BIOFILM FORMATION BY STAPHYLOCOCCI AND STREPTOCOCCI: STRUCTURAL, FUNCTIONAL AND REGULATORY ASPECTS AND IMPLICATIONS FOR PATHOGENESIS

EDITED BY: Joan A. Geoghegan and Pietro Speziale PUBLISHED IN: Frontiers in Cellular and Infection Microbiology

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ISSN 1664-8714 ISBN 978-2-88919-563-3 DOI 10.3389/978-2-88919-563-3

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## **BIOFILM FORMATION BY STAPHYLOCOCCI AND STREPTOCOCCI: STRUCTURAL, FUNCTIONAL AND REGULATORY ASPECTS AND IMPLICATIONS FOR PATHOGENESIS**

Topic Editors:

**Joan A. Geoghegan,** Trinity College, Dublin, Ireland **Pietro Speziale,** University of Pavia, Pavia, Italy

Scanning electron micrograph image of Streptococcus mutans biofilm on an apatitic surface

Members of the genus Staphylococcus and Streptococcus are the causative agnets of many human and animal diseases. Over the past decade the complete sequencing of many staphylococcal and streptococcal genomes has promoted a significant advance in our knowledge of these important pathogens. The pathogenicity of these bacteria is due to the expression of a large variety of virulence factors. Such determinants, which are cell wall-associated and secreted proteins, include adhesins that confer to the pathogen the ability to attach to extracellular matrix/plasma and host cell

surfaces, proteins that contribute to host cell invasion and intracellular survival and soluble factors that decrease phagocytosis and modulate the immune response. Furthermore, these Gram-positive cocci in many natural environments (heart valve, lung, oral cavity, throat) and infections on implanted devices live in matrix-encased groups known as biofilms. Biofilms are specialized bacterial communities with high order organization analogous to that of a tissue in multicellular organism that adhere to abiotic or biological substrata and produce an exopolymeric matrix composed of polysaccarides, proteins, DNA or combination thereof. Bacteria within a biofilm persist in adverse conditions, show resistance to killing by antibiotics and to host immune defences and are difficult to eradicate and treat clinically. Therefore, understanding the mechanisms of biofilm development will allow us to effectively combat staphylococcal/streptococcal biofilm-based infections.

This Research Topic will focus on the molecular components involved in biofilm formation by staphylococci and streptococci, the role they play in the development, maturation and dispersal of biofilm and on the regulatory aspects of such complex processes. The implication for the pathogenesis of infective diseases and potential therapeutic strategies against biofilmbased infections will be also discussed. The articles will highlight both the recent advances and future challenges inherent in this rapidly evolving area.

**Citation:** Geoghegan, J. A., Speziale, P., eds. (2015). Biofilm Formation by Staphylococci and Streptococci: Structural, Functional and Regulatory Aspects and Implications for Pathogenesis. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-563-3

# Table of Contents

*17 Structural basis of* **Staphylococcus epidermidis** *biofilm formation: mechanisms and molecular interactions*

Henning Büttner, Dietrich Mack and Holger Rohde

Ryan P. Gilley and Carlos J. Orihuela

## Biofilm formation by staphylococci and streptococci: structural, functional, and regulatory aspects and implications for pathogenesis

Pietro Speziale1 \* and Joan A. Geoghegan2 \*

1 Unit of Biochemistry, Department of Molecular Medicine, Università Degli Studi di Pavia, Pavia, Italy, 2 Department of Microbiology, School of Genetics and Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin, Ireland

Keywords: Staphylococcus, Streptococcus, biofilm, structure, regulation, pathogenesis

Members of the genus Staphylococcus and Streptococcus are commensals and opportunistic pathogens that cause a variety of infections in human and animals. Biofilm formation is an important survival strategy adopted by these Gram-positive cocci. Biofilms are surface-associated, specialized multicellular bacterial communities embedded in a self-produced matrix of polysaccharides, proteins, DNA or combinations thereof. Biofilm growth allows bacteria to persist in vivo by resisting host immune defenses and antibiotics meaning that these infections are difficult to eradicate and treat clinically. Biofilms form in many natural niches occupied by staphylococci and streptococci (such as the nasopharynx, heart valves, lungs, and oral cavity) and are important during the infection of implanted medical devices.

Biofilms develop in multiple stages to form highly ordered multicellular communities (O'Toole et al., 2000; Otto, 2013). Initially the bacteria must adhere to a surface or host tissue (primary attachment phase) before they proliferate to form multicellular aggregates (accumulation phase). During the maturation stage, channels and mushroom-shaped structures are created to allow nutrients to penetrate the deeper layers of the biofilm (O'Toole et al., 2000). Finally, during the dispersal stage, bacteria detach from the biofilm and disseminate to new sites (Otto, 2013).

In this series of articles, the authors provide an overview of the molecular components involved in biofilm formation by staphylococci and streptococci. Particular emphasis is placed on the mechanisms of development and dispersal of biofilm and the regulation of biofilm formation. Novel therapeutic strategies for the prevention and eradication of medical biofilms are also discussed.

Arciola et al. (2015) describe our evolving understanding of the enzymes involved in the biosynthesis of polysaccharide intracellular adhesion (PIA), a polymer that mediates staphylococcal biofilm accumulation. They discuss the genetic control of factors involved in biofilm formation and disruption. McCarthy et al. (2015) explore the different mechanisms used by methicillinsensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) to form biofilm and the impact of methicillin resistance on PIA production and virulence in vivo. Speziale et al. (2014) describe proteins of staphylococci that contribute to primary attachment and biofilm accumulation. The current understanding of the mechanistic basis of cell wall anchored surface protein-dependent biofilm accumulation is described. Büttner et al. (2015) capture recent advances in our understanding of the contribution of Staphylococcus epidermidis multifunctional surface proteins to the primary attachment and accumulation phases of biofilm formation. They also illustrate the integration of biofilm-promoting factors into regulatory networks and emphasize how these factors contribute to the adaptation of S. epidermidis to changing environmental conditions. The review by Le et al. (2014) presents evidence of the critical activity of phenol soluble modulins (PSMs) as mediators of biofilm structuring and dispersal, stressing their role

#### Edited and reviewed by:

Yousef Abu Kwaik, University of Louisville School of Medicine, USA

#### \*Correspondence:

Pietro Speziale and Joan A. Geoghegan<EMAIL_ADDRESS><EMAIL_ADDRESS>
Received: 16 March 2015 Accepted: 18 March 2015 Published: 08 April 2015

#### Citation:

Speziale P and Geoghegan JA (2015) Biofilm formation by staphylococci and streptococci: structural, functional, and regulatory aspects and implications for pathogenesis. Front. Cell. Infect. Microbiol. 5:31. doi: 10.3389/fcimb.2015.00031 in the formation of biofilm channels and dissemination of clusters of biofilm to distal organs in vivo. In keeping with the theme of dispersal, Lister and Horswill (2014) report the current understanding of enzymatic dispersal mechanisms (proteases, nucleases, and dispersin B) and the newly described broad-spectrum effects of D-amino acids and synthetic cationic peptides.

Gilley and Orihuela (2014) describe the reasons why biofilmforming Streptococcus pneumoniae colonizing the nasopharynx of humans are not invasive. The bacteria in the biofilm have a decreased rate of metabolism, produce less capsular polysaccharide and less pneumolysin than their planktonic counterparts. The authors suggest that this may explain long-term, asymptomatic nasopharyngeal colonization by S. pneumoniae in humans. Chao et al. (2015) describe the increased propensity of S. pneumoniae in a biofilm to exchange genetic material. Mechanisms involved in dispersal during colonization and disease are also considered.

Fiedler et al. (2015) review the clinical relevance of biofilm formation by the human pathogen Streptococcus pyogenes, in particular during oto-nasopharyngeal and skin infection. Moreover, they report the involvement of capsule carbohydrates, pili,

## References

surface proteins, and secreted enzymes and the regulatory aspects of the biofilm lifestyle of S. pyogenes. Rosini and Margarit (2015) discuss the role of pili and other surface components and the influence of environmental conditions on biofilm production by Streptococcus agalactiae. Klein et al. (2015) present evidence of how Streptococcus mutans assembles a cariogenic biofilm in the mouth and how extracellular polysaccharide provides mechanical stability and facilitates the creation of highly acidic microenvironments inside the matrix. Extracellular DNA enhances the local synthesis of extracellular polysaccharide, increases the adherence of S. mutans to saliva-coated apatitic surfaces and increases the cohesive properties of biofilm.

In conclusion, these reviews summarize one of the most vital areas of research in the pathogenesis of staphylococcal and streptococcal infections. The limitations of in vitro techniques, and the urgent need to advance our understanding of biofilm formation using in vivo models, are considered. The knowledge generated from this research has the potential to lead to novel approaches to diagnosing and treating biofilm-related infections, and it lays the groundwork for deeper investigation into the basic biology of these diseases.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Speziale and Geoghegan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects

## *Carla Renata Arciola1,2\*, Davide Campoccia1, Stefano Ravaioli 1,2 and Lucio Montanaro1,2*

*1 Research Unit on Implant Infections, Rizzoli Orthopaedic Institute, Bologna, Italy*

*2 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy*

#### *Edited by:*

*Pietro Speziale, University of Pavia, Italy*

#### *Reviewed by:*

*Michael L. Vasil, University of Colorado School of Medicine, USA Michael Otto, National Institute of Allergy and Infectious Diseases, USA*

#### *\*Correspondence:*

*Carla Renata Arciola, Rizzoli Orthopaedic Institute, via di Barbiano 1/10, 40136 Bologna, Italy e-mail<EMAIL_ADDRESS>
*Staphylococcus aureus* and *Staphylococcus epidermidis* are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-*N*-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the *icaADBC* locus. DNA sequences homologous to *ica* locus are present in many coagulase-negative staphylococcal species, among which *S. lugdunensis*, however, produces a biofilm prevalently consisting of proteins. The product of *icaA* is an *N*-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-*N-*acetylglucosamine. The product of *icaD* gives optimal efficiency to IcaA. The product of *icaC* is involved in the externalization of the nascent polysaccharide. The product of *icaB* is an *N*-deacetylase responsible for the partial deacetylation of PIA. The expression of *ica* locus is affected by environmental conditions. In *S. aureus* and *S. epidermidis ica*-independent alternative mechanisms of biofilm production have been described. *S. epidermidis* and *S. aureus* undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within *icaC*. A role is played by the *quorum sensing* system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the *QS* system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of *S. aureus* favors opsonophagocytosis and is a potential candidate for immune-protection.

**Keywords:** *Staphylococcus***, biofilm, ica locus, Polysaccharide intercellular adhesin (PIA), poly-β(1-6)-***N***-acetylglucosamine (PNAG), anti-PIA vaccine**

## **INTRODUCTION**

The recognition of "slime" as a mucilaginous material elaborated by certain microrganisms such as molds and bacteria represents a very early discovery in the history of microbiology. Impressively, the first papers describing the ability of bacterial species to form slime date back to the beginning of the 20th century. The description of specific extracellular polymeric components that structurally contribute to slime composition started with the second half of the century, when Wilkinson (1958) and Catlin and Cunningham (1958) began to report the existence of extracellular polysaccharides and deoxyribonucleic acids.

The medical importance of bacterial biofilm was for the first time enlighten by Bill Costerton, recognized as the "Father of Biofilm," who, in 1978 established an extraordinarily new microbiological paradigm, the "biofilm theory." In an article, published in Scientific American, he asserted that bacteria stick on available surfaces in glycocalyx-enclosed biofilms and that the sessile bacterial population becomes predominant particularly in medical ecosystems (Costerton et al., 1978). Costerton's observations shifted the medical research from the attention to microbial cell-wall structures, which are the interface of planktonic bacteria with the environment, to the biofilm, which is the interface of sessile bacteria with their environment (Costerton, 1989). The introduction of the *biofilm theory* opened two lines of research: the study of biochemistry and genetics of biofilms and their formation and, on the other side, the improvement of the medical diagnosis and treatment of biofilm-centered infections.

The profound influence that the Costerton's insight exerted on the bio-molecular knowledge of bacterial adhesion and on the need of appropriate medical methods for diagnosis and treatment of biofilm-related infections is analyzed in a review written by one of us together with the Director of the Costerton's Institute. The review was thought in the sad circumstance of the passing away of Bill, remembered as a charming *Maestro* for a large number of colleagues and students (Ehrlich and Arciola, 2012).

The early experimental works on slime producing bacteria were just a prelude to the more elaborated concept of biofilm (Mack et al., 1975; Costerton et al., 1978), where the sessile lifecycle phase was progressively associated to the social behavior of communicating and mutually interacting bacterial cells (Stoodley et al., 2002), forming communities encased within a protective extracellular matrix (Costerton et al., 1987) derived from the elaboration of an exocellular slime (the glycocalix) (Gristina and Costerton, 1984).

Over the years, progressive light was cast on the importance of glycocalix elaboration and biofilm formation not only as a fundamental mechanism of adhesion and colonization of biomaterial surfaces (Jacques et al., 1987), but also as a critical virulence mechanism enabling bacteria to escape the host immune-response and resist medical antibiotic chemotherapies (Gilbert et al., 1997; Costerton et al., 1999). The possibility for bacteria with a planktonic phenotype, otherwise susceptible to the host defenses and medical treatments, to switch to a sessile form of life more adapt to survive to the aggressive environment of host tissues, assumed a specific meaning in the pathogenesis of clinical infections, especially those associated to implant materials.

The great difficulty to eradicate microbial infections generated by biofilm-forming bacteria in presence of implant materials has received increasing attention over the last decades. Indeed, once established on a biomaterial surface, biofilm-forming strains are capable to resist and survive common antibiotic regimens formulated against and active on planktonic bacteria.

Soon after the launch of the *biofilm theory*, *Staphylococcus epidermidis* was early discovered to be one of the principal actors of biomaterial-associated infections and, certainly, its ability to colonize implant surfaces and produce resistant biofilms was recognized as a key factor in determining its success.

With the studies on slime/biofilm production by clinical strains, the biofilm was progressively demonstrated to be an important mechanism in bacterial adherence and pathogenesis of infections associated to biomaterials surfaces (Christensen et al., 1982). The production of biofilm was searched both by phenotypic methods, such as the microtiter plate (MtP) test (Christensen et al., 1985), the Congo red agar (CRA) plate test (Freeman et al., 1989) and its optimization (Arciola et al., 2002b), and by molecular detection of the *ica* locus, that had been identified as the genetic basis of biofilm production in *S. epidermidis* (Heilmann et al., 1996). The presence of *ica* locus in *S. epidermidis*, detected together with the positivity of phenotypic evidence of biofilm production, was then proposed as a marker of virulence of *S. epidermidis* strains responsible for implant-associated infections (Arciola et al., 2002a,b). PIA, besides described in *S. epidermidis* strains (Mack et al., 1996), was also described in *S. aureus* and the role of *ica* locus recognized also in this species (Cramton et al., 1999). The *ica* locus was then found widespread present in biofilm producing *S. aureus* strains responsible for catheter and implant infections (Ammendolia et al., 1999; Montanaro et al., 1999; Arciola et al., 2001).

The great attention that staphylococcal biofilms have received in recent years is justified by the large prevalence of *S. aureus* and *S. epidermidis*, but also of other emerging coagulase-negative staphylococci, as etiological agents of implant-infections. For instance, in orthopedics epidemiological studies have shown that staphylococci are the primary cause of implant-infections, causing nearly 80% of all prosthetic infections (Arciola et al., 2005, 2014; Montanaro et al., 2011).

Bacteria in biofilms can resist antibiotics at concentrations up to 1000 times higher than those active on the same bacteria in the planktonic state (Ceri et al., 1999). Antibiotic substances targeting the biofilm phenotype are therefore urgently needed. In search for the most efficacious antibiotic treatments active on biofilms, it has become common to assay antibiotic substances not only on planktonic bacteria to extrapolate minimal inhibitory concentrations (M.I.C.), but also screen them on sessile bacteria to achieve relevant information on the minimum biofilm eradication concentration (M.B.E.C.) (Ceri et al., 1999; Parra-Ruiz et al., 2010).

Apart from the efforts to identify the most active combinations of conventional antibiotics to eradicate bacterial biofilms, increased attention is being paid to identify new antibacterial molecules specifically targeting the biofilm state and therefore defined anti-biofilm substances. The great interest to counteract biomaterial-associated infections caused by biofilm-producing bacterial species has therefore seen the proliferation of studies screening different classes of compounds ranging from herb extracts, natural antimicrobial peptides, molecules of microbial origin to synthetic molecules. The intent has become not just the treatment but also the prevention of biofilm formation. This important goal can be achieved by the better knowledge of the molecular mechanisms of biofilm production and control and by the development of anti-biofilm biomaterial surfaces (Arciola et al., 2012; Campoccia et al., 2013). In this context, the knowledge of biofilm pathophysiology plays a fundamental role, as different strategies can be developed to contrast bacterial colonization and biofilm formation. They all rely on a deep understanding of the mechanisms implicated in biofilm production and of the fine mechanisms that rule the expression of the biofilm phenotype and govern biofilm formation. Two main antibiofilm strategies are presently under consideration. One is based on biofilm disaggregating agents, such as enzymes that attack the PNAG (Dispersin B) or the extracellular DNA (DNase I) or the biofilm proteins (proteinase k) (Arciola, 2009; Kaplan, 2009; Arciola et al., 2012).

The other strategy is based on the modulation of the effector molecules, an approach called *quorum quenching* (QQ), which consists in interfering with the intercellular bacterial communications, with the aim at artificially inducing bacteria to assume a planktonic rather than sessile phenotype. In other words, the latter target consists in fooling the *quorum sensing* (QS) system. Based on signal molecules often referred to as *pheromones* or *autoinducers*, the QS system enables bacteria to sense their own density in the *milieu* and modify their phenotype accordingly. This involves ruling the expression of distinct traits for the specific cellular phase of growth and/or *milieu* colonization.

While some of the principal components of the QS system in *S. aureus* and *S. epidermidis* have been unveiled, much still remains to be elucidated as significant functional differences exist between the two bacterial species and, even, among different strain types, which often happen to compete in the environment just using different allelic forms of the signal molecules. These different alleles of signal molecules often exert the function of QS interference in the competition among different types of strains.

A brief description of some fundamental aspects of staphylococcal biofilm pathophysiology concerning the ultrastructural composition of the extracellular biofilm matrix, and the genetic mechanisms governing biofilm formation will be depicted in the next paragraphs.

## **CHEMISTRY OF PIA AND ALTERNATIVE FORMS OF BIOFILM**

The investigation of the chemical nature of the extracellular biofilm matrix began rather early around the years '50 s. However, in spite of many efforts made to elucidate its composition, the difficult purification of bacterial extracellular matrix and the multiplicity of analytical techniques adopted, often generating artifacts, initially led to divergent results. Thus, depending on their different experimental approach, different authors described different main exopolysaccharide components: the capsular polysaccharide-adhesin (PS/A) (Muller et al., 1993), the polysaccharide intercellular adhesin (PIA) (Mack et al., 1996), poly-β(1-6)-*N*-acetylglucosamine (PNAG) (Maira-Litrán et al., 2002), and *S. aureus* exopolysaccharide (SAE) (Joyce et al., 2003). In 2005, was finally demonstrated that PIA from *S. epidermidis* was structurally identical to the poly-β(1-6)-*N*acetylglucosamine from the PS/A-overproducing strain *S. aureus* MN8m (Sadovskaya et al., 2005), definitively solving a dilemma lasted for more than a decade on the real chemical formula of the main exopolysaccharide component of the biofilms of most *S. aureus* and *S. epidermidis* clinical strains.

Currently, poly-β(1-6)-*N*-acetylglucosamine, alternatively named with the synonymous terms PIA or PNAG, has therefore been identified as the main exopolysaccharide component of staphylococcal biofilm matrix. Interestingly, the same exocellular polysaccharide has recently been identified even in numerous other gram-negative bacterial species members of the *Proteobacteria* family, including *Escherichia coli*, *Yersinia pestis*, *Pseudomonas fluorescens*, *Bordetella* spp., *Xenorhabdus nematophila*, *Aggregatibacter actinomycetemcomitans*, and *Actinobacillus pleuropneumoniae* (Ganeshnarayan et al., 2009), suggesting a convergent evolution in phylogenetically diverse bacteria.

Although PIA certainly represents a main mechanism of biofilm formation in *S. aureus* and *S. epidermidis*, numerous advancements in the study of biofilm have shown the existence, especially for *S. aureus*, of alternative forms of biofilm that are PIA-independent. The observation that a minor proportion of *S. aureus* strains can form biofilm even in the absence of the *ica* locus and that certain strains carrying such locus continue anyway to produce biofilm even after deletion of the locus suggested the existence of *ica*-independent pathways (O'Gara, 2007). The assumption of the possibility of alternative mechanisms to produce biofilms was long debated despites some very early studies had already suggested the complexity of biofilm architecture and the inclusion of extracellular polymeric substances different than PIA, such as e-DNA and teichoic acids, in the biofilm matrix.

A number of proteins localized in the extracellular matrix of biofilms have been identified that can generate PIA-independent biofilms. The biofilm-associated protein (Bap), a 2276-amino acid surface protein, is one of these proteins enabling biofilm production even in the absence of production of the exopolysaccharide component (Cucarella et al., 2004; Tormo et al., 2005). The Bap protein seems to play a role prevalently in human staphylococcal infections caused by coagulase-negative staphylococcal (CoNS) species. The *bap* gene encoding for Bap has been identified in *S. epidermidis*, *S. chromogenes*, *S. xylosus* and a few other CoNS species, where it is often carried in a putative composite transposon (Cucarella et al., 2004; Tormo et al., 2005). However, as far as *S. aureus* is concerned, the role of Bap seems less relevant in isolates from human infections (Vautor et al., 2008; Tang et al., 2013) and, up to now, exclusively strains of veterinary origin (e.g., bovine mastitis) have been found to harbor the *bap* gene (Valle et al., 2012). However, the functions of Bap are still far from being totally understood and are not limited to biofilm formation. Indeed, it has been found that Bap not only promotes the adhesion, but also prevents the entry of *S. aureus* into epithelial cells (Valle et al., 2012). Bap involvement in the pathogenesis of infections is therefore more complex as this protein influences the extent of bacterial internalization into host cells and consequently tissue invasiveness.

In the production of biofilm more clinically significant is the role of another protein factor, namely the accumulation associated protein (Aap). Among biofilm-forming *S. epidermidis* isolates from total hip or total knee infected arthroplasties, up to 27% of the strains were found to be endowed with this surface protein and negative to the *ica* locus (Rohde et al., 2007).

Differently, surface proteins such as SasG (Geoghegan et al., 2010), SasC (Schroeder et al., 2009), Protein A (Merino et al., 2009), and two fibronectin-binding proteins, namely FnBPA and FnBPB (O'Neill et al., 2008), have been documented to contribute to biofilm formation in *S. aureus*. Interestingly, *S. aureus* clinical strains are all generally endowed with the *ica* locus. These alternative mechanisms of biofilm formation can probably concur and could be switched on in different phases of the pathogenesis of infections, adapting the characteristics of the biofilm extracellular matrix in response to external stimuli (Houston et al., 2011), in order to colonize and establish the infection in host tissues, while evading the immune response and the effects of antibiotic treatments.

The protein components of staphylococcal biofilms are discussed in depth in a recent review that illustrates how a multitude of proteins intervene at different stages of the biofilm formation, with certain proteins contributing to the biofilm accumulation and others mediating the primary attachment to the surfaces (Speziale et al., 2014).

## **THE SYNTHESIS OF PIA**

PIA, as described above, is now well-established to consist of poly-β(1-6)-*N*-acetylglucosamine, a linear glucosaminylglycan that plays a fundamental function in mediating intercellular adhesion of bacterial cells. In addition to bacterial aggregation, this exopolysaccharide has important structural functions in the biofilm matrix architecture, and is implicated in bacterial adhesion to biomaterial surfaces as well as evasion from host immune-response (Vuong et al., 2004). PIA synthesis is mediated by the *icaADBC* locus, first discovered and investigated by Heilmann et al. (1996). The *icaADBC* locus was initially described in *S. epidermidis*, but few years later its presence was confirmed even in many other species of the *Staphylococcus* genus, *S. aureus* included (Cramton et al., 1999). The *ica* locus is however part of the "accessory genes" genome and not of the so-called "core" genome, meaning that it is not found in all bacterial strains. Its presence is exclusively observed as part of the virulon of exopolysaccharide-based biofilm-forming staphylococcal strains. In early studies, the *ica* locus was reported in a large proportion of staphylococcal strains isolated from implant related-infections (catheter-associated infections) (Arciola et al., 2001). Important differences in the prevalence of *ica*-positive strains had previously been observed also comparing clinical isolates (blood cultures) with saprophytic strains (85% vs. 6%) (Ziebuhr et al., 1997).

Further evidence of the important role of PIA as a virulence factor came also from experimental studies in rat models of intravascular catheter-associated infections with isogenic mutant strains deficient in PIA production (Rupp et al., 1999, 2001).

The *icaADBC* locus consists of four genes. The first two genes of this gene cluster, respectively *icaA* and *icaD*, exert a primary role in the exopolysaccharide synthesis. The former gene encodes for a transmembrane enzyme with *N*-acetylglucosaminyl transferase activity, necessary for the synthesis of the poly-*N*acetylglucosamine polymer. However, the enzymic activity of the product of *icaA* becomes significant and olygomers longer than 20 residues are synthesized only when coexpressed with the product of the *icaD* gene (Gerke et al., 1998). Conversely, the product of the *icaC* gene appears to translocate the poly-*N*acetylglucosamine polymer to the bacterial cell surface, while the *icaB* product operates the deacetylation of the molecule (Vuong et al., 2004). Deacetylation of poly-*N*-acetylglucosamine polymer is relevant for the structural development of exopolysaccharidebased biofilm, enabling the fixation of the polymer to the outer bacterial surface. The negative regulator termed intercellular adhesin locus regulator (*icaR*) gene governs the expression of the *ica* locus under the influence of SarA and the stress sigma σB (Cerca et al., 2008).

While DNA-sequences homologous to the *ica* locus have been identified also in many staphylococcal species other than *S. aureus* and *S. epidermidis*, up to now they have not been reported for two staphylococcal species, respectively *S. haemolyticus* and *S. saprophyticus*. Species such as *S. lugdunensis*, although endowed with the *ica* locus, appear to produce a biofilm whose matrix prevalently consists of protein material (Frank and Patel, 2007). By CLSM and specific staining with fluorescent dyes we have observed that the biofilm of *S. lugdunensis* is composed of both PIA, which is stained with FITC-WGA, and proteins, stained with Sypro®-Ruby, specific for the protein component (**Figure 1**).

## **PHASE VARIATION IN STAPHYLOCOCCI FOR THE BIOFILM FORMATION**

An interesting question is the phase variation of staphylococci, which consists in a process of switching on/off for the expression/silencing of the *ica* locus, leading to a quantitative variation of biofilm production. In *S. epidermidis*, the phase variation for biofilm formation is thought to be a mechanism of persistence and relapse. The insertion/excision of the insertion sequence IS*256* in the gene *icaA* or in *icaB*, or, more frequently, in *icaC* as a possible mechanism of the phase variation was hypothesized by Ziebuhr et al. (1999). These Authors observed the phase variation in two *S. epidermidis* reference strains that had been subjected to repeated *in vitro* subcultures. In a different way, the natural occurrence of the IS*256* insertion element either in *ica* locus or in the genomic DNA of clinical strains of *S. epidermidis* - just as they had been isolated, that is without artificial manipulations to induce phase variations - was searched by Arciola et al. (2004). The search for *ica* genes was carried out in 120 *S. epidermidis* isolates from prosthesis-associated infections and in 4 *S. epidermidis* reference strains and was compared with the bacterial phenotypes (ability/inability to produce biofilm). Moreover, two biofilmnegative RP62A-derived acriflavin mutants (D9 and HAM892) were analyzed. The four genes of the *ica* locus appeared, in all cases of that collection, strictly linked each other, so that they were either all present or all absent, nor were detected gene deletions within the *ica* locus. IS*256* was present in eight out of the 69 *ica*-negative strains and in 34 out of the 51 *ica*-positive strains. However, when IS*256* was found in the bacterial genomic DNA, it was never found within the *ica* locus, this observation suggesting that the insertion/excision of IS256 is not a natural occurring mechanism for off/on switching the biofilm production. In the same study, for the first time, two RP62A-derived acriflavin mutants, D9 and HAM892, unable to produce biofilm, were shown to harbor within their *icaC* genes, at the base 3319, a 1300-bp insertion corresponding to IS*256*. Although the insertion was found within *icaC*, as in the study of Ziebuhr et al. (1999), it was traced in a position rather different from that described by Ziebuhr et al. (1999). The different point of insertion was ascribed by the Authors to the different mutagenesis conditions, namely repeated subcultures in the experiments reported by Ziebuhr et al. (1999) and, instead, the chemical mutagenesis by acriflavin in the study of Arciola et al. (2004). However, interestingly, in none of the 120 *S. epidermidis* clinical isolates from prosthesis associated infections the IS256 insertion element was found within the *ica* operon, neither in some strains that, although *ica*positive, were biofilm *non*-producers. Thus, the insertion/excision of IS*256* in *ica* operon does not appear as a natural occurring mechanism for switching off/on the biofilm production, but rather as the consequence of either a chemical mutagenesis or of manipulative mutations. Afterwards, in the same 2004 year, only some months later, the phase variation of biofilm formation by an insertion sequence was described also in *Staphylococcus aureus* by Kiem et al. (2004). A biofilm-negative phase-variant *S. aureus* mutant was detected from six strains subjected to repeated subcultures. Again, the *ica*C gene of the phase-variant strain was found to be inactivated by the insertion sequence IS*256*.

More recently, a mechanism of phase variation of the poly-Nacetylglucosamine expression in *Staphylococcus aureus* has been described that does not involve the insertion/excision of IS*256* but the expansion or contraction of a simple tetranucleotide tandem repeat housed within *icaC*. Inactivation of IcaC by the expansion

or contraction of this tetranucleotide tandem repeat results in a PIA/PNAG-negative phenotype (Brooks and Jefferson, 2014). And indeed, the expansion (or contraction) of a 4-nt tandem "ttta" repeat shifts the reading frame of *icaC* and leads to a premature stop codon, truncating the IcaC protein at 303 amino acids, 47 amino acids shorter than the full-length protein. The Authors suggest that, under certain conditions, the loss of PIA/PNAG production may be advantageous during infection. Inactivation of *icaC* is therefore a mechanism of phase variation for PIA/PNAG expression and *icaADB* may contribute to the bacterial fitness, by a mechanism still unknown and involving the absence of an intact *icaC* gene and of PIA/PNAG production. All these findings indicate that the mutation of i*caC* is the preferred "off switch" for PIA/PNAG production.

## **GENETIC CONTROL OF BIOFILM METABOLISM**

The numerous studies on the genetic control of biofilm production in staphylococci have led to consider the expression of the biofilm forming phenotype very complex. This complexity is in part because there is a multiplicity of factors contributing to the biofilm extracellular matrix, these varying with the bacterial species but also, within the same species, with the strain type. In addition, biofilm production derives from a complicated equilibrium of production of extracellular polymeric substances, including amyloid fibrils and polymerized phenol soluble modulins (PSMs), and their catabolism determined by expression of enzymes such proteases, nucleases and soluble, still non-polymerized, PSM peptides (Schwartz et al., 2012).

A fine control of sessile and planktonic phenotypes is highly required to explicate a well coordinate and efficacious action during the invasive phase. The expression of biofilm is therefore governed by mechanisms of collective coordination. These mechanisms enable not only the sensing of environmental stimuli, but also the density of bacterial cells belonging to the same group and sharing the same pheromone system.

Diverse studies have documented as *ica*-positive *S. epidermidis* easily express their biofilm-forming phenotype under *in vitro* conditions. Differently, in *S. aureus*, whose strains are generally all endowed with *ica* locus, the biofilm production is not always fully expressed *in vitro* and often requires a modified atmosphere (anaerobiosis) or supplementation of the growth broth with nutrients in order to be fully manifested (Arciola et al., 2001; Cramton et al., 2001; Stepanovic et al., 2003, 2007 ´ ).

*Vice versa*, *S. aureus* strains manifestly express increased biofilm production *in vivo* (Ammendolia et al., 1999; O'Gara, 2007). *In vitro* stress conditions induced by iron limitation, starvation, thermal stress or subinhibitory concentrations of ethanol, salt and some antibiotics have also been found to increase the amount of biofilm produced.

Therefore, a series of stimuli from the environment and from bacterial density are sensed by bacterial cells and influence the expression of biofilm-forming phenotype. As far as the response to environmental stimuli is concerned, important regulators of staphylococcal biofilm production are represented by the twocomponent signal transduction systems (TCSs) and σ*B*. TCSs mediate a diverse range of adaptive responses in response to environmental stresses. First identified in *S. aureus* (Fournier and Hooper, 2000), the presence of ArlRS TCS was later confirmed also in *S. epidermidis* (Zhu et al., 2010). Recent data show that ArlRS plays an important role in the regulation of *S. epidermidis* biofilm formation, and acts in an *ica*-dependent manner distinct from the role of ArlRS in *S. aureus* biofilm formation, which was found to be *ica*-independent (Wu et al., 2012). In addition to influencing biofilm formation, ArlRS is also involved in the modulation of bacterial autolysis and, consequently, of e-DNA release contributing to the biofilm extracellular matrix.

In *S. aureus* another two-component regulatory system is the *lytSR* operon that affects murein hydrolase activity and autolysis (Brunskill et al., 1996). The LytS sensor component, interacting with its cognate response regulator LytR, activates the transcription of genes under its control. The target of the system is the *lrg/cid* operon, which has been shown to be a regulator in the control of cell death and lysis during biofilm development (Rice et al., 2003; Rice and Bayles, 2008). The *cidA* gene encodes a putative holin protein that is an effector of murein hydrolase activity and cell lysis, while *lrgA* encodes a putative antiholin that is an inhibitor of these processes (Rice et al., 2007), Thus, the biological function of the *cid* and *lrg* operons is to provide a source of extracellular genomic DNA (eDNA) for interweaving and strengthening with this molecule the scaffold of the matrix of biofilm.

The complex intervening factor influencing biofilm production and, in particular PIA synthesis, are depicted in **Figure 2**.

It has been earlier anticipated that biofilm is also influenced by bacterial density. Intercellular communication in staphylococci is enabled by the so-called *quorum sensing* (QS) systems.

The role of QS system in controlling staphylococcal biofilm dispersal and structuring is presented in details in another review of this series (Le et al., 2014) and will be treated here only summarily.

In staphylococci, the system that regulates the expression of virulence factors in response to cell density is the accessory gene regulator (*agr*) system (Recsei et al., 1986; Novick et al., 1993). The importance of the *agr* in biofilm formation in both *S. aureus* and *S. epidermidis* was first shown by the group of Michael Otto (Otto, 2001), who highlighted that, while *agr* leads to increase expression of toxins, serine protease, DNase, fibrinolysin, enterotoxin B, and toxic shock syndrome toxin-1, it decreases the expression of colonization factors and biofilm formation. Since biofilm is one of the most important virulence factors in chronic bacterial infections, the use of *agr*-inhibiting substances for antistaphylococcal treatment has been proposed, but it may have severe drawbacks, as it might turn an acute into a chronic infection by promoting the expression of colonization factors (Otto, 2001).

Among the peptides strictly controlled by the *agr* locus, a family of short staphylococcal peptides, Phenol-Soluble Modulins (PSMs), characterized by amphipathic a-helical structure conferring surfactant-like properties, have been shown to be key effector molecules in biofilm structuring, dispersal and dissemination, by a mechanism consisting in the disruption of non-covalent interactions between biofilm matrix macromolecules (Otto, 2013). These peptides have been demonstrated to be relevant in the pathogenesis of *S. aureus* and *S. epidermidis* biofilm-associated infection, under both *in vitro* and *in vivo* models (Le et al., 2014).

## **THE DEBATED PROMISE OF QUORUM QUENCHING (QQ)**

In the introduction, quorum quenching (QQ), alternatively known as quorum interference, has been mentioned as one of the latest strategies to counteract staphylococcal infections. In nature, QQ is a strategy pursued by antagonistic bacterial strains competing for the environment (Geisinger et al., 2009). QQ can be achieved by alternative tactics, these including quorum sensing disruption through inhibition of signal molecule biosynthesis, signal molecule inactivation and blockade of signal transduction (Kiran et al., 2008; Rampioni et al., 2014). A weakness of the QQ approach resides in the fact that not only QS systems markedly differ between Gram-positive and Gram-negative bacteria, but also their functions are different in the same *genus* and sometimes even in diverse strain types of the same species (Geisinger

**FIGURE 2 | Scheme of the complex network of interactions governing biofilm formation and disruption in** *S. aureus* **based on current scientific evidences.** The right side of the figure illustrates the anabolic phase of biofilm with the production of some fundamental extracellular polymeric substances (EPS) such as PIA, extracellular-DNA (eDNA) and amyloid fibrils. The *lytSR* operon with its target genes *lrg/cid*, which affects murein hydrolase activity, is also represented. The center of the figure reports the protein membrane

components implicated in biofilm formation, these including the FnBPs adhesins, the Biofilm associated protein (Bap), SasG and Spa. Conversely, in the left side of the figure, the molecules playing a role in biofilm catabolism and extracellular biofilm matrix disruption, such as phenol-soluble modulins (PSMs) and extracellular proteins, are reported. Agr QS system, σB factor and SarA appear to act as central regulators, orchestrating the bacterial behavior in response to stress factors, cellular densities and cell cycle phases.

et al., 2012). This means that it may result hard to find universal key molecules able to exert their desired action on all pathogens and that what can inhibit biofilm-formation in a species could stimulate it in another.

However, while inhibiting *agr* would be counterproductive and the use of QS inhibitors is debatable for combatting biofilmassociated infections by staphylococci, *vice versa*, QS interference is expected to become a powerful strategy to control virulence and antibiotic tolerance of Gram-negative bacteria.

To date, methods that can be used to disrupt quorum sensing include: (1) antagonizing signal binding to LuxR-family receptor, (2) inhibiting signal production, (3) degrading signals, (4) trapping signals, and (5) suppressing synthase and receptor activities, stabilities or productions (Hirakawa and Tomita, 2013).

Overall QQ appears an important strategy for applications in medicine (but also veterinary and agriculture), opening new horizons for preventive/therapeutic measures alternative or adjuvant to conventional antibiotics (Costerton et al., 2007).

## **PIA IN IMMUNOTHERAPEUTIC STRATEGIES TO COMBAT STAPHYLOCOCCAL INFECTIONS**

The poly-*N*-acetyl-β-(1,6)-glucosamine, PNAG, also designed as PIA, is a high-profile candidate for a vaccine that could possibly provide protection against both coagulase-positive and coagulase-negative staphylococci. As outlined above, the native form of PIA/PNAG is partially de-*N*-acetylated (dPNAG). It has been observed that only antibodies to the deacetylated epitopes of PNAG are able to give protection, favoring opsonization of *S. aureus* and killing by human neutrophils (Kelly-Quintos et al., 2005, 2006).

Antibodies that bind to PNAG with either low (*<*15%) or high (*>*90%) levels of acetate were shown to have a superior opsonic and protective activity than antibodies that bind to PNAG with only high levels (*>*70%) of acetate (Cerca et al., 2007). Both in *S. epidermidis* and in *S. aureus*, IcaB is a deacetylase that causes partial deacetylation of PNAG and thus ensures a better surface retention of PNAG and optimal biofilm formation.

By studying the acetylation of PNAG by *icaB* negative *S. aureus* mutants, Cerca et al. (2007) observed that less PNAG was associated to the bacterial surface, and this strain was highly susceptible to antibody-independent killing by neutrophils. A *S. aureus* mutant with over-expression of *icaB* producing primarily surfaceassociated PNAG, was more susceptible to opsonophagocytosis with antibody to deacetylated PNAG. The higher retention of deacetylated PNAG on the surface of *S. aureus* provides a molecular mechanism explaining the superior opsonic and protective activity of antibody to dPNAG (Cerca et al., 2007).

While there is a large consensus on the need of a vaccine protecting people from staphylococcal infections, there is much less clarity about the choice of efficacious candidates for a component vaccine. Projan et al., in discussing the possible targets of an anti-*Staphylococcus* vaccine, list almost eight diseases and about thirty possible molecular targets (Projan et al., 2006).

The choice of an appropriate antigen for immunotherapy should attentively consider the issue of the immune evasion. In fact, PIA/PNAG, in addition to its role in intercellular adhesion and biofilm formation, has been pointed out to play a role just in the immune evasion by bacteria. Evidence suggests that antibodies against PIA/PNAG often recognize the secreted PIA/PNAG rather than its surface-associated form, this behavior resulting in an ineffective immune response (Cerca et al., 2007). An effective immune response against surface-associated PIA/PNAG has been evoked by a conjugate vaccine, composed of the *Staphylococcus aureus* PNAG and Clumping Factor A, which has been proved successful in eradicating infection (Maira-Litrán et al., 2012). Thus, PIA/PNAG protects the bacteria from immune defenses but, otherwise, it could actually be the target of an effective immune response.

Although this field is rich of interests and researches, and progress have been made, further experimental, epidemiological, pre-clinical and clinical studies are required before an efficacious vaccine could be achieved.

## **ACKNOWLEDGMENTS**

A financial contribution by "5 per mille" grants for Health Research to the Rizzoli Orthopedic Institute of Bologna is acknowledged.

## **REFERENCES**

cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. *Infect. Immun.* 65, 890–896.

Ziebuhr, W., Krimmer, V., Rachid, S., Lössner, I., Götz, F., and Hacker, J. (1999). A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. *Mol. Microbiol.* 32, 345–356. doi: 10.1046/j.1365-2958.1999. 01353.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 03 November 2014; accepted: 14 January 2015; published online: 10 February 2015.*

*Citation: Arciola CR, Campoccia D, Ravaioli S and Montanaro L (2015) Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects. Front. Cell. Infect. Microbiol. 5:7. doi: 10.3389/fcimb.2015.00007*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2015 Arciola, Campoccia, Ravaioli and Montanaro. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Henning Büttner 1, Dietrich Mack2 and Holger Rohde1 \**

*1 Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany 2 Mikrobiologie/Infektiologie, Bioscientia Labor Ingelheim, Institut für Medizinische Diagnostik GmbH, Ingelheim, Germany*

#### *Edited by:*

*Pietro Speziale, University degli Studi di Pavia, Italy*

#### *Reviewed by:*

*Chia Lee, University of Arkansas for Medical Sciences, USA Carla Renata Arciola, Rizzoli Orthopaedic Institute, Italy*

#### *\*Correspondence:*

*Holger Rohde, Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany e-mail<EMAIL_ADDRESS>
*Staphylococcus epidermidis* is a usually harmless commensal bacterium highly abundant on the human skin. Under defined predisposing conditions, most importantly implantation of a medical device, *S. epidermidis*, however, can switch from a colonizing to an invasive life style. The emergence of *S. epidermidis* as an opportunistic pathogen is closely linked to the biofilm forming capability of the species. During the past decades, tremendous advance regarding our understanding of molecular mechanisms contributing to surface colonization has been made, and detailed information is available for several factors active during the primary attachment, accumulative or dispersal phase of biofilm formation. A picture evolved in which distinct factors, though appearing to be redundantly organized, take over specific and exclusive functions during biofilm development. In this review, these mechanisms are described in molecular detail, with a highlight on recent insights into multi-functional *S. epidermidis* cell surface proteins contributing to surface adherence and intercellular adhesion. The integration of distinct biofilm-promoting factors into regulatory networks is summarized, with an emphasis on mechanism that could allow *S. epidermidis* to flexibly adapt to changing environmental conditions present during colonizing or invasive life-styles.

**Keywords:** *Staphylococcus epidermidis* **biofilm formation, regulation, PIA, Aap, Embp, AtlE, primary attachment, biofilm accumulation**

## **MEDICAL RELEVANCE OF** *STAPHYLOCOCCUS EPIDERMIDIS*

*Staphylococcus epidermidis*, member of the group of coagulasenegative staphylococci, belongs to the commensal skin flora of every human individual (Kloos, 1980, 1997; Kloos and Schleifer, 1986). In its natural niche, the species is of significant importance for maintenance of local homoeostasis—a role that is so far understood only superficially (Grice and Segre, 2011). Only recently light was shed onto the potential importance of *S. epidermidis* to interfere with colonization with *Staphylococcus aureus* via expression of a serine-type protease termed Esp, thereby interfering with colonization mechanisms of *S. aureus*, e.g., biofilm formation (Iwase et al., 2010; Sugimoto et al., 2013). Given the tremendous abundance of *S. epidermidis* on the skin, it is not surprising that *S. epidermidis* is the most common cause of contamination in clinical specimens, and it is a challenge for medical microbiologist to reliably identify true invasive isolates (Mack et al., 2006). Improved abilities to discriminate between clinically relevant and contaminating *S. epidermidis* isolates is of utmost importance, as the species is today one of the most important bacteria related to hospital acquired infections. If the invasive behavior of *S. epidermidis* follows a clear pathogenic strategy that extends its colonizing abilities (Mack et al., 2009), or merely can be regarded as an accident during which mechanisms usually contributing to commensalism turn into virulence features (Otto, 2009), is still a matter of debate. Nevertheless, without doubt *S. epidermidis* is a true opportunistic pathogen that only causes disease in patients with predisposing factors. These include individual patient characteristics (i.e., premature birth, congenital immune defects) or concomitant medical conditions—like HIV infection, immunosuppression after bone marrow or solid organ transplantation and chemotherapy related neutropenia (Goldmann and Pier, 1993; Rupp and Archer, 1994). Most significantly, *S. epidermidis* is the leading organism isolated from foreign material related infections (FMRI) (Darouiche, 2004; Geipel and Herrmann, 2005) such as infected prosthetic joints, central venous catheters (CVC), cerebrospinal fluid shunts, intracardiac devices, artificial heart valves, and vascular grafts (Mack et al., 2006; Rogers et al., 2009). Use of implanted medical devices increases in number, and certainly, this will further propel the importance of *S. epidermidis* as an important human pathogen.

*S. epidermidis* is responsible for the vast majority of nosocomial catheter related blood stream infections (CRBSI) in the United States (Wisplinghoff et al., 2003; Hidron et al., 2008) and also in German intensive care units (ICUs) (Geffers and Gastmeier, 2011). Evaluation of a multicenter international data collection calculated a risk of 6.8 CRBSI per 1000 central line-days in ICUs (Rosenthal et al., 2014). Results from the Surveillance and Control of Pathogens of Epidemiological Importance (SCOPE) indicate that coagulase-negative staphylococci are the most frequent cause of nosocomial blood stream infections (Wisplinghoff et al., 2003). Species discrimination identified eighty percent of CoNS from these infections as *S. epidermidis* (Jukes et al., 2010).

CoNS rank as third most common infective agent in native (NVIE) and first in prosthetic valve infective endocarditis (PVIE) clearly demonstrating the importance of CoNS in these clinical entities (Murdoch et al., 2009). Among CoNS isolates, about eighty percent were identified as *S. epidermidis* (Chu et al., 2004, 2009). Evaluation of the results of the International Collaboration on Endocarditis database showed a significantly higher rate of complicated clinical courses of PVIE due to CoNS with respect to heart failure compared to *S. aureus* or viridans streptococci (Lalani et al., 2006).

*S. epidermidis* is a significant cause of infections of prosthetic joint implants. The lack of non-invasive curative treatment options for joint implant infections often necessitates surgical intervention including replacement surgery. In the UK, CoNS and *S. epidermidis* were isolated in 36% of total hip and 49% of total knee arthroplasty infections (Phillips et al., 2006; Nickinson et al., 2010). In another study of infected total hip and knee arthroplasties about 77% of the isolated CoNS were confirmed *S. epidermidis* (Rohde et al., 2007).

First evidence suggesting a pathogenetic link between foreignmaterial implantation and *S. epidermidis* infection came from early electron microscopic analysis of explanted central venous catheters. Here, bacteria were found to colonize artificial material in large agglomerations, embedded into an amorphous material (Peters et al., 1982). While this specific mode of growth was first referred to as "slime," today it is termed biofilm formation (Götz, 2002). In fact, there is significant evidence connecting the biofilm mode of growth to the general persistent course of *S. epidermidis* foreign-material infections (Scherr et al., 2014a), and to the regular failure to eradicate infections by antimicrobial therapies (Lewis, 2005; Mack et al., 2009; Otto, 2009; Rohde et al., 2010). The latter aspect has been subject to extensive review recently (Lewis, 2010). Although *S. epidermidis* infections are regarded as prototypic biofilm infections (Costerton et al., 1999; Otto, 2009), it must be noted that it is by far not clear that biofilms observed under *in vitro* conditions indeed correlate with the biofilm growth evident *in vivo*. While this is most probably the case in central venous catheter (Peters et al., 1981) or cerebrospinal fluid shunt (Kockro et al., 2000) infections, there is some doubt that this model can be easily transferred to infections occurring at the interface of an implant and the surrounding tissues, e.g., prosthetic joint infections (Broekhuizen et al., 2008; Zaat et al., 2010). Clearly, much needs to be learned with regard to the exact spatial organization of *S. epidermidis* in implant infections, e.g., by making use of *ex vivo* or *in vivo* imaging approaches.

## **STRUCTURAL FACTORS CONTRIBUTING TO** *S. EPIDERMIDIS* **BIOFILM FORMATION**

Traditionally, the process of biofilm formation is divided into at least three steps. During the phase of primary attachment, bacteria adhere to the surface to be colonized, while during the accumulative phase, bacteria initiate the establishment of a three dimensional, multi-cellular and multi-layered architecture in which, intriguingly, most bacteria do not have direct contact to the surface (Mack et al., 2009; Otto, 2009; Rohde et al., 2010). *S. epidermidis*, then, is able to disassemble the biofilm structure again, and liberated cells are believed to allow *S. epidermidis* to colonize additional body sites. The process of biofilm formation needs a wide range of functional activities, ranging from molecules mediating binding to native or conditioned (i.e., host extracellular matrix covered) surfaces, over glue-like factors fostering cell-cell aggregation, to activities that break down matrix components (Otto, 2009). The tremendous diversity of specific functional requirements during biofilm formation is on the bacterial side significantly mirrored by the expression of a plethora of different, highly specialized factors characterized by very distinct profiles of biological functions. Thus, related to their specialized functions during *S. epidermidis* biofilm morphogenesis, specific factors are assigned to groups representing mechanisms being active either during primary attachment or during biofilm accumulation, respectively.

## **FACTORS INVOLVED IN PRIMARY ATTACHMENT**

Tight binding of bacteria to foreign-materials is a pivotal step toward establishment of a device-associated infection. Not unexpectedly, factors specifically involved in mediating bacterial– surface interactions were identified and further characterized. Some genetic evidence suggests that bacterial binding to unmodified polystyrene is fostered by the *S. epidermidis* autolysin AtlE (Heilmann et al., 1997). AtlE is a 115 kDa protein, which belongs to a group of bacterial peptidoglycan (PGN)-hydrolases playing a pivotal role in the degradation of the bacterial cell wall (Biswas et al., 2006). The protein consists of an N-terminal signal peptide, a propeptide, a catalytic domain with *N*-acetylmuramyl-L-alanine amidase activity, three repeats (R1–3), and a C-terminal catalytic domain with *N*-acetylglucosaminidase activity (Schlag et al., 2010; Zoll et al., 2010). In addition to its general role in cell wall turnover, AtlE also is of importance for binding to unmodified polystyrene, as demonstrated by the defect of an *atlE*::Tn*917* transposon mutant of *S. epidermidis* O-47 that lost its ability to adhere to plastic surfaces (Heilmann et al., 1997). Intriguingly, the importance of distinct domains for recruitment of AtlE to the bacterial cell wall (i.e., internal repeats) and enzymatic activities have been identified (Zoll et al., 2010). High resolution structural information is available, however so far it remains unclear, which exact AtlE domains are relevant for the primary attachment process and stable bacterial surface binding. In fact, at present it appears possible that expression and functional activation of AtlE induces significant changes in cell surface hydrophobicity, and thus, the AtlE effect on primary attachment might be secondary (Otto, 2014). In addition, a significant role of AtlE in eDNA mediated *S. epidermidis* biofilm formation is apparent (Qin et al., 2007; Christner et al., 2012).

While interactions between *S. epidermidis* and unmodified artificial surfaces most likely does not involve specific receptorligand interactions, it is well-known that *S. epidermidis*, similar to *S. aureus*, expresses cell surface proteins that mediate specific interactions with host extracellular matrix (ECM) components (Patti et al., 1994). Proteins with ECM-binding activity are believed to be of significant importance for the initiation of a device infection, since foreign materials become, as soon as they are inserted into the body, covered by ECM material (e.g., FN, fibronectin; Fg, fibrinogen; Vn, vitronectin; Cn, collagen) (Arrecubieta et al., 2006; Mack et al., 2009). In fact, *S. epidermidis* can use AtlE to adhere to surface organized Vn, while the lipase GehD is involved into interactions with collagen (Bowden et al., 2002). In addition to these proteins, for which their enzymatic activities might be of primary importance for *S. epidermidis* physiology, *S. epidermidis* also expresses proteins with a primary and dedicated function in bacterial-ECM interactions. These proteins belong to the group of serine-aspartate repeat (Sdr) proteins (McCrea et al., 2000), a widely investigated protein family of microbial surface components recognizing adhesive matrix molecules (MSCRAMM) (Josefsson et al., 1998; Foster et al., 2014). In *S. epidermidis*, three Sdr proteins referred to as SdrF, SdrG, and SdrH have been identified (Josefsson et al., 1998). SdrG, a LPXTG-motif containing protein covalently attached to the bacterial cell surface, is crucial for *S. epidermidis* adherence to fibrinogen-coated surfaces. It is therefore also being referred to as fibrinogen binding protein of *S. epidermidis* (Fbe) (Nilsson et al., 1998; Pei et al., 1999; Hartford et al., 2001). The gene encoding Fbe/SdrG is common in clinical *S. epidermidis* isolates (Nilsson et al., 1998; Rohde et al., 2004, 2007). Fbe/SdrG protein contains five distinct regions: an N-terminal export motif sequence, an A region that contains the Fg binding activity, a B region of so far unknown function, and the R region containing serine-aspartate repeat sequences. Fbe/SdrG specifically binds to a peptide sequence of 14 amino acids found in the N-terminus of the β-chain of Fg (Ponnuraj et al., 2003), and structural analysis of the interaction revealed a unique "dock, lock, and latch" mechanism ensuring a particularly strong interaction (Bowden et al., 2008; Herman et al., 2014). SdrF, sharing overall organizational similarity with SdrG, has been shown to mediate *S. epidermidis* binding to collagen I (Arrecubieta et al., 2007). In contrast to Fgbinding properties of Fbe/SdrG, the collagen binding epitopes of SdrF are located within the B repeat region (Arrecubieta et al., 2007). So far no specific functionality has been attributed to the N-terminal A domain of SdrF. A single B domain repeat of SdrF was sufficient to interact with collagen I, and apparently, this binding occurs via interactions with the α1 and α2 chains of type I collagen (Arrecubieta et al., 2007). Using a *Lactococcus lactis* heterologous expression system and a murine infection model evidence was generated that SdrF may contribute to cardiac assist device driveline infections (Arrecubieta et al., 2009). SdrF also mediates binding to unmodified Dacron surfaces covering drivelines. In contrast, *L. lactis* expressing GehD bound only weakly to driveline surfaces (Arrecubieta et al., 2009). Anti-SdrF inhibited *S. epidermidis* 9491 binding in the *in vivo* model only by roughly 50%, indicating that additional *S. epidermidis* collagen binding factors may be involved (Arrecubieta et al., 2009).

Extensive work has addressed the role of extracellular DNA (eDNA) in *S. epidermidis* and *S. aureus* biofilm formation. Data confirm that eDNA is a structural component of the biofilm matrix in both species, although evidence anticipates that eDNA has, at least partially, different functions in both species. Several independent studies have demonstrated that eDNA is released through increased cell lysis (Allesen-Holm et al., 2006; Rice et al., 2007; Christner et al., 2012). In *S. epidermidis* autolysis is determined to a large extent by the activity of the major autolysin AtlE (Biswas et al., 2006). A role for eDNA in *S. epidermidis* 1457 during primary attachment was deduced from observations showing that addition of DNase I abrogated bacterial attachment to glass surfaces. These findings were confirmed in additional, genetically independent *S. epidermidis* backgrounds (i.e., RP62A) (Qin et al., 2007). In extent to its effect on primary attachment, eDNA functions as an intercellular adhesin contributing to the stabilization of biofilms (Whitchurch et al., 2002). Based on the finding that DNase I has biofilm disintegrating activity when added within the first 6 h of biofilm accumulation currently it is believed that eDNA mediated intercellular adhesion is critical especially during the early accumulative phase (Qin et al., 2007). A role of eDNA in earlier stages of staphylococcal biofilm formation has recently been underpinned by observations showing that during *S. aureus* surface colonization under flow conditions eDNA, while not having impact on primary attachment, is critical during the transition from attachment to accumulation (Moormeier et al., 2014). It should again be stressed that functional differences of eDNA during *S. epidermidis* and *S. aureus* biofilm formation are apparent (especially with respect to the function during accumulation) (Izano et al., 2008; Christner et al., 2012), and observations in one species cannot easily be extrapolated to the other. This is especially true for the role of eDNA as a target during biofilm detachment events. By *saeRS* regulated expression of nuclease Nuc *S. aureus* can remodel the biofilm ultrastructure and control the release of bacteria from established biofilms (Mann et al., 2009; Olson et al., 2013). The lack of nuclease activity questions if this biofilm-escape mechanism is, in addition to *agr*-mediated biofilm dispersal (Vuong et al., 2003; Wang et al., 2011a), of relevance in *S. epidermidis*.

## **MECHANISMS OF** *S. EPIDERMIDIS* **BIOFILM ACCUMULATION**

The hallmark of the accumulative phase is expression of intercellular adhesive properties, ultimately leading to cell aggregation and subsequent development of a multicellular, multilayered biofilm architecture (Costerton et al., 1995). Parallel to the discovery of factors with dedicated functions during primary attachment, the nature of intercellular adhesins, functioning as the "biofilm glue," was partially unraveled. Based on the early electron microscopic studies, showing *S. epidermidis* cells embedded in an amorphous extracellular matrix (Peters et al., 1981), focus was set onto the biochemical analysis of biofilm matrix components. These efforts ultimately resulted in the discovery of the polysaccharide intercellular adhesin (PIA), which is at present the most extensively studied intercellular adhesin (Mack et al., 1994b).

Structural analysis of PIA and comparison of PIA isolated from *S. epidermidis* and *S. aureus* has been recently reviewed (Mack et al., 2013). The structure of PIA was first described for biofilm-forming *S. epidermidis* 1457 and RP62A. PIA was extracted from the cells by sonication after the strains had been cultured in trypticase soy broth, which revealed the existence of both a major polysaccharide I (>80%), and a minor polysaccharide II (<20%), which are structurally closely related and could be separated due to differing ionic properties (Mack et al., 1996). Chemical analyses and NMR spectroscopy have demonstrated that polysaccharide I is a linear homoglycan of β-1,6-linked 2-amino-2-deoxy-D-glucopyranosyl residues. Approximately 80– 85% of them are *N*-acetylated; the rest are non-*N*-acetylated and carry a positive charge. Polysaccharide II of PIA has a lower proportion of de-N-acetylated 2-amino-2-deoxy-D-glucopyranosyl residues and is modified by ester-linked succinate residues rendering it anionic (Mack et al., 1996). Despite a high apparent molecular weight indicated by elution in the void volume of Sephadex G200 (Mack et al., 1994a, 1996) or Sephacryl S300 columns (C. Fischer and D. Mack, unpublished results), the ratio of reducing terminal sugar residues to total sugar residues was shown by methylation analysis to be 1:130, implying an average Mr of 30,000 for PIA polysaccharide chains (Mack et al., 1996). This implies aggregation of PIA polysaccharide chains in solution. PIA was shown to function also as the hemagglutinin of *S. epidermidis* (Rupp and Archer, 1992; Fey et al., 1999; Mack et al., 1999). Production of a functionally active PIA molecule requires expression of all four *icaADBC* genes (Gerke et al., 1998). The process has been the subject of detailed study in recombinant strains of *S. carnosus* which expressed different combinations of the *icaADBC* genes and with UDP-GlcNAc as a sugar donor (Gerke et al., 1998). IcaA belongs to the glycosyltransferase 2 family. It is an integral membrane protein with 412 aa and four predicted transmembrane domains (Heilmann et al., 1996; Gerke et al., 1998; Gill et al., 2005), and directs the synthesis of β-1,6 linked GlcNAc oligosaccharides of up to 20 GlcNAc units. IcaD is required for full activity of IcaA *in vitro*. It is a 101 aa integral membrane protein with two potential membrane spanning domains: it may be a chaperone directing folding and membrane insertion of IcaA and may act as a link between IcaA and IcaC (Gerke et al., 1998). Also essential for the synthesis of fully functioning PIA is IcaC, a 355 aa integral membrane protein with 10 predicted transmembrane domains, which may be involved in externalization and elongation of the growing polysaccharide (Gerke et al., 1998). IcaB is a member of the polysaccharide deacetylase family, including, for example, chitin deacetylases or the chitooligosaccharide deacetylase NodB of *Rhizobium melioti*. In its mature form it is a 259 aa secreted protein with a predicted signal sequence, responsible for the de-*N*-acetylation of PIA, and crucial for PIA activity in biofilm formation and for virulence in *S. epidermidis* (Vuong et al., 2004a). In *icaB*-mutants, where the *icaB* gene has been deleted, PIA is poorly retained on the cell surface as it does not contain non-*N*-acetylated GlcNAc (Vuong et al., 2004a).

Early observations made by biochemical analysis of biofilm matrix extracts not only showed the presence of(poly-)saccharides but pointed toward the additional presence of proteins and nucleic acids (Hussain et al., 1991). Specific proteins have been identified and characterized. Apart from biofilm associated protein Bap (Tormo et al., 2005a), that is only rarely found in invasive *S. epidermidis* from human infections (Tormo et al., 2005a; Rohde et al., 2007; Piessens et al., 2012), SesC has been proposed to play a role in biofilm formation (Shahrooei et al., 2009). SesC is an LPXTG motif-containing 68 kDa surface protein of *S. epidermidis* distantly related to clumping factor A of *S. aureus* and is expressed more strongly in biofilm-associated as compared to planktonic *S. epidermidis* 1457 and 10b cells *in vitro* and *in vivo* (Shahrooei et al., 2009; Lam et al., 2014). Rabbit anti-SesC inhibited biofilm formation of a number of *S. epidermidis* isolates *in vitro*, which may be related to changes in primary attachment to fibrinogen-coated surfaces in the presence of anti-SesC. All of 105 *S. epidermidis* isolates recovered from nose swabs or infections were in possession of the *sesC* (Shahrooei et al., 2009). Passive and active immunization using SesC as a target protein was shown to decrease *S. epidermidis* biofilm formation in an *in vivo* model of central venous catheter infections (Shahrooei et al., 2012). A specific role of SesC as an intercellular adhesin in biofilm accumulation remains to be demonstrated.

## **MULTIFUNCTIONAL PROTEIN FACTORS IN** *S. EPIDERMIDIS* **BIOFILM FORMATION**

Although having partially additional enzymatic functions (e.g., AltE, GehD), it is a common feature of many factors contributing to *S. epidermidis* surface colonization that they carry out functions either during the primary attachment or accumulative phase of biofilm formation (Rohde et al., 2006; Mack et al., 2009; Otto, 2009). With the increasing interest in protein factors contributing to staphylococcal biofilm accumulation, it became apparent that, at least in *S. aureus*, many factors (e.g., FnBPA, ClfA) must be regarded as multifunctional proteins not having an exclusive role in either primary attachment or accumulation (Foster et al., 2014). This concept of multifunctional proteins with important roles during several phases of biofilm formation and surface colonization is now also evolving in *S. epidermidis*, with the accumulation associated protein (Aap) and the extracellular matrix binding protein (Embp) being the most prominent factors.

Embp and its ortholog in *S. aureus* designated Ebh were almost simultaneously identified during studies aiming at identifying *S. epidermidis* or *S. aureus* protein factors with Fn binding activities (Clarke et al., 2002; Williams et al., 2002). By using a phage display approach a phage was isolated, which exhibited fibronectin binding activity and contained a DNA fragment from a 30,500 bp open reading frame (ORF) coding for a 10,203 aa protein that was referred to as extracellular matrix binding protein Embp (Williams et al., 2002) (**Figure 1**). Using bioinformatics the architecture of Embp was predicted to consist of an N-terminal YSIRK-motif containing export signal (aa 58–84), followed by an unordered region of approximately 2500 amino acids (aa 85–2586). The overall architecture of Embp mainly is characterized by 21 repetitive "Found In Various Architectures" (FIVAR) repeats (aa 2587–4500) and 38 alternating "G-related Albumine-binding" (GA) motifs and FIVAR repeats—termed the FIVAR-GA repeats—(aa 4501–9443) that span roughly 7000 amino acids in the central proportion of the Embp protein. Finally the C-terminus consists of four domains of unknown function (DUF1542) (aa 9444–9841), followed by a potential transmembrane motive (aa 10,070–10,088) (Christner et al., 2010) (**Figure 1**). Although the *S. aureus* homolog Ebh displays at least functional homologies with respect to fibronectin binding activity (Clarke et al., 2002) some functional predictions found in Ebh, e.g., an N-terminal hyperosmolarity resistence domain (Kuroda et al., 2008; Tanaka et al., 2008) have not been identified in the Embp. The overall structural organization of the Ebh protein seems to be more or less identical to Embp, but in

detail gradual differences are apparent, e.g., only seven FIVAR motifs but 12 additional FIVAR-GA repeats and four additional DUF1542 repeats compared to Embp were predicted (Tanaka et al., 2008). In fact, it appears that among different *S. aureus* strains the number of repetitive modules (FIVAR and FIVAR-GA) within Ebh is variable, while available sequence data shows no variability of these features in Embp from *S. epidermidis* RP62A, ATCC12228 or 1585.

Crystallization of two 126 amino acid FIVAR-GA repeats from EbhA of *S. aureus* MU50 (termed EbhA-R7-R8) revealed a triple α-helical structure interconnected by a continuous alpha helical string displaying an elongated shape (Sakamoto et al., 2008; Tanaka et al., 2008). In fact, the corresponding FIVAR-GA repeat of Embp exhibits a very similar, if not identical structure (Büttner, Perbandt, Rohde, unpublished results). Additional preliminary structural analysis of repetitive FIVAR regions (Büttner, Perband, Rohde, unpublished results) or DUF1542 repeats (Linke et al., 2012) suggest that overall Embp constitutes an elongated rod-like conformation.

In collections of clinical significant *S. epidermidis* isolates*embp* was detected in more than 90% of strains (Rohde et al., 2004, 2007). In addition, evidence for the expression of Embp *in vivo* resulted from investigations showing the presence of anti-Embp antibodies in patients with confirmed *S. epidermidis* prosthetic joint infections (Mack, Büttner, Rohde, unpublished). Strikingly, using a flow cell model of biofilm formation, anti-Embp antibodies were shown to inhibit *S. epidermidis* 1457 biofilm formation (Lam et al., 2014), making Embp a potential candidate for preventive strategies (Götz, 2004).

Experimental evidence primarily suggested a role of Embp in primary attachment. Importantly, over-expression of Embp did not alter binding to unmodified polystyrene, but only boosted bacterial adherence to Fn-coated surfaces (Christner et al., 2010), and Embp–Fn interactions were necessary for biofilm accumulation on plastic surfaces that otherwise did not promote bacterial binding (Christner et al., 2010). Results from phage display suggested that FIVAR-GA repeats were relevant to the Fn-binding activity of Embp (Clarke et al., 2002; Williams et al., 2002). This assumption was later validated by biochemical analysis showing direct evidence for interactions between a recombinant protein containing FIVAR-GA repeats (Christner et al., 2010). In addition, these studies also found evidence that FIVAR-modules alone are capable of binding to immobilized Fn (Christner et al., 2010) (**Figure 1**). The vast majority of bacterial Fn-binding proteins bind to Fn via interactions with the N-terminal Fn type I domains (Bingham et al., 2008; Chagnot et al., 2012). This is especially true for the *S. aureus* Fn binding protein FnBPA (Meenan et al., 2007). Although the exact mechanism of Embp–Fn interaction awaits definitive molecular analysis, it is already clear that Embp uses an mechanism for Fn interactions independent of type I Fn modules, but involving Fn type III modules located at the C-terminus of Fn (Bustanji et al., 2003), most likely FnIII12−14 (Christner et al., 2010). This type of interaction has only rarely been described in bacterial pathogens (Kingsley et al., 2004; Dabo et al., 2006).

In addition to its function in primary attachment, Embp is also functional as an intercellular adhesin. The intercellular adhesive properties and biofilm inducing activity of Embp was first detected in a laboratory derived strain *S. epidermidis* 1585v that, by a spontaneous chromosomal rearrangement, overexpressed a truncated isoform of Embp referred to as Embp1 (Christner et al., 2010). A transposon insertion within Embp1 resulted in abolished biofilm formation.  Of notice, upregulation of *embp* is also associated with resistance against uptake by professional phagocytes (Schommer et al., 2011). Studies on the overall impact of Embp on *S. epidermidis* cell wall assembly and its relation to immune-escape will shed light on the question if in this species, the giant protein carries similar functions as compared to Ebh in *S. aureus* (Cheng et al., 2014).

While at the time of its identification Embp appeared primarily as a factor mediating primary attachment, Aap was initially, and as already suggested by its designation, thought to confer intercellular adhesion and thereby to contribute to biofilm accumulation.  The 584 aa A domain harbors a N-terminal export signal, several imperfect, 16 amino acid repeats, and a globular 212 amino acid region with predicted α-helical and β-sheet content. The 212 amino acid region is highly conserved between Aap and its *S. aureus* ortholog SasG, and bioinformatical analysis predicts that this domain possesses lectin-like activity (Schaeffer et al., 2015). The B domain consists of a varying number of repetitive 128 amino acid repeats (Rohde et al., 2007). Variations of B repeats not only exist between independent *S. epidermidis* strains (e.g., reference strain RP62A possess 13 repeats, while *S. epidermidis* 1457 only harbors seven repeats) (Monk and Archer, 2007; Schaeffer et al., 2015), but are also encountered in clonally identical clinical isolates subsequently recovered during the course of device infections from individual patients (Rohde et al., 2007). This observation lead to the hypothesis that Aap B repeat variations could represent a mechanism contributing to *S. epidermidis* immune escape through modification of major cell surface epitopes (Rohde et al., 2007).

Aap can be detected on the bacterial cell wall, where it is most likely retained by covalent linkage to the peptidoglycan via its C-terminal gram-positive anchor region (Hussain et al., 1997; Rohde et al., 2005; Schommer et al., 2011; Conlon et al., 2014). A more detailed analysis using confocal microscopy demonstrated that within living, three dimensional *S. epidermidis* biofilms, Aap strictly localizes to the bacterial cell surface, while only minimal amounts are released into the biofilm matrix itself (Schommer et al., 2011). These results are underpinned and extended by electron-microscopic studies showing that Aap forms elongated fibers that project 120 nm away from the cell wall in localized tufts (Banner et al., 2007). Recently, using a structural biology approach, the molecular basis of this intriguing spatial organization was determined. An X-ray crystallography derived high resolution model of different recombinant proteins from the B region of *S. epidermidis* Aap (Conrady et al., 2013) or *S. aureus* SasG (Gruszka et al., 2012) showed that each B repeat consists of two regions, an approximately 80 aa G5 domain and an approximately 50 aa linker region referred to as E-region that interconnects repetitive G5 domains (Gruszka et al., 2012). The G5 domains each comprise two successive three-stranded βsheets connected by triple-helix-like regions, while the E region is composed of two β-sheets (Gruszka et al., 2012; Conrady et al., 2013). E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. Although E and G5 domains lack a compact hydrophobic core, G5 domain and multidomain constructs thereof have thermodynamic stabilities only slightly lower than globular proteins of similar size, explaining why Aap could form protruding fibers even under harsh environmental conditions (Gruszka et al., 2012; Conrady et al., 2013).

The functional importance of Aap for *S. epidermidis* biofilm formation was first recognized during studies in which chemically derived, biofilm-negative mutant M7 of *S. epidermidis* RP62A was analyzed (Hussain et al., 1997). Mutant M7 failed to express Aap on the cell surface, and antibodies raised against Aap were able to inhibit biofilm formation in biofilm-positive parent strain RP62A (Hussain et al., 1997). Later, Aap was independently picked up in experiments in which cell surface proteins of a clinically significant but biofilm-negative *S. epidermidis* wild-type strain 5179 were compared with those isolated from a laboratory derived, biofilm-positive revertant of that strain, referred to as 5179-R1. In protein preparations from 5179-R1 reduced amounts of full length Aap were detected, while in parallel, a shorter, roughly 140 kDa Aap isoform became apparent (Rohde et al., 2005). By using mass spectrometry and N-terminal sequencing evidence was created that the 140 kDa isoform mainly consists of repetitive B domain, mapping to aa 596 of the mature Aap protein. Rabbit antiserum raised against recombinantly expressed B domain inhibited biofilm formation by strain 5179-R1, not only directly supporting the idea of a functional involvement of Aap, but moreover indicating the crucial importance of B domain during this process. Indeed, genetic studies corroborated this hypothesis, showing that *in trans* expression of B domain in biofilm-negative *S. epidermidis* 1585 and surrogate host *S. carnosus* TM300 was sufficient to induce a biofilm-positive phenotype (Rohde et al., 2005). Importantly, expression of B domain did not alter the primary adherence properties, but resulted in cell aggregation, showing that in fact, Aap can be regarded as an intercellular adhesin (Rohde et al., 2005; Conrady et al., 2008; Geoghegan et al., 2010). Indeed, the importance of B domain for intercellular adhesion was also described for SasG in an *S. aureus* background (Geoghegan et al., 2010). The intercellular adhesive properties can be partially explained by Zinc-dependent homodimerization of B domains (Conrady et al., 2008, 2013), however, some evidence suggests the existence of additional, heterotypic interactions involved in Aap B domain mediated biofilm accumulation (Decker et al., unpublished results).

Importantly, expression of full length mature Aap is not sufficient to mediate intercellular adhesion during the second, accumulative phase of biofilm formation, but to become functionally active as an intercellular adhesin, Aap requires proteolytic processing, resulting in the removal of the A domain (Rohde et al., 2005; Geoghegan et al., 2010). Since Aap processing does not regularly occur under *in vitro* growth conditions (Rohde et al., 2005; Schaeffer et al., 2015), this phenomenon is a reasonable explanation for the finding that Aap-expressing *S. epidermidis* not necessarily form a biofilm (Rohde et al., 2007).

Although the intercellular adhesive Aap properties were recognized first, there is now mounting evidence supporting a significant role of Aap also in primary attachment to natural epithelial cells or artificial surfaces (Macintosh et al., 2009; Conlon et al., 2014; Schaeffer et al., 2015). Intriguingly, early work already pointed toward a role of domain A in this process, in which Aap B domain most likely is of only minor functional importance (Macintosh et al., 2009). Binding of Aap expressing *S. epidermidis* NCTC 11047 to squamous epithelial cells was partially inhibited by the addition of recombinant Aap A domain, as was binding of several additional, Aap-positive clinical *S. epidermidis* strains (Macintosh et al., 2009). Moreover, *in trans* expression of A domain in surrogate host *L. lactis* enabled the bacteria to more efficiently adhere to corneocytes as compared to a *L. lactis* strain that expressed B domain alone (Macintosh et al., 2009), thus providing genetic evidence for a potential role of A domain in colonization of natural skin surfaces. In extension to the work of Macintosh and co-workers, the role of Aap A domain in primary attachment was further refined in two studies addressing the question as to which extent the A domain could also contribute to colonization of abiotic surfaces (Conlon et al., 2014; Schaeffer et al., 2015). In a clinical *S. epidermidis* isolate CSF41498, expressing an unprocessed mature, i.e., A domain containing Aap on the surface, attachment to polystyrene was almost completely abolished after deletion of *aap*. In addition, attachment was significantly inhibited by an antiserum raised against Aap A domain, whereas anti-Aap domain B antiserum had no significant impact on adherence capacities of that strain (Conlon et al., 2014). Intriguingly, deletion of *aap* in *S. epidermidis* 1457, expressing a processed Aap isoform devoid of the A domain, did not render the adherence capacities of that strain (Schaeffer et al., 2015). Moreover, anti-Aap A domain antiserum had no effect on binding to the surface of *S. epidermidis* 1457, supporting the idea that indeed, the presence of A domain, i.e., expression of a mature unprocessed Aap, is essential for Aap-mediated surface adherence. Indeed, *in trans* expression of full length Aap in 1457*aap* resulted in improved bacterial binding (Schaeffer et al., 2015). Direct genetic evidence for an involvement of Aap A domain in staphylococcal plastic adherence is demonstrated by *in trans* expression of A domain fused to the C-terminal cell wall anchor in *S. carnosus* TM300, which significantly improved bacterial binding as compared to the wild-type strain or a strain expressing the Aap B domain (Schaeffer et al., 2015). Thus, a new picture of Aap evolved in which the protein plays a bifunctional role in both, the very early primary attachment phase as well as the later accumulative phases of biofilm formation, and that Aap's inherent functionalities are represented by separated and structurally distinct domains within the protein (Rohde et al., 2005; Macintosh et al., 2009; Conlon et al., 2014; Schaeffer et al., 2015).

## **MOLECULAR INTERACTIONS AND REGULATORY EVENTS DURING** *S. EPIDERMIDIS* **BIOFILM FORMATION**

The detailed analysis of functional molecules contributing to *S. epidermidis* biofilm formation has clearly revealed that primary attachment as well as biofilm accumulation is mediated by redundantly organized factors with remarkable exclusive properties, but certainly broadly overlapping functional characteristics. The latter aspect is especially true for molecules active during the accumulative phase: production of PIA, Aap or Embp all efficiently induce cell aggregation, ultimately leading to assembly of a biofilm consortium. However, given the common observation that in clinical *S. epidermidis* isolates genes encoding for the PIA-synthesis machinery (*icaADBC*), *aap*, *embp*, and additional adherence-associated factors are all widely distributed, with a large percentage of strains carrying all three genes (Frebourg et al., 2000; Galdbart et al., 2000; Klug et al., 2003; Arciola et al., 2004; Rohde et al., 2004, 2007; Petrelli et al., 2006), the question is put forward if and how these mechanisms interact, and which consequences co-expression of distinct intercellular adhesins might have for *S. epidermidis* biofilm accumulation.

Indeed, the idea that distinct *S. epidermidis* intercellular adhesins cooperate during biofilm assembly is supported by epidemiological work identifying that strains being positive for *icaADBC* and *aap* appear to form stronger biofilms compared to strains being positive only for *icaADBC* or *aap* (Stevens et al., 2008). Bioinformatic analysis of *aap* and *embp* indeed suggests that direct interaction with PIA might be possible. The G5 domains of the Aap B domain are postulated to have Nacetylglucosamine binding activity (Bateman et al., 2005), and similarly, FIVAR regions of Embp are likewise predicted to have sugar binding potential (Christner et al., 2010), making the direct interaction between proteinacous intercellular adhesins and Nacetylglucosamine containing PIA possible. However, so far no experimental data are available that would support this important and interesting hypothesis. At present, it rather appears that parallel expression of specific intercellular adhesins introduces functional redundancy into biofilm accumulation, i.e., the intercellular adhesive properties of distinct adhesins functionally substitute for each other. Experiments, in which the susceptibility of biofilms of Embp-producing *S. epidermidis*strain 1585v against treatment with proteases was tested, revealed that, while being naturally sensitive against protease activity, additional *in trans* expression of *icaADBC* and PIA production protected the *S. epidermidis* 1585v biofilm from proteolytic breakdown (Christner et al., 2010). On the other hand, while PIA-dependent biofilms are readily disrupted by PIA-degrading enzyme DspB (Kaplan et al., 2004; Rohde et al., 2007), parallel expression of a proteinacous intercellular adhesin Embp and PIA rescued a biofilm-positive phenotype even in the presence of DspB (Christner et al., 2010). Functional substitution is also evident for Aap and PIA: the inactivation of *aap* in a PIA-producing genetic background had no apparent effect on biofilm formation in *S. epidermidis* 1457 as tested by conventional crystal violet biofilm assays or confocal laser scanning microscopy, probably because biofilm formation is already maximal in the various assay systems when PIA is expressed alone (Schaeffer et al., 2015). On the other hand, *in trans* expression of Aap B domain, being sufficient for induction of cell aggregation, induces biofilm formation in a PIA-negative, *icaADBC* mutant 1457-M10 (Henke and Rohde, unpublished results).

The interpretation of functional redundancy in intercellular adhesive molecules as a simple means allowing *S. epidermidis* to form as robust biofilms as possible, however, might be an inappropriate oversimplification of their actual functional importance during different *S. epidermidis* live styles, ranging between colonization and (foreign-material associated) host invasion. In that respect, it is important to acknowledge that a more detailed morphological analysis of PIA-, Aap- or Embp dependent biofilms revealed that these biofilm types differ significantly in their morphological properties. While in PIA-dependent biofilms, *S. epidermidis* cells are embedded into meshwork of PIA-containing extracellular matrix fibers, Aap production induces formation of densely packed cell layers that evenly cover the surface. In Embp-dependent biofilms, bacteria produce small amounts of Embp-containing extracellular matrix structures, however, these biofilms differ from PIA-dependent cell consortia by the lack of towers and clusters (Schommer et al., 2011). Overall, PIA-dependent biofilms are significantly more stable against washing procedures as compared to protein-dependent biofilms, indicating their inherent, pronounced mechanical robustness. Thus, the specific biological properties of a given intercellular adhesin could constitute a way how *S. epidermidis* can cope with varying challenges during colonization and infection (Schommer et al., 2011). Analysis of invasive *S. epidermidis* strains from various types of infections supports the idea that in fact, the ability to differentially make use of specific intercellular adhesins equips *S. epidermidis* to specifically adapt to changing environments with potential fundamentally different requirements, e.g., presence of mechanical or osmotic stress, or exposure to effectors of the host immune system (Otto, 2014). Specifically, *S. epidermidis* strains from central venous catheter infections, i.e., a situation with significant exposure to mechanical stress and cellular and soluble factors of innate immunity (e.g., complement factors) are more likely to carry *icaADBC* compared to strains from prosthetic joint infections, i.e., an infection setting characterized by static conditions at the implant–tissue interface (Ziebuhr et al., 1997; Arciola et al., 2004; Rohde et al., 2004, 2007; Stevens et al., 2008; Mack et al., 2013).

Additional evidence suggesting that usage of specific intercellular adhesins indeed follows an adaptive program results from the observation that PIA-dependent biofilm formation on one hand, and Aap- and Embp-dependent biofilm formation on the other, are, at least under *in vitro* growth conditions, mutually exclusive (Rohde et al., 2005, 2007; Christner et al., 2010). In fact, under standard growth conditions in trypticase soy broth (TSB), neither Aap nor Embp-dependent biofilm formation would have been identified, since here Aap is not properly processed, while *embp* is not expressed at all (Rohde et al., 2005; Christner et al., 2010). The characterization of these intercellular adhesins only was possible by analysis of laboratory derived, spontaneous mutants or by introduction of artificial promoters allowing for inducible gene expression, respectively (Christner et al., 2010; Rohde et al., 2005). Thus, PIA- and Aap- or Embp-dependent modes of biofilm formation are obviously under the control of opposed regulatory circuits. For PIA-dependent biofilm formation, a very detailed picture of how *icaADBC* expression is integrated into a complex superimposed regulatory network has evolved. Others have recently reviewed this field in detail (Mack et al., 2004; Cue et al., 2012). In brief, several regulators of *icaADBC* expression and PIA synthesis were identified (Xu et al., 2006; Sadykov et al., 2011; Wang et al., 2011b), with sigma factor B and staphylococcal accessory regulator SarA being the most prominent (Fluckiger et al., 1998; Knobloch et al., 2001, 2004; Tormo et al., 2005b; Handke et al., 2007). Especially, negative regulator of *icaADBC* expression, IcaR, has been elucidated in great detail (Conlon et al., 2002). However, the regulation of PIA-independent mechanism of *S. epidermidis* biofilm formation remained obscure.

More recently, in an attempt to shed light onto potential negative regulators interfering with biofilm formation in clinical *S. epidermidis* isolates under *in vitro* conditions, a transposon mutant library established in biofilm-negative *S. epidermidis* 1585 was screened for biofilm-positive mutants (Christner et al., 2012). Independent biofilm-positive mutants were identified, and further analysis showed that all carried Tn*917* insertions in *sarA*. Intriguingly, inactivation of *sarA* induced a strong upregulation of *embp* expression, and additional experimental work proved that Embp was necessary for 1585*sarA* biofilm formation. Moreover, higher eDNA amounts were present in biofilms of 1585*sarA*, and this finding was related to increased autolysis, itself being a result of over-production of metalloprotease SepA (Lai et al., 2007) and subsequent processing and functional activation of AtlE (Christner et al., 2012). Of notice, protease-mediated autolysin processing, augmented autolysis and subsequent eDNA release and biofilm formation has also been described in *Enterococcus faecalis* as part of a fratricidal mechanism (Thomas et al., 2008, 2009).

The results of the study by Christner and co-workers appear to contradict previous findings, showing that SarA is a positive regulator of *S. epidermidis* biofilm formation by augmenting *icaADBC* expression (Tormo et al., 2005b; Handke et al., 2007). However, a more detailed analysis of SarA function in PIApositive background of *S. epidermidis* 1457 showed that in this strain, inactivation of *sarA* does not completely abolish biofilm formation (Handke et al., 2007). Even inactivation of *icaADBC* in 1457*sarA* did not render the strain biofilm-negative, and in fact, the biofilm of 1457-M10*sarA* is Embp- and eDNA-dependent (Christner et al., 2012). In conclusion, the study by Christner and co-workers establishes a key role for SarA in controlling the mode of biofilm formation in *S. epidermidis*: up-regulation of *sarA* shifts *S. epidermidis* toward production of a PIA-dependent type of biofilm, whereas down-regulation of *sarA* supports formation of PIA-independent types of biofilm formation (**Figure 2**). Clearly, the regulation involves transcriptional effects with direct consequences for intercellular aggregation (i.e., *embp* and *icaADBC* up- or down-regulation), but also post-translational mechanisms of regulation exerted via up- or down-regulation of metalloprotease SepA with subsequent proteolytic processing and functional modification of cell surface proteins (e.g., AtlE). Importantly, through the latter mode of action SarA could also influence Aapdependent biofilm formation by boosting proteolytic removal and functional activation of Aap B-domain (**Figure 2**). In the future it will be of major interest as to which extent the SarA regulatory circuit and interrelated additional regulators, e.g., *agr* (Vuong et al., 2004b), *ssrAB* (Wu et al., 2015), *saeRS* (Lou et al., 2011), or *codY* (Batzilla et al., 2006) as well as the levels of proteolytic activity influence the balanced formation of PIA-dependent or -independent types of biofilm formation.

Certainly, the highly artificial character of studies related to the function of SarA makes it difficult to draw general conclusions on the relevance of this mechanism during *in vivo* biofilm accumulation. In that respect, it becomes clearer that that more appropriate models of biofilm formation are generally needed in order to reflect the *in vivo* situation during infection and colonization. These models must take into account the potential inappropriateness of TSB as a growth medium that obviously introduces a significant bias toward PIA-dependent biofilm formation into the experimental system. The finding that *embp*, but also additional factors involved in *S. epidermidis* biofilm formation (e.g., Fbe/SdrG) are only expressed in the presence of serum (Sellman et al., 2005, 2008; Christner et al., 2010), clearly argues

for experimental set-ups that mimic environmental conditions in ecological niches usually encountered by *S. epidermidis*, e.g., within the vestibulum nasi (Krismer et al., 2014) or on the skin (Ohnemus et al., 2007; Olson et al., 2014).

## **RELEVANCE OF** *S. EPIDERMIDIS* **BIOFILM FORMATION FOR THE PATHOGENESIS OF FOREIGN-MATERIAL ASSOCIATED INFECTIONS** *IN VIVO*

In general, studies aiming at elucidating the clinical relevance of biofilm formation and the relative contribution of specific factors to foreign-material colonization and establishment of a chronic persistent infection are either studied using cell culture models (Vuong et al., 2004a,c; Schommer et al., 2011), *Caenorhabditis elegans* (Begun et al., 2007) or animal models of device infections, e.g., central venous catheter or prosthetic device infection models (Wang et al., 2011a; Odekerken et al., 2013; Scherr et al., 2014b; Schaeffer et al., 2015). In early studies on the importance of biofilm formation *in vivo* using animal models and genetically unrelated, biofilm-positive or biofilmnegative *S. epidermidis* isolates failed to demonstrate conclusive evidence that biofilm forming isolates are more virulent compared to biofilm-negative strains. However, subsequent studies using genetically defined, isogenic pairs of biofilm-positive wildtype and biofilm-negative mutants were more conclusive. In a subcutaneous catheter infection model in mice and a central venous catheter infection model in rats biofilm-positive, PIA-producing *S. epidermidis* 1457 was more virulent than its isogenic biofilm-negative transductant 1457-M10 (Rupp et al., 1999a,b). An AtlE-deletion mutant of this strain was also attenuated (Rupp et al., 2001). In a rat central venous catheter model, expression of *icaRADBC* in *icaADBC*-negative *S. epidermidis* strains also led to increased virulence (Li et al., 2005). A *Caenorhabditis elegans* infection model was used to study biofilmpositive *S. epidermidis* 9142, showing attenuation of an *icaA* insertion mutant compared to its isogenic, PIA producing parent strain 9142 (Begun et al., 2007). Virulence was restored to wildtype in the biofilm-negative mutant by complementation with cloned *icaADBC* (Begun et al., 2007). However, in a collection of *S. epidermidis* infective endocarditis isolates, PIA expression and pathogenicity for *C. elegans* was not closely associated (Monk et al., 2008). More recently, using a catheter infection model in which realistic colonization modalities were chosen (i.e., infection after catheter insertion and not usage of pre-colonized materials) the inactivation of *icaADBC* had no apparent effect on colonization, while *aap* inactivation almost completely abolished the ability of *S. epidermidis* to establish an infection (Schaeffer et al., 2015).

A significant reason for the impaired pathogenicity of *icaADBC*-negative mutants in animals models is the improved ability of the innate immune system to clear biofilm-negative *S. epidermidis* (Schommer et al., 2011). There is significant experimental evidence from cell culture assays that indeed biofilm positive strain 1457 was less susceptible to killing by antimicrobial peptides and also displayed decreased phagocytosis and killing by polymorphonuclear granulocytes (PMNs) compared to its isogenic *icaA* mutant 1457-M10 (Vuong et al., 2004c). When *S. epidermidis* 1457 was either grown in a static biofilm or planktonic culture, the organism grown in a biofilm was less susceptible to phagocytic killing after opsonisation with normal human serum, as was an isogenic biofilm-negative *icaA*-insertion mutant (Kristian et al., 2008). PIA-dependent biofilm formation also interferes with host complement activation. Biofilm-positive wild-type bacteria pre-opsonised with normal human serum were more resistant to complement-dependent killing than the corresponding isogenic biofilm-negative bacteria (Kristian et al., 2008). There is, moreover, evidence that *S. epidermidis* biofilm formation interferes with phagocytic up-take and with pro-inflammatory activation of macrophages. This effect was irrespective of the intercellular adhesin used (Schommer et al., 2011). These phenotypes clearly could additionally contribute to the chronic persistent, low-grade inflammatory course of a *S. epidermidis* infection.

It is important to stress that, since *S. epidermidis* is an opportunistic pathogen, mechanisms of pathogenicity which are important in some types of device-related infection might be less crucial in others. For example, in the guinea pig tissue cage model (Zimmerli et al., 1982) there was no difference in virulence between a biofilm-positive wild-type *S. epidermidis* 1457 and its isogenic *icaA*-insertion mutant, and no difference between *icaADBC*-positive and -negative clinical isolates (Francois et al., 2003; Chokr et al., 2007). Nonetheless PIA was expressed *in vivo* in the tissue cages, and when animals were infected with both strains at the same time, the wild-type out-competed the mutant (Fluckiger et al., 2005). This may be because phagocytes are severely impaired in tissue cages (Zimmerli et al., 1984), masking the expected advantage of the wild-type.

*S. epidermidis* produces a number of pro-inflammatory peptides called phenol-soluble modulins (PSMs), which are produced in a strictly *agr*-controlled manner (Mehlin et al., 1999; Yao et al., 2005). PSM-δ rapidly lyses neutrophils, supporting the idea that the peptide is of relevance to the pathogenesis of *S. epidermidis*. However, PSM-δ is expressed only at low levels by *S. epidermidis* 1457, in line with a low overall cytolytic activity of *S. epidermidis* (Cheung et al., 2010). PSM-δ is expressed only at very low levels in *S. epidermidis* 1457 biofilms as compared to planktonic cells (Wang et al., 2011a). PSM-β peptides promote *S. epidermidis* biofilm structuring and detachment *in vitro* and dissemination of infection during catheter colonization *in vivo*, thereby providing the first mechanism of biofilm detachment in *S. epidermidis* (Wang et al., 2011a).

## **OUTLOOK AND FUTURE DIRECTIONS**

Over the past two decades, significant progress has been made in our understanding of the specific pathogenic nature of *S. epidermidis* in foreign material-associated infections. A molecular picture evolved showing that *S. epidermidis* virulence is linked to biofilm formation, a phenotype that depends on a wide variety of different factors which carry distinct and tightly regulated functions during surface colonization and interactions with host immune responses. Thus, *S. epidermidis* biofilm research has reached a turning point, at which on one hand additional *in vitro* evidence for the involvement of dedicated mechanisms in surface colonization can easily be accumulated, but on the other hand the question of the *in vivo* relevance of a given factor or process certainly arises. Thus, a major future challenge will be to translate findings from highly artificial, simple *in vitro* biofilm analysis systems into complex (organ-) models that more appropriately reflect the *in vivo* infection settings. Moreover, it is of urgent importance to validate findings from *in vitro* models in relevant animal models of device infections. These approaches should involve not only state of the art molecular biology, biochemical and immunological methods but also time-resolved *in vivo* and *ex vivo* imaging technologies, allowing to create a more distinct picture of the invasive *S. epidermidis* life style in different and extremely variable environmental conditions. Using the new armament of technologies, including three-dimensional cell culture techniques and tissue engineering, efforts are necessary to study the role of *S. epidermidis* as a beneficial skin commensal more intensively.

## **REFERENCES**

States hospitals: epidemiology, clinical features and susceptibilities. *Pediatr. Infect. Dis. J*. 22, 686–691. doi: 10.1097/01.inf.0000078159.53132.40

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 December 2014; accepted: 26 January 2015; published online: 17 February 2015.*

*Citation: Büttner H, Mack D and Rohde H (2015) Structural basis of Staphylococcus epidermidis biofilm formation: mechanisms and molecular interactions. Front. Cell. Infect. Microbiol. 5:14. doi: 10.3389/fcimb.2015.00014*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2015 Büttner, Mack and Rohde. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## *Streptococcus pneumoniae* biofilm formation and dispersion during colonization and disease

## *Yashuan Chao1, Laura R. Marks 2, Melinda M. Pettigrew3 and Anders P. Hakansson1,2\**

*1 Division of Experimental Infection Medicine, Department of Laboratory Medicine, Lund University, Malmö, Sweden*

*2 Department of Microbiology and Immunology, University at Buffalo, The State University of New York, Buffalo, NY, USA*

*3 Department of Epidemiology and Microbial Diseases, Yale School of Public Health, New Haven, CT, USA*

#### *Edited by:*

*Pietro Speziale, Università degli studi di Pavia, Italy*

#### *Reviewed by:*

*Odile Tresse, French National Institute for Agricultural Research, France Gregory T. Robertson, Colorado*

#### *State University, USA \*Correspondence:*

*Anders P. Hakansson, Division of Experimental Infection Medicine, Department of Laboratory Medicine, Lund University, Wallenberg Laboratory Floor 5, Inga Marie Nilssons Gata 43, Malmö SE-20502, Sweden e-mail: Anders\_P.Hakansson@ med.lu.se*

*Streptococcus pneumoniae* (the pneumococcus) is a common colonizer of the human nasopharynx. Despite a low rate of invasive disease, the high prevalence of colonization results in millions of infections and over one million deaths per year, mostly in individuals under the age of 5 and the elderly. Colonizing pneumococci form well-organized biofilm communities in the nasopharyngeal environment, but the specific role of biofilms and their interaction with the host during colonization and disease is not yet clear. Pneumococci in biofilms are highly resistant to antimicrobial agents and this phenotype can be recapitulated when pneumococci are grown on respiratory epithelial cells under conditions found in the nasopharyngeal environment. Pneumococcal biofilms display lower levels of virulence *in vivo* and provide an optimal environment for increased genetic exchange both *in vitro* and *in vivo*, with increased natural transformation seen during co-colonization with multiple strains. Biofilms have also been detected on mucosal surfaces during pneumonia and middle ear infection, although the role of these biofilms in the disease process is debated. Recent studies have shown that changes in the nasopharyngeal environment caused by concomitant virus infection, changes in the microflora, inflammation, or other host assaults trigger active release of pneumococci from biofilms. These dispersed bacteria have distinct phenotypic properties and transcriptional profiles different from both biofilm and broth-grown, planktonic bacteria, resulting in a significantly increased virulence *in vivo*. In this review we discuss the properties of pneumococcal biofilms, the role of biofilm formation during pneumococcal colonization, including their propensity for increased ability to exchange genetic material, as well as mechanisms involved in transition from asymptomatic biofilm colonization to dissemination and disease of otherwise sterile sites. Greater understanding of pneumococcal biofilm formation and dispersion will elucidate novel avenues to interfere with the spread of antibiotic resistance and vaccine escape, as well as novel strategies to target the mechanisms involved in induction of pneumococcal disease.

**Keywords: biofilm, colonization, streptococcus, virus, virulence**

## **BACKGROUND**

*Streptococcus pneumoniae* colonizes the upper respiratory tract in humans. Colonization occurs on the mucosal surface of the nasopharynx during childhood and persists asymptomatically in healthy individuals into adulthood (Gray et al., 1981; Hogberg et al., 2007). Pneumococcal carriage rates are greater in children compared to adults, with approximately 20–50% carriage rate in children and 5–20% in adults in higher resourced countries while even higher rates are seen in resource poor settings where up to 90% of children and over half of adults are colonized (Gray et al., 1980; Revai et al., 2008; Huang et al., 2009; Pebody et al., 2009; Mackenzie et al., 2010; Korona-Glowniak and Malm, 2012; Adegbola et al., 2014). Despite a low attack rate, transition from asymptomatic colonization to disease occurs often enough that the pneumococcus remains a leading cause of acute otitis media, pneumonia, sepsis, and meningitis globally (Sleeman et al., 2006; O'brien et al., 2009; Black et al., 2010). In 2011, *S. pneumoniae* caused an estimated 2,858,000 severe pneumonia episodes and 411,000 deaths worldwide in children under the age of 5 (Walker et al., 2013). The burden of disease is highest in resource poor settings where the lack of nutrition, antibiotics, and vaccines make the population particularly susceptible to disease.

## **PROPERTIES OF PNEUMOCOCCAL BIOFILMS INTRODUCTION**

Biofilms are highly-structured communities of cells that produce an extracellular matrix and adhere to abiotic or biological surfaces (Costerton et al., 1999; Donlan and Costerton, 2002; Stoodley et al., 2002). Antibacterial resistance is an inherent characteristic of biofilms and the protective biofilm matrix enables evasion of host immune responses, facilitating persistence, and dissemination of bacteria (Costerton et al., 1999; Donlan and Costerton, 2002; Chole and Faddis, 2003; Lewis, 2008; Sanchez et al., 2011b). In this context, resistance refers to an increased tolerance to antibacterials rather than a decreased susceptibility due to changes in the genome, such as mutations or obtaining antibiotic resistance genes. Pneumococcal colonization precedes disease and has been known to be more challenging to eradicate than invasive disease in patients as treatment with antimicrobial agents do not eliminate the majority of bacteria carried in the nasopharynx (Cohen et al., 1997, 1999; Dabernat et al., 1998; Dagan et al., 1998, 2001; Varon et al., 2000; Garcia-Rodriguez and Fresnadillo Martinez, 2002). Thus, a reasonable explanation for the decreased sensitivity of pneumococci to antimicrobial treatment during carriage is the formation of biofilm communities in the nasopharynx (Waite et al., 2001; Oggioni et al., 2006; Munoz-Elias et al., 2008; Trappetti et al., 2009; Sanchez et al., 2011b).

The original literature investigating pneumococcal biofilm formation *in vivo* detected biofilms during disease states such as otitis media, chronic rhinosinusitis, with some evidence for clustering of bacteria also during pneumonia (Hall-Stoodley et al., 2006; Sanderson et al., 2006; Hoa et al., 2009; Reid et al., 2009; Sanchez et al., 2011b). More recent data indicate that biofilm bacteria detected at disease sites represent asymptomatic colonization and, therefore, the presence of biofilms at sterile sites during disease presumably form a reservoir from which virulent bacteria may seed off under the right conditions, resulting in a role for biofilm bacteria in the disease process (Oggioni et al., 2006; Weimer et al., 2010; Sanchez et al., 2011b).

The vast majority of *in vitro* studies have been performed primarily on abiotic surfaces (Moscoso et al., 2006; Oggioni et al., 2006; Garcia-Castillo et al., 2007; Munoz-Elias et al., 2008; Domenech et al., 2009; Parker et al., 2009; Trappetti et al., 2009, 2011b,c; Sanchez et al., 2010, 2011a; Tapiainen et al., 2010; Camilli et al., 2011)., mimicking the classical models set up for organisms that confer problems in patients by producing biofilms on abiotic surfaces associated with medical devices. The extent of relevance these *in vitro* studies have *in vivo* is unclear as most of the biofilm formation experiments were conducted over short periods of time on abiotic surfaces that, as far as we know, are not major natural environments for the pneumococcal life cycle. For the same reason, *in vitro* studies on abiotic surfaces conducted for longer periods of time have unclear *in vivo* implications (Allegrucci et al., 2006; Allegrucci and Sauer, 2007; Vandevelde et al., 2014). Additional studies utilizing clinical isolates to study biofilms with longer biofilm formation times have been unable to show any association between the ability to produce *in vitro* biofilms on abiotic surfaces and *in vivo* virulence (Lizcano et al., 2010; Tapiainen et al., 2010). Furthermore, controversy exists in the literature regarding the correlation between biofilms grown *in vitro* on abiotic surfaces and their infectivity *in vivo* where investigators have suggested that biofilm bacteria are more likely (Trappetti et al., 2011b) or less likely (Sanchez et al., 2011b) to cause invasive disease. Our data at this point support the notion that biofilm bacteria are less virulent in invasive disease models (Marks et al., 2013). The virulence of biofilm bacteria will be covered in more depth in a separate review in this topic series by Orihuela et al. (Cross-reference to Orihuela review) (Gilley and Orihuela, 2014).

While these studies have been essential in building our understanding of pneumococcal accretion and biofilm formation, studies with more complex model systems that include physiological conditions and components modeling host–pneumococcal interactions have only recently shed more light on the phenotype of biofilm bacteria. In a study by Parker et al. bacteria recovered after adhering to epithelial cells had an increased ability to form biofilms on abiotic surfaces compared to bacteria with no previous exposure to epithelial cells (Parker et al., 2009). Also, Sanchez et al. found that biofilm bacteria grown on abiotic surfaces adhered better to epithelial cells than planktonic, broth grown bacteria (Sanchez et al., 2011b). These two studies, supported by studies in other human pathogens (Konkel et al., 1997; Sulaeman et al., 2012), demonstrate a relationship between epithelial cell adherence and biofilm formation, however, the studies have not investigated the role of this relationship during pneumococcal colonization.

## **FORMATION OF WELL-ORGANIZED AND STRUCTURED BIOFILMS DURING NASOPHARYNGEAL COLONIZATION**