Antibiotics As Growth Promotants: Mode Of Action
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 of the 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 of the 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.
Numerous physiological, nutritional, and metabolic responses to feed-grade antibiotics have been reported, as summarized in Table 1 (Commission on Antimicrobial Feed Additives, 1997). From a production standpoint, feed antibiotics have been consistently shown to improve pig weight gain and feed efficiency as summarized in Figure 1. The data demonstrate that growth and feed efficiency are improved to a greater extent in young pigs than in older animals. Also, the enhancement of body weight gain is greater than the improvement in feed efficiency, indicating that feed intake is increased in animals receiving antibiotic-supplemented feed. However, antibiotics increase gain and feed efficiency even at constant feed intake (Jones and Tarrant, 1982), consistent with a direct effect on growth which is independent of feed intake. These growth responses are associated with improved nitrogen metabolism, including an increase in apparent nitrogen digestibility (3.0%), increased nitrogen retention (5.8%), and reduced nitrogen excretion (10%) in pigs fed tylosin (10 ppm to 50 ppm; Table 2). Growth-promoting antibiotics also benefit protein metabolism irrespective of protein concentration in the diet (Roth and Kirchgessner, 1993). Mechanisms by which antibiotics enhance growth should thus be consistent with their demonstrated effects on growth, feed efficiency, nitrogen metabolism, and with the relatively greater responses observed in younger animals.
Table 1. Summary of reported physiological, nutritional and metabolic effects of growth promoting antibiotics (Modified from Commission on Antimicrobial Feed Additive, 1997).
|+ Nutrient absorption||+ Energy retention||+ Liver protein synthesis|
|+ Feed intake||+ Nitrogen retention||+ Gut alkaline phosphatase|
|— Food transit time||+ Vitamin absorption||— Ammonia production|
|— Gut wall diameter||+ Trace element absorption||— Toxic amine production|
|— Gut wall length||+ Fatty acid absorption||— Aromatic phenols|
|— Gut wall weight||+ Glucose absorption||— Bile degradation products|
|— Fecal moisture||+ Calcium absorption||— Fatty acid oxidation|
|— Mucosal cell turnover||+ Plasma nutrients||— Fecal fat excretion|
|— Gut energy loss||— Gut microbial urease|
|— Vitamin synthesis|
Proposed Mechanisms for Antibiotic-Mediated Growth Enhancement
At least four mechanisms have been proposed as explanations of antibiotic mediated growth enhancement: 1) inhibition of sub-clinical infections, 2) reduction of growth-depressing microbial metabolites, 3) reduction of microbial use of nutrients, and 4) enhanced uptake and use of nutrients through the thinner intestinal wall associated with antibiotic-fed animals (Francois, 1962; Visek, 1978; Anderson et al., 2000). These share the common postulate that intestinal bacteria, whether commensal or pathogenic, depress animal growth, either directly or indirectly, through their metabolic activities. Perhaps the best support of this hypothesis is the observation that oral antibiotics do not enhance the growth of germ-free animals (Coates et al., 1963), while inoculating germ-free animals with GI bacteria depresses growth (Coates, 1980). A clear difference between germ-free and conventional animals is a thinner wall of the small intestine, with a reduction in connective tissue and lymphoid elements (Coates, 1980). Microscopic evaluation of germ-free intestine reveals a more regular and slender villus structure, with a thinner lamina propria. Further, the rate of renewal of epithelial cells is slower in germ-free animals (Coates, 1980), which may have a beneficial effect on basal energy expenditure and the efficiency of nutrient utilization. These observations are consistent with the view of Reeds and coworkers (1993) that in rapidly growing young animals, the GI tract and skeletal muscle draw from the same limited supply of nutrients and, in effect, compete for nutrients.
Table 2. Effect of antibiotics on nitrogen metabolism in swine.1
|Parameter||Control||Tylosin||Relative Percent Improvement|
|Apparent Nitrogen Digestibility
(% of Nitrogen intake)
G N retained / day)
|Relative Nitrogen Excretion
(g N excreted / g N retained)
|Nitrogen Utilization Efficiency
(N retained as % of N intake)
The Intestinal Microbiota is Competitive with the Host in the Small Intestine but Cooperative in the Large Intestine
Most of the attention given the pig intestinal microbiota has focused on the large intestine. Indeed, the large intestine (cecum and colon) is a major site of microbial colonization because of slow digesta turnover, and it is characterized by large numbers of bacteria (1010 – 1011 per g or ml of content), low redox potential, and relatively high short chain fatty acid (SCFA) concentrations. The composition of the hindgut microbiota is both diverse and stable. Several hundred anaerobic bacterial species and strains appear to coexist without one or a few becoming dominant (Fewins et al., 1957; Smith and Jones, 1963; Allison et al., 1979; Robinson et al., 1981; Robinson et al., 1984; Varel et al., 1987; Anugwa et al., 1989; Butine and Leedle, 1989; Pryde et al., 1999). Microbial activity in the cecum and colon appears to benefit the host (Hedde and Lindsay, 1986), with estimates up to 5-20% of the pig’s total energy being provided by fermentation end-products (Friend et al., 1963). However, the small intestine is the principal site of nutrient and energy absorption, and thus is the region in which bacterial activity is likely to have the greatest influence on the efficiency of growth (Figure 2; Anderson et al., 2000; Gaskins, 2001). In the proximal small intestine, the rate of digesta flow and thus the rate of bacterial washout, exceeds the maximal growth rates of most bacterial species. Accordingly, this intestinal region is colonized typically by bacteria that adhere to the mucus layer or epithelial cell surface. Acid-tolerant lactobacilli and streptococci are thought to predominate in the upper small intestine (Fewins et al., 1957). The distal small intestine (ileum) maintains a more diverse microbiota and higher bacterial numbers (108 per g or ml of contents) than the upper intestine, and is considered a transition zone preceding the large intestine. In a study of microbial gas production in various gastrointestinal regions, Jensen and Jørgensen (1994) reported that the highest H2 concentrations and production rates were found in the distal small intestine in pigs. This indication for significant microbial activity in the pig ileum was substantiated in their study by dense populations of culturable anaerobic bacteria and the detection of high ATP concentrations.
Culture-based studies have shown that small intestinal bacteria tend to compete with the host for energy and amino acids (Hedde and Lindsey, 1986). For example, as much as 6% of the net energy in pig diets can be lost due to bacterial utilization of glucose in the small intestine (Vervaeke et al., 1979). Bacterial use of glucose to produce lactic acid reduces the energy available to the host epithelium (Saunders and Sillery, 1982). Lactic acid also enhances peristalsis, thus increasing the rate of nutrient transit through the intestine (Saunders and Sillery, 1982). Amino acids, which are also degraded by small intestinal bacteria, are made unavailable to the pig and produce toxic metabolites such as amines, ammonia, phenols and indoles (Macfarlane and Macfarlane, 1995). Microbial deconjugation and dehydroxylation of bile acids impair lipid absorption and produces toxic degradation products (Hylemon, 1985). Mucolytic activities of intestinal bacteria are likely key for intestinal colonization but compromise the mucosal barrier and indirectly affect the efficiency of growth via stimulation of additional mucus production (Deplancke and Gaskins, 2001).
It is interesting to note that although different types of bacteria may generate one or more of the metabolites mentioned, the Gram-positive facultative anaerobes, which are oxygen-tolerant and predominant in the small intestine, often contribute more than one toxic catabolite (Gaskins, 2001). Further, the small intestinal microbiota consists predominantly of Gram-positive bacteria (Stewart, 1997), and most growth-promoting antibiotics target Gram-positive organisms (Table 3). These observations are consistent with involvement of small intestinal Gram-positive bacteria in growth depression.
It is curious that a class of organisms that appear to depress growth, namely Gram-positive facultative anaerobes including strains of Lactobacillus and Enterococcus, are also often used as probiotic organisms for enhancing health and promoting growth in livestock (reviewed in Jonsson and Conway, 1992). The growth-promoting effect of probiotics in livestock is less consistent than that observed with antibiotic supplementation (Jonsson and Conway, 1992). Supplementation of animals and humans with certain probiotic bacteria has been shown to provide protection against intestinal, diarrhea-producing pathogens (reviewed in McCracken and Gaskins, 2000). Therefore, probiotics may promote growth under situations in which certain pathogens are present; however, these same organisms in a cleaner facility may suppress growth via the mechanisms discussed above.
Table 3. Growth-promoting antibiotics and their antibacterial mode of action.1
|Class||Trade Name||Generic Name||Antimicrobial Spectrum||Mechanism of Action||Orally Absorbed2|
|Diterpene||Tiamulin||tiamulin||Gram +||Protein synthesis inhibition||Yes|
|Glycopeptide||Avotan||avoparcin||Gram +||Cell wall synthesis inhibition||No|
|Lincosaminides||Lincomix||lincomycin||Gram +||Protein synthesis inhibition||Yes|
|Macrolide||Tylan Spira 200||tylosin spiramycin||Gram +||Protein synthesis inhibition||Yes|
|Oligosaccharide||Maxus||avilamycin||Gram +||Protein synthesis inhibition||No|
|?-lactam||Penicillin||penicillin||Gram +||Cell wall synthesis inhibition||Yes|
|Peptides||Bacitracin Zn Bacitra||bacitracin bacitractin||Gram +||Cell wall synthesis inhibition||No|
|Streptogramin||Stafac||virginiamycin||Gram +||Protein synthesis inhibition||Yes|
|Phosphoglycolipid||Flavomycin||bambermycin||Gram +||Cell wall synthesis inhibition||No|
|Polyether||Salocin Monteban||salinomycin nerasin||Gram +||Membrane alterations||No|
|Quinoxalines||Mecadox Bayonox||carbadox olaquindox||Broad||DNA synthesis inhibition||Yes|
|Sulfonamides||Sulfamethazine Sulfa thizole||sulfamethazine sulfathiazole||Broad||Metabolic inhibition||Yes|
|Tetracycline||Aureomycin Terramycin||chlortetracycline oxytetracyline||Broad||Protein synthesis inhibition||Yes|
1Modified from O’Connor (1980), Commission on Antimicrobial Feed Additives (1997), and Lawrence (1998)
2Commonly accepted property. “No” does not necessarily mean 0 percent absorption nor is “Yes” equal to 100 percent absorption.
The combined potential of microbial activities to negatively impact intestinal functions clearly supports the hypothesis that certain bacterial populations commonly inhabiting the pig small intestine, though not necessarily pathogenic, cause a depression in growth which is reversed when the responsible organism(s) are metabolically inhibited or eliminated by feed-grade antibiotics (Figure 3). However, the basic questions relating to the effects of microbial production of toxic catabolites in the pig intestine remain mostly unanswered, including the taxonomy, ecology, and metabolic properties of target bacteria. Because of the profound bias introduced by cultivation-based techniques, these questions will be defined most efficiently through the use of novel molecular ecology techniques.
Novel Methods for Intestinal Microbial Ecology Studies
Several limitations are associated with cultivation-based microbiological techniques, particularly for surveying the intestinal ecosystem (Vaughan et al., 2000). In addition to being time- and labor-intensive, the use of selective media for different types of bacteria imposes an a priori bias on the types of bacteria that can be enumerated. Further, only 20-40% of bacterial species from the mammalian GI tract can be cultured and identified using current cultivation techniques. In other words, 60-80% of intestinal bacterial species may be overlooked (Vaughan et al., 2000; Langendijk et al., 1995; Suau et al., 1999). In view of this, we have defined the utility and limitations of a cultivationindependent technique that uses the phylogenetic information contained in 16S rDNA to objectively study the intestinal microbiota (Simpson et al., 1999; Simpson et al., 2000; McCracken et al., 2001).
Denaturing gradient gel electrophoresis (DGGE) is a polymerase chain reaction (PCR)-based technique in which DNA from a mixed sample is amplified using conserved 16S rDNA bacteriadomain primers (Muyzer et al., 1998). Although all PCR products are of approximately equal size, when electrophoresed on a polyacrylamide gel containing an increasing gradient of DNA denaturants, individual amplicons cease to migrate as the double-stranded products denature according to their G+C content (Freter, 1983). Thus, this approach allows separation of individual sequences based on 16S rDNA G+C content, corresponding to the different microbial species within the sample. Banding patterns from mixed samples are compared to evaluate the relative similarity of microbial communities from different habitats or treatments. Further, following electrophoresis, individual bands can be excised from the gel for sequencing and phylogenetic identification. This allows the characterization of complex microbial populations independent of bacterial cultivation.
Effects of Antibiotics on the Normal Microbiota
We have recently used 16S rDNA PCRDGGE to examine the effects of feed grade antibiotics on the intestinal microbiota in ilealcannulated pigs. Two antibiotic regimes were compared with a standard nonmedicated diet: 1) continuous administration of a single antibiotic and 2) an antibiotic rotation sequence in which a different antibiotic was used each week. Newly weaned pigs were fed control (no antibiotic) or medicated diets for five weeks. Ileal microbiota profiles were compared by DGGE-PCR analysis as demonstrated in the preliminary data in Figure 4 (Collier et al., unpublished observations). Similar gels were generated from all pigs. Through the use of statistical measures of community diversity, we observed that the ileal microbiota is more homogenous in anti-biotic-treated pigs. This finding reflects the fact that bacterial community profiles generally varied among individual animals and did so to a greater extent in the control pigs that were weaned to a non-medicated diet. Other molecular-based ecology studies, by us and others, in mice, pigs, and humans also demonstrate that, although the intestinal bacterial community within a single individual is relatively stable over time, bacterial populations from different individuals vary significantly (Simpson et al., 1999; McCartney et al., 1996; Kimura et al., 1997; Zoetendal et al., 1998). Individual variation in bacterial community profiles is consistent with the possibility that genetically encoded chemical epitopes expressed in mucus may dictate which bacteria colonize specific intestinal segments. This has significant implications for intestinal health issues and may also partly explain individual differences in the realization of growth potential in livestock species.
The inclusion of antibiotics also eliminated particular bacterial groups as evidenced by the disappearance of certain 16S rDNA PCR bands on the DGGE gels (Fig. 5). Individual bands that were eliminated by antibiotics were excised from DGGE gels and cloned and sequenced to identify the bacteria affected. Thus far, six Lactobacillus and two Streptococcus species have thus far been identified through this process as being selectively depleted in antibiotic-treated pigs. Interestingly, lactobacilli are thought to be one of the main contributors to microbial bile acid biotransformation in the small intestine (Tannock et al., 1989). Indeed, Feighner and Dashkevicz (1987, 1988) proposed that an important mechanism of growth-promoting antibiotics is the inhibition of microbial bile acid biotransformation in the intestine. Microbial deconjugation and dehydroxylation of bile impairs lipid absorption by the host animal (DeSomer et al., 1963; Eyssen, 1973) and produce toxic degradation products that can impair growth (Eyssen and DeSomer, 1963a). Bile acids are not deconjugated in the germfree intestine, demonstrating the important role of commensal bacteria in this process (Madsen et al., 1976). The particular contributions of the lactobacilli to bile acid biotransformation in the intestine is demonstrated by evidence that ileal bile salt hydrolase activity in conventional mice is reduced 86% by the elimination of lactobacilli from the microbiota, and by greater than 98% when both lactobacilli and enterococci are eliminated (Tannock et al., 1989). Using chicks, Eyssen and DeSomer (1963b) first suggested that bile acid transformation products might be responsible for the growth depression caused by intestinal bacteria. Additional evidence from chick studies showed that bile acid deconjugation by intestinal bacteria causes growth depression that is reversible by antibiotic supplementation (Fuller et al., 1984). Further, Feighner and Dashkevicz (1987, 1988) have shown an inverse relationship between the level of cholyltaurine hydrolase activity in the small intestine and growth rate in broiler chickens fed antibiotics. Antibiotictreated pigs also demonstrated decreased concentrations of lithocholic acid and corresponding increases in average daily gain and feed efficiency, consistent with work reported in poultry (Tracy and Jensen, 1987). While additional studies are required to verify the contribution of bile biotransformation to growth depression, these data clearly demonstrate the utility of molecular ecology to study the effects of antibiotics on the intestinal microbiota.
It is proposed that antibiotic modification of the small intestinal microbiota of swine permits more efficient intestinal and therefore whole animal growth. Recent advances in swine growth research have been directed primarily at affecting growth, feed efficiency, and body composition by altering the metabolism of muscle and fat (Moody et al., 2000; Beermann and DeVol, 1991). An understanding of the inter-relationship of intestinal physiology, microbiology, and immunology to swine growth will become increasingly important to critically evaluate the impact of commensal bacteria on animal growth. In that regard, it is particularly exciting that molecular techniques are now available that allow a better understanding of how the intestinal microbial profile is changed with the use of antibiotics. These advances will allow the development of novel technologies, management systems, and modified nutrition to optimize intestinal health and animal growth.
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H. R. Gaskins, Ph.D., University of Illinois
Rex Gaskins, a native of coastal North Carolina, obtained B.S. and M.S. degrees in animal science from North Carolina State University and a Ph.D. in nutritional sciences from The University of Georgia. From 1989-92, he completed postdoctoral studies in immunology at The Jackson Laboratory in Bar Harbor, Maine. Dr. Gaskins has been on the faculty of the University of Illinois since 1992 where he is now Professor of Immunobiology in the Departments of Animal Sciences and Veterinary Pathobiology. Research in his laboratory focuses on host-intestinal microbe interactions and is supported by the National Institutes of Health, the USDA-NRI Competitive Grants Program, and private industry.