Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited

Abstract

The phylogenetic diversity of the intestinal bacterial community in pigs was studied by comparative 16S ribosomal DNA (rDNA) sequence analysis. Samples were collected from a total of 24 pigs representing a variety of diets, ages, and herd health status. A library comprising 4,270 cloned 16S rDNA sequences obtained directly by PCR from 52 samples of either the ileum, the cecum, or the colon was constructed. In total, 375 phylotypes were identified using a 97% similarity criterion. Three hundred nine of the phylotypes (83%) had a <97% sequence similarity to any sequences in the database and may represent yet-uncharacterized bacterial genera or species. The phylotypes were affiliated with 13 major phylogenetic lineages. Three hundred four phylotypes (81%) belonged to the low-G+C gram-positive division, and 42 phylotypes (11.2%) were affiliated with the Bacteroides and Prevotella group. Four clusters of phylotypes branching off deeply within the low-G+C gram-positive bacteria and one in the Mycoplasma without any cultured representatives were found. The coverage of all the samples was 97.2%. The relative abundance of the clones approximated a lognormal distribution; however, the phylotypes detected and their abundance varied between two libraries from the same sample. The results document that the intestinal microbial community is very complex and that the majority of the bacterial species colonizing the gastrointestinal tract in pigs have not been characterized.

FIG. 1.

Dendrogram showing the phylogenetic affiliation of OTUs in the Clostridium botulinum group (RDP reg. no. 2.30.9.2) and four deep-branching clusters of OTUs from the porcine GI tract. Near-full-length 16S rDNA sequences were aligned to the Ribosomal Database Project small subunit rRNA alignment version 7.1 and added to the matching phylogenetic tree without changing the topology of the tree by using the constrained maximum-parsimony method provided in the ARB software. 16S rDNA sequences from the bovine rumen (superscript a [47] and b [54]) and the GI tract of a pig (superscript c [33]) were added to this tree. The scale bar represents a 10% estimated sequence divergence. For each OTU the similarity to the closest related known bacteria and the number of clones belonging to that phylotype are indicated. The most closely related bacteria in the database and the mean similarity for the deep-branching clusters were as follows: CLUSTER__B, Acetivibrio cellulolyticus (84.1%); CLUSTER__C, Acetivibrio cellulolyticus (86%); CLUSTER__D, Eubacterium plautii (84.6%); CLUSTER__E, STR.16SX-1 (81.7%).

FIG. 1.

Dendrogram showing the phylogenetic affiliation of OTUs in the Clostridium botulinum group (RDP reg. no. 2.30.9.2) and four deep-branching clusters of OTUs from the porcine GI tract. Near-full-length 16S rDNA sequences were aligned to the Ribosomal Database Project small subunit rRNA alignment version 7.1 and added to the matching phylogenetic tree without changing the topology of the tree by using the constrained maximum-parsimony method provided in the ARB software. 16S rDNA sequences from the bovine rumen (superscript a [47] and b [54]) and the GI tract of a pig (superscript c [33]) were added to this tree. The scale bar represents a 10% estimated sequence divergence. For each OTU the similarity to the closest related known bacteria and the number of clones belonging to that phylotype are indicated. The most closely related bacteria in the database and the mean similarity for the deep-branching clusters were as follows: CLUSTER__B, Acetivibrio cellulolyticus (84.1%); CLUSTER__C, Acetivibrio cellulolyticus (86%); CLUSTER__D, Eubacterium plautii (84.6%); CLUSTER__E, STR.16SX-1 (81.7%).

FIG. 2.

Dendrogram showing the phylogenetic affiliation of OTUs from the porcine GI tract in the Clostridium leptum subgroup (RDP reg. no. 2.30.9.1.3). See the legend to Fig. 1 for explanation.

FIG. 3.

Dendrogram showing the phylogenetic affiliation of OTUs from the porcine GI tract in the Sporomusa and relatives group (RDP reg. no. 2.30.3). See the legend to Fig. 1 for explanation.

FIG. 4.

Dendrogram showing the phylogenetic affiliation of OTUs from the porcine GI tract in the Eubacterium and relatives group (RDP reg. no. 2.30.4) and in the C. purinolyticum group (RDP reg. no. 2.30.5). See the legend to Fig. 1 for explanation.

FIG. 5.

Dendrogram showing the phylogenetic affiliation of OTUs from the porcine GI tract in the Clostridium coccoides group (RDP reg. no. 2.30.4.1). See the legend to Fig. 1 for explanation.

FIG. 5.

Dendrogram showing the phylogenetic affiliation of OTUs from the porcine GI tract in the Clostridium coccoides group (RDP reg. no. 2.30.4.1). See the legend to Fig. 1 for explanation.

FIG. 6.

Dendrogram showing the phylogenetic affiliation of OTUs from the porcine GI tract in the Mycoplasma and relatives (RDP reg. no. 2.30.8). 16S rDNA sequences from the bovine rumen (superscript a, from Tajima et al. [47]) and feces from an adult human (superscript b, from Suau et al. [45]) were added to this tree. The most closely related bacterium in the database to the phylotypes in CLUSTER__A was Mesoplasma seiffertii (81.5%) affiliated with the spiroplasmas. See the legend to Fig. 1 for explanation.

FIG. 7.

Dendrogram showing the phylogenetic affiliation of OTUs from the porcine GI tract in the Bacteroides group (RDP reg. no. 2.15.1.2). See the legend to Fig. 1 for explanation.

FIG. 7.

Dendrogram showing the phylogenetic affiliation of OTUs from the porcine GI tract in the Bacteroides group (RDP reg. no. 2.15.1.2). See the legend to Fig. 1 for explanation.

FIG. 8.

The relative abundance of phylotypes in two 16S rDNA clone libraries from one DNA extraction sampled from the colon of pig SPF-A. Only phylotypes representing >1% of the clones in each library are shown. Twenty-four phylotypes in lib_1 and 43 phylotypes in lib_2 were found. The names of the most closely related known bacterial species are indicated for some of the abundant OTUs.