Figure 1.
Frequency of recovery of toxigenic Clostridium difficile from stool samples of vancomycin and metronidazole-treated patients.
In addition to acquisition of toxigenic strains, 2 vancomycin-treated patients acquired colonization with nontoxigenic C. difficile between 8 and 14 days after discontinuation of therapy.
Figure 2.
Concentrations of vancomycin and metronidazole in stool during and after Clostridium difficile infection (CDI) treatment.
The concentration of vancomycin was measured using an AxSym II fluorescence polarization immunoassay that was modified for stool samples by including an extraction of the sample in dilute ammonia to prevent nonspecific binding of vancomycin to protein in the stool. The limit of detection of the assay was 2 µg of vancomycin per ml. The concentration of metronidazole in stool was measured using a bioassay with Clostridium perfringens as the indicator organism. Samples from patients receiving concurrent systemic antibiotics for indications other than CDI were excluded.
Figure 3.
In vitro assay of colonization resistance during and after oral vancomycin (A) or metronidazole (B) treatment.
Growth of Clostridium difficile was assessed in homogenized fresh stool specimens inoculated with 104 colony-forming units (CFU) of vegetative C. difficile. Loss of colonization resistance was defined as >1 log10CFU increase in the C. difficile concentration. To assess whether residual metronidazole or vancomycin in stool samples could account for suppression of growth of inoculated C. difficile, growth of the C. difficile isolates was concurrently assessed in sterile filtrates of the stool suspensions.
Figure 4.
Relative abundance of bacterial phyla in stool of 8 patients treated with oral vancomycin.
Stool specimens collected before or after completion of vancomycin therapy for Clostridium difficile infection (CDI), stratified by those with intact (i.e., C. difficile growth suppressed) or disrupted (i.e., C. difficile growth supported) colonization resistance based on the in vitro colonization assay. Micriobiota of each stool specimen was evaluated by deep 16S rRNA sequencing. Each column represents total phyla distribution of one sample. Columns are labelled with sample ID and timing of stool collection in comparison to vancomycin treatment.
Figure 5.
Microbiota signature of bacteria associated with intact versus disrupted colonization resistance to Clostridium difficile.
(A) Phylogenetic tree display of the bacteria identified from stool samples of 8 vancomycin-treated patients with intact (i.e., C. difficile growth suppressed) or disrupted (i.e., C. difficile growth supported) colonization resistance based on the in vitro colonization assay. Clades of bacteria that significantly support or suppress C. difficile growth are highlighted in green or red, respectively. Circle diameter at each phylogenetic level is proportional to the corresponding taxon’s abundance. (B) Histogram of the Linear Discriminant Analysis (LDA) score of key bacteria abundance in suppressive and supportive groups arranged according to their effect size. Positive (green bars) and negative (red bars) LDA scores represent supportive and suppressive bacteria, respectively.
Figure 6.
Surveillance of bacterial family relative abundance before, during, and after vancomycin treatment in one patient.
Stool samples were collected before (Day 0), during, and after vancomycin treatment at the indicated time points. Changes in the abundance (abundance >0.05%) of major bacterial families are shown at each time point.