Figure 1.
Transmission electron microscopy of obstructive mucin from CRS aspirates.
In some cases (A), the mucin either formed a layered structure, with relatively intact cells towards the outer layers (black arrow) and more degraded cellular material further in (white arrow). (B) Alternatively, samples had little clear structure and the mucin was punctuated by pockets. The outermost layer of each sample is indicated by dark arrowheads.
Figure 2.
Confocal laser scanning microscopy of surface associated bacteria on mucosa removed from patients diagnosed with CRS.
Bacterial DNA (green) was visualized using an EUB338 PNA-FISH probe, and epithelial cell nuclei (blue) were counterstained with DAPI. Maximum projection images are shown. In some fields, epithelial cells were observed in the absence of bacteria (A), and in other fields bacterial biofilm was evident (B). B includes z-stacks oriented from the outside of the mucosal biopsy specimen (labelled ‘top’) to the deeper layers (indicated by a thick white arrow). Small white arrows indicate patches of diffuse staining, consistent with the presence of extracellular nucleic acids.
Table 1.
Bacteria isolated from CRS aspirates.
Table 2.
Biofilm formation and NucB sensitivity of selected isolates from CRS aspirates.
Figure 3.
Confocal laser scanning microscopy of Streptococcus constellatus FH20
biofilms with or without NucB treatment. Biofilms were formed on glass surfaces and were visualised with CLSM using BacLight LIVE/DEAD stain, which stains compromised (dead) cells red and live cells green. (A) Biofilms treated with buffer alone, and (B) biofilms treated with NucB.
Figure 4.
Scanning electron microscopy of Streptococcus constellatus FH20 biofilms treated with NucB or buffer control.
Biofilms were visualised with SEM after treatment for 1 h with buffer (A) or with NucB (B). At higher magnification, extracellular material (white arrow) was observed in the absence of NucB treatment (C), but was not seen in NucB-treated biofilms (D).
Figure 5.
The visualization and quantification of eDNA from CRS isolates.
(A) Intracellular DNA (i) or eDNA (e) was purified from bacterial biofilms of S. aureus FH7, S. constellatus FH20 or S. salivarius FH29, and analysed by agarose gel electrophoresis. High molecular weight chromosomal DNA is indicated by a black bracket; low molecular DNA or RNA is highlighted at the bottom of the gel by a black arrow. M; size marker. (B) The concentration of DNA in the intracellular (grey bars) and extracellular (white bars) fractions from bacterial biofilms was measured by NanoDrop spectrophotometry. Bars represent means of three independent extracts, and SEs are indicated. (C) Extracellular DNA concentration in biofilms was also visualised for another 19 isolates (see Table 1 for species names). In many cases, distinct bands were observed with an apparent migration at approximately 30 kbp. The total DNA concentration was measured by NanoDrop spectrophotometry, bars represent the average of three replicates and error bars are S.E.