Fecal samples human Amplicon-Sequencing 16S. human gut metagenome

Clostridium difficile infections are an emerging health problem in the modern hospital environment. In about 20-30% of cases with antibiotic-associated diarrhea (AAD), 50-75% of cases of antibiotic-associated colitis, and more than 90% of cases with pseudomembranous colitis Clostridium difficile can be implicated as the causing pathogen. Pathophysiologically, severe alterations of the gut microbiom with loss of colonization resistance against C. difficile are supposed to be the major trigger for C. difficile infection, but there is no clear concept of how C. difficile infection evolves and which microbiological factors are involved. In our study we have investigated three groups of patients: (i) fecal samples of control patients (no antibiotic therapy); (ii) patients under antibiotic therapy (penicillins, cephalosporins, and fluoroquinolones with C. difficile infection or (iii) patients under antibiotic therapy without C. difficile infection. The intestinal microbiom was sequenced using a molecular approach based on FLX-technology with more than 500.000 sequences. We compared the effects of the three different antibiotic classes on the intestinal microbiome and the alterations of the gut microbiome towards C. difficile infection on both the DNA and rRNA (transcriptionally active microbiota) level. We observed significant differences in the microbiota composition generally between antibiotic groups and controls on both RNA and DNA level. Comparison of different antibiotic classes showed significantly different results for cephalosporins vs. fluoroquinolones and for penicillins vs. fluoroquinolones, but not between cephalosporins and penicillins at DNA level. At RNA level comparison between fluroquinolones, cephalosporins and penicillins were not significantly different. In the overall analysis of patients under antibiotics with/without developing C. difficile infection we found no significant differences on DNA level and on RNA level. But in an indicator analysis we could identify single species up- or down regulated in patients under antibiotics with/without developing C. difficile infection. Compared to previous studies, which exclusively used DNA, we found no significant differences in the global composition of transcriptionally active gut microbiota associated with C. difficile infection.