ACTION OF INTESTINAL MICROORGANISMS ON ASCORBIC ACID' respond to a diet rich in vitamin C; however, upon intramuscular injection of

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1 ACTION OF INTESTINAL MICROORGANISMS ON ASCORBIC ACID' RAYMOND M. YOUNG AND L. H. JAMES Department of Bacteriology, University of Maryland, College Park, Maryland Received for publication, July 26, 1941 EFFECT OF PURE AND MIXED BACTERIAL CULTURES ON ASCORBIC ACID Marin (1936) described a clinical case of hemorrhagic scurvy which did not respond to a diet rich in vitamin C; however, upon intramuscular injection of a solution of the vitamin, the patient recovered rapidly. Hetenyi (1935) gave patients suffering from colitis gravis and exhibiting avitaminosis daily intravenous injections of 150 mg. of ascorbic acid; recovery was complete in several cases. The failure of response to a diet rich in vitamin C and the positive effects of intramuscular and intravenous injections of this substance suggest bacterial destruction of ascorbic acid in the gastro-intestinal tract before absorption can take place. The possibility of microbial decomposition of the vitamin has not been overlooked. Stepp and Schroder (1935) found a strain of Escherichia coli that decomposed ascorbic acid, and Kendall and Chinn (1939) reported the isolation of members of the Mucosus capsulatus and Enterococcus groups which fermented the vitamin. Kendall and Chinn showed that bacterial decomposition of this substance is more a strain than a species characteristic, for some strains in these groups failed to show any destructive effect. A strain of the Mucosus capsulatus group, one of the Flexner bacillus and one of Alcaligenes inhibited atmospheric oxidation of ascorbic acid. Glucose exerted a sparing action on the fermentation of ascorbic acid by intestinal strains of enterococci and members of the Mucosus capsulatus group. Because of the importance of the interrelationship between vitamins, bacterial growth and human nutrition, the present research was undertaken to investigate the influence of several pure and mixed cultures of normal intestinal bacteria on the stability of ascorbic acid, and to study various factors involved in the action. It must be assumed that recognition of vitamin C deficiency resulting from microbic destruction in the gastro-intestinal tract must depend, to some degree at least, upon actual isolation of the agents involved and on chemical proof of the destructive action of ascorbic-acid-fermenting bacteria on the vitamin in artificial medium. EXPERIMENTAL WORK Methods Ascorbic acid nutrient broth was employed. The broth was prepared as follows: a weighed amount of crystalline ascorbic acid was dissolved in a small amount of water, filtered through a Berkefeld filter and the sterile filtrate added to sterile standard nutrient broth, giving a final concentration of A portion of the data submitted by the senior author as a thesis in partial fulfillment of the requirements for the degree of Master of Science. 75

2 76 RAYMOND M. YOUNG AND L. H. JAMES mg per ml of broth. This concentration of ascorbic acid was decided upon after consideration of the results of preliminary studies, in which it was observed that ascorbic acid in a concentration as low as 0.01 per cent at times would be completely oxidized by atmospheric oxygen before the bacteria had multiplied sufficiently to attack the vitamin. On the other hand, concentrations of ascorbic acid above 0.14 per cent definitely inhibited growth of the intestinal bacteria. Thus it was considered undesirable to use either very high or low concentrations of the vitamin. Under the conditions existing in the intestinal tract ascorbic acid is present only in relatively small amounts. If it could be demonstrated that intestinal bacteria are capable of oxidizing ascorbic acid in amounts several times the concentration commonly found in the intestinal contents, the evidence for microbial decomposition of vitamin C would be more conclusive. When carbohydrates were used in the medium they were present in a final concentration of 1 per cent, unless indicated otherwise in this paper. The ph for the mediums used was adjusted to Preliminary experiments indicated no significant acceleration or inhibition of oxidation of vitamin C in uninoculated nutrient broth at several ph values ranging from 3 to 8. After removing an aliquot portion for immediate analysis, the ascorbic acid nutrient broth was distributed in 100 ml. portions in 180 ml. bottles which were inoculated with the various organisms studied. All cultures and an uninoculated control were incubated at 37CC. Titrations were made frequently, using a modified procedure of Tillman's sodium 2,6-dichlorophenolindophenol method described by Bessey and King (1933). Duplicate portions of 1 ml. each were removed from the cultures and control to 150 ml. flasks containing 1 ml. glacial acetic acid in 50 ml. of distilled water. The samples were titrated with freshly standardized s6dium, 2, 6-dichlorophenolindophenol, 1 ml. of which was equivalent to 0.22 mg. of ascorbic acid. The end-point was considered reached when a faint pink coloration appeared and remained foratleast 20 seconds. The titration was completed within two minutes, and all determinations were carried out in duplicate or triplicate. For control purposes, all of the cultures employed were tested for ability to produce a dye-reducing substance in ascorbic-acid-free nutrient medium and glucose broths. In no instance was the presence of such a substance detected. Unless indicated otherwise, a heavy inoculum of 0.5 ml. of an 18-hour-old nutrient broth culture was used in the following studies in order to obtain a large bacterial population in a short time. Under these conditions luxuriant growth was evident in all of the cultures within a few hours. In experiments conducted under anaerobic conditions reduced oxygen tension was obtained by use of a vacuum incubator which provided a 30-inch vacuum. The temperature was adjusted to 370C. and was maintained evenly in all parts of the incubator by means of a pump which circulated heated oil around the sides of the incubator.

3 ACTION OF INTESTINAL NaCRO6RGANISMS ON ASCORBIC ACID 77 ACTION OF PURE CULTURES AND MIXTURES OF ENTERIC NaCRO6RGANISMS UPON ASCORBIC ACID Several pure cultures of the Proteus, Alcaligenes and Escherichia genera were inoculated into ascorbic acid nutrient broth. The Proteus cultures included the following strains: OXK, X-19, X-2 and Iowa. The Alcaligenes strains employed were Alcaligenes fecalis and Alcaligmene fecalis var. Iowa. Three varieties of Escherichia coli were used, communis, communior, and H-52. Under aerobic conditions the three varieties of Escherichia coli employed decomposed the ascorbic acid completely within hours, whereas an uninoculated control contained 63 and 34.4 per cent respectively of the initial amount of vitmin C after the same time intervals. The loss of ascorbic acid in the uninoculated controls was due, we must assuime, to atmospheric oxida- TABLE 1 Action of pure cultures and a mixture of pure cultures of intestinal bacteria upon ascorbic acid in nutrient broth under aerobic conditions CULTURES INCUBATION PERIOD IN HOURS Escherichia coli var. communis * E8cherichia coli var. communior Escherichia coli var H Proteus OXK Proteus X Proteus X Proteus vulgari var. Iowa Alcaligenes fecalis Alcaligeneefecalis var. Iowa Mixtures of the above pure cultures Uninoculated control * Milligrams ascorbic acid per ml. tion. Undoubtedly some atmospheric oxidation of the vitamin occurred in the treated cultures, but this does not affect the interpretation of the results, since the control still contained some vitain C several hours after the vitamin had completely disappeared from the broth cultures of Esche7richia coli (see table 1). Unlike the action of Escherichia coli, Proteus and Alcaligenes organisms did not decompose ascorbic acid but markedly inhibited atmospheric oxidation of the substance. As indicated in table 1, the vitamin was not completely oxidized even after 192 hours in the presence of several species or varieties of Proteus and Alcaligenes, whereas the uninoculated control had lost all of the substance between 32 and 72 hours of incubation. The protective action was quite similar with the species of both genera and was operative even in an alkaline ph of from 7.3 to 7.72.

4 78 RAYMOND M. YOU&G AND L. H. JAMES When the protective Alcaligenes and Proteus organisms were mixed with the ascorbic acid decomposers the protective action was not operative; in a mixture of the nine pure cultures listed in table 1 the ascorbic acid was completely destroyed during 14 hours incubation. Since anaerobic conditions prevail in the intestine of man, it was desirable to determine whether the oxidation of vitamin C by intestinal bacteria is effective in the absence of oxygen. Accordingly, ascorbic acid nutrient broth cultures of Escherichia coli, including the communis and communior varieties and a few strains isolated from human feces, together with a typical fecal strain of Aerobacter aerogenes and a strain of Citrobacter, were incubated under anaerobic conditions. The results presented in table 2 show that vitamin C is oxidized by intestinal strains of bacteria also under anaerobic conditions. The oxidation was completed within 14 hours, with the exception of the Citrobacter strain TABLE 2 Action of pure cultures of intestinal bacteria upon ascorbic acid under anaerobic conditions CULTURES INCUBATION PERIOD IN HOURS Citrobacter * Aerobacter aerogenes var. Y Escherichia coli var. communis Escherichia coli var. communior Escherichia coli (typical strains isolated from human feces) Y S B F A A A Uninoculated control * Milligrams ascorbic acid per ml. which oxidized the vitamin completely within 22 hours. The uninoculated control contained 95.4 per cent of the original amount of vitamin after 14 and 22 hours. Small losses of vitamin C from uninoculated controls held under anaerobic conditions are due to unavoidable exposure of the samples to air during sampling and titration. ACTION OF PURE CU1UURES AND OF MIXTURES OF ENTERIC MICROORGANISMS UPON ASCORBIC ACID IN THE PRESENCE OF FERMENTABLE CARBOHYDRATE This study had the following objectives: (1) to determine if the presence of readily fermentable carbohydrate will prevent the oxidation of vitamin C; (2) to learn if the action is similar under both aerobic and anaerobic conditions; and (3) to observe the effect of varying concentrations of glucose upon the oxidation of the vitamin by Escherichia coli var. communss.

5 ACTION OF INTESTINAL MICROORGANISMS ON ASCORBIC ACID One per cent glucose and lactose broths containing 0.41 mg. of ascorbic acid per ml. were prepared and portions inoculated with the communis, communior,and H-52 varieties of Escherichia coli. Uninoculated controls were run with each carbohydrate broth; a second series of controls was employed consisting of nutrient broth containing 0.41 mg. of ascorbic acid per ml. but no glucose. All test samples were placed under aerobic conditions. The data in table 3 give evidence that both glucose and lactose not only spare vitamin C from bacterial oxidation but also protect the vitamin from atmospheric oxidation in actively growing cultures of Escherichia coli. After 48 hours all of the cultures of Escherichia coli in glucose and lactose broth contained at least 80.5 per cent of the initial amount of the vitamin, while the same cultures which had no glucose or lactose present in the medium lost all of the vitamin within 22 hours. Bacterial destruction of the vitamin is evidenced by the presence of 32.2 per cent of the initial amount in the uninoculated con- TABLE 3 Action of pure cultures of enteric bacteria upon ascorbic acid in nutrient, glucose and lactose broths incubated under aerobic conditions CULTURES NUJTRIETwNT GLUCOSE LACTOSE Hours Escherichia coli var. communis * Escherichia coli var. communior Escherichia coli var. H Uninoculated control * Milligrams ascorbic acid per ml. trol after 22 hours, when the sugar-free cultures of Escherichia coli had lost all of the vitamin. Protection from atmospheric oxidation is observed by comparing the amount of vitamin retained in the lactose and glucose broth cultures of Escherichia coli with the vitamin content of uninoculated controls. The former after 48 hours contained at least 80.5 per cent of the initial amount of vitamin C, while the vitamin in uninoculated controls had been completely oxidized by atmospheric oxygen. If no protection from atmospheric oxidation were offered, the glucose and lactose broth cultures at any momentduringthe incubation would contain the same amount of vitamin C as the uninoculated controls for atmospheric oxidation. Since bacteria grow under anaerobic conditions in the intestine of man, it was desirable to determine whether the carbohydrate-sparing action on vitamin C as observed under aerobic conditions would operate under anaerobic conditions. Accordingly, the experimental conditions used in the previous experiment were duplicated with the exception of placing the cultures under anaerobic 79

6 Of0 RAYMOND M. YOUNG AND L. H. JAMES conditions. The bacterial cultures employed included the communiu, communior and Y-12 varieties of Escherichia coli, Aerobacter aerogenes var. Y-12, and a strain of Citrobacter. The results of these tests indicated that ascorbic acid is spared completely from bacterial oxidation also under anaerobic conditions. No decomposition of the vitamin had occurred in any of the cultures containing glucose or lactose when the experiment was concluded after 144 hours incubation whereas the vitamin in the sugar-free nutrient broth control cultures was completely oxidized within 14 hours. In order to determine the effect of concentration of fermentable sugar upon the sparing of vitamin C, a series of tests were carried out in which ascorbic acid broths with varying concentrations, 1 per cent, 0.1 per cent and 0.03 per cent glucose were employed. Escherichia coli var. communis was used as the test culture, and incubation was carried out under anaerobic conditions. Bacterial counts were made during the growth of the culture to ascertain if ascorbic acid stimulates growth which may be observed by comparing the growth curve of bacteria in the presence of vitamin C with the growth curve of bacteria developing in the same medium without the vitamin. The amount of glucose present was determined with each titration of ascorbic acid, and the quantitative platings. The method used for glucose determination was a micro method especially designed by Stiles, Peterson and Fred (1926) for estimating glucose in bacterial cultures. The results of the tests in which glucose was employed are shown in figures 1, 2 and 3. One per cent and 0.1 per cent concentrations of glucose in the presence of Escherichia coli var. communis spared vitamin C to the extent of 99 per cent of the original amount after 60 hours incubation, while in the sugar-free control inoculated with the same organism ascorbic acid was oxidized completely within 25 hours. After 60 hours incubation, the one per cent glucose broth contained 58 per cent of the initial amount of glucose, while the 0.1 per cent broth retained only 15 per cent of the initial amount of glucose. Glucose in the 0.03 per cent broth was entirely utilized after 43 hours; as is evident in figure 3, sparing of vitamin C was operative until the glucose content began to be depleted. The vitamin was spared to the extent of 97 per cent of the initial amount during the first 24 hours of incubation, while in the sugar-free control it was completely oxidized within this period. However, after the glucose had been utilized the destruction of the vitamin was rapid, being completed at 60 hours incubation. The uninoculated control contained 60 per cent of the vitamin after 60 hours, indicating that the bacteria were responsible for rapid decomposition of ascorbic acid after the sparing action of glucose ended. The effect of ascorbic acid upon growth of Escherichia coli var. communis in varying concentrations of glucose also is represented in figures 1, 2, and 3. It will be not.ed that in general the amounts of bacterial growth in the different concentrations were quite similar and that, although the initial counts in all tubes were alike, approximating 200 organisms per ml., vitamin C definitely inhibited bacterial growth to such an extent that at no time did the growth of bacteria in broths containing the vitamin exceed that of the vitamin-free broths, except near the end (fig. 3).

7 ACTION OF INTESINAL CRO6RGANISMS ON ASCORBIC ACID u U I: 54-2Z 0 J 0 goo 9 80 U 50 C d L.. 20 l go 10 o lr 0 INCtJBATION PENOD IN HOURS FIG. 1. EFFECT OF A 1 PER CENT GLuCOSE BROTH UPON THE SPARING OF ASCORBIC ACID IN THE PRESENCE OF ESCHERICHIA COLI VAR. COMMUNIS A-Vit. C in vitamin-glucose culture; B-Vitamin C in uninoculated control; C-Vitamin C in glucose-free culture; D-Glucose in vitamin-glucose culture; E-Bacterial growth in plain broth; F-Bacterial growth in vitamin-glucose broth. OAn 20 I0 4 S I- -.~~~~~~~~~~~ '.. U ~~~El,f *1 -I ~ ~~~~~~~~~~ J1~~~~~~~~~~ INCUBATION PERIOD IN HOURS FIG. 2. EFFECT OF A 0.1 PER CENT GLuCOSE BROTH UPON TEB SPARING OF ASCORBIC ACID IN THE PRESENCE OF ESCHERICHIA COLI VAR. COMMUNIS A-Vit. C in vitamin-glucose culture; B-Vit. C in uninoculated control; C-Vit. C in glucose-free culture; D-Glucose in vitamin-glucose culture; E-Bacterial growth in plain broth; F-Bacterial growth in vitamin-glucose broth.

8 82 RAYMOND M. YOUNG AND L. H. JAMES These studies concerning the effect of varying concentrations of glucose upon the sparing of vitamin C suggest that ascorbic acid, a carbohydrate, is utilized as a source of carbon when the available supply of fermentable sugar is depleted. ACTION OF FECAL CUIrURES UPON ASCORBIC ACID IN PLAIN NUTRIENT AND IN CARBOHYDRATE-CONTAINiNG BROTHS Having observed that pure stock cultures of Escherichia coli and pure cultures of the same species isolated from human feces were able to decompose vitamin C, an experiment was planned with the purpose of determining if normal intestinal flora of a few individuals would exhibit the same destructive in vitro action. 0.~~~~~~~~~~~~~~~~~~~~~~~~~h O INCUBATINPERIODINHOUS FIG. 3. EFFECT OF A 0.03 PER CENT GLUCOSE BROTH UPON TH SPARING OF ASCORBIC ACID IN THE PRESENCE OF ESCHERICHIA COLI VAR. COMMJNIS A-Vit. C in vitamin-glucose; B-vit. C in uninoculated control; C-vit. C in glucosefree culture-d4glucose in vitamin-glucose culture; Bacterial growth in plain broth; F-Bacterial growth in vitamin-glucose broth. The use of mixed fecal cultures more nearly duplicates microbial conditions in the intestines than the use of isolated pure cultures of predominating types. A small fecal sample from each of four human subjects was added to each of four dilution blanks. The suspensions were well shaken, allowed to settle, and a loop of the suspended microorganisms from each was transferred to nutrient broth and the culture incubated. Mfter luxuriant growth was evident inoculations were made in duplicate into glucose and lactose nutrient broths, each tube containing 0.36 mgm. of ascorbic acid per mi. of broth. Incubation was carried out at 370 under both aerobic and anaerobic conditions. The data obtained indicated that under both aerobic and anaerobic conditions sugar-free cultures from the four fecal specimens oxidized ascorbic acid com-

9 ACTION OF INTESTINAL MICRO6RGANISMS ON ASCORBIC ACID pletely within 24 hours, while after the same period 0.16 mg. of ascorbic acid per ml. of broth remained in an uninoculated control under aerobic conditions, and 0.33 mg. per ml. remained in an uninoculated anaerobic control, indicating again that atmospheric oxygen is not necessary in the oxidation of ascorbic acid by intestinal bacteria. As was observed previously with the pure cultures of ascorbic acid oxidizing bacteria, glucose and lactose in concentrations of 1% completely protected the vitamin from bacterial oxidation under both aerobic and anaerobic conditions. DISCUSSION The outstanding observation from the studies of this investigation is the apparent ease with which ascorbic-acid-fermenting bacteria may be isolated from human feces without having to resort to special selection or acclimatization technique. Strains of E8cherichia coli and Aerobacter aerogenes isolated from the intestinal contents of several human subjects were found to destroy ascorbic acid rapidly in nutrient broth after good growth was evidenced. These findings suggest that symptoms of vitamin C deficiency in certain individuals, in spite of a diet adequate in the vitamin, might be traced to an intestinal flora high in ascorbic-acid-fermenting bacteria. Although the point of absorption of ascorbic acid is not well-known as yet, a possibility that significant loss of the vitamin occurs through overgrowth of ascorbic-acid-fermenting bacteria in the alimentary tract has been suggested by several investigators. Abt and Farmer (1938) reported that the normal individual does not excrete over 6-10 mg. of vitamin C daily, even when large amounts are taken by mouth. Under abnormal conditions (diarrhea and colitis) the amount varied according to number and type of stools. Wilder and Wilbur (1937) reported that variable amounts of vitamin C are lost in the stool or destroyed in the gastro-intestinal tract even under normal conditions. Bacterial fermentation in the stomach commonly proceeds unchecked in achlorhydric patients. Einhauser (1936) reported that conditions of hypoacidity were accompanied in several cases by disturbed absorption and destruction of vitamin C. Since gastro-intestinal catarrh also accompanied the vitamin deficiency symptoms, Einhauser believed that the vitamin was oxidized and destroyed by the gastro-intestinal flora. The hypochlorhydric subjects improved markedly after oral administration of large quantities of lemon juice. The destruction of ascorbic acid by bacteria in the intestine of man cannot be of serious consequence in most people or there would be more extensive evidence of vitamin C deficiency. However, with some individuals who have an intestinal flora high in ascorbic-acid-fermenting microorganisms symptoms of scurvy such as lowered resistance, anemia, internal hemorrhage, pains in the joints, and edema in the extremities may be present and wrongly attributed to another cause; thus the possibility of this factor playing a significant part in Added significance the health of some individuals should not be overlooked. is attached to disorder involving anacidity, since this condition favors growth ascorbic-acid-fermenting bacteria of intestinal bacteria in the stomach; and, if 83

10 84 RAYMOND M. YOUNG AND L. H. JAMES are present much or all of the vitamin may be decomposed before it reaches the absorbing tissues of the intestines. The presence of even small amounts of glucose definitely delays the decomposition of ascorbic acid until the sugar is used up. It seems quite possible that the juices of citrus fruits, which are rich in sugars and are natural sources of vitamin C, would deter microbic attack upon ascorbic acid so long as these sugars are present in the digestive tract. The failure of the oral administration of vitamin C in certain conditions in which parental injection yields positive experimental or clinical results, and the favorable results following its use in extremely large doses when small doses were unavailing present a problem which invites further study. SUMMARY Several direct human fecal cultures and isolated cultures of Escherichia coli and Aerobacter aerogenes were found to decompose vitamin C under both aerobic and anaerobic conditions. Organisms of the Proteus and Alcaligenes groups failed to attack. the vitamin and definitely protected the substance from atmospheric oxidation, but the protective action was not operative in the presence of vitamin-c-decomposing bacteria. The presence of glucose or lactose in ascorbic acid nutrient medium definitely "spared" the vitamin from oxidation by the fecal cultures and pure cultures of Escherichia coli. The sparing action was operative under aerobic and anaerobic conditions. The vitamin disappeared rapidly from the medium as soon as the sugar had been completely utilized. REFERENCES ABT, A. F., AND FARMER, C. J Pharmacology and Therapeutics of Vitamin C. J. Am. Med. Assoc., 111, BESSEY, 0. A., AND KING, C. G The Destruction of Vitamin C in Plant and Animal Tissues and its Determination. J. Biol. Chem., 103, EINHAUSER, M Vitamin C and Gastro-Enteritis. Z. ges. exptl. Med., 98, HETENYI, G Treatment of Colitis Gravis with Ascorbic Acid. Orvosi Hetilap, 79, KENDALL, A. I., AND CHINN, H The Decomposition of Ascorbic Acid by Certain Bacteria. Studies in bacterial metabolism. J. Infectious Diseases, 62, MARIN, P Scurvy from the Intestinal Bacterial Destruction of Vitamin C. Minerva med. II, STEPP, W., AND SCHRODER, H The Fate of Vitamin C in the Intestinal Canal. I. The effect of intestinal bacteria on vitamin C. Klin. Wochschr., 14, STILES, H. R., PETERSON, W. H., AND FRED, E. B A Rapid Method for the Determination of Sugar in Bacterial Cultures. J. Bact., 12, WILDER, R. M., AND WILBUR, D. L Diseases of metabolism and nutrition. Arch. Internal Med., 59,