Spatial metabolic insights of Pseudomonas aeruginosa biofilm treatments using stable isotope labeling with DESI mass spectrometry imaging

Bacteria form biofilms on diverse substrates, and these communities can cause persistent infections owing to their elevated antibiotic resistance. Pseudomonas aeruginosa is a major cause of chronic infections because of its ability to form biofilms, which makes it difficult to eradicate with conventional antibiotics. The metabolic response of P. aeruginosa biofilms to antibiotic treatment, however, remains only partially understood. Here, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was used to map the spatial metabolic responses of mature and developing P. aeruginosa biofilms to ciprofloxacin and two ciprofloxacin-nitroxide hybrids. Stable isotope labeling of metabolites was used to support the spatial interpretation of quorum-sensing (QS) metabolites within the chemically complex and dynamic spatial environment of biofilms and to understand their biosynthetic pathway. Ciprofloxacin induced localized metabolic changes and selective reorganization of quorum-sensing and redox-associated metabolites in biofilms. Both hybrids underwent heterogeneous in situ reduction to their inactive hydroxylamine forms within biofilms. These results indicate that DESI-MSI with stable isotope labeling can resolve growth-stage-dependent colony biofilm metabolism and characterize the metabolic response of biofilms to antibiotic treatment.