Biofilm Formation in Borrelia

For the advanced reader- here is a great article discussing the biofilm slime that covers and protects the Lyme bateria from dying when antibiotics are introduced. It’s got some pictures! http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0048277

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2 responses to this post.

  1. Thank you for posting this important and ground-breaking study. For readers who have trouble with some of the scientific terminology, I have done a layperson’s summary:

    Summary of Biofilm paper by Sapi et al at PLOS ONE Oct 2012 written by Elena Cook

    Biofilms are highly organised structures, created by microbes, which notoriously protect them from unfavourable conditions in their surroundings. Examples of the latter include an environment which is too acidic/alkaline; a change in temperature; and chemicals toxic to the microbe, such as antibiotics. Biofilms are known to be involved in serious chronic infection.

    The researchers in this study set out to discover whether Borrelia burgdorferi (Bb), bacteria which cause Lyme disease, are capable of forming biofilms in the test tube.

    They point out that Bb is well known to possess various strategies for survival and persistence in the infected host. For example, Bb has been proven to transform itself from its familiar spiral shape into other forms known as “Cystic”, “Granular” and “Cell-wall Deficient” varieties, which are adaptations to environmental conditions which might otherwise destroy it.

    The authors note that organised collections, or “aggregates” of Bb containing not only spirals, but also Cystic and Granular forms have been seen both in the test tube and in living infected tissue. The formation of these aggregates dramatically impacts Bb’s ability to survive in unfriendly environments.

    There are certain characteristics which are considered to be hallmarks, or “markers”, of biofilms. These include:

    • Ongoing re-arrangement of structure and shape during development of the biofilm

    • Ability to grow on a variety of living and non-living surfaces

    • Secretion of EPS. [EPS stands for Extracellular Polymeric Substances. This is non-living material, distinct from the bacteria themselves, which envelopes the structure as a protective layer. Certain components of the EPS such as Alginate, Calcium and extracellular DNA are commonly seen in Biofilm EPS]

    The researchers studied Bb cultures with a variety of microscopic techniques to determine which, if any, of the above Biofilm markers were present. They grew three sub-types of Bb in culture in polystyrene tubes (Bb 297, B31 and a variety of B31 genetically altered to glow fluorescently green for easier identification).

    While initially they saw only individual spiral Borrelia, once the concentration had reached ten million bacteria per ml, aggregates began to appear.

    Structural Re-arrangements

    The authors describe, with accompanying photographs, changes in shape and structure that appeared over time. Using the technique of Darkfield Microscopy, they saw a semi-spherical shape flatten to become film-like, then further change into a rigid structure with defined “hills, valleys and cracks”.

    Examination of the same cultures using another technique, DIC microscopy, clarified that the “film” was not uniform but had a diverse composition. FITC staining demonstrated that spiral-shaped Bb create the original structure, and can be found throughout it after a few days, but some days later most of the Bb are in the form of “Round Bodies” (cysts).

    The team next used the revolutionary technique of Atomic Force Microscopy (AFM). This allowed them to visualise the structural changes during the development of the Bb aggregates on an unprecedented nano-scale [ie one billionth of a metre; the diameter of atoms]. [Modern electron microscopes can achieve nano-scale resolutions, but the necessary preparation of the specimen by freezing or chemical techniques kill the organisms, so that the development in time could not be seen, and/or may damage the structure. Using AFM eliminated these drawbacks.]

    The team observed spiral –shaped Bb combining in pairs, which later assembled themselves into a web-like mesh. In the centre they saw spiral Borrelia folding in on themselves, and Cystic forms throughout. Individual meshes then went on to combine together to form a progressively larger network, with new features appearing such as arches and loops. These latter were of uniform size, indicating that a highly organised and deliberate activity was taking place.

    As time went on, more re-arrangements to the structure occurred, with pits and protrusions appearing. The authors have included 3D images documenting these changes.

    Ability to Grow on Varied Surfaces

    The New Haven team attempted to grow aggregates of Bb on a wide range of substances, including materials that are non-living ( glass and plastic) and those derived from living tissue, such as rat collagen and Matrigel (a product derived from mouse tumour ). Bb was able to attach to, and form aggregates on, all these materials, even when its density was only 5000 cells per ml. The authors also found that Bb could form free-floating aggregates, and have included a video demonstrating this.

    EPS

    The team biochemically analysed the covering of the structure to see if it conformed with the known composition of Biofilms. Using a staining method known as the Spicer and Meyer technique, they found that the material was either alginate or an alginate-like compound.

    Alginate is a substance found in nature in brown algae, but is also a well-known constitutent of EPS in Biofilms. Tests with antibodies that bind specifically to Alginate proved that it was this.

    Presence of Calcium on the surface of the EPS is another marker of Biofilm. Alginate reacts with Calcium to form an insoluble compound, Calcium Alginate. Using a specific stain for Calcium (Alizarin Red-S), it was shown that the centre of the aggregates were positive for presence of Calcium.

    Another hallmark of Biofilm formation is the occurrence of extracellular DNA (eDNA) on the surface. This plays several roles, including helping microbes to attach to surfaces, and enabling them to resist unfavourable conditions. Using the chemical stain DDAO, the team found that eDNA was indeed present on the surface of the aggregates.

    The researchers point out that eDNA bearing a negative electric charge is known to be able to chelate certain minerals such as Magnesium and Calcium. This can play a role in bacterial resistance to antibiotics. They hope to explore this connection with regard to Bb in the future.

    Borrelia is part of the bacterial classification of Spirochetes, and other spirochetes are known to make biofilms, some of which are involved in disease.

    The team note that Treponema denticola, a spirochete found in biofilms along with other microbes, is involved in gum disease. This bacterium also produces biofilm in the test tube.

    Another spirochete, Leptospira, forms free-floating biofilms. Certain leptospira cause Weil’s Disease, a serious and often fatal malady transmitted by contact with rat urine.

    Atomic Force Microscopy allowed the researchers to see channels running through the Bb aggregate structure, similar to those known to occur in Biofilms made by Leptospira and other bacteria. These channels may function to supply nutrients and oxygen to the bacteria resident in the Biofilm, and carry away waste products, much like the circulatory system in a human.

    The team discuss the possibility of Bb forming Biofilms in living organisms, and the question of whether these might serve other functions in addition to protecting the bacteria.

    Bb aggregates have been found in nymphal (juvenile) ticks feeding on blood, and these may play a role in transmission of disease. Bb in non-motile form (unable to move as individual bacteria) have nevertheless been shown to advance as “networks” inside the gut of the tick. They hope to re-examine Bb aggregates in feeding adult and nymphal ticks to see whether hallmarks of Biofilm are present there.

    Taken together, the weight of all the evidence above proves that Bb forms Biofilms under test tube conditions. This is the first study to demonstrate this conclusively. The researchers now look forward to investigating the prospect of Bb Biofilm formation in the living infected host, and its possible role in chronic Lyme disease.

    Full paper including images and video at link http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0048277

    Reply

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