A biofilm is a structured community of microbial cells, such as bacteria and fungi, that adhere to a surface and are encased in a self-produced polymeric matrix. This matrix protects the microbes from external threats, including the host immune system and conventional antibiotics. Biofilms contribute to persistent, chronic infections and significant industrial fouling. Since up to 80% of all bacterial infections are linked to this microbial lifestyle, researchers are focusing on strategies that disrupt the biofilm’s structure and communication. This involves targeting the collective behaviors and protective scaffolding rather than simply trying to kill individual cells.
Inhibiting Cell Communication
Bacteria coordinate their collective behavior within a biofilm using quorum sensing (QS), which relies on chemical signaling molecules called autoinducers. These signals accumulate as the bacterial population density increases, allowing the community to sense its numbers. Once a concentration threshold is reached, the signals trigger a coordinated change in gene expression, leading to the formation of the protective matrix and the production of virulence factors.
Quorum sensing inhibition (QSI) aims to interfere with this cell-to-cell communication, preventing the coordinated expression of biofilm-related genes. QSIs work by blocking receptor sites, mimicking signals to send a false message, or enzymatically degrading the signal molecules themselves. For instance, in Gram-negative bacteria, quorum quenching enzymes like AHL-lactonase can degrade \(N\)-Acyl homoserine lactones (AHLs), a common autoinducer.
This approach offers an advantage over traditional antibiotics because it does not directly kill the bacteria. By disabling the community’s ability to organize, QSIs reduce the selective pressure that drives antibiotic resistance. Disrupting QS also makes uncoordinated bacteria more susceptible to immune clearance and existing antimicrobial treatments. Small molecule inhibitors are being developed to target specific autoinducers, such as the AI-2 molecule, which mediates inter-species communication, offering broad-spectrum anti-biofilm potential.
Enzymatic and Chemical Matrix Dissolution
To breach an established biofilm, scientists target the extracellular polymeric substance (EPS) matrix, the physical barrier protecting the embedded microorganisms. The EPS is a complex scaffolding primarily composed of polysaccharides, proteins, and extracellular DNA (eDNA). Breaking down these specific components with targeted agents is an effective strategy for biofilm disruption.
Enzymes are highly specific tools for this task, with different classes targeting different matrix polymers. Deoxyribonucleases (DNases), such as DNase I, hydrolyze the eDNA component, a structural element of the biofilm. Glycoside hydrolases, like Dispersin B, target the polysaccharide backbone, breaking down poly-\(N\)-acetylglucosamine (PNAG) polymers. Proteases, such as Proteinase K, degrade the structural proteins within the matrix, further weakening the biofilm’s integrity.
The degradation of the EPS exposes microbial cells, making them vulnerable to traditional antibiotics and disinfectants. Chemical agents are also employed to destabilize the matrix structure. Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that binds to divalent metal ions like calcium and magnesium. These metal ions are necessary for EPS structural stability and bacterial outer membrane integrity. By sequestering these ions, EDTA weakens the matrix and enhances antibiotic penetration by up to 15-fold.
Harnessing Biological Agents
The use of living organisms or their active components represents a powerful biological approach to biofilm control. Bacteriophages (phages) are viruses that specifically target and infect bacterial cells. Phage therapy involves applying these viruses to a biofilm, where they penetrate the matrix, multiply inside the bacteria, and cause the host cell to lyse.
This process releases new phages to attack neighboring cells and releases phage-encoded enzymes that actively degrade the EPS matrix. These enzymes, like depolymerases and endolysins, break down structural polysaccharides and the bacterial cell wall, allowing phages to penetrate deep into the biofilm. Phage treatments can be combined with antibiotics in a synergistic therapy, where phage-mediated disruption allows the antibiotic to reach previously protected cells.
Predatory bacteria, such as Bdellovibrio bacteriovorus, are also being explored as a living antibiotic. This tiny, highly motile bacterium preys on other Gram-negative bacteria, penetrating their cell wall and replicating between the inner and outer membrane. Bdellovibrio is capable of penetrating both Gram-negative and Gram-positive biofilms, where its rapid movement and lytic action physically disrupt the microbial structure. Researchers are exploring genetically engineering Bdellovibrio to deliver therapeutic agents, such as antibiotic-loaded liposomes, directly into the core of the biofilm, improving treatment efficacy.
Physical and Material Surface Strategies
A major focus of biofilm prevention is engineering surfaces to limit the initial attachment of microorganisms. Anti-fouling surfaces are designed to minimize the adhesive forces between the substrate and the microbial cell. One successful strategy involves creating “fouling-release” surfaces using low-surface-energy polymers like poly(dimethylsiloxane) (PDMS). These materials are slippery, requiring only a small shear force, such as fluid flow, to remove weakly adhered microbial cells.
Alternatively, “non-fouling” surfaces utilize highly hydrophilic polymers, such as poly(ethylene glycol) (PEG) or polyzwitterions, which resist protein adsorption and cell adhesion. These materials create a dense, structured layer of water molecules at the surface interface, preventing bacteria from making the close contact necessary for irreversible adhesion. Physical topography can also be manipulated, as surfaces patterned with micro- and nano-scale features interfere with the bacteria’s ability to settle and spread.
In addition to surface modification, physical forces disrupt established biofilms. Electroceuticals utilize low-level direct electrical currents (DC) to either kill bacteria or enhance the effect of antimicrobials. This “bioelectric effect” is often achieved with very low amperages (e.g., 2 μA to 10 μA), which are imperceptible and safe for humans. The electric current disrupts the electrical charges on the bacterial cell surface and the EPS matrix, promoting cell detachment. This allows antibiotics to penetrate more effectively, potentially involving localized electroporation or the electrochemical generation of oxidants.