Metagenomic And Metabolomic Approach Studying The Diversity Of Gut Microbiota In Patients With Clostridium Difficile Infection

Part 1 Molecular epidemiology and risk factors of Clostridium difficile infection in a tertiary care hospital of ChinaClostridium difficile, an anaerobic, Gram-positive, spore-forming Bacillus, is component of normal human gut flora. Clostridium difficile infection (CDI) is most frequently associated with administration of antibiotics, which disturbs the gastrointestinal tract microbiota and in turn reduces resistance against toxin-producing C. difficile. Infection with C. difficile has surpassed that with methicillin-resistance Staphylococcus aureus as the most common cause of nosocomial and healthcare-associated infectious diarrhea in developed countries. CDI accounts for 20-30% of antibiotic-associated diarrhoea and is the causative agent of about 90% of pseudomembranous colitis. Over the last decade, CDI has increased markedly in both healthcare and community settings and the mortality rate of CDI is much higher with the emergence of hypervirulent strains of C. difficile ribotype 027. With the increasing elderly population and the well-recognized problem of over prescription of antibiotics in China, persistent surveillance in specific and targeted populations is essential.In this study, the molecular epidemiology and ward distribution of Clostridium difficile in a tertiary hospital of China is reported. A total of 161 toxigenic strains were isolated from 1,845 patients originating from different wards and the strains were characterized by toxin profile and multi-locus sequence typing (MLST). Variable rates of isolations were observed in different wards and the occurrence was higher in ICU and geriatric wards. Results of the toxin profile at the gene level revealed that out of the 161 isolates 134 (83.2)%were positive for both toxin A(tcd A) and toxin B (tcd B) (A+B+) followed by toxin A-negative and B-positive (A-B+) (16.8%). However, only three of the toxigenic strains (1.9%) were positive for both the cdt A and cdt B genes. Based on the molecular epidemiology study, a total of 31 different sequence types (STs), including one new ST, were distinguishable. ST-54 was the most prevalent (23.0%) followed by ST-35 (19.3%) and ST-37 (10.0%). None of the isolates belonged to ST-1 (ribotype 027) or ST-11 (ribotype 078). Combining the toxin profile with the molecular epidemiological data showed that all the ST-37 clades were toxin type A-B+, which accounted for 59.3% of all type A-B+isolates. Meanwhile the calde ST-54 was widely distributed among the geriatric, infection and haematology wards. There was no outbreak of Clostridium difficile infection during our study.Meanwhile we evaluated the risk factors and epidemiology associated with CDI in patients with hematologic malignancies by studying patients’hospitalization data in a tertiary care hospital of China. CDI was diagnosed in 21 hematology-oncology ward patients and 14 marrow transplantation service patients for a cumulative incidence of 1.89/1,000 and 3.69/1,000 patient-days, respectively. Univariate analyses showed that patients who received etoposide and Fluoroquinolone had an increased risk of CDI. Thirteen sequence types were identified, of which ST-3 and ST-54 were the most common; the hypervirulent ST-1 (ribotype 027) and ST-11 (ribotype 078) C. difficile strains were not detected in the patients in this study. Approximately 80% of the CDI cases occurred during the peritransplant period within 30 days of HSCT, which remind the physicians to be aware of this global infection. Our research provided reference rationales for rational use of antibiotics, prevention and control of CDI.Part 2 Molecular Analysis of the Diversity of Gut Microbiota Associated with Patients with Clostridium difficile InfectionAlthough standard antibiotic therapy (e.g., oral vancomycin or metronidazole treatment) is highly effective in treating C. difficile infection (CDI),10 to 30%of patients experience the relapse within 3 months following antibiotic treatment. Fecal microbial transplantation (FMT), the most effective treatment with cure rates of over 80% or 90% for recurrent CDI, may restore gut microbial community structure and function of the recipient to effectively prevent C. difficile colonization and growth. However, FMT is not widely practiced due to multiple practical barriers, including esthetic concerns, lack of long term efficacy and safety research, etc. Therefore, it is necessary to identify the key taxa involved in the protective effect against CDI to enable selective probiotic therapy for prevention, treatment and cure of human CDI.In this study, using high throughput sequencing of the 16S ribosomal RNA V3 region and real-time quantitative polymerase chain reaction, we identified CDI-associated key taxa by comparing the fecal microbiota composition of 15 adult patients with CDI with those of 18 individuals with C. difficile-negative nosocomial diarrhea (CDN) and 25 healthy control subjects. Reduced fecal bacterial diversity and dramatic shifts of intestinal microbial composition in CDI and CDN groups were observed compared with healthy controls. Putative butyrate-producing anaerobic bacteria, such as Ruminococcaceae and Lachnospiraceae families were significantly depleted, whereas endotoxin-producing opportunistic pathogens and lactate-producing phylotypes increased dramatically in patients with CDI compared with healthy controls. Meanwhile, the antiinflammatory commensal bacterium Clostridium leptum group and the most representative Faecalibacterium prausnitzii were significantly depleted in the CDI and CDN patients compared with the healthy subjects. These changes of microbial communities may increase the susceptibility to C. difficile colonization. Our findings suggested that the CDI altered key taxa may provide a guideline for development of probiotic therapies that are more effective than FMT for the prevention and treatment of the infectious disease.Part 3 Metabolomic study of fecal supernatant in Patients with Clostridium difficile InfectionIt is well recognized that the intestinal microecology is the most important ecosystem of human, which is regarded as an important metabolic "organ". Our knowledge of the correlation between the gut microbial composition of Clostridium difficile infection (CDI) patients and host metabolites is currently limited. In the study, we try to explore the relationship between the gut microbiota of CDI patients and host metabolism in details.The fecal extracts from CDI patients, C. difficile-negative nosocomial diarrhea patients (CDN) and healthy controls were investigated by Ultra-performance Liquid Chromatography-Mass Spectroscopy technique. Themetabolic profiling of fecal samples were analyzed by principal component analysis, partial least squares discriminant analysis and orthogonal partialleast squares discriminant analysis methods. Our research found that the metabolites of fecal samples were changed between healthy controls, CDI and CDN patients. Principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) models showed robust discrimination from healthy controls, CDI and CDN patients. A total of 20 characteristic metabolites including bile acids, bile pigments, some amino acids, fatty acids and other metabolites were identified by OPLS-DA loading plot. Compared with healthy controls, levels of primary bile acids, some amino acids and fatty acids metabolites were increased significantly but levels of the secondary bile acid and bile pigments were decreased significantly in CDI patients. We considered that changes in the structure and function of the gut microbial community might create a metabolic environment that favors C. difficile germination and growth. However, further studies underlying the regulatory mechanisms of intestinal microbiota are essential.