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Current and Future Approaches to the Prevention and Treatment of Staphylococcal Medical Device-Related Infections S. Hogan1, N.T. Stevens1, H. Humphreys1,2, J.P. O’Gara3 and E. O’Neill1,4* 1 Department of Clinical Microbiology, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland; 2Department of Microbiology, Beaumont Hospital, Dublin 9, Ireland; 3Microbiology, School of Natural Sciences, National University of Ireland Galway, Ireland; 4Department of Microbiology, Connolly Hospital, Dublin 15, Ireland

Abstract: Staphylococci, in particular Staphylococcus aureus and Staphylococcus epidermidis, are a leading cause of healthcareassociated infections. Patients who have a medical device inserted are at particular risk of an infection with these organisms as staphylococci possess a wide range of immune evasion mechanisms, one of which being their ability to form biofilm. Once embedded in a biofilm, bacteria are inherently more resistant to treatment with antibiotics. Despite advances in our understanding of the pathogenesis of staphylococcal biofilm formation, medical devices colonised with biofilms frequently require removal. New and novel approaches to prevent and treat biofilm infections are urgently required. In recent years, progress has been made on approaches that include antiadhesive strategies to prevent surface adhesion or production of bacterial adhesins, dissolution of already established biofilm, targeting of biofilm matrix for degradation and interference with biofilm regulation. Several obstacles need to be overcome in the further development of these and other novel anti-biofilm agents. Most notably, although in vitro investigation has progressed over recent years, the need for biofilm models to closely mimic the in vivo situation is of paramount importance followed by controlled clinical trials. In this review we highlight the issues associated with staphylococcal colonisation of medical devices and potential new treatment options for the prevention and control of these significant infections.

Keywords: Biofilm, staphylococcus, prophylaxis, management. INTRODUCTION Implantable medical devices, such as prosthetic joints, heart valves and intravascular catheters have revolutionised the delivery of modern medical care. However, infection of these devices by surface-adhering bacteria can result in significant patient morbidity and mortality. Such device-related infections (DRIs) now represent a very common cause of healthcare-associated infection (HCAI). Evidence that DRIs are due to biofilm formation is well documented. Indeed, up to 80% of human bacterial infections are said to involve biofilm associated microorganisms [1]. Device related infections occur when microorganisms coat or enter into the lumen of a device (e.g. an intravascular catheter (IVC)). Nutrient rich blood products being passed through or around the device provide optimum growth conditions for bacteria. Microorganisms on medical devices are present in one of two forms: the planktonic free-floating form where organisms disseminate over the device or catheter surface and the sessile form where organisms have become embedded in an attached biofilm [2]. It is the latter in which the majority of microbes reside. Biofilms are structures interspersed with aqueous channels that facilitate transport of nutrients and waste products. In biofilm communities extracellular polymeric materials or surface associated adhesins anchor the cells to a surface and glue the bacteria together [3]. The biofilm shields the bacterial cells from physical and chemical attack and acts as a nutrient/water trapping device enabling microorganisms to survive desiccation and extended periods of time in low nutrient environments [4]. Characterisation of biofilm adhesins and extracellular matrices has been the focus of extensive research as to the first steps in the development of effective biofilm treatments [5-8]. After attachment of bacterial cells to a surface, the morphology and phenotype of the microorganism changes profoundly. Most cells within a biofilm exist in a stationary phase-like state, where transcription, translation and cell division are dramatically reduced [9]. The adaptive and genetic changes of *Address correspondence to this author at the Dept. of Clinical Microbiology, RCSI Education & Research Centre, Beaumont Hospital, Beaumont, Dublin 9, Ireland; E-mail: [email protected] 1873-4286/15 $58.00+.00

the cells within the biofilm make them highly resistant to conventional therapeutic doses of antimicrobial agents and clearance by the host response, often requiring device removal and placing the patients at increased risk of clinical complications [10]. Biofilm formation leads to persistent infections resistant to conventional antimicrobial treatment and is today a major cause of treatment failure. Substantial healthcare costs are caused by biofilm infections due to their high frequency, their resistance to antibiotic treatment and the need to remove the infected foreign body to cure the infection. Staphylococci are recognised as the most frequent cause of biofilm-associated infections. Amongst staphylococci, Staphylococcus aureus is regarded as the most virulent species due to its wide array of secreted and cell surface associated virulence factors, mechanisms of immune evasion and toxin production [11]. Over recent decades, coagulase-negative staphylococci, notably Staphylococcus epidermidis, have become increasingly recognised as opportunistic pathogens in patients with medical devices in situ. Indeed the majority of staphylococcal bloodstream infections are due to DRIs and many of these infections are associated with IVCs. Systemic antibiotics are usually administered to treat DRIs but, although generally effective in eliminating circulating planktonic bacteria, they frequently fail to sterilize the device or IVC, leaving the patient at a continuing risk of complications or recurrence. In this review, we present and discuss some of the key issues in relation to the management and treatment of staphylococcal DRIs and highlight potential new approaches to the prevention and/or treatment of these clinically significant infections. PATHOGENESIS OF STAPHYLOCOCCAL BIOFILM FORMATION In order to assess future treatment strategies, a clear understanding of the pathogenesis of the staphylococcal biofilm needs to be elucidated. There has been significant progress in this area in recent years resulting in a clearer understanding of stages involved in staphylococcal biofilm attachment and maturation. Biofilm formation by staphylococci has been proposed to occur in two stages, the © 2015 Bentham Science Publishers

Approaches to Staphylococcal Medical Device Related Infections

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first being the initial attachment to a surface and the second involving cellular proliferation and accumulation of a multi-layered biofilm (see Fig. 1). Cells within the biofilm are then released and may revert to planktonic state or they may have the potential to seed and form biofilms in other body sites [6, 12].

acids of staphylococci influence the net charge of the cell surface as well as playing a role in primary attachment [18] and interactions with epithelial cells [19]. Staphylococci contain two types of teichoic acid, peptidoglycan bound wall teichoic acids and membrane anchored lipoteichoic acids [19, 20].

Initial Attachment Initial attachment may involve specific and non-specific interactions of the bacterial cell with the surface being colonised. It is often the non-specific interactions such as cell surface hydrophobicity that influence adhesion to implanted medical devices [13]. The staphylococcal major autolysin, Atl, which is a bi-functional protein, along with teichoic acids is believed to be a significant contributor to cell surface hydrophobicity and has been implicated in primary attachment to surfaces. Atl is a wall-anchored peptidoglycan hydrolase and is the predominant peptidoglycan hydrolase in staphylococci [8, 14]. Houston et al. [8] demonstrated that an atl mutation reduces primary attachment rates and impairs biofilm formation on both hydrophilic and hydrophobic surfaces in methicillin resistant S. aureus (MRSA) isolates while only impairing methicillin sensitive S. aureus (MSSA) biofilm formation on hydrophobic surfaces. Staphylococci with atl null mutations have been reported to grow in clusters, have a rough cell surface and have impaired turnover of cell wall peptidoglycan [15]. Another important factor in primary attachment is surface characteristics of the implanted catheter. Following insertion into the patient, biomaterials are rapidly coated with a conditioning film comprising of host-derived matrix proteins, such as fibronectin, fibrinogen and collagen, some of which are known to act as receptors for bacterial attachment [16]. Staphylococcal binding to extracellular matrix proteins is mediated by a group of exposed surface proteins referred to as MSCRAMMs (microbial surface components recognising adhesive matrix molecules) [17]. These are cell surface ligand binding surface protein adhesins that allow staphylococci to readily bind to a variety of blood and extracellular matrix molecules [13]. Host cell wall components that interact with MSCRAMMs in S. aureus include the fibronectin binding proteins (FnBPA and FnBPB), the collagen binding protein (Cna) and the fibrinogen binding protein (ClfA), amongst others [13]. Teichoic

Biofilm Accumulation After initial attachment of bacterial cells to a surface, intracellular accumulation and biofilm maturation occurs. Clinical isolates of S. aureus are capable of producing at least two distinct types of biofilm mediated by either the genes in the intercellular adhesion operon (icaADBC) that encode the enzymes involved in the synthesis of polymeric N-acetyl-glucosamine (PNAG) / polysaccharide intercellular adhesion (PIA) (ica-dependent biofilm) or the fibronectin-binding proteins (FnBPs) (ica-independent biofilm) [21]. Under continuous-flow culture conditions PNAG/PIA and FnBP mediated biofilm results in significantly differing microscopic biofilm appearances of the extracellular matrix (see Fig. 2). For S. epidermidis biofilm formation PIA has been reported to be essential [22], but there have been a number of S. epidermidis PIA negative infections reported, with Rohde et al. reporting a small number of strains with the ability to form proteinatious biofilm [23]. ica-dependent biofilm: Production of PNAG/PIA, by icaADBC-encoded enzymes, is a well-defined mechanism of biofilm development in S. aureus and S. epidermidis [16, 24, 25]. The ica operon consists of four genes: icaA, icaB, icaC and icaD. The icaA gene encodes a transmembrane protein with homology to N-acetyl-glucosaminetransferases and requires IcaD for optimal activity. N-acetyl-glucosamine monomers produced by IcaAD reach a maximum length of 20 residues and it is only when IcaAD is coexpressed with IcaC that chain elongation occurs [26]. icaC encodes an integral membrane protein required for translocation of the polysaccharide through the cytoplasmic membrane. It is also likely to be involved in the translocation of the growing polysaccharide to the cell surface [16]. Finally, IcaB is a secreted protein, involved in the deacetylation of PIA/PNAG leading to positively charged Nacetyl–glucosamine residues [10]. ica-independent biofilm: Although the ica locus is maintained and expressed in 100% of S. aureus clinical isolates [27] recent

A: Immunisation

Patient B: Surface Coatings

D: Target biofilm matrix

C: Target attachment proteins

Skin Vessel

1: Attachment

3: Dispersal

2: Accumulation

Fig. (1). Model of staphylococcal biofilm development on intravascular medical devices and associated treatment strategies. Biofilm formation occurs in three stages: (1) Free floating planktonic cells attach to the catheter surface, (2) Cells accumulate to form a biofilm and (3) Cells disperse from biofilm to regain planktonic characteristics. There are several preventative/treatment strategies available (A) immunisation (B) coating the catheter with various agents to prevent against bacterial attachment (C) targeting the bacterial proteins associated with primary attachment and finally (D) targeting the biofilm matrix itself. Image adapted from Heilmann and Gotz [54].

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a)

Hogan et al.

b)

Fig. (2). Scanning electron microscopy images, at 30, 000 magnifications, of biofilm formation of a clinical MRSA strain under continuous-flow conditions in TSB-NaCl (a) and TSB-Glucose (b) media after 24 hours at 370C. Image a) is of an fnbAB mutation in this strain and represents PNAG/PIA dependent biofilm; whilst image b) is an ica mutation in this strain and represents a FnBP mediated biofilm. Image reproduced from Vergara-Irigaray et al. [175].

reports have highlighted the ability of S. aureus to produce icaindependent biofilm [6, 28]. Such reports have also emerged for a number of S. epidermidis strains [23]. In S. aureus this type of biofilm is mediated by the FnBPs, as well as other cell surface components such as teichoic acids and MSCRAMMs [21]. FnBPs were identified based on their ability to bind to fibronectin, however they have also shown to be capable of binding to elastin and fibrinogen [29]. Two FnBPs have been identified, namely FnBPA and FnBPB, and previous work in our laboratory has shown their importance for the accumulation phase of biofilm development in MRSA isolates. FnBPA and FnBPB can functionally substitute for each other as single mutations in fnbA or fnbB, are closely located but independently transcribed, and do not impair biofilm formation whereas an fnbAB double mutation abolishes biofilm [30] [31]. The FnBPs are large multifunctional proteins and recent analysis to investigate the mechanism of FnBP-mediated biofilm has identified an essential role for subdomains N2N3 of FnBPA A domain [32]. Ica-independent biofilm formation in S. epidermidis has been shown to be strongly linked to two cell wall associated proteins in particular, the accumulation associated protein (Aap) and a biofilm associated protein (BAP) homologue, termed Bap-homologue protein (Bhp) [33]. Biofilm Regulation Staphylococci, in particular S. aureus, express a wealth of pathogenic determinants or virulence factors. The regulation of these virulence factors is tightly controlled [34]. The bacteria must constantly adapt to changing environments. Production of virulence factors is metabolically expensive and therefore appropriate regulation is vital to ensure survival of the pathogen [35]. The main staphylococcal global regulators are described below. Accessory gene regulator (agr): Cells within a biofilm are an integrated community. This community uses quorum signalling (cell-to-cell signalling) to coordinate gene expression involved in attachment, maturation and detachment of the cells in the biofilm. These occur in response to environmental stimuli. The best characterised quorum sensing system in staphylococci is the agr system. The agr locus was first identified and described by Recsei et al. [36]. About 150 genes have been shown to be regulated by a putative multi-component signal transduction system encoded by the agr locus [37], with at least 16 of them being associated with virulence genes [38]. The agr locus facilitates communication between bacteria by producing and sensing a molecule called an autoinducing peptide (AIP). The agr locus consists of two divergent promoters P2 and P3 which modulate transcription of two transcripts RNAII and RNAIII respectively. The RNAII transcript contains four genes agrB, agrD, agrC and agrA. The RNAIII transcript acts as the effector molecule of the agr locus [39].

Activation of the agr system has been shown to increase the production of extracellular proteases via the effector molecule RNAIII. These proteases in turn can effect biofilm development and detachment. Recent studies have shown that upon dispersal from the biofilm, bacteria display high levels of agr activity whereas agr expression is repressed in cells that remain within the biofilm [6]. Activation of the Agr system and increased levels of protease activity degrades protein adhesins that cement the cell within the biofilm of ica- and polysaccharide-independent biofilms. Thus, mutations in agr can promote ica-independent biofilm while having a neutral effect on ica-dependent types of biofilm [6, 40-42]. We recently reported that in S. aureus, expression of high level methicillin resistance is accompanied by repression of both the agr and ica loci [35], which is consistent with the expression of protein adhesion mediated biofilm in clinical MRSA isolates. Staphylococcal accessory regulator (sarA): The staphylococcal accessory regulator, SarA, is a central regulatory element that controls the expression of staphylococcal virulence factors [43]. It was shown by Beenken et al. that sarA plays an important role in the expression of both surface-associated and secreted virulence factors [5]. It can also affect the expression of up to 120 additional genes, including upregulation of cell wall proteins and selected exoproteins and the down regulation of protein A and various proteases [5, 44]. Staphylococci with sarA mutations have been demonstrated to impair both ica-dependent and ica-independent biofilm formation [5, 30]. Alternative sigma factor B (B): Alternative sigma factors allow bacteria to adapt to metabolic and environmental stresses. SigB is an alternative sigma factor of S. aureus, and is homologous to SigB of Bacillus subtilis [45]. The staphylococcal sigB operon consists of four genes encoding the RsbU, RsbV, RsbW and SigB proteins. RsbUVW are regulatory proteins that control the activity of SigB [46, 47]. SigB controls over 250 genes involved in a diverse range of cellular processes. Many adhesins are upregulated by SigB while transcription of various exoproteins and toxins are repressed suggesting that sigB is an important modulator of virulence [48]. CURRENT STRATEGIES TO PREVENT AND TREAT STAPHYLOCOCCAL DEVICE-RELATED INFECTIONS Several approaches have been used in an attempt to decrease the incidence of DRIs with multiple national and international guidelines published on the subject. For example, epic2: National evidence based guidelines for preventing HCAI in National Health Service hospitals in England, provide comprehensive recommendations on hand hygiene, the use of personal protective equipment and the prevention of infections with the use of indwelling urethral


catheters and central venous access devices [49]. In recent times the use of “care bundles”, which can be defined as “the bundling together of several scientifically grounded elements essential to improve clinical outcome” have been developed and used in the prevention of DRIs [50]. Care bundles provide a method for establishing evidence-based best clinical practice. However, a recent Cochrane review on adherence to guidelines for the prevention of DRIs found insufficient evidence to determine, with certainty, which interventions are most effective [51]. Examples of other techniques used in the prevention of DRI include the use of chlorhexidine impregnated sponges for the prevention of catheter-related infections [52] and the application of prophylactic antibiotic ointment at a device exit site [53]; all of which have been used with various success rates. Treatment of staphylococcal DRIs remains a significant challenge. In the human host, bacteria form a biofilm on a biotic (e.g. an epithelial cell) or an abiotic (foreign body) surface [54, 55]. The abiotic surface is usually coated with host matrix proteins, including fibronectin, fibrinogen and collagen [55]. Staphylococci contain numerous cell wall bound surface proteins that bind to these matrix proteins (also referred to as the previously described MSCRAMMs) [56]. Mature biofilms are highly resistant to the action of the innate and adaptive immune defence systems, and to the action of antimicrobial agents. Numerous research groups have demonstrated the ineffectiveness of antibiotics against bacteria within a biofilm compared to their effectiveness versus the planktonic counterparts. Hence, antibiotics have limited success in achieving device sterility and the resolution of biofilm and infection [57, 58]. Indeed a study by Olson et al. demonstrated a one thousand times increase in the minimum inhibitory concentration (MIC) of bacteria to antibiotics when embedded in a biofilm [59]. This is due to the requirement for antibiotics to penetrate through the biofilm and have activity against biofilms cells with an altered growth rate and metabolism. Antibiotics such as oxazolidinones, tetracyclines, glycopeptides and lipopeptides have been used alone or in combination with variable success [60-62]. Rifampicin, which is an inhibitor of RNA synthesis, has been shown in a number of studies to effectively penetrate biofilms [63-65]. However, rifampicin should always be used in combination with other antimicrobials as monotherapy accelerates the emergence of rifampicin resistant strains [66]. In relation to IVCs, the use of antibiotic/antiseptic locks to prevent or treat these biofilm-related IVC infections has generated considerable interest. Antimicrobial lock solutions are used to fill the lumen of the IVC for a defined period of time before removal in order to prevent or eradicate biofilm formation [67]. This technique provides very high concentrations of antimicrobial agents at the site of infection/potential infection, with a low incidence of systemic toxicity from these antibiotics. However, concerns that their widespread use would lead to the selection of resistant organisms have thus far limited their application in the treatment of DRIs. A metaanalysis evaluating the use of antimicrobial lock therapies (ALTs) for the prevention of IVC-related infections in haemodialysis patients showed that the use of antibiotic locks was associated with a reduction in rates of catheter-related bloodstream infections (CRBSIs). However, a final recommendataion on which antimicrobial lock solution to choose (different antimicrobial agents were assessed in the meta-analysis, i.e. vancomycin, gentamicin, minocycline) was not addressed [68]. The Infectious Diseases Society of America guidelines on the management of CRBSIs recommend the use of ALTs for the salvage of an IVC associated with CRBSI [69]. However, the choice of which antibiotic should be used in the ALT is often based on the in vitro susceptibilities using conventional susceptibility testing, which may not necessarily indicate that the antibiotic is active against the same organisms embedded in the biofilm [59]. Another effective technique involves filling indwelling catheters with a solution that consists of antibiotics and anticoagulants. This has been shown to prevent bacteria from colo-


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nizing the surface of the catheter and thus preventing catheterrelated infections [70]. Heparin, or heparinised saline, has also been used in many institutions as a catheter locking solution. This solution prevents thrombosis effectively. However bacteria such as staphylococci may survive in heparin-locked catheters because of the limited antimicrobial activity of heparin, and in some studies it has even been shown to actually stimulate staphylococcal growth [71]. Other ALTs such as sodium citrate and ethanol solutions have been investigated by several groups [72-75]. Sodium citrate is the sodium salt of citric acid. There are three sodium salts, of which the most common is trisodium citrate (often simply referred to as sodium citrate). Traditionally trisodium citrate was used as an anticoagulant as the citrate chelates calcium ions in the blood disrupting the blood clotting process [74]. In recent years, data has emerged highlighting the antimicrobial properties of this sodium citrate solution both by itself and in combination with other agents [76]. It is the main component of two commercially available antibiotic lock solutions (ALSs), namely Duralock-CTM and TuraLockTM. Duralock-CTM is a 46.7% solution of sodium citrate, while TuraLockTM comprises of 4% sodium citrate and 1.35 % taurolidine, a modified amino acid with antimicrobial properties. While both solutions have proved effective against CRIs, when compared to heparin [77], enough data has not been given to justify long-term or widespread use [78]. A wider study comparing these solutions to a broader range of potential ALSs in the prevention or treatment of staphylococcal biofilm infections may help determine if its use should be recommended. Ethanol has long been reported to have antibacterial activity associated with surface protein denaturation and solubilisation of lipids [79], and has been shown to be an effective ALS, reducing infection rates in a number of studies [80, 81]. Despite this, there are a number of potential drawbacks to the use of ethanol as it may not be compatible with all catheters. For example, one study suggests an incompatibility with polyurethane [82] and it has also been associated with patient side-effects such as “inebriated like” symptoms. Further work is needed to examine this solution and its suitability for use in sterilising an IVC. Chlorohexidine, is a cationic antiseptic compound with two strong basic groups, both biguanides. At low concentrations it has little toxicity and it has been reported to be suitable for human use [83]. Chlorohexidine acts by destabilizing the bacterial cell wall, of both gram-positive and gramnegative bacteria, and then attacks the bacterial cytoplasmic membrane (inner membrane), resulting in leakage of cell components and cell death [84]. It has been suggested that due to its broad spectrum antimicrobial properties and relatively low toxicity it would make a suitable ALS. Currently the decision to use antimicrobial lock prophylaxis/treatment and the choice of antimicrobial agent to be used follows a positive microbiology culture and is often decided on an individual patient basis in conjunction with the infection and pharmacy specialists. There is as yet no consensus on what is the best approach, partly because there is insufficient insight and knowledge about which agent, or combination of agents, is likely to be most efficacious in vivo against biofilm-forming bacteria [9]. NEW AND EVOLVING APPROACHES TO THE PREVENTION AND TREATMENT OF STAPHYLOCOCCAL BIOFILM INFECTIONS Strategies aimed at interrupting staphylococcal adhesion and accumulation will keep the bacteria in the less virulent planktonic growth phase, which restores the efficacy of antibiotics and reduces the risk of staphylococcal DRIs. Targeting the early stages of biofilm development represents an attractive prophylactic strategy, which could potentially involve the use of antibodies, or other agents that inhibit biofilm formation, or it could involve treatment with compounds that will block the initial attachment. Whereas,


Table 1.

Hogan et al.

Examples of preventative and therapeutic strategies, along with suggested mode of action, for the management of staphylococcal device related infection.

Therapeutic/Prophylactic Strategy

Mode of action

Immunisation PNAG/PIA

Target polysaccharide mediated biofilm

rClfA/rCLFB

Stimlates antibody production to the surface protein, clumping factor A and B

rIsdB

Vaccine versus conserved iron-sequestering protein in staphylococci

cna-FnBP

Fusion of fnb and cna to create recombinant protein to prevent staphylococcal biofilm adhesion.

SecC

Recombinant vaccine to surface exposed protein C in S. epidermidis

Charge modification

Hydrophilic negatively charged surfaces repel bacteria and prevent attachment.

Metals e.g. silver, copper

Prevent bacterial adhesion.

Hydrophilic polysaccharides/resins

Bind to biomaterial to create hydrophilic coating preventing attachment.

Attachment

Enzymatic disruption Lysostaphin

Cleaves cross linking pentaglycine bridges, causing cell lysis

Dispersin B

Glycoside hydrolase enzyme, degrades PIA/PNAG

DNase I

Degrades cell surface-associated nucleic acids directly or indirectly

V8

Serine protease which cleave peptide bonds, such as glutamic acid, to disrupt protein mediated biofilm

Photodynamic treatment TBO

Generation of free radicals and cytotoxic reactive oxygen species

Chlorin (e6)

Generation of free radicals and cytotoxic reactive oxygen species

N-acetyl-L-cysetine (NAC)

Disrupts disulfide bonds and inhibits amino acid utilization.

Ethanol

Membrane disruption and protein denaturation.

Chlorohexidine

Destabilizes the bacterial cell wall and targets bacterial cytoplasm.

Apo-transferrin

Inhibits staphylococcal biofilm attachment to surfaces

EDTA

Chelator of metal ions with high affinity for magnesium

EGTA

Chelator of metal ions with high affinity for calcium

PGG

Chelates iron and enhances expression of iron-regulated genes

Antiseptics

Chelators

Quorum sensing targets AIP

Induces activation of agr system with increased levels of proteases and PSMs

PSMs

Surfactant properties to disrupt staphylococcal biofilm

Small molecules/Sortase inhibitors C2DA

Chemical messenger which signals prevention and dispersion of biofilm

Diarylacrylonitriles

Sortase inhibitor; inhibition of staphylococcal biofilm formation

Aryl ethyl ketones

Sortase inhibitor; inhibition of staphyloccal biofilm formation

Vinyl sulfones

Inhibition of sortaseA mediated linkage of fibronectin to the cell surface



105

(Table 1) Contd…. Therapeutic/Prophylactic Strategy

Mode of action

Bacteriophage Bacteriophages

Degrade cell wall

Antimicrobial peptides Cathelicidin

Cytoplasmic membrane

LL-37

Cytoplasmic membrane, inhibit initial attachment

treatment of the established biofilm requires targeting of, later stages in biofilm development such as enzymatic disruption, quorum sensing, etc. These and other approaches are summarised in Table 1. Immunisation Although the majority of staphylococcal immunotherapy approaches target acute disease and bacteria in the planktonic stage the use of immunisation or vaccines to prevent staphylococcal DRIs is an evolving area of research. In the context of biofilm infections, two targets exist, namely the bacterial cells within the biofilm and the biofilm matrix itself [85]. The staphylococcal biofilm matrix is composed of polysaccharide, protein or eDNA depending on species and strain. As previously highlighted, PNAG/PIA, is a surface polymer produced by both S. aureus and S. epidermidis that promotes biofilm formation via an ica-dependant route. Active or passive immunisation with PNAG/PIA has been shown to be protective against S. aureus infection in a kidney infection model [86]. However, research has indicated that only one component of PIA is immunogenic, and responses to this antigen are variable [87]. Furthermore, not all S. aureus or S. epidermidis produce PNAG/PIA, and further research is required to determine the effectiveness of PNAG/PIA-based vaccines. Surface proteins of staphylococci, such as MSCRAMMS, represent a prototype of targets because of their exposed location and role in virulence [88]. Recombinant clumping factor A (rClfA) containing the fibrinogen binding domains, has been shown to be partially effective when used in an animal model of septic arthritis [89]. Immunisation with recombinant clumping factor B (rClfB) has been shown to lead to lessened colonisation of murine nares by S. aureus [90]. Vaccination with iron-regulated surface determinant B led to increased survival rates of 20-40% in a murine sepsis model [91] and a fusion protein consisting of the binding regions of collagen-binding protein and fibronectin-binding protein showed some protection in a mouse intraperitoneal model [92]. A recent study examined four S. aureus protein targets (glucosaminidase, an ABC transporter lipoprotein, a conserved hypothetical protein and a conserved lipoprotein) that were found to be immunogenic and upregulated during a chronic biofilm infection and report that a quadrivalent vaccine with these components, co-administered with the antibiotic vancomycin, significantly reduced MRSA osteomyelitis in a rabbit model of infection [93]. Shahrooei et al. also reported that active vaccination with the recombinant S. epidermidis surface exposed protein C (SesC) inhibited S. epidermidis biofilm formation in a rat model of subcutaneous foreign body infection [94]. These may represent promising targets in the future for antibody mediated strategies against staphylococcal biofilm. Altering Adhesion to Indwelling Medical Devices The materials used to produce medical devices, the surface of which are mostly chemically inert, has also been an area of research in recent times. As one of the requirements for staphylococcal biofilm infection is adherence to the polymer surface, efforts have been made to find materials that prevent adherence. Anti-adhesives

prevent bacterial adhesion by forming a highly hydrated layer on the surface of the biomaterial. They do this by binding to the biomaterial and then taking up free water molecules that then become trapped in hydrophilic chains [95]. Therefore, hydrophilic surfaces and negative charges on the biomaterial generally show less bacterial adhesion than hydrophobic ones [96]. Strategies to prevent biofilm formation include physiochemical modification of the biomaterial surface to create anti-adhesive surfaces, incorporation of antimicrobial agents into medical device polymers, mechanical design alternatives and release of antibiotics. Medical devices or catheters have been coated or impregnated with various antibiotics (e.g. minocycline, rifampicin), antiseptics (e.g. chlorhexidine, silver sulfadiazine) and metals (e.g. silver, platinum) [97]. Others, have shown that polypropylene mesh sleeves coated with rifampicin and minocycline prevented S. aureus biofilm on pacemakers in a rabbit endocarditis model [98]. However, the success of these alternatives has been varied, mainly due to the various environments into which devices are placed, the diversity of ways in which organisms can colonise surfaces and the mounting concerns regarding the emergence of antimicrobial/antiseptic resistance. Nano-structured reservoirs, which can be integrated with antibacterials (e.g. silver) for surface coating or as a cement additive for bone implants, is also an emerging promising area allowing controlled drug release over a prolonged period of time [99, 100]. Fiedler et al. recently reported that copper and silver ion implantation on Ti6AIV4 alloy surfaces stimulates osteoblast proliferation and prevents bacterial contamination [101]. Since bacterial adhesion arises from non-specific passive adsorption of proteins of the extracellular matrix, such as fibronectin and collagen, the concept of preventing protein adsorption by a material surface, able also to favour osseointegration, may have a significant impact on the prevention of orthopaedic implant infections. Antiadhesives have also been the focus in other areas of biofilm research for the prevention of staphylococcal infections. Carpobol 934, is a hydrophilic resin that is a cross-linked polyacrylate polymer, which prevents adhesion [102]. Hyaluronan is a natural antiadhesive hydrophilic polysaccharide that is found in the body fluids of all vertebrates. It is found at particularly high concentrations in connective tissue and it can form a covalent bond with the biomaterial after contact [103]. Hyaluronan is a particularly attractive option for prevention of DRI as it has also been shown to have bacteriastatic properties against a range of bacteria including S. aureus [104]. Both carpobol 934 and hyaluronan have been shown to exhibit great potential to prevent biofilm formation [102]. Further research is warranted to determine their effect on bacteria embedded within a biofilm and to test the ability of these molecules to coat different biomaterials such as catheters. Enzymatic Disruption of Mature Biofilm When a mature staphylococcal biofilm has formed on a device, conventional antimicrobial treatment has a limited role in the eradication and treatment of DRIs. However, there are a wide variety of agents and enzymes that have the ability to depolymerise compo-


nents in a formed staphylococcal biofilm and have been the subject of many recent research approaches aimed towards investigating their effectiveness in removing a biofilm from a surface. However, it must be acknowledged that one of the main concerns with enzyme based biofilm therapy is the risk of dispersal and seeding of bacteria to other organs. Therefore, such therapies should be used in combination with systemic antimicrobial agents. Nevertheless, enzymatic degradation of the biofilm matrix represents a promising group of agents which may be used in the future. Lysostaphin has been shown to be effective in the disruption of staphylococcal biofilms [105]. It causes lysis of staphylococci by cleaving the cell wall peptidoglycan pentaglycine bridges and has been demonstrated to have in vitro anti-biofilm properties within hours of exposure [105-107]. Kun et al. demonstrated eradication of established S. aureus biofilms from implanted catheters and sterilized heart and liver infections of S. aureus infected mice using an animal model of infection with high dose lysostaphin (15mg/Kg three times a day) in combination with systemic nafcillin. Furthermore, a single pre-instillation of 10 mg/Kg lysostaphin in catheters was shown to completely protect catheterised mice from a subsequent biofilm infection [107]. Lysostaphin has also been used with clarithromycin to eradicate biofilm [108]. Lysostaphin is thought not to have a direct enzymatic effect on eukaryotic cells and has little toxicity [107] but further studies are required. Recent research has investigated dispersin B, which is a glycoside hydrolase enzyme produced by Aggregatibacter actinomycetemcomitans, a human periodontal pathogen, with preliminary data indicating non-toxicity to human cells [109]. It has been shown to have the capability to hydrolyse and degrade the PIA/PNAG produced by a number of staphylococci during biofilm formation, even at concentrations as low as 40 ng/ml [110, 111]. The effectiveness of dispersin B against PNAG/PIA-independent S. epidermidis and S. aureus biofilms is less clear but it could be used in combination with agents targeting other constituents of staphylococcal biofilm such as eDNA and surface proteins. eDNA has been shown to be a major component of the biofilm matrix [110]. DNase I is an endonuclease that nonspecifically cleaves DNA and has been shown to have a role in inhibition and detachment of staphylococcal biofilm [112]. Inhibition of biofilm formation by DNase I is thought to occur by degradation of cell surface-associated nucleic acids that function as surface adhesins [110, 113] or by direct degradation of eDNA [114]. With regard to biofilm detachment, studies have shown DNase I to be more effective in detaching recent rather than established biofilm [113]. Pulmozyme® is a recombinant human DNase I administered by inhalation, without toxic side effects, to treat Pseudomonas aeruginosa infections in patients with cystic fibrosis. Its potential role in the possible prevention or treatment of staphylococcal device related infections remains unclear. The role of proteases in biofilm formation and dispersal is acknowledged and these may represent another prophylactic or therapeutic option. In S. aureus deletion of the genes encoding the extracellular proteases Aureolysin and Spl resulted in a significant increase in biofilm formation in a flow study, suggesting a key role for protease activity in biofilm dispersal [40]. A neutral protease, termed V8, is also produced by S. aureus. V8 is a serine protease implicated in the adhesion of S. aureus cells to each other and to surfaces during the course of infection [115]. This protease has been shown to inhibit biofilm formation of staphylococcal isolates that typically produce a proteinaceous biofilm [30]. Its ability to inhibit and perhaps disrupt proteinaceous biofilm can be attributed to the cleaving of glutamic acid, the most abundant amino acid of FnBP [115, 116]. Other enzymatic biofilm dispersal agents such as proteinase K, trypsin and pancreatin have also been examined; however, the action of these agents is limited to proteinaceous biofilms [117-120].

Hogan et al.

Overall the potential to enzymatically disrupt biofilm should be examined in greater detail. Enzymatic therapies may have the potential to be used in combination, for example combining an agent such as Dispersin B with a protease to target both polysaccharide and protein mediated biofilm may be a viable biofilm treatment option in the future. Photodynamic Treatment of Biofilm An interesting and novel approach to the treatment of staphylococcal biofilm infections is the use of photodynamic therapy (PDT), a process where microorganisms are treated with a photosensitizer drug and then with low-intensity visible light of an appropriate wavelength [121]. This process generates a cytotoxic reactive oxygen species and free radicals that are able to exert a bactericidal effect that can inactivate staphylococcal biofilms adhering to medical devices, without affecting eukaryotic cells. Sharma et al. described the significant dose dependent inactivation of a S. epidermidis and a MRSA strain exposed to toluidine blue O (TBO) and laser treatment [122]. A recent study demonstrated the activity of highly pure chlorin, which is widely used as a second generation photosensitizer, in the eradication of S. aureus biofilm in a mouse model of infection [123]. A greater photo-efficiency for killing bacteria is obtained when the photosensitizer molecule is able to penetrate deeper into the bacterial membranes. PDT has been used in the eradication of biofilm in dental plaques and on oral implants [124]. In the future PDT may become a complementary therapy in clinical practice depending on various pharmacokinetics of the photosensitizer and on the accessibility to the device site. Antiseptic Agents and Chelators Given the large range of antiseptics available in modern medicine, many of these have been shown to be safe for human use as mouthwashes and for topical application. With many demonstrating a broad spectrum of antimicrobial properties, being either bacteriostatic or bactericidal, and having a non-toxic side effect profile there is a potential to further expand these agents for use in staphylococcal biofilm treatment. Drago et al. reported the ability of Nacetyl-L-cysteine to disaggregate biofilm produced by S. aureus on polyethylene and titanium discs after three hours of incubation, suggesting a possible role for N-acetyl-L-cysteine in the treatment of orthopaedic prosthetic infections caused by S. aureus [125].Indeed, there have been a number of other studies evaluating a range of agents in recent times [126-129]. These agents may have been applied topically on wound dressings or systemically. A clear understanding of the mechanism of action and cytotoxicity profile is of significant importance in evaluating the potential use of such compounds. The role of iron in staphylococcal biofilm formation appears multi-factorial. Several studies have demonstrated that iron positively regulates staphylococcal biofilm formation. For instance, a biological iron chelator, apo-transferrin, inhibits adhesion of S. aureus and S. epidermidis to polystyrene, polyurethane and silicone surfaces [130]. Furthermore, catecholamine inotropes that extract iron from plasma iron-binding proteins, stimulate biofilm formation by S. epidermidis, which suggests that S. epidermidis requires iron to adhere to solid surfaces and form biofilms [131]. However, Johnson et al. demonstrated that iron prevents biofilm formation by S. aureus strain Newman [132]. Chelators such as ethylenediaminetetraacetic acid (EDTA), which is a chelator of metal cations, has previously been approved for the treatment of mercury and lead poisoning. The effect of EDTA on bacterial cells has been shown to be bacteriostatic by destabilising divalent cations in bacterial cell membranes. This subsequently leads to the release of various cell components and has been demonstrated, on several occasions, to be effective on staphylococcal biofilms in vitro [133-135]. EDTA may have the potential to be used in treatment of DRIs and has been used in com-


bination with antibiotics in ALT in the treatment of catheter-related infections [135]. Another less well known chelating agent, similar to EDTA, is ethelyene glycol tetraacetic acid (EGTA). This agent has a much higher affinity for calcium than magnesium ions and proved very effective against staphylococcal biofilm [136]. However, another study reported an increase in biofilm, which was linked to the surface adhesion clumping factor B (ClfB) [136]. Lin et al. recently reported the role of iron in S. aureus biofilm development and that the plant extract 1, 2, 3, 4, 6-Penta-O-galloyl-D-glucopyranose (PGG), which has a low cytotoxicity profile, inhibits biofilm formation by S. aureus. This study highlighted the potential for coating PGG on polystyrene and silicon rubber surfaces to prevent biofilm formation [137, 138]. In addition, PGG chelates iron and enhances the expression of S. aureus ironregulated genes. Overall, while chelating agents have been shown to be effective in some studies, more in depth research is needed to help determine their safety and efficacy. Quorum Sensing System and Biofilm Dispersal In recent years, research has been undertaken in the area of targeting the quorum sensing mechanism of staphylococcal communication [6, 41, 139]. Staphylococcal quorum sensing systems may impact on the pathogenesis of staphylococcal biofilm infections by: influencing global gene expression via the agr-system and the effector molecule RNAIII, decreasing the expression of several cell surface MSCRAMMs or up-regulating the expression of many secreted virulence agents such as extracellular proteases (e.g. aureolysin, serine and cysteine proteases) resulting in detachment by degrading protein adhesins that cement the staphylococcal cell within PNAG/PIA-independent biofilms [6, 40-42]. In addition to proteases, other agr regulated factors contributing to biofilm detachment include toxins such as -toxin [140]. The activation of the agr quorum sensing system occurs in response to the extracellular concentration of an autoinducing peptide (AIP) signal that binds to the membrane-bound receptor domain of the AgrC histidine kinase. Therefore, the use of AIP signal molecule analogues have been indicated as a potential antibiofilm strategy [6]. To support this, Boles et al. reported that bacteria are dispersed from a staphylococcal biofilm when they display high levels of agr activity. Conversely, agr expression is repressed in cells that remain within the biofilm. Furthermore, the addition of exogenous AIP was shown to activate the agr system throughout a mature S. aureus biofilm leading to complete disassembly and conversion of bacteria to a planktonic state [6]. However, there are a number of challenges to overcome if this is approach is ultimately to be used in the treatment of staphylococcal DRIs. The activation of agr may have an impact on the functioning of host innate and adaptive immunity or convert the staphylococcal strain into a more invasive pathogen [9, 141]. Furthermore, across the staphylococcal species there are a variety of agr systems with each type recognising a unique AIP (see Fig. 3a for an example of the chemical structure of AIP-1). A universal agr activator has to be selected and produced before a therapeutic approach based on AIP analogues can be used on patients. Other influences on the agr system in relation to staphylococcal biofilm pathogenesis include inducing the expression of phenolsoluble modulins (PSMs). PSMs are a family of amphipathic helical peptides including the well documented staphylococcal toxin [142, 143], which have been suggested to have surfactant properties [144]. PSMs have recently been shown to be involved in biofilm detachment in a mouse model of S. epidermidis DRI and it has therefore been suggested that exploiting this mechanism to target the bacteria could have therapeutic potential [145]. Mannoprotein, a cell wall component of Saccharomyces cerevisiae, also has surfactant properties and has been used as an inhibitor of S. aureus and S. epidermidis biofilm formation [146].


107

Small Molecules and Sortase Inhibitors The potential role of small molecules such as fatty acids and amino acids, and sortase inhibitors in the treatment or prevention of staphylococcal biofilm has also been investigated recently. The fatty acid messenger cis-2-decenoic acid (C2DA) and 2aminoimidazole containing compounds have been of particular interest. C2DA is a medium-chain fatty acid chemical messenger produced by bacteria to signal prevention and dispersion of biofilms for multiple types of bacteria [147]. Reports of several other medium- and long-chain fatty acids studies have indicated that C2DA may also have growth inhibitory or bactericidal effects [148]. A recent study reported that higher concentrations of C2DA inhibit bacterial growth and biofilm formation, and that the combination of C2DA with antibiotics may have had additive or synergistic effects on biofilm inhibition [149]. Therapeutic interventions with C2DA could be in the form of local delivery of the fatty acid directly to potentially contaminated tissue. Biofilm studies on fatty acids such as oleic acid, which is reported to be the predominant bactericidal unsaturated fatty acid naturally present in staphylococcal abscesses and on the skin surface, have shown that oleic acid inhibited primary adhesion but, of concern, it increased biofilm production in eight S. aureus strains tested [150]. Synthetically modified compounds with 2-aminoimidazole containing scaffolds have been shown to exhibit increased ability to inhibit and disperse bacterial biofilms at non-microbicidal concentrations [151-153]. A recent study reported the ability of Nsubstituted derivatives of a 2-aminoimidazole containing compound to inhibit biofilm formation by MRSA; as well as synergistic activity with oxacillin against planktonic MRSA [154]. Overall, this is an evolving area, where there is limited information on the precise mechanism of action or toxicology data for many of these agents. Due to the key role of sortase enzymes, including anchoring MSCRAMMS to the bacterial cell wall in S. aureus and survival within phagosomes [155], the effect of their inhibition on biofilm has also been studied with numerous molecules identified as potential inhibitors of sortase enzymes. Indeed, deletion of srtA in clinical MRSA isolates impaired biofilm formation [30]. Furthermore, the localisation of SrtA within the cell membrane of staphylococci offers an advantage in terms of ease of access to this target. Oh et al. have reported on a novel class of S. aureus sortase inhibitors, the diarylacrylonitriles [156], with a derivative of this molecule ((Z)-3-(2, 5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA)) effective in treating bone and joint infections in a mouse model of S. aureus infection (see Fig. 3b for an example of the chemical structure of DMMA) [157]. Also, the aryl (B-amino) ethyl ketones and pyridazinone and pyrazolethione analogues, have been shown to reversibly inhibit expression of srtA [158, 159]. Frankel et al. reported the potential of small molecule vinyl sulfones for the treatment of S. aureus infections via inhibition of SrtA-mediated linkage of the FnBPs to the cell surface, highlighting the potential use of these agents to prevent adhesion to and colonization of host tissues during S. aureus infection [160]. Of concern the inhibition of sortase enzymes, by preventing the display of surface antigens, may dampen the host immune response which is required for bacterial clearance. Detailed in vivo evaluation of these and other inhibitors should be performed to assess therapeutic response and toxicity. Novel Delivery Mechanisms Novel delivery mechanisms for treating biofilm with bactericidal agents are under investigation. Houry et al. discovered novel deep seeded channels within the biofilm matrix [161]. These channels are not the water channels formed in late stage biofilm but instead are transient tunnels created by “stealth swimmers” identified in highly motile bacteria. Here a small number of bacteria were observed to pass through the biofilm through small pores that were quickly closed after the “swimmer” had passed through [161]. This


Hogan et al. b) DMMA

a) AIP-1 MeO

O

S

S

Me

CN

Tyr Ser

Thr

Phe

OMe

NH

Asp

N H

Ile

O

MeO

Fig. (3). Example of the chemical structure of a) staphylococcal autoinducing peptide 1 (AIP-1), reproduced from Williams et al. [141] and b) (Z)-3-(2, 5dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA), reproduced from Oh et al. [156].

can have a number advantages for the biofilm as it facilitates the circulation of macro nutrients [162]. The study by Houry et al. investigated the possibility of using motile bacteria such as Bacillus thuringiensis to treat S. aureus biofilm, whereby B. thuringiensis was added to a pre-existing S. aureus biofilm before treatment with benzalkonium chloride, with resulting increased biofilm susceptibility of up to one hundred fold. Bacillus spp. strains were also modified to express lysostaphin, an enzyme that facilitates biofilm detachment, to enable the bacteria to deliver lysostaphin into the inner parts of the biofilm with resultant eradication of a pre-existing biofilm [161]. This area of research shows potential for motile bacteria to be used with drugs that may usually have difficulty in penetrating the biofilm, however it should be noted that the motile bacteria to be added must not be pathogenic or have the ability to aid biofilm formation. Bacteriophage Therapy Bacteriophages represent a novel approach to prevention or treatment of staphylococcal DRIs. Although used historically, their clinical use fell out of favour in many countries with the introduction of antibiotics. The ever increasing problem of antimicrobial resistance has re-focused interest on to these agents [163, 164]. Conflicting studies have been recently published on the effect of phage therapy in animal models. Yilmaz et al. report dissolution of staphylococcal biofilm using species specific phage in conjunction with antibiotics in a rat model of implant related MRSA osteomyelitis, with the effect on S. aureus more pronounced than that on Pseudomonas aeruginosa [60]. Seth et al. reported a benefit of topical bacteriophage treatment in a rat model of soft tissue infection but only if physical disruption (debridement) of the biofilm occurred before phage treatment, perhaps indicating that access of the phage to the biofilm cells was a challenge [163, 165]. Furthermore concerns remain regarding the optimum dose, route of administration, optimal duration of treatment, as well as the potential side effects on the normal bacterial flora of higher doses of bacteriophages. A comprehensive study investigating the potential of bacteriophage therapy as prophylaxis and treatment of staphylococcal biofilm infections is required before their full role can be elucidated. Antimicrobial Peptides Antimicrobial peptides (AMPs), commonly known as host defence peptides, are a group of peptides that are part of the host immune response and have properties which may make them promising candidates for use in the prevention or treatment of staphylococcal DRIs. The mechanism of action of this unique and diverse group of molecules is biophysical rather than biochemical, where the target is the cytoplasmic membrane structure itself [166]. Fitzgerald-Hughes et al. highlighted the potential of natural cationic peptides as anti-staphylococcal agents [166]. The potential of AMPs in bacterial infections, including biofilm related infections, may be realised through their development as systemic agents. Although, most AMPs in clinical trials to date are for topical applica-

tion, the targeted delivery of AMPs may further improve the therapeutic potential of these molecules. Two of the best studied AMPs, due to their antimicrobial activities, are the cathelicidin, LL-37, and the human-beta defensins. These peptides have served as templates for the development of derivatives with improved potential for therapeutic application and lower potential for toxicity than natural peptides. Cathelicidins have also been shown to inhibit staphylococcal biofilm. LL37, which is a 37 amino acid AMP found in humans, is of particular interest as it has been shown to inhibit initial attachment and biofilm formation in S. epidermidis [167, 168]. Other AMPs have recently been shown to inhibit S. aureus biofilms including synthetic peptides indolicidin, KABT-AMP and nicin [169-171]. Despite their broad spectrum rapid killing potential, there are a number of concerns with regard to the use of AMPs as systemic therapies. The main areas for concern include their potential toxicity or immunogenicity, their stability at various pH and osmotic conditions, the development of resistance and the unknown effect on innate responses to infection [172]. Appropriate doses required to exert an effect on staphylococcal biofilms need to be determined as a recent study has highlighted that secreted proteases may play a role in resistance to these compounds [173]. Nevertheless, these agents represent a novel, and possibly viable, option for preventing or treating staphylococcal DRIs and they should be explored further via appropriately designed studies. CONCLUSION HCAIs affect between 5-10% of patients admitted to acute hospitals [174]. Recognised risk factors associated with HCAIs are the use of invasive medical devices (e.g. peripheral and central vascular catheters, prosthetic joints, etc.); leading to DRI and as previously highlighted, staphylococci are the major cause of DRI due to biofilm formation. Although our understanding of the complexities of the staphylococcal matrix remains incomplete, significant progress has been made over the last number of years in our understanding of the complexity of the various stages involved in staphylococcal biofilm attachment, formation, regulation and disassembly. Progress in applying this growing knowledge base, along with a plethora of emerging experimental therapeutic approaches to both the prevention and treatment of staphylococcal biofilm has been considerable. Novel approaches focusing on anti-adhesion compounds, new medical device designs, immunisation with “biofilm vaccines”, matrix degrading enzymes and therapies and antistaphylococcal biofilm agents such as peptides and small molecules are advancing with considerable progress. However, the patient must remain at the centre of future studies in order to identify safe and different approaches to prevent or treat staphylococcal biofilm. Ultimately, the aim must be to reduce or prevent staphylococcal DRIs and their associated morbidity and mortality for our patients. Within the patient population there are differences in the types of DRIs, the host’s response to the foreign body, differences in drug pharmacokinetics and stability at varying environmental conditions, as well as varying patient co-morbidities, which all represent challenges to the introduction of novel biofilm


therapeutics. There must be continued focus on in vivo experimental studies with efforts made to mimic the in vivo situation. Also, undesired consequences for the patient, such as dissemination of staphylococci to other organs after use of biofilm degrading therapies, effects of novel compounds on host tissues and the unknown effect on innate responses to infection must be thoroughly investigated before their use can be translated into clinical practice. Ongoing progress and investment in novel approaches to the prevention and treatment of staphylococcal biofilm infection with clear patient benefit must be maintained. This will in the future lead to clear patient benefit and augment our current antimicrobial approach for these very significant infections. CONFLICTS OF INTEREST HH has received research support from Pfizer & has also recently received lecture & other fees from Novartis, Astellas and AstraZeneca. Other authors declare no conflicts of interest ACKNOWLEDGEMENTS Siobhan Hogan is supported by a research grant (Major Grant Award 2011) from the Healthcare Infection Society and Eoghan O’Neill, James O’Gara and Hilary Humphreys are funded by the UK Healthcare Infection Society and the Irish Health Research Board.

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Received: March 10, 2014

Accepted: August 27, 2014


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