Researchers develop better methods for studying antimicrobial resistance

Disease and treatment 17. okt 2020 4 min Head of Section, Professor Anja Boisen Written by Kristian Sjøgren

Studying how bacteria act when they form biofilms in the lungs of a person with cystic fibrosis is difficult. Danish researchers have solved this problem with a new invention.

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Bacteria in the lungs of people with cystic fibrosis form biofilms that clump together in a layer of mucus.

They remain dormant and do not absorb very many nutrients or even grow.

When the bacteria form biofilms, they are very difficult to eliminate, and this is generally the worst news for people with cystic fibrosis.

Further, studying bacterial pathogens in biofilms in the laboratory and thereby identifying the best options for eliminating them is very difficult.

However, Danish researchers have found a solution to this problem, and this may mean much better opportunities for developing and testing antibiotics for people with cystic fibrosis and many other diseases, in which pathogenic bacterial biofilms play a role.

“Biofilms are one reason why treating people with cystic fibrosis properly is so difficult. Screening for potential treatments in the laboratory is extremely difficult because we cannot recreate the conditions in the lungs, and even if these conditions can be recreated, the process takes a long time and is very cumbersome. We have developed a method that makes it much easier to study the formation of pathogenic bacterial biofilms and how to kill them,” explains a researcher behind the new study, Anja Boisen, Professor and Head of Section, Department of Health Technology, Technical University of Denmark.

The research, which is the result of collaboration between the Department of Health Technology and Novo Nordisk Foundation Center for Biosustainability of the Technical University of Denmark and Rigshospitalet in Copenhagen, has been published in Analytical Chemistry.

Difficult to recreate biofilms in the laboratory

Anja Boisen and colleagues have come up with a solution to the problem.

When bacteria form a biofilm, they secrete a matrix of mucus, comprising protein, sugar, DNA and fat.

The matrix enables the bacterial colony to attach to a surface, protecting the bacteria from the surrounding environment.

Biofilms could be described as bacterial fortresses that antibiotics have difficulty penetrating.

Biofilms do not form exclusively in the lungs of people with cystic fibrosis but are present in many places, including teeth, chronic wounds and the inside of the tubes and catheters used in hospitals.

With the great medical interest in counteracting biofilm formation, many doctors and researchers clearly want to take biofilms from the lungs of people with cystic fibrosis and transfer these to a plate in the laboratory and then try to influence them with antibiotics or examine them with a microscope.

However, this is easier said than done.

“The problem is to recreate something that realistically reproduces the bacterial environment. In the lungs, they grow in a three-dimensional structure, and access to nutrients flows over the biofilm. Growing the bacteria on a flat agar plate with the nutrients coming from below does not help because it does not create a realistic environment and is therefore not suitable for studying biofilms,” explains Anja Boisen.

Compact disc-like platform for cultivating biofilms under realistic conditions

Despite the obstacles, researchers have developed various techniques that enable insight into what the bacteria in a biofilm do. But as Anja Boisen says:

The chip on which the bacteria grow may be very small, but then there are 500 tubes and hoses in the background, which makes it all extremely cumbersome to operate.

The innovative platform that Anja Boisen and her team have developed is a new way of studying bacteria cultured in a biofilm.

The researchers make compartments and openings in a plastic disc that resembles a compact disc, to create an environment where biofilms can grow.

When the disc is rotated very slowly, nutrients – food for bacteria – are moved from the centre towards the edge of the disc that has small chambers in which bacteria can grow and form biofilms.

This means that when the researchers add a nutrient-rich medium, it flows across the disc and the bacterial film, which absorbs the amount the bacteria need to grow.

The bacteria on the compact disc-like platform are also easy to study under a microscope, and then the entire set-up can be placed in a warming cabinet at 37°C to give the bacteria optimal growing conditions.

“They only need a continual supply of fluid and food to grow, and they prefer to grow this way with a flow over them. This resembles the human body, which is also in 3D and with flowing nutrients,” says Anja Boisen.

Discovering which antibiotics are most effective for people with cystic fibrosis

This method enables the researchers to study not only how biofilms form but also the effects of treating a biofilm with different types of antibiotics and in different combinations.

The method can be used in research aiming to develop new antibiotics or study how bacteria respond to an antibiotic or in direct treatment.

For example, Anja Boisen envisions that, based on the sputum of a person with cystic fibrosis, the bacteria would be cultured in a biofilm using the new method and then many antibiotics could be tested on the exact type of bacteria each person has in the lungs.

An antibiotic or combination thereof that seems especially effective can be immediately applied to the patient.

“This will enable treatment to be quickly adapted, because growing the bacteria in a biofilm and testing treatments takes just a few days. Each disc has eight chambers, so you can try several treatments at the same time,” explains Anja Boisen.

A patented discovery

Anja Boisen also envisions various ways to apply the new method in basic research.

She is researching new methods for delivering antibiotics precisely where they will optimally harm bacteria.

The researchers have already tested this method on biofilms cultured on discs, and the results will be published soon.

“We can also place a camera on the disc so we can monitor the bacterial growth in real time. We can also measure this using various instruments and get much better insight into what happens when the bacteria change from acting one way as individual bacteria to acting completely differently in a biofilm,” says Anja Boisen.

She also says that the new laboratory equipment has already been patented and that the researchers are negotiating with an industrial partner about mass production and global distribution.

Bacterial cell cultures in a lab-on-a-disc: a simple and versatile tool for quantification of antibiotic treatment efficacy” has been published in Analytical Chemistry. Researchers from the Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby co-authored the article.

Anja Boisen is working on micro- and nano sensor development and microdevices for drug delivery. The sensor projects are focusing on micro- and nanose...

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