目的  探究艰难梭菌序列型11（ST11）克隆群菌株的致病性，从体外实验和体内实验两个角度研究引起艰难梭菌感染（Clostridioides difficile infection, CDI）的原因和相关致病差异。采用树鼩作为艰难梭菌感染的新型实验动物，并研究其肠道菌群在CDI过程中的特征及作用。

方法  基于前期艰难梭菌流行病学监测，研究组从中国老年患者中分离并鉴定了艰难梭菌核糖体型078（RT078）菌株（CD21062），还包括同属于ST11克隆群的两个新核糖体型（RTs）菌株（CD10010和CD12038），并对这些菌株的基因组特征进行了解析。本研究通过分析上述3个ST11克隆群艰难梭菌的体外芽孢形成能力、芽孢萌发试验、毒素基因和调节基因的mRNA表达、毒素蛋白的定量分析、菌株的运动能力与鞭毛特性、对细胞的粘附性以及RNA-seq初步探究艰难梭菌ST11克隆群菌株的体外致病性和致病差异；同时，采用树鼩作为艰难梭菌的实验动物感染模型，开展体内致病性研究，经过抗生素喂养和芽孢灌胃的方式建立3个艰难梭菌的动物模型，观察树鼩的临床症状和体征、检测感染后树鼩粪便中的毒素定量水平、HE染色和组织病理学检查其肠道的病理变化、16S rRNA扩增子测序研究在感染过程中树鼩肠道菌群的改变以及分析3个菌株引起树鼩肠道菌群变化的各自特点；将树鼩模型与经典的小鼠实验模型进行比较，分析树鼩应用于艰难梭菌动物感染模型的可用性和有效性。

结果  体外研究中，3个艰难梭菌菌株之间的芽孢形成和萌发能力没有统计学差异。随着培养时间的延长，三个菌株的芽孢形成率逐渐增加，在第7天，平均芽孢形成率大约为60％；毒素和调节基因的mRNA相对表达研究表明，体外培养48小时之前，CD21062的tcdA、tcdB、tcdC、tcdE和tcdR的表达水平低于CD10010和CD12038。CD10010和CD12038之间的tcdA和tcdB基因表达水平仅在12 h时才发现差异。此后，这些基因的表达水平没有发现统计学差异。在72小时，所有菌株的毒力和调节基因的转录水平趋于一致；毒素蛋白定量结果表明，CD10010在24小时显示出比CD21062更高的TcdB水平。尽管CD10010中的TcdA和TcdB蛋白水平比其他菌株略高，但差异无统计学意义；3个艰难梭菌菌株产生的毒素导致Vero细胞出现了CPE，并在12、24、48和72小时时间点的CPE滴度是一致的；基于这3个艰难梭菌菌株基因组的测序分析表明，CD21062菌株中缺失了大多数编码鞭毛及其相关基因。而新的RTs型别菌株显示二者具有负向的鞭毛开关（flager switch OFF）。PCR实验证实CD21062丢失了鞭毛开关基因下游的早期鞭毛合成操纵子flgB和sigD这两个关键基因。在swim琼脂中培养时，新的RTs菌株鞭毛开关的OFF方向转变为ON，CD10010和CD12038在swim琼脂中出现了运动能力，同时，PCR结果显示，出现游动的菌株此时的鞭毛相变由OFF转变成了ON状态（flager switch ON）。透射电镜进一步证实，CD10010和CD12038在flg OFF状态下没有鞭毛，而两个新的RTs菌株在flg ON状态下观察到了鞭毛的出现。qRT-PCR显示，当鞭毛开关的方向从OFF转变为ON时，CD10010和CD12038下游鞭毛基因flgB和sigD均被上调表达。此外，当鞭毛开关进行相变时，两个新RTs型别菌株的tcdA和tcdB的表达也被上调。Western blot结果证明TcdA和TcdB毒素均与其mRNA表达的情况相一致；3个艰难梭菌菌株粘附Hep-2细胞的能力有所不同，其中CD12038表现出最强的细胞粘附能力。RNA-seq结果提示，CD12038较强的细胞粘附能力可能与其cwpV和cwp26等细胞壁蛋白基因的表达上调密切相关。体内实验研究结果显示，3个艰难梭菌菌株均能够定植在树鼩的胃肠道中，引起腹泻和体重减轻；组织病理学检查表明感染的树鼩回肠和结肠粘膜和粘膜下层细胞破坏、结构异常、粘膜溃疡和坏死。在粘膜和粘膜下层观察到腺体和隐窝破坏，以及杯状细胞数量的减少和炎性细胞浸润。炎症评分显示，CD12038感染后在回肠和结肠组织中引起最高程度的炎症损伤，其次分别是CD21062和CD10010；在灌胃感染后第5天，部分树鼩粪便的TcdA和TcdB毒素都达到了较高的水平，之后，毒素的浓度相对稳定；树鼩肠道微生物群落在感染前具有高度多样性特征，并以厚壁菌门（Firmicutes）、梭杆菌门（Fusobacteria）、拟杆菌门（Bacteroidetes）和变形菌门（Proteobacteria）为主要分类单元。抗生素的施用大幅度减少了其肠道菌群的多样性，并导致了乳杆菌属（Lactobacillus）的相对丰度的增加。艰难梭菌感染后，树鼩肠道中的变形菌门（Proteobacteria）、γ-变形菌纲（Gammaproteobacteria）、肠杆菌目（Enterobacteriales）、毛螺菌科（Lachnospiraceae）、肠杆菌科（Enterobacteriaceae）、埃希氏菌属（Escherichia）和布劳特氏菌属（Blautia）等菌群分类单元的相对丰度增加。这些细菌分类单元可作为艰难梭菌定植抵抗的生物标记；按照3个艰难梭菌菌株分组的肠道菌群变化表明，CD12038显示出动物灌胃后梭菌属的测序reads数最高，表明该菌株在树鼩胃肠道中具有最强的生长和复制功能。RNA-seq结果提示，涉及菌株生长复制、硫代谢和半胱氨酸代谢通路的cysK（乙酰化丝氨酸硫醇基裂解酶）和cysE（乙酰化丝氨酸转移酶）基因在CD12038菌株中高表达，提示二者可能在新发菌株的体内致病机制中起到重要作用。BALB/c小鼠的动物感染模型实验结果与树鼩结果类似，炎症评分结果同样显示CD12038炎症评分最高，其次为CD21062和CD10010。

结论  艰难梭菌ST11克隆群中代表性菌株RT078和新发菌株的芽孢形成能力和萌发能力相似；新发菌株的毒素和调节基因在体外培养过程的早期高表达，后续基因的表达情况趋于一致；RT078菌株缺失鞭毛及其合成的关键基因，而新发菌株在体外培养过程中经历了鞭毛的相变，由负向鞭毛开关转换成正向，这一过程上调了艰难梭菌的主要毒素的表达，增强了菌株的致病性。动物体内研究显示，树鼩感染艰难梭菌后的疾病症状、组织病理学和肠道菌群变化与人类相似，提示树鼩可作为艰难梭菌的动物模型开展相关研究；小鼠动物模型验证了树鼩感染艰难梭菌模型的可用性和有效性；新发菌株引起的肠道炎症反应和损伤强于典型的RT078菌株。因此，本研究中新的核糖体型别菌株，特别是CD12038，在体内和体外均表现出比典型的RT078菌株更高的致病能力。基于上述研究结论，我们认为后续流行病学监测和研究中应更加关注这种新的艰难梭菌菌株。

Objective To explore the pathogenicity of Clostridioides difficile sequence type 11 (ST11) clonal strains, and analyze the causes and related pathogenic factors of Clostridioides difficile infection (CDI) from both in vitro and in vivo perspectives. The tree shrew was used as an alternative laboratory animal model for Clostridioides difficile infection, and the characteristics and role of its gut microbiota in the process of CDI were studied.

Methods Based on the previous epidemiological surveillance of C. difficile, our research team has isolated and identified the C. difficile ribotype 078 (RT078) strain (CD21062) from elderly patient in China, and also included two new ribotypes (new RTs) isolates that belonged to the ST11 clonal group (CD10010 and CD12038), the genome features of these strains were also analyzed. In this study, the spore-formation ability, spore germination test, mRNA expression of toxin genes and regulatory genes, quantitative analysis of toxin proteins, motility and flagella characteristics of the strains, and adhesion to cells of three ST11 clones of C. difficile were investigated in vitro. RNA-seq was used to explore the pathogenicity and pathogenic difference of C. difficile ST11 clonal group mentioned above. The tree shrew was used for C. difficile infection to carry out pathogenicity research in vivo. Three animal models of C. difficile were established through antibiotic treatment and spore gavage to observe the clinical symptoms and signs of tree shrews, detect the quantitative level of toxins in the feces of tree shrews after infection, HE staining and histopathological detection of their intestinal tissues. 16S rRNA amplicon sequencing was used to study the changes in the gut microbiota of tree shrews during the infection process, and also analyzed the gut microbiota change features caused by three strains in this study; mice laboratory animal models and tree shrew models were both used to compare and analyze the availability and effectiveness of tree shrews in animal infection models of C. difficile.

Results There was no statistical difference in the spore formation and germination ability between the three C. difficile strains in vitro. With the extension of the culture time, the rate for sporulation of the three strains gradually increased. On the 7th day, the average sporulation rate was about 60%; the relative mRNA expression of toxin and regulatory gene showed that before 48 hours culture, the expression levels of CD21062 of tcdA, tcdB, tcdC, tcdE and tcdR were lower than those of CD10010 and CD12038. The differences in the expression levels of tcdA and tcdB genes between CD10010 and CD12038 were only found at 12 h. Since then, the expression differences of these genes had no statistical significance. At 72 hours, the virulence of all strains and the transcription levels of regulatory genes tended to be simialr; the quantitative results of toxin protein showed that CD10010 showed a higher level than CD21062 at 24 hours for TcdB level. Although the TcdA and TcdB protein levels in CD10010 were slightly higher than those of other strains, no statistical difference was found; the toxins produced by the three C. difficile strains caused CPE in Vero cells, and the titers of CPE among three strains were consistent at 12, 24, 48, and 72 hour time points; based on the genomic sequencing of three C. difficile strains, CD21062 showed the lost of most flagella-related genes. While, the new RTs strains showed that they had flagella switch OFF (flager switch OFF). PCR experiments confirmed that CD21062 lost downstream of the flagellar switch gene flgB and sigD, the two key genes of the early flagellar synthesis operon. When cultured in swim agar, the OFF direction of the flagella switch of the new RTs strain changed to ON status, CD10010 and CD12038 showed motility ability in swim agar. At the same time, PCR results showed that the flagella phase transition of the swimming strain was changed from OFF turns into ON status (flager switch ON). Transmission electron microscopy further confirmed that CD10010 and CD12038 bacteria had no flagella in flg OFF status, while the appearance of flagella was observed in the two new RTs strains in flg ON status. qRT-PCR results showed that when the direction of the flagella switch changed from OFF to ON, the downstream flagellar genes flgB and sigD of CD10010 and CD12038 were both up-regulated. In addition, when the flagellar switch underwent a phase transition, the expressions of tcdA and tcdB of the two new RTs strains were also up-regulated. Western blot results proved that both TcdA and TcdB toxins were consistent with their mRNA expression; the three C. difficile strains had different ability to adhere to Hep-2 cells, and CD12038 showed the strongest cell adhesion ability. RNA-seq results suggested that the strong cell adhesion ability of CD12038 might be closely related to the up-regulation of cell wall protein genes such as cwpV and cwp26. The results of in vivo experiments showed that all the three C. difficile could colonize the gastrointestinal tract of tree shrews, causing diarrhea and weight loss; histopathological examination showed that the infected tree shrews’ ileum and colon mucosa and submucosal cells were destroyed and structured. Abnormalities, mucosal ulcers and necrosis were also found out. Destruction of glands and crypts, as well as goblet cell reduction and inflammatory cell infiltration were observed in the mucosa and submucosa. The inflammation score showed that CD12038 infection caused the highest degree of inflammatory damage in the ileum and colon tissues, followed by CD21062 and CD10010, respectively; on the 5th day after gavage infection, the TcdA and TcdB toxins in the feces of some tree shrews reached higher levels. Since then, the concentration of toxins had been relatively stable; the gut microbiota of tree shrew was highly diverse before infection, and dominated with Firmicutes, Fusobacteria, Bacteroidetes and Proteobacteria. The administration of antibiotics reduced the diversity of their intestinal flora and led to an increase in the relative abundance of Lactobacillus. After C. difficile infection, the Proteobacteria, γ-Proteobacteria, Enterobacteriales, Lachnospiraceae, Enterobacteriaceae, Escherichia and Blautiain of tree shrews had increased. These bacterial taxa could be used as biomarkers for colonization resistance of C. difficile; changes in the gut microbiota grouped by three C. difficile strains revealed that CD12038 showed highest numbers sequence reads of Clostridium genus after animal gavage, which indicated that this strain had the strongest growth and replication ability in the gastrointestinal tract of tree shrews. RNA-seq results suggested that cysK (acetylated serine thiol lyase) and cysE (acetylated serine transferase) genes involved in strain growth replication, sulfur metabolism and cysteine metabolism pathways were highly expressed in the CD12038 strain, which suggested two genes might play an important role in the in vivo pathogenic mechanism of new strains. In addition, the results of BALB/c mice were similar to those of tree shrews. The inflammation score results also showed that CD12038 was the highest, followed by CD21062 and CD10010.

Conclusion The representative strain RT078 in the clonal group of C. difficile ST11 had similar sporulation and germination ability to the new RT strains; the toxin and regulatory genes of the new RT strains were highly expressed in the early stage of the in vitro culture, and the subsequent gene expression tended to be consistent; The RT078 strain lacked flagella and its key genes for synthesis, while the new RT strains underwent a phase transition of flagella during in vitro culture, switched from a negative flagella switch to a positive one. This process up-regulated the expression of the main toxin of C. difficile, enhanced the pathogenicity of the strains. In vivo studies in animals showed that the disease symptoms, histopathology, and gut microbiota changes of tree shrews infected with C. difficile were similar to those of humans, which suggested that tree shrews could be used as animal models for C. difficile; mice animal models validated the availability and effectiveness of the tree shrews infection model of C. difficile; the intestinal inflammatory response and damage caused by the new strain was stronger than that of the typical RT078 strain. Therefore, the new ribotype strains, especially for CD12038, showed higher pathogenicity than the typical RT078 strain both in vitro and in vivo. Based on the above research conclusions, we believe that follow-up epidemiological surveillance and research should pay more attention to this new C. difficile strain.