Hey my name is Morten Alhede welcome to this session about biofilm properties. There are many definitions of bacterial biofilms but the most common definition is “an aggregation of bacteria on a surface embedded in a self-produced matrix”. In order to understand biofilms we have to study them. And there are many ways to study biofilms in laboratory. Assays vary from very simple and cheap models to complex but also more realistic models. In the simple end, we have the crystal violet assay. This assay is a very cheap and simple biofilm model. The assay is only assessing the quantity of attached bacteria in wells of a plate by applying and measuring a soluble stain bound to the bacteria. Another simple way to study biofilms is filter biofilms. Here the biofilms are grown on top of a micropore filter, which is lying on top of an agar plate. The filter enables transfer of the bacteria onto fresh agar plates when needed and hence the bacteria can be supplied with fresh nutrients, allowing mature biofilms to form. At third model, is a model we developed here at the department of international health, immunology and microbiology. We call it the semi solid model. Here the biofilms are not attached to a surface but instead kept in a semi solid matrix composed of digested meat powder, horse Blood and serum. The idea of this model is to provide a more realistic biofilm-model where host components are included. The importance of host components is dual. Firstly, the formed biofilms are different to biofilms raised in simple media – but secondly, the host components effectively bind antimicrobial molecules and hence must be considered when evaluating anti-biofilm treatments. There are as mentioned numerous of biofilm models, however the gold standard has always been the flow-cell system. This system utilizes a continuous flow on top of the bacteria growing in a chamber. The idea is to keep the bacteria supplied with a low but constant supply of nutrients and flush out non-attached bacteria, and hence to form a biofilm attached to the bottom off the chamber. Many extremely beautiful images and videos of biofilm formation have been produced using this assay. From a distance it looks like a simple stepwise process. First the bacteria attaches to surface then they grow into huge towers looking like mushrooms and lastly the bacteria within the mushrooms disperse into the surrounding media to form new biofilms and colonize new surfaces. However, lets take a closer look into the mushroom-looking towers First the stalk is formed by clonal growth – meaning that the bacteria divide and multiply to form the stalk. In contrast, the cap is dependent on motility – see here how the motile bacteria crawl on top of the biofilm to form the top part of the biofilm. During this stage the so-called matrix is produced. The matrix is a slime substance composed of mainly water, but also on all available macromolecules such as i. Polysaccharides ii. DNA iii. Protein These large structures are meant to be interspersed by channels to ensure nutrient supply. However, when measuring oxygen concentration within a biofilm it is obvious that the supply is not sufficient. The oxygen is rapidly consumed by the bacteria in the outer layers and subsequently, the middle part is almost depleted from oxygen. Due to this lack of oxygen and possibly also other nutrients the growth rate within a biofilm is dramatically lower than their planktonic counterparts The matrix components, the lack of oxygen and subsequent the slow growth, are all contributing to the hallmark of the biofilm – namely: extreme tolerance towards antimicrobials and immune cells. Due to these factors the biofilm can simply resist doses of antimicrobials that would otherwise be lethal to the bacteria. Recall that antibiotics mostly target metabolic process, so if they once penetrate the matrix there are no targets to inhibit. Interestingly, it is only physiologically dependent processes that make the biofilm tolerant – therefore it is not a genetic or inherited trait as is antibiotic resistance. Hence the all traits making the biofilm tolerant are reversible and can be circumvented! However, are these matrix-embedded mushrooms found outside a flow chamber? Are they found at the site of chronic infections and thus being important? So I guess the important question is: “are flow cell biofilms a realistic model that resembles what is going on during infections?” In a recent publication we looked into that question. Interestingly we found that biofilms found in the body are actually very small – ranging from 5 to 100um, and none of them looked like the enormous mushrooms found in the flow cells. In addition to being very small the biofilms are often also very heterogeneously distributed – meaning they can be extremely hard to find and diagnose. Another interesting observation was that even though Biofilms are per definition attached to a surfaced. It has been shown that aggregates are equally tolerant to antimicrobials and immune cells as surface attached bacteria. This conclusion is important since many in vitro assays are evaluating the antibiofilm properties with surface attachment as an endpoint… What if the compound is just releasing the tolerant aggregate into the blood stream? This stresses the need for more realistic biofilm models! So to sum up: We now that the biofilms are aggregates of bacteria embedded in a matrix of various compounds. The hallmark of the biofilm is to be extremely tolerant to outside threads. The tolerance is reversible and thus dependent on physiological properties rather than genetic and inherited traits. That is why biofilms is NOT a part of the resistance problem we are also facing in our environment, but needs its own research! Biofilms can be studied in numerous of different models in the lab, but it is important to know that all models have drawbacks and no models resembles what is found in the nature.
