Undergraduate Student Report 2021

 Willemijn Kuiters worked with Dr Ryan Kean, Glasgow Caledonian University, to investigate the effect of co-infection with the bacterium S. aureus on the ability of C. albicans to form biofilms.

It is well known that Candida albicans and the bacterial pathogen Staphylococcus aureus are able to physically interact to cause infection, but little is known on potential chemical interactions between these two pathogens. Therefore, the aim of my studentship project was to investigate this potential chemical relationship between C. albicans and S. aureus and how this relationship is able to affect biofilm formation and drug susceptibility.

To start off the project, I started by growing and assaying C. albicans and S. aureus biofilms grown using conventional techniques such as Crystal Violet and XTT assays which can give an indication of the biofilm biomass and viability. Following this, three different biofilms were grown in bulk (Candida only, Staphylococcus only and a mixed culture) after which the supernatants were extracted, filtered and used to supplement the growth of C. albicans biofilms at different concentrations (5/10/20%). By filtering the supernatants, this meant that there were no cells present, only any microbial factors which might be secreted by the biofilm. The effect of these supernatants on biofilm formation was assayed again using the crystal violet and XTT assays together with colony forming unit counting and fluorescent microscopy. Biofilms grown with supernatants were also treated with different concentrations of the antifungal micafungin to investigate any potential increased drug tolerance of these biofilms. Finally, given the differences in biomass and morphological as shown in figure 1, qPCR was sued to assess the expression of three key C. albicans biofilm genes (Als3, Hwp1 and Zap1).

The clinical isolate of C. albicans used does not normally form a good biofilm with regards to biomass, but by growing the biofilm in presence of the bacterial supernatants, the biofilm forming properties were enhanced. The results demonstrated that there is an increase in biomass but not in viable cells after growing the Candida biofilms with the bacterial supernatants, indicating a much denser biofilm (Figure 1), likely due to an increase in extracellular matrix (ECM) production, a defining feature of biofilms. The qPCR results highlighted that the Zap1 gene of C. albicans gets highly upregulated in the presence the supernatant which is a gene that is known to be involved in the production of the ECM.  There is an increase in drug tolerability to Micafungin when growing the biofilms with the treated media (supernatants).

Figure 1. C. albicans biofilm grown in the absence (A) and presence (B) of bacterial supernatant. Biofilms were stained with calcofluor white and imaged using an EVOS fluorescence microscope.

Some experimental difficulties were encountered but we were able to troubleshoot so that we didn’t have to deviate from the original plan. One example was that we did start off using two different C. albicans strains, but following the initial Crystal violet and XTT assays and also microscopy, it was clear one of these strains would be more appropriate for this project as it was demonstrating better biofilm formation.

Given some of the differences which my data demonstrated, it would be great to look in more detail at what molecule(s) within the bacterial supernatant are causing the biofilms of C. albicans to increase. To identify these, we could use metabolomics of the supernatants to identify any potential metabolites which are highly present in the supernatant samples. Given the frequency of these organisms causing hospital-acquired infections, there is a medical interest to better understand the biology and relationship of these species.

Throughout the eight weeks I could feel myself becoming more and more confident in the lab and it was great to engage with other undergraduate, MSc and PhD students. My technique and skills within the lab have developed and I have got a clearer idea of what working in a research environment is like. My studentship has confirmed for me that my future after my undergraduate degree at university is in research and I am keen to pursue a postgraduate degree in research. I have loved working in the lab over the summer and I am so grateful to have been given this opportunity, so a massive thank you to the Society for giving me this opportunity.

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