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.

传统的革兰氏染色法是一个多世纪以前发明的，用于将细菌区分为革兰氏阳性或革兰氏阴性，但仍在微生物学中广泛应用。但是，革兰氏染色存在几个问题，这些问题会影响诊断的准确性。在这里，我们报告了一种新的革兰氏阴性特异性荧光探针，该探针基于窄谱抗生素十三烷A1，可以选择性染色不同固定细菌样品中的革兰氏阴性细菌。固相肽合成用于制备具有单一结构的十三烷A1-荧光团偶联物。通过针对一组细菌物种进行测试，确认了探针对革兰氏阴性细菌的标记选择性。通过结合使用先前报道的革兰氏阳性特异性荧光探针，然后，我们进一步展示了这种新探针在多种复杂细菌样品的差异标记中的能力，这些样品包括体外培养的小鼠肠道菌群以及从人口腔，土壤和原油中收集的菌群。在大多数样品中观察到高标记选择性和覆盖率。该方法提供了一种具有确定结构的新型革兰氏阴性特异性探针，该探针可实现基于荧光的革兰氏阳性细菌和革兰氏阴性细菌的轻松分化，以进行进一步的微生物研究。