Approaches to combating methicillin-resistant Staphylococcus aureus (MRSA) biofilm infections

KEYWORDS:

• Biofilm infection
• inhibitors
• MRSA
• Staphylococcus aureus

1. Approaches to combating methicillin-resistant Staphylococcus aureus (MRSA) biofilm infections

Using the earliest microscopes, scientists have already observed that bacteria and fungi exist not only as individual cells but part of microbial communities. Their three-dimensional texture led to these microbial communities being referred to as ‘bacterial slime’ in early bacteriology manuscripts. It was only after observing similar communities of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients that J. W. Costerton recognized their pertinence to human disease and coined the phenotype as ‘biofilms’ [1]. Biofilms have since been implicated in colonization and infection states of virtually all human organ systems. With the increased frequency of catheter utilization and implantation of medical devices and prostheses, the skin commensal staphylococci in particular have emerged as the most common group of pathogens implicated in human biofilm infections [2].

Staphylococcal biofilms, including those involving methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) isolates, have been implicated in infective endocarditis, chronic wounds, osteomyelitis, and infections of foreign bodies such as catheters, cardiovascular implantable electronic devices (CIEDs), and orthopedic prostheses. Staphylococcal biofilm infections are extremely difficult to eradicate, and current management approaches remain hindered by prolonged courses of antimicrobial therapy combined with multistage surgical interventions, often necessitating complete removal of the infected materials. These therapies impose undue patient morbidity and mortality, are costly, and promote antimicrobial resistance [2]. Given the dire need for preventive and therapeutic measures to combat staphylococcal biofilm infections, some efforts have been devoted to understanding biofilm determinants and identify approaches to fighting these infections.

Staphylococcal biofilm infections are difficult to treat due to their mechanical and metabolic properties. In biofilms, staphylococci are embedded within an extracellular matrix consisting of polysaccharides, proteins, enzymes, and extracellular DNA, molecules that are secreted by the bacteria or released to the surrounding environment upon bacterial cell death [2]. This extracellular matrix serves as a physical barrier to shield the S. aureus community from the effects of antibiotics, antimicrobial peptides, and the host immune response, including phagocytes [3]. Moreover, S. aureus within the deep layers of the biofilm is metabolically inactive and may include persister or small-colony variant phenotypes that further render biofilms intrinsically resistant to antibiotics. In these settings, prolonged courses of antimicrobials will inevitably fail and promote antimicrobial resistance. These characteristics underscore the role of biofilm as a major virulence factor behind S. aureus’ success as a leading human pathogen and shed light on the need to identify novel approaches to S. aureus biofilm prevention and eradication.

Despite the recognition of the importance of S. aureus biofilm infections, research identifying antibiofilm therapies remains largely in the experimental stages and may be categorized into approaches that interfere with biofilm formation or target developed biofilms. One approach to preventing biofilm formation is through preventing attachment of S. aureus to the device surfaces through altering their physical characteristics. This includes the incorporation of polymers and metals into the device surface material, the alteration of surface physical characteristics such as charge, hydrophobicity or topography, or the embedding of antibiotics into the device that are gradually released. However, all these approaches remain largely at the in vitro or initial animal infection study stages [4]. Among the rare human clinical trials that have been performed, the physical enclosure of CIEDs within a protective antibiotic envelope at the time of device implantation has been shown to reduce the perioperative infection rate from 1.2% to 0.6% [5]. Of note, the long-term efficacy of these approaches might be limited by the ability of host proteins to coat these modified surfaces and serve as attachment sites for S. aureus in later infection.

Vaccination specifically targeting S. aureus biofilm is challenged by the dynamic antigen expression of bacteria in planktonic and biofilm modes of growth, and to date has only been attempted by one group utilizing a multivalent vaccine targeting planktonic S. aureus surface antigens [6]. A 3-log reduction in biofilm and complete biofilm clearance was observed in 87.5% of the animals in an S. aureus osteomyelitis model when vaccination was administered in conjunction with antibiotic therapy [6].

Most investigative efforts toward treating existing S. aureus biofilms have been devoted to evaluating the effects of natural products [7], which might interfere with bacterial adhesion, or disruption of existing biofilm three-dimensional structure. Most remain in pre-clinical phases and are limited to in vitro assays, with largely undetermined translational value.

Antimicrobial peptides (AMPs) are compounds that are bactericidal and may also enhance the activity of antibiotics or interfere with signaling [8]. AMPs have been demonstrated, for example, to reduce preformed S. aureus biofilm in a rodent catheter lock therapy model [9] and a mouse subcutaneous biofilm catheter infection model [10] and generally appear to be promising as biofilm therapeutics. However, the exact anti-biofilm mechanism remains to be defined, and AMP therapy remains limited by cytotoxicity and host inflammatory responses, and might be subjected to similar resistance mechanisms as antibiotics, such as the physical barrier posed by the extracellular matrix, microbial surface charge repulsion, proteolytic inactivation, sequestration, and efflux [11].

Bacteriophage therapy [12] has received renewed attention owing to ongoing antimicrobial resistance challenges. Some approaches have been proposed as potential salvage therapy options against S. aureus biofilm infection in case series [12]. There are also strategies using phage-encoded peptidoglycan hydrolases (PGHs/lysins) [13], which target bacterial peptidoglycans, including of S. aureus, and have activity against biofilms, persisters, and small-colony-variants. Considered nontoxic, PGHs have been shown to demonstrate high substrate specificity and can further be engineered and co-administered with antibiotics. Like phage therapy, PGH therapy faces resistance challenges including target alteration, efflux, inactivation by cytoplasmic enzymes, inactivation by host immunity, and limited efficacy against intracellular infections [13]. While efficacy against S. aureus biofilm has been demonstrated in vitro, ongoing clinical trials involving lysins targeting S. aureus are not designed to specifically evaluate biofilm infection [13].

Matrix-degrading enzymes such as proteases, dispersin B, and DNAse 1 have been utilized to facilitate penetration of antibiotics into biofilms, and some have activity in biofilm infection models [14]. However, this sort of approach triggers the release of the bacteria into the bloodstream, which despite increased sensitivity to co-administered antibiotics may lead to more serious sepsis and dissemination of bacteria to distal sites.

Interfering with regulatory mechanism through quorum-sensing blockade has been proposed early as an anti-biofilm therapeutic approach, based on initial findings in P. aeruginosa. However, it was soon discovered that this approach does not work for staphylococci as reduced activity of the staphylococcal quorum-sensing system Agr leads to increased rather than decreased biofilm formation, and accordingly, Agr-deficient isolates are often obtained from biofilm-associated staphylococcal infections [15].

In conclusion, biofilm infection remains a persistent problem, including for one of its most frequent causes, S. aureus, and warrants further research. Many modern strategies remain in their investigational or early pre-clinical stages, and all come with intrinsic problems. Most likely, there will be no general anti-biofilm drug and even drugs that efficiently target the basis of biofilm formation in specific pathogens or even only different strains of one pathogen, will need to be administered together with antibiotics. As for antibiotic development, there may be hope in attempting to find more antibiotics or antibiotic-like substances like AMPs that are efficient in treating biofilm infections, once the underlying antibiofilm mechanism is defined. Rifampicin, for example, appears to be efficient against staphylococcal biofilms and is frequently used in the clinic for that purpose. However, it has remained unclear why rifampicin works better than other antibiotics against biofilms. Furthermore, there is rapid development of resistance to rifampicin by alteration of its target, RNA polymerase, which is why it is usually given in combination with other antibiotics. Nevertheless, understanding and improving these types of biofilm-active antibiotics may represent an additional avenue to improve the clinical treatment of staphylococcal biofilm infections.

Declaration of interests

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants, or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (Project No. ZIA-AI001080) and the Young Scholar Program/Chairman’s Circle Award of the New York University Grossman School of Medicine.