Out of control: The need for standardised solvent approaches and data reporting in antibiofilm assays incorporating dimethyl-sulfoxide (DMSO)

Bacteria in biofilm formations are up to 1000 times less susceptible to antibiotics than their planktonic counterparts. Recognition of the role of biofilms in ∼80% of chronic infections, their contribution to bacterial tolerance and development of antimicrobial resistance, and thus the search for compounds with antibiofilm properties, has increased greatly in recent years. The need for robust experimental methods is therefore critical but currently undermined by inappropriate controls when dimethyl-sulfoxide (DMSO) is used to enhance test compound solubility. DMSO is known to have a limited effect on planktonic growth, but emerging data indicates that the solvent can affect biofilm formation even at low concentrations. Here, we present both a literature review on the application of DMSO in in vitro antibiofilm studies, as well as a series of experiments and Bayesian hormetic dose-response modelling to define the effects of DMSO alone and in combination with standard antibiotics using two clinically important biofilm-forming bacteria. DMSO has been used in 76 published studies to solubilise a wide variety of synthesised and natural products, including plant extracts, isolated secondary metabolites, modified lead molecules and proteins, in in vitro antibiofilm assays. DMSO solvent concentrations to which biofilms were exposed ranged between <1 and 100% but unfortunately, 35% of articles did not specify the DMSO concentrations used, 50% of articles did not include solvent controls and, of those that did, 26% did not specify control concentrations, 47% did not report or discuss control data, and 53% omitted media controls. In a further 12 studies, DMSO is used as a biofilm treatment, demonstrating the antibiofilm properties of this solvent at higher concentrations. We provide evidence that DMSO (between 0.03 and 25%) significantly inhibits biofilm formation in Pseudomonas aeruginosa, but not Streptococcus pneumoniae, and acts synergistically with standard antibiotics at very low concentrations (<1%). Interestingly, intermediate concentrations of DMSO (∼6%) strongly promote the growth of P. aeruginosa biofilms. As the research community strives to identify bioactive antimicrobial compounds, there is a need for increased scientific rigour when using DMSO as a solvent in antibiofilm assays.