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.