自1673年人类首次发现细菌以来，科学家们便尝试用各种方法对其进行分类，但其核心问题尚未解决，那就是如何界定细菌的物种。目前标准是DNA杂交率≥70%和16S rRNA序列相似度≥97%。但是这两项指标由于都是连续性的，因此无法划定出清晰的细菌物种间的界限。我们基于前期工作，提出了细菌物种的半封闭基因池概念以及与之对应的3Cs标准（同一物种成员之间亲缘关系密切、近缘但不同物种的细菌之间有清晰的遗传边界、同一物种成员具有共同的生物学特性）。本研究拟通过实验来验证3Cs标准。我们的实验模型是代表性沙门菌，主要方案包括基于基因组物理图和序列分析证实同一物种的所有成员亲缘关系密切，通过同源基因序列的歧化度找出物种间的遗传界限，以及利用细菌的生物学特性来进行表型区分。此3Cs标准基于细菌的自然关系，反映细菌基因组和生物学特性，因此是真正意义上的客观标准，其最直接的社会意义是指导临床诊断和治疗。

Since the discovery of bacteria in 1673, scientists have attempted classifying them by a variety of methods, all with defining bacterial species based on objective criteria as the core and yet unsolved issue. The current criteria include DNA-DNA re-association rate of ≥70% and 16S rRNA sequence similarity of ≥97%. However, because both kinds of data are continuous, clear-cut genetic boundaries can hardly be determined among bacteria to define then as species. As a result, the current bacterial species tend to contain genetically as well as biologically quite diverse bacteria, making a name of bacteria meaningless. We have recently put forward a "semi-closed gene pool" concept to define phylogenetic units of bacteria as biological species with a novel set of standards, the triple C criteria (close relatedness among all members of the same unit, clear-cut genetic boundaries among different units of bacteria, and common biological features shared only by members of the same unit). In this application, we propose to experimentally validate these criteria. We use Salmonella as a model and work on a selected set of serovars, involving isolates of diverse sources. To begin with, we tentatively treat the individual Salmonella serovars as separate biological species based on the triple C criteria in an attempt to validate these criteria. We shall first validate the close relatedness among members within the selected Salmonella serovars by physical mapping and genomic sequencing, compare sequence identity of homologous genes among different Salmonella serovars to detect clear-cut genetic boundaries among them, and correlate the biological features of the bacteria (host range, infection types, etc.) with their genomic clustering. These triple C criteria are based on both genomic (evolutionary) and biological (pathogenic) properties and therefore are objective rather than subjective. Once applied to studies of bacterial systematics and taxonomy, these triple C criteria would help restructure the bacterial classification systems and facilitate clinical practice.