Targeting microbial biofilms: current and prospective therapeutic strategies

Abstract

Biofilm formation is a key virulence factor for a wide range of microorganisms that cause chronic infections. The multifactorial nature of biofilm development and drug tolerance imposes great challenges for the use of conventional antimicrobials and indicates the need for multi-targeted or combinatorial therapies. In this Review, we focus on current therapeutic strategies and those under development that target vital structural and functional traits of microbial biofilms and drug tolerance mechanisms, including the extracellular matrix and dormant cells. We emphasize strategies that are supported by in vivo or ex vivo studies, highlight emerging biofilm-targeting technologies and provide a rationale for multi-targeted therapies aimed at disrupting the complex biofilm microenvironment.

Figure 1. Opportunities for therapeutic intervention during various stages of the biofilm life-cycle

Biofilm formation proceeds as a developmental process with distinct stages: “initial adhesion” where microorganisms bind to host or medical device surfaces through cell surface associated adhesins; “early biofilm formation” where they begin to divide and produce EPS which enhances adhesion, while forming the matrix that embeds the cells; “biofilm maturation” where 3D structures develop in which the EPS matrix provides a multi-functional and protective scaffold which allows heterogeneous chemical and physical microenvironments to form where microorganisms co-exist within polymicrobial and social interactions (competitive and synergistic); and finally “dispersal” where cells leave the biofilm to re-enter the planktonic phase. Biofilms can be targeted at these various stages. a) The initial phase of biofilm formation can be disrupted, for example, by preventing the attachment of microorganisms by interrupting the interactions between the microorganism and the surface, by targeting cell surface associated adhesins (appendages, proteins and EPS). b) The inhibition of early stages of biofilm development includes targeting the production of EPS and cellular division. c) Disruption of formed biofilms could be achieved by physical removal, the degradation of the EPS-matrix, targeting the establishment of pathogenic microenvironments (low pH or hypoxia) and social interactions (in polymicrobial biofilms) as well as elimination of dormant cells. d) Finally, biofilm dispersion can be induced by EPS matrix remodelling or activation to dispersal mechanisms.

Figure 2. Targeting the EPS

Disruption of EPS components, and the underlying mechanisms that are responsible for the production and secretion of EPS components, represent attractive targets for the development of biofilm-targeting strategies, some of which have potential efficacy across microbial species. One approach includes the degradation of the EPS. Treatments have been developed that directly target the eDNA (DNases), exopolysaccharides (dispersin B, glycoside hydrolases, monoclonal antibody vaccines), and protein (DNABII family antibodies) components of the matrix. EPS adhesin-binding antibodies or inhibitors and phage-encoded peptidoglycan hydrolases have been developed to target bacterial adhesion and biofilm initiation. Inhibitors of EPS synthesis and the secretion systems have also shown promise to disrupt biofilm accumulation. Endogenous pathways that induce biofilm dispersal can also be targeted, including the regulation of c-di-GMP and c-di-AMP levels using exogenous NO and inhibitors, or targeting quorum sensing using various inducing peptides and messenger molecules. Importantly, all of these treatment strategies, alone or in combination, can lead to inhibition of biofilm formation, disrupt biofilm integrity and/or promote the release of individual bacterial cells that are more susceptible to conventional antibiotic treatment enhancing clinical efficacy.

Figure 3. Technological approaches to combat biofilms

Recent advances in material science and nanotechnology enabled the engineering of a wide array of biofilm-targeting strategies. a) The material and surface properties of medical devices, such as surface charge, hydrophobicity, roughness, topography and chemistry among others, can be modified to prevent bacterial attachment and therefore attenuate or block biofilm formation. Additionally, ‘smart’ or stimuli-triggered responsive surfaces can be constructed that elicit their effect only in response to physical contact with cell-wall or membrane associated adhesins or chemical cues (i.e. secreted EPS, metabolites) of the bacteria. b) Advancement in nanoparticle synthesis has led to the development of diverse approaches to combat biofilms. Inorganic metallic (silver, copper etc.) and organic nanoparticles (liposomes, aptamers etc.), have been increasingly evaluated to improve their anti-biofilm efficacy, as well as their biocompatibility to reduce toxic effects on the host. Nanoparticles can be used to form nanocoatings, be incorporated into materials as composites or fillings or combined together with conventional antimicrobials and other approaches designed to physically disrupt or remove the biofilm. Furthermore, antimicrobial peptides (AMPs) and aptamers also display specific biofilm-targeting properties that can be also used to enhance specificity and efficacy of nanoparticles (hybrid nanoparticles). c) New technologies for physical biofilm removal, including mechanical, energy- and light-based disruption, may further improve biofilm intervention strategies. Given the multifaceted nature of biofilm formation and the complex microbial interactions with the surrounding physical and chemical environment, a combination of these approaches may be required to successfully combat biofilm-mediated disease.

Figure 4. Multi-targeting approach to combat biofilms

The physical and biological complexity of biofilms and tolerance to antimicrobials render them less susceptible to conventional therapeutic approaches. Biofilm targets include microbial cells (often polymicrobial communities) and the EPS matrix, and therapeutics can be delivered from the overlying surrounding biological fluid as well as the surfaces of the medical devices themselves. We envision exogenous approaches (such as adhesion-targeting materials and coatings, and adhesin-blocking agents) to complement or synergize with endogenous activation (such as immunity modulation) to prevent microbial attachment to host or abiotic surfaces in patients. Likewise, a combination of approaches that degrade the protective matrix, activate dispersal, and target the resident pathogens, persisters and dispersed cells without affecting commensals may be required to eliminate existing biofilms. Long-term effects of modified surfaces in the presence of biological fluids as well as enhanced drug penetration properties and a decrease in toxicity or allergic reactions are required for in vivo efficacy. These combined with clinically relevant treatment regimen (either topical or systemic) and long-term effect assessment should help successfully translate the hypothetical concepts into the clinic. The grey arrows indicate that biofilm bacteria and EPS can move or interact between the surface and fluid phases.