Metabolic fingerprinting of bacteria by fluorescence lifetime imaging microscopy (original) (raw)

Bacterial populations exhibit a range of metabolic states influenced by their environment, intra-and interspecies interactions. The identification of bacterial metabolic states and transitions between them in their native environment promises to elucidate community behavior and stochastic processes, such as antibiotic resistance acquisition. In this work, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to create a metabolic fingerprint of individual bacteria and populations. FLIM of autofluorescent reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been previously exploited for label-free metabolic imaging of mammalian cells. However, NAD(P)H FLIM has not been established as a metabolic proxy in bacteria. Applying the phasor approach, we create FLIM-phasor maps of Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis at the single cell and population levels. The bacterial phasor is sensitive to environmental conditions such as antibiotic exposure and growth phase, suggesting that observed shifts in the phasor are representative of metabolic changes within the cells. The FLIMphasor approach represents a powerful, non-invasive imaging technique to study bacterial metabolism in situ and could provide unique insights into bacterial community behavior, pathology and antibiotic resistance with sub-cellular resolution. Bacteria are found in diverse environments, and their ability to modulate metabolic processes in response to adverse conditions gives them unique survival advantages. In the natural environment, multiple species interact in complex niches and exhibit a range of community behavior 1-4. In the medical environment, these metabolic changes make bacterial infections difficult to eliminate. Treatment of bacterial infections are often hindered by emergence of multi-drug resistant phenotypes 5, 6. A major source of antibiotic resistance and tolerance are slow growing 'persister' phenotypes, which are often associated with biofilm formation and chronic infections 7-9. Mapping the metabolic activity of bacteria within natural communities and medical infections, therefore, can provide insights into the role of metabolism in determining bacterial community behavior. Fluorescence spectroscopy is a non-invasive technique which has been extensively used for metabolic imaging in mammalian cells. Fluorescence lifetime imaging microscopy (FLIM), in particular, is a powerful label-free method to probe the local environment and molecular conformation of endogenous fluorophores 10. The FLIM signature of an autofluorescent metabolic coenzyme, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), has been employed as an endogenous biomarker for metabolic activity of mammalian cells in cancer biology and the detection of stem cell differentiation 11-17. NAD(P)H in its free state has a significantly shorter fluorescence lifetime (0.4 ns), due to self-quenching, than its protein bound state and is thus easily discernable by FLIM 18. FLIM measurements allow mapping of NAD(P)H lifetimes with sub-cellular resolution. The relationship between the NAD(P)H fluorescence lifetime and metabolic activity, however, has not been established in bacteria. As in the case of mammalian cells, bacteria produce many endogenous fluorescent molecules with distinct spectral characteristics 19, 20 , some unique to specific bacteria 21, 22 , rendering them as promising probes for identification and characterization. Fluorescence spectroscopy of some intrinsic fluorophores has been previously explored in bacteria for the detection, differentiation, and characterization of various species 23-27. The relationship between cytosolic concentrations of NAD(P)H in bacteria and their metabolic activity has been previously