The Bigger Picture on Dual-Species Biofilms
Dr Katherine Baxter returned to research through a three-year-sponsored fellowship from Medical Research Scotland with Professors Gail McConnell and Paul Hoskisson, looking at the biofilm formation of the fungus Candida albicans and bacteria Staphylococcus aureus.
Here, Katherine shares the latest discoveries from her research.
Biofilm research has gathered pace over the last few decades, and the concept has also evolved. For instance, a biofilm used to be defined as a film simply adhering a micro-organism community to a surface – attaching, maturing and disseminating. However, a far more complex picture is now emerging. Small aggregates of unattached biofilm contribute to secondary infections, and the way the micro-organisms themselves interact further adds to this complexity. The multifactorial nature of biofilms makes it challenging for both understanding and predicting their development.
My research focuses on the formation of dual-species biofilm structures, in mixed species communities of the fungus Candida albicans and the bacterium Staphylococcus aureus. Infections from these micro-organisms are far more serious when combined, compared to infections with either micro-organism alone. They result in greater virulence, and a poorer patient outcome. Understanding how these dual biofilms form and possibly contribute to the synergy between the micro-organisms is therefore crucial in understanding their role in antimicrobial resistance and infection.
Observing Biofilm Development
Following the biofilm development over time involves mixing the two cultures together, pipetting them onto a surface, and following the different structures emerging in the biofilm using time-lapse fluorescence microscopy.
Biofilm formation is followed over 12 hours after deposition, at three-hour time points, using the unique Mesolens microscope system (Figure 1). This bespoke imaging system has four times magnification and a numerical aperture of 0.47, combining a wide field of view with sub-cellular resolution. We can visualise whole structures within the biofilm with high resolution, and zoom in on individual cells. Other forms of microscopy restrict the field of view and you can’t see the structures in the context of the bigger picture, making it difficult to understand what is happening. This isn’t a problem with such a representative sample area from the Mesolens.
Multichannel fluorescence is also possible, and the CoolLED pE-4000 fluorescence LED Illumination System provided excitation for GFP and mCherry fluorophores. The illumination was very straightforward to control, especially with the manual pod. Before I started this project, it was several years since I had worked with fluorescence microscopes, and I have found the Mesolens with the pE-4000 straightforward to use, and I have been grateful for that.
Figure 1: A Valuable Tool for Biofilm Analysis: Combining a wide field of view with sub-cellular resolution, the Mesolens is a bespoke imaging system with four times magnification and a numerical aperture of 0.47. Image courtesy of David Blatchford.
Insights from the Mesolens
Candida is a dimorphic fungus, which can either exist as yeast form cells or hyphal form cells. Following biofilm development, we observed greater biomass formation and species interaction in the hyphal form. The hyphae at the surface catch any yeast form C. albicans and S. aureus cells to form aggregates. Over time, they grow into large rope-like structures, leading to greater potential for interaction from an earlier stage.
My findings have produced evidence to show that S. aureus clusters around hyphae, and the biofilm matrix protects the bacteria from anti-microbial drugs. This aligns with the literature, which provides many further examples of synergies between C. albicans and S. aureus that promote virulence.
Biofilms in Four Dimensions
For imaging under the Mesolens, cell samples are spotted onto a thin pad of agar and left to dry. This creates a ‘coffee ring’ effect due to the flow dynamics of a drying droplet. The ring around the periphery transitions into specific structures between both the yeast and the hyphae, while the core of the biofilm looks very different.
This suggests that the biophysical aspects are also potentially significant, where interaction between the two species is derived from fluid dynamics around the cell shape. At the start of this project, we did not expect to observe such complex structures and different zones.
Figure 2: Time-lapse of Dual-Species Biofilm Formation. Using the Mesolens with fluorescence illumination from the CoolLED pE-4000, C. albicans was visualised with GFP (cyan), and S. aureus with mCherry (magenta) in biofilms. Images show structures forming from the time of inoculation (a), 6 hours after inoculation (b) and at 12 hours after inoculation (c).
Next Steps for Biofilm Research
Biofilms process the information around them in order to produce an optimal structure for those given conditions, and the next step for my research is to understand this in greater detail. For example, conducting a stepwise analysis under a variety of different conditions to identify which biofilm components are produced in response, effectively dissecting how the communities form a biofilm in a systematic fashion. In the future, the hope is to analyse the data and utilise machine learning for predicting biofilm formation under different environmental conditions.
As standard antibiotics become less effective due to antimicrobial resistance, a stratified medicine approach could utilise the information to devise methods to modulate microbial responses to drugs in a way to avoid resistance. Data could one day help determine a biofilm’s structure and how it will develop, guiding the use of more effective treatments known to work for a given structure.
Helping scientists return to academia
The Daphne Jackson Trust is an organisation who supports people who have had to leave academic research. After leaving academia due to childcare responsibilities, and working part-time as a science lecturer, Katherine started looking for ways to get back into science.
If you have had a leave of absence from research for two years because of caring reasons or health reasons, The Daphne Jackson Trust offers two-year fellowships and also three-year-sponsored fellowships. Similar initiatives exist, such as the Dorothy Hodgkinson scheme with the Royal Society.
