Research On Clostridium Difficile Infection: Clinical Features, Molecular Epidemiology And Gut Microbiota

Objective: This study aimed to analyze the clinical features and assess the risk factors associated with Clostridium difficile infection(CDI) in elderly hospitalized patients, to discover the difference between clinical isolates and community-derived isolates in terms of their toxin types, genotypes and antimicrobial susceptibility, and to explore the alteration of gut microbiota in C. difficile infection and asymptomatic C. difficile colonization, in order to provide evidence for the prevention, treatment and surveillance of C. difficile associated diseases.Methods: In Section One, we collected the medical records of 202 elderly hospitalized patients(aged>60) in a Chinese tertiary hospital from December 2010 to May 2013. Fifty-two CDI patients and 150 randomly selected non-CDI patients were included in this retrospective case-control study. Clinical features, laboratory results and medications were compared between CDI patients and non-CDI patients by appropriate statistical tests. Logistic regression analyses were carried out on a series of factors to determine the risk factors for CDI among the elderly hospitalized patients. In Section Two, 45 clinical C. difficile isolates from CDI patients and 12 community-derived isolates from asymptomatic C. difficile carriers(57 strains in all) were detected for toxin genes, followed by multilocus sequence typing to analyze the genotypes of the strains. Antimicrobial susceptibility testing of eight antibiotics, including clindamycin, tetracycline, cefoxitin, cefotaxime,moxifloxacin, imipenem, metronidazole and vancomycin, was conducted by the agar dilution method. In Section Three, we performed gut microbiota analysis on the fresh fecal samples from eight CDI patients, eight asymptomatic C. difficile carriers and nine healthy subjects using 16 S r RNA gene high-throughput pyrosequencing. The phylum-level and the genus-level differences in microbial community composition were compared among the three sample groups with the help of bioinformatics technology.Results: Among the elderly hospitalized patients, those with CDI were characterized by higher leukocyte counts, lower serum albumin levels, longer duration of hospital stay and higher mortality compared to the non-CDI patients. Multivariate analysis indicated that serum creatinine(OR 1.004; 95% CI 1.001-1.008), the number of comorbidities(OR 2.573; 95% CI 1.353-4.892), surgical intervention(OR 6.132; 95% CI 2.594-14.493), gastrointestinal disease(OR 4.670; 95% CI 2.002-10.895) and antibiotic use(OR 6.718; 95% CI 2.846-15.859) were significantly associated with CDI in the elderly hospitalized patients. Clinical C. difficile isolates were all toxigenic, predominantly toxin A-positive, toxin B-positive(A+B+) strains. Genotype ST-37 was the epidemic clone accounting for 33.3% of the clinical isolates. Meanwhile, non-toxigenic strains were found in community-derived isolates, some of which shared the same genotype with the clinical isolates. Besides, the multi-drug resistance rate turned out to be higher in the clinical C. difficile isolates than that in the community-derived isolates, and especially the resistance to fluoroquinolones was detected in a larger proportion of the clinical isolates. CDI patients and asymptomatic C. difficile carriers demonstrated intestinal flora dysbiosis of varying degree, showing reduced microbial richness and diversity compared with the healthy subjects, accompanied with a paucity of Bacteroidetes and Firmicutes as well as an overabundance of Proteobacteria. Moreover, some normally commensal butyrate-producing bacteria were depleted in CDI patients and asymptomatic carriers. Principal coordinate analysis revealed significant differences in microbial community structure between CDI patients and asymptomatic C. difficile carriers.Conclusions: In this study, we found that elevated serum creatinine level, increased number of comorbidities, surgical intervention, gastrointestinal disease and exposure to antibiotics were risk factors for CDI in the elderly hospitalized patients. Multi-drug resistance may be one of the important reasons for the wide spread of CDI. Individuals asymptomatically colonized with C. difficile in the community population propably serve as a reservoir for clinical CDI. Alteration of gut microbiota can not only affect the occurrence of CDI, but also be regarded as a potential factor for the surveillance of C. difficile associated disease state.