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Microbes and Infection xx (2015) 1e7 www.elsevier.com/locate/micinf

Extracellular DNA in oral microbial biofilms Nicholas S. Jakubovics a,*, J. Grant Burgess b b

a School of Dental Sciences, Newcastle University, UK School of Marine Science and Technology, Newcastle University, UK

Received 6 February 2015; revised 25 March 2015; accepted 26 March 2015 Available online ▪ ▪ ▪

Abstract The extracellular matrix of microbial biofilms is critical for surface adhesion and nutrient homeostasis. Evidence is accumulating that extracellular DNA plays a number of important roles in biofilm integrity and formation on hard and soft tissues in the oral cavity. Here, we summarise recent developments in the field and consider the potential of targeting DNA for oral biofilm control. © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Biofilm matrix; Competence; Dental plaque; eDNA; Membrane vesicles; Oral streptococci

1. Introduction Microorganisms in nature tend to congregate at interfaces, where they form robust mixed-species biofilm communities. A key feature of biofilms is the presence of an extracellular macromolecular matrix that surrounds cells and retains small molecules. The matrix is composed of many different macromolecules including polysaccharides, proteins, lipids and extracellular DNA (eDNA) [1]. The role of eDNA in particular has received a great deal of attention recently, and many different microorganisms have been shown to depend upon eDNA to form biofilms in laboratory monocultures [2]. In view of these observations, there is growing optimism that deoxyribonucleases (DNases) may be used to disrupt the biofilm matrix and thereby control biofilm growth. However, before this can be achieved it is necessary to understand the functions of eDNA in mixed-species biofilms, and here there is still much work to be done. All humans carry a rich and complex microbiota on the skin, and within the gut, nose, oral cavity and the genito* Corresponding author. School of Dental Sciences, Centre for Oral Health Research, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4BW, UK. Tel.: þ44 191 208 6796; fax: þ44 191 208 8807. E-mail address: [email protected] (N.S. Jakubovics).

urinary tract. At each of these locations, there are different niches that harbour distinct microbial populations. For example, in the oral cavity there are marked differences in the diversity of microorganisms present on gums, cheeks, tongue, palate or teeth [3,4]. Biofilms on teeth can be subdivided into supragingival dental plaque, which forms on exposed surfaces above the gumline and is exposed to saliva and oxygen, and subgingival dental plaque that grows below the gum margin and is fed by gingival crevicular fluid, a serum exudate. In areas of the teeth that are protected from mechanical forces and oral hygiene measures, such as cracks, fissures or exposed cementum in tooth roots, dental plaque matures into a highly complex microbial community. Frequent sugar consumption selects for acidogenic and aciduric microorganisms such as mutans streptococci, lactobacilli and certain bifidobacteria. The metabolic activity of these microorganisms converts dietary sugars into acid and promotes dental caries development. At the same time, oral streptococci convert sucrose into polymers of glucose or fructose (glucans and fructans) which provide a bulky (large volume) extracellular matrix. Whilst the potential contributions of glucans and fructans to the caries process have received a great deal of attention [5], far less research has been directed towards the composition of the extracellular matrix in early supragingival dental plaque. Similarly, the extracellular matrix of subgingival dental

http://dx.doi.org/10.1016/j.micinf.2015.03.015 1286-4579/© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Jakubovics NS, Grant Burgess J, Extracellular DNA in oral microbial biofilms, Microbes and Infection (2015), http:// dx.doi.org/10.1016/j.micinf.2015.03.015

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plaque, or of oral biofilms on epithelial surfaces, has not been thoroughly defined. This review will briefly describe our current understanding of the structure of the oral biofilm extracellular matrix, and will discuss recent evidence that eDNA may play many critical and defined roles as a key matrix component. We will highlight areas for future research and discuss the potential of targeting eDNA for oral biofilm control. 2. Structure of the extracellular matrix in oral biofilms Throughout the 1960s and 1970s, a series of studies described the application of transmission electron microscopy (TEM) or scanning electron microscopy (SEM) to define the ultrastructure of supragingival and subgingival dental plaque. Sample preparation for conventional electron microscopy requires dehydration, resulting in shrinkage and condensation of the extracellular matrix. Nevertheless, TEM images showed very clearly that there was space between cells in dental plaque and that this space was filled with extracellular material [6]. Using SEM, an amorphous layer covering the cells was observed [7]. On more modern scanning electron microscopes, and using critical point drying to prepare samples, extracellular material can clearly be seen as strands between the cells (Fig. 1). It should be noted that the structure of these strands is likely to be somewhat different from the natural matrix prior to fixing and dehydration for SEM imaging. Dehydration can be avoided completely by optically sectioning biofilms using confocal laser scanning microscopy, which shows dental plaque as an open and heterogeneous structure of matrix-enclosed cells permeated by channels and voids [8]. The channels through the biofilm appear to be fluid filled and are accessible

Fig. 1. Scanning electron microscopy of subgingival dental plaque on the extracted tooth of a periodontitis patient. The sample was dehydrated and dried at the critical point of CO2. Strands of condensed extracellular material are highlighted by large arrows, and small arrows indicate vesicles of varying sizes apparently being released from the surface of cells. Image kindly provided by R. Holliday and L. Bowen.

to fluorescein. Nevertheless, it is difficult to eliminate the possibility that they contain small amounts of extracellular polymer. There is evidence that the dental plaque matrix provides a barrier to the free diffusion of substrates, since exogenously added fluoride and triclosan are retained in the outer layers of dental plaque [9]. Therefore, physical or chemical treatments that open up the dental plaque matrix may enhance the delivery of antimicrobials. The key polymeric constituents of the dental plaque extracellular matrix have been studied in some detail, and attention has particularly focussed on the carbohydrates produced by oral streptococci. By dry weight, supragingival dental plaque contains approximately 10e20% glucan and 1e2% fructan, and these concentrations vary with plaque maturation and dietary sugar intake [10]. Recent work on Streptococcus mutans has provided new evidence that biofilm matrix polysaccharides may be important in the caries process. In S. mutans, glucans are produced by three glucosyltransferases encoded by gtfB, gtfC and gtfD genes. Of these, gtfB and gtfC are required for microcolony formation [11]. The expression of gtfB and gtfC is up-regulated in the presence of Streptococcus oralis or Actinomyces naeslundii, and glucan production results in low-pH microcolonies that are dominated by S. mutans and are protected from antimicrobial agents [12]. Therefore, it is likely that glucans play a role in shaping the architecture of natural supragingival dental plaque. Proteins are also considered an important component of the extracellular dental plaque matrix. There is evidence that the protein content in plaque fluid increases by >50% following exposure to sucrose [13], and changes in the proteome composition have been observed in response to sucrose [14]. However, it is not yet clear what effects these changes in protein content of the plaque matrix have on the structure and function of dental plaque. Several different lines of evidence provide a clear indication that eDNA is also present in dental plaque. One approach to investigate eDNA is by the addition of propidium monoazide combined with bright light, which results in crosslinking to DNA. The cross-linked DNA is then inhibited from PCR amplification. Approximately two thirds of S. mutans DNA in dental plaque or carious dentin was inhibited from qPCR-amplification following the application of propidium monoazide and bright light [15]. Propidium monoazide does not cross the membrane of viable cells, and therefore the cross-linked DNA must have been extracellular, or at least present in dead cells. There is also evidence from electron microscopy images that membrane vesicles are abundant in dental plaque [Fig. 1; reference [6]]. Membrane vesicles are often loaded with DNA [16,17], and the presence of vesicles in dental plaque indicates that DNA is likely to be present outside the cells. Additionally, indirect evidence for eDNA comes from analysis of the genomes of oral bacteria such as Streptococcus sanguinis, S. mutans and S. oralis, which all show extensive evidence of recombination [18e20]. Oral streptococci are naturally transformable and it is likely that recombination has arisen from the uptake and incorporation of eDNA. Despite these intriguing indications that eDNA is

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present in oral biofilms, there is a paucity of data on the extent of eDNA in different biofilms, and on its importance for biofilm structure and function. 3. Sources of eDNA in oral biofilms Within mixed-species biofilms it is likely that several distinct pathways contribute to the overall accumulation of eDNA in the extracellular matrix. Perhaps the most obvious mechanism is the death and lysis of microbial cells within the biofilm. Passive lysis from cell aging or due to external insults including antimicrobial agents may increase the levels of eDNA within biofilms. In addition, several mechanisms have been described whereby bacteria actively kill neighbouring cells of different species or of the same species. For example, oral streptococci produce lactic acid and hydrogen peroxide (H2O2) at bactericidal concentrations. Lactic acid can permeabilise Gram-negative bacterial cells [21], and the production of H2O2 during batch culture has been shown to release DNA from the early dental plaque colonisers Streptococcus gordonii and S. sanguinis [22]. It is not clear whether cell lysis is required for release of eDNA from oral streptococci such as S. gordonii. There is evidence that a murein hydrolase, LytF, and the major autolysin AtlS are involved in lysis and release of DNA from subpopulations of S. gordonii cells [23,24]. On the other hand, LytF and AtlS have been shown to be important for DNA release from cells in the apparent absence of extensive cell lysis [25]. It is likely that differences in the strains employed in these studies or the cultures conditions used have resulted in the apparently conflicting data. It will be important to determine whether cell lysis is required for DNA release in natural dental plaque. Bacteriocins also apparently have complex roles contributing to the release of eDNA and competence development in oral bacteria. Bacteriocins are peptides produced by some oral bacteria that induce lysis in target cells. For example, S. mutans produces a range of bacteriocins known as mutacins that each target different species [26]. Of these, mutacin V has been shown to contribute to the accumulation of eDNA in biofilms [27]. In addition to its lytic activity against a relatively narrow range of bacterial species, Mutacin V (also known as NlmC or CipB) accumulates intracellularly within a proportion of cells in an S. mutans population and triggers autolysis through activation of the murein hydrolase LytFSm [27,28]. This autolytic activity is critical for competence development and genetic exchange. It is currently unclear how a proportion of cells in a population are able to resist the activation of LytFSm and subsequent cell lysis, and thus are able to benefit from the eDNA that is released into the biofilm. Other possible mechanisms for the release of DNA from bacterial cells include active secretion by vesicles or by secretory apparatus. Vesicles are produced by both Gramnegative and Gram-positive bacteria [29]. Recently, it has been shown that the well-characterised strain S. mutans UA159 produces membrane vesicles that contain eDNA [30]. Other oral bacteria such as the periodontal pathogens Porphyromonas gingivalis and Aggregatibacter

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actinomycetemcomitans are well known to produce vesicles [31,32], but currently their contribution to DNA release is unclear. In Neisseria gonorrhoeae, DNA is secreted into the surrounding medium through an unusual Type IV secretory apparatus that, unlike other Type IV secretion systems, does not channel secretion directly into a host cell [33]. At present, homologous systems for DNA release have not been demonstrated in other bacteria. However, there is some evidence that secretion systems may be utilised by oral bacteria for DNA export since strains disrupted in yidC1, yidC2 or ffh genes in S. mutans UA159, which encode secretion system components, are impaired in DNA release from cells [30]. Other potential sources of eDNA in oral biofilms include host cells and viral particles. At present, the oral virome has not been characterised in great detail, but it is clear that the majority of viruses are bacteriophage which may have roles in shaping the oral bacterial community [34]. On soft tissues in the mouth, bacteria are in close contact with epithelial cells, and it is possible that host DNA from epithelial cell lysis contributes to the biofilm. Epithelial cells can be observed close to the enamel surface in the early stages of dental plaque development, but are less common in mature dental plaque [35]. However, in subgingival dental plaque, bacteria are in close proximity to the gum tissue. Neutrophils are a key component of the host response to subgingival bacteria and DNA released from neutrophils in the form of neutrophil extracellular traps has been observed in crevicular fluid and on pocket epithelium [36]. Studies conducted so far suggest that oral biofilms contain eDNA from a variety of different sources. A detailed understanding of the composition of this eDNA will require a great deal more work. Distinguishing between bacterial, viral and human DNA is relatively easy with qPCR or metagenomic sequencing. Active release mechanisms such as secretion or entrapment of DNA in vesicles will likely result in the release of DNA fragments which may be double stranded or single stranded. Small fragments of DNA may be distinguished from intact chromosomes by Southern blotting. However, it is likely that much of the chromosomal DNA released by cell lysis will be degraded to some extent by DNase enzymes in biofilms and therefore it will be difficult using this approach to assess whether DNA was intact or degraded at the point of release. Nevertheless, a detailed analysis of the eDNA present in biofilms is urgently needed in order to shed light on the key sources of biofilm matrix eDNA, and the pathways that lead to the release of DNA from cells. 4. Functions of eDNA in biofilms Studies in the 1950's identified eDNA as the major component of the slimy pellicle produced by a variety of different bacteria including Actinomyces viscosus [37,38]. Even at such an early stage, it was speculated that eDNA may play roles in addition to the carriage of genetic information between cells, such as protection against desiccation or radiation, avoiding overcrowding or slowing the movement of cations towards cells [38]. Another intriguing function of

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eDNA was identified in A. actinomycetemcomitans, where eDNA was shown to mediate adhesion of the leukotoxin to bacterial cell walls [39]. Even though the concept of biofilms was introduced in the 1970's, it was not until 2002 that a clear functional role of eDNA in biofilms was established. Working on the model biofilm organism Pseudomonas aeruginosa, Whitchurch et al. [40] found that treatment of early biofilms with DNase I caused cells to disperse from the surface, indicating that eDNA was critical for biofilm adhesion. Since then, many studies have identified eDNA in a wide variety of different monospecies biofilms, and occasionally in mixedspecies biofilms [2,41]. Surprisingly few of these studies have focussed specifically on oral bacteria, even though oral bacteria are essentially obligate biofilm formers and dental plaque is one of the most intensively studied biofilm systems. Nevertheless, evidence is gradually accumulating for several distinct functional roles of eDNA in oral bacterial biofilms.

that eDNA may interact with glucans to enhance adhesion [30]. In S. mutans UA159, eDNA forms nanofibers that strengthen the biofilm. Digestion of these nanofibers with DNase I reduces biofilm formation [30]. Extracellular DNA has been purified from monospecies Fusobacterium nucleatum and P. gingivalis biofilms, but treatment with DNase I did not significantly reduce biofilm formation, and the function of eDNA within these biofilms is therefore unclear [49]. Oral bacteria are often isolated from other body sites, and a study of chronic rhinosinusitis isolates identified eDNA in a number of biofilms produced by common oral streptococci including S. anginosus, S. constellatus, S. intermedius and Streptococcus salivarius [50]. With the exception of one strain of S. salivarius, all biofilms were significantly reduced following a 1-h incubation with the DNase NucB from Bacillus licheniformis, indicating that eDNA was essential for biofilm stability (Fig. 2).

4.1. Adhesion and biofilm structure

4.2. Protection against antimicrobial agents

Extracellular DNA is a sticky molecule that associates with a number of different surfaces. For example, in the marine environment eDNA forms part of the matrix of adhesive freefloating cell aggregates termed ‘protobiofilm’ [42]. Aggregates of P. aeruginosa growing in batch culture also contain eDNA [43]. Aggregation of different oral bacteria, termed coaggregation, has been shown to occur between many different oral bacteria and may form the basis for early colonisation and biofilm formation [44]. Coaggregation studies in vitro often employ washed cells from fresh laboratory cultures, which may have relatively little eDNA on the surface. It would be interesting to investigate whether eDNA is retained for prolonged periods on the surface of bacterial cells, and whether it is present in natural coaggregates within the mouth. It has been shown that eDNA in the biofilm matrix also binds to the external surface of membrane vesicles produced by biofilm cells of P. aeruginosa [45]. Similarly, it is the interaction of eDNA with membrane vesicles and bacterial cell surfaces that traps A. actinomycetemcomitans leukotoxin and prevents it diffusing freely into the culture supernatant [39]. Enterococcus faecalis is commonly found in endodontic infections, and there is evidence that eDNA enhances the adhesion of E. faecalis cells to dentin since adhesion is significantly reduced by treatment with DNase I [46]. The adhesion of S. mutans LT11 cells with or without DNase treatment to hydrophilic and hydrophobic surfaces has been measured by time-lapse phase contrast microscopy or atomic force microscopy. The rate of bacterial deposition on both types of surfaces was increased in the presence of eDNA, and eDNA promoted the development of surface-associated cell aggregates, particularly on hydrophilic surfaces [47]. Further, eDNA increased the strength of binding to surfaces, and eDNA-mediated adhesion was significantly stronger on hydrophobic than hydrophilic surfaces [48]. Using saliva-coated hydroxyapatite as a substrate, eDNA was shown to promote the adhesion of S. mutans UA159 only in the presence of glucan polymers, indicating

There is now strong evidence that eDNA in the biofilm matrix protects microbial cells from a variety of antimicrobial agents. For example, DNase I increases the susceptibility of nontypeable Haemophilus influenzae to ampicillin and ciprofloxacin and partially sensitises biofilm cells of Mycobacterium tuberculosis to isoniazid [51,52]. Vancomycin binds 100 times more tightly to DNA than to its cellular target, the dAla-d-Ala C terminus of the peptidoglycan pentapeptide, and eDNA protects Staphylococcus epidermidis biofilms from vancomycin [53]. Extracellular DNA protects P. aeruginosa biofilm cells from tobramycin [54]. Protection is diminished by the addition of cationic agents, indicating that eDNA may retard penetration of tobramycin into biofilms through ionic interactions. However, treatment of Burkholderia cepacia complex biofilms with recombinant human DNase did not increase sensitivity to tobramycin [55]. It is possible that polysaccharides are more important for interactions with cationic agents in B. cepacia complex biofilms, or that the eDNA within B. cepacia complex biofilms is protected from DNase digestion by DNA-binding proteins. In the oral cavity, antibiotics are used for some soft tissue infections and very occasionally in periodontal therapy. In addition, antimicrobial agents are incorporated into a variety of oral hygiene agents. However, there is very little evidence regarding the contribution of eDNA to oral biofilm resistance against antimicrobial agents. A single study has shown that E. faecalis biofilms are sensitised to 2% chlorhexidine by treatment with either dextranase or DNase I [46]. It would be interesting to assess the impact of combinations of DNase enzymes with chlorhexidine or other commonly used antimicrobials against mixed-species oral biofilms in vitro or, ideally, in vivo. It is also possible that eDNA protects against natural antimicrobial agents produced in the oral cavity. For example, eDNA from H. influenzae has been shown to bind human bdefensin-3, an antimicrobial peptide that is expressed in oral tissues and is upregulated in periodontitis [56,57].

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Fig. 2. Effect of the Bacillus licheniformis DNase, NucB, on biofilms formed by Streptococcus salivarius FH29 [50]. Biofilms were cultured on glass coverlips and treated at 37  C for 1 h with buffer alone (control, left panel) or with 5 mg ml1 NucB (right panel). After NucB treatment, only scattered cells remained on the surface. Scale bar ¼ 20 mm. Image provided by Robert Shields.

4.3. Nutrient storage Extracellular DNA is a major source of phosphorous for microorganisms in deep sea sediments, and can provide essential phosphate for the growth of nuclease enzymeproducing P. aeruginosa batch cultures in the laboratory [58,59]. Like many marine bacteria, oral bacteria also live a ‘feast and famine’ existence, and nutrient stores are important for maintaining growth during periods of fasting. In supragingival dental plaque, extracellular fructans are thought to be important extracellular carbohydrate stores for mutans streptococci that produce fructanases [60]. Many oral bacteria produce extracellular DNase enzymes and potentially could utilise eDNA as a source of phosphate, fixed nitrogen and carbon [61]. In addition, eDNA binds cations such as trace metals, and it is possible that eDNA helps to retain essential metal ions within biofilms. High affinity metal scavenging systems such as the S. gordonii manganese transporter ScaCBA are up-regulated in the presence of saliva, indicating that metal ions are relatively scarce in the oral environment [62]. Further work is needed to establish the role of eDNA in providing a source of both phosphate and cations for oral biofilm bacteria. 4.4. Genetic exchange The ability of streptococci to uptake and incorporate exogenous DNA has been known since the early experiments of Griffith and others on rough and smooth types of Streptococcus pneumoniae nearly 100 years ago [63]. By contrast, it is only relatively recently that natural transformation has been demonstrated in P. gingivalis [64]. Competence for genetic transformation is a tightly controlled process, and incorporation of novel genetic material is a relatively rare event. Nevertheless, the genomes of many oral bacteria including streptococci and P. gingivalis show evidence of extensive genetic recombination and horizontal gene transfer, indicating that eDNA uptake has likely driven the evolution of many different oral bacteria [65]. Worryingly, penicillin resistance in S. pneumoniae appears to have arisen from the transfer of portions of the pbp2x gene from S. mitis and/or S. oralis [66]. In a model system, genomic DNA from Veillonella dispar was

shown to transfer tetracycline resistance to S. mitis [67]. It is important to establish more clearly the transfer of genetic information via eDNA within oral biofilms in order to ensure that we minimise the potential for the transfer of antibiotic resistance to pathogenic strains. 5. Targeting eDNA for biofilm control Since eDNA appears to be important for the structural integrity and the antimicrobial resistance of many different biofilms, treatment with exogenous DNase enzymes is an attractive option for biofilm control. The possibilities for treating biofilms in this way have been considered in detail in a recent review [68]. In the oral cavity, strategies to deliver DNase enzymes would need to ensure that (i) the enzyme is safe for use in humans, (ii) the enzyme is compatible with other components of the delivery system such as fluoride, sodium dodecyl sulphate or EDTA, (iii) that the enzyme reaches the oral biofilm effectively, (iv) that the enzyme is active when in contact with the oral biofilm, (v) that activity is retained for long enough to perturb the biofilm, and (vi) that the enzyme is stable during storage for a reasonable period of time. These are significant demands, and clearly it will take some time to translate current research on in vitro biofilms into an enzyme that can be used in the clinic. Nevertheless, there is some hope that DNase enzymes could potentially be used clinically, since there are already precedents for the use of enzymes in human healthcare. For example, recombinant human DNase I (Pulmozyme, or dornase alpha) is used for the treatment of cystic fibrosis [69]. However, mammalian DNase enzymes require glycosylation for activity and therefore cannot be expressed in bacterial production systems. The advantages of bacterial enzymes such as NucB from B. licheniformis in combatting clinically relevant biofilms have recently been highlighted [50,68]. A mouthwash or dentifrice would perhaps be the simplest approach for delivering DNase enzymes, but an alternative would be to coat artificial surfaces that are introduced into the mouth such as dentures or implants, in order to delay or prevent biofilm accumulation. Swartjes et al. [70] have developed a functional DNase I coating on a polymethylmethacrylate (PMMA) material that inhibits colonisation by P. aeruginosa and S. aureus for up to

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14 h. Although this study was aimed at developing orthopaedic implant materials, PMMA is also widely used for dentures, and potentially a functional coating could help to reduce problems of dental caries or denture stomatitis. At present the coating would be most appropriate for inhibiting early colonization since the DNase I coating lost activity between 8 and 24 h. However, approaches to stabilize or replenish the enzyme activity could potentially lead to broader applications. 6. Conclusions There is a great deal of evidence that eDNA is present in oral biofilms, and that it plays a number of important roles in colonisation and biofilm formation. At the same time, oral biofilms are mixed-species microbial communities and there are many unanswered questions about the nature of eDNA in such complex ecosystems. It is not clear what proportion of eDNA is microbial in origin, and how much is derived from the host. Is the microbial-produced eDNA similar in structure to chromosomal DNA? Do all bacteria in a mixed biofilm rely on eDNA to the same extent, or could some species be selectively removed from the biofilm by DNase treatment? It is important to note that many isolated oral bacteria produce DNase enzymes [50,61]. At present, it is not clear how the activity of these endogenous biofilm DNases is controlled. Studies on DNase production by individual oral bacteria will be critical for determining how these enzymes function in natural communities, and for providing insight into how exogenous enzymes can be delivered effectively for oral biofilm control. Conflict of interest Newcastle University is the assignee of the UK granted patent ‘Bacterial deoxribonuclease compounds and methods for biofilm disruption and prevention’, publication number GB201002396. Acknowledgements We are very grateful to Richard Holliday (Newcastle University) and Leon Bowen (Durham University) for providing the image in Fig. 1. We thank Robert Shields (University of Florida) for providing images and for critically reading the manuscript. References [1] Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010;8:623e33. [2] Jakubovics NS, Shields RC, Rajarajan N, Burgess JG. Life after death: the critical role of extracellular DNA in microbial biofilms. Lett Appl Microbiol 2013;57:467e75. [3] Segata N, Haake SK, Mannon P, Lemon KP, Waldron L, Gevers D, et al. Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples. Genome Biol 2012;13:R42. [4] Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012;486:207e14. [5] Koo H, Falsetta ML, Klein MI. The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm. J Dent Res 2013;92:1065e73.

[6] Frank RM, Houver G. An unltrastructural study of human supragingival dental plaque formation. In: McHugh WD, editor. Dental plaque. Dundee: D. C. Thomson & Co. Ltd.; 1970. p. 85e108. [7] Eastcott AD, Stallard RE. Sequential changes in developing human dental plaque as visualized by scanning electron microscopy. J Periodontol 1973;44:218e24. [8] Wood SR, Kirkham J, Marsh PD, Shore RC, Nattress B, Robinson C. Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy. J Dent Res 2000;79:21e7. [9] Robinson C. Mass transfer of therapeutics through natural human plaque biofilms: a model for therapeutic delivery to pathological bacterial biofilms. Arch Oral Biol 2011;56:829e36. [10] Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res 2011;45:69e86. [11] Koo H, Xiao J, Klein MI, Jeon JG. Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms. J Bacteriol 2010;192:3024e32. [12] Xiao J, Klein MI, Falsetta ML, Lu B, Delahunty CM, Yates 3rd JR, et al. The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm. PLoS Pathog 2012;8:e1002623. [13] Gao XJ, Fan Y, Kent Jr RL, Van Houte J, Margolis HC. Association of caries activity with the composition of dental plaque fluid. J Dent Res 2001;80:1834e9. [14] Paes Leme AF, Bellato CM, Bedi G, Cury AA, Koo H, Cury JA. Effects of sucrose on the extracellular matrix of plaque-like biofilm formed in vivo, studied by proteomic analysis. Caries Res 2008;42:435e43. [15] Yasunaga A, Yoshida A, Morikawa K, Maki K, Nakamura S, Soh I, et al. Monitoring the prevalence of viable and dead cariogenic bacteria in oral specimens and in vitro biofilms by qPCR combined with propidium monoazide. BMC Microbiol 2013;13:157. [16] Perez-Cruz C, Delgado L, Lopez-Iglesias C, Mercade E. Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria. PLoS ONE 2015;10:e0116896. [17] Sahu PK, Iyer PS, Oak AM, Pardesi KR, Chopade BA. Characterization of eDNA from the clinical strain Acinetobacter baumannii AIIMS 7 and its role in biofilm formation. Scientific World J 2012;2012:973436. [18] Do T, Gilbert SC, Clark D, Ali F, Fatturi Parolo CC, Maltz M, et al. Generation of diversity in Streptococcus mutans genes demonstrated by MLST. PLoS One 2010;5:e9073. [19] Do T, Gilbert SC, Klein J, Warren S, Wade WG, Beighton D. Clonal structure of Streptococcus sanguinis strains isolated from endocarditis cases and the oral cavity. Mol Oral Microbiol 2011;26:291e302. [20] Do T, Jolley KA, Maiden MC, Gilbert SC, Clark D, Wade WG, et al. Population structure of Streptococcus oralis. Microbiology 2009;155:2593e602. [21] Alakomi HL, Skytta E, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander IM. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl Environ Microbiol 2000;66:2001e5. [22] Itzek A, Zheng L, Chen Z, Merritt J, Kreth J. Hydrogen peroxidedependent DNA release and transfer of antibiotic resistance genes in Streptococcus gordonii. J Bacteriol 2011;193:6912e22. [23] Berg KH, Ohnstad HS, Havarstein LS. LytF, a novel competenceregulated murein hydrolase in the genus Streptococcus. J Bacteriol 2012;194:627e35. [24] Liu Y, Burne RA. The major autolysin of Streptococcus gordonii is subject to complex regulation and modulates stress tolerance, biofilm formation, and extracellular-DNA release. J Bacteriol 2011;193:2826e37. [25] Xu Y, Kreth J. Role of LytF and AtlS in eDNA release by Streptococcus gordonii. PLoS One 2013;8:e62339. [26] Hossain MS, Biswas I. Mutacins from Streptococcus mutans UA159 are active against multiple streptococcal species. Appl Environ Microbiol 2011;77:2428e34. [27] Perry JA, Jones MB, Peterson SN, Cvitkovitch DG, Levesque CM. Peptide alarmone signalling triggers an auto-active bacteriocin necessary for genetic competence. Mol Microbiol 2009;72:905e17.

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N.S. Jakubovics, J. Grant Burgess / Microbes and Infection xx (2015) 1e7 [28] Dufour D, Levesque CM. Cell death of Streptococcus mutans induced by a quorum-sensing peptide occurs via a conserved streptococcal autolysin. J Bacteriol 2013;195:105e14. [29] Dorward DW, Garon CF. DNA is packaged within membrane-derived vesicles of gram-negative but not gram-positive bacteria. Appl Environ Microbiol 1990;56:1960e2. [30] Liao S, Klein MI, Heim KP, Fan Y, Bitoun JP, Ahn SJ, et al. Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery. J Bacteriol 2014;196:2355e66. [31] Mantri CK, Chen C, Dong X, Goodwin JS, Pratap S, Paromov V, et al. Fimbriae-mediated outer membrane vesicle production and invasion of Porphyromonas gingivalis. Microbiologyopen 2015;4:53e65. [32] Demuth DR, James D, Kowashi Y, Kato S. Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin. Cell Microbiol 2003;5:111e21. [33] Hamilton HL, Dominguez NM, Schwartz KJ, Hackett KT, Dillard JP. Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. Mol Microbiol 2005;55:1704e21. [34] Pride DT, Salzman J, Haynes M, Rohwer F, Davis-Long C, White 3rd RA, et al. Evidence of a robust resident bacteriophage population revealed through analysis of the human salivary virome. ISME J 2012;6:915e26. [35] Tinanoff N, Gross A. Epithelial cells associated with the development of dental plaque. J Dent Res 1976;55:580e3. [36] Vitkov L, Klappacher M, Hannig M, Krautgartner WD. Extracellular neutrophil traps in periodontitis. J Periodontal Res 2009;44:664e72. [37] Catlin BW. Extracellular deoxyribonucleic acid of bacteria and a deoxyribonuclease inhibitor. Science 1956;124:441e2. [38] Catlin BW, Cunningham LS. Studies of extracellular and intracellular bacterial deoxyribonucleic acids. J Gen Microbiol 1958;19:522e39. [39] Ohta H, Kato K, Kokeguchi S, Hara H, Fukui K, Murayama Y. Nucleasesensitive binding of an Actinobacillus actinomycetemcomitans leukotoxin to the bacterial cell surface. Infect Immun 1991;59:4599e605. [40] Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA required for bacterial biofilm formation. Science 2002;295:1487. [41] Okshevsky M, Meyer RL. The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms. Crit Rev Microbiol 2013 [In press]. http://dx.doi.org/10.3109/1040841X.2013. 841639. [42] Bar-Zeev E, Berman-Frank I, Girshevitz O, Berman T. Revised paradigm of aquatic biofilm formation facilitated by microgel transparent exopolymer particles. Proc Natl Acad Sci U S A 2012;109:9119e24. [43] Alhede M, Kragh KN, Qvortrup K, Allesen-Holm M, van Gennip M, Christensen LD, et al. Phenotypes of non-attached Pseudomonas aeruginosa aggregates resemble surface attached biofilm. PLoS One 2011;6:e27943. [44] Katharios-Lanwermeyer S, Xi C, Jakubovics NS, Rickard AH. Mini-review: microbial coaggregation: ubiquity and implications for biofilm development. Biofouling 2014;30:1235e51. [45] Schooling SR, Hubley A, Beveridge TJ. Interactions of DNA with biofilm-derived membrane vesicles. J Bacteriol 2009;191:4097e102. [46] Li W, Liu H, Xu Q. Extracellular dextran and DNA affect the formation of Enterococcus faecalis biofilms and their susceptibility to 2% chlorhexidine. J Endod 2012;38:894e8. [47] Das T, Sharma PK, Busscher HJ, van der Mei HC, Krom BP. Role of extracellular DNA in initial bacterial adhesion and surface aggregation. Appl Environ Microbiol 2010;76:3405e8. [48] Das T, Sharma PK, Krom BP, van der Mei HC, Busscher HJ. Role of eDNA on the adhesion forces between Streptococcus mutans and substratum surfaces: influence of ionic strength and substratum hydrophobicity. Langmuir 2011;27:10113. [49] Ali Mohammed MM, Nerland AH, Al-Haroni M, Bakken V. Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K. J Oral Microbiol 2013;5. [50] Shields RC, Mokhtar N, Ford M, Hall MJ, Burgess JG, ElBadawey MR, et al. Efficacy of a marine bacterial nuclease against biofilm forming

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58] [59]

[60]

[61]

[62]

[63] [64]

[65]

[66]

[67]

[68] [69]

[70]

7

microorganisms isolated from chronic rhinosinusitis. PLoS One 2013;8:e55339. Ackart DF, Hascall-Dove L, Caceres SM, Kirk NM, Podell BK, Melander C, et al. Expression of antimicrobial drug tolerance by attached communities of Mycobacterium tuberculosis. Pathog Dis 2014;70:359e69. Cavaliere R, Ball JL, Turnbull L, Whitchurch CB. The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin. Microbiologyopen 2014;3:557e67. Doroshenko N, Tseng BS, Howlin RP, Deacon J, Wharton JA, Thurner PJ, et al. Extracellular DNA impedes the transport of vancomycin in Staphylococcus epidermidis biofilms preexposed to subinhibitory concentrations of vancomycin. Antimicrob Agents Chemother 2014;58:7273e82. Tseng BS, Zhang W, Harrison JJ, Quach TP, Song JL, Penterman J, et al. The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin. Environ Microbiol 2013;15:2865e78. Messiaen AS, Nelis H, Coenye T. Investigating the role of matrix components in protection of Burkholderia cepacia complex biofilms against tobramycin. J Cyst Fibros 2014;13:56e62. Jones EA, McGillivary G, Bakaletz LO. Extracellular DNA within a nontypeable Haemophilus influenzae-induced biofilm binds human beta defensin-3 and reduces its antimicrobial activity. J Innate Immun 2013;5:24e38. Liu J, Chen J, Du X, Hu L, Chen L. The expression of hBDs in the gingival tissue and keratinocytes from healthy subjects and periodontitis patients. Arch Oral Biol 2014;59:193e8. Dell'Anno A, Danovaro R. Extracellular DNA plays a key role in deepsea ecosystem functioning. Science 2005;309:2179. Mulcahy H, Charron-Mazenod L, Lewenza S. Pseudomonas aeruginosa produces an extracellular deoxyribonuclease that is required for utilization of DNA as a nutrient source. Environ Microbiol 2010;12:1621e9. Burne RA, Chen YY, Wexler DL, Kuramitsu H, Bowen WH. Cariogenicity of Streptococcus mutans strains with defects in fructan metabolism assessed in a program-fed specific-pathogen-free rat model. J Dent Res 1996;75:1572e7. Palmer LJ, Chapple IL, Wright HJ, Roberts A, Cooper PR. Extracellular deoxyribonuclease production by periodontal bacteria. J Periodontal Res 2012;47:439e45. Jakubovics NS, Smith AW, Jenkinson HF. Expression of the virulencerelated Sca (Mn2þ) permease in Streptococcus gordonii is regulated by a diphtheria toxin metallorepressor-like protein ScaR. Mol Microbiol 2000;38:140e53. Griffith F. The significance of pneumococcal types. J Hyg (Lond.) 1928;27:113e59. Tribble GD, Rigney TW, Dao DH, Wong CT, Kerr JE, Taylor BE, et al. Natural competence is a major mechanism for horizontal DNA transfer in the oral pathogen Porphyromonas gingivalis. MBio 2012;3. Roberts AP, Kreth J. The impact of horizontal gene transfer on the adaptive ability of the human oral microbiome. Front Cell Infect Microbiol 2014;4:124. Chi F, Nolte O, Bergmann C, Ip M, Hakenbeck R. Crossing the barrier: evolution and spread of a major class of mosaic pbp2x in Streptococcus pneumoniae, S. mitis and S. oralis. Int J Med Microbiol 2007;297:503e12. Hannan S, Ready D, Jasni AS, Rogers M, Pratten J, Roberts AP. Transfer of antibiotic resistance by transformation with eDNA within oral biofilms. FEMS Immunol Med Microbiol 2010;59:345e9. Okshevsky M, Regina VR, Meyer RL. Extracellular DNA as a target for biofilm control. Curr Opin Biotechnol 2015;33:73e80. Goss CH, MacNeill SJ, Quinton HB, Marshall BC, Elbert A, Knapp EA, et al. Children and young adults with CF in the USA have better lung function compared with the UK. Thorax 2015;70:229e36. Swartjes JJTM, Das T, Sharifi S, Subbiahdoss G, Sharma PK, Krom BP, et al. A functional DNase I coating to prevent adhesion of bacteria and the formation of biofilm. Adv Funct Mater 2013;23:2843e9.

Please cite this article in press as: Jakubovics NS, Grant Burgess J, Extracellular DNA in oral microbial biofilms, Microbes and Infection (2015), http:// dx.doi.org/10.1016/j.micinf.2015.03.015