RESULTS
Patient Demographics
Nasal swabs were taken from Forty-four patients who were enrolled in the
study. In total 17 control, 10 CRSsNP and 17 CRSwNP patients
participated. S. aureus was cultured in 4 control, 4 CRSsNP and 3
CRSwNP patients. There was a higher proportion of asthmatic patients
within the CRSwNP group which was found to be statistically significant
at 58.8% compared with 17.6% in the control and 20% in the CRSsNP
group (p=0.023). There was a larger number of smokers in the control
group (23.5%) with no smokers in the CRSsNP or CRSwNP groups(p=0.034).
Other demographics showed no statistically significant differences
between the groups (Table 1). A subgroup analysis was performed on
subjects from whom S. aureus was cultured. There was no
significant difference in demographics including age, procedure,
antibiotic use, steroid use, airborne allergies, asthma presence and
smoking status (Table 2).
Bacterial genome
sequencing
Illumina short read sequencing was completed for 11 S. aureusstrains identified from 4 control subjects, and 4 CRSsNP and 3 CRSwNP
patients. Two previously collected, well characterised S. aureusstrains from a patient without CRS (Control 1) and a CRSwNP strain
(CRSwNP 1) were also sequenced. Each paired read sequence was
reconstructed and aligned using bacterial alignment and analysis
pipeline, Bactopia. Each assembled genome spanned between 2.67 and 2.77
mega base pairs.
Virulence genes
Bi-component gamma haemolysin, hlgA/B and hlgB/C ,
polysaccharide intracellular adhesin biosynthesis/export protein
(icaC ) and aureolysin (aur ) genes were ubiquitous
throughout the groups. Reduced carriage of delta haemolysin (hld )
and staphylococcal complement inhibitor (scn ) genes were observed
in the CRSwNP group when compared with the control and CRSsNP cohort.
CRSsNP strains appeared to have reduced carriage of virulence genes with
none exhibiting leukocidin E/D (lukD/E) , serine protease A/B
(splA, splB) , enterotoxins n,u (sen, seu ) or enterotoxin
like protein X (selX ), and only one exhibiting Sei/m . In
contrast 75% of CRSwNP strains carried lukE/D genes and serine
protease (splA, splB) (Figure 1).
Intracellular localisation and
survival
Control 1 and CRSwNP 1 which closely matched the virulence gene carriage
patterns of control and CRSwNP groups were selected for further study of
intracellular survival in a S. aureus -LAD2 mast cell infection
model. Intracellular survival increased until 6 hours after which there
were less viable bacteria. There were marked differences between the two
strains, with no viable bacteria noted at 3 hours in the reference
strain but growth in the CRSwNP strain. There was a significant increase
in the CRSwNP strain survival at 6 and 9 hours of 1.89 (p=0.0087) and
9.84 (p=0.0088).
Given the reduction in intracellular bacteria after 6 hours which
appeared to be due to mast cell death (unpublished data), we eradicated
extracellular bacteria using lysostaphin and cultured cells for 24
hours. We noted persistence and a significant increase in intracellular
CFUs in both the control (97-fold) and CRSwNP (2570-fold) strains. The
CRS strain showed an 11.4-fold increase in intracellular survival when
compared with that of the control strain (p=0.035) (Figure 2).
Confocal microscopy
Following infection for 6 hours, cells were stained with BacLight
Live/Dead stain and imaged using confocal microscopy (Figure 3). The
images confirmed intracellular localisation of the bacteria within the
cytoplasm of mast cells. For the CRSwNP strain there was a large number
of bacteria within both live mast cells and apoptotic cell bodies when
compared to the control strain (Figure 3). The commensal organism
infected between 14-25% of cells while the pathogenic strain infected
52-57% of cells (p=0.18).