Biofilms are communities of bacteria that exist in a matrix composed of Extra Polymeric Substances (EPS): polysaccharides, proteins, extracellular DNA (eDNA), and other minor substances.
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Biofilms exist in nature to confer great protection to bacteria from competing microbes, predators, and to provide protection in times of stress (changes in temperature, pH, salinity, pressure, nutrient abundance).
Biofilms have been observed in many inhospitable environments. The matrix and the bacterial colony have evolved symbiotically to adapt to their niches. Biofilms are of greatest concern in industrial and clinical settings.
Patients who are exposed to intermittent doses of antibiotics are at a higher risk of developing resistance, and the development of biofilms. Industries such as pharmaceuticals and food are susceptible to biofilm contamination due to formation in internal systems such as water and food processing.
Why do biofilms develop?
Biofilms confer great protection to bacteria than “free-floating” or planktonic cells. It protects against predation, harsh conditions, and it mitigates the risk of interaction with exogenous substances such as antibiotics.
Greater expression of efflux pumps has been found in species of bacteria that exist in biofilms compared to their planktonic counterparts: important for toxin egress like antibiotics. The matrix generated in a biofilm prevents the penetration of immune cells such as neutrophils. It also protects it from shear force, affording it greater protection.
How are they formed?
Its formation occurs in a stepwise manner. Planktonic cells interact freely with (a)biotic surfaces through weak intermolecular forces. The subsequent engagement of extracellular bacterial components such as cilia, flagella, and membrane adhesion factors with the surface drive stronger hydrophobic and polar interactions.
Over time, the cells in the colony replicate and produce EPSs, accelerating the development of the biofilm. Maturation involves the development of a 3-Dimensional structure capable of coordinating the delivery of nutrients and regulation of gene expression. Ultimately, the biofilm undergoes a recycling process: cells are detached to colonize new environments.
Quorum Sensing plays an important role in the formation of biofilms: the community of bacterial cells can regulate gene expression in response to cell density. It can regulate the gene expression of more than 10% of genes in Pseudomonas aeruginosa, including factors that regulate biofilm formation.
Nucleotide second messenger signaling also plays an important role in the development of biofilms. Examples include cyclic dimeric guanosine 3′,5′-monophosphate (c-di-GMP) and cyclic adenosine 3′,5′-monophosphate (cAMP). c-di-GMP plays an important role in the regulation of adhesion factors such as cilia and flagella. cAMP can control the biofilm formation in Vibrio cholerae and biofilm dispersal in P. aeruginosa.
Biofilms in a clinical setting
Biofilms present a serious concern for immunocompromised patients or those with chronic wounds. Antibiotic resistance has played a critical role in this. Intermittent exposure to antibiotics generates a niche that allows “tolerant” and “persister” cells to exist safely in biofilms.
The mechanism of action for most antibiotics is to target replication or growth; cells with genes that allow for slower growth phases have an advantage. Subminimal inhibitory concentrations of antibiotics can potentiate the biofilm to generate greater volumes of extracellular factors.
Persister cells adapt to the intermittent exposure to antibiotics, facilitating their survival. The populations of tolerant and persister cells are relatively small, but as faster-growing cells die off they play a greater role.
Clinical treatment of biofilms depends on their location. Mechanical debridement or capillary scraping methods are utilized for chronic wounds. Internal biofilms are treated using chemicals that enable the dispersal of biofilm components; this acts as an adjuvant to antibiotic treatment. Dispersin B is approved for wound care and coating of medical devices. Other methods attempt to reactivate cells or promote dispersal to promote antibiotic activity.
Biofilms in the food industry
Biofilms in the food industry are a serious concern. A large number of processes and surfaces provide ample opportunity for different disease-causing bacteria to develop.
Biofilms facilitate the expression of recalcitrant bacteria which are more resistant to the conventional decontamination processes in the food industry. This has resulted in a series of serious disease outbreaks in the past 20 years.
Some of the major culprits include Escherichia coli, Bacillus cereus, Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus. The type of surface, temperature, pH, and pressure can all affect the bacteria present in the biofilm and resistance to conventional decontamination processes.
A variety of processes have been established to assist in the detection of biofilms and prevent their formation. As it is often not feasible to use conventional methods like agar plating, PCR detection, or mass spectrometry, other means have been created to facilitate biofilm detection.
Many of these techniques detect outputs from the biofilm such as heat variations, electrochemical outputs (BIOX/BIOGEORGE), or detect changes in the vibration signal on the surfaces (quartz crystal microbalance).
Many of the conventional decontamination processes are chemical - substances that interfere with the development of biofilms or growth of bacteria (sodium hypochlorite, sodium hydroxide solutions, hydrogen peroxide, peracetic acid) or physical - processes that disrupt the biofilm (steam, ultrasonication).
Enzymes are often used that degrade components of the biofilm; proteases and glycosidases are typically used in detergents. Modifying the affected surfaces can also regulate biofilm formation: treated stainless is more effective for mitigating formation. More novel techniques have been trialed such as bacteriophages, bacteriocins, and QS inhibitors.
Bacteriophages are bacteria-specific viruses: Listeria phage 100 has been approved in the USA for use in meat processing plants. Bacteriocins are chemicals produced by bacteria to inhibit the growth of competing species. Nisin has been approved for its microbial activity. QS inhibitors disrupt the formation of biofilms as discussed above. These treatments are much more specific and thus provide greater efficacy. However, these still warrant many optimizations and scaling to prove truly effective.
Sources:
- Galié, S. et al. (2018) ‘Biofilms in the Food Industry: Health Aspects and Control Methods ’, Frontiers in Microbiology, p. 898. Available at: https://www.frontiersin.org/article/10.3389/fmicb.2018.00898.
- Yan, J. and Bassler, B. L. (2019) ‘Surviving as a Community: Antibiotic Tolerance and Persistence in Bacterial Biofilms’, Cell host & microbe, 26(1), pp. 15–21. doi: 10.1016/j.chom.2019.06.002.
- Yin, W. et al. (2019) ‘Biofilms: The Microbial “Protective Clothing” in Extreme Environments’, International journal of molecular sciences, 20(14), p. 3423. doi: 10.3390/ijms20143423.
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