Alteration of the Ileal Microbiota of Weanling Piglets by the Growth-Promoting Antibiotic Chlortetracycline

Antibiotics such as chlortetracycline (CTC) have been used to promote growth of pigs for decades, but concerns over increased antibiotic-resistant infections in humans have prompted the development of alternative strategies. Developing alternatives to antibiotic growth promoters (AGPs) could be informed by information on the mechanisms of growth promotion, notably, how AGPs affect the microbial populations of the gastrointestinal tract. Pigs from three sows were aseptically delivered by cesarean section. Six piglets were distributed to each of two foster mothers until weaning, when piglets were fed a diet with or without 50 mg/kg CTC for 2 weeks. The ileal bacterial microbiota was characterized by using a cultivation-independent approach based on DNA extraction, PCR amplification, cloning, and sequencing of the 16S rRNA gene pool. The ileal and mucosal communities of these growing pigs were dominated by Lactobacillus bacteria, various members of the family Clostridiaceae, and members of the poorly known genus Turicibacter. Overall, CTC treatment resulted in three shifts: a decrease in Lactobacillus johnsonii, an increase in L. amylovorus, and a decrease in Turicibacter phylotypes. The composition of the microbiota varied considerably between individual pigs, as revealed by shared operational taxonomic units (OTUs) and similarity (SONS) analysis (θ_YC values). While the observed variation between untreated pigs obscured the possible effect of CTC, ∫-LIBSHUFF and SONS analyses of pooled libraries indicated a significant shift due to CTC in both the lumen and the mucosa, with some OTUs unique to either treated or control ileum. DOTUR analysis revealed little overlap between control and treated communities at the 3% difference level, indicating unique ileal communities in the presence of CTC.Antibiotics have been used to promote animal growth for over 50 years. Antibiotic growth promoters (AGPs) such as tylosin, bacitracin, virginiamycin, and chlortetracycline (CTC) have been fed to pigs, chickens, and other animals to promote growth through increased feed intake, weight gain, and improved herd health (7, 36). Use of AGPs has come under increasing pressure with the growing consensus that their use leads to increased antibiotic-resistant infections in humans via generation of reservoirs of antibiotic-resistant bacteria that may enter the food chain through contamination (38, 46). The increasing concerns about antibiotic resistance have raised questions about whether the potential risks are worth the beneficial effects (44). Development of non-antibiotic-based alternative strategies to promote animal growth may benefit through increased understanding of AGP mechanisms of growth promotion.The growth-promoting impact of antibiotics was first described in the 1940s, and their use soon became routine (29, 35). The gastrointestinal (GI) tract harbors a great diversity of bacteria at a very high density (27). The increased growth and feed efficiency promoted by AGPs may be due to alteration of the microbiota of the GI tract. Early hypotheses focused on the suppression of pathogenic bacteria (19), but the broad-spectrum antibiotics used as growth promoters do not target specific species. Suggested mechanisms of action have included suppression of subclinical infections, a decrease in the levels of growth-depressing bacterial metabolites, decreased consumption of nutrients by intestinal microbiota, and improvement of nutrient uptake due to a thinner intestinal wall (14, 48). Data on the effect of AGPs on pig intestinal microbiota are needed in order to determine the relative contributions of the various proposed mechanisms. Much of the evidence available points to the action of antibiotics on intestinal bacteria as the main component responsible for the growth effect on animals (17, 20, 36).Traditional culture methods have provided some insights into pig GI microbiota, but culture-independent techniques utilizing analysis of rRNA genes have revealed a far greater diversity. Culture-independent methods have also helped to further our understanding of bacterial population dynamics and the complex interplay between the host and pathogenic and nonpathogenic bacteria. The construction of a large 16S rRNA bacterial clone library from the pig GI tract identified 375 phylotypes by using a similarity criterion of 97% (27). Studies utilizing denaturing gradient gel electrophoresis have shown the microbial variances between compartments of the pig intestinal tract, the effect of the diet on microbial communities of the colon, and the ileal microbiota changes produced by the use of several types of AGP (5, 28, 45). Each technique can hold its own bias or limitation, but combinations of fingerprinting and PCR techniques have led to a greater understanding of the composition of pig GI microbiota and their ecology (16, 49, 50).Studies on the effect of antibiotics on intestinal microbiology have focused on colonic or fecal microbiota because bacterial densities are highest (14) and sampling is noninvasive, allowing temporal studies. Yet, nutrient uptake occurs primarily in the small intestine, the region where bacterial activity would therefore have the greatest influence on growth (14). Demands on the GI tract to respond to bacteria by increased mucus production occur primarily in the small intestine (13). The main growth-promoting effect of antibiotics is therefore more likely to occur in the small intestine, specifically in the ileum, where bacterial numbers have reached a high density. One study showed that AGPs, including bacitracin, CTC, and tylosin, caused a shift in the ileal microbial profile of pigs (5). In that study, only one pig was used per treatment, so the basal variation in microbiota between individuals was not taken into account.The objective of this study was to examine how the AGP CTC affects the microbial community of the porcine ileum. To account for variation in the intestinal microbiota as influenced by both antenatal and postnatal environment, pigs from three separate sows were aseptically delivered by cesarean (C) section and distributed to two foster mothers until weaning, when piglets were fed a diet either with or without the AGP CTC. A cultivation-independent approach based on DNA extraction, PCR amplification, and cloning and sequencing of the 16S RNA gene was taken to characterize the pig ileal microbiota.