depression in monogastric animals (12, 16, 17). Fuller et al. (8, 23, 24) have further suggested that growth depression in

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb p /87/ $02.00/0 Copyright 1987, American Society for Microbiology Vol. 53, No. 2 Subtherapeutic Levels of Antibiotics in Poultry Feeds and Their Effects on Weight Gain, Feed Efficiency, and Bacterial Cholyltaurine Hydrolase Activity SCOTT D. FEIGHNER* AND MICHAEL P. DASHKEVICZ Depa)rtment oj Animnal Druiig Disco?very, Merc k Shar-p & Doh/ne Research Labh)rtorries, RalhvaV, News Jersey Received 26 September 1986/Accepted 21 November 1986 A radiochemical method was developed to estimate cholyltaurine hydrolase potentials and rates of cholyltaurine hydrolysis in chicken intestinal homogenates. This method was used to monitor the effects of antibiotic feed additives on cholyltaurine hydrolase activity. Avoparcin, bacitracin methylenedisalisylic acid, efrotomycin, lincomycin, penicillin G procaine, and virginiamycin improved rate of weight gain and feed conversion of chicks and decreased cholyltaurine hydrolase activity in ileal homogenates relative to those of nonmedicated control birds. The results provided the first evidence that feeding selected antibiotics at subtherapeutic levels can affect bile acid-transforming enzymes in small-intestinal homogenates. The inverse relationship between growth performance and cholyltaurine hydrolase activity raises the possibility that specific inhibitors of this enzyme may promote weight gain and feed conversion in livestock and thereby reduce or eliminate the need for antibiotic feed additives. Since the discovery that fermentation solids contained a component capable of promoting growth in seemingly healthy monogastric animals (25, 53, 57), antibiotics have been fed at subtherapeutic levels to improve weight gain and efficiency of feed utilization (4). This husbandry technique has been practiced since the 1950s. Antibiotics promote improved growth responses because of an effect on the autochthonous microflora in the gastrointestinal tract. This view is supported by results of studies comparing the growth responses of conventional and germfree chickens (7, 21). Germfree chickens grow faster and more efficiently than do conventional chickens, and they do not respond to growth-permittant antibiotics. In addition, conventional chickens fed antibiotics grow and exhibit feed efficiencies approaching those achieved by germfree chickens. It is also relevant that the extent of growth improvement elicited by subtherapeutic levels of growth-permittant antibiotics has not diminished substantially over years of use (6, 31, 32). This supports the suggestion that an infectious agent per se is probably not responsible for reduced growth of conventional chickens. Other studies (9, 15, 50; R. W. Kelly, Ph.D. Thesis, Virginia Polytechnic Institute and State University, Blacksburg, 1982) have failed to demonstrate significant changes at the genus level in the microbiological composition of the intestinal tract when antibiotics are fed. These results strongly implicate a role for the normal autochthonous microflora in the growth depression of conventional animals. The combined functional activities of the gastrointestinal microflora on endogenous or exogenous nutrients may account for growth depression in conventional animals. For example, Visek has proposed that the production of ammonia from urea by the gastrointestinal microflora is responsible for growth depression in conventional animals. Numerous studies (10, 28, 55) have provided evidence to support this hypothesis, although other studies are contradictory (40, 56). Eyssen was the first investigator to suggest that bacterial transformation products of bile acids may cause growth * Corresponding author. depression in monogastric animals (12, 16, 17). Fuller et al. (8, 23, 24) have further suggested that growth depression in chickens is due to deconjugation of bile salts by Streptococc(US fjaeciurm adhering to duodenal epithelial cells. Accordingly, they maintain that antibiotic-containing feeds reduce the levels of S. faeciirn in the gastrointestinal tract and consequently lower bile salt-deconjugat4ing activity. This proposal, however, assumes growth depression in conventional animals is in response to a single agent. The potential role of bile acids in growth depression, however, has not been defined. The mechanism by which antibacterial agents improve growth performance is not known, but several theses have been proposed. (i) Nutrients are more efficiently absorbed because of a thinner small-intestinal epithelium (3, 18, 22, 38, 39, 48, 58); (ii) nutrients are spared since competing microorganisms are reduced (13, 44); (iii) microorganisms responsible for subclinical infections are reduced or eliminated (2, 16, 18, 19, 49); or (iv) production of growth-depressing toxins or metabolites by intestinal microflora is reduced (10, 35). Although each proposal has merit when viewed in the light of a particular criterion, none explains the entire growth phenomenon. Bile acid deconjugation and dehydroxylation (Fig. 1) are transformations catalyzed exclusively by bacterial enzymes (30, 36). The present report describes the effect of antibiotics on the transforming potentials and specific activities of cholyltaurine hydrolase in the small intestinal tracts of young chickens and correlates these activities with growth performance. (Portions of this work were presented at the Annual Meeting of the American Society for Microbiology, March 1986, Washington, D.C. [Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, Q58, p. 293].) MATERIALS AND METHODS Chicken growth experiments. Chicken growth and feed conversion experiments were conducted with a soybean protein-sucrose-based diet (54). Day-old male (Arbor Acre x Peterson) broiler chicks were housed overnight and fed a 331

2 332 FEIGHNER AND DASHKEVICZ APPL. ENVIRON. MICROBIOL. Taurocholic Acid Cholic Acid Deoxycholic Acid Taurochenodeoxycholic Acid Chenodeoxycholic Acid Lithocholic Acid FIG. 1. Bacterial bile acid transformation reactions. Steps: A, Bile salt hydrolase; B, 7o-dehydroxylase. nonmedicated practical diet (Ralston Purina [St. Louis, Mo.] Lab Chick Chow S-G no. 5065). On the following day the chicks were sorted and blocked on a weight basis and fed the experimental diets containing antibiotics for 9 days. Each diet was fed to four replicate groups of seven birds. Feed and water were available ad libitum. The chicks were maintained in battery brooders on raised floors and subjected to continuous lighting. To simulate growth conditions on litter, chicks were exposed to normal intestinal flora via the water supply, which was inoculated with 0.5% of a 1:1 (wt/vol) aqueous homogenate of hen excreta at the start of the experiment. Fresh water was added as needed daily. On day 5, the entire water supply was replaced with fresh, uninoculated water. Chicks were weighed by pens at 5 and 9 days. Feed consumption was determined for each pen and corrected for mortality. At termination of the experiment, two chicks from each pen whose body weights were nearest the mean pen weight were retained and combined with similarly selected birds within the treatment group. Five birds were randomly selected from the eight retained birds for preparation of intestinal homogenates. Preparation of intestinal contents for bile salt hydrolase transformation activities. The chickens were asphyxiated with C02, the small intestines were rapidly excised, and the various anatomical sections were dissected and placed into tared specimen cups. The segment from the gizzard to the bile ducts was defined as the duodenum, that from the bile ducts to the yolk sac stalk was defined as the jejunum (upper ileum), and that from the yolk sac stalk to the ileocecal junction was defined as the ileum. Gross weights were recorded, and the specimens were introduced into an anaerobic glove box (Coy Laboratory Products, Ann Arbor, Mich.) containing an atmosphere of 10% H2 and 5% CO2 in nitrogen. Anaerobic acetate buffer (5 mm, ph 5.6) containing 0.336% EDTA and 0.156% 2-mercaptoethanol was added to each segment (1:10 [wt/vol]), which was then homogenized in a Waring blender. The homogenates were centrifuged at 2,000 x g for 5 min to remove large feed particles and tissue debris. Samples (10 ml) of the supernatant fluid, representing bacteria attached to intestinal epithelial cells and bacteria in intestinal contents, were removed and retained at 4 C in the anaerobic glove box. The initial dilution normalized the estimated bile salt hydrolase potential to a grams of wet tissue weight basis. Bile salt hydrolase transformation activity. Glycine and taurine released from conjugated bile salts by bacterial bile salt hydrolase enzymes have been measured spectrophotometrically by using the ninhydrin reaction (37, 45). Because this assay procedure is based on measuring the formation of an amino acid-ninhydrin complex, its usefulness for measuring activities in intestinal homogenates was restricted because of high levels of background ninhydrin complexes formed. Thus, an alternative assay for measuring bacterial cholyltaurine hydrolase activity in intestinal contents was sought. Bile salt hydrolase activity in chicken intestinal homogenates was measured radiochemically by quantitating the amount of 14C-labeled carboxyl-cholic acid (CA) hydrolyzed from 14C-labeled carbonyl-taurocholic acid (TCA). Biotransformation potentials were determined by mostprobable-number (MPN) analysis (three tubes, seven dilutions). Reaction mixtures were prepared in 1-dram (1 fluidram = ml) screw-cap vials containing 1.0 ml of the anaerobic acetate buffer described above and supplemented with 0.4 mm TCA and sufficient [14C]TCA to yield ~ici/p.mol. Reactions were initiated by addition of 1.0 ml of diluted intestinal contents. The inoculated vials were incubated anaerobically for 24 h at 41 C in the glove box incubator. Uninoculated vials served as negative controls, and reactions incubated with commercial Clostridium perfringens cholylglycine hydrolase (Sigma Chemical Co., St. Louis, Mo.; 10 U per tube) served as a positive control. Radioactivity measured in the organic phase was corrected for background extraction of [14C]TCA. Corrected counts per minute exceeding 1% of the initial counts added were scored as positive (52), and MPN estimates were made from tables (11). [14C]TCA biotransformation rates in ileal homogenates of chicks were measured essentially by the method described above for estimating biotransformation potentials. The unlabeled substrate concentration was increased to a final concentration of 2.0 mm TCA, and the specific radioactivity was FCi/jimol. Preliminary experiments using ileal homogenates from nonmedicated chickens demonstrated that rates were linear over 2-h incubation period at 41 C. Routinely, TCA transformation rates were determined after a 30-min incubation at 41 C. All reactions were terminated by lowering the ph to 2.0 with 6 N HCl. The pka of TCA is less than 2.0, and that of CA is 4.6 (5). Thus, at ph 2.0, TCA is ionized and soluble in aqueous phases, and CA is protonated and soluble in ethyl acetate. Ethyl acetate (1.0 ml) was added to partition the [14C]CA into the organic phase, and 0.5-ml samples were removed and added to 10 ml of Aquasol-2 (DuPont) in glass

3 VOL. 53, 1987 BILE SALT HYDROLASE AND ANTIBIOTIC FEED ADDITIVES 333 scintillation vials. Radioactivity was measured in an LKB no Rack-beta II liquid scintillation counter. Counts per minute were corrected with an external-standard channel ratio and a "'C quench curve. Statistics. Chicken growth and performance data were subjected to a one-way analysis of variance, and significance was determined by calculating the least-significant difference (51). [14C]TCA transformation rate data were transformed as the natural logarithm to normalize the distribution. The transformed data were subjected to the same analysis by calculating the least-significant difference. Chemicals. All chemicals used were at least reagent grade. [14C]TCA (55.7 mci/mmol) and [14C]CA (52 mci/mmol) were obtained from Amersham Corp., (Arlington Heights, Ill.). Stock solutions were prepared in methanol and stored at 0 C. Bacitracin methylenesalicylic acid was purchased from A. L. Laboratories (Englewood Cliffs, N.J.), virginiamycin was obtained from SmithKline Animal Health Products (Philadelphia, Pa.), avoparcin was obtained from American Cyanamid Co. (Pearl River, N.Y.), and lincomycin and polymyxin B were purchased from Sigma. RESULTS A study was designed to investigatethe extraction efficiencyof [14C]CA as a function of the ratio of conjugated-to-free bile acids. Conditions were chosen to represent reaction mixtures in which up to 7.5,umol of [14C]TCA was transformed to [14C]CA (i.e., 0 to 100% biotransformation). This was accomplished by keeping the sum of the [14C]TCA and [14C]CA concentrations equal to 7.5 mm, varying the [14C]CA concentration from 0 to 7.5 mm, and incorporating a constant amount of [14C]CA (approximately 50,000 dpm) in each test mixture. The simulated reaction mixtures were acidified, the CA was extracted into an ethyl acetate phase, and the radioactivity was determined. The results indicated that extraction [14C]CA by ethyl acetate was nearly 100% from 0.1 to 7.5 mm (data not shown). Less than 2% of the initial radioactivity in control reaction mixtures was extracted into the organic layer. Furthermore, 100% of the radioactivity was recovered as [14C]CA in the organic layer after 24 h of incubation of [14C]TCA with C. perfringens cholylglycine hydrolase (data not shown). The influence of subtherapeutic levels of antibotics on weight gain and feed conversion of chicks during a 9-day continuous-feeding treatment are shown in Table 1. Efrotomycin and bacitracin methylenesalicylic acid improved weight gain and feed conversion compared with TABLE 1. Nine-day growth performance of chickens fed a semisynthetic soybean protein-sucrose-based diet supplemented with subtherapeutic levels of antibiotics % Change (vs control)a in: Treatment (level [mg/kg of diet]) Weight Feed/gain gain ratio Efrotomycin (4) Bacitracin methylenedisalicylic c acid (50) Polymyxin (50) (200) -10.7c +6.7 a The average weight gain of controls was g, and their feed/gain ratio was b Significantly different from control at P s c Significantly different from control at P s TABLE 2. Three-tube MPN evaluation for cholyltaurine hydrolase potential from the ilea of nonmedicated control chicks Dilution cpm/rxna Corrected Avg % Scorec cpmb transformation ,404 49, ,196 49, ,344 49,244 + l0-4 29,312 28, ,408 29, ,112 28, ,676 1, ,828 1, ,032 1, < < a rxn, Reaction mixtures contained 1.0 ml of the anaerobic acetate buffer, 0.4 mm TCA, and sufficient [14C]TCA to yield 0.125,uCi/,umol. Transformation potentials were initiated by addition of 1.0 ml of diluted intestinal homogenate. b cpm/rxn - 1,100 (620 cpm of TCA extracted background cpm for >1% transformation). c MPN for cholyltaurine hydrolase potential from the ilea of nonmedicated control chicks was 20 x 104. those of nonmedicated controls. Polymyxin B decreased weight gain and feed conversion in a dose-dependent manner Ėnumeration of the bile salt hydrolase potential in different intestinal segments from chicks fed control and antibiotic-supplemented soybean protein-sucrose diets was based on a three-tube MPN method. Table 2 shows the radioactivity recovered and the MPN score for the bile salt hydrolase potential in the ileum of a nonmedicated bird. Figure 2 shows the MPN estimation of the bile salt hydrolase potential in duodenum, jejunum, and ileum from chicks fed subtherapeutic levels of antibiotics a. C) C) Duodenum Jejunum Ileum Intestinal Segment Polymyxin B Control 0 Efrotomycin x 0 o o / Bacitracin-MD o 0 FIG. 2. MPN-estimated cholyltaurine hydrolase potentials in intestinal segments of chicks treated with antibiotics.

4 334 FEIGHNER AND DASHKEVICZ TABLE 3. Growth parameters and ileal cholyltaurine hydrolase transformation activities in chickens fed antibiotic-amended diets Treatment % Change (vs control)" in: Cholyltaurine (level [mg/kg]) Wt gain Feed/gain hydrolause ratio activity" Polymyxin B (50) ,539 Efrotomycin (4) ' -6.2" 380 Virginiamycin (10) ' -11.9' 539 Penicillin G procaine (25) +25.2' -12.3' 11' Avoparcin (10) +27.1' -10.0' 464 Lincomycin (4) +28.0' -17.5' 330 Bacitracin methylenedisalicylic ' ' 626 acid (50) afor controls, the average weight gain was g, the average feed/gain ratio was , and the average cholyltaurine hydrolyase activity was Rates were calculated by using geometric means; n = 5 (nanomoles of cholic acid released per 30 min per gram [wet weight] of ileum). ' Significantly different from control at P d Significantly different from control at P c 0.1. " Significantly different from control at P c Data demonstrated that bacteria capable of catalyzing the hydrolysis of TCA were located throughout the small intestines of antibiotic-fed and nonmedicated birds (Fig. 2). In addition, MPN analysis of cholyltaurine hydrolysis potentials in chicken small-intestinal contents provided preliminary data to suggest an association between the enzyme level and improved growth performance. However, MPN measurements do not provide an estimate of the rate of bile salt hydrolysis, and they are not suitable for investigations involving large numbers of birds and treatments. Therefore, the specific activity of cholyltaurine hydrolase in ileal homogenates was used. Additional antibiotics used in the poultry industry to enhance weight gain and feed conversion were tested, and the influence these feed additives exerted on the bile salt hydrolase transformation rates in ileal homogenates from treated and control chickens was measured. The effect of 9 days of continuous feeding of subtherapeutic levels of the growth-permittant antibiotics bacitracin methylenedisalicylic acid, virginiamycin, avoparcin, lincomycin, penicillin G procaine, and efrotomycin and the non-growth-permittant antibiotic polymyxin B on weight gain and feed conversion are shown in Table 3. Growth-permittant antibiotics improved gain (P < 0.05) and feed intake per unit of weight gain (P < 0.1). In contrast, polymyxin B reduced weight gain and feed conversion compared with nonmedicated controls. The specific activity of cholyltaurine hydrolase was decreased in ileal homogenates of birds fed those antibiotics that improved weight gain and feed conversion. Conversely, decreased weight gain and feed conversion were associated with increased cholyltaurine hydrolase activity in the ilea of chickens fed the polymyxin B-supplemented diet. These results confirmed and extended the trends observed from the MPN estimates of cholyltaurine hydrolase potentials in intestinal homogenates from chicks fed a medicated or nonmedicated diet. DISCUSSION The continuous use of antibiotics at subtherapeutic levels in animal feeds to promote improved weight gain and feed conversion in monogastric animals has become a controversial issue because of concerns over antibiotic resistance development (1, 34, 43). Understanding how antibiotics APPL. ENVIRON. MICROBIOL. improve performance in monogastric animals may permit identification of a specific biochemical target or a microbial activity which could be modified with nonantibacterial feed additives, thereby allowing conventional animals to grow at rates comparable to those of germfree animals. The mode of action of the antibiotics used as feed additives to promote improved weight gain and feed conversion must account for the observed changes in animal physiology and gastrointestinal microbial ecology, as well as the known antibiotic pharmacokinetics. Bile acids are synthesized in the liver from cholesterol; conjugated with glycine or taurine to facilitate biliary excretion; concentrated in the gallbladder; and secreted as bile into the intestinal lumen. Conjugated bile salts aid in digestion, emulsification, and absorption of fats and lipids from the small intestine. In chickens, the bile salt component of bile is composed predominantly of taurochenodeoxycholic acid, TCA, and tauroallocholic acid, accounting for approximately 80, 15, and 5%, respectively (29). Once in the intestinal lumen, the conjugated bile acids are extensively biotransformed by the endogenous gastrointestinal microflora. The biotransformation reactions of bile acids considered important for growth promotion include deconjugation and dehydroxylation (Fig. 1). Bacteria capable of catalyzing the hydrolysis of bile acid conjugates (Fig. 1, step A) include members of the genera Clostridiium, Lactobacillius, Peptostreptococcus, Bifidobacterilum, Fusobacteriurn, Eiubacteriium, Streptococcus, and Bac teroides (30, 36, 37). Salanitro et al. (47) showed that the small intestines of chickens were colonized with a significant anaerobic flora including anaerobic cocci, and members of the genera Eubacteriurm, Propionibacteriirm, Clostridiuim, Gemmiger, and Fiusobacterium, as well as Streptococcus and Lactobacilllls. The transformation of primary bile acids to secondary bile acids (Fig. 1, step B) has been reported to be catalyzed by members of Clostridiium and Eiubacterium (27, 30, 33). Lithocholic acid, a secondary bile acid produced from chenodeoxycholic acid (46), is hepatotoxic (20, 41), causes inflammation of the intestinal epithelium, and impairs nutrient uptake (14, 20). Bile acid profiles of gallbladder bile and feces from germfree animals demonstrate the complete absence of unconjugated bile acids and secondary bile acids, thus strongly implicating a role for the endogenous gastrointestinal microflora of conventional animals in these reactions in vivo (26, 42). Antibiotics developed as feed additives for growthpermittant activity include polypeptide, glycopeptide, macrolide, lincosamide, and,-lactam classes of antibiotics. The majority of growth-permittant antibiotics share two characteristics: they are not absorbed from the gastrointestinal tract, and they display activity against gram-positive microorganisms. The fact that antibacterial agents used as growth-permittant antibiotics may (e.g., lincomycin) or may not (e.g., bacitracin) be absorbed argues against the importance of alleviating subclinical infections as a primary mechanism of action for growth-permittant antibiotics. The apparent gram-positive spectrum of growth-permittant antibiotics may represent a uniformly common attribute of this group of feed additives. The gram-positive spectrum of growthpermittant feed additives and the predominantly grampositive representation of bile acid-transforming gastrointestinal microflora may be a related association regarding cause and effect of antibacterial feed additives. Continuous feeding of subtherapeutic levels of antibiotics affects the potential for deconjugating cholyltaurine as estimated by an MPN technique (Fig. 2). Deconjugation poten-

5 VOL. 53, 1987 BILE SALT HYDROLASE AND ANTIBIOTIC FEED ADDITIVES 335 tials were reduced in the ileum 25- and 50-fold after treatment with the growth-permittant antibiotics efrotomycin and bacitracin methylenedisalicylic acid, respectively. In contrast, the deconjugating potential in ilea from chickens fed polymyxin, a non-growth-permittant antibiotic, increased 4.5-fold compared with that of nonmedicated controls. In addition, feed additives which promote weight gain and feed conversion in chickens also reduced bile salt hydrolase activity in ileal homogenates (Table 3). These results provide the first evidence of antibiotic feed additives affecting the transformation potential and specific activity of bile acidtransforming enzymes from intestinal contents. These results support the hypothesis that modifying the activity of bacterial bile salt hydrolase enzymes in intestinal contents from poultry, and presumably other livestock, would result in improved animal growth performance. Modification of the bacterial enzymatic activity could possibly be achieved with specific inhibitors, thereby reducing the use of antibiotics as feed additives for growth promotion. ACKNOWLEDGMENTS We thank Joy Gillett and Bridget Butler for technical assistance in conducting the growth phase of the experiments reported in Table 3. We also thank Karen Rosa for typing the manuscript. LITERATURE CITED 1. Baldwin, R.A The development of transferable drug resistance in Salmonella and its public health implications. J. Am. Vet. Med. Assoc. 157: Barnes, E. M., G. C. Mead, C. S. Impey, and B. W. Adams The effect of dietary bacitracin on the incidence of Streptococcus faecalis subspecies liquefaciens and related streptococci in the intestines of young chicks. Br. Poult. Sci. 19: Boyd, F. M., and H. M. Edwards, Jr Fat absorption by germ-free chicks. Poult. Sci. 46: Bunyan, J., L. Jeffries, J. R. Sayers, A. L. Gulliver, and K. 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