Strains and Plasmids used.

<p> <i>Km^R: Kanamycin resistance, CmR: Chloramphenicol resistance, Ery^R: Erythromycin resistance. cds: Coding sequence.</i></p

Primers used in this study.

<p>(Restriction sites are underlined).</p

Comparison between NucB and DNaseI mediated biofilm dispersal.

<p>Efficiency of dispersal of 24 hour old <i>B. licheniformis</i> DSM13 biofilms by the tested nucleases in decreasing concentrations. Dispersal of the target biofilm was determined using a 96 well microtitre plate setup, using a concentration range of either <i>B. subtilis</i> supernatant containing NucB or commercially available DNaseI. For every data point, the average of at least 6 independent wells was taken, and the experiment was repeated three times.</p

Dispersal efficiency in time and concentration.

<p>Efficiency of dispersal of <i>B. licheniformis</i> DSM13 24 hour old biofilm by AMS supernatant (sup.) visualised as remaining CV stain as measured by plate reader. Incubation time in minutes (') and seconds (”) indicated on the left, concentration of AMS supernatant indicated on top. The biofilm remaining is indicated with both a colour scale (dark blue: no dispersal, white: full dispersal) and as a percentage of non-dispersed biofilm (red numbers).</p

Dispersal of several bacterial species by AMS supernatant.

<p>Typical examples of dispersal of several 26 hour grown biofilm forming strains by AMS supernatant. Remaining biofilm visualised by CV staining after 30 minutes incubation with dispersal compound. x = control (only medium added), 10% = 10% of AMS supernatant added, 5% = 5% of AMS supernatant added.</p

Heterologous overexpression of NucB in <i>B. subtilis</i> NZ8900.

<p>Lane <b>m</b> = Invitrogen Novex Sharp Pre-stained Marker, band sizes indicated in kDa. Lanes <b>A–C</b>: 20 fold concentrated TCA precipitated supernatant of strain <i>B. subtilis</i> NZ8900+pNZ8901-<i>nucB</i>, loaded 20 µl (<b>A</b>), 10 µl (<b>B</b>), 5 µl (<b>C</b>). Lane <b>D</b>: loaded 20 µl unprocessed supernatant. Arrow indicates NucB position.</p

Efficiency of different AMS supernatant fractionation methods.

<p><b>A</b>: total supernatant of the AMS culture; <b>B</b>: active fraction obtained after rotary evaporation followed by Sephadex G50 gel filtration; <b>C</b>: active fraction obtained after freeze-drying by Sephadex-LH20 gel filtration, <b>D+E</b>: Active fraction obtained by TCA precipitation followed by Superose 12 gel filtration. <b>m</b> = Invitrogen Novex Sharp Pre-stained Marker, band sizes indicated in kDa. Arrows indicate bands 1, 2 and 3 cut out for peptide mass fingerprinting, as described in text.</p

Confocal laser scanning microscopy of surface associated bacteria on mucosa removed from patients diagnosed with CRS.

<p>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 <i>z</i>-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.</p

Efficacy of a Marine Bacterial Nuclease against Biofilm Forming Microorganisms Isolated from Chronic Rhinosinusitis

<div><p>Background</p><p>The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS). Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS.</p> <p>Methods/Principal Findings</p><p>Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, <i>Staphylococcus aureus</i> and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation <i>in vitro</i> using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of <i>Bacillus licheniformis</i>. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from <i>in vitro</i> biofilms.</p> <p>Conclusion/Significance</p><p>Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.</p> </div

The visualization and quantification of eDNA from CRS isolates.

<p>(A) Intracellular DNA (i) or eDNA (e) was purified from bacterial biofilms of <i>S. aureus</i> FH7, <i>S. constellatus</i> FH20 or <i>S. salivarius</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055339#pone-0055339-t001" target="_blank">Table 1</a> 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.</p