HIHHIHIWIHII‘HWWHI I ‘ NIH I 7 | W 144 490 HTHS um.’ THE 13.0133 {35‘ AN {fi3’55?.§‘25ii Sm gaff-QC “News GR6$ 3233.!!!“ €33. 1235 ’{WQ 3%" G? "$35.33 FEE $3.5» “A‘Givcfi. £3. Tiwsis m: an: Dagwe 42:! 33.5.1. MEG 2333333 ‘ ”SHE CCELL 3‘3 ,k. . . 3"..4: ‘ .'- f'l 2 1!: 313326? 2*: gm: 3 3 1:11; E958 This is to certify that the thesis entitled "The Role of an Intestinal Streptococcus Organism in the Nutrition of Rats Fed an Evaporated Milk Diet" presented by Shirley V. Bring has been accepted towards fulfillment of the requirements for Master of Science degree in Foods and Nutrition QYWQW Major professor fl Date Decemb 0-169 THE ROLE OF.AN INTESTINAL STREPTOCOCCUS ORGANISM IN THE NUTRITION OF RATS FED AN EVAPORATED MILK DIET by Shirley Virginia gring A.THESI8 Submitted to thw School of Graduate Studies of Michigan State College of Agriculture and.Applied Science in partial fulfillment of the requirements for the degree of HASTEB.OF SCIENCE Department of Foods and Nutrition School or Home Economics 1950 THtlSlu ' we/ 5'! ACKNOWLEDGMENT The author wishes to express her sincere thanks to Dr. Dena C. Oederquist and Ir. Elbert 3. Churchill for their guidance and constructive criticism; to Mrs. Annanell G. Jubb for her helpful suggestions; to Dr. William D. Baten for his counsel; and.tc Mrs. Grace M. Burns and Mrs. Carol L. Frank for their assistance in the laboratory. 20 -...Jl 23 “a .5) TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . 3 The Effect of Diet on Intestinal Flora . . . . 3 The Effect of Streptomycin on Intestinal Flora. 6 EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . 11 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 21 SUMMARI.......................32 LITERATURE CITED . . . . . . . . . . . . . . . . . . 3h APPENDIX . . . . . . . . . . . . . . . . . . . . . . Number I II TABLES Title ORGANISMS ISOLATED FROM LOWER JEJUNUM OF EACH RAT FED MILK WITH AND WITHOUT STREPTOMYCIN AVERAGE WEIGHT GAINED BI'RATS FED MILK WITH AND WITHOUT STREPTOMICIN 23 28 Number 1 FIGURES Title Page Average total bacteria expressed as logarithmic values per gram of intestinal contents in the duodenum, upper and.lower JeJunum, upper and lower ileum, and cecum of rats fed milk with.and without streptomycin. 25 Relation of duration of treatment to the average total bacteria expressed as logarithmic values per gram of intestinal contents of the duodenum, lower JeJunun, and cecum of rats fed milk supplemented.with streptomycin. 27 Composite growth.curves of rats fed.milk with and without streptomycin. 3O INTRODUCTION INTRODUCTION The fundamental purpose of all nutrition research is to determine the adequate food needs of humans throughout their life span. Once it was thought that this objective could be realized simply by discovering the food nutrients necessary for growth, reproduction, and lactation. However, further studies revealed interrelationships between many of these factors. Varying the intake of one factor often changed the requirement for another factor. Still other investigations showed that some of the bacterial flora of the intestine contributed to the nutrition of the heat by synthesizing some of the required factors, and by aiding in the breakdown of complex food materials. The bacterial flo- ra has been shown to change with the diet, further compli- cating the requirements of the host. In the main, previous workers, studying the relation of diet to the intestinal flora, have investigated the role of the lactobacilli, streptococci, and coliforl organisms, as these organisms have been the most readily isolated and cultivated. Investigators have thus far reported the con- centration of these organisms (differential counts) in the intestinal material, ignoring the high concentration of un- identified organisms which complete the total bacterial -2. population. Also these earlier studies revealed no appar- ent relationship between the total and differential counts. More information is needed relative to the unidentified or- ganisms, as these may prove to be of vital nutritional im- portance. l The purpose of this study was to isolate and identify one of the organisms found in relatively large numbers in the intestinal tract of the milk-fed rat and to study its nutritional importance to this animal. REVIEW OF LITERATURE REVIEW OF LITERATURE The Effect of Diet on Intestinal Flora Studies have been made on many species of animals, including horses, cows, sheep, cats, dogs, rats, mice, non- keys, and humans, demonstrating that diet does affect the intestinal flora. From these studies it appears that the alimentary bacterial flora of both man and animals is essen- tially alike. Early studies revealed that in the intestinal flora of animals fed a high protein diet proteolytic or putrefactive organisms predominated, while in those fed a high carbohy- drate diet, aciduric or fermentative organisms predominated (Porter and Rettger, 1940; Winblad, 19M; and Call, _e_t _a_l_., l9h8a). It was found that a variation in the kind of carbohy- drate induced a change in the intestinal flora. Nath, 31; gl_. (19%) reported that the total aerobic and anaerobic plate counts, as well as the differential counts of coli- forms and lactic-acidpproducing organisms were higher in the ceca of rats on lactose diets than on dextrin or sucrose. It was found that ten times as many Lactobacillus acidophilus colonies were cultivated from the feces of rats fed a starch 4+- diet than were cultivated from the same quantity of feces of rats fed a sucrose diet (Winblad, l9ll-l). That a change in kind of carbohydrate does cause a change in the intesti- nal flora is supported by the work of Gall, _e__t al_. (1948a) , who found that the cecal contents of rats on a dextrose diet always contained many large cocci, occurring in pairs and short chains. The ceca of animals on a dextrin diet contained many elongated cocci in pairs, tiny cocci in long chains, and highly curved rods. Fredericia first described refection in 1926 as the synthesis of vitamin B by rats on vitamin B-deficient diets. Subsequent work reported by Ken and Porter (19h?) showed that refection depended on the presence of undigested starch and starch-splitting organisms in the cecum of the rat. Heller, gt a}; (1925) presented evidence that a spore- forming organism was responsible for the synthesis of vitamin B. More recently, Gall, gt _a_l__. (191le) demonstrated that coliforms isolated from the alimentary tract of the mouse synthesized riboflavin, niacin, biotin, folic acid, and pantothenic acid in tubes of broth deficient only in each respective vitamin. The elongated cocci, characteristic of the flora found in mice fed a dextrin diet, were demon- strated in the same way to have synthesized all of these B vitamins, while the round cocci, found in animals fed a dextrose diet, were shown to have synthesized all of these vitamins except folic acid. (Q Other investigations have established the fact that vitamin K may also be synthesized by rats either in the co- cum or other parts of the intestinal tract (Day, at a_l_., 193-3; ElvehJem, 1911-6). Nath, g_t_ 5;. (19%) found that the total bacterial counts per gram of cecal material and the total weights of the ceca from animals receiving lactose as a source of car- bohydrate were greater than the bacterial counts and weights of the ceca from rats receiving dextrin or sucrose. Kath, 33 9.1... (1948, p. 786) stated, "Thus the total numbers of organisms per cecum was higher in rats fed lactose than in those fed sucrose or dextrin.‘ The amount of protein in the diet seems to influence the intestinal flora. legner, gt gl_. (1913.1) found that increasing the amount of nitrogen in the feed of a cow caused no bacterial synthesis of riboflavin, thiamin, pan- tothenic acid, biotin, and folic acid, and depressed the synthesis of niacin. Czackes and Guggenheim (19%) reported one-third as many bacteria per gram of fecal material from rats fed a high protein diet as from those fed a low protein diet. Boutwell and coworkers (19%) investigated the effect of different fats on the growth of rats. They found that butterfat and lard induced superior growth to corn oil, coco- nut oil, cottonseed oil, soybean oil, peanut oil, and hydro- genated cottonseed oil when lactose was the carbohydrate source. Butterfat and lard supported slightly better growth than oleomargarine of animal fat origin and decidedly better growth than oleomargarine of vegetable fat origin. Growth was the same for all oils and fats when fed with a mixed carbohydrate diet, including sucrose, starch, dextrin, dex- trose, and lactose. The author suggests that these changes in growth may be caused by changes in the intestinal flora. When a high level of corn oil was added to a sucrose diet (Hath, _e_’§ 91., 19%), the total aerobic and anaerobic plate counts and the coliform counts on a differential medium were decreased in rat ceca. The ceca of rats fed butterfat diets weighed more and exhibited higher total bacterial counts than ceca of rats fed corn oil diets. The Effect of Streptomycin on Intestinal Flora Pratt and DuFrency (l9lt9, p. 2) define an antibiotic as, '... a metabolic product of one micro-organism that is detrimental or inimical to the life activities of other micro-organisms, usually even when present in extremely low concentrations.“ These metabolic products may also be pro- duced by higher plants and animals. The phenomenon is called antibicsie and was employed 30 or more centuries ago by the Chinese. At the present time, many antibiotic substances have been identified and studied. Among them are penicillin, streptomycin, streptothricin, aureomycin, chloromycetin, neomycin, tyrothricin, and terramycin. Even the acid pro- duced by the lactic acid bacteria in the intestine can be considered an antibiotic, as it is antagonistic toward putrefactive bacteria. Stokstad (19%) suggested three possible actions of antibiotics that reach the intestine after oral administra— tion. First, they may prevent some organisms from competing with the animal for nutrients in the diet; second, certain types of organisms may be allowed to grow in the presence of the antibiotics, which, in turn, synthesize important nutrients used by the animal; or third, the antibiotics may suppress certain bacteria that normally produce toxic substances. To date, most investigations of the effects of anti- biotics on intestinal flora have been made with aureomycin and streptomycin. However, McGinncs (1950) reported that when terramycin, aureomycin, streptomycin, or penicillin was added to the feed of turkeys, their growth rate was greatly improved. Mortality and stunted growth of poults were reduced markedly and the feed efficiency was increased. Streptomycin combats gram-negative, acid fast, and some gram-positive organisms in 1.1.33.9. and in 1112. It is pro- duced by the fungus, Actinomzces ggiseus, which occurs uni- versally. Streptomycin is water soluble, hydroscOpic, and very stable in both solution and the dried state under -S~ ordinary storage conditions. Reimann (l9k9, p. #60) states, “Streptomycin given orally is poorly absorbed and no toxic effects have been reported.“ Its mode of action is unknown. Ycumans and Fisher (19t9) report that streptomycin may act either as a bactericstatic or a bactericidal agent ig‘zitgg. The predominance of either activity seems to depend, in part, on the time of contact with.streptomycin, the organism in- volved, the concentration of streptomycin, the size of the inoculum, temperature, pH, and.the age of the culture. Smith and Robinson (l9fl5) found.that the coliform con- centration of the feces of mice fed 30,000 units of strepto- mycin per kilogram of body weight decreased from a count of 100,000 to 100 bacteria per three milligrams of feces in an hours. This low coliform count was maintained through, ' out the three-week period with.little fluctuation. When the diet was supplemented with 300,000 units of strepto- mycin per kilogram of body weight, coliforms disappeared from the feces within an hours. This higher dosage also eliminated all other gram-negative organisms, leaving only a small number of gram-positive spore-formers. The lower dosage caused a similar change, but with a less complete elimination of gram-negative organisms. Emerson and.Smith (19%) found a marked diminution in the numbers of coli- forms as well as in the total intestinal bacteria of rats fed streptomycin. They found doses as high as 580,000 and 875,000 units per'kilogram caused symptoms similar to uthose observed in biotin-deficient rats. By cultural methods Shaw and Dalldorf (1950) showed that after seven days, streptomycin (average dose: 5,500 units per mouse) inhibited all but the staphylococci and a few coliforms of the total flora; after twenty days (average dose: 11,250 units per mouse) this antibiotic inhibited all but staphylococci and spore-formers. During oral administration of streptomycin to humans, bacteria sensitive to streptomycin, including Streptococcus faecalis, usually began to diminish twelve hours after the first dose and.when at least 600 units of streptomycin per gram of feces were present (Weinstein, l9h9). Growth.of the organism was usually reestablished an to #8 hours after the last dose, or when the stool no longer contained‘bacterio- static amounts of the drug. Smith and.Robinson (1945) found normal counts were delayed six days after cessation of streptomycin supplements. Pratt and DuErenoy (19h9) state that streptomycinp resistant strains of bacteria may develop with.astonishing rapidity from cultures of organisms. Emerson and.Smith (1935) found the bacterial pepulation returned to normal in seven to twelve days, because of the development of streptomycin-fastness within the first an hours of therapy. Smith and Robinson (1935) assumed that no resistance devel- Oped.iglzgzg as the organisms appearing in the feces before -10- and during therapy were isolated and tested for sensitivity to streptomycin. It was found that organisms most sensitive to streptomycin in 2-332 were eliminated first in 1112 and those most resistant g 2132 remained in the feces. The ultimate purpose of studying the intestinal flora and its control through diet and antibiotics is to direct that flora to the greatest advantage of the host animal. EXPERIMENTAL PROCEDURE EXPERIMENTAL PROCEDURE Previous investigations conducted in these laboratories (McClure, 19h9; Katainen, 19h9) have demonstrated the great differences between the number of differentiated bacteria and.the total number of bacteria in the intestine of both milk and stock-fed.rats. It was hoped that one of the un_ identified organisms in the intestine of a milk-fed rat, in which.a characteristic flora had been established, could be isolated and identified and its nutritional importance to the animal studied. White male rats1 were fed,‘§d.libitum, the basal diet of reconstituted evaporated milk fortified with iron, copper, and.manganese. Water was given‘gg.libitum. All animals were housed in wire bottomed.raised cages to prevent copra- phasy- Preliminary examination of the intestinal flora of the milkpfed rat showed that a large coccus predominated.the flora of the ileum and.the cecum. This same coccus also appeared in the lower JeJunum, but in much smaller numbers. It was decided that the lower JeJunum was the best site for 1Sprague-Dawley strain, Madison, Wisconsin -12... the isolation of this organism. is total counts were very low in this section, there seemed to be less chance for in- terference from other bacteria. The isolation was made from the intestinal tract of a growing rat that had been on this basal diet for a suffi- ciently long time to establish a characteristic intestinal flora. The animal was chloroformed, the abdominal surface was swabbed with Boccal solution,2 and the abdominal cavity opened with sterile3 scissors and forceps. The cavity was flushed with Roccal,2 and the organs were kept moist by in. termittent flushing with this disinfectant. Sterile} scis- sors were used to make a transverse section of the lower Jejunum, and its contents were sampled with a sterile plat- inum loop. The sample was transferred to double strength Bacto-S 1' medium (Difco)? and was incubated at 37°C. for 48 hours. This culture was plated with Bactc-TGE agar (Dif- co) in a concentration that allowed the growth of isolated colonies under the conditions of incubation described above. The isolated colonies were picked and inoculated into litmus 2access diluted 1:5000 33terilized by autoclaving 20 minutes at 15 pounds pressure and 121°C. ”Double strength S 1" medium contained 0.1% sodium azide to inhibit growth of gram-negative organisms but to allow growth of fecal streptococci. -13- milk and enriched tryptose medium.5 The purified culture was carried on TGE agar slants and transferred bi-weekly. The isolated culture was tested by the methods recom- mended by the Committee of Bacteriological Technic of the Society of American Bacteriologists (19%). It was iden- tified as Streptococcus faecalis by the Bergey classifica- tion, 6th edition (Breed, gt _a_l_., 1911-8). The plan of studying the nutritional role of the iso- lated organism was altered because the nutritional require- ments and reactions of g. faecalis have been studied exten- sively. Workers have reported that g. faecalis requires most of the essential growth factors (Bellamy and Gunsalus, 19nd; Shankman, gingl, 19u7; and Sahyun, 19nd). Investiga- tions made to date, suggest that this organism could con- tribute to the nutrition of the milk—fed rat by converting lactose to lactic acid, and peptones to ammonia. Because the reactions and nutritional requirements of _S_. M have been studied extensively, it was decided to determine the role this organism played in the well-being of the milk- fed rat by studying the effect which would be obtained if _S_. faecalis were removed from the intestinal tract. By the use of an antibiotic it was believed that this could be 5Enriched tryptose medium contained 1% Bacto-tryptose, 0.5% Bacto-yeast extract, and 1% glucose and was adjusted to a pH of 7.0. ~111— accomplished. If the use of an antibiotic resulted in a marked decrease in the number of g. faecalis present, with.- out any detrimental effect on the animal, then it could be postulated that any other microorganism which replaced g. faecalis in the tract would have the ability to carry out the same nutritional function as g. faecalis. Preliminary investigation verified other reports (Pratt and DuFrenoy, 1911-9) that penicillin, orally administered, is destroyed or absorbed in the intestine. No penicillin was present in the cecal contents of rats fed an average of ten units per milliliter of milk. This antibiotic was found to be ineffective in inhibiting this strain of _8_. faecalis _i_n 11.2.9.2 which agrees with reports of Herrell (19%) and Pratt and DuFrenoy (193-9). §_. faecalis was inhibited by streptomycin both in 1.1.3.12 (Youmans and Fisher, 1949) and is m (Ravdin and Zintel, 1911-9). The results of other work underway in this labora- tory, involving the effect of streptomycin on _S_. faecalis in the rumen of the cow, together with the results of a preliminary investigation carried out by the author agree with the published reports. Therefore streptomycin was chosen for use in this study. In determining the correct dosage, it was found that 30,000 units per kilogram of body weight produced an average concentration of 160 units of streptomycin per milliliter of stomach contents and 1150 units per milliliter of cecal contents. This concentration was considered more than ade- quate to inhibit S. faecalis, as the range of sensitivity of this organism to streptomycin has been reported as 1.0 to 100.0 units per milliliter (Knap, 1946; Harrell, Herndon, Gillikin, and Aikawa, 19h7; and Brooke, 1947). The paper disc method described by Loo, 25 21. (19u5) was used to assay the streptomycin content, which was determined from a standard curve previously prepared.(Figure 1., Appendix). The following variations of the method of Loo, 33 El- (19u5) were used. Bacto-mycin assay agar was used for both the base and seeded media. Each paper disc was held with for- cops so that it Just touched.the surface of the mixed solu- tion being assayed and.was saturated by capillary action. The disc was then touched to the side of the container to eliminate the excess amount clinging to the point of con- tact. Two experimental groups of rats were placed on the basal milk diet supplemented with 30,000 units of strepto- mycin per kilogram of body weight daily for 30 days. Group I consisted of twelve 38 day old rats, housed in two cages. Two of these animals, one from.each cage, were sacrificed every five days and.the contents of the duodenum, upper and lower JeJunum, upper and lower ileum, and cecum were sam- pled for total bacterial counts. Material was also obtained for culturing the flora of the lower JeJunum in S P mediumll and tryptose medium,5 as described on page 12. Group II -16- consisted of seven 2h day old rats, one of which was sacri- ficed and sampled, as described, on each of the first five days. The two remaining animals were sacrificed and sampled at the end of the 30 day period. Six 23 day old control animals received the basal milk diet supplemented with minerals for the 30 day period. These were sacrificed at the end of the experimental period and sampled.by the same method used for the experimental animals. Daily records were kept of the total weight of food consumed, the total weight of the experimental animals, and the streptomycin intake. Control animals were weighed on two consecutive days at five-day intervals. Fresh.milk was given daily except on Sunday. Double rations were given on Saturday. The streptomycin6 was added to the weighed milk by syringe and the mixture inverted 20 times in a glass Jar to insure thorough.mixing. The amount of strep- tomycin actually consumed was calculated from the amount of food eaten. The average intake for Group I was 26,07u units of streptomycin per kilogram and for Group II, 25,015 units per kilogram. 6The crystalline streptdmycin calcium chloride com- plex in a rubber-topped bottle was diluted to a concentra- tion of 500,000 units/m1. with 0.5M sodium citrate. I. -17- Each.rat was prepared.for sampling as described on page 12 with.the following changes in technique: The au- topsy board was swabbedwithRoccal,2 and.the instruments were immersed in the same disinfectant and.returned to the Roccalz when not in use. Scissors were dried on a clean gauze to avoid diluting the intestinal contents with liquid adhering to the disinfected instruments. The outer skin of the abdominal cavity was removed with scissors, the perito- neum was removed, and the cavity floodedwithBoccal.2 Each section of the alimentary tract was clamped off with two small straight mosquito forceps. Prior te sampling, a transverse section was made of each.segment followed by a longitudinal incision, which.exposed.the intestinal contents for easy sampling. The duodenum included the pyloric sphincter and the point where it tapered to the even-bored JeJunum. It was usually one to two inches long. The length of the ileum was considered approximately equivalent to the length of the duodenum and JeJunum combined. Samples taken from each section were transferred.with a sterile platinum loop to sterile ccttcnpplugged 75 x 10 millimeter tubes, which had been dried (180°C. for 18 hours) to constant weight, and stored in a dessicator until used. The tubes containing the samples were wiped clean of fin- ger marks and weighed immediately following the sampling of each animal. One milliliter of 7% formalin was pipetted -18.. into each tube after weighing to serve as a preservatiye. Cork or rubber stoppers wrapped in cellophane were then substituted for the cotton plugs previously used and.the samples were refrigerated until total bacterial counts were made. The cecum of each animal was weighed.befcre it was sampled. The method of Simmons (1935) with.adaptations by Bortree and Smith? was used in determining total bacterial counts. Crystal violet stain was used to stain the organs isms and was prepared.as follows: One milliliter of crys- tal violet-saturated alcohol solution diluted to 50 milli- liters with distilled water was heated to simmering and filtered. The first dilution contained one part of sample in formalin to one part of stain in eight parts of distilled water. Higher dilutions were made when the total number of bacteria in the sample was too high to allow easy counting. Debris was worked out by carefully agitating the cover slip in a rotating motion, exercising care that contact with.the solution was not broken. 7AlfredL. Bortree, Veterinary Department, Pennsylva- nia State College, State College; and Clyde Smith, School of Veterinary Medicine, Michigan State College, East Lansing, personal communication -19- The organisms were counted under oil immersion.8 .A strongly lighted background showed.up the violet stained organisms for easiest identification. A.tota1 of 200 squares were counted for each sample in the Petroff-Hausser chamber. Clumps of organisms were counted as one bacterium. The number of bacteria per gram of intestinal sample was calculated by the formula: Number of Bacteria x Dilution Factor x.20 000 000(9) moms. Countemmm A sample was taken from the lower Jejunum and kept in sterile physiological saline solution at the same time the sample was taken for total bacterial counts. .A smear of this material was made immediately and gram stained for examination of the flora. Double strength 8 F mediumg was inoculated with this sample in order to isolate‘g. faecalis and tryptose medium5 was inoculated.with.this sample to grow possible substituting organisms. TGE agar plates were streaked.with.samp1es from tubes showing growth. .After in- cubation for #8 hours, isolated colonies were picked.and 8A 1:1 dilution of cedar oil with.xylene was used to prevent breaking the seal between cover slip and chamber with.movement of the objective. (9)Twenty million is a constant figure accounting for depth of chamber (1/50 mm.), square of sides (each sub- square 1/20 mm.), etc. -20.. transferred into litmus milk and lactose broth fermentation tubes to observe their reaction in these media. RESULTS AND DISCUSSION RESULTS AND DISCUSSION The organism isolated from the lower JeJunum of a milk_ fed rat and identified.as Streptococcus faecalis had these morphological characteristics: It was a gramepositive coccus, spherical to oval in shape, and appeared mostly in clusters, frequently in pairs, and sometimes singly and in chains. Older cultures exhibited.1ess tendency toward clustering. The size of this coccus ranged from 1 x 1 to 2 x 2 microns. Gram stains of the flora from the lower Jejunum10 of milk-fed and of streptomycin-supplemented.animals revealed no difference in types of organisms. The general flora con. sisted of tiny gram-negative and gramppositive cocci (they were so tiny the actual shape was not clear; medium-sized gram—positive oval cocci, occurring mostly in pairs; large gramepositive cocci, occurring singly and in pairs; large gram-positive yeast-like organisms; gramppositive rods, varying in length and shape and appearing mostly in chains; and single gram-positive slim rods. The significant differ- ence between the flora of the milk-fed and streptomycin- 10Suspended in 0.85% saline solution -22- supplemented rats was that the basal flora in the milk-fed rats was predominated.by the large gram-positive cocci with only a small proportion of the other organisms present, while the large cocci appeared in a much lower proportion in the streptomycin-treated flora. In contrast, Porter and Rettger (19h0) found very few cocci in any portion of the intestinal tract in rats fed.12 different diets. However, they found yeast-like organisms present in the stomach, du- odenum, Jejunum, upper ileum, lower ileum, and cecum. Shaw and Dalldorf (1950) reported these predominating types in the intestines of mice fed.Purina Laboratory Chow: Lacto- bacilli, coliforms, enterococci, proteus, and spore-formers. .As already reported,.§. faecalis was isolated from the lower JeJunum of the milk—fed rat in double strength.S F medium.” .After animals were treated with streptomycin, two organisms could be isolated in double strength S r mediumll (Table I): A.large gram-positive cocci; and a rod that varied in length, sometimes assumed irregular shapes and occurred most frequently in chains. This rod was usually gramppositive but sometimes parts of the longer rods stained gram-negative. The large coccus did not always survive streptomycin therapy, while the rod always survived. The morphology of the large coccus was similar to the identified g. faecalis. However, neither the coccus nor the rod reacted with.1itmus milk or fermented lactose as did'g.‘£gggglig. Evidently these organisms were not duplicating the role of -23- TABLE I ORGANISIS ISOLATED FROM.LOWER.JEJUNUM OF EACH RAT FED MILK WITH AND WITHOUT STREPTOMICIN Rat Group Treatment No. of Organism Isolated No. Days on from Lower Jejunum- Experiment of Rat Spore-forming rod 1} I; Streptggycin 1 L335: cgggggg rod* 1A II Streptogycin pg, Large coccus 15 ’II Streptomycin _3 Spore-formingrod 16 II’ Streptomycin _j£ Spore-fErming r657 17 II Strgptomyoip £1 gpore—fbrming rod pore-form ng ro 1 I Streptomycin 45 gargegocggs d pore- or ng ro I Streptomycin #5 Large cocg¥s *“d pore- or ng ro 3 I Streptomycin 10 gar e cccggs pore-for ng rod H I Strepto cin :;Q_ Large coccus 5 I Streptomyc n _;5 gpore-fbrmi r6d , pore-forming rod Large coccus 6 I Streptogycin .lfil §%S;:i%g;;§:gcgils 7 I Strepto cin 20 Large coccus £13 5”' SporeRTSrmIng rod 8 I Streptomypin gggp Larggpccccus .9' I Stre to c.n fig; Sporeéfdrml’ rod. 10* I Streptomyo.n 25, Spore-formIng_rod II wI: Streptomycin 36, Sporeeforming rod 12 I ‘Streptomycin 30 gporeegorgingrog pore- or ng ro 18 II Streptogycin 30 garge%occgs *df pore- orm ng ro 19 II Streptomyoin Large coccus 20* Cbfitrol o tre tom cin 30 ‘Large coccus 21 *Control No Stre tom sin ‘0 Large coccus 22’ Control 0 treptomycin 30 Large coccus 2 Control NEAStreptomygin 30 Large coccus ‘Contro. INS StreptomyEin #39 ‘Large coccus 25' Contro. No treptomycin 30 Large coccus -211- the _S_. faecalis in the breakdown of lactose. Further in- vestigation in this laboratoryll revealed that after repeat- ed culturing, the coccus reassumed all the characteristic reactions of g. faecalis. The rod was identified as B3931.— _2l_._t_1_g subtilis.u By cultural methods Shaw and Dalldorf (1950) found that the intestinal flora of mice, fed Purina Labora- tory Chow supplemented for seven days with 5,500 units of streptomycin per' animal, was predominated by staphylococci and a few coliforms; while the intestinal flora of mice, fed the same basal diet supplemented for 20 days with 11,250 units of streptomycin per mouse, was predominated by staph- ylococci and spore-formers. .- The total number of bacteria per gram of intestinal contents at the sampled points in the control and strepto- mycin-supplemented rats were similar and are expressed as logarithmic numbers in Figure 1. In general, both the milk- fed and the streptomycin-supplemented rats showed an increase in the average number of bacteria from the duodenum to the cecum. The report of Porter and Rettger (19%) agrees with these findings. They reported low microbial counts from the stomach to the JeJunum of the rat with a gradual in- crease in the number of bacteria in the upper and lower il- eum, and the highest microbial counts in the cecum. It llElbert S. Churchill, and Joseph Nichols, Department of Bacteriology and Public Health, Michigan State College, East Lansing Total Number of Bacteria Expressed as Logarithmic Numbers per Gram of Intestinal Contents 10.6 H O F H O '1" '3 i’ 9.84 9.64 9.h— 9.2. 9.0q 8.84 -25- Without streptomycin .._._1 With streptomycin Duodenum Upper JeJunum Upper Ileum Lower Ileum Cecum Lower Jejunum Figure 1. Average total bacteria expressed as logarithmic values per gram of intestinal con- tents in the duodenum, upper and lower JeJunum, upper and.1cwer ileum, and cecum of rats fed.milk with.and.without streptomycin. -26- should be noted in Table II of the Appendix that individu- al variations, as well as group variations, increased as the total number of bacteria per gram of sample increased. During streptomycin therapy,the greatest depression in number of bacteria occurred near the twentieth day in the duodenum, the fifteenth day in the lower JeJunum, and the tenth day in the cecum (Figure 2.). After the twenti- eth.day, there was a gradual increase in.the average total number of bacteria.per gram of fresh.material in the duo- denum and lower JeJunum but a continued decrease in total bacteria in the cecal flora. Emerson and.Smith.(l9#5) re- ported that the bacterial population of rats returned to normal seven to twelve days after streptomycin.therapy, because of the development of streptomycin fastness. The average weight gained during the 30 day experimen- tal period by the milkpfed and the two groups of streptomycin- supplemented rats is presented in Table II. Both.groups receiving streptomycin gained more weight per rat (63.7 and.81.6 grams) than those not receiving the antibiotic (33.7 grams). It should be noted that only two animals were continued through the 30 day period in each.expcrimen~ tal group, while six control animals were continued through. the same period. The rats of Group 1, beginning the exper- imental period at 38 days of age, gained less than the rats of Group II, beginning the experimental period at 2A days of age. -27- . 11e2 ‘5 O ‘3 10.8— "\ :3 . . “3 104s / \-/“\~\ . t - _ _ -__. me-i up 38 10.0.. A: HH ’fi‘fl-‘1 “H 906- l \\ e-IO I \ a 3 l \\ fig 9e2'1 A " \ g I ‘38, 8.8-. s.m use a; 8.11— 25: 1,, 8.0.. ‘33 a __...__ Cecum fl 7.6- ...... Lower JeJunum g Duodenum u) 3 7'2 U I l r ‘r 5 10 15 20 25 30 Days Rats Ped.Milk and Streptomycin Figure 2. Relation of duration of treatment to the average total bacteria expressed as logarithmic values per gram of intestinal contents of the duodenum, lower JeJunum, and cecum of rats fed.milk supplemented with.streptomycin. ~28- ~.nn ~.mm 0.0m w maohaopmomam oz flamenco ~.mw o.mma e.~m m naeasouaesam HH m.am o.~ma m.nm m uneasewmcnsm H macaw macaw macaw ceases cams on caused no phone: do u as usanaamem we spam no emcnob4. unmask o ebd. unmask omcao><_ mopsnz «couscous macaw ZHOHSoammmam gomaHt n34 EH3 MAHI Em.— madm Hm EH46 9mg”: Hagd HH Hag -29- Figure 3. shows the composite growth.curves of the three groups of animals. the growth of the milkpfed con- trol rats was slightly superior to that of the strepto- mycinpsupplemented rats of the same age (Group II) for the first six days. After this initial period, the experimen- tal animals grew more rapidly than those receiving only the basal diet. The elder rats (Group I) grew at a more rapid rate than the control animals at the same age period, but at a lesser rate than the rats of Group II, which.had re- ceived streptomycin for 1h days before reaching the compa— rable.age of 38 days. the mean weight of ceca of the control rats was 5.96 grams and of the streptomycin-supplemented.rats, 7.50 grals. Only the cecal weights of the four experimental animals fed streptomycin for the full 30 day period were included in the average figure presented. Since the inhibition of the characteristic functions of g. faecalis in the lower JeJunum of the rat by strepto- mycin therapy did.not cause a depression of the growth.cf the animals, it may be concluded that this organism did not contribute to the nutrition of the rat. 0n the contrary, the evidence suggests that the animals in which.the char- acteristic reactions of‘g. faecalis were inhibited exhibi- ted.a more rapid rate of growth.for the 30 day period. McGinnes (1950) reported similar growth.improvement and in- -30.. fiche—evacua- paonpdk one A»: Md? can and." no mega £2.98 033980 .m 0563 when 3” and o~ ow om ow on cm s om cache—canon»- naah HH 9.98 lllll \ , cache—each: #3.. H 9.8.5 ll-|u row mach-Sagan gonna: Ill: ION Tom / \ \\ \\\ too." \\\ \\\ \ \\ IOHH \\ \\ .s \\ IONH \ \ \\ A\\~.\\\ roma \ gsfi \\ \\ noma \ , oma Funds at suit-n -31- creased feed efficiency in turkeys fed streptomycinp supplemented diets. This evidence suggests the possibil- ity that g, faecalis may compete with.the rat for nutrients in the intestinal tract. At any rate, it seems that this strain of g. faecalis does not improve the nutrition of milhpfed rats, as its absence does not depress growth, nor is it replaced by another organism or organisms that dupli- cate the reactions of‘g. faecalis. This field of research.requires much.mcre investigation before the complete role of microorganisms in the nutrition of man can be fully understood. It seems desirable to iden- tify all the microorganisms that can be identified in the intestinal tract, and.study their possible contribution to the animal on different diets. Not only should.the possible nutritional function of each.microorganism be studied, but also the availability of any products of synthesis to the host animal. SW SUMMARY A large coccus was isolated from the lower JeJunum of milk-fed white male rats, and the organism was identified as Streptococcus faecalis. Ninteen white male rats were fed the basal milk diet supplemented with an average daily oral dose of 25,5’4-5 units of streptomycin per kilogram of body weight. It was demon- strated that this antibiotic inhibited the characteristic functions of g. faecalis within the first 21} hours of ther- apy. This inhibitory effect was continued throughout the 30 day experimental period, but did not cause a depression of growth in the animals. On the contrary, the average weight gain (except for the first six days) was greater in the streptomycin-supplemented animals than in the six mill:— fed animals. II'he evidence suggests that this organism does not con- tribute tc the nutrition of the rat, and may even be detri- mental to the well-being of this animal by possibly compet- ing with the host for nutrients. It was also demonstrated that no other organism in the lower Jejunum of streptomycin-supplemented rats replaced g. faecalis since the reactions of E. faecalis were not du- plicated by other organisms. It was found.that the average total number of bacteria per gram of intestinal contents in the duodenum, upper and lower JeJunum, upper and lower ileum, and cecum was similar in the streptomycin-supplemented and.milk-fed rats, deter- mined‘by the direct microscopic count method. Both groups showed an increase in the total number of bacteria from the duodenum to the cecum. LITERATURE CITED LITERATURE CITED Bellamy, W. D., and.Gunsalus, I. G. 19uu Tyrosine decar- boxylation by Streptococci: Growth.requirements for active cell production. ‘J. Bact. 5g: 191-199 Boutwell, R. K., GeKer, B. P., ElvehJem, G. A., and Hart, E. B. 19 3 Further studies on the comparative value of butter fat, vegetable oils, and oleomargarine. J. Nutrition.g§: 601-609 Breed, R. S., Murray, E. G. D., and.Hitchens, A. P. 6th ed. l9#8 Bergey's manual of determinative bacteriology. The Williams & Wilkins Company, Philadelphia Brooke, W. S. 19”? Streptomycin and parachlorophenol in surgical infections. Arch. Surg. 55: 305-315. Cited by Waksman, l9h9, 9%. Committee of Bacteriological Technic of the Society of American Bacteriologists. 1946 Manual of methods for pure culture study of bacteria. Biotech.Publications, Goneva, N e Ye Czackes, J. W., and.Guggenheim, K. l9h6 The influence of diet on the riboflavin metabolism of the rat. J. Biol. Chem. 162: 267-27h Day, H. G., Wakim, K. G., Krider,.M. M., and.G'Banion, E. E. l9#3 Effects of cecectomy, succinylsulfathiazole, and p-aminobenzoic acid on vitamin K synthesis in the in- testinal tract of rats. J. Nutrition g9: 585-600 Difco manual of dehydrated.oulture media and.reagents for microbiological and.clinical laboratory procedures. 8th ed. 19GB Difco Laboratories, Inc., Detroit Elvehjem, C. A. l9n6 The role of intestinal bacteria in nutrition. J. Am. Diet. Assn.‘g§: 959-963 Emerson, G. A., and Smith, D. G. 1946 A comparison of the effects of streptomycin in the nutrition of the rat and the mouse. Fed. Proc. of Am. Soc. for Exp. Biol. 5: 177 -35- Gall, L. 8., Fenton, P. F., and Cowgill, G. R. 1948a The nutrition of the mouse. II. Effect of diet on the bacterial flora of the intestine and the cecum. J. Nutrition 35: 13-25 Gall, L. 8., Illingsworth, B. A., Cowgill, G. R., and Fenton, P. F. 19M8b The nutrition of the mouse. III. Relation of diet to the synthesis activity of the predominating flora isolated from the small intestine and cecum. J. Nutrition‘35: 27-38 Harrell, G. T., Herndon, E. G., Gillikin, C. M., and Aikawa, J. K. 1947 A bacteriologic study of the factors affecting the efficacy of streptomycin therapy of urinary tract infections. J. Clin. Invest. gg: 577- 589. Cited by‘Waksman, 19u9, 9h. Heller, V. G., McElroy, C. H., and Garlock, B. 1925 The effect of the bacterial flora on the biological test for vitamin B. J. Biol. Chem. 65: 255-26h Harrell, W. E. l9h6 Penicillin and other antibiotic agents. W. B. Saunders Company, Philadelphia Katainen, V. O. 19u9 Riboflavin and thiamine content of the intestinal tract of oecectomized and non-cecectomized white rats. unpublished.M. S. Thesis. East Lansing, Michigan, Michigan State College Library Knop, C. Q. 19%6 Experimental study of the development of resistance to streptomycin by some bacteria commonly found in urinary infections. Proc. Staff Meet. Mayo Clinic g1: 273-276. Cited by Waksman, l9fl9, 9%. Ken, S. K., and Porter, J. W. G. 1947 The role of micro- flora of the alimentary tract. 5. The synthesis vita- mins in relation to requirements. Nutr. Abst. Rev. 11: 31-37 Loo, I. H., Skell, P. S., Thornberry, H. H., Ehrlich, J., MbGuire, J. M., Savage, G. M., and.Sylvester, J. C. l9h5 Assay of streptomycin by the paper-disc plate method. J. Bact. 52; 701-709 McClure, S. 19u9 Influence of milk and stock diets on the intestinal flora of cecectomized and normal rats. Unpublished M. S. Thesis. East Lansing, Michigan, Michigan State College Library McGinnes, J. July, 1950 The antibiotics make good feeds bett ere Turkey World 11, 56, 57 Nath, H., Barki, V. H., Sarles, W. B., and.E1vehJem, C. A. 1948 Microorganisms in the cecal contents of rats fed various carbohydrates and fats. J. Bact. 56: 783-793 Porter, J. B., and.Rettger, L. F. 1940 Influence of diet on the distribution of bacteria in the stomach, small intestine and cecum of the white rat. J. Infect. Dis. ‘66: 104-110 Pratt, H., and DuFrenoy, J. 1949 Antibiotics. J. P. Lip- pincott Company, Philadelphia Ravdin, I. 8., and Zintel, H. A. Chap. 32. Cholangiche atitis and peritonitis. Waksman, S. A., Editor. 19 9 Streptomycin, nature and practical applications. The Williams & Wilkins Company, Baltimore Reimann, H. A. Chap. 31. Intestinal infections. Waksman, S. A., Editor. 1949 Streptomycin, nature and practical applications. The Williams & Wilkins Company, Baltimore Sahyun, M. 2nd ed. 1948 Outline of amino acids and proteins. Reinhold.Publishing Corporation, New York Shankman, 8., Camien, M. N., Block, H., Merrifield, R. B., and Dunn, M. S. 1947 Vitamin requirements of twenty- three lactic acid bacteria. J. Biol. Chem. 1.68; 23-31 1950 Investigations of the fecal bacteria of mice, with reference to the presence of mouse encephalomyelitis virus. Shaw, M., and Dalldorf, G. Je Bacte _6_O_: 175-183 Simmons J. 8., and Gentzkow, C. J., Editors. 19411 5th Ode Laboratory methods of the‘United.States Army. Lea & Febiger, Philadelphia Smith, D. G., and.Robinson, H. J. 1945 The influence of streptomycin and streptothricin on the intestinal flora of mice. J. Bact. 5g: 613-621 Stokstad, E. L. R. 1950 The effect of aureomycin on animal nutrition. Foodstuffs fig No. 28: 17-18, Waksman, S. A., Editor. 1949 Streptomycin, nature and practical applications. The Williams & Wilkins Company, Baltimore Wegner, M. I., Booth, A. N., ElvehJem, C. A., and Hart, E. B. 1941 Rumen synthesis of the vitamin B complex on gatuial rations. Proc. Soc. Exp. Biol. Med.'41: 0-9 Weinstein, L. Chap. 41. Alterations in normal bacterial flora of man and animals and secondary infections during streptomycin therapy. Waksman, S. A., Editor. 1949 Streptomycin, nature and practical applications. The Williams & Wilkins Company, Baltimore Winblad, S. 1941 variations in the bacterial flora of the intestine of white rats in different carbohydrate diets. Acta Pathologioa et Microbiologica Scandina- vica gs; 204-224 Youmans, G. P., and Fisher, M. W. Chap. 7. .Aotion of streptomycin on microorganisms in vitro. Waksman, S. A., Editor. 1949 Streptomycin, nature and.practical applications. The Williams & Wilkins Company, Baltimore APPENDIX TABLE I STREPTOMYCIN CONTENT or was cecum or EACH MILK. FED an suppnmmmn ORALLI WITH AN AVERAGE or 25,545 UNITS or STREPTOMICIN PER KILOGRAM or sop: WEIGHT pea DAY AND DETERMINED BY METHOD or LOO, g3. 59. (1945) Group No. of Days Units of Fed Streptomycin Streptomycin per M1. of Cecal Contents I 5 208 I 5 180 I 10 280 I 10 275 I 15 188 I 15 275 I 20 215 I 20 250 I 25 380 25 1 3o 2 E I 30 2 II 2 190 II a 156 II 220 II 5 295 II 30 - II 30 - ”.mmn.m: H.mmH.mH o.mmm H.omm «.mH: o.onm on eHoHaopaos-m HH . mm. H m.mmo.m w.m m m.omw H. «H m.mmm on aHoHso-assom HH H.mmm.mH H.mm:.m m.H m.~m m.nm m.mm om eHoHao-eos-m H H.mmm.mH H.mam.m m.sas.m m.mo .H H.mmm m.H~m on eHoHsopaos-n H H.oHH.om 0.0mm. 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