Effective recovery of bacterial DNA and percent-guanine-plus-cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens

Abstract

A DNA-based, direct method for initial characterization of the total bacterial community in ileum and cecum of the chicken gastrointestinal (GI) tract was developed. The efficiencies of bacterial extraction and lysis were >95 and >99%, respectively, and therefore the DNA recovered should accurately reflect the bacterial communities of the ileal and cecal digesta. Total bacterial DNA samples were fractionated according to their percent G+C content. The profiles reflecting the composition of the bacterial community were reproducible within each compartment, but different between the compartments of the GI tract. This approach is independent of the culturability of the bacteria in the consortium and can be used to improve our understanding of how diet and other variables modulate the microbial communities of the GI tracts of animals.

FIG. 1

Cell removal from digesta material () and cumulative recovery () of bacteria from ileal digesta samples during the bacterial recovery process. The data were obtained by direct microscopic enumeration of bacterial cells in the supernatant following each round of differential, low-speed centrifugation. Percent removal is based on the number of cells present in the starting ileal digesta sample. Error bars indicate 1 standard error of the mean (n = 4).

FIG. 2

Profiles of the bacterial community in ileal (A) and cecal (B) digesta material from 4-week-old chickens. Data were obtained as a continuous stream from the UV absorbance flow cell and are presented as percent G+C content versus relative abundance. The solid line in each panel indicates the mean of two replicate samples. The shaded areas in each panel indicate the standard error of the mean.

FIG. 3

Ranges of percent G+C content in bacterial genera present in the GI tract of the chicken. Boxes indicate ranges, which accommodate 80% of the species within a given genus, and the vertical line in each box is the median of that genus. The values in parentheses show the number of species included in the survey. The figure is based on the literature data (13, 19, 22, 34, 37).

FIG. 4

Profiles of the cecal bacterial community in 10-day-old chickens. Data were obtained as a continuous stream from the UV absorbance flow cell and are presented as percent G+C content versus relative abundance. Each line represents the profile obtained from pooled cecal samples of six broiler chickens. Solid line (——), uninoculated control; dotted line (........), inoculation with 10⁸ cecal bacteria from commercially raised, free-range chickens; dashed line (–––), inoculation with 10⁵ C. perfringens cells.