Biofilms & Cyst Forms in Lyme Video

Eva Sapi Ph.D.

Associate Professor

University of New Haven

Department of Biology and Environmental Sciences

Dodds Hall 314

300 Boston Post Road, West Haven CT 06516

Telephone: (203) 479-4552

Fax: (203) 931-6097

The University of New Haven established a Lyme Disease Research group six years ago. To date, over forty graduate students have received training in Lyme disease related research. One of our recent projects studies the different formations of Borrelia burgdorferi, the Lyme disease bacteria. With coauthor Dr. Alan MacDonald, we recently suggested that Borrelia burgdorferi is capable of forming an organized structure called biofilm1. Our proposal is based on recently published2 and several unpublished images of Borrelia. Those images, like the image presented in this article (Figure 1) show structures that strongly resemble biofilm formation.

What is biofilm? “Biofilm is a self-made protective environment for microbial populations in which they adhere to each other or to living or inert surfaces”. In the biofilm, single or multiple types of organisms can surround themselves with a complex matrix, better known as “slime” 3-4. 

The main purpose of the biofilm structure is to allow microbes to survive various environmental stresses, including the presence of attacking immune cells like phagocytes, or antibacterial agents. While conventional antibiotic therapy is usually effective against free-floating bacteria, it is frequently ineffective once pathogens have formed biofilms, because biofilm colonies can be up to 1,000-times more resistant to antibiotics. 

Furthermore, even if a biofilm-related infection appears to respond to antibiotics, it could relapse weeks or even months later and turn into a very difficult to treat chronic infection. The National Institutes of Health estimate that nearly 80 percent of chronic microbial infections in the human body are due to biofilms, such as chronic lung infection in cystic fibrosis patients, catheter infections, chronic urinary and middle ear infections, gingivitis, sinusitis and even fatal endocarditis6.

What do we know about biofilm? Biofilms start as just a few microorganisms adhering to each other or to a surface, and then begin to communicate3-4. This communication will initiate a change in gene expression and cells start to produce an exopolysaccharide, which will become the protective matrix 3.

The colonies then can develop into complex, three dimensional structures housing millions of individual microbes. Like cities, they have towers, columns, bridges and channels for the flow of nutrients. A mature biofilm is usually composed of three layers: an inside film layer that binds the biofilm to the surface; another film made up of colonies of single or multiple species of bacterial and/or fungal organisms; and the surface film from which free-floating microorganisms can be released as individual cells that can colonizing other places3.

So what are the mechanisms by which bacteria can evade the therapeutic interventions in biofilm? The first studies suggested that the bacteria deep within the biofilm live in an environment where diffusion of antibiotics might be difficult. 

There are major differences in the chemical composition of the biofilm such as low pH and anaerobic condition etc, which can either inactivate the antibiotics or render bacteria inactive so the antibiotics cannot kill them6. However, recent publications suggested that the main reason for antibiotics resistance is the changes in the gene expression profile of microbial cells harboring in biofilms. 

For example, researchers identified mutant bacteria that are capable of forming biofilm but are not resistant to antibiotics7. The differential expression of a large number of genes is known to occur in the initial steps of biofilm formation8, such as the upregulation of exopolysaccharide synthesis9.

So how about the immune system? Why can’t they recognize and destroy the biofilm? Multiple studies demonstrate that phagocytes can be found attached to biofilm but they are not able to eliminate it3. 

To answer this very puzzling question German scientists used marine bacteria as a model and studied the defensive mechanism against their environmental enemy, which is a phagocyte (called amoebae). They identified that this marine bacterial biofilm can release a paralyzing agent that deactivates and even kills the amoeba10. So clearly biofilm is not just a defensive fortress, it can also fight back.

So how about chronic Lyme disease? Can it be explained by a biofilm formation of Borrelia burgdorferi? If yes, the possibility that Borrelia burgdorferi is capable of forming a biofilm can change the way we think about Lyme disease, especially in patients where it seems to be a persistent disease, despite long term antibiotic treatment11. 

The elucidation of the molecular mechanisms responsible for the switch from free-living growth to a biofilm phenotype, with the development of antibiotic resistance, should provide novel therapeutic targets in chronic Lyme disease. 

Despite the potential importance of this hypothesis, to date there has been no studies attempted to determine whether Borrelia burgdorferi is indeed capable of biofilm formation and whether such a formation results in increased antibiotic resistance. Borrelia burgdorferi sensitivity to antimicrobial agents has traditionally been studied in the free-living state12. Conclusions drawn from many of these studies, therefore, need to be revalidated.

We have recently established an in vitro model to study biofilm formation of Borrelia burgdorferi and proposed to use this system to evaluate the antimicrobial sensitivity of Borrelia burgdorferi in biofilm. Our goal is to test several known antibiotic agents frequently used in the Lyme disease treatment, as well as several natural agents, for the ability to interfere with or destroy biofilm production. This project recently received a generous support grant from the Turn the Corner Foundation.

As mentioned above, the formation of a biofilm begins with the attachment of free-floating microorganisms to each other or to a surface. In the case of Borrelia burgdorferi, our preliminary results show that Borrelia burgdorferi is capable of forming biofilm. Figure 1 shows a dense culture of Borrelia spirochete surrounded by a very dominant matrix. In this protective matrix, Borrelia burgdorferi can be found in spirochete and cystic form.

We have monitored several environmental conditions for the initiation of biofilm structure and found that that cell density is one of the most important factors. When Borrelia burgdorferi reach a certain density, they started to “stick” to each other and start to form an organized structure. 

Our working hypothesis for this finding is that nutrient depletion is the main stress factor for the initiation of biofilm. It was previously demonstrated that organisms within biofilms could withstand nutrient deprivation better than free-floating counterparts3. Furthermore, as we presented at the 2007 ILADS conference, we have seen similar changes after exposure of Borrelia burgdorferi to penicillin. In the penicillin treated samples, as early as 24h, we observed formation of a granual/cystic form covered by a biofilm-like substance13.

In our next set of experiments we will test other stressors that can initiate Borrelia burgdorferi biofilm formation, including different temperatures, pH, oxidative radicals, heavy metals and of course several synthetic and natural antibacterial agents. Our final goal is to identify antibacterial agents that are effective in killing Borrelia burgdorferi without inducing biofilm, or even capable of destroying Borrelia burgdorferi in biofilm.

In summary, if we can demonstrate that biofilm structure of Borrelia burgdorferi renders them resistant to antibiotics, it could provide a logical explanation as to why extensive antibiotic treatment for patients with a tick-bite history could fail. The end result from our study could provide novel therapeutic approaches for Lyme literate physicians to explore for chronically ill patients.

We would like to thank the Turn the Corner foundation for the generous support for this project and the Schwartz Foundation for donating a “state of the art” microscope for our research. For further information about UNH Lyme Disease Research Group please visit our website: www.unh-lyme.org.