Antibiotics-based fluorescent probes for selective labeling of Gram-negative and Gram-positive bacteria in living microbiotas

Gram-staining distinguishes bacteria into two major groups, Gram-positive and Gram-negative bacteria, and has become an essential technique in microbiology. However, Gram-staining is not compatible with living cells and thus limits its applications. Here, we report the development of a polymyxin B-based fluorescent probe that enables selective labeling of Gram-negative in the living microbiota samples. We first synthesized the polymyxin B-Cy3 conjugate and confirmed its specificity for labeling Gram-negative bacteria. In combination with a previously developed Gram-positive-specific fluorescent probe, we demonstrate two-color imaging of Gram-positive and Gram-negative bacteria in various kinds living microbiotas, including mouse gut, human oral, soil, and crude oil microbiotas, with high selectivity and coverage. Finally, a pilot use of the probes in staining bacteria on heat-fixed sputum smear was also demonstrated, showing its potentials in clinical microbiology. Our method provides a versatile tool for distinguishing Gram-positive and Gram-negative bacteria in both basic research and clinical settings.