FIG 1.
Comparative analysis of metabolites present in CDI-positive and CDI-negative stools by preparative high-performance liquid chromatography using a C18 column. Stools (1 g each) from 20 diarrhea patients infected with C. difficile and 20 diarrhea patients without C. difficile were pooled, suspended in PBS, and acetate precipitated. The precipitated material was resuspended in PBS and filtered through a centrifugal filter with a 3-kDa-cutoff membrane, and the filtrate was concentrated in a SpeedVac (Thermo Fisher Scientific, Waltham, MA). The concentrated filtrate was injected into a preparative Econosil C18 column (250 mm by 10 mm; Alltech) and purified using a Shimadzu Prominence HPLC system (Shimadzu Scientific Instruments, Columbia, MD). Each purification run was performed by injecting 1 ml of the sample and washing with buffer A (97.5% [vol/vol] acetic acid-H2O; pH 3.8), followed by gradient elution with buffer B (80%:20% [vol/vol] acetonitrile/H2O). The red arrow indicates the unique indole peak. *, peak contained the autoinducing quorum signaling peptide (TI signal) associated with C. difficile toxin regulation. A representative chromatogram from multiple runs is shown. mAU, milli-arbitrary units.