Bacteroides sp. strain D8, the first cholesterol-reducing bacterium isolated from human feces

Abstract

The microbial community in the human colon contains bacteria that reduce cholesterol to coprostanol, but the species responsible for this conversion are still unknown. We describe here the first isolation and characterization of a cholesterol-reducing bacterium of human intestinal origin. Strain D8 was isolated from a 10(-8) dilution of a fresh stool sample provided by a senior male volunteer with a high capacity to reduce luminal cholesterol to coprostanol. Cholesterol-to-coprostanol conversion by strain D8 started on the third day, while cells were in stationary phase, and was almost complete after 7 days. Intermediate products (4-cholesten-3-one and coprostanone) were occasionally observed, suggesting an indirect pathway for cholesterol-to-coprostanol conversion. Resting-cell assays showed that strain D8 could reduce 1.5 mumol of cholesterol/mg bacterial protein/h. Strain D8 was a gram-negative, non-spore-forming, rod-shaped organism identified as a member of the genus Bacteroides closely related to Bacteroides vulgatus, based on its morphological and biochemical characteristics. The 16S rRNA gene sequence of strain D8 was most similar (>99.5%) to those of two isolates of the recently described species Bacteroides dorei. Phylogenetic tree construction confirmed that Bacteroides sp. strain D8 clustered within an independent clade together with these B. dorei strains. Nevertheless, no cholesterol-reducing activity could be detected in cultures of the B. dorei type strain. Based on Bacteroides group-specific PCR-temporal temperature gradient gel electrophoresis, there was no correlation between the presence of a band comigrating with the band of Bacteroides sp. strain D8 and cholesterol conversion in 11 human fecal samples, indicating that this strain is unlikely to be mainly responsible for cholesterol conversion in the human population.

FIG. 1.

Chemical structures of cholesterol and coprostanol.

FIG. 2.

Scanning electron micrograph of strain D8 grown on BHI-YH medium enriched with cholesterol.

FIG. 3.

Neighbor-joining tree showing the phylogenetic position of Bacteroides sp. strain D8 among recognized members of the genus Bacteroides based on 16S rRNA gene sequences. The 16S rRNA gene sequence of Prevotella melaninogenica was used as an outgroup to root the tree. Bootstrap values are indicated at branch points of interest. A GenBank database accession number is included for each sequence in the phylogenetic tree. Bar = 10% sequence divergence.

FIG. 4.

Growth and kinetics of cholesterol-to-coprostanol conversion by Bacteroides sp. strain D8 in SBM at 37°C. Symbols: ▪, cholesterol content; □, coprostanol content; ▴, log CFU per ml.

FIG. 5.

Cholesterol-to-coprostanol conversion by nondisrupted cells (cross-hatched bars), disrupted cells (dotted bars), and supernatants (open bars) produced from 24-h-old Bacteroides sp. strain D8 cultures in BHI-YH medium. The data are means ± standard errors of experiments performed in triplicate. The conditions used for the resting-cell assays are described in Materials and Methods.

FIG. 6.

Bacteroides spp. group-specific TTGE profiles of fecal samples from 11 individuals with different cholesterol conversion patterns. Lanes 1 to 3, low or inefficient cholesterol converters; lanes 4 to 6, intermediate cholesterol converters; lanes 7 to 11, high cholesterol converters; lane 12, Bacteroides sp. strain D8; lane 13, B. dorei 175^T. The arrow indicates the main band obtained with Bacteroides sp. strain D8 and B. dorei 175^T. Circles indicate the bands comigrating with this band. Lane M contained markers corresponding to mixed PCR products from pure cultures.