A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called ‘Pig intestinal bacterial collection’ (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota.

The tree was constructed based on 400 marker genes in PhyloPhlan (v0.99). The accession numbers of genomes used for the analysis are indicated in brackets next to species names. Novel taxa are written in bold letters. Colors indicate phyla.     Gordonibacter pamelaeae T 7-10-1-b (AM886059)

* The proposition to accomodate Eubacterium rectale into the genus Agathobacter has been thereafter refuted by genome analysis, which indicated that the species is located deeply inside the Roseburia cluster. There is no genome available for Agathobacter ruminis, preventing further analysis. The taxonomic status of Agathobacter spp. is thus ambiguous and requires amendment.
Suppl. Fig. 4l 16S rRNA gene-based phylogenetic tree of Eisenbergiella porci sp. nov., Velocimicrobium porci gen. nov., sp. nov. and Waltera intestinalis gen. nov., sp. nov. (2) Cells were processed by high pressure freezing (protocol 2; see methods sec�on). (1) Cells fixed with 2% (v/v) formaldehyde and 2.5% (v/v) glutaraldehyde (protocol 1 with ruthenium red).
(2) Cells were processed by high pressure freezing (protocol 2; see methods sec�on).
(1) Nega�ve staining of cells observed by TEM indica�ng the presence of an extracellular matrix surrounding cells.
(2) Cells were processed by high pressure freezing (protocol 2; see methods sec�on).    To assess the coverage of sequence-based diversity by all PiBAC isolates in relation to parameters such as diet, age, and gut locations, we processed three datasets: Dataset 1: Published data 5 investigating the impact of dietary protein content on the fecal microbiota of castrated male Duroc pigs at the age of 165 days; Datasets 2: Stool samples from ten German landrace pigs from the animal facility of Thalhausen (TU Munich, Germany) at the age of 8, 24, and 52 weeks; Dataset 3: Samples from six wildtype pigs from the MIDY biobank, 6 including ve locations within the gastro-intestinal tract.
For study 2 and 3, samples were processed as previously described (see also method section). 7 All sequencing data were processed using IMNGS 8 and Rhea. 9 Cultured fractions were determined by searching for matches between the amplicon sequences of all study-speci c OTUs (those occurring at a relative abundance of ≥0.25% in at least one sample within the respective dataset) and 16S rRNA gene sequences of the isolates using blastn (E-value <1e-25, 80% query coverage) at two di erent sequence identity thresholds: 97% (as proxy for species level) and 95% (genus level). Data are shown in a box plots, center line: median, bounds of box: quartile, whiskers: Tukey.

C in Hungates or Petri dishes
Freshly collected feces Fecal biomass was collected directly from the rectum of pigs using sterile gloves.

EXPERIMENTAL STEPS
2--Close the lid and hand-shake vigourusly until fecal material is re-suspended. Remember, the environment now contains oxygen, so be quick (<1 min). 3--Let to stand for 1 min to sediment debris. Collect 5 ml of fecal slurry using a syringe with sterile needle (>1 mm ø) via the rubber stopper (avoid particles). 4--1:100 is obtained. Mix gently to homogenize. 5--1:5,000 is obtained. Alternatively, dilution series can be plated directly onto agar plates.