A tridecaptin-based fluorescent probe for differential staining of Gram-negative bacteria

  • Wei Wang
  • Yingyan Wang
  • Liyuan Lin
  • Yanling Song
  • Chaoyong James YangEmail author
Part of the following topical collections:
  1. New Insights into Analytical Science in China


The traditional Gram-staining method, which was invented more than a century ago for differentiating bacteria as Gram positive or Gram negative, is still widely practiced in microbiology. However, Gram staining suffers from several problems which can affect the accuracy of the diagnosis. Here, we report a new Gram-negative-specific fluorescent probe, which is based on a narrow-spectrum antibiotic, tridecaptin A1, and allows selective staining of Gram-negative bacteria in different fixed bacterial samples. Solid-phase peptide synthesis was used to prepare the tridecaptin A1–fluorophore conjugate with a single structure. Labeling selectivity of the probe toward Gram-negative bacteria was confirmed by testing against a panel of bacterial species. By combining the use of a previously reported Gram-positive-specific fluorescent probe, we then further showed the capability of the new probe in differential labeling of a number of complex bacterial samples, which included a mouse gut microbiota cultured in vitro, as well as microbiotas collected from the human oral cavity, soil, and crude oil. High labeling selectivity and coverage were observed in most samples. This method offers a new Gram-negative-specific probe with a defined structure, which allows facile fluorescence-based differentiation of Gram-positive and Gram-negative bacteria for further microbial studies.


Microbiota imaging Gram staining Tridecaptin A1 Differential bacterial labeling Antibiotic-based probe 


Funding information

This work is supported by the National Natural Science Foundation of China (21735004, 21775128, 21705024, 21807070).

Compliance with ethical standards

Oral microbiota donor had given informed consent for the experiment. All animal experiments were carried out in accordance with guidelines approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine, and the use of human microbiota samples was approved by the Ethics Committee at Renji Hospital of Shanghai Jiao Tong University School of Medicine.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Beveridge TJ. Use of the Gram stain in microbiology. Biotech Histochem. 2001;76:111–8.CrossRefGoogle Scholar
  2. 2.
    Madison BM. Application of stains in clinical microbiology. Biotech Histochem. 2001;76:119–25.CrossRefGoogle Scholar
  3. 3.
    Forster S, Snape JR, Lappinscott HM, Porter J. Simultaneous fluorescent Gram staining and activity assessment of activated sludge bacteria. Appl Environ Microbiol. 2002;68:4772–9.CrossRefGoogle Scholar
  4. 4.
    Holm C, Jespersen L. A flow-cytometric Gram-staining technique for milk-associated bacteria. Appl Environ Microbiol. 2003;69:2857–63.CrossRefGoogle Scholar
  5. 5.
    Gram C. The differential staining of Schizomycetes in tissue sections and in dried preparations. Fortschr Med. 1884;2:185–9.Google Scholar
  6. 6.
    Mcclelland R. Gram’s stain: the key to microbiology. Med Lab Obs. 2001;33:20–2.Google Scholar
  7. 7.
    Mason DJ, Shanmuganathan S, Mortimer FC, Gant VA. A fluorescent Gram stain for flow cytometry and epifluorescence microscopy. Appl Environ Microbiol. 1998;64:2681–5.PubMedPubMedCentralGoogle Scholar
  8. 8.
    Clauß M, Springorum AC, Hartung J. Comparison of different fluorescence and non-fluorescence staining techniques for rapid detection of airborne micro-organisms collected on room temperature vulcanizing (RTV) silicones from generated aerosols and from ambient air. Aerosol Sci Technol. 2012;46:818–27.CrossRefGoogle Scholar
  9. 9.
    Sizemore RK, Caldwell JJ, Kendrick AS. Alternate Gram staining technique using a fluorescent lectin. Appl Environ Microbiol. 1990;56:2245–7.PubMedPubMedCentralGoogle Scholar
  10. 10.
    Kajiwara H, Toda M, Mine T, Nakada H, Wariishi H, Yamamoto T. Visualization of sialic acid produced on bacterial cell surfaces by lectin staining. Microbes Environ. 2010;25:152–5.CrossRefGoogle Scholar
  11. 11.
    Wang W, Chen X. Antibiotics-based fluorescent probes for selective labeling of Gram-negative and Gram-positive bacteria in living microbiotas. Sci China Chem. 2018;61:792–6.Google Scholar
  12. 12.
    Shoji J, Hinoo H, Sakazaki R, Kato T, Wakisaka Y, Mayama M, et al. Isolation of tridecaptins A, B and C (studies on antibiotics from the genus Bacillus. XXIII). J Antibiot. 1978;31:646–51.CrossRefGoogle Scholar
  13. 13.
    Cochrane SA, Findlay B, Bakhtiary A, Acedo JZ, Rodriguezlopez EM, Mercier P, et al. Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II. Proc Natl Acad Sci U S A. 2016;113:11561–6.CrossRefGoogle Scholar
  14. 14.
    Wang W, Zhu Y, Chen X. Selective imaging of Gram-negative and Gram-positive microbiotas in the mouse gut. Biochemistry. 2017;56:3889–93.CrossRefGoogle Scholar
  15. 15.
    Hancock R. The bacterial outer membrane as a drug barrier. Trends Microbiol. 1997;5:37–42.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Wei Wang
    • 1
  • Yingyan Wang
    • 2
  • Liyuan Lin
    • 1
  • Yanling Song
    • 1
  • Chaoyong James Yang
    • 1
    • 3
    Email author
  1. 1.Institute of Molecular Medicine, Renji HospitalShanghai Jiao Tong University School of MedicineShanghaiChina
  2. 2.Department of Neurology, Shanghai Children’s Medical CenterShanghai Jiao Tong University School of MedicineShanghaiChina
  3. 3.Collaborative Innovation Center of Chemistry for Energy Materials, The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical EngineeringXiamen UniversityXiamenChina

Personalised recommendations