Isolation and Characterization of Cytotoxic, Aggregative Citrobacter freundii

Citrobacter freundii is an infrequent but established cause of diarrhea in humans. However, little is known of its genetic diversity and potential for virulence. We analyzed 26 isolates, including 12 from human diarrheal patients, 2 from human fecal samples of unknown diarrheal status, and 12 from animals, insects, and other sources. Pulsed field gel electrophoresis using XbaI allowed us to divide the 26 isolates into 20 pulse types, while multi-locus sequence typing using 7 housekeeping genes allowed us to divide the 26 isolates into 6 sequence types (STs) with the majority belonging to 4 STs. We analyzed adhesion and cytotoxicity to HEp-2 cells in these 26 strains. All were found to adhere to HEp-2 cells. One strain, CF74, which had been isolated from a goat, showed the strongest aggregative adhesion pattern. Lactate dehydrogenase (LDH) released from HEp-2 cells was evaluated as a measure of cytotoxicity, averaging 7.46%. Strain CF74 induced the highest level of LDH, 24.3%, and caused >50% cell rounding, detachment, and death. We named strain CF74 “cytotoxic and aggregative C. freundii.” Genome sequencing of CF74 revealed that it had acquired 7 genomic islands, including 2 fimbriae islands and a type VI secretion system island, all of which are potential virulence factors. Our results show that aggregative adherence and cytotoxicity play an important role in the pathogenesis of C. freundii.弗氏柠檬酸杆菌（Citrobacter freundii）是一种虽不常见但已被证实的人类腹泻致病菌。然而目前学界对其遗传多样性及致病潜力的了解仍十分有限。本研究共分析26株分离菌株，其中12株来自腹泻患者，2株来自腹泻状态未知的人类粪便样本，剩余12株分离自动物、昆虫及其他来源。采用XbaI限制性内切酶的脉冲场凝胶电泳（Pulsed field gel electrophoresis, PFGE）可将26株菌株划分为20种脉冲型；而基于7个管家基因（housekeeping genes）的多位点序列分型（multi-locus sequence typing, MLST）则将其分为6种序列型（sequence types, STs），其中绝大多数菌株隶属于4种序列型。本研究对上述26株菌株的HEp-2细胞黏附活性与细胞毒性进行了检测，结果显示所有菌株均可黏附HEp-2细胞。其中一株分离自山羊的菌株CF74呈现出最强的聚集性黏附模式。以HEp-2细胞释放的乳酸脱氢酶（Lactate dehydrogenase, LDH）水平作为细胞毒性检测指标，所有菌株的平均细胞毒性率为7.46%。菌株CF74诱导的LDH释放水平最高，达24.3%，并可导致超过50%的细胞变圆、脱落乃至死亡。研究团队将菌株CF74命名为“具有细胞毒性与聚集性黏附特性的弗氏柠檬酸杆菌”。对CF74的全基因组测序结果显示，该菌株携带7个基因组岛（genomic islands），包括2个菌毛岛与1个VI型分泌系统（type VI secretion system, T6SS）岛，上述元件均为潜在致病因子。本研究结果表明，聚集性黏附与细胞毒性在弗氏柠檬酸杆菌的致病过程中发挥重要作用。