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.