Method comparison for the direct enumeration of bacterial species using a chemostat model of the human colon.
- 作者列表："Moura IB","Normington C","Ewin D","Clark E","Wilcox MH","Buckley AM","Chilton CH
BACKGROUND:Clostridioides difficile infection (CDI) has a high recurrent infection rate. Faecal microbiota transplantation (FMT) has been used successfully to treat recurrent CDI, but much remains unknown about the human gut microbiota response to replacement therapies. In this study, antibiotic-mediated dysbiosis of gut microbiota and bacterial growth dynamics were investigated by two quantitative methods: real-time quantitative PCR (qPCR) and direct culture enumeration, in triple-stage chemostat models of the human colon. Three in vitro models were exposed to clindamycin to induce simulated CDI. All models were treated with vancomycin, and two received an FMT. Populations of total bacteria, Bacteroides spp., Lactobacillus spp., Enterococcus spp., Bifidobacterium spp., C. difficile, and Enterobacteriaceae were monitored using both methods. Total clostridia were monitored by selective culture. Using qPCR analysis, we additionally monitored populations of Prevotella spp., Clostridium coccoides group, and Clostridium leptum group. RESULTS:Both methods showed an exacerbation of disruption of the colonic microbiota following vancomycin (and earlier clindamycin) exposure, and a quicker recovery (within 4 days) of the bacterial populations in the models that received the FMT. C. difficile proliferation, consistent with CDI, was also observed by both qPCR and culture. Pearson correlation coefficient showed an association between results varying from 98% for Bacteroides spp., to 62% for Enterobacteriaceae. CONCLUSIONS:Generally, a good correlation was observed between qPCR and bacterial culture. Overall, the molecular assays offer results in real-time, important for treatment efficacy, and allow the monitoring of additional microbiota groups. However, individual quantification of some genera (e.g. clostridia) might not be possible without selective culture.
背景: 艰难梭菌感染 (CDI) 具有较高的复发性感染率。粪便微生物移植 (FMT) 已成功用于治疗复发性 CDI，但人类肠道菌群对替代疗法的反应仍有许多未知。在这项研究中，抗生素介导的肠道菌群失调和细菌生长动力学通过两种定量方法进行了研究: 实时定量 PCR (qPCR) 和直接培养计数, 在人结肠的三级恒化器模型中。3 种体外模型暴露于克林霉素诱导模拟 CDI。所有模型用万古霉素治疗，2 例接受 FMT。细菌总数，拟杆菌属。乳酸杆菌属,肠球菌属,双歧杆菌属,使用两种方法监测艰难梭菌和肠杆菌科细菌。通过选择性培养监测总梭菌。使用 qPCR 分析，我们另外监测了普氏普氏菌的种群。,球壳梭菌组，和麻风梭菌组。 结果: 两种方法均显示万古霉素 (和更早的克林霉素) 暴露后结肠菌群破坏加剧，且恢复更快 (4 天内) 接受 FMT 的模型中的细菌群体。QPCR 和培养也观察到艰难梭菌增殖，与 CDI 一致。Pearson 相关系数显示结果之间的相关性，从拟杆菌属的 98% 变化到肠杆菌科的 62%。 结论: 一般情况下，qPCR 和细菌培养之间存在良好的相关性。总的来说，分子检测提供了实时、对治疗疗效很重要的结果，并允许监测额外的微生物群。然而，如果没有选择性培养，一些属 (如梭菌属) 的个体定量可能是不可能的。
METHODS::Chronic diseases, including inflammatory bowel disease (IBD) urgently need new biomarkers as a significant proportion of patients, do not respond to current medications. Inflammation is a common factor in these diseases and microbial sensing in the intestinal tract is critical to initiate the inflammation. We have identified ELMO1 (Engulfment and Cell Motility Protein-1) as a microbial sensor in epithelial and phagocytic cells that turns on inflammatory signals. Using a stem-cell-based "gut-in-a-dish" coculture model, we studied the interactions between microbes, epithelium and monocytes in the context of IBD. To mimic the in-vivo cell physiology, enteroid-derived monolayers (EDMs) were generated from the organoids isolated from WT and ELMO1-/- mice and colonic biopsies of IBD patients. The EDMs were infected with the IBD-associated microbes to monitor the inflammatory responses. ELMO1-depleted EDMs displayed a significant reduction in bacterial internalization, a decrease in pro-inflammatory cytokine productions and monocyte recruitment. The expression of ELMO1 is elevated in the colonic epithelium and in the inflammatory infiltrates within the lamina propria of IBD patients where the higher expression is positively correlated with the elevated expression of pro-inflammatory cytokines, MCP-1 and TNF-α. MCP-1 is released from the epithelium and recruits monocytes to the site of inflammation. Once recruited, monocytes require ELMO1 to engulf the bacteria and propagate a robust TNF-α storm. These findings highlight that the dysregulated epithelial ELMO1→MCP-1 axis can serve as an early biomarker in the diagnostics of IBD and other inflammatory disorders.
METHODS:BACKGROUND:Peripheral blood eosinophilia (PBE) is a biomarker of an aggressive multiyear natural history in adults with inflammatory bowel diseases (IBDs). Additionally, PBE at diagnosis is associated with higher disease activity in pediatric-onset IBD. We sought to determine if PBE can function as a biomarker of long-term disease severity in pediatric-onset IBD patients who are followed into adulthood. METHODS:We analyzed a consented, prospective, natural history IBD registry at an adult tertiary center from 2009 to 2018. Prevalence of PBE was evaluated in both pediatric- and adult-onset IBD patients. Demographics, clinical characteristics, and health care utilization data were compared in patients with and without PBE. RESULTS:Among 2800 adult IBD patients, 23.4% had pediatric-onset disease. PBE was found in 34% of the pediatric-onset patients compared with 26.8% of the adult-onset IBD patients (P < 0.001). In the pediatric-onset IBD cohort, PBE was associated with higher rates of allergies (P < 0.0001), but not of asthma, allergic rhinitis, or primary sclerosing cholangitis. In the adult IBD patients with pediatric-onset disease, PBE was associated with higher rates of C-reactive protein elevation (P < 0.0001), erythrocyte sedimentation rate elevation (P < 0.0001), higher health care utilization, and higher average health care charges per year (P < 0.00001). CONCLUSIONS:Peripheral blood eosinophilia was more prevalent in adult IBD patients with pediatric-onset compared with adult-onset disease. Among all IBD patients with long-term follow-up, PBE defined a subgroup with more severe illness. These data suggest that PBE may be a biomarker for a high-risk subgroup with high cost trajectory and long-term severity in pediatric-onset IBD that persists into adulthood.
METHODS::Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex pathogenesis, affecting people of all ages. They are characterized by alternating phases of clinical relapse and remission, depending on the fine balance between immune cells and the gut microbiota. The cross talk between cells of the immune system and the gut microbiota can result in either tolerance or inflammation, according to multifactorial triggers, ranging from environmental factors to genetic susceptibility. Glucocorticoid (GC) administration remains the first-line treatment for IBDs, although long-term use is limited by development of serious adverse effects. Recently, new alternative pharmacological therapies have been developed, although these are not always effective in IBD patients. There is a constant demand for effective new drug targets to guarantee total remission and improve the quality of life for IBD patients. The glucocorticoid-induced leucine zipper (GILZ) has been implicated as a promising candidate for this purpose, in view of its powerful anti-inflammatory effects that mimic those of GCs while avoiding their unwanted adverse reactions. Here we present and discuss the latest findings about the involvement of GILZ in IBDs.