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Response of the Gut Microbiota During the Clostridioides Difficile Infection

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Response of the Gut Microbiota During the Clostridioides Difficile Infection Gu et al. BMC Microbiology (2020) 20:260 https://doi.org/10.1186/s12866-020-01943-z RESEARCH ARTICLE Open Access Response of the gut microbiota during the Clostridioides difficile infection in tree shrews mimics those in humans Wenpeng Gu1,2, Wenge Li3, Wenguang Wang1, Dexuan Kuang1, Wenzhu Zhang3, Caixia Lu1,NaLi1, Pinfen Tong1, Yuanyuan Han1, Xiaomei Sun1, Jinxing Lu3, Yuan Wu3* and Jiejie Dai1* Abstract Background: Clostridioides difficile is a major cause of antibiotic associated diarrhea. Several animal models are used to study C. difficile infection (CDI). The tree shrew has recently been developed as a model of primate processes. C. difficile infection has not been examined in tree shrews. We infected tree shrews with hyper-virulent C. difficile strains and examined the alterations in gut microbiota using 16S rRNA gene sequencing. Results: C. difficile colonized the gastrointestinal tract of tree shrew and caused diarrhea and weight loss. Histopathologic examination indicated structures and mucosal cell destruction in ileal and colonic tissues. The gut microbial community was highly diversity before infection and was dominated by Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria. Antibiotic administration decreased the diversity of the gut microbiota and led to an outgrowth of Lactobacillus. The relative abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Lachnospiraceae, Enterobacteriaceae, Escherichia, Blautia, and Tyzzerella increased following C. difficile infection. These taxa could be biomarkers for C. difficile colonization. Conclusions: In general, the disease symptoms, histopathology, and gut microbiota changes following C. difficile infection in tree shrews were similar to those observed in humans. Keywords: Clostridioides difficile, Tree shrew, Gut microbiota Background outcomes, including asymptomatic colonization, diarrhea, Clostridioides difficile is a major cause of antibiotic asso- life threatening pseudomembranous colitis, or intestinal ciated diarrhea in patients after hospitalization and anti- obstruction [2]. CDI is the most common cause of biotic administration [1]. C. difficile infection (CDI) healthcare-associated diarrhea and colitis, and is respon- caused by toxigenic isolates can result in a wide range of sible for significant morbidity and substantial healthcare costs worldwide [3, 4]. The primary pathogenic mechan- * Correspondence: [email protected]; [email protected] ism of C. difficile is the production of enterotoxin A 3 State Key Laboratory of Infectious Disease Prevention and Control, (TcdA) and cytotoxin B (TcdB); some C. difficile strains Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and can produce a binary toxin, encoded by the cdtA and cdtB Prevention, Chinese Center for Disease Control and Prevention, Chang bai genes, that increases pathogenesis [5]. Since emerging in Road 155, Chang ping District, Beijing 102206, China North American in 2005, hyper-virulent C. difficile isolates 1Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, (PCR ribotype RT027) have caused several infections and Yunnan Key Laboratory of Vaccine Research and Development on Severe outbreaks in the United States of America, Canada, and Infectious Diseases, Yunnan Innovation Team of Standardization and most European countries [6]. Recently, C. difficile RT078 Application Research in Tree Shrew, Zhao zong Road 66, Kunming 650118, China has been reported from both hospitalized patients and Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gu et al. BMC Microbiology (2020) 20:260 Page 2 of 13 environmental surfaces in eastern China [7]. This emer- not a statistically significant difference in weight loss be- gent strain also causes severe disease, especially in elderly tween groups (each strain represented a group) (Fig. 1.E, patients [8]. F = 0.115, P = 0.892; average weights 13.35 ± 2.79 g for Several animal models have been used to study differ- CD21062, 12.68 ± 1.71 g for CD10010, and 12.92 ± 2.13 g ent aspects of CDI, including pathophysiology, for CD12038). No animals died during the experiment. colonization, transmission, recurrence, and the impact of Fecal samples were all positive for tcdA, tcdB, cdtA, and strain variability [9]. Small animals, such as mice, ham- cdtB two days after infection. Fecal samples were toxin sters, and rats, as well as larger animals, such as foals, negative before infection. The control group for gross gnotobiotic piglets, and rhesus monkeys have been used anatomy of the abdominal cavity showed normal gut to study CDI [10]. Each laboratory animal has its own structure of the tree shrew, without any congestion, application limitations and does not fully reflect the en- bleeding or inflammation (Additional file 1.A). However, tire pathophysiological process of C. difficile infection in the infected group of animal indicated the typical in- humans. For example, primates were rarely used due to flammation for anatomy of the abdominal cavity, the their ethical restrictions; the lesion sites of rodents in- mesenterium of the tree shrew emerged bled or conges- fected by C. difficile were different from human patients; tion, and the gut structure disorders could be found germfree animals didn’t really show the ecological niche (Additional file 1.B). of human intestine. The utility of the animal model mainly depends on the similarities in pathological char- Elisa acteristics with humans. The tree shrew (Tupaia belan- TcdA toxin could be detected at day 4 after oral gavage geri) belongs to the family Tupaiidae and order of the animals, shown as Fig. 2. A-C. CD10010 and Scandentia, and is widely distributed in South and CD12038 strain produced TcdA toxin from two tree Southeast Asia [11]. Compared with rodents, tree shrews shrews at day 4, while toxin could be detected at day 5 were considered as an alternative animal model for non- for CD21062 strain. At experimental day 5, most of the human primates. Simultaneously, use of the tree shrew animal feces could be identify the TcdA. Similar results as a laboratory animal has increased in recent years due of TcdB toxin were showed from Fig. 2. D-F. At day 5, to its low cost of maintenance, life span, small body size, both TcdA and TcdB reached the higher levels for some short reproductive cycle, and close relationship to pri- of the tree shrews, after that, the concentration of toxins mates [12]. Several human diseases models have been were relatively stable. constructed by using this animal [13]. In view of advan- tages of tree shrews mentioned above, we attempted to Histopathology use this animal as a novel model for CDI, compared the Histopathology changes were detected in the ileal and characteristics of infection with other animals, and ex- colonic tissues of all animals. In healthy ileal and colonic plored its feasibility and applicability. tissues, mucosal cells and structures were arranged In addition, disruption of the gut microbiota is another neatly and lacked congestion, edema, and inflammatory important component of CDI [14]. A number of animal infiltration (Figs. 3.A to D). Pathology following infection models have been developed to examine the relationship showed evidence of epithelial cell destruction, structural between the gut microbial community and C. difficile infec- disorders, mucosal ulcers, and necrosis in the mucosa tion [15, 16]. However existing models do not fully capture and submucosa of the ileum. Gland and crypt destruc- the relationship between the human gut microbiota and C. tion, as well as goblet cell reduction and inflammatory difficile pathogenesis. Therefore, we further analyzed the cell infiltration, was observed in mucosal and submuco- gut microbiota changes of tree shrews during CDI process sal layers (Figs. 3.E and F). There was an increase in eo- by using 16SrRNA gene sequencing in this study. The fea- sinophil cells, as well as an increase in the infiltration of tures of intestinal microbial communities among different lymphocyte and plasma cells into colonic tissues in the animals and humans were compared, and the usability of mucosal and submucosal layers (Figs. 3. G and H). tree shrews as CDI model was further estimated. Taxonomy of the gut microbiota Results 3,794,301 reads were obtained from 45 fecal samples. 3, Clinical features 690,628 reads survived merging and quality trimming. Nine of 15 infected animals showed clinical signs of The average read length was 423.62 ± 6.93 bp and ranged CDI, including diarrhea and weight loss (Fig. 1). The from 408 to 430 bp. Reads were mapped to 3675 OTUs. remaining animals did not exhibit signs of diseases des- At baseline (TSCDB group), Firmicutes (64.98%), pite being colonized with C. difficile. Four days after in- Fusobacteria (11.06%), Bacteroidetes (9.28%), Proteobac- fection, the animals began suffering diarrhea (i.e., loose teria (7.79%), and Epsilonbacteraeota (4.29%) were the stools containing mucous) (Fig. 1.A to D). There was most abundant phyla (Fig.
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