Biofilm is an organized structure of microorganisms that adhere to a surface and form a protective matrix. The matrix is mainly composed of polysaccharides, nucleic acids, proteins, and lipids, which protect the microorganisms embedded within it from physical and chemical stressors, including the host’s immune system, antibiotics, and disinfectants. There is a growing concern that biofilm formation contributes to the persistence of infectious diseases and antibiotic resistance. In this article, we will explore the mechanisms by which biofilms confer antibiotic resistance and how we can potentially break down this resistance.
Biofilm antibiotic resistance mechanisms
Biofilms are known to exhibit up to 1000 times higher antibiotic resistance than their planktonic counterparts. This resistance has been attributed to several factors, including slow growth rates, limited diffusion of antibiotics through the matrix, and adaptive genetic changes. The following are the known mechanisms by which biofilms confer antibiotic resistance.
Reduced antibiotic penetration
Biofilms form a complex network of EPS (Extracellular Polymeric Substance) that restricts antibiotic penetration. EPS comprises extracellular DNA (eDNA), proteins, and polysaccharides, among others. The matrix acts as a physical barrier that hinders antibiotic diffusion to inner layers of the biofilm. Furthermore, the matrix increases the viscosity of the area, making it challenging for the antibiotic molecules to diffuse, and they eventually get diluted and inactivated.
Slow growth and dormant cells
Biofilm cells have a slower growth rate compared to their planktonic counterparts. Biofilm cells are metabolically active only on the surface of the biofilm and can create a gradient of nutrient and oxygen within the biofilm. Cells located at the center of the biofilm often experience a decreased metabolic rate and enter a dormant state characterized by decreased metabolism, low nutrient acquisition, and minimal growth. Dormant cells are less susceptible to antibiotics due to low metabolic rates and cellular activity. Therefore, antibiotics that target growing cells might not be effective against biofilm cells because of their reduced susceptibility.
Efflux pumps are ubiquitous resistance mechanisms in bacteria. They assemble in the bacterial membrane and pump out drugs that enter the cell, rendering the antibiotic ineffective. Efflux pumps are present in both planktonic and biofilm cells, and they play a crucial role in antibiotic resistance. Several classes of efflux pumps have been identified, including efflux pumps that expel only specific classes of antibiotics, such as tetracyclines, macrolides, and quinolones.
Biofilm-specific efflux pumps, such as AdeABC, CmeABC, and MexAB-OprM, have been identified to confer antibiotic resistance specifically in biofilm cells. These efflux pumps can remove a wide range of antimicrobial agents, including those that target cell wall, protein synthesis inhibitors, quinolones, and beta-lactams. The overexpression of efflux pump genes in biofilm cells has also been linked to biofilm maturation stages. This observation suggests that biofilm cells upregulate efflux pumps to resist antibiotic stress and that the activity of efflux pumps may be controlled by factors that regulate biofilm development.
Bacteria have developed several ways to modify antibiotics through enzymatic or chemical means. Such modifications include antibiotic acetylation or phosphorylation, which render the antibiotic ineffective. Enzymes that degrade antibiotics, such as beta-lactamases, can inactivate beta-lactam antibiotics.
Certain bacteria embedded within the biofilm have a unique ability to modify or inactivate antibiotics. A case in point is the Pseudomonas aeruginosa, which enzymatically inactivates β-lactams and aminoglycosides through the expression of β-lactamases, aminoglycoside-modifying enzymes (AMEs), and proteases. The transcription of β-lactamase genes in P. aeruginosa biofilms can be enhanced by the overexpression of MexAB-OprM efflux pumps.
Horizontal gene transfer
Horizontal gene transfer (HGT) is the process by which bacteria acquire new genes. HGT can take place through three mechanisms: transformation, transduction, and conjugation. Through HGT, bacteria can acquire antibiotic resistance genes and exchange these genes among different species. This transfer of genes can result in rapid evolution, leading to the emergence of antibiotic-resistant populations.
Biofilms are known to harbor different bacterial species, and HGT can occur within the biofilm. The transfer of antibiotic resistance genes among different species within the biofilm can contribute to the emergence of antibiotic-resistant biofilms. Moreover, biofilms can act as potential reservoirs for antibiotic resistance genes, which can be transferred to other environments.
Breaking down the biofilm antibiotic resistance
The critical question is how we can overcome biofilm antibiotic resistance mechanisms. The following are a few strategies that have shown some promise in breaking down biofilm antibiotic resistance.
Attacking the matrix
Breaking down the biofilm matrix can potentially increase antibiotic penetration into the biofilm and ultimately increase the efficacy of antibiotics. Specific agents that disrupt biofilm EPS have been identified, including enzymes, surfactants, and other chemical compounds. Enzymes such as dispersin, DNase, and alginate lyase can break down the EPS matrix leading to biofilm dispersal. Alternatively, surfactants such as sodium dodecyl sulfate (SDS) and bleach can dissolve the matrix, leading to an increase in antibiotic penetration.
Targeting the dormant cells
Attacking dormant cells in the biofilm can potentially increase antibiotic susceptibility in biofilm cells. Several drugs that target dormant cells have been identified, such as 5-Nitroimidazole and nitrofurans. These drugs target the oxygen-deprived conditions that favor biofilm dormancy and subsequently activate nitroreductase enzymes that break down the drug leading to oxygen depletion and sterilization of the biofilm.
Inhibiting efflux pumps
Inhibitors that target efflux pumps have the potential to enhance the efficacy of antibiotics in biofilms. Some inhibitors have been proposed as adjuvants, enhancing antibiotics’ effectiveness by blocking efflux pumps specifically in the biofilm. Though several broad-spectrum efflux pump inhibitors have been identified, the challenge is identifying inhibitors that can penetrate the biofilm matrix and inhibit efflux pumps that are overexpressed in biofilms.
Development of new antibiotics
The development of new antibiotics that can target biofilm cells specifically could be a game-changer in overcoming biofilm antibiotic resistance. The discovery and development of new antibiotics are a response to the emergence of antibiotic-resistant bacteria. However, we need to explore alternative approaches to antibiotic therapy, including bacterial interference and phage therapy, which are potential biofilm-targeted therapies.
Biofilm antibiotic resistance mechanisms have been explored extensively and have been shown to result in persisting infections that are challenging to treat. The biofilm antibiotic resistance mechanisms discussed above, including reduced antibiotic penetration, slow growth, and dormant cells, efflux pumps, antibiotic modification, and HGT, have far-reaching effects on health care.
The strategies proposed, including breaking down the matrix, targeting dormant cells, inhibiting efflux pumps, and developing new antibiotics, offer exciting potentials to overcome biofilm antibiotic resistance. The research should not stop here, and scientists should continue exploring alternative antimicrobial agents that could help mitigate the growing antimicrobial resistance problem.