(154b) Invited Talk 2: Repeatability of Metabolic Profiles in Multispecies Biofilms – Toward Metrics for Biofilm Comparability

Novel materials and approaches to control microbial biofilms are typically tested in vitro to evaluate their efficacy. Consistency among control biofilms within an experiment, across experiments, and among laboratories and operators is needed to enable repeatability within a lab and reproducibility and comparability across labs. In vitro model biofilm systems are increasingly multispecies rather than monospecies to better represent naturally occurring biofilms. However, reproducibility in multispecies biofilms can be daunting due to their complexity including genotypic, phenotypic, physical, chemical, biological, and mechanical heterogeneities on multiple length scales. We hypothesize that biofilm function can be an indicator of biofilm repeatability and reproducibility, and metrics based on metabolomic studies can help establish criteria to demonstrate a biofilm is within expected limits, thus enabling comparison among laboratories.

The objective of this work was to evaluate the repeatability of metabolic profiles for a two-species model biofilm through small molecule mass spectral analysis. The biofilms consisted of Pseudomonas aeruginosa and Staphylococcus aureus, two common pathogens often co-located in human disease states including medical device infections, chronic wound infections, and chronic lung infections of cystic fibrosis (CF) patients. The species were inoculated as monocultures or ≈50:50 co-cultures and grown for 18 h in artificial sputum medium to mimic nutrient conditions found in CF lung infections. Experiments were performed on three separate days with three biological replicates and three technical replicates. Cells were quantified via plating on selective media and DNA extraction followed by quantitative polymerase chain reaction. Spent culture medium and cells were quenched and extracted to obtain exo-metabolites and endo-metabolites, respectively. Global metabolic profiles were obtained using liquid chromatography tandem-mass spectrometry (LC-MS/MS) and analyzed using in-house software platforms.

Co-cultures consisted predominantly of P. aeruginosa, as expected based on literature. All metabolic profiles had an acceptably low variability over three levels (day, biological replicate, and technical replicate). Principal component analysis indicated that metabolic profiles of P. aeruginosa, S. aureus, and the co-culture were statistically differentiable (n = 9), with exo-metabolic profiles of the co-culture having over 500 unique features as compared to monocultures. All cultures had over 500 compounds in common but at different quantities, suggesting up- and down-regulation of pathways in the co-culture. These results suggest that LC-MS/MS metabolic analysis holds promise to monitor repeatability and reproducibility in polymicrobial biofilms. Additional studies are needed to determine robustness including across laboratories, culture conditions, and timepoints, and also to identify unique features of interest. Overall, our approach has the potential to enable comparison and combination of data from multiple laboratories to improve characterization of material effects on biofilms.