Effect of High-Fiber and High-Oil Diets on the Fecal Flora of Swine

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1987, p Vol. 53, No /87/ $02.00/0 Effect of High-Fiber and High-Oil Diets on the Fecal Flora of Swine W. E. C. MOORE,'* L. V. H. MOORE,' E. P. CATO,' T. D. WILKINS,' AND E. T. KORNEGAY2 Department of Anaerobic Microbiology' and Department of Animal Science,2 Virginia Polytechnic Institute and State University, Blacksburg, Virginia Received 12 November 1986/Accepted 13 April 1987 Six pairs of pigs were fed a basal diet, a high-fiber diet, and a diet high in corn oil in different sequences to minimize the carry-over effect of diet. After 2 months on each diet, a fecal specimen from each pig was cultured on nonselective medium in roll tubes. Fifty colonies were randomly selected from each fecal sample, and isolates were characterized to identify a representative cross section of the fecal flora. The bacterial composition of the fecal flora differed between basal and high-fiber diets (P = 0.002) and between high-fiber and high-oil diets (P = 0.015). However, the floras were not significantly different between the basal and the high-oil diets (P = 0.135), nor were the floras of the 12 individual pigs (each on all three diets) statistically different (P = 0.103). Only 14 of the 160 observed taxa have been detected in the human fecal flora, and only 159 of 1,871 total isolates (8.5%) were members of described species. The most common isolate was a Streptococcus species similar to that reported by Robinson et al. (I. M. Robinson, S. C. Whipp, J. A. Bucklin, and M. J. Allison, Appl. Environ. Microbiol. 48: , 1984), which was found in 34 of 36 samples and which represented 27.5% of all isolates. Lactobacillus, Fusobacterium, Eubacterium, Bacteroides, and Peptostreptococcus species were the next most common bacteria. Escherichia coli represented 1.7% of all fecal isolates, which is somewhat higher than the 0.1 to 0.6% observed in human feces cultured similarly with prereduced anaerobically sterilized media. As in human feces, Bacteroides fragilis was low (36th) on the list of species ranked according to frequency. Epidemiological data indicate that the incidence of colorectal cancers may be related to diet (for reviews, see Van Tassell and Wilkins [19] and Wilkins and Van Tassell [24]). One mechanism by which diet can influence colon cancer and possibly other types of cancer is through production of mutagens and carcinogens by intestinal bacteria (1, 13), particularly some strains of Bacteroides thetaiotaomicron, B. fragilis, B. ovatus, B. uniformis, and B. caccae (18, 19, 24) (formerly designated the Bacteroides sp. strain 3452A homology group [6]). No ideal animal model is available with which to study the effects of diet on microbial production of mutagens in humans. An animal that shows great potential as a model, for physiological and economic reasons, is the pig, as has been proposed by Pertel and Sellers (R. Pertel and R. A. Sellers, Fed. Proc. 45:970, 1986, abstr. no. 4748). There are only a few studies (14-16) that have reported on the species of bacteria in feces or lower intestinal tracts of normal pigs as determined with nonselective medium (16) or by techniques (22) that are adequate to recover obligately anaerobic intestinal bacteria. There has been no previous attempt to correlate the effect of increased amounts of corn oil or wheat bran on the swine fecal flora by using either the anaerobic chamber or roll tube technique and nonselective media. As a basis for future studies, it is critical to have a well-defined data base of the colonic fecal microflora of swine under the influence of various diets which have been shown to be involved with the incidence of colon cancer. The purpose of this study was to provide such a data base. MATERIALS AND METHODS Animals and rations. The pigs were cross-bred females produced from Yorkshire, Hampshire, and Duroc dams mated to purebred Yorkshire or Duroc boars. The pigs were 4 to 6 months of age and weighed 150 to 250 lb (68 to 114 kg) * Corresponding author. when they entered the 1-month quarantine stage of the experiment. During quarantine they were fed the basal diet and housed in the same quarters that were used during the following 6 months of feed trials. Pigs received 2.5 lb (1.135 kg) of diet twice daily. Diets were mixed in 300-lb (136 kg) amounts as needed and stored at room temperature (between 18 and 24 C). The formulas for the three types of diets are given in Table 1, and other characteristics of the diets are given in Table 2. The feeding schedule for the different pairs of pigs is given in Table 3. Media and culture methods. After 2 months on each diet, a stool was obtained from each animal for bacterial culture. Fecal dilutions, isolation, and characterization were as described by Holdeman et al. (4). In brief, immediately after defecation, the entire fecal stool from an animal was placed in a plastic bag. The bag was flushed with carbon dioxide and the stool was kneaded. Approximately 1 g (wet weight) of the mixed feces was placed in a tared tube containing 9 ml of prereduced salts diluent and three or four glass beads (diameter, ca. 3 mm). The tube was shaken to break up the fecal pellet, and 10-fold dilutions were prepared from the resulting suspension. Duplicate tubes of 30% rumen fluidglucose-cellobiose agar (M-RGCA) or 30% pig fecal extractglucose-cellobiose agar (PFE) (4), each with 0.5% peptone added, were inoculated with 107, 108, or 109 dilutions and incubated for 5 days. To prepare pig fecal extract, 1 part feces and 2 parts water (wt/wt) were mixed, filtered through a double layer of cheesecloth, and allowed to settle. The decanted supernatant was autoclaved and clarified by centrifugation. Colonies were counted under x10 magnification, and colonies were picked in a randomized pattern from tubes that contained 30 to 300 colonies. Each resulting culture was restreaked, and colonies with different morphological characteristics were subcultured. Thus, if the original colony was not pure, each different species in the original colony was identified. Subcultures were stained to determine morphological characteristics and purity. Each isolate was subjected

2 VOL. 53, 1987 SWINE FECAL FLORA 1639 TABLE 1. Diet formulations Amt (lb [kg]) in the following diets: Ingredient Basal High-fiber High-oil Ground corn (37.77) (30.71) (24.40) Soybean meal (44% crude protein) (5.90) (2.52) (11.42) Dicalcium phosphate (0.77) (0.25) (1.13) Ground limestone (0.43) (0.67) (0.40) Sodium chloride (0.17) (0.16) (0.21) Trace mineral premixa (0.09) (0.08) (0.11) Vitamin premixb (0.22) (0.20) (0.27) Lysine hydrochloride (0.07) (0.11) None Corn oilc None None (7.47) Wheat bran None (10.69) None a Composition of trace mineral premix, in grams per 100 g, was as follows: FeSO4 7H2O, ; ZnSO4 7H,O ; MnSO4 5H20, 2.191; CuS04 5H,O, 0.702; KI, ; Na2SeO3, ; corn meal (carrier), b Composition of vitamin premix, in amount per 454 g, was as follows: vitamin A (as retinyl palmitate or acetate), 361,193 IU; vitamin D3, 18,060 IU; vitamin E (as a-tocopherol acetate), 903 IU; vitamin K (menadione sodium bisulfite complex), 903 mg; riboflavin, 271 mg; niacin, 903 mg; pantothenic acid (as calcium pantothenate), 1,104 mg; vitamin B12, mg; biotin, 10 mg; thiamin (as thiamin hydrochloride or mononitrate), 100 mg; folic acid, 100 mg; vitamin B6 (as pyridoxine hydrochloride), 100 mg; choline (as choline chloride), g; corn meal carrier to bring the mixture to 1 lb (454 g). c Mazola (Best Foods, CPC International, Inc., Englewood Cliffs, N.J.), 100% food grade. to polyacrylamide gel electrophoresis by the method described by Moore et al. (9). Cultures that gave identical protein banding patterns were assumed to belong to the same species. Cultures that gave different protein banding patterns were characterized by biochemical tests in prereduced media and by chromatographic analysis of short-chain volatile and methylated acids (4). Statistical analyses. Data were analyzed by using the lambda analysis described by Good and co-workers (3, 12) and by using minimum similarity coefficients and random permutations of sample associations. Estimation of the percent coverage by the observed species was by formula Hg described by Good (2), in which [1 - (number of taxa seen once/total number of isolates)] x 100 = percent coverage. The percent cultural recovery of isolates was estimated by comparing the direct microscopic clump count of Gramstained smears (4) with the colony count (under x 10 magnification) of roll tubes that contained between 30 and 300 colonies each. RESULTS The cultural recovery from the fecal samples from two pigs on the fiber diet (pigs 5 and 6, Table 3) was exceptionally low. This low recovery of 0.87 and 2.1% for pigs 5 and 6, respectively, was attributed to inhibitory rumen fluid. Bacteria from the 14 subsequent samples were isolated from medium containing 30% pig fecal extract rather than 30% TABLE 2. Other characteristics of the diets Characteristic Basal High-fiber High-oil Feeding level (kg) Crude protein (g) Digestible energy 7,620 7,094 9,253 (kcal) CP:DE ratio" Calcium (g) Phosphorus (g) Sodium (g) Lysine (g) Total fat (estimated %) Neutral detergent fiber (estimated %) " CP:DE, Crude protein:digestible energy. rumen fluid. Cultural recoveries are given in Table 4. The relatively larger amount of particulate matter in pig feces as compared with human feces probably contributed to a high level of inaccuracy of the direct microscopic clump counts, which gave considerable variation in the percent cultural recovery. The percent coverage (2) of the 36 samples was This means that the 160 species of bacteria isolated accounted for approximately 97% of the cells in the cultivable flora of the feces of these pigs. The remaining 3% of the cells, however, might represent many additional species that occur in very low concentrations. The results of the cultural counts are summarized in Table 5. Counts from PFE cultures were higher than were counts from M-RGCA cultures, but there was no significant difference in cultural counts from feces of pigs fed the three different diets. The fecal flora recovered on the three diets is shown in Tables 6 and 7. Lambda analysis (3, 12) showed that the compositions of the floras of the 12 different pigs (each on three diets) were not significantly different from each other (P = 0.103). However, the bacterial composition of the fecal flora of pigs on basal diet differed from that of pigs on the high-fiber diet (P = 0.002), and the bacterial composition of the fecal flora of pigs on the high-fiber diet differed from that of pigs on the high-oil diet (P = 0.015). The bacterial composition of the fecal flora of pigs on the basal diet was not significantly different from that of pigs on the high-oil diet (P = 0.135). When the two fiber diet samples with low cultural recovery were omitted from the statistical comparison, the flora of pigs on basal diet still differed from the flora of pigs on high-fiber diet (P = 0.010), and the flora of pigs on the high-fiber diet still differed from that of pigs on the TABLE 3. Feeding schedule Diet administered during the following mo: Pig no. 1" and 2 Basal Basal Fiber Oil 3 and 4 Basal Fiber Oil Basal 5 and 6 Basal Basal Oil Fiber 7 and 8 Basal Basal Fiber Oil 9 and 10 Basal Fiber Oil Basal 11 and 12 Basal Basal Oil Fiber " Quarantine period for acclimation to the housing conditions used during the trial.

3 1640 MOORE ET AL. TABLE 4. Percent recovery' No. of Culture % Recovery Sample samples medium Avg Range All 36 Both All except two cultured on 34 Both inhibitory RFb medium Cultured on good RF medium 20 M-RGCA Cultured on PFE medium 14 PFE Basal diet 8 M-RGCA PFE High-fiber diet 8 M-RGCA c M-RGCA PFE High-oil diet 6 M-RGCA PFE a Cultural count divided by direct microscopic clump count from the Gram stain. Phase-contrast microscopic clump counts with a Petroff-Hauser counting chamber yielded comparable counts. For the 10 specimens counted microscopically both ways, the cultural count was 131% of the Gram-stained microscopic count and 111% of the phase microscopic count. b RF, rumen fluid. c Recovery on M-RGCA with the two bad samples omitted. high-oil diet (P = 0.044). The distribution of bacterial species that exceeded 0.5% of the total isolates is shown in Table 6. The concentrations of additional species are listed in Table 7. Of the 1,871 total isolates, 39 Bacteroides species represented 8.2% of the flora; 7 Bifidobacterium species, 3.6%; 6 Butyrivibrio species, 3.0%; Clostridium perfringens, 2.6%; 6 Coprococcus species, 0.6%; Escherichia coli, 1.7%; 5 Streptococcus species, 28.7%; 28 Eubacterium species, 11.7%; 14 Fusobacterium species, 12.0%; 12 Lactobacillus species, 13.6%; 2 Leptotrichia species, 0.2%, 3 Propionibacterium species, 0.2%; 15 Peptostreptococcus species, 4.9%; 1 Ruminococcus species, 0.2%; 8 Selenomonas species, 3.7%; 4 Succinivibrio species, 1.4%; and 8 miscellaneous species, 0.9%. The remainder (3.2%) failed to survive through characterization and identification. DISCUSSION Varel et al. (20, 21) reported a decrease in the total bacterial counts per gram (dry weight) of intestinal contents of pigs fed a high-fiber diet compared with those of pigs on control diets. We did not determine the percent dry matter of the feces. On a wet weight basis, we found no significant difference in the total bacterial counts in feces from pigs on the three diets (Table 5). From the host animal standpoint, it seems to us that the actual bacterial concentration on a wet weight basis bears more relationship to the physiological conditions within the gut than do dry matter calculations that artificially increase or decrease the relative bacterial count. From a bacterial standpoint, the only way that counts from different animals fed different diets could be compared (by either dry matter or wet weight calculations) would be to determine the total fecal output per day so that the total bacterial load or productivity could be compared. Our pigs were on a restricted diet intake, but the effect of this factor on relative counts is difficult to estimate because the total fecal output was not measured. The mean cultural count for 34 samples from the pigs was 9.7 x x 109 (standard error of the mean [SEM])/g (wet weight) which is higher than the mean count for 25 samples from humans which, in medium without added peptone, was 7.8 x x 109 (SEM)/g (wet weight) (5). The percent recovery in the human studies without peptone added to the medium (87 ± 12.8% [SEM]) also was lower than that obtained in this study (95 ± 15.7% [SEMI). In general, the bacteria isolated from the pigs belonged to the same genera as bacteria isolated from humans, but few of the species or the proportions that were observed were the same. Percent coverage was in the range observed in human fecal bacteriological studies (1, 5). Percent cultural recovery was higher than that reported by previous investigators (15, 16) who used similar media and methods (Table 8). Both Salanitro et al. (16) and Robinson et al. (14) reported that streptococci were the predominant bacteria in colon and fecal contents, at 44 to 47% of the flora, which is 15 to 18% more than we found. Salanitro et al. (16) reported more eubacteria than we did (36 versus 12%), but it is possible that they grouped isolates of bifidobacteria with eubacteria. Robinson et al. (14) reported the isolation of about 3 times as many bacteroides as we did (22 versus 8%) and fewer lactobacilli and eubacteria. In this study and those by Salanitro et al. (16) or Robinson et al. (14), the high numbers of peptostreptococci (peptococci) reported by Russell (15) were not confirmed. Russell (15) reported that Peptostreptococcus (Peptococcus) asaccharolyticus (at 8.33% of the flora) was the predominant species. Of these four studies, only that of Russell (15) was done in an anaerobic chamber. It is possible that cultures in the anaerobic chamber did not recover as many of the more fastidious obligately anaerobic bacteria as did cultures in roll tubes (14, 16; this study). However, the more important reasons for the differences between the results presented by Russell (15) and those published subsequently probably are media (rumen fluidglucose-cellobiose without additional peptone supplementation) and incubation time (3 versus 5 to 7 days). After only 3 days of incubation, many of the nonfermentative species may not have attained sufficient colony size to be recognized. This also might account for the lack of isolations of the fastidious fusobacteria and eubacteria in the study described by Russell (15). Other than Streptococcus intestinalis (I. M. Robinson, J. M. Stromley, V. H. Varel, and E. P. Cato, Int. J. Syst. Bacteriol., submitted), which is group U-2 in the study by Robinson et al. (14), none of our isolates had characteristics of the species groups described by Robinson et al. (14). Strains in our Bacteroides P01 and P03 groups are similar to the Bacteroides ruminicola type 1 strains described by Medium TABLE 5. APPL. ENVIRON. MICROBIOL. Cultural counts on M-RGCA medium and PFE medium' Cultural counts (109 + SEM/g [wet wt]) from samples of pigs fed the following diets: Basal (N) High-fiber (N) High-oil (N) M-RGCA Mean (8) 6.8 ± 1.7 (6) 7.0 ± 2.1 (6) Range (8) (6) (6) PFE Mean 13.8 ± 5.0 (4) 11.2 ± 1.8 (4) 13.1 ± 2.1 (6) Range (4) (4) (6) a Average for 20 samples cultured with M-RGCA was 7.5 x x 109; average for 14 samples cultured with PFE = 12.8 x 109 ± 1.65 x 109.

4 VOL. 53, 1987 SWINE FECAL FLORA 1641 TABLE 6. Predominant bacteria of swine feces % of 36 % + SEM of isolates from pigs on the following diet": Taxon Rank" samples Total High-fiber High-oil positive (1.871)' Basal (639)' (613)' (619)' Streptococcus intestinalisd ± ± ± Lactobacillus P01" ± ± ± ± 4.7 Eubacteriumn P02' ± ± ± ±3.6 Fusobacterium P02f ± ± ± ± 1.0 Did not survive to be identified ± ± ± 1.1 Peptostreptococcus POlf ± ± Clostridium perfringens ± Fusobacterium P03f ± ± ± ± 0.6 Eubacterium POlf ± ± ± ± 1.4 Bifidobacterium thermophilum ± ± ± ± 0.4 Selenomonas P ± ± ± ± 1.5 Lactobacillus P ± ± ± Escherichia coli ± ± ± ± 0.3 Fusobacterium P01f ± ± ± ± 0.2 Bacteroides P0Sf ± ± ± ± 0.6 Peptostreptococcus P03' ± ± ± ± 0.7 Fusobacterium P ± ± ± ± 0.6 Bifidobacterium boum ± ± ± ± 0.4 Succinivibrio P ± ± ± 2.4 Bacteroides P04f ± ± ± ± 0.5 Fusobacterium P05f ± ± ± ± 0.6 Selenomonas P ± ± ± Streptococcus P ± ± ± Bacteroides P09f ± ± ± 0.2 Butyrivibrio P03f ± ± 0.2 Butyrivibrio P02f ± ± ± Eubacterium Pll ± ± ± 0.4 Butyrivibrio P ± ± Bacteroides P ± ± Eubacterium P ± ± ± 0.2 Fusobacterium P06f ± ± Lactobacillus P ± ± ± ± 0.3 Butyrivibrio P ± ± ± ± 0.5 " Ranked as a percentage of 1,871 total isolates. btotal sample number = 36, number of pigs on basal diet = 12, number of pigs on fiber diet = 12. number of pigs on oil diet = 12. Number of isolates. d Group U-2 described by Robinson et al. (14; submitted). e Undescribed species are designated by P and two numbers. J Species that did not ferment carbohydrates. Terada et al. (17), and our isolates of Bacteroides RP are fermented by oiiur isolates. Mitsuoka (8) has reported that L. similar to their B. ruminicola type 2 isolates. None of our fermentum IVb is the predominant lactobacillus from swine, isolates had characteristics of the B. ruminicola type 3, 4, or followed by L. acidophilus groups VIa and IVb. The groupby Mitsuoka (8) were done on the basis of 5 groups reported by Terada et al. (17). Varel et al. (21) ings described reported an increase in xylanolytic and cellulolytic bacteria morphological aand biochemical characteristics, as compared from feces of adult pigs on a high-fiber (40% alfalfa meal) with type or refference strains. Subsequently, Johnson et al. diet. They identified three strains of xylanolytic bacteria that (7) reported ornly negligible (21 to 29%) DNA homology closely resembled B. ruminicola. Five of our saccharolytic between most of the isolates from hogs labeled L. species groups of Bacteroides fermented xylan: Bacteroides acidophilus thaat they studied and the type strain of L. RP (0.37% of isolates), Bacteroides P03 (0.43% of isolates), acidophilus. Ncone of our isolates of lactobacilli had characto those described by Ward et al. (23) for the Bacteroides P07 (0.06% of isolates), Bacteroides P21 (0.11% teristics similar of isolates), and Bacteroides P33 (0.15% of isolates). Of strain isolated lfrom swine feces that decarboxylated paraacetic acid. these, only Bacteroides P03 increased in concentration in the hydroxyphenyl; high-fiber over basal diet (0.81 versus 0.32% of flora), and At the speci ies level, the flora of the pigs was grossly only Bacteroides P03 was detected in feces from pigs on the different from tthat of humans. Few of the species found in high-oil diet (0.16%). humans (5, 10 )) were found in the pigs (Table 9). The Our isolates of Lactobacillus P01 were most similar to the predominant sppecies in humans are primarily fermentative Lactobacillus acidophilus IVb or VIa groups described by species. Of thee 35 most numerous species in one human Mitsuoka (8). Our Lactobacillus P05 group corresponds to study (5), 29 Nwere fermentative, and 33 of the 34 most Lactobacillus fermentum ("fermenti") group IVb described numerous spec ies in another study were fermentative (10). by Mitsuoka (8), except that melibiose and xylose were not Predominant feermentative species in both human studies

5 1642 MOORE ET AL. TABLE 7. Minority flora of 36 pigs on three different diets Rank" Concn Taxon (± SEM of concn)" Bacteroides P03 (±+0.18), Bifidohacteriumn P02 (+0.24) Eubacteiiun P15 (+0.21) Bacteroides fiagilis (±+ 0.20), EubacteIriumi P03 (+0.20) Bacteroides RP (±0.20), Fuisobacteriumn P11 (±0.17) Selenoinonas P07 (±0.22) Eubactrium P26 (±0.17) Bacteroides P08 (±+0.14), Butrit'ibrio P05 (±0.14), Eubacteriin P12 (±0.12), Fu.sobacterium P04 (±+ 0.11) Selenomonas P05 (±0.19) Anaerovibrio P01 (±0.13), Bacteroides P11 (±0.11), Bacteroides P28 (±0.10), Lactobacillus P03 (±0.10), Peptostreptococcuis P11 (±0.13) Facultative positive rod P04 (±0.21), Lactobacillus P08 (±0.21) Bacteroides P01 (±0.10), Bacteroides P18 (±0.10) Selenomontis P08 (±0.19) Bacteroides P06 (±0.09), Eubacteriumn P14 (±0.09), Eubaccteiiun P25 (±0.17), Fusobacteriuin P08 (±0.09) Bacteroides P10 (±0.09), Bacteroides P12 (±0.09), Bacteiroides P15 (±0.16), Bac teroides P20 (±0.09), Bacteroides P30 (±0.12), Strepto- COCCUS P04 (±0.12), Eubacterium P13 (±0.12), Eubacteriuln P18 (±0.09), Fusobacterilmn praiusnitzii (± 0.09), Peptostreptococcus P04 (±0.09), Rurminococcius P05 (±0.09), Suicciniv,ibrio P03 (±0.09), Succinit ibrio P04 (±0.17) Coprococcus P02 (±0.09), Coprococcus P03 (±0.08) Bacteroides P16 (±0.08), Bacteroides P21 (±0.07), Bacteroides P26 (±0.07), Bacteroides P31 (±0.08), Enterococcus hirae (±0.08), Eubacteriuon P04 (±0.08), Eubacteri,mn P05 (±0.08), Eubacterium P06 (±0.11), Eubacteriuin P07 (±0.08), Fiusob(icteriumn P12 (±0.08), Fusobacterium P13 (±0.07). Lactobacillus P07 (±0.08), Propionibacteriumn acnes (±0.08), Selenoionaos P03 (±0.08), Streptococculs boriis (±0.11), Succinizibrio P01 (±0.08) Bacteroides P14 (±0.07), Bacteroides P23 (±0.07), Coprococcus P04 (±0.07), Coproc occus P06 (±0.07), Leptotrichia P01 (±0.10), Peptostreptococcus P09 (+0.07), Peptostreptococcus P10 (±0.07) Bacteroides uniformis (±0.06), Bacteroides P07 (±0.06), Bacteroides P35 (±0.06), Bifidobacterium choerinum (±0.06), Bifidobacterium P01 (±0.06), Eubacterium P08 (±0.06), Eubacterium P16 (±0.06), Eubacterium P19 (±0.06), Eubacteriumn P23 (±0.06), Lactobacillus fermentum (±0.06), Peptostreptococcus P02 (±0.06), Peptostreptococcus P06 (±0.06), Peptostreptococcus P07 (±0.06), Peptostreptococcus P16 (± 0.06), Selenomonas P04 (±0.06) Bacteroides P02 (±0.06), Bacteroides P13 (±0.05), Bacteroides P17 (±0.05), Bacteroides P19 (±0.05), Bacteroides P22 (±0.05), Bacteroides P27 (±0.06), Bacteroides P29 (±0.05), Bacteroides P32 (±0.05), Bacteroides TABLE 7-Continued APPL. ENVIRON. MICROBIOL. Rank' Concn Taxon (t SEM of concn)' (%_ P33 (±0.05), Bacteroides P34 (±0.06), Bacteroides P36 (±0.06), Bacteroides P37 (±0.06), Bifidobacterium catenulatum (±0.05), Bifidobacterium pseudolongum (+0.06), Butyrivibrio P06 (±0.05), Coprococcus P01 (±0.05), Coprococcus P07 (±0.06), Streptococcus P02 (±0.06), Eubacterium P10 (±0.05), Eubacterium P17 (±0.05), Eubacterium P20 (±0.05), Eubacterium P21 (±0.06), Eubacterium P22 (±0.05), Eubacterium P24 (±0.06), Eubacterium P27 (±0.06), Eubacterium P28 (±0.06), facultative positive rod P01 (±0.05), Facultative positive rod P02 (±0.06), facultative positive rod P05 (±0.06), Fusobacterium P10 (±0.05), Fusobacteriumn P14 (±0.06), Lactobacillus buchneri (±0.05), Lactobacillus plantarum (±0.05), Lactobacillus P04 (±0.05), Lactobacillus P06 (±0.05), Lactobacillus P10 (±0.06), Leptotrichia P02 (±0.06), Peptostreptococcus P08 (±0.06), Peptostreptococcus P12 (±0.06), Peptostreptococcus P13 (±0.06), Peptostreptococcus P14 (±0.06), Peptostreptococcus P15 (±0.06), Propionibacterium granulosum (±0.05), Propionibacterium P01 (±0.06), Selenoinonas P06 (±0.05), Staphylococcus warneri (±0.06), Veillonella dispar (±0.06) "Ranked as a percentage of 1,871 total isolates. Undescribed species are designated by P followed by two numbers. included Bacteroides vulgatus, Bifidobacterium adolescentis, Eubacterium aerofaciens, Peptostreptococcus productus, Bacteroides uniformis, Bacteroides thetaiotaomicron, and Eubacterium rectale. Fewer (17 of 33) of the predominant species in pigs were fermentative (Table 6). There were slightly more gram-positive isolates from pigs (66%) than from humans (59%), but fewer than the report (16) of 90% gram-positive isolates from swine fecal flora. It is somewhat disconcerting that the flora of pigs appears to be so different from that observed in humans. This observation is further evidence that each animal species carries a flora distinct from that found in other animals, with little overlap in flora composition. Not only are many species of the human flora undescribed but there is increasing evidence that most of the flora of our domestic animals also is not described. Distinct bacterial species in different animals might be a product of natural selection, which provides protection for different animals. The fewer the bacterial species that different animals share, the less the likelihood of disease transmission between animal species. The lack of similarity of floras raises concern about the suitability of animals as models for human intestinal disease. Bacteroides thetaiotaomicron, B. fragilis, B. ovatus, and B. caccae of the human flora are stimulated by bile and are known to produce fecapentaene mutagens (18). Of this group, only B. fragilis was detected in the pigs, but its concentration did not increase in pigs on the high-oil diet; rather, it increased in pigs on the high-fiber diet. The high-oil diet should have stimulated bile flow in the pigs, but corn oil may not be as effective as animal fat would have been.

6 VOL. 53, 1987 SWINE FECAL FLORA 1643 TABLE 8. Comparison of results with those reported previously Results of': Characteristic Present Salanit Roboinso study et al. et al14) Russell (15) (16) eta No. of pigs Area sampled Feces Feces Proximal Large colon intestine Incubation time (days) No. of colonies NR NR picked/sample % Recovery NR 56 No. of isolates 1, Bacteria isolated (%) Streptococci Lactobacilli Eubacteria 12 36b 6 26 Fusobacteria Bacteroides Peptostreptococci Bifidobacteria 4 ob 0 2 Selenomonads Clostridia Butyrivibrios <1 Escherichia Ruminococci < Succinivibrios Leptotrichia < Propionibacteria < Coprococci < Veillonellae < Megasphaera a Abbreviations: NR, Not reported;+, less than 7.8% of the flora. b Some bifidobacteria may have been grouped with the eubacteria. Because some epidemiological data suggest that animal fat intake correlates with colon cancer, animal fat rather than vegetable oil might have been the preferred source of fat for TABLE 9. this study. For the current experiment, however, it was impractical to mix the diet with lard or other animal fat as a major constituent. If the flora of the pig, albeit composed of different bacterial species, functions in a manner similar to that in humans, the statistically significant change in flora composition between basal and high-fiber diets might indicate that coarse fiber itself, rather than the simple lack of fat, causes a change in the composition of the flora. Further study may be warranted to examine this effect. The low cultural recovery from the two fecal samples from pigs on the fiber diet probably was caused by an unusual sample of rumen fluid that was used to make the isolation medium. The striking effect on the composition of the flora as detected by the isolation medium is evident from the effect of those samples on the statistical analyses. B. fragilis was detected in those two samples with low cultural recovery from fecal samples from pigs on the fiber diet (2 and 6% of the flora) and also in two samples from other pigs (2% of the flora in one fed fiber and 4% of the flora in one fed the basal diet). Lactobacillus P09 represented 28 to 32% of the isolates on each of the two low-recovery fiber diet fecal samples and was only found (8% of the flora) in one other sample from another pig on the basal diet. These results suggest that the inhibitory rumen fluid medium may have been somewhat selective, and therefore, the results of those two samples may not be truly representative of the flora composition. Although no medium duplicates the environment of a mixed flora, in which there is often interdependence of species, results with higher percent recoveries provide the more reliable estimate of flora composition. In a comparison of the flora of two fecal samples following the failure of M-RGCA, PFE produced about 20% higher counts than M-RGCA made with a different lot of rumen fluid. Replacement of the rumen fluid with pig fecal extract generally improved cultural recovery (Table 5). Salanitro et al. (16) have reported that hog cecal extract in place of rumen fluid increased recovery by 4.5% but that counts were not significantly different from those obtained with the M98.5 medium used in their studies. Comparison with species that have been reported to be in human fecal flora Taxon % of 36 samples % of isolates from pigs with the following diets: (no. of isolates): Rank in: positive Total (1,871)' Basal (639)" High-fiber (613)' High-oil (619)' Humansb Pigs Bacteroides fragilis Bacteroides uniformis Bifidobacterium catenulatum > Clostridium perfringens >113 6 Escherichia coli Fusobacterium prausnitzii Lactobacillus buchneri > Lactobacillus fermentum > Lactobacillus plantarum > Propionibacterium acnes Propionibacterium > granulosum Streptococcus bovis > Staphylococcus warneri > Veillonella dispar ? a Number of isolates. b Data from references 5 and 10 and unpublished data (W. E. C. Moore, L. V. H. Moore, and E. P. Cato). c Differentiation of Veillonella parvula from Veillonella dispar was not possible when the human intestinal data were collected. However, because Veillonella dispar is occasionally isolated from the human gingival crevice (11), it, along with Veillonella parvula, probably does occur in the human fecal flora.

7 1644 MOORE ET AL. ACKNOWLEDGMENTS This research was supported by contract from the U.S. Food and Drug Administration administered by Booz, Allen, and Hamilton, Inc. We gratefully acknowledge the help of Pauletta C. Atkins and Dianne M. Wall in the bacteriological studies, Ann P. Donnelly for chromatographic and electrophoretic analyses, Sarah Owens for the care and feeding of animals, Roger Cook for the collection of fecal specimens, Robert Moore for diet formulation, and Faye Smith for technical support. LITERATURE CITED 1. Bruce, W. R., A. J. Varghese, A. J. Furrer, and P. C. Land A mutagen in the feces of normal humans. Cold Spring Harbor Conf. Cell Proliferation 4: Good, I. J The population frequencies of species and the estimation of population parameters. Biometrika 40: Good, I. J An index of separateness of clusters and a permutation test for its significance. J. Stat. Comp. Sim. 15: Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.) Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, Va. 5. Holdeman, L. V., I. J. Good, and W. E. C. Moore Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl. Environ. Microbiol. 31: Johnson, J. L., W. E. C. Moore, and L. V. H. Moore Bacteroides caccae, sp. nov., Bacteroides merdae sp. nov., and Bacteroides stercoris, sp. nov. isolated from human feces. Int. J. Syst. Bacteriol. 36: Johnson, J. L., C. F. Phelps, C. S. Cummins, J. London, and F. Gasser. i980. Taxonomy of the Lactobacillus acidophilus group. Int. J. Syst. Bacteriol. 30: Mitsuoka, T Vergleichende Untersuchungen uber die Laktobazillen aus den Faeces von Menschen, Schweinen und Huhnern. Zentralbl. BakterioJ. Parasitenk. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 210: Moore, W. E. C., D. E. Hash, L. V. Holdeman, and E. P. Cato Polyacrylamide slab gel electrophoresis of soluble proteins for studies of bacterial floras. Appl. Environ. Microbiol. 39: Moore, W. E. C., and L. V. Holdeman Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl. Microbiol. 27: Moore, W. E. C., L. V. Holdeman, E. P. Cato, R. M. Smibert, APPL. ENVIRON. MICROBIOL. J. A. 1urmeister, K. G. Palcanis, and R. R. Ranney Comnparative bacteriology of juvenile periodontitis. Infect. Immun. 48: Moore, W. E. C., L. V. Holdeman, R. M. Smibert, I. J. Good, J. A. Burmeister, K. G. Palcanis, and R. R. Ranney Bacteriology of experimental gingivitis in young adult humans. Infect. Immun. 38: Reddy, B. S Bile salts and other constituents of the colon as tumor promoters. Banburry Rpt. 7: Robinson, I. M., S. C. Whipp, J. A. Bucklin, and M. J. Allison Characterization of predominant bacteria from the colons of normal and dysenteric pigs. Appl. Environ. Microbiol. 48: Russell, E. G Types and distribution of anaerobic bacteria in the large intestine of pigs. Appl. Environ. Microbiol. 37: Salanitro, J. P., I. G. Blake, and P. A. Muirhead Isoiation and identification of fecal bacteria from adult swine. Appl. Environ. Microbiol. 33: Terada, A., K. Uchida, and T. Mitsuoka Die Bacteriodaceenflora in den Faeces von Schweinen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 234: Van Tassell, R. L., R. M. Schram, and T. D. Wilkins Microbial biosynthesis of fecapentaenes, p In I. Knudsen (ed.), Genetic toxicology of the diet. Alan R. Liss, Inc., New York. 19. Van Tassell, R. L., and T. D. Wilkins The precursors of fecapentaenes. A preliminary report. Ann. Ist. Super. SanitA 22: Varel, V. H., W. G. Pond, J. C. Pekas, and J. T. Yen Influence of high-fiber diet on bacterial populations in intestinal tracts of obese- and lean-genotype pigs. Appl. Environ. Microbiol. 44: Varel, V. H., I. M. Robinson, and H.-J. G. Jung Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs. Appl. Environ. Microbiol. 53: Vervaeke, I. J., and C. J. Van Nevel Comparison of three techniques for the total count of anaerobes from intestinal contents of pigs. Appl. Microbiol. 24: Ward, L. A., K. A. Johnson, I. M. Robinson, and M. T. Yokoyama Isolation from swine feces of a bacterium which decarboxylates p-hydroxyphenylacetic acid to 4- methylphenol (p-cresol). Appl. Environ. Microbiol. 53: Wilkins, T. D., and R. L. Van Tassell Production of intestinal mutagens, p In D. Hentges (ed.), Human intestinal microflora in health and disease. Academic Press, Inc., New York.