Dietary Effects on the Composition of Fecal Flora of Rats

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1977, p Copyright X) 1977 American Society for Microbiology Vol. 33, No. 3 Printed in U.S.A. Dietary Effects on the Composition of Fecal Flora of Rats KING-THOM CHUNG,* GEORGE E. FULK, AND SIDNEY J. SILVERMAN' Frederick Cancer Research Center, National Cancer Institute, Frederick, Maryland Received for publication 30 August 1976 A long-term animal feeding experiment was conducted to compare the effect of meat and Wayne laboratory chow diets on the composition of rat fecal flora. Fecal bacteria were enumerated on selective media under both aerobic and anaerobic conditions. Intrarectal administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) affected the count only on the phenylethylalcohol and veillonella-neomycin agars, whereas a slightly higher number of anaerobes appeared in the feces of rats that were treated with MNNG as compared with those obtained in the feces of untreated rats on the meat diet. In the absence of MNNG, feces of meat-fed rats yielded higher bacterial counts on aerobically incubated MacConkey agar, deoxycholate agar, and Pfizer selective enterococcus agar as well as higher numbers of clostridia on anaerobic egg yolk agar than did feces of rats on the Wayne diet. Feces of the group fed the Wayne diet produced more colonies on aerobic mitis-salivarius agar and lactobacillus agar as well as on anaerobically incubated phenylethylalcohol agar, veillonellaneomycin agar, bifidobacteria agar, fusobacterium (Nissui) agar, and kanamycin-vancomycin blood agar. These differences were consistent throughout the 1- year feeding period. It has been postulated that colon cancer may be induced by environmental factors (1, 7, 17, 18). The factor most common in countries with a high incidence of the disease is that large quantities of beef are consumed (4, 6). It has been postulated that the effects of diet on colon carcinogenesis may be mediated through changes in the composition of intestinal microflora and that specific microflora may convert dietary components into carcinogens and/or cocarcinogens (12). Some studies have indicated that patients with colon cancer may have different microflora in their intestinal tracts as compared with normal healthy individuals (8). It is not known whether diet and/or the disease affect the composition of the microflora. An animal model system was employed to test the effects of beef on fecal flora of rats. Although N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is known as a direct-acting carcinogen (12), its effects on the microflora in the gut are unclear. The objective of this study was to determine the effects of beef and MNNG on the composition of fecal microbes of rats. MATERIALS AND METHODS Animals and preparation of diets. Six-week-old male and female Fischer rats (F-344/N) were used 1 Present address: Department of Biology, Hood College, Frederick, MD after a 2-week quarantine period. Animals were housed four per cage, and cages were changed twice a week. Acidified water (ph 2.5) was available continuously from bottles. Normal Wayne diet was offered three times a week; meat diet was offered fresh daily in sanitized glass food jars (3/4 full) before 4:00 p.m. The autoclaved beef and fat diet with supplements was prepared as follows. A 44.7-kg amount of meat, consisting of 25% fat and 75% lean ground beef (head meat), was prepared weekly. The meat was divided into 10 batches of 4.47 kg each, autoclaved at 1210 C for 30 min, and allowed to cool at 6 C for about 2 h. A mineral mixture and vitamin mixture were added to give a vitamin fortification per kilogram of cooked meat of 5,000 IU of vitamin A, 750 IU of vitamin D, 25 mg of vitamin E, mg of vitamin K, 450 mg of choline chloride, 12.5 mg of niacin, 7.5 mg of calcium pantothenate, 2.5 mg of riboflavin, 6 mg of thiamine, 0.25 mg of biotin, 1.0 mg of folic acid, 15 mg of vitamin B,2, and 2.0 mg of pyridoxine. The mineral fortification per kilogram of cooked meat was 16.0 g of dicalcium phosphate, 4 mg of copper (copper sulfate), 0.15 mg of iodine (potassium iodate), 12.5 mg of iron (iron sulfate), 0.5 g of magnesium (magnesium oxide), 37.5 mg of manganese (manganous oxide), 1.0 g of sodium (sodium chloride), and 12.5 mg of zinc (zinc oxide). Each batch of fortified meat was stored in appropriate amounts in plastic bags at - 10 C until time of use. The Wayne diet chow was called Wayne Sterilizable Lab-Blox Meal. It contains corn and wheat flakes, wheat middlings, soy bean meal, ground corn, fish meal, brewers' dried yeast, soybean oil, animal liver meal, vitamin A palmitate, D-activated 654

2 VOL. 33, 1977 animal sterol, vitamin E supplement, menadione sodium bisulfite, riboflavin supplement, niacin, calcium pantothenate, choline chloride, thiamine, and minerals. The MNGG (8 mg/ml) was suspended in a 1% aqueous solution of sodium carboxymethylcellulose in 0.1 M acetate buffer (ph 5.5). The meat and normal diet feedings were started when the animals were 6 weeks old. A total of 100 males and 100 females were employed in each dietary group. After 1 month one-half of the males and one-half of the females from each group were given a single rectal instillation of 0.5 ml of the MNNG solution. Animals were lightly anesthetized with ether, and an intubation needle was inserted into the rectum. Sampling. All animals were housed in a building that was specially equipped for animal experiments (i.e., proper air flow and filtration, precautions against contamination, etc.). Animal caretakers, technicians, or anyone entering the animal holding area had to take a shower and dress in a special suit, including cap, face mask, and shoe covers, to minimize the possibility of contamination. Tables 2 and 4 show culture counts of fecal samples collected at approximately 3-month intervals. Two samples were collected in March-April, two samples in June-July, and two samples in October- November periods (1975). In other cases, the exact dates of sampling were indicated in each table. Fecal samples were collected by transferring each rat to a clean cage and lifting the rat by its tail to induce defecation. At least one fresh pellet was collected with sterile forceps from each rat and immediately put into a small sterile jar gassed with carbon dioxide prior to closing with a rubber stopper. Fresh pellets from at least eight rats (one-half male, one-half female) on each diet were combined and transferred to the laboratory within 30 to 60 min. One gram of fecal pellets was put into 99 ml of prereduced diluent (9) in an Eberbach semi-microblending container mounted on a Waring blender, gassed with a heavy flow of CO2, and blended for 5 min. After blending, 1 ml of fecal suspension was anaerobically transferred into 9 ml of diluent and serially diluted to A 0.5-ml amount of dilutions 10-6 to 10-1 was inoculated into roll tubes containing 4.5 ml of appropriate agar media. The aerobic agar plates were inoculated with 0.1 ml from dilutions of 10-2 to 10-5 and/or The original fecal suspensions were examined microscopically, and direct counts were obtained by using a Petroff-Hausser chamber. Dry weight determination. Fecal dry weights were determined by a previously published method (3) Ṁedia. Dehydrated agar media, including Pfizer selective enterococcus (PSE), mitis-salivarius (Difco), lactobacillus (BBL), and MacConkey (BBL), were prepared according to the directions of the manufacturers. Blood agar base (Difco) to which 5% fresh sheep blood was added was used for the enumeration of total facultative organisms. Fresh sheep blood was obtained from the animal farm at the Frederick Cancer Research Center. For DIETARY EFFECTS ON FECAL FLORA OF RATS 655 anaerobic media, blood was aseptically pipetted into a sterilized tube that was gassed with 02-free CO2 and closed with a sterile rubber stopper. Sodium deoxycholate agar was prepared by adding 5 mg of hemin and 15 g of agar to 900 ml of thioglycolate broth fbbl). A total of 100 ml of 1.0% sodium deoxycholate (Difco) in water was sterilized by membrane filtration (0.45 Mm; Millipore Corp.) and added to the medium aseptically before the agar was dispensed into plates. Rumen fluid-glucose-cellobiose agar (RGCA) was prepared by the procedure of Holdeman and Moore (9). The medium contained 1% NaHCO3 rather than 0.1% as recommended by these authors. Wadsworth Anaerobe Laboratory all-purpose blood agar medium, prereduced and anaerobically sterilized (WAL APM PRAS), and the differential media (phenylethylalcohol agar [BBL], bifidobacterium agar, veillonella-neomycin agar, and kanamycin-vancomycin blood agar) were prepared by the method of Sutter et al. (15). Fusobacterium (Nissui- Seiyaku) agar was prepared as directed on the bottle, excepting the addition of 1% NaHCO3, and sterilized by autoclaving. All anaerobic media were prepared as anaerobic roll tubes (9). Kanamycin was included before autoclaving, whereas neomycin (10 mg/ml) and vancomycin 375 (,ug/ml) were dissolved in 02-free deionized water and sterilized by membrane filtration (0.45,m; Millipore Corp.) anaerobically. Neomycin (0.05 ml) and 0.1 ml of vancomycin were injected through the rubber stoppers with a hypodermic syringe into the veillonella-neomycin agar (4.45 ml) and kanamycinvancomycin blood agar (4.4 ml), respectively. The agar tubes were melted and placed in a 47 C water bath before the antibiotics were injected. The composition of egg yolk agar was according to Sutter et al. (15), but an anaerobic roll tube procedure was employed (9). A 0.5-ml amount of egg yolk enrichment (BBL) was injected through the rubber stopper with a hypodermic syringe into a culture tube containing 4.0 ml of egg yolk agar. The inoculum for egg yolk agar was heated at 80 C for 5 min before inoculation. Incubation. All plates and roll tubes were incubated at 37 C. Colonies on aerobic plates were obtained within 1 to 3 days; those on anaerobic roll tubes were counted within 5 to 10 days. The results given were the average of duplicate tubes or plates that showed 25 to 300 colonies. Statistical analyses. Where appropriate, the data were analyzed by the analysis of variance that examined the effects of time, type of diet, and presence or absence of MNNG. In Tables 1, 2, and 4 no consistent trends over time were discernible; therefore, the culture counts of each medium were grouped together. The individual means of diet-mnng combination (i.e., meat alone, Wayne alone, meat and MNNG, and Wayne and MNNG) were compared and tested by the Least Significant Difference. These results are presented in each table. RESULTS The studies revealed that there was no difference in the moisture content of the feces of rats

3 656 CHUNG, FULK, AND SILVERMAN on the two diets. One gram of wet feces contained dry materials averaging 0.27 g in animals on the beef and fat diet, 0.26 g in the animals on the Wayne diet, 0.26 g in animals on the beef and fat diet treated with MNNG, and 0.26 g in animals on the Wayne diet treated with MNNG. The total counts of fecal bacteria are shown in Table 1. The direct microscopic counts of the fecal microflora averaged 2.8 x 1011/g of dry weight. There were no significant differences in total numbers of anaerobic organisms between the feces of rats on either diet. Colony counts obtained on the nonselective all-purpose medium showed that an average of only 16% of the direct count could be cultivated on WAL APM PRAS medium, whereas 24 to 43% could be isolated on RGCA medium. More colonies were obtained from the feces of rats on the Wayne diet than from the feces of those on the meat diet. Sabouraud dextrose agar with chloramphenicol (50,g/ml) was used for the cultivation of yeasts and molds (15). The results of four experiments did not show a single colony of yeast or mold appearing in the 10-3 dilution. Therefore, if present, they constitute a very small fraction (<103/g) of the total fecal microflora of these animals. It was decided to discontinue work on the enumeration of yeasts and molds. Colony counts of various types of bacteria are recorded in Tables 2 through 5. The bacterial population appeared to be affected by MNNG only as indicated by results with the. phenylethylalcohol agar and veillonella-neomycin agar, which are reported to be selective for gram-positive and -negative cocci (15). Growth on blood agar plates (Table 2) indicated that facultative microbes constituted 0.5% of the total direct count. No significant difference was found in numbers of facultative organisms in the feces of rats on either of the two diets. Recovery of enteric bacteria was consistently 1 log greater from feces of rats on the TABLE 1. meat diet than from feces of rats on the Wayne diet. This was noted on both sodium deoxycholate agar and MacConkey agar. Table 2 also shows the number of group D streptococci colonies grown on PSE agar (14). The count was about 1 log greater on cultures of feces from meat-fed than from chow-fed rats. On mitissalivarius agar, however, more colonies were obtained from the Wayne diet-fed rats. It is interesting to note that the combined counts of streptococci from feces ofrats on the Wayne diet ranged from 4.3 x 109 to 4.5 x 109/g of dry weight, about 1.6% of the total direct count, and are greater than those in the feces of rats on the meat diet. The most significant finding among the facultative aerobes was observed in the difference in the occurrence of lactobacilli in the two groups of animals. Cultures of feces from Wayne diet-fed animals consistently yielded 107 to 108 colonies/g of dry weight on lactobacillus agar. Those of the beef-diet group were initially 2 logs lower and, during the 11 months of culturing, dropped to less than 104 colonies/g ofdry weight of feces (Table 3). The colony counts of strict anaerobes on selective media are shown in Table 4. Total counts of anaerobes on RGCA as well as those anaerobes grown on the phenylethylalcohol agar, veillonella agar, bifidobacteria agar, Nissui-fusobacterium medium, and kanamycinvancomycin blood agar appeared to be slightly, but significantly, higher in the feces of rats on the Wayne diet than those in the feces of rats on the beef diet (in the absence of MNNG). To determine the validity of phenylethylalcohol agar for the isolation of anaerobic cocci, colonies were picked at random from a roll tube inoculated with a high fecal dilution. Gram stains of 24-h cultures were examined for cell morphology. Of the 106 colonies picked from cultures of both groups of rats, all but 13 were gram-positive cocci. The thirteen colonies were gram-positive, rod-shaped organisms. On this Comparison of direct and culture counts of fecal microbes of ratsa APPL. ENVIRON. MICROBIOL. Counting method Medium Type of diet Counts/g of dry wt % (log,,) Direct Meat 11.4 ± 0.03c 100 Wayne 11.4 ± Culture RGCA Meat 10.8 ± Wayne 11.1 ± Culture WAL APM PRAS Meat Wayne a Rats were not treated with MNNG. b Counts per gram of dry weight are the averages of four determinations that were carried out on 23, 24, and 30 January 1975 and 3 February e Standard error.

4 VOL. 33, 1977 TABLE 2. DIETARY EFFECTS ON FECAL FLORA OF RATS 657 Culture counts of aerobes in the feces of rats Culture counts ± standard error (log1o) (per g of dry wt) Medium Major organism Diet MNNG Absent Present Blood agar Facultatives and aerobes Meat a 8.9 ± 0.2 Wayne ± 0.1 MacConkey agar Enterics Meat 7.8 ± ± 0.1 **b ** Sodium deoxycholate Enterics Wayne Meat 6.9 ± ± ± ** ** Wayne 6.8 ± ± 0.2 PSE Streptococci type D Meat **~~~~~~~~ Mitis-salivarius Streptococci Wayne Meat 7.8 ± ± ± ± 0.2 ** ** Wayne 9.6 ± ± 0.1 a, No significant differences. b Significant differences at the significance level < TABLE 3. Culture counts on lactobacillus agar plates incubated aerobically Sample date Culture counts (log10) (per g of dry wt) Meat Wayne Meat+ Wayne + MNNG MNNG 1/ NDa ND 1/ ND ND 2/ ND ND 3/ / /24 < < / /22 < < / / Average SEb a ND, Not determined. b SE, Standard error. basis, phenylethylalcohol agar was considered to be satisfactory for the comparative study. The results show a significantly higher count of gram-positive cocci in the feces of rats on the Wayne diet. The number of gram-positive anaerobic cocci represented 29.6% of the direct microscopic count, making these the predominant organisms in the feces of this group. The number of clostridia found in the feces of rats on the meat diet was greater than that observed in the feces of animals on the Wayne diet and remained fairly constant over the time period of the study. The number of anaerobic sporeformers in the feces of the latter group dropped about 1 log during the 11 months of study. DISCUSSION Although the differences were statistically significant in culture counts on many differential media, errors involved in this type of study involving dilutions of 9 logs might raise a question as to whether the statistical differences are biologically significant. The most obvious differences in the fecal flora of rats on the two diets were detected on egg yolk agar and kanamycin-vancomycin blood agar incubated anaerobically and aerobically on lactobacillus agar, sodium deoxycholate, MacConkey, and PSE agars. Those differences that were about 1 log or more can be considered significant, even without statistical analyses. Diet had a significant influence on the composition of fecal flora of the two groups. The most impressive differences were with clostridia and lactobacilli. A meat diet contains a high concentration of protein; it is unlikely that all of the oligopeptides and amino acids are absorbed completely by the upper part of the gastrointestinal tract, so some protein and amino acids are carried over into the colon. In this respect, it has been shown that feces of rats on a lean-meat diet contain a higher tryptophan concentration than feces of rats on a chow diet (3). Since clostridia subsequently isolated from feces of rats on a meat diet are proteolytic (K.-T. Chung, G. E. Fulk, and K. Fowler, unpublished data), their presence in higher numbers in feces of animals on a meat diet is perhaps explained by the availability of more protein and/or amino acids. Lactobacilli were more abundant in the feces of rats on the regular

5 658 CHUNG, FULK, AND SILVERMAN APPL. ENVIRON. MICROBIOL. TABLE 4. Culture counts of anaerobes in the feces of rats Culture counts ± standard error (log0o) (per g of dry wt) Medium Major organisms Diet MNNG Absent Present RGCA Total anaerobes Meat a 10.4 ± 0.1 **b ** Wayne 11.0 ± Phenylethylalcohol Gram-positive cocci Meat 10.3 ± 0.1 * 10.5 ± 0.03 agar ** ** Wayne 10.9 ± ± 0.1 Veillonella agar Gram-negative cocci Meat 9.8 ± 0.04 * 10.1 ± 0.1 veillonella ** Wayne 10.2 ± Bifidobacteria agar Bifidobacteria Meat 9.5 ± ** ** Wayne 10.1 ± ± 0.2 Fusobacteria (Nis- Fusobacteria Meat 9.5 ± sui) agar ** Wayne 9.9 ± ± 0.2 Kanamycin-vanco- Bacteroides Meat 8.4 ± mycin blood agar ** ** Wayne ± 0.2 a, No significant differences. b*, Significant differences at the significance level P < e, Significant differences at the significance level P < TABLE 5. Enumeration of clostridia in the feces of rats Culture counts on egg yolk agar (log,0) (per g of dry wt) Sample date Meat Wayne Meat + Wayne + Mea Wane MNNG MNNG 1/ ND" ND 2/ ND ND 2/ ND ND 3/ / / / / Average ± 8.7 ± ± ± ± 0.4 SEb a ND, Not determined. b Significant differences were observed between rats on the meat versus the Wayne diet or meat + MNNG versus Wayne + MNNG at the significant level P < No significant differences were observed between rats on either diet with or without MNNG. Wayne diet, and their numbers decrease gradually in the feces of rats on the meat ration. This could possibly be due to the limited availability of readily usable carbohydrates since lactobacilli are saccharolytic. MNNG, a known carcinogen, did not affect the number of bacteria on any of the various selective media, except phenylethylalcohol agar and veillonella-neomycin agar. However, in the present analysis, species were not determined. In studies involving human subjects on different diets, Reddy et al. (13) reported that in feces the total anaerobes, as well as the counts of bacteroides, bifidobacteria, peptococci, and anaerobic lactobacilli, were significantly higher during the period of consumption of a high-meat mixed Western-type diet as compared with the counts during the nonmeat consumption period. Our results do not support this observation, since the numbers of total anaerobes, as well as those on specific selective media, were in fact lower for most of the above genera in the feces of rats on the meat diet. The mixed Western diet of the subjects studied by Reddy et al. also included considerable amounts of carbohydrates, vegetables, coffee, and sugar in addition to the protein and fat. However, a comparison with this study is not necessarily valid because of marked differences between the fecal flora of rats and humans. In addition, diets of the two studies were not comparable. Dietary effects on the composition of fecal microflora may lend support to the observed phenomenon by Weinstein et al. (16) that the colonic contents of rats on a meat diet have greater infectivity in inducing the intra-abdominal abscesses as compared with the colonic contents of rats on a conventional chow diet.

6 VOL. 33, 1977 Bornside and Cohn (2) showed that in human subjects on a low-liquid diet, the fecal microbial composition did not deviate from normal flora, and Maier et al. (11) also found that a meat diet did not strongly influence the composition of human fecal microflora. In humans, microbial populations may be established by age 10 (10); dietary variations may have little, if any, influence on the microbial population in the ecosystem of the large intestine. On the other hand, the fact that the animals were placed on dietary controls at a young age might be quite critical in microflora colonization in the gastrointestinal tract. The present study indicates that diet has a pronounced effect on the composition of fecal microflora in rats. ACKNOWLEDGMENTS This research was supported by Public Health Service contract NO1-CO with Litton Bionetics, Inc., from the National Cancer Institute. We sincerely thank J. J. Knapka for providing the vitamin and mineral formulations used in our experiment. We are deeply indebted to M. W. Slein for his part in planning the basic protocol followed in this study and for offering helpful professional advice during the course of the experiments. Grateful acknowledgments are also due B. Ulland, E. Gordon, and R. Cypher for planning and executing the handling and care of the animals. We also wish to express our appreciation to L. Thibault for her statistical analysis of the data. LITERATURE CITED 1. Berg, J. W., M. A. Howell, and S. J. Silverman Dietary hypothesis and diet-related research in the etiology of colon cancer. Health Serv. Rep. 88: Bornside, G. H., and I. Cohn Stability of normal human fecal flora during a chemical defined, low residue liquid diet. Anim. Surg. 18: Chung, K.-T., G. E. Fulk, and M. W. Slein Tryptophanase of fecal flora as a possible factor in the etiology of colon cancer. J. Natl. Cancer Inst. 54: Drasar, B. S., and D. Irving Environmental factors and cancer of the colon and breast. Br. J. Cancer 27: DIETARY EFFECTS ON FECAL FLORA OF RATS Fuschs, A. R., and G. J. Bonde The nutritional requirement of Clostridium perfringens. J. Gen. Microbiol. 16: Gregor, O., R. Toman, and F. Prusova Gastrointestinal cancer and nutrition. Gut 10: Hanszel, W., J. W. Berg, and M. Segi Largebowel cancer in Hawaiian Japanese. J. Natl. Cancer Inst. 51: Hill, M. J., B. S. Drasar, R. F. 0. Williams, T. W. Meade, A. G. Cox, J. E. P. Simpson, and B. C. Morson Faecal bile acids and clostridia in patients with cancer of the large bowel. Lancet i: Holdeman, L. V., and W. E. C. Moore (ed.) Anaerobe laboratory manual. Virginia Polytechnic Institute and State University, Blacksburg. 10. Levitt, M. D., and J. H. Bond, Jr Volume, composition and source of intestinal gas. Gastroenterology 59: Maier, B. R., M. A. Flynn, G. C. Burton, R. K. Tsutakawa, and D. J. Hentges Effects of a high-beef diet on bowel flora: a preliminary report. Am. J. Clin. Nutr. 27: Narisawa, T., N. E. Magadia, J. H. Weisburger, and E. L. Wynder Promoting effects of bile acids on colon carcinogenesis after intrarectal instillation of N-methyl-N'-nitrosoguanidine in rats. J. Natl. Cancer Inst. 53: Reddy, B. S., J. H. Weisburger, and E. L. Wynder Effects of high risk and low risk diets for colon carcinogenesis on fecal micro-flora and steroids in man. J. Nutr. 105: Sabbaj, J., V. L. Sutter, and S. M. Finegold Comparison of selective media for isolation of presumptive group D streptococci from human feces. Appl. Microbiol. 22: Sutter, V. L., H. R. Attebury, J. E. Rosenblatt, K. S. Bicknell, and S. M. Finegold Anaerobic bacteriology manual. Anaerobic Bacteriology Laboratory, Wadsworth Hospital Center, Veterans Administration, Los Angeles, Calif. 16. Weinstein, W. M., A. B. Onderdonk, J. G. Bartlett, and S. L. Gorbach Experimental intra-abdominal abscesses in rats: development of an experimental model. Infect. Immun. 10: Wynder, E. L., T. Kajitan, S. Ishikawa, H. Dodo, and A. Takano Environmental factors of cancer of the colon and rectum. II. Japanese epidemiological data. Cancer 23: Wynder, E. L., and T. Shigematsu Environmental factors of cancer of the colon and rectum. Cancer 20: