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 grampositive 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.The microbial ecology of gastrointestinal tract ecosystems is not well understood due to the inadequacy of classical, culturedependent microbiological methods. Two decades ago, substantial efforts were put into characterizing the intestinal microbiota of pigs by using microbiological methods based on culturing and phenotypic analysis of the isolates (1,28,36,37,39,40). These studies showed that the majority of the culturable bacteria are gram-positive, strict anaerobic streptococci, lactobacilli, eubacteria, clostridia, and peptostreptococci, while the gram-negative part of the microbiota is dominated by Bacteroides. Because culture-based methods are very time-consuming, thereby limiting the number of samples that can be processed, no information on the population dynamics or community responses to perturbations was obtained.Detailed information of the microbial community composition in natural systems can be gained from the phylogenetic analysis of 16S ribosomal DNA (rDNA) sequences obtained directly from samples by PCR amplification, cloning, and sequencing, although this procedure may be biased as well (9,35,46,49,50). 16S rDNA cloning and sequencing has been applied to analyze the intestinal bacterial community in humans (45, 56) and in a pig (33) and compared to culture-based methods. The results showed that the microbial community is complex and that the bacterial diversity cannot be comprehended by culturing. However, these studies were limited, as only a single individual was sampled, the number of clones analyzed was small relative to the expected di...

Impact of chicory inclusion in a cereal-based diet on digestibility, organ size and faecal microbiota in growing pigs

“…TRF 262 was stimulated by chicory forage, whereas TRF 163 and 264 was lower in the diets with chicory forage. Leser et al (2002) found that Prevotella is one of the dominating commensal bacteria in the LI of the pig, but the sequence similarities with identified bacteria were few. Moreover, we know little about how this genus responds to different diets in pigs.…”

“…The microbial ecosystem in the gastrointestinal (GI) tract is characterized by a high population density, wide diversity and complexity of hostmicrobe and microbe-microbe interactions (Zoetendal et al, 2004). However, the knowledge about the gut microbial diversity is still far from complete and the use of molecular techniques has revealed that many of the pig gut microbial communities have not previously been cultured (Leser et al, 2002;Castillo et al, 2007;Patterson et al, 2010).…”

The effects of supplementing varying molecular weights of chitooligosaccharide on performance, selected microbial populations and nutrient digestibility in the weaned pig

“…The pigs fed the diets supplemented with different MW COS had a lower faecal score, which may support the improvement in daily gain and gain to feed ratio achieved during the weaner period. As a large number of intestinal bacterial species are unculturable (Leser et al, 2002), lactic acid bacteria were enumerated as a reflection of changes in the population structure of beneficial bacteria. The relevance of measuring E. coli populations as an indicator of pathogenic bacteria is debated; however, increased coliform counts were recorded in the intestine of scouring pigs (Muralidhara et al, 1977) and the density of coliforms in the gastrointestinal tract are used as an indicator of E. coli in pigs (Jørgensen et al, 1999;Hojberg et al, 2003;Mikkelsen et al, 2004).…”

Effects of feeding finisher pigs with chicory or lupine feed for one week or two weeks before slaughter with respect to levels of Bifidobacteria and Campylobacter

“…Extracted total DNA was adjusted using a spectrophotometer to a concentration of 5 mg/ml of DNA. Four replicate 50-ml PCR mixtures were made from each sample using the universal bacterial 16S rRNA gene primers S-D-Bact-0008-a-S-20 (5 0 -AGAGTTTGATCMTGGCTCAG-3 0 ) and S-D-Bact-0926-a-A-20 (5 0 -CCGTCAATTCCTTTRAGTTT-3 0 ; Leser et al, 2002). Primer S-D-Bact-0926lam-a-A-20 was 5 0 FAM (carboxy-fluorescein-N-hydroxysuccinimide-ester-dimethylsulfoxide) labelled.…”