Table_4_Comparative Genomic Analysis of Citrobacter and Key Genes Essential for the Pathogenicity of Citrobacter koseri.xlsx

Citrobacter species are opportunistic bacterial pathogens that have been implicated in both nosocomial and community-acquired infections. Among the genus Citrobacter, Citrobacter koseri is often isolated from clinical material, and has been known to cause meningitis and brain abscess in neonates and immunocompromised individuals. The virulence determinants of Citrobacter, however, remain largely unknown. Based on traditional methods, the genus Citrobacter has been divided into 11 species, but this has been problematic. Here, we determined an improved, detailed, and more accurate phylogeny of the genus Citrobacter based on whole genome sequence (WGS) data from 129 Citrobacter genomes, 31 of which were sequenced in this study. A maximum likelihood (ML) phylogeny constructed with core genome single-nucleotide polymorphisms (SNPs) classified all Citrobacter isolates into 11 distinct groups, with all C. koseri strains clustering into a single group. For comprehensive and systematic comparative genomic analyses, we investigated the distribution of virulence factors, resistance genes, and macromolecular secretion systems among the Citrobacter genus. Moreover, combined with group-specific genes analysis, we identified a key gene cluster for iron transport, which is present in the C. koseri group, but absent in other the groups, suggesting that the high-pathogenicity island (HPI) cluster may be important for the pathogenicity of C. koseri. Animal experiments showed that loss of the HPI cluster significantly decreased C. koseri virulence in mice and rat. Further, we provide evidence to explain why Citrobacter freundii is less susceptible than C. koseri to several antibiotics in silico. Overall, our data reveal novel virulence clusters specific to the predominantly pathogenic C. koseri strains, which form the basis for elucidating the virulence mechanisms underlying these important pathogens.

柠檬酸杆菌属（Citrobacter）物种为条件致病细菌，既与医院获得性感染相关，也可引发社区获得性感染。在该属中，科氏柠檬酸杆菌（Citrobacter koseri）常从临床样本中分离得到，已知可导致新生儿及免疫功能低下人群罹患脑膜炎与脑脓肿。然而，柠檬酸杆菌属的毒力决定因子仍大多尚未明确。基于传统分类方法，柠檬酸杆菌属曾被划分为11个物种，但该分类方式存在局限。本研究基于129株柠檬酸杆菌的全基因组序列（whole genome sequence, WGS）数据构建了该属更完善、细致且准确的系统发育树，其中31株的基因组序列为本研究测定。基于核心基因组单核苷酸多态性（single-nucleotide polymorphisms, SNPs）构建的最大似然（maximum likelihood, ML）系统发育树，将所有柠檬酸杆菌分离株划分为11个独立类群，所有科氏柠檬酸杆菌菌株均聚类为单一类群。为开展全面系统的比较基因组学分析，本研究探究了柠檬酸杆菌属内毒力因子、耐药基因及大分子分泌系统的分布特征。此外，结合类群特异性基因分析，本研究鉴定出一个关键的铁转运基因簇：该基因簇仅存在于科氏柠檬酸杆菌类群中，其余类群均不含该序列，提示高致病岛（high-pathogenicity island, HPI）簇可能对科氏柠檬酸杆菌的致病性至关重要。动物实验结果显示，缺失该HPI簇可显著降低科氏柠檬酸杆菌对小鼠和大鼠的毒力。此外，本研究还通过计算机模拟（in silico）分析阐释了弗氏柠檬酸杆菌（Citrobacter freundii）对多种抗生素的敏感性低于科氏柠檬酸杆菌的原因。综上，本研究揭示了主要致病的科氏柠檬酸杆菌菌株所特有的新型毒力基因簇，为阐明这类重要病原菌的毒力机制奠定了坚实基础。