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). doi:10.1371/journal.pone.0055339.g004 

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). doi:10.1371/journal.pone.0055339.g004 

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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...

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Context 1
... The production of extracellular DNase enzymes by clinical isolates was assessed using DNase test agar and staining with toluidine blue. In total, 13 of the 75 isolates (17%) produced extracellular DNase (Table 1). All S. aureus isolates produced extracellular DNase, and other producers were Streptococcus anginosus group ( S. anginosus / S. constellatus / S. intermedius ) strains (80% of strains), Staphylococcus lugdunensis (33% of strains) and Streptococcus salivarius (33% of strains). Extracellular nuclease producers were isolated from 11 out of 20 (55%) patients. In only two cases, more than one nuclease producing organism was isolated from the same patient sample. Twenty-four bacteria, isolated from patient specimens, were grown in 96-well microtiter plates to assay for biofilm formation. Representative strains of all species that produced extracellular DNase were selected for these studies, along with a similar number of non-producing organisms. Following incubation for 20 h in microtitre wells, all isolates had grown in the planktonic phase to OD 600 . 0.1 with the exception of three strains: S. anginosus FH19, S. constellatus FH21 and S. pneumoniae FH26 (Table 2). Nevertheless, all of these strains produced biofilms that were detectable by crystal violet staining. In fact, S. pneumoniae FH26 produced a relatively strong biofilm ( A 570 = 1.87). Growth rates of each strain in BHY medium were determined in planktonic cultures (Table 2). No correlation was seen between the maximum growth rate of strains and the capacity to form biofilms. Generally, there was extensive variation in the extent of biofilm formation between different species and between different strains of the same species. For example, M. catarrhalis FH3 produced a very weak biofilm ( A 570 = 0.77), whereas M. catarrhalis FH4 formed extensive biofilms ( A 570 = 2.78). Of the strains tested, Streptococcus anginosus FH19 produced the least abundant biofilms ( A 570 = 0.22). The mean extent of biofilm formation by non-nuclease producers ( A 570 = 1.51, S.E. 0.19, n = 15) was not significantly different from that of nuclease producers ( A 570 = 1.48, S.E. 0.32, n = 9). To assess the importance of eDNA in maintaining the structural integrity of biofilms, pre-formed biofilms were incubated for 1 h in the presence of the microbial DNase NucB (Table 2). Biofilms formed by 9 out of 9 (100%) nuclease producing strains were significantly reduced by NucB (T test comparing NucB treatment with buffer control, p , 0.05, n = 3). By contrast, only 5 out of 15 (33%) of the biofilms produced by non-nuclease producing bacteria were dispersed by NucB. In addition, 2 out of 15 (13%) non-nuclease producers had slightly increased levels of biofilm following incubation with NucB than without the enzyme. To assess whether NucB had detrimental effects on the cells themselves, four different isolates, S. aureus FH7, S. constellatus FH20, S. salivarius FH29 and M. catarrhalis FH4, were cultured to mid-exponential phase in THYE broth and challenged with 5 g ml 2 1 NucB. These isolates were selected as representative Gram- positive and Gram-negative organisms to assess the general toxicity of NucB for bacterial cells. Since the production of extracellular nucleases by bacteria is widespread, it seemed unlikely that DNase activity itself would be toxic to bacteria. Nevertheless, it was important to assess whether the NucB protein could inhibit the growth of bacteria. No effects were observed on the growth rate of cells following the challenge (data not shown). The number of viable cells in each culture continued to increase following NucB addition, and 1 h after adding NucB there was no difference in the number of viable cells in cultures containing NucB compared with control cultures without the enzyme. Overall, these data suggest that eDNA is an important component of the EPS for over 50% of the CRS isolates, including strains that produce extracellular DNase enzymes, and that addition of NucB dislodges cells without killing or inhibiting bacteria. To obtain more detailed information about the effects of NucB, biofilms of selected organisms were cultured on glass coverslips and analysed by CLSM and SEM. This work focussed on staphylococci and streptococci, since these were the genera most commonly isolated from CRS patients. In the absence of NucB treatment, biofilms formed by S. constellatus FH20 were relatively thin and consisted primarily of a single cell layer that covered most of the surface (Figure 3). In places, clusters of cells projected from the surface to a depth of , 12 m m. Using BacLight Live/Dead stain, both live cells (green) and dead cells (red) were observed in biofilms. Biofilms that had been treated with NucB were clearly less extensive than the untreated controls, and consisted of sparsely distributed single cells or very small aggregates of , 10 cells (Figure 3B). To obtain higher resolution images, similar biofilms were analysed by SEM (Figure 4). Again, in the absence of NucB, cell aggregates were evident and a relatively large proportion of the surface was covered by micro-organisms (Figure 4A). By contrast, NucB-treated biofilms almost exclusively contained isolated cells or small clusters of cells (Figure 4B). In addition, extracellular material was apparent in untreated biofilms under high resolution SEM (Figure 4C), that was not seen in biofilms incubated with NucB (Figure 4D). Biofilms formed on glass surfaces by S. aureus FH7 or S. intermedius FH22 were also visualised by SEM (not shown). As with S. constellatus FH20, biofilms that were treated with NucB contained far less biomass than those incubated in buffer alone. However, extracellular polymers were not observed in these organisms. To quantify levels of eDNA in model biofilms, eDNA and intracellular DNA (iDNA) was extracted from biofilm cultures of S. aureus FH7, S. constellatus FH20 and S. salivarius FH29. The eDNA was analysed by agarose gel electrophoresis (Figure 5). Sharp bands migrating at an apparent size of 30 kbp were observed in eDNA fractions of S. aureus FH7 and S. constellatus FH20. However, no high molecular eDNA bands were seen in S. salivarius FH29. Intracellular DNA from all three organisms appeared as a smear of high molecular weight fragments, probably due to binding of chromosomal DNA to cell wall fragments. In addition to the high molecular weight fragments, small fragments of DNA or RNA were seen at the bottom of the gel. Nucleic acids in each fraction were quantified using the Nanodrop spectrophotometer (Figure 5B). For each strain, eDNA represented approximately 5–10% of the total DNA present in the biofilm. To account for the possibility that samples may have contained RNA in addition to DNA, nucleic acids were also quantified using PicoGreen dye, which is strongly selective for double stranded DNA. No significant differences were observed between the total amount of eDNA in S. salivarius FH29 biofilms and eDNA in biofilms formed by the other two strains. Therefore, despite the lack of a clear band by agarose gel electrophoresis, it appears that eDNA was present in S. salivarius FH29 biofilms. To assess whether eDNA was present in biofilms formed by other CRS isolates, biofilms of each strain were cultured in 6-well plastic dishes and eDNA was purified as described in the Methods section. Only two strains were omitted from this analysis: S. constellatus FH21 grew very poorly in biofilms and it was not possible to extract eDNA, and S. salivarius FH28 was prone to contamination and, after several attempts, it was decided not to pursue DNA purification from this strain. By agarose gel electrophoresis, sharp bands corresponding to high molecular weight eDNA products were observed in all Staphylococcus spp., S. constellatus FH20 and in S. intermedius FH22 (Figure 5C). By contrast, similar bands were not detected from Corynebacterium spp., S. anginosus or S. pneumoniae . Only M. catarrhalis and S. salivarius had inter-strain differences in the production of eDNA. Thus, eDNA was not visualised in M. catarrhalis FH3 biofilm extracts, whereas eDNA was clearly present in M. catarrhalis FH4 (Figure 5C). Similarly S. salivarius FH29 did not produce a band of eDNA on an agarose gel, whereas a sharp band was seen in S. salivarius FH27. The concentrations of eDNA in samples were determined using the Nanodrop spectrophotometer. Concentrations of eDNA in the extracts ranged from 206 ng m l 2 1 to 917 ng m l 2 1 (Figure 5C). Interestingly, there appeared to be little correlation between the concentration of eDNA and the presence or absence of a band on the agarose gel. For example, M. catarrhalis FH3 produced one of the highest concentrations of eDNA, but no band on the gel. Conversely, the eDNA concentration from S. intermedius FH22 was just 325 ng m l 2 1 even though this strain clearly produced a band of eDNA on a gel. The production of an extracellular nuclease did not correlate with the presence of a band of eDNA on a gel. All strains of S. aureus (nuclease-positive) and S. epidermidis (nuclease- negative) produced clear bands of eDNA, for example. With the exception of S. anginosus FH18 and S. anginosus FH19, all strains that failed to produce a clear band of eDNA on an agarose gel were insensitive to NucB treatment. There is mounting evidence that microbial biofilms growing within paranasal sinuses are a major factor in the pathogenesis of CRS [14]. Bacterial biofilms have been most commonly detected on the sinus mucosa, whereas fungi tend to be more easily detected within the sinonasal mucous. Fungal growth is often accompanied by mucous secretions containing large numbers of intact or degraded eosinophils, known as ‘eosinophilic mucin’ or ‘allergic mucin’ [43–45]. The eosinophils appear to migrate intact from the tissues, and degrade or degranulate upon reaching the mucin, possibly in order to target fungi growing within the mucin. Allergic mucin may be ...
Context 2
... patients during CRS treatment. We aimed to establish whether isolated CRS bacteria form biofilms in vitro and, further, whether eDNA contributes to the integrity of the biofilm. In total, 24 isolated strains were tested for biofilm formation in a microplate model system, and all strains produced biofilms to some extent. The ability to form biofilms was not closely related to the growth rate or yield in planktonic cultures. These data are in line with previous studies on Listeria monocytogenes or Salmonella enterica strains, which also found no correlation between the growth rate or yields of individual strains and their capacities to form biofilms in microplate model systems [50,51]. Representative strains of many of the species found in this study have been shown to produce DNase I-sensitive biofilms including, for example, S. aureus [52,53], S. pneumoniae [54,55], Neisseria spp. [56,57], P. aeruginosa [29] and E. coli [53]. We have recently identified a novel DNase enzyme, NucB, from a marine strain of Bacillus licheniformis that has potent anti-biofilm activity against a number of bacteria including E. coli and M. luteus [33]. This enzyme is smaller in size ( , 12 kDa) than many other nucleases, including DNase I, and appears to be well adapted to breaking up bacterial biofilms even at low concentrations [33]. A key goal of this study was to establish whether freshly isolated CRS-associated bacteria produce biofilms that are sensitive to NucB. Overall, . 50% of the strains tested produced biofilms that were reduced upon treatment with NucB. In fact, the vast majority of staphylococci (8 of 10 strains tested) and streptococci (6 of 9 strains) produced NucB-sensitive biofilms. In contrast, two Corynebacterium spp. and two M. catarrhalis strains made biofilms that were not removed by NucB. In one case the M. catarrhalis biofilm was slightly, but significantly, increased by NucB treatment. Whilst eDNA commonly promotes adhesion and biofilm formation by bacteria, in rare cases eDNA has been shown to inhibit bacterial settlement [58]. It is possible that eDNA may be inhibitory to M. catarrhalis adhesion and that NucB- mediated eDNA degradation would therefore promote adhesion by this organism. This hypothesis requires further investigation. The production of extracellular DNase enzymes by bacteria may influence the structure of biofilms. For example, isogenic nuclease-deficient mutants of S. aureus , Neisseria gonorrhoeae or Vibrio cholerae form thicker biofilms than their wild-type progenitor strains [57,59,60]. However, using in vitro or in vivo models of catheter biofilms, Beenken et al. [61] found that the total number of viable cells in biofilms of the clinical osteomyelitis isolate S. aureus UAMS- 1 was not affected by mutation in either of two extracellular nuclease-encoding genes. Therefore, it is not clear whether microbial nucleases contribute to the gross biofilm structure in clinically relevant situations. Production of extracellular DNase enzymes has been reported for several of the genera isolated here. S. aureus is well-known to produce DNases, and DNase production is often used as a phenotypic test to differentiate S. aureus from coagulase-negative staphylococci. However, the test must be interpreted with caution, since some coagulase-negative staphylococci such as S. lugdunensis can produce nucleases [62]. In fact, one of the S. lugdunensis strains isolated here was found to produce DNase. Corynebacterium diphtheriae and N. gonorrhoeae have also been reported to be able to produce extracellular DNase enzymes [57,63], but DNases were not detected in any of the Corynebacterium spp. or Neisseria spp. identified in this study. Production of DNases is variable in a -haemolytic streptococci [64], and 5 of the 9 streptococci isolated here produced DNase activity. Representative strains of all species that produced nuclease were tested in biofilm assays. Interestingly, all 9 nuclease-producing strains made biofilms that were reduced by treatment with the exogenous addition of NucB. These data provide clear evidence that the ability of a strain to produce extracellular nucleases does not preclude the formation of biofilms that are stabilised by eDNA. The production of extracellular DNases is tightly regulated in bacteria. For example, in S. aureus , nuclease production is regulated by the stress response sigma factor B [59]. Within biofilms, nucleases may be produced at low levels or by only a small proportion of the cells. Here, direct evidence for the presence of eDNA in biofilms formed by 22 CRS isolates was provided by extraction and quantification of eDNA. All strains produced significant amounts of eDNA that could easily be measured in the Nanodrop spectrophotometer. A more detailed analysis was conducted on three different CRS isolates, including two that produce nucleases ( S. aureus FH7 and S. constellatus FH20). In S. constellatus FH20 biofilms, extracellular material was observed by SEM (Figure 4). Extracellular DNA purified from S. aureus FH7 and S. constellatus FH20 biofilms was visualised as sharp high molecular weight bands on agarose gels with an apparent migration similar to that of intracellular chromosomal DNA. However, eDNA from S. salivarius FH29 was not detected by this technique. Nevertheless, quantitative measures indicated that extracellular nucleic acids were present in S. salivarius FH29 biofilms. Interestingly, in contrast to S. aureus FH7 and S. constellatus FH20, S. salivarius FH29 biofilms were not sensitive to NucB. Therefore, it appears that S. salivarius FH29 does not rely on large fragments of eDNA to stabilise biofilms. A broader analysis of the CRS isolates identified six other strains that did not produce defined bands of eDNA when analysed on agarose gels. Of these, four strains were insensitive to NucB indicating that, like S. salivarius FH29, these strains do not utilise large eDNA fragments for biofilm stabilisation. The two strains of S. anginosus did not produce visible bands of eDNA on gels, even though both strains were sensitive to NucB. It is possible that eDNA from S. anginosus was partially degraded, to the point where it did not form a defined band on a gel, but was still present in sufficient quantities to be utilised for maintaining the biofilm structure. Improving the surgical treatment of CRS requires new methods for controlling microbial biofilms in the paranasal sinuses. The data presented here demonstrate that many CRS-associated bacteria produce biofilms that can be reduced by treatment with a microbial nuclease NucB in vitro . Given the high prevalence of CRS, even a 50% reduction in the colonization of sinus mucosa by micro-organisms would be predicted to have significant clinical benefits on a population level. Of course, the current study has focussed on in vitro work and it is acknowledged that translating the findings to the clinic will require further investigations in animal models and ultimately in patients. Before this can be done, the safety of NucB for clinical use must be established. We are currently in the process of testing the safety of NucB with a view to conducting clinical trials in future. In addition, it would be interesting to determine whether matrix-degrading enzymes act synergistically with antibiotics to control biofilm growth since this would present additional therapeutic possibilities. Ultimately, the utility of DNase enzymes to aid the treatment of CRS will depend upon in vivo data. Nevertheless, we have shown that NucB has clear potential for the control of biofilms formed by clinically important strains of bacteria. Figure S1 Three-dimensional rotation showing micro-organisms associated with the outer layer of a mucosal biopsy. Bacterial DNA was hybridized with the EUB338 PNA-FISH probe, and appears green in the image. Host cell nuclei were counterstained blue. Bacterial cells (punctate green staining) are seen interacting with cells on the surface of the biopsy. ...

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... mellea DSM 3731 [59] or A. mellea ELDO17 [60] available at the fungal genomics resource MycoCosm [61]. Intriguingly, the N-terminal amino acid sequence of melleatin shows a high identity (>88 %; Fig. S4) with several non-specific endonucleases belonging to the His-Me finger endonucleases superfamily [62]. Structurally, the core of this endonuclease superfamily consists of a β-hairpin followed by an α-helix, forming a binding site for a single catalytic metal ion, generally Mg 2+ [32]. ...
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