BIOFILMS: CHARACTERISTICS, FORMATION, TYPES AND EXAMPLES - BIOLOGY - 2025

The biofilms or biofilms are communities of microorganisms attached to a surface, living in a matrix of extracellular polymeric substances self - generated. They were initially described by Antoine von Leeuwenhoek, when he examined the "animalcules" (thus named for him), on a plate of material from his own teeth in the 17th century.

The theory that conceptualizes biofilms and describes their formation process had not been developed until 1978. It was discovered that the ability of microorganisms to form biofilms appears to be universal.

Figure 1. Biofilm produced by Staphylococcus aureus in a catheter. Source: CDC / Rodney M. Donlan, Ph.D.; Janice Carr (PHIL # 7488), 2005. via

Biofilms can exist in environments as varied as natural systems, aqueducts, water storage tanks, industrial systems, as well as in a wide variety of media such as medical devices and devices for hospital patients (such as catheters, for example).

Through the use of scanning electron microscopy and confocal scanning laser microscopy, it was discovered that biofilms are not homogeneous, unstructured deposits of cells and accumulated silt, but rather complex heterogeneous structures.

Biofilms are complex communities of associated cells on a surface, embedded in a highly hydrated polymeric matrix whose water circulates through open channels in the structure.

Many organisms that have been successful in their survival of millions of years in the environment, for example species of the genera Pseudomonas and Legionella, use the biofilm strategy in environments other than their native native environments.

Characteristics of biofilms

Chemical and physical characteristics of the biofilm matrix

-The polymeric extracellular substances secreted by biofilm microorganisms, polysaccharide macromolecules, proteins, nucleic acids, lipids and other biopolymers, mostly highly hydrophilic molecules, cross over to form a three-dimensional structure called the biofilm matrix.

-The structure of the matrix is ​​highly viscoelastic, has rubber properties, is resistant to traction and mechanical breakdown.

-The matrix has the ability to adhere to interface surfaces, including internal spaces of porous media, through extracellular polysaccharides that act as adherent gums.

-The polymeric matrix is ​​predominantly anionic and also includes inorganic substances such as metal cations.

-It has water channels through which oxygen, nutrients and waste substances circulate that can be recycled.

-This matrix of the biofilm works as a means of protection and survival against adverse environments, a barrier against phagocytic invaders and against the entry and diffusion of disinfectants and antibiotics.

Ecophysiological characteristics of biofilms

-The formation of the matrix in non-homogeneous gradients, produces a variety of microhabitats, which allows biodiversity to exist within the biofilm.

-Within the matrix, the cellular life form is radically different from the free life, not associated. Biofilm microorganisms are immobilized, very close to each other, associated in colonies; this fact allows intense interactions to occur.

-The interactions between the microorganisms in the biofilm include communication through chemical signals in a code called “quorum sensing”.

-There are other important interactions such as gene transfer and the formation of synergistic micro-consortia.

-The phenotype of the biofilm can be described in terms of the genes expressed by the associated cells. This phenotype is altered with respect to growth rate and gene transcription.

-The organisms within the biofilm can transcribe genes that do not transcribe their planktonic or free life forms.

-The biofilm formation process is regulated by specific genes, transcribed during initial cell adhesion.

-In the confined space of the matrix, there are mechanisms of cooperation and competition. Competition generates constant adaptation in biological populations.

-A collective external digestive system is generated, which retains the extracellular enzymes near the cells.

-This enzymatic system allows to sequester, accumulate and metabolize, dissolved, colloidal and / or suspended nutrients.

-The matrix functions as a common external recycling area, storage of the components of lysed cells, also serving as a collective genetic archive.

-The biofilm functions as a protective structural barrier against environmental changes such as desiccation, the action of biocides, antibiotics, host immune responses, oxidizing agents, metal cations, ultraviolet radiation and is also a defense against many predators such as phagocytic protozoa and insects.

-The matrix of the biofilm constitutes a unique ecological environment for microorganisms, which allows a dynamic way of life for the biological community. Biofilms are true microecosystems.

Biofilm formation

Biofilm formation is a process in which microorganisms go from a free-living, single-celled, nomadic state to a multicellular sedentary state, where subsequent growth produces structured communities with cell differentiation.

Biofilm development occurs in response to extracellular environmental signals and self-generated signals.

Researchers who have studied biofilms agree that it is possible to construct a generalized hypothetical model to explain their formation.

This model of biofilm formation consists of 5 stages:

1. Initial adhesion to the surface.
2. Formation of a monolayer.
3. Migration to form multilayer microcolonies.
4. Production of the polymeric extracellular matrix.
5. Maturation of the three-dimensional biofilm.

Figure 2. Process of formation of a biofilm. Source: D. Davis, via Wikimedia Commons

Initial adhesion to the surface

The formation of the biofilm begins with the initial adhesion of microorganisms to the solid surface, where they are immobilized. It has been discovered that microorganisms have surface sensors and that surface proteins are involved in the formation of the matrix.

In non-mobile organisms, when the environmental conditions are favorable, the production of adhesins on their external surface increases. In this way, it increases its cell-cell and cell-surface adhesion capacity.

In the case of mobile species, individual microorganisms are located on a surface and this is the starting point towards a radical change in their way of life from nomadic free mobile, to sedentary, almost sessile.

The ability to move is therefore lost in the formation of the matrix, different structures such as flagella, cilia, pilus and fimbria participate, in addition to adhesive substances.

Then, in both cases (mobile and non-mobile microorganisms), small aggregates or microcolonies are formed and a more intense cell-cell contact is generated; adaptive phenotypic changes to the new environment occur in clustered cells.

Formation of a monolayer and microcolonies in multilayers

The production of extracellular polymeric substances begins, the initial formation in monolayer occurs and the subsequent development in multilayer.

Production of the polymeric extracellular matrix and maturation of the three-dimensional biofilm

Finally, the biofilm reaches its maturity stage, with a three-dimensional architecture and the presence of channels through which water, nutrients, communication chemicals and nucleic acids circulate.

The biofilm matrix retains cells and holds them together, promoting a high degree of interaction with intercellular communication and the formation of synergistic consortia. The cells of the biofilm are not completely immobilized, they can move inside it and also become detached.

Types of biofilms

Number of species

According to the number of species participating in the biofilm, the latter can be classified into:

• Biofilms of a species. For example, biofilms formed by Streptococcus mutans or Vellionela parvula.
• Biofilms of two species. For example, the association of Streptococcus mutans and Vellionella parvula in biofilms has also been discovered.
• Polymicrobial biofilms, made up of many species. For example, dental plaque.

Training environment

Also depending on the environment where they are formed, biofilms can be:

• Natural
• Industrial
• Domestic
• Hospitable

Figure 3. Biofilms of thermophilic bacteria in Mickey Hot Springs, Oregon, USA. Source: Amateria1121, from Wikimedia Commons

Type of interface where they are generated

On the other hand, according to the type of interface where they are formed, it is possible to classify them into:

• Solid-liquid interface biofilms, such as those formed in aqueducts and tanks, pipes and water tanks in general.
• Solid-gas interface biofilms (SAB for its acronym in English Sub Aereal Biofilms); which are microbial communities that develop on solid mineral surfaces, directly exposed to the atmosphere and solar radiation. They are found in buildings, bare desert rocks, mountains, among others.

Examples of biofilms

-Dental plaque

Dental plaque has been studied as an interesting example of a complex community that lives in biofilms. The biofilms of dental plates are hard and not elastic, due to the presence of inorganic salts, which give rigidity to the polymeric matrix.

The microorganisms of dental plaque are very varied and there are between 200 to 300 associated species in biofilm.

Among these microorganisms are:

• The genus Streptococcus; made up of aciduric bacteria that demineralize enamel and dentin, and initiate dental caries. For example, the species: mutans, S. sobrinus, S. sanguis, S. salivalis, S. mitis, S. oralis and S. milleri.
• The genus Lactobacillus, made up of acidophilic bacteria denaturing dentin proteins. For example, the species: casei, L. fermentum, L. acidophillus.
• The genus Actinomyces, which are aciduric and proteolytic microorganisms. Among these, the species: viscosus, A. odontoliticus and A. naeslundii.
• And other genera, such as: Candida albicans, Bacteroides forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetecomitans.

-Biofilms in black water

Another interesting example is domestic wastewater, where nitrifying microorganisms that are oxidizing ammonium, nitrite and autotrophic nitrifying bacteria live in biofilms attached to pipes.

Among the ammonium oxidizing bacteria in these biofilms, the numerically dominant species are those of the genus Nitrosomonas, distributed throughout the biofilm matrix.

The majority components within the group of nitrite oxidants are those of the Nitrospira genus, which are located only in the internal part of the biofilm.

- Subaerie biofilms

Subaerie biofilms are characterized by patchy growth on solid mineral surfaces such as rocks and urban buildings. These biofilms present dominant associations of fungi, algae, cyanobacteria, heterotrophic bacteria, protozoa, as well as microscopic animals.

In particular, SAB biofilms possess chemolytotrophic microorganisms, capable of utilizing inorganic mineral chemicals as energy sources.

Chemolithotrophic microorganisms have the ability to oxidize inorganic compounds such as H ₂, NH ₃, NO ₂, S, HS, Fe ²⁺ and take advantage of the electrical potential energy produced by oxidations in their metabolisms.

Among the microbial species present in subaerial biofilms are:

• Bacteria of the genus Geodermatophilus; cyanobacteria of the genera C hrococcoccidiopsis, coccoid and filamentous species such as Calothrix, Gloeocapsa, Nostoc, Stigonema, Phormidium,
• Green algae of the genera Chlorella, Desmococcus, Phycopeltis, Printzina, Trebouxia, Trentepohlia and Stichococcus.
• Heterotrophic bacteria (dominant in subaerial biofilms): Arthrobacter sp., Bacillus sp., Micrococcus sp., Paenibacillus sp., Pseudomonas sp. and Rhodococcus sp.
• Chemoorganotrophic bacteria and fungi such as Actynomycetales (streptomycetes and Geodermatophilaceae), Proteobacteria, Actinobacteria, Acidobacteria and Bacteroides-cytophaga-Flavobacterium.

-Bio films of causative agents of human diseases

Many of the bacteria known as causative agents of human disease live in biofilms. Among these are: Vibrio cholerae, Vibrio parahaemolyticus, Vibrio fischeri, Vellionela parvula, Streptococcus mutans and Legionella pneumophyla.

-Bubonic plague

Of interest is the transmission of bubonic plague by flea bites, a relatively recent adaptation of the bacterial agent responsible for this disease, Yersinia pestis.

This bacterium grows as a biofilm attached to the vector's upper digestive system (the flea). During a bite, the flea regurgitates the biofilm containing Yersinia pestis in the dermis, thus initiating the infection.

-Hospital venous catheters

Organisms isolated from biofilm on explanted central venous catheters include an astonishing array of Gram-positive and Gram-negative bacteria, as well as other microorganisms.

Several scientific studies report as Gram-positive bacteria of biofilms in venous catheters: Corynebacterium spp., Enterococcus sp., Enterococcus faecalis, Enterococcus faecium, Staphylococcus spp., Staphylococcus aureus, Staphylococcus epidermidis, Stppreptococcus spp. and Streptococcus pneumoniae.

Among the Gram-negative bacteria isolated from these biofilms, the following are reported: Acinetobacter spp., Acinetobacter calcoaceticus, Acinetobacter anitratus, Enterobacter cloacae, Enterobacter aerogens, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas putppida spp.. and Serratia marcescens.

Other organisms found in these biofilms are: Candida spp., Candida albicans, Candida tropicalis and Mycobacterium chelonei.

-In the industry

Regarding the operation of the industry, biofilms generate pipe obstructions, equipment damage, interferences in processes such as heat transfers when covering exchangers surfaces, or corrosion of metal parts.

Food industry

Film formation in food industry can create significant public health and operational problems.

Associated pathogens in biofilms can contaminate food products with pathogenic bacteria and cause serious public health problems for consumers.

Among the biofilms of pathogens associated with the food industry are:

Listeria monocytogenes

This pathogen uses in the initial stage of biofilm formation, flagella and membrane proteins. Forms biofilms on the steel surfaces of slicing machines.

In the dairy industry, biofilms of Listeria monocytogenes can be produced in fluid milk and milk products. Dairy residues in pipes, tanks, containers and other devices favor the development of biofilms of this pathogen that uses them as available nutrients.

Pseudomonas

Biofilms of these bacteria can be found in food industry facilities, such as floors, drains, and on food surfaces such as meats, vegetables, and fruits, as well as low-acid derivatives of milk.

Pseudomonas aeruginosa secretes several extracellular substances which are used in the formation of the polymeric matrix of the biofilm, adhering to a large amount of inorganic materials such as stainless steel.

Pseudomonas can coexist within the biofilm in association with other pathogenic bacteria such as Salmonella and Listeria.

Salmonella

Salmonella species are the first causal agent of zoonoses of bacterial etiology and outbreaks of food poisoning.

Scientific studies have shown that Salmonella can adhere as biofilms to concrete, steel, and plastic surfaces in food processing plant facilities.

Salmonella species possess surface structures with adherent properties. Additionally, it produces cellulose as an extracellular substance, which is the main component of the polymeric matrix.

Escherichia coli

It uses flagella and membrane proteins in the initial step of biofilm formation. It also produces extracellular cellulose to generate the three-dimensional framework of the matrix in the biofilm.

Resistance of biofilms to disinfectants, germicides and antibiotics

Biofilms offer protection to the microorganisms that make them up, to the action of disinfectants, germicides and antibiotics. The mechanisms that allow this feature are the following:

• Delayed penetration of the antimicrobial agent through the three-dimensional matrix of the biofilm, due to very slow diffusion and difficulty in reaching the effective concentration.
• Altered growth rate and low metabolism of microorganisms in the biofilm.
• Changes in the physiological responses of microorganisms during biofilm growth, with altered resistance gene expression.

References

1. Bacterial Biofilms. (2008). Current Topics in Microbiology and Immunology. Tony Romeo Editor. Vol. 322. Berlin, Hannover: Springer Verlag. pp301.
2. Donlan, RM and Costerton, JW (2002). Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews. 15 (2): 167-193. doi: 10.1128 / CMR.15.2.167-193.2002
3. Fleming, HC and Wingender, F. (2010). The biofilm matrix. Nature Reviews Microbiology. 8: 623-633.
4. Gorbushina, A. (2007). Life on the rocks. Environmental Microbiology. 9 (7): 1-24. doi: 10.1111 / j.1462-2920.2007.01301.x
5. O'Toole, G., Kaplan, HB and Kolter, R. (2000). Biofilm formation as microbial development. Annual Review of Microbiology. 54: 49-79. doi: 1146 / annurev.microbiol.54.1.49
6. Hall-Stoodley, L., Costerton, JW and Stoodley, P. (2004). Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews Microbiology. 2: 95-108.
7. Whitchurch, CB, Tolker-Nielsen, T., Ragas, P. and Mattick, J. (2002). Extracellular DNA required for bacterial biofilm formation. 259 (5559): 1487-1499. doi: 10.1126 / science.295.5559.1487