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.
microbiota imaging Gram staining polymyxin B differential bacterial labeling
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This work was supported by the National Natural Science Foundation of China (21672013, 21425204, 21521003) and the National Key Research and Development Projects (2016YFA0501500). We thank Dr. Xiaolei Wu for providing the crude oil microbiota and the National Center for Protein Sciences Beijing (Peking University, Beijing, China) for help with flow cytometry analyses.
1.Institute of Molecular Medicine, Renji Hospital, School of MedicineShanghai Jiao Tong UniversityShanghaiChina
2.College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, Peking-Tsinghua Center for Life Sciences, Synthetic and Functional Biomolecules Center, and Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of EducationPeking UniversityBeijingChina