Staphylococcus aureus is a gram-positive bacterium that is commonly found in the human population, colonizing approximately 30% of humans in the nasal passages (Lister and Horswill, 2014), as well as the skin and gastrointestinal tract (Raineri et al., 2022). S. aureus is an important human pathogen since it causes skin infections, bacteremia, osteomyelitis, pneumonia, and endocarditis and leads to nearly 20,000 deaths per year in the US (Kavanagh, 2019). S. aureus also exhibits a high level of antibiotic resistance, with many strains demonstrating resistance even to last-resort antibiotics (Guo et al., 2020).

S. aureus forms biofilms, which are surface-associated assemblages of bacteria embedded in a self-produced extracellular matrix. These aggregations are an ideal way for bacteria to evade the immune system and to survive in nutrient-poor locations (De La Fuente-Núñez et al., 2013; Yin et al., 2019). Biofilms are of particular concern in medical settings due to the extreme difficulty in treating them (Gebreyohannes et al., 2019). This is due in large part to the limited or delayed diffusion of some antibacterial agents through the biofilm matrix (Singh et al., 2010), as well as the presence of persister cells (Craft et al., 2019) and antibiotic-resistant bacteria (Balcazar et al., 2015). Persister cells are non-dividing cells that exhibit transient antibiotic resistance during antibiotic challenge (Chang et al., 2020). Each of these defense mechanisms make biofilms hard to target with traditional antibiotic regimens.

Biofilm formation in S. aureus follows several well-studied steps, including attachment, maturation, and dispersal (Schilcher and Horswill, 2020). First, free-floating S. aureus bacteria attach to a surface by hydrophobic interactions, hydrogen bonds, ionic bonds, and/or protein-mediated attachment (Jiang et al., 2021). The bacteria then multiply into a confluent mat of cells (Moormeier and Bayles, 2017). This is followed by a period of exodus where a subpopulation of bacteria is released, allowing for the development of metabolically diverse microcolonies (Grande et al., 2014; Moormeier et al., 2014; Moormeier and Bayles, 2017). The microcolonies grow rapidly, and finally, quorum sensing initiates the dispersal of cells, which begin new biofilms in additional locations (Moormeier and Bayles, 2017).

Biofilms consist of cells surrounded by an extracellular matrix. The composition of this matrix in S. aureus is highly strain, time, and condition-dependent (Sugimoto et al., 2018; Lade et al., 2019; Ball et al., 2022). The main components of this self-generated matrix are proteins, polysaccharides, lipids, and extracellular DNA (eDNA) (Karygianni et al., 2020). These components are important attachment and structural components of the biofilm (Moormeier and Bayles, 2017). Extracellular RNA may also be present in S. aureus biofilms, where it is hypothesized to associate with eDNA and provide structural support (Chiba et al., 2022). However, the low stability of the RNA molecule and constraints in available extraction protocols have made it difficult to study (Mugunthan et al., 2023). Therefore, this review will focus on eDNA. Although much attention has focused on the protein and polysaccharide biofilm matrix constituents (Hobley et al., 2015; Moormeier and Bayles, 2017), the vital role of eDNA is less-well appreciated. The idea that eDNA was a critical component of the biofilm matrix was first suggested by Whitchurch et al., 2002. They showed that DNase I prevented Pseudomonas aeruginosa from forming biofilms, suggesting the importance of eDNA as a structural component (Whitchurch et al., 2002). Since that time, further research has shown that the presence of eDNA in biofilms is nearly universal across bacterial species (Campoccia et al., 2021). In S. aureus biofilms, eDNA plays important roles in attachment, structure, and stability. The purpose of this review is to describe recent breakthroughs in our understanding of the characteristics of S. aureus eDNA as well as its mechanism of release, roles within the S. aureus biofilm, and potential methods of targeting eDNA to disrupt biofilm formation.

In S. aureus biofilms, eDNA is released by lysing a subfraction of the bacterial population in a process that depends on murein hydrolases (Rice et al., 2007). Murein (peptidoglycan) hydrolases cleave covalent bonds in peptidoglycan for a variety of purposes (Vollmer et al., 2008). Autolysis-independent mechanisms of eDNA release have been shown in some species of bacteria such as E. coli and P. aeruginosa, but not yet in S. aureus (Fischer et al., 2014). The process of autolysis relies on several important effector and regulatory proteins. After the murein hydrolases degrade the peptidoglycan barrier, the cell lyses and DNA is released into the surrounding area. The now-extracellular DNA is then able to become part of the biofilm matrix.

The eDNA in S. aureus biofilms is composed of genomic DNA released from lysed cells (Mann et al., 2009; Svarcova et al., 2021). Therefore, it is presumed to contain all chromosomally encoded genes (Fischer et al., 2014), and likely also includes extrachromosomal plasmid DNA. Indeed, amplified fragment length polymorphism comparison of eDNA to genomic DNA reveals high similarity between the two in S. aureus (Svarcova et al., 2021). This is in contrast to some other bacterial species, which may incorporate eDNA into their biofilm matrices in a sequence-specific manner (Jakubovics et al., 2013). Sequence-specific eDNA incorporation may also occur in mixed-species biofilms containing S. aureus (Steinberger and Holden, 2005), due to either differences in eDNA release or post-release DNA modifications.

Matrix eDNA varies in molecular weight and may take on different roles as it is enzymatically or otherwise modified following release. Addition of restriction enzymes to produce fragments <10 kb resulted in near-complete biofilm detachment while fragments of 11–24 kb caused partial detachment (Izano et al., 2008). This suggests that only fragments >11 kb can function as intercellular adhesins (Izano et al., 2008). In support of these results, additional studies have described the presence of high molecular weight eDNA in S. aureus biofilms (Kiedrowski et al., 2011; Kavanaugh et al., 2019).

eDNA plays a vital role in biofilm attachment and early development. Multiple studies have shown that the application of DNase I during early biofilm development results in a reduction in biomass (Mann et al., 2009; Das et al., 2010). Other studies suggest that while eDNA is important for early biofilm formation, its role may not be easily elucidated by DNase I treatment. One group found that in very early biofilm formation (0–8 hours) eDNA is present in the biofilm but protected from nuclease activity until about 4–6 hours into development (Moormeier et al., 2014). Similarly, another report found that DNase I treatment during early biofilm formation did not affect the total number of cells in the biofilm (Grande et al., 2014). Although no difference in biofilm structure was found after two hours of incubation between DNase I and control treatments, differences in biofilm architecture and morphology were noted at 24 and 72 hours, indicating that eDNA is an important component of biofilm structure (Grande et al., 2014). The authors note that some eDNA remained in DNase I-treated biofilms, indicating that some of the eDNA may have been protected from DNase I digestion (Grande et al., 2014). Although results on whether eDNA is vital for early biofilm formation vary, they indicate that it is likely that eDNA plays an important role in biofilm stability under certain conditions.

At one time eDNA was thought to be mainly important for bacterial attachment and early biofilm formation (Houston et al., 2011). However, many studies have found that DNase I can significantly affect and even dissolve older biofilms in vitro (Izano et al., 2008; Tetz et al., 2009; Kaplan et al., 2012b; Moormeier et al., 2014; Ball et al., 2022). DNase I treatment was also found to inhibit early (24 hour) biofilm formation in an in vivo rabbit model of empyema, a condition marked by pockets of pus collecting in body cavities, particularly the pleural space (Deng et al., 2022).

Less research has been done into the role of eDNA in mature biofilms. One report that investigated P. aeruginosa biofilms found that DNase I was capable of reducing biofilm size at 12, 36, and 60 hours, but not at 84 hours (Whitchurch et al., 2002). This could either indicate that eDNA is less important in a mature biofilm, or that it is protected from DNase activity in an older biofilm.

One of the key theories of the role of eDNA in the S. aureus biofilm is the electrostatic net model. In this model, negatively charged eDNA facilitates cell-cell adhesion by interacting with positively charged matrix proteins, which can interact with negatively charged cell surface molecules such as teichoic acids ( Figure 2 ) (Dengler et al., 2015). An additional study found that in the acidic conditions of biofilms, the proteins of the extracellular matrix are strongly positively charged, supporting the model (Graf et al., 2019).

Some other groups have shown that proteins from the cytoplasm of lysed cells may be recycled to act as biofilm matrix proteins (Foulston et al., 2014), binding to eDNA and protecting it from nuclease activity. Such moonlighting proteins associate with cells in biofilms due to the drop in pH found in biofilm interiors (Dengler et al., 2015). These proteins found in the extracellular matrix have a high isoelectric point and therefore in the acidic milieu of the interior of a biofilm will be strongly positively charged (Graf et al., 2019). This result corroborates those found by Dengler et al., suggesting that the negatively charged eDNA can act as an electrostatic net, connecting positively charged proteins and anionic cell surfaces (Dengler et al., 2015). Furthering this paradigm, work by Kavanaugh et al. confirmed the presence of these positively charged proteins in addition to showing that membrane-attached lipoproteins can interact with matrix eDNA. These lipoproteins can function as anchors between matrix eDNA and cell surfaces (Kavanaugh et al., 2019).

Interestingly, while it is well known that the interior of biofilms is acidic, one group found that in P. aeruginosa, eDNA may be partially responsible for the acidification of the biofilm (Wilton et al., 2016), and a similar mechanism may be in play for S. aureus. The acidic environment is then ideal for eDNA to act as a stabilizing electrostatic net.

eDNA is known to associate with cells in a biofilm in very specific ways. A study by Dengler et al. degraded proteins in the matrix of S. aureus biofilms and observed that eDNA was freed from the cell surface. This indicates that the specific methods by which eDNA interacts with biofilm cells depend on the proteins within the matrix (Dengler et al., 2015). Such a claim would suggest that eDNA interactions are complex and largely not understood and can change depending on the protein composition of the biofilm (Dengler et al., 2015). Nevertheless, it is suggested that eDNA serves to hold cells in place by creating a web of interactions with the matrix proteins (Dengler et al., 2015).

There have been several studies into the interactions of eDNA with specific biofilm matrix components. Beta toxin is a neutral sphingomyelinase that belongs to the DNase I superfamily (Huseby et al., 2007). A study by Huseby et al (Huseby et al., 2010). showed that rather than simply degrading eDNA, co-incubation of beta toxin and DNA resulted in the formation of a precipitate of beta toxin oligomers. Thus, beta toxin cross-links in the presence of DNA, forming an insoluble matrix that stimulated biofilm formation in vivo. This points to a molecular mechanism for a structural framework for some staphylococci biofilms, but many strains of S. aureus do not express beta toxin, indicating that many other mechanisms must be at work (Huseby et al., 2010). This research is also similar to studies in other bacterial species showing that proteins with DNA-binding activity may be important biofilm matrix components (Kavanaugh et al., 2019).

However, not all proteins with DNA-binding activity appear to contribute to biofilm structure or formation in all strains (Mackey-Lawrence et al., 2009). IsaB is a protein that was discovered to have DNA-binding capabilities but its deletion did not result in changes to biofilm biomass (Mackey-Lawrence et al., 2009). Further studies determined that in a different strain that had previously reported higher levels of secreted IsaB, a 2-fold reduction in biofilm formation was found in an isaB mutant (Kavanaugh et al., 2019). Further, they determined that deletion of both IsaB and another DNA-binding protein, Eap resulted in a reduction of eDNA, suggesting that in some cases eDNA-binding proteins may act redundantly to bind eDNA in the biofilm matrix (Kavanaugh et al., 2019).

S. aureus also produces phenol-soluble modulins (PSMs) that are involved in biofilm structure and dissemination (Schilcher and Horswill, 2020). PSMs can disperse biofilms but they can exist in a polymerized, amyloid-like form in stable biofilms (Zheng et al., 2018). PSMs were found to attach to eDNA and were found in some cases to provide resistance against DNase digestion (Zheng et al., 2018), though earlier research had postulated that the presence of eDNA promoted amyloid formation by PSMs (Schwartz et al., 2016).

In addition to binding to proteins to stabilize the biofilm matrix, eDNA interacts with the poly-N-acetylglucosamine (PNAG) polysaccharide to stabilize the biofilm (Mlynek et al., 2020). It was once thought that S. aureus produced one of two possible biofilm morphologies based on either PNAG or eDNA/protein. However, these morphologies are not mutually exclusive, as discussed above, and isolates that produce large amounts of polysaccharide also produce eDNA (Sugimoto et al., 2018). In biofilms, PNAG carries a net positive charge and thus may directly interact with eDNA as part of the electrostatic net model (Mlynek et al., 2020). Due to an expanding understanding of S. aureus biofilm matrix composition, the relationship and interaction between PNAG and eDNA is an area of active research. Further research into eDNA interactions with polysaccharides needs to be done to conclude whether an interaction between them is a widespread phenomenon important to biofilm structure.

The mechanical strength and structure of a biofilm is affected by the amount of eDNA present. Biofilms are both viscous (resistant to flow) and elastic (returning to their original shape and size when force is removed) (Peterson et al., 2015). These properties help the bacteria within the matrix to survive various stresses such as fluid flow or mechanical detachment. In one study, biofilms of S. aureus and several other species were mechanically deformed, and the stress relaxation was quantified. Principle component analysis revealed that eDNA contributes to viscoelastic relaxation, the ability of the biofilm to rebound after stress is placed upon it (Peterson et al., 2013). Even relatively small changes in biofilm viscoelasticity may impact a biofilm’s resistance to phagocytosis as well as the time required for effective phagocytosis (Wells et al., 2023). From a mechanical standpoint, eDNA is considered to be an effective construction material, participating not only in biofilm structure but also in biofilm remodeling. This is due to environmental mechanical forces such as shear. Biofilm mechanics in response to shear and compressive forces were found to depend on the concentration of eDNA and the eDNA-to-cell ratio (Lysik et al., 2022).

In addition to its role as a mechanical stabilizer, in some bacteria eDNA also acts as a mechanism for horizontal gene transfer (Panlilio and Rice, 2021). Although the S. aureus genome has competence genes, S. aureus displays natural competence only under certain conditions. One study has found that in S. aureus biofilms, horizontal gene transfer of the SCCmec gene could occur between heat-killed cells and living cells. These results could suggest the existence of horizontal gene transfer involving eDNA in S. aureus biofilms in other environments (Maree et al., 2022). Additionally, microaerobic conditions may induce natural competence in S. aureus (Feng et al., 2023). Oxygen-poor microenvironments can be found in biofilms, which may provide the proper environment for horizontal gene transfer in the form of transformation to take place.

Not all of the roles of eDNA are helpful to the biofilm—it can also act as a pathogen-associated molecular pattern (PAMP). Bacterial eDNA can be recognized by the innate immune system by toll-like receptors, particularly TLR9, which is triggered upon phagocytosis of eDNA (Knuefermann et al., 2007; Campoccia et al., 2023). One group found that the treatment of P. aeruginosa biofilms with DNase I reduced the ability of the biofilm to upregulate neutrophil activation markers and reduced the release of neutrophil proinflammatory cytokines (Fuxman Bass et al., 2010). However, another study of in vivo S. aureus showed that these biofilms were capable of evading detection by both TLR9 and TLR2 (Thurlow et al., 2011).

A better understanding of the role of eDNA in biofilms can lead to the development of treatments for biofilm-related infections that target eDNA ( Table 1 ). DNase treatment has previously been found to prevent biofilm formation (Mann et al., 2009), but results have been mixed as to when during biofilm development it may be effective, or whether it is effective at all (Grande et al., 2014; Moormeier et al., 2014). This is possibly due to either proteins that protect eDNA from DNase I treatment (Grande et al., 2014), or the potential accumulation of Z-form DNA (Ramesh and Brahmachari, 1989; Buzzo et al., 2021). As discussed above, both conditions result in eDNA that is not a favorable substrate for DNase I treatment.

However, DNase may be a possible treatment for some applications. DNase I has shown promise in empyema models both in vitro and in vivo (Deng et al., 2022). When DNase was used in conjunction with tissue plasminogen activator, the combination therapy resulted in undetectable S. aureus levels in about 90% of patients without the need for surgery (Piccolo et al., 2015; Mehta et al., 2016), as well as improving pus viscosity (Kacprzak et al., 2013) and pleural drainage (Rahman et al., 2011). DNase may be a potential therapeutic for other disease models. Potential avenues include the administration of DNase in combination with antibiotic therapy (Li et al., 2023) as well as DNase pre-treatment of medical implants (Aktan et al., 2022).

Not all therapeutic anti-biofilm efforts revolve around the application of DNase. The DNABII family of proteins is ubiquitously expressed in all eubacterial species (Goodman and Bakaletz, 2022). They are small, basic proteins that bind to bent DNA and have been found to contribute greatly to the end structure of eDNA in biofilms of many pathogens including non-typable H. influenzae (Goodman et al., 2011), E. coli (Devaraj et al., 2015), and Streptococcus gordonii (Rocco et al., 2017), by acting as a binding agent at the vertices of the eDNA (Goodman and Bakaletz, 2022). DNABII proteins are also present in the biofilms of S. aureus (Goodman et al., 2011).

Both of the two members of the DNABII protein family, integration host factor (IHF) and histone-like protein (HU) do not have any known homologs in mammalian species (Rogers et al., 2022). S. aureus does not code for the integration host factor, but does contain the gene for the histone-like protein (Rogers et al., 2022). HU binds to and bends double-stranded DNA in a non-sequence-specific manner, but it has a high affinity for highly structured dsDNA, such as Holliday junctions (Goodman et al., 2011) and DNA bent at various angles (Swinger and Rice, 2004). Since biofilm eDNA contains structural Holliday junction orthologs (Devaraj et al., 2019), it is unsurprising that proteins such as IHF and HU are important to biofilm stability.

It has been proposed that disruption or depletion of DNABII proteins is a potential therapeutic treatment against biofilms (Novotny et al., 2016; Rocco et al., 2017; Goodman and Bakaletz, 2022), and preclinical ex vivo data suggest that it could be effective against multiple pathogens, including S. aureus (Gustave et al., 2013; Idicula et al., 2016). Treatment of in vitro S. aureus biofilms with a monoclonal antibody treatment against HU resulted in a dose- and time-dependent disruption of the biofilm within about 15 minutes, continuing to increase until a 60-minute time point (Kurbatfinski et al., 2022).

This treatment also resulted in the released cells from the biofilm being more susceptible to antibiotic treatment (Kurbatfinski et al., 2022). This increase in antibiotic susceptibility could be due to either increased exposure of cells within the biofilm as the structure was degraded, and/or due to increased release of bacteria from the biofilm into the planktonic state (Goodman et al., 2011). In a mouse implant infection model using S. aureus, the addition of anti-DNABII antibodies and daptomycin significantly reduced both biofilm and planktonic bacteria compared to the administration of daptomycin alone (Estelles et al., 2016).

One potential mechanism for the ability of anti-DNABII-antibody-induced biofilm collapse suggests that the antibodies bind to free DNABII proteins in the biofilm environment. This causes an equilibrium shift between free and eDNA-bound DNABII proteins, resulting in a release of DNABII proteins from the biofilm. The loss of the DNABII proteins results in biofilm structural collapse (Brockson et al., 2014) ( Figure 3 ). This collapse releases bacteria into the surrounding environment, rendering them more susceptible both to antibiotic treatment and to clearance by the host immune system (Rogers et al., 2022).

eDNA has a pivotal role in the development and architecture of S. aureus biofilms. The eDNA found in S. aureus biofilms is composed of DNA released from lysed cells during biofilm formation. This cell lysis is facilitated by the murein hydrolase, Atl, which is regulated by the holin/antiholin CidA/LrgA system. The process of cell lysis is also potentially influenced by factors such as cyclic-di-AMP levels and other hydrolases. The release of eDNA is contingent upon various factors including culture conditions, individual strain characteristics, and the presence of subinhibitory antibiotics.

Functioning as a crucial structural component within biofilms, eDNA significantly impacts biofilm adhesion, as evidenced by the substantial effects of DNase I on biofilm size and attachment. Acting as an electrostatic net, eDNA binds proteins together, facilitating cell-cell connections. Due to its strong negative charge, it can interact with positively charged proteins in the biofilm matrix, which in turn interact with negatively charged cell surface molecules. This results in a strong adhesion between various biofilm components, which protect the biofilm from removal agents.

The ubiquity of eDNA in S. aureus isolates suggests its potential as a general target for biofilm eradication. DNase, commonly used to study the effects of eDNA in biofilms, has potential as a therapeutic agent, especially in combination with other therapies. However, its efficacy may be limited by mechanisms that protect eDNA from DNase activity. An alternative approach involves targeting the DNABII family of proteins that bind to and stabilize bent DNA. This method demonstrates versatility against a wide variety of biofilm-forming pathogens and enhances the effectiveness of concurrent antibiotic treatments.

Despite these improved insights into the role of eDNA in S. aureus biofilms, there are many areas that warrant further investigation. One critical aspect is an improved understanding of the mechanisms behind the observed variations in the outcomes of DNase I treatment of S. aureus biofilms. Since DNase I is one of the most common methods of studying eDNA production in S. aureus biofilms, the limitations of this approach influence our current understanding of eDNA.

Another area of study that requires more in-depth study is the impact of subinhibitory antibiotics on eDNA production, as well as their influence on biofilm formation and stability. Understanding these interactions is important in the development of effective strategies for biofilm-associated infection management. Additionally, more exploration of the relationship between glucose metabolism and eDNA production is warranted. Glucose is widely used as an additive in culture media to increase biofilm formation, and also has relevance to research on diabetes-associated infections. Furthermore, the investigation into the proteins that bind eDNA opens avenues for potential therapeutic interventions. Continued research in this domain may reveal novel approaches for treating biofilm-associated infections. This area of research includes exploring the viability of targeting the DNABII family of proteins in actual patients. These unresolved aspects of eDNA in biofilm formation emphasize the ongoing challenges and opportunities behind understanding S. aureus biofilms.

eDNA plays an indispensable role as a structural component of S. aureus biofilms. This makes it a promising target for treatment strategies against a pathogen associated with significant morbidity and mortality. The exploration of innovative approaches to manipulate eDNA holds potential for advancing biofilm eradication efforts and improving therapeutic outcomes.

LB: Writing – review & editing, Writing – original draft, Supervision, Conceptualization. JF: Writing – original draft. BJ: Writing – original draft. BB: Writing – review & editing, Project administration, Supervision.