm the Staphylococcus genus. Positive S. aureus cultures were grown to the logarithmic growth phase in Rosewell Park Memorial Institute Medium 1640 (RPMI 1640; Life Technologies, Paisley, UK) at 37oC and frozen in the presence of 25% glycerol (VWR, Leicestershire, UK) at -80oC. All 11 collectedS. aureus strains and 2 well characterised strains previously collected from a control and CRSwNP patient were prepared in RNA Shield (Zymo Research, Freiburg im Breisgau, Germany) and underwent 30x Illumina short read sequencing (MicrobesNG, Birmingham, UK).

Bioinformatics approach

Paired end DNA sequencing reads (contigs) were assembled using the Bactopia pipeline using S. aureus specific datasets from NCBI AMRFinderPlus, Ariba’s getref reference datasets, RefSeqMashSketch and Genbank Sourmash Signatures.19, 20 Post alignment, the assembled sequences were interrogated using AMRFinder to determine virulence factor presence.21 Only genes demonstrating greater than 90% sequence coverage and 90% sequence homology were included in the results.

Intracellular survival assays

Well characterised reference S. aureus were grown in RPMI1640 at 37oC in the presence of 5% CO2 until the exponential growth phase was reached. Absorbance at 600nm was calculated and extrapolated to a known colony forming unit (CFU) concentration.
LAD2 cells (a kind gift from Dr AS Kirshenbaum, Laboratory of Allergic Diseases, Bethesda, MD) were grown in antibiotic free STEMPRO-34 media (Life Technologies, Paisley, UK) containing 0.1 mM stem cell factor (SCF; PreproTech, London UK). S. aureus (1x106CFUs) was added at an MOI ratio of 1:1 LAD2 cells in 2 ml cell culture medium. The co-cultures were incubated for 3, 6 or 9 hours then centrifuged at 250 g for 10 minutes. The cell pellet was then resuspended with 0.5ml 20µg/ml lysostaphin (Sigma Aldrich, Dorset, UK) for 60 minutes. LAD2 cells were then pelleted at 250g for 10 minutes and washed in 1ml antibiotic free media three times. The washed LAD2 cells were then centrifuged at 250g for 10 minutes and the supernatant was streaked on a Columbia blood agar plate (Oxoid, Basingstoke, UK) to ensure no growth. The remaining pellet was resuspended in STEMPRO-34 media containing 0.1 mM SCF and 0.5% Triton X (Sigma Aldrich, Dorset, UK) and vortexed for 10 minutes. The remaining supernatant was used to perform serial CFU assessments.
Analysis of intracellular survival at 24 hours was conducted by infecting LAD2 cells as above, and culturing for 6 hours. Cells were then washed with 20µg/ml lysostaphin for 60 minutes to eradicate extracellular bacteria as above and the cells washed in antibiotic free media three times. Co-culture continued until the 24-hour timepoint at which point the cells were resuspended in medium with 0.5% Triton X-100 and vortexed for 10 minutes. The lysate was subjected to serial CFU enumerations as described above.

Confocal microscopy

LAD2 cells were co-cultured with the reference control and CRSwNPS. aureus strains as above for 6 hours. Cells were resuspended in 0.5ml 20 µg/ml lysostaphin for 60 minutes and washed in calcium and magnesium free phosphate buffered saline (PBS) three times. Cells were then resuspended in 15 µM Syto9 and 40µM propidium iodide in 1 ml PBS (Thermo-Fisher, Basingstoke, UK). A 50µl aliquot of each suspension was placed on an Ibidi 8-well glass bottom slide (Thistle Scientific, Glasgow, UK) and imaged using a Leica TCS SP5/8 inverted confocal microscope (Lecia Microsystems, Milton Keynes, UK) using a 63x glycerol immersion lens. Images were collected with Leica LAS-AF software and analysed with Fiji 2 (22).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc, San Diego, Ca, USA) and SPSS (IBM, Portsmouth, UK). Data was assessed for normality using histogram plots and normality tests. Pearson Chi-squared and one-way Anova tests were used to compare demographic data. Paired t-tests were used for to compare the differences between S. aureus strain co-cultures.