Optimising biofilm production of Clostridioides difficile for downstream applications: potential for methodological bias

Clostridioides difficile named an ‘Urgent threat’ by the CDC in 2019, causes the most common healthcare-associated infection in the USA (CDC 2019). The DnaK molecular chaperone responsible for protein folding is involved in the heat stress response in Clostridioides difficile (Jain et al. 2011). Disruption of the dnaK gene in strain 630Δerm resulted in a DnaK deficient strain with 50% cell elongation, increased hydrophobicity, and increased biofilm production (Jain et al. 2011). Investigation into biofilm forming abilities of weak and strong biofilm forming strains of C. difficile has revealed a plethora of techniques with various modifications which result in different findings.
C. difficile wild type strain 630, parent strain 630Δerm and dnaK mutant strain 630Δerm:dnaK biofilms were grown in microtiter plates, on glass coverslips and on semi-permeable membranes. Biofilm properties on each media were investigated using crystal violet assays, TTC assays, VCC and BacLight staining where appropriate.
Unsurprisingly, biofilms grown in closed systems were less sustainable, whereas replenished media biofilms were viable for extended periods of time. Furthermore, biofilms that were grown within a planktonic system were also more susceptible to disruption through washing techniques, making them less representative of a mature biofilm system. In order to fully characterise biofilm features of a specific bacterium different methods should be applied to control for technique variation, or a standardised approach created.