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...

SummaryMammals live in a homeostatic symbiosis with their gastrointestinal microbiota. The mammalian host provides the microbiota with nutrients and a stable environment; whereas the microbiota helps shaping the host's gut mucosa and provides nutritional contributions. Microorganisms start colonizing the gut immediately after birth followed by a succession of populations until a stable, adult microbiota has been established. However, physiological conditions differ substantially among locations in the gut and determine bacterial density and diversity. While Firmicutes and Bacteroidetes dominate the gut microbiota in all mammals, the bacterial genera and species diversity is huge and reflects mammalian phylogeny. The main function of the gastrointestinal epithelium is to absorb nutrients and to retain water and electrolytes, yet at the same time it is an efficient barrier against harmful compounds and microorganisms, and is able to neutralize antagonists coincidentally breaching the barrier. These processes are influenced by the microbiota, which modify epithelial expression of genes involved in nutrient uptake and metabolism, mucosal barrier function, xenobiotic metabolism, enteric nervous system and motility, hormonal and maturational responses, angiogenesis, cytoskeleton and extracellular matrix, signal transduction, and general cellular functions. Whereas such effects are local at the gut epithelium they may eventually have systemic consequences, e.g. on body weight and composition.

The microbial communities of the ileum and cecum of broiler chickens from a conventional and an organic farm were investigated using conventional culture techniques as well as cloning and sequencing of 16S rRNA genes. Eighty-five percent of the 557 cloned sequences were <97% related to known cultured species. The chicken ileum was dominated by lactobacilli, whereas the cecum harbored a more diverse microbial community. The cecum was dominated by a large group of bacteria with hitherto no close cultured relatives but most closely related to Faecalibacterium prausnitzii. Approximately 49 and 20% of the cecal clones belonged to this cluster in conventional and organic broiler chickens, respectively. We were, however, able to recover a number of these phylotypes by cultivation, and the isolates were shown to be butyric acid producers. The investigation was a descriptive rather than a comparative study of 2 different rearing systems; however, several differences were observed. For instance, Clostridium perfringens was found in significantly higher numbers in the birds from the organic farm compared with the conventional broilers, probably due to the addition of salinomycin to the conventional feed. In the ileum, the abundance of the different Lactobacillus species differed between the 2 broiler types. The culture-based and culture-independent techniques complemented each other well. Strengths and limitations of the different methods are discussed.

A feeding experiment was carried out over 42 d with four groups of broiler chickens fed experimental diets formulated to provide no supplementation, 20 mg zinc bacitracin, 60 mg salinomycin, or both feed additives in combination. During the fifth week of the experiment, four chickens from each pen were killed, and the contents of gizzard, duodenum, jejunum, ileum, ceca, and rectum were separately collected and pooled. In all intestinal segments, the pH and the concentration of lactic acid were measured, and the numbers of anaerobic bacteria, coliforms, lactic acid bacteria, lactobacilli, enterococci, and Clostridium perfringens were counted. In homogenates of pancreas obtained from four animals, the activities of amylase, lipase, trypsin, and chymotrypsin were measured. A significant growth-promoting effect was observed in the group receiving zinc bacitracin in combination with salinomycin. Zinc bacitracin significantly reduced the number of coliform bacteria in the ileum and increased the activities of amylase and lipase in pancreas homogenates. Supplementation with salinomycin and zinc bacitracin, alone or in combination, resulted in significantly lower counts of C. perfringens as well as Lactobacillus salivarius, which was a dominant lactic acid bacterium found in broiler intestinal contents. High numbers of these lactobacilli may play a role in broiler growth depression related to competition in nutrient uptake or impaired fat absorption due to bile acid deconjugation.

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with Brachyspira hyodysenteriae, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.The gastrointestinal tract of pigs is densely populated with bacteria, and the intestinal microbiota has important influence on animal health and growth performance. The lumenal contents of the colon support between 10 10 and 10 11 culturable bacteria per g (wet weight) (1,6,21). The majority of the culturable bacteria in the pig colon are gram-positive, strict anaerobic streptococci, lactobacilli, eubacteria, clostridia, and peptostreptococci (21,27,28). The gram-negative organisms comprise about 10% of the total culturable bacteria. Most isolates belong to the Bacteroides and Prevotella groups (27). The gastrointestinal tract bacterial community structure is susceptible to changes in the diet of the animal. For example, the bacterial community will adapt to the introduction of high levels of dietary fibers by increased growth of bacteria with cellulolytic and xylanolytic activities (8,21,32). Other dietary treatments that are known to affect the intestinal bacterial community are the addition of organic acids to the feed (25) and prefermentation of commercial dry feeds (26). However, the effects of these treatments on the structure of the bacterial community are unknown. Pathogenic bacteria and bacteria that are part of the indigenous microbiota in the pig intestine may interact. Resistance to colonization by pathogens is well known (4), while synergistic relations with one or more indigenous intestinal bacterial species are a prerequisite for the pathogenicity of Brachyspira hyodysenteriae (34) and Lawsonia intracellularis (16). Both species cause severe intestinal disorders. On the other hand, it has been shown previously that the onset of swine dysentery causes a dramatic disturbance of the intestinal bacterial community (24). All these studies are based on culturing of the bacteria. However, comparisons with direct microscopic counts have shown that only part of the intestinal bacteria can...