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How Bacteria Stick to Implants and Become Tolerant to Antibiotics

Andet forskningsprojekt | 21/09/2017

Biomedical implants pose a life-long risk to their owners because they provide an opportunity for bacteria to attach to their surface and form biofilms. Bacteria in biofilms are encased in a protective matrix, and treating biofilm infections with antibiotics is in most cases a lost cause. Many researchers have tried to develop implant materials that repel bacteria, but this has so far been unsuccessful. Human proteins quickly cover the implant surface, and bacteria are equipped with specific receptors that bind to these proteins. We propose that the key to preventing biofilms lies not in preventing protein adsorption, but in controlling the conformation (shape) that adsorbed proteins will take, as bacteria will only bind to these proteins when they are in the right conformation.

This project aims to understand how the conformation of human proteins like fibrinogen and fibronectin affects the way bacteria interact with them. This knowledge is the basis of design and development of new implant materials that direct protein conformations to prevent bacterial attachment but promote tissue integration. We believe that the outcome of this project can lead to a new way of thinking in implant materials design, which is required to tackle the rising problem of implant-associated infections. 

Every year millions of people have their quality of life improved as a result of implanted medical devices such as pacemakers, cardiovascular stents, and orthopedic implants. Due to the shift in demographics towards an older population, we are currently seeing a dramatic increase in the use of implants, but along with the benefits comes a life-long risk of complicated and life-threatening infections caused by bacteria that attach to the implant surface and form biofilms.

Figure 1: Staphylococcus epidermidis biofilm (bacterial cells = blue) showing that vancomycin (green/cyan), which binds to the cell wall during growth, does not bind equally well to all of the cells in the biofilm. The cells in the top of the image are at the edge of the biofilm. Image courtesy of Sandra Skovdal.

Bacterial Biofilms Cause Persistent Infections

Most bacteria have two alternating lifestyles: They can live freely as single-celled organisms, or come together in a biofilm, which means that a community of cells is attached to a surface and encased within a matrix of organic molecules. Life in a biofilm provides many advantages for the bacteria. The matrix protects its inhabitants from predators, such as the body’s immune cells, and from antibiotics that are bound by the extracellular matrix. 

Furthermore, the sessile life in a biofilm requires a very low level of cellular activity, and these inactive cells are highly tolerant of many antibiotics that target cellular processes used for growth. Figure 1 shows a biofilm of Staphylococcus epidermidis (blue) and illustrates that bacteria deep in the biofilm bind are less susceptible to binding the antibiotic vancomycin (green).

The emergence of antibiotic resistance among important human pathogens is of great concern. However, such resistance is usually limited to a specific group of antibiotics, and - if discovered in time - shifting to a different drug can treat the infection. This option does not exist for biofilms. There is currently no effective antibiotic therapy for biofilm infections, because the lethal dose is so high that it would kill the patient along with the biofilm. The most effective treatment is therefore surgical removal and replacement of the implant. 

Such revision surgery often involves the risk of injury or death to the patient, it has a high rate of reinfection, and it is much more expensive than inserting the original implant. Because implant-associated infections can develop years after the implant was inserted, the increasing number of implants currently in use poses a growing drain on the health care system. Finding a solution for tackling implant-associated infections through prevention or better treatment therefore benefits both patients and the community.

“Biofilm infections remain one of the major unsolved challenges in infection biology, and we need a better understanding of this serious problem,” says Professor Thomas Bjarnsholt from Copenhagen University.

Preventing Biofilm Infections Through Development of Novel Implant Materials

Preventing biofilm infections by developing implant materials that prevent bacterial attachment is an attractive strategy, but cannot help patients who already have implanted devices. My research group is therefore working together with researchers from the fields of physics, chemistry and medicine to confront this problem both by: 1) developing coatings that prevent bacterial attachment and therefore biofilm formation, and 2) developing more effective drug formulations that combine several therapeutic strategies or concentrate antibiotics at the site of infection.

Figure 2: Bacteria attach to implant surfaces via the interaction with human proteins adsorbed to the implant surface. The same proteins are present in solution, but it is the adsorbed proteins that promote attachment. Adsorption of proteins induces conformational changes to their structure, and we propose that these changes enable their interaction with bacteria.

Both of these developments require intricate knowledge about how biofilms form and develop a tolerance of antibiotics. We therefore study the role of specific matrix components and adhesive molecules in surface attachment. The biofilm matrix was originally assumed to consist mostly of polysaccharides, but we now know that proteins and even DNA are equally important. For example, we have shown that amyloid protein fibers, otherwise famous for their role in Parkinson’s disease, can secure the biofilm structure by providing mechanical strength (1). We have also shown that DNA on the cell surface is used for the attachment to a wide variety of materials, and is critical for biofilm development in many different bacteria (2,3).  DNA is also the culprit of why bacteria can attach to anti-adhesive polymer coatings that are repellent to proteins. The 2 nanometer wide DNA strands extend from the bacterial cell surface, and are small enough to wriggle their way in between the polymer brushes of the coating to facilitate attachment of the cell to the underlying material. Such a polymer brush coating can therefore only repel bacteria when brushes are packed closer together than 2 nm (4). This example illustrates the importance of understanding the biological mechanisms of biofilm formation.

In the human body, colonization of implant surfaces happens via bacterial cell-surface proteins, which bind to specific human proteins, such as fibronectin, adsorbed to the implant surface. Preventing protein adsorption entirely is not always an option because implants also have to integrate with the human tissue. We therefore need to better understand the interaction between bacteria and the adsorbed human proteins, and how the implant material affects this interaction. In a new project funded by The the Carlsberg Foundation, we will study how human proteins that adsorb to implant surfaces promote bacterial attachment, and how the conformation (shape) of these proteins affect their interaction with bacteria (Figure 2).  It is well known that conformational changes of adsorbed proteins affect how human cells interact with the implant surface, and we hypothesisze that similar effects will be seen for bacteria. The question is, then, if the properties of the implant material can direct these protein conformations in a way that promotes tissue integration while preventing bacterial attachment.

Biofilms Tolerate High Concentrations of Antibiotics

- Bacteria attach to surfaces  

- Shortly after attachment, the cell changes its gene expression, and antibiotic resistance emerges through yet unknown mechanisms 

- After some time, the growth and accumulation of cells within an extracellular matrix results in biofilm formation. At this stage, the biofilm is highly tolerant of antibiotics due to 1) lack of penetration through the matrix, and 2) lack of effectiveness against inactive cells in the biofilm

Rikke Louise Meyer about the Grant from the Carlsberg Foundation

The Distinguished Associate Professor Fellowship is vital for my research because it allows me to maintain the right balance between applied and fundamental research projects in my laboratory. Keeping this balance is important because it is - in my experience - the knowledge we generate in fundamental research projects, which breed the ideas for next year’s applied research direction. Doing fundamental and applied research side by side, shortens the route from generating conceptual knowledge that seeds ideas which then develop into applications in collaboration with industry for the benefit of society:

“It is my vision that this conceptual knowledge will change how we think about the design and development of implant materials, and that the knowledge we generate will be translated into design of better and safer implants,” says associate professor Rikke Meyer.

Publications that Relate to this Topic

1. Zeng, G., B. S. Vad, M. S. Dueholm, G. Christiansen, M. Nilsson, T. Tolker-Nielsen, P. H. Nielsen, R. L. Meyer* and D. E. Otzen* (2015). "Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness." Frontiers in Microbiology 6 (OCT), 1099
2.  Okshevsky, M., Viduthalai R. Regina and Rikke L. Meyer*. (2015) Extracellular DNA as a target in biofilm control. Current Opinion in Biotechnology. 33:73-80
3. Regina V. R., A. R. Lokanathan, J. J. Modrzyński, D. S. Sutherland, and R. L. Meyer*. (2014) Surface physicochemistry and ionic strength affects eDNA’s role in bacterial adhesion to abiotic surfaces. PLOS One, 9 (8), e105033.
4. Zeng, G., R. Ogaki and R. L. Meyer (2015). "Non-proteinaceous bacterial adhesins challenge the antifouling properties of polymer brush coatings." Acta Biomaterialia 24, 3747, 64-73.
5. Meyer, R.L.*, A. Arpanaei, S. Pillai, N. Bernbom, J.J. Enghild, Y.Y. Ng, L. Gram, F. Besenbacher, P. Kingshott. (2013)  Physicochemical characterization of fish protein adlayers with bacteria repelling properties. Colloids and Surfaces B: Biointerfaces 102, 504– 510