Biofilms? The Microscopic Shields Bacteria Build to Survive

 

A coherent cluster of bacterial cells embedded in a matrix that are more tolerant to most microbial and host defenses than planktonic bacterial cells are called biofilms.

The best example to understand the structure of biofilms is a mushroom-like tree. At the top, there will be more oxygen, resulting in more growth, while on the inner surface, there will be less oxygen and slower growth. So, in conclusion, the outer part is more tolerable and the main support for biofilm compared to the inner part. If any Abx succeeds in entering inside the biofilm, there is no chance for an encounter.

Bacteria first multiply to form a matrix (have DNA, proteins, and LPS) and then form biofilms. Hosts respond to biofilms in three ways:

• non-inflammatory response
• immune response
• inflammatory response

The non-inflammatory defense involves permanent mechanisms that maintain the self-cleaning capacity of the organism, such as the skin and the respiratory tract. This defense system is continuous by default, which means that it is constitutively active and can be modulated by infectious microorganisms. The immune response is the body’s response to antigens. Antigens include pathogen-associated molecular patterns such as extra-cellular DNA, various toxins, LPS, and other microbial components. 

The inflammatory response is a reaction to tissue damage, and is not necessarily, as with the activation of the immune system, caused by antigens. Inflammation is characterized by pain, swelling, heat, redness, and by loss of functionality.

Ways to grow biofilms:

There are numerous approaches to studying biofilms. But in vitro, assays use more realistic models, ranging from basic, inexpensive models to more complex ones. Four types of assays are mostly used:

• Crystal violet assay
• Filter biofilms 
• Semi-solid assay
• Flow cell system

Crystal violet assay is a very cheap and simple biofilm model. The assay assesses the number of attached bacteria in the wells of a plate but it does not discriminate between live and dead bacteria.

Filtering biofilms is a straightforward method of studying biofilms. In this instance, the biofilms are developed on top of an agar plate that is resting on top of a micropore filter. When necessary, the filter allows the bacteria to be transferred onto new agar plates so they can receive fresh nutrients and create a biofilm.

In a semi-solid assay, the biofilms are preserved in a semi-solid matrix made of digested beef powder, horse blood, and serum rather than being attached to a surface. The purpose of this model is to offer a biofilm model that is more accurate by including host components. The host's components are crucial on two levels. First, the produced biofilms differ from biofilms generated in simple media. Second, the host components effectively bind antimicrobial molecules and must be taken into consideration when evaluating anti-biofilm treatments.

Flow cell systems are the gold standard for biofilm in vitro growth. In this system, there is a continuous flow of nutrients on top of bacteria growing in a chamber. The idea of this system is to provide a low but constant supply of nutrients to bacteria and to flush out non-attached bacteria from the chamber.

Tolerance:

The tolerance of biofilms is a part of its definition. All types of biofilms are tolerant to all types of Abx. Fresh and young biofilms are more susceptible to Abx than old biofilms. The tolerance of bacteria in biofilms is a result of three tolerant mechanisms:

• Physical
• Quorum sensing
• Physiological 

Physical tolerance depends on the three-dimensional structure of the biomass consisting of bacterial cells and matrix. Some of the matrix components such as DNA and polysaccharides (alginate) bind some types of antibiotics such as tobramycin and colistin. This binding leads to a delayed diffusion of the antibiotic molecules in the biofilm.

Quorum sensing is a cell-to-cell communication system that synchronizes gene expression in response to population cell density. It plays a role in tolerance by making extracellular DNA that inhibits Abx.

The metabolic condition of the bacteria developing in biofilms determines physiological tolerance. The ability to synthesize proteins, known as the metabolic rate, can only be found near the biofilm's air-liquid interface.