FLIM Phasor Fingerprint of Bacterial Metabolic State

Abstract

While a large number of bacteria are deemed beneficial, there is an increasing number of strains associated with pathology, including life-threatening diseases. Moreover, during the last decade, a large number of new infectious strains have been discovered. Treatment is often hindered by emergence multi-drug resistant bacterial strains. Bacteria adapt to adverse environmental conditions by changing their metabolic activity. Further, there also exists metabolic diversity within the same population. Non-invasive assessment of bacterial metabolism could shed light on their physiological status. In this work we employ two-photon fluorescence lifetime imaging microscopy (FLIM) of live, clinically relevant bacteria. FLIM of autofluorescent metabolic coenzyme reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been extensively exploited for label-free metabolic imaging of mammalian cells to study cancer and other diseases. However, it has not been explored to similar extents in bacteria. We apply phasor analysis to FLIM and create FLIM-phasor fingerprints of bacteria species Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis. The FLIM-phasor method also allows metabolic fingerprinting of individual bacteria within a population. We demonstrate that the NAD(P)H FLIM-phasor fingerprint of bacteria is sensitive to bacteriostatic and bactericidal antibiotics treatment. Furthermore, it varied with cell growth phase of planktonic cultures, suggesting that characteristic shifts in the NAD(P)H FLIM-phasor are representative of modulation of metabolic state of the cells. The FLIM-phasor approach represents a powerful non-invasive imaging technique to study bacterial metabolism and characterize the phasor fingerprint of bacteria under various conditions. This method could prove important in understanding bacteria related pathology, drug response and therapy as well as emergence of newer drug-resistant bacterial strains, all in a label-free manner.Work supported in part by NIH grants P50GM076516,P41GM103540