| dc.description.abstract | Staphylococcus aureus infections pose a serious threat to human health with the increasing incidence of microbial resistance to last-line antibiotics such as daptomycin. Many S. aureus infections present as a biofilm wherein molecular heterogeneity of bacterial subpopulations has been observed, further confounding their burden on human health. Here, the utility of matrix-assisted laser desorption/ionization trapped ion mobility spectrometry imaging mass spectrometry (MALDI TIMS IMS) was assessed for the multi-omic spatial investigations of S. aureus biofilm layer heterogeneity. To achieve high spatial resolution molecular investigation of S. aureus biofilms by MALDI TIMS IMS, a workflow was first developed to prepare these samples for interrogating lipids, peptides, and intact proteins. Most lipids were observed to localize primarily to a single biofilm layer. Methods for on-biofilm trypsin digestion of bacterial proteins for bacterial tryptic peptide analysis were developed and applied toward spatial interrogation of S. aureus biofilms. Next, development of novel instrumental methods for high-mass analysis by MALDI TIMS IMS resulted in the detection of bacterial proteins up to m/z ~16,000. Both spatial proteomic approaches uncovered heterogeneity between biofilm layers, further underscoring the molecular differences between these biofilm subpopulations. Finally, biofilms grown with daptomycin-susceptible and -resistant clinical isolates of S. aureus were found to exhibit differences in abundance and distributions of lysyl-PG lipids between layers under normal growth conditions. Furthermore, MALDI TIMS IMS revealed a dose-dependent bacterial response to daptomycin treatment only in the daptomycin-resistant clinical isolate when exposed to varying doses of antibiotic. Intriguingly, although lysyl-PG lipids are associated with the proposed mechanism of daptomycin-resistance, these spatial investigations uncovered a shift in localization of these lipid species away from antibiotic penetration. Further work to confirm bacterial viability within the biofilm indicated that these shifts in spatial distribution may be the result of a coordinated localized biofilm response to the antibiotic. This elucidation of a coordinated biofilm response to antibiotic may provide a new avenue for localized biofilm treatment. | |
