Recent concerns about the use of growth-promoting antibiotics in pig diets have renewed interest in the immunologic and growth-regulating functions of the gastrointestinal (GI) tract. The numerically dense and metabolically active microbiota ofthe pig GI tract represents a key focal point for such questions. The intestinal microbiota is viewed typically as a beneficial entity for the host. Intestinal bacteria provide both nutritional and defensive functions for their host. However, the host animal invests substantially in defensive efforts to first sequester gut microbes away from the epithelial surface, and second to quickly mount immune responses against those organisms that breach epithelial defenses. The impact of host responses to gut bacteria and their metabolic activities require special consideration when viewed in the context of pig production in which efficiency of animal growth is a primary objective. Here, we summarize the working hypothesis that antibiotics improve the efficiency of animal growth via their inhibition of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs ofthe GI system. In addition, novel molecular ecology techniques are described that can serve as tools to uncover the relationship between intestinal microbiology and growth efficiency.

The gastrointestinal (GI) microbiota of mammals is characterized by its high population density, wide diversity and complexity of interactions. While all major groups of microbes are represented, bacteria predominate. Importantly, bacterial cells outnumber animal (host) cells by a factor of ten and have a profound influence on nutritional, physiological and immunological processes in the host animal. Our knowledge of the molecular and cellular bases of host-microbe interactions is limited, though critically needed to determine if and how the GI microbiota contributes to various enteric disorders in humans and animals. Traditionally, GI bacteria have been studied via cultivation-based techniques, which are labor intensive and require previous knowledge of individual nutritional and growth requirements. Recently, findings from culture-based methods have been supplemented with molecular ecology techniques that are based on the 16S rRNA gene. These techniques enable characterization and quantification of the microbiota, while also providing a classification scheme to predict phylogenetic relationships. The choice of a particular molecular-based approach depends on the questions being addressed. Clone libraries can be sequenced to identify the composition of the microbiota, often to the species level. Microbial community structure can be analyzed via fingerprinting techniques, while dot blot hybridization or fluorescent in situ hybridization can measure abundance of particular taxa. Emerging approaches, such as those based on functional genes and their expression and the combined use of stable isotopes and biomarkers, are being developed and optimized to study metabolic activities of groups or individual organisms in situ. Here, a critical summary is provided of current molecular ecological approaches for studying the GI microbiota.

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Cultivation-independent microbial molecular ecology approaches were used to examine the effects of antibiotic growth promoters on the pig ileal microbiota. Five-week-old barrows were fitted with a simple T-cannula at the distal ileum. Three diets meeting or exceeding the minimum nutrient requirements were fed for 5 wk and supplemented as follows: 1) negative control (no antibiotic; n = 5), 2) continuous tylosin administration (n = 5), and 3) an antibiotic rotation sequence (wk 1, chlorotetracycline sulfathiazole penicillin; wk 2, bacitracin and roxarsone; wk 3, lincomycin; wk 4, carbadox; wk 5, virginiamycin; n = 5). Ileal luminal contents were collected for DNA isolation at the end of each of the 5 wk of the testing period. The V3 region of 16S rDNA was amplified by PCR and analyzed via denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR). Resulting PCR-DGGE band numbers (bacterial species) were counted, and the banding patterns analyzed by calculating Sorenson's pairwise similarity coefficients (C(S)), an index measuring bacterial species in common among samples. Band numbers and total bacterial DNA concentrations decreased (P < 0.05) temporally in antibiotic-treated pigs compared with controls. Comparisons between treatments yielded low intertreatment C(S) indices, indicating treatment-dependent alterations in banding patterns, whereas intratreatment comparisons revealed increased homogeneity in antibiotic-treated vs. control pigs. Sequence analysis of treatment-specific bands identified three Lactobacillus, one Streptococcus, and one Bacillus species that were diminished with antibiotic rotation treatment, whereas tylosin selected for the presence of L. gasseri. Lactobacillus-specific qPCR was performed and analyzed as a percentage of total bacteria to further evaluate the effects of antibiotic administration on this genus. Total bacteria were decreased (P < 0.05) by tylosin and rotation treatments, whereas the percentage of lactobacilli increased (P < 0.05) by d 14 and through d 28 in tylosin-treated pigs. The decrease in total bacteria by antibiotics may reduce host-related intestinal or immune responses, which would divert energy that could otherwise be used for growth. Conversely, the ability of tylosin to improve animal growth may relate to its apparent selection for lactobacilli, commensals known to competitively exclude potentially pathogenic species from colonizing the intestine.

Supplementation of infant formulas with prebiotic ingredients continues the effort to mimic functional properties of human milk. In this double-blind, controlled, 28-day study, healthy term infants received control formula (control group; n ‫؍‬ 25) or control formula supplemented with polydextrose (PDX) and galactooligosaccharide (GOS) (4 g/liter) (PG4 group; n ‫؍‬ 27) or with PDX, GOS, and lactulose (LOS) (either 4 g/liter [PGL4 group; n ‫؍‬ 27] or 8 g/liter [PGL8 group; n ‫؍‬ 25]). A parallel breast-fed group (BF group) (n ‫؍‬ 30) was included. Stool characteristics, formula tolerance, and adverse events were monitored. Fecal bacterial subpopulations were evaluated by culture-based selective enumeration (Enterobacteriaceae), quantitative real-time PCR (Clostridium clusters I, XI, and XIV, Lactobacillus, and Bifidobacterium), and fluorescence in situ hybridization (FISH) (Bifidobacterium). Fecal bacterial community profiles were examined by using 16S rRNA gene PCR-denaturing gradient gel electrophoresis. The daily stool consistency was significantly softer or looser in the BF group than in all of the groups that received formula. The formulas were well tolerated, and the incidences of adverse events did not differ among feeding groups. Few significant changes in bacterial subpopulations were observed at any time point. The bacterial communities were stable; individual profiles tended to cluster by subject rather than by group. Post hoc analysis, however, demonstrated that the bacterial community profiles for subjects in the BF, PG4, PGL4, and PGL8 groups that first received formula at a younger age were less stable than the profiles for subjects in the same groups that received formula at an older age, but there was no difference for the control group. These data indicate that formulas containing PDX, GOS, and LOS blends are more likely to influence gut microbes when administration is begun in early infancy and justify further investigation of the age-related effects of these blends on fecal microbiota.Nondigestible food ingredients called prebiotics pass into the lower gastrointestinal tract and, by definition, may be selectively metabolized by mutualistic microorganisms, such as Lactobacillus spp. and Bifidobacterium spp., which in turn contribute to improved host health (12, 34). After lactose and lipids, oligosaccharides, which have prebiotic activity, are the third largest component of human breast milk (5 to 10 g/liter), and there are as many as 200 distinct molecular structures (5, 26). Lactobacilli and bifidobacteria are the predominant bacteria in the intestinal microbiota of breast-fed infants, whereas infants who receive cow's milk-based infant formulas, which naturally contain low levels of oligosaccharides, often have higher concentrations of potentially pathogenic bacteria, such as Enterobacteriaceae and clostridia, in their intestinal microbiota (4,15,17).Clinical investigations of infant formulas supplemented with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) at a range of concent...