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).