Bile Acid inhibition of the intestinal microflora—A function for simple bile acids?

Vol. 61, No.2 Printed in U.S.A.

GASTROK~TEROLOGY

Copyright © 1971 by The Williams & Wilkins Co.

BILE ACID INHIBITION OF THE INTESTINAL MICROFLORA-A FUNCTION FOR SIMPLE BILE ACIDS? MARTIN H. FLOCH, M.D ., WILLIAM GERSHENGOREN, SYLVIA ELLIOTT, AND HowARD M. SPIRO, M .D .

Section of Gastroenterology, Department of Medicine, Yale University School of Medicine, The Yale Affiliated Gastrointestinal Program , and Th e Department of Medicine, The Norwalk Hospital

The predominant groups of intestinal bacteria were tested in vitro for sensitivity to concentrations of bile acids that occur in the intestine. Strains of anaerobic Bacteroides and lactobacilli were easily inhibited by the simple bile acids, cholic and deoxycholic acid, whereas aerobic organisms were less sensitive. Inhibition of streptococci varied with the strain tested but coliforms were not inhibited by simple bile acids. Anaerobic and aerobic organisms alike were rarely inhibited by the conjugated bile acids, taurocholic or glycocholic acid. Growth of common anaerobic intestinal bacteria in vitro is apparently inhibited by simple bile acids but not by conjugated bile acids, a phenomenon in which the possibility that unconjugated bile acids may play a role in controlling bacterial populations within the small and large intestinal lumen is raised. Bile, or its component acids exhibit anti· bacterial activity depending upon the concentration of bile and the susceptibility of a given organisms. 1 In general, bile acids inhibit gram-positive organisms, but have little effect against gram-negative organisms. In man, the primary bile acids, cholic acid and chenodeoxycholic acid, are produced by the liver and secreted into the duodenum as conjugates of glycine and taurine. 2 Deoxycholic acid and lithocholic acid are produced in the ileum by bacterial transformation of the primary acids and enter the enterohepatic circulation to be conjugated and excreted. Bile acids are important end products of liver detoxification and cholesterol metabolism. 2 The only

significant direct function thus far attributed to conjugated bile acids has been the solubilizing of fats. Other than an intermediate biochemical step, no function of free bile acids has been ascertained. In an initial report we described cholic acid inhibition of certain strains of human intestinal bacteria, 3 and here report the effect of free and conjugated bile acids on aerobic and anerobic intestinal organisms in vitro which suggests an additional function for free bile acids in man.

Materials and Methods Intestinal bacteria. Eighteen strains of intestinal organisms were studied (see table 1) . Bacteria were either freshly isolated and identified from human feces • or from American Type Colony Culture (Lactobacillus bifidus no. 11146). Aerobic organisms were maintained in brain heart infusion broth (BHI) and used within 1 week of isolation. Anaerobic organisms were transferred to thioglycolate media enriched with ascitic fluid (2 ml per 20 ml) after isolation, maintained in nitrogen-carbon dioxide

Received May 15, 1970. Accepted February 26, 1971. Address requests for reprints to: Dr. Martin H. Floch, The Norwalk Hospital, Norwalk, Connecticut 06856. This research was supported in part by United States Public Health Service Grant No. 7817 from the Institute of Allergy and Infectious Disease. 228

August 1971

229

BILE ACID INHIBITION OF INTESTINAL MICROFLORA TABLE

1. Concentration of bile acids inhibiting bacterial growth" Bile acids

Bacteria

Gram-negative aerobes Klebsiella aerogenes Escherichia coli Gram-positive aerobes Streptococcus faecalis Streptococcus (group N) Streptococcus (group A) Staphlycoccus aureus Gram-negative anaerobes Bacteroides species Gram-positive anaerobes Streptococcus species Lactobacillus acidophilus Lactobacillus bifidus (Bifidobacterium) Lactobacillus species

No. of strains tested

1 1

ments

2 4

Cholic

Deoxycholic

N• N

N

2 1 1 1

12

2

1.0-N N 0.1-N N

3

15

0.1-1.5

1 1 3

2 6 12

0.5 0.1-1.5 0 . 1- 0.5

3

12

0.1-0.5

4 4

Conjugated

Simple

No. of ex peri-

Taurocholic

Glycoholic

N N 0.5 0.5

0.05-0.5

N N 2.5

N

N

0.05 N 0.1-N 0.1

a Concentration equals g per 100 mi. • N, no inhibition noted.

atmosphere with an oxygen absorbing pad, • and similarly used within 1 week of isolation. Bile acids solutions. Cholic acid (Mann Research Laboratories) was dissolved 3 to maintain a stock solution of 25 mg per ml at a pH of 7.7. Deoxycholic acid (Mann Research Laboratories) was prepared fresh before use by dissolving 2.5 g in 100 ml of Tris buffer at pH 7.7 at 25 C. Taurocholic acid (Mann Research Laboratories) and glycocholic acid (Mann Research Laboratories) stock solutions containing 25 mg per ml were made by dissolving in Tris buffer of pH 7. 7 at 25 C. All test solutions were assayed by thin layer chromatography to evaluate the relative degree of purity. Only minute contamination with deoxycholic acid was noted (see fig. 1). Chenodeoxycholic acid (Mann Research Laboratories) solution was prepared and tested but proved to be heavily contaminated, and results obtained with this bile salt were not considered meaningful. Bacteria-bile acid test system. A series of tubes containing either 0.1 ml of concentrated BHI (12.5 g per 25 ml distilled water 3 for aerobes, lactobacilli, and streptococci), or 1.0 ml of enriched thioglycolate broth solution (0.1 ml ascitic fluid to 0.9 ml thioglycolate for Bacteroides and 0.1 ml BHI to this for Lactobacillus bifidus) and 0.05 ml of the test organism culture at ap-

proximately 18 hr growth were added to the tubes containing stock bile acid solution and sterile Tris buffer (pH 7.2 to 7.9), so that the series of tubes contained 0.0 (control), 0.1, 0.5, 1.0, 5, 10, 15, or 25 mg per ml of the bile acid to be tested. The pH of the test system at the onset of incubation varied from 7.0 to 8.0. The series of tubes were then incubated either aerobically or anaerobically for 24 to 72 hr depending upon the organism to be tested. An aliquot from each tube was then serially diluted 4 and plated on either Schaedler's enriched A media 4 or a selective media in order to obtain a quantitative assessment of the amount of growth at each level of bile acid concentration. The control tube had no bile acid and consequently documented the concentration of the test organism. Inhibition was considered significant when there was at least a depression of growth of 2 logs. A pilot control study was also run to assess whether the phases of bacterial growth of an organism affected the susceptibility to a given bile acid. Two-, 4-, 8-, 12-, 18-, and 24-hr growth of lactobacillus species were tested in a cholic acid system as described above. There was no significant variation in the inhibition of the lactobacilli by cholic acid. This indicates that the growth phase did not affect cholic acid inhibition. However, more detailed experiments

230

FLOCH ET AL.

Vol. 61, No.2

FIG. 1. Thin layer chromatograph of deoxycholic, cholic, and taurocholic acid solutions. Numbers 1, 2, 3, 7, and 10 represent 0.1, 0.5, 1.0, 1.0, and 2.5% taurocholic acid solution (1.0, 5, 10, and 25 mg per ml as described in "Methods"). Numbers 4, 5, 6, 8, and 9 represent 0.1, 0.5, 1.0, 1.0 and 2.5% cholic acid solution. Number 11 represents a 1% deoxycholic acid solution. Note the minimal contamination of the cholic acid solution and relative purity of the taurocholic acid solution.

with other organisms are needed to fully assess this relationship. We selected approximately 18-hr cultures, stationary phase, of the freshly isolated intestinal bacteria for the tests.

Results The growth of 15 of the 18 strains of intestinal bacteria tested was inhibited by one or more of the bile acids. In the circumstances of our experiments all strains of the anaerobes tested were inhibited to some degree by bile acids. None of the gram-negative aerobes, but some grampositive aerobes were inhibited by bile acids. The following reaction of the test system was typical for the inhibition caused by the bile acids described later. 1. Concentration of bile acid required to inhibit growth. Figure 2 demonstrates the inhibition caused by a simple bile acid, cholic acid, against an enterococcus and the lack of effect of a conjugated acid, taurocholic. Control tubes of Streptococci had 10 6 growth which persisted in the presence of graded concentrations of taurocholic acid; but at 0.1 % concentration of cholic acid the growth decreased to 10<, at 0.5%

108

f

107 10 6 105 STREPTOCOCCI 10• GROWTH/ml 10 3

TAUROCHOLIC ACID \

·--------·~.----·~............

b, ',

''0

',

10 2 10

'\

',\ I

CHOLIC ACID

.

0----~----~----~

I

5

10

15

20

25

CONCENTRATION OF BILE ACID (mg/ml)

FIG. 2. Concentrations of cholic acid (abscissa) causing inhibition of streptococcal (Enterococcus) growth (logarithmic colony counts on ordinate). Note that inhibition occurred at 1 mg per ml, a 0.1% solution, and complete inhibition of growth at 10 mg per mi. Taurocholic acid produced no inhibition.

fell to 10 3 , and at 1.0% was completely inhibited. This curve of inhibition was noted in all experiments. In table 1 the concentration of bile acids causing at least a 10 2 depression (two tubes equals 2 logs) of growth are listed for each group of organisms tested. In all experiments the growth was completely

August 1971

BILE ACID INHIBITION OF INTESTINAL MICROFLORA

inhibited at 5 to 10 times the concentration at which initial inhibition was noted (fig. 2). 2. Effects of individual bile acids. Cholic acid. Anaerobic Bacteroides, Lactobacillus, and Streptococcus were all sensitive to this bile acid. Although a 0.1 % solution usually produced inhibition, occasionally the same strain tolerated a higher concentration and inhibition did not occur until a 0.5 or 1.0% solution was used, presumably as a result of variation in the number of organisms inoculated, or because of the phase of growth at the time of inoculation. Although this phenomenon was observed, inhibition was invariably complete at a 0.5% solution. Deoxycholic acid. Aerobic and anaerobic streptococci, Bacteroides, and some freshly isolated lactobacilli were inhibited. Most strains of L. acidophilus and L. bifidus were not inhibited. Conjugated bile acids. Both taurocholic and glycocholic acids were ineffective in inhibiting the intestinal organisms. Only in one experiment was some inhibition noted where a concentration of 2.5% of taurocholic acid inhibited a {3 hemolytic Group A streptococcus organism. Discussion This study shows that physiologic concentrations of simple bile acids 5 readily inhibit anaerobic human intestinal bacteria in vitro, but conjugated bile acids rarely do. As the strains of bacteria employed in these experiments were freshly isolated from human feces and subcultured on bile free media, bile sensitivity could not have developed in vitro. Consequently the bile sensitivity presumably may exist in vivo. The fresh strains of bacteria tested were those most commonly recovered from the small and large intestine of man. 4 • 6 The predominant anaerobes of man, Bacteroides species and lactobacilli species (including the Bifidobacterium) were easily inhibited by cholic and deoxycholic acid. Coliform organisms, the common aerobes were not inhibited; streptococci showed the greatest variability of reaction, some strains demonstrating sensitivity while others were completely resistant. The phenomena cif

231

enteric bacterial strain sensitivity to cholic acid has been observed but not studied in detail by others. 7 • 8 In a recent series of experiments, it was demonstrated that a 0.5% solution of cholic acid often inhibited bacterial degradations of cholate, but, when the concentration of the bile acid was lowered, the bacteria easily performed transformations. 9 All bile acids have a lytic action on the bacterial wall which release enzymes that may dissolve the cells, as occurs with pneumococci, or kill the cell destroying the enzyme so that lysis does not occur, as with streptococci. 1 • 10 Deoxycholic acid, cholic acid, and some unsaturated fatty acids have the highest degree of this type of bacteriocidal activity. 11 Differences in antibacterial activity are related to the number and position of the hydroxyl radical of the steroid nucleus. 1 2 Because of the initial description of lysis of pneumococci 11 most research in this field has been performed on organisms isolated from the mouth or respiratory tracts. There has been little research concerning these phenomenon as they relate to the common anaerobic intestinal organisms, but recent clinical studies have stressed the role of bacteria in deconjugating and transforming bile acids. 7 • 13 - 1 6 Past studies revealed that intestinal bacteria increase the turnover and metabolic rate of cholic acid in germ-free animals 17 • 1 8 ; they also produce the enzyme necessary to transform bile acids by oxidizing hydroxyl groups at C-3, C-7, and C-12 bonds of the steroid nucleus as well as to deconjugate and free taurine and glycine. 1 9 Aerobic enterococci and anaerobic Clostridia were first shown to deconjugate bile acids. 2 0 • 2 1 Now it is clear that a wide variety of intestinal bacteria including anaerobic Bacteroides and lactobacilli can degrade and transform bile salts. 7 - 9 • 19 - 21 What controls the growth of bacterial populations in the small bowel? Certainly it is a very complex system . The jejunum is ordinarily relatively free from any significant bacterial growth. 22. 23 In the ileum, the flora becomes abundant 2 4 · 25 where deconjugation and transformation of bile acids occur. Disorders of the small bowel permit bacterial overgrowth in the jejunum and re-

232

Vol. 61, No.2

FLOCH ET AL.

suit in disturbed bile acid metabolism. 14 • 16 Gastric acidity 26 • 27 and normal peristalsis 28 are partially responsible for the relative sterility of the upper small bowel. In the presence of achlorhydria, stasis of the small bowel, or operative resection, the duodenum and jejunum are contaminated with large numbers of organisms which normally exist only in the lower small bowel and colon. 27 ' 33 Other factors such as mucus, 34 lysozyme, 35 bacterial by-products (such as "colicines") 32 • 36 and fatty acids 37 • 38 have been suggested as possible mechanisms which control the intestinal flora. Probably all of these, plus numerous as yet unknown factors, act in a complex system to maintain the balance between host and flora. This in vitro study suggests that simple bile acids may be one more mechanism which contributes to the control of intestinal bacterial populations. Gastric acidity and normal motility probably play a major role in keeping upper small bowel bacterial populations at very low levels; whereas chemical antimicrobial agents as fatty acids, colicines, and bile acids may have selective effects in the lower small bowel and colon to maintain the quantitative relationships (rather than sterility) that are so reproducibly measured from the colon of a given individual. 4 • 6 Bacteroides, lactobacilli, coliforms, and streptococci are invariably recovered in descending frequency with the former at a concentration of 10 s- 11 colonies per g of wet feces and the latter 10 4 • 7 depending on the subject. It is postulated that many intraluminal intestinal chemical factors, in combination, bile acids being one, help maintain this reproducible bacterial balance. The experiments in this study were performed in vitro at bile acid concentrations noted in bile. More information is needed on the effects of: (1) physical and chemical factors within the gut lumen, (2) combinations of bile acids with other chemicals, and (3) bile acid deficiency states, on intestinal bacteria before this study can be more meaningful. At present our observations are introductory. The in vitro results do not accurately compare to in vivo intraluminal

conditions and should be accepted as only suggestive that simple bile acids have an in vivo effect on intestinal bacterial populations. REFERENCES 1. Stacey M, Webb M: Studies on the antibacterial properties of the bile acids and some compounds derived from cholanic acid. Proc Roy Soc 134: 523-537, 1947 2. Harper HA: Review of physiological chemistry. Lange Medical Publications, 1967, p 207-208 3. Floch MH, Gershengoren W, Diamond S, et a!: Cholic acid inhibition of intestinal bacteria. Amer J Clin Nutr 23:8- 10, 1970 4. Floch MH, Gershengoren W, Freedman LR: Methods for the quantitative study of the aerobic and anaerobic intestinal bacterial flora of man. Yale J Bioi Med 41 :50-61, 1968 5. Nakayama F: Quantitative microanalysis of bile. J Lab Clin Med 69:594-609, 1967 6. Gorbach SL, Nahas L, Lerner PI, eta!: Studies of intestinal microflora. I. Effects of diet, age, and periodic sampling on numbers of fecal microorganisms in man. Gastroenterology 53:845-855 1967 7. Drasar BS, Hill MJ, Shiner M : The deconjugation of bile salts by human intestinal bacteria. Lancet 1:1237-1238, 1966 8. Hill MJ, Drasar BS: Degradation of bile salts by human intestinal bacteria. Gut 9:22-27, 1968 9. Aires V, Crowther JS, Drasar BS, eta! : Degradation of bile salts by human intestinal bacteria. Gut 10:575-576, 1969 10. Dubos RJ: Mechanism of the lysis of pneumococci by freezing and thawing, bile, and other agents. J Exp Med 66:101-112, 1937 11. Kozolowski A: Comparative studies of the action on the pneumococcus of bile acids and unsatu· rated fatty acids, found in bile in the form of soaps. J Exp Med 42:453-463, 1925 12. Downie AW, Stent L, White SM : The bile solubility of pneumococcus, with special reference to the chemical structure of various bile salts. Brit J Exp Path 12:1- 9, 1931 13. Rosenberg IH, Hardison WG, Bull DM: Abnormal bile salt patterns and intestinal bacterial overgrowth associated with malabsorption. New Eng J Med 276:1391-1396, 1967 14. Tabaqchali S, Booth CC : Jejunal bacteriology and bile salt metabolism in patients with intestinal malabsorption. Lancet 2:12- 15, 1966 15. Floch MH: Bacteria, absorption and malabsorption. Amer J Clin Nutr 20:1244-1248, 1967 16. Shimada K, Bricknell KS, Finegold SM: Deconjugation of bile acids by intestinal bacteria: Re-

August 1971

BILE ACID INHIBITION OF INTESTINAL MICROFLORA

view of literature and additional studies. J Infect Dis 119:273-280, 1969 17. Gustafsson BE, Norman A, Sjovall J: Influence of E. coli infection on turnover and metabolism of cholic acid in germ-free rats. Arch Biochem Biophys 91 :93-100, 1960 18. Gustafsson BE, Bergstrom S, Lindstedt S, et a!: Turnover and nature of fecal .bile acids in germ free and infected rats fed cholic acid-24- 1 'C bile acids and steroids. Proc Soc Exp Bioi 94:967-971, 1957 19. Midtvedt T, Norman A: Bile acid transformation by microbiol strains belonging to genera found in intestinal contents. Acta Path Microbiol Scand 71:629-638, 1967 20. Norman A, Grubb R: Hydrolysis of conjugated bile acids by clostridia and enterococci. Acta Path Microbiol Scand 36:537-547, 1955 21. Norman A, Bergman S : The action of intestinal microorganisms on bile acids. Acta Chern Scand 14:1781-1789, 1960 22. Kaiser MH, Cohen R, Artega I, et al: Normal viral and bacterial flora of the human small and large intestine. New Eng! J Med 274:500-505, 558-563, 1966 23. Gorbach SL, Plaut AG, Nahas L, eta!: Studies of intestinal microflora. II. Microorganisms of the small intestine and their relations to oral and fecal flora. Gastroenterology 53:856-867, 1967 24. Donaldson RM Jr: Normal bacterial populations of intestine and their relation to intestinal function. New Eng J Med 270:938-946, 944- 1000, 1050-1056, 1964 25. Gorbach SL, Nahas L, Weinstein L, eta!: Studies of intestinal microflora IV. The microflora of ileostomy effluent: a unique microbial ecology. Gastroenterology 53 :874-880, 1967 26. Drasar BS, Shiner M, McLeod GM: Studies on

27.

28. 29.

30.

31.

32. 33.

34.

35.

36. 37.

38.

233

the intestinal flora. I. The bacterial flora of the gastrointestinal tract in healthy and achlorhydric persons. Gastroenterology 56:71-79, 1969 Franklin MA, Skoryna SC: Studies on natural gastric flora: I. Bacterial flora of fasting human subjects. Canad Med Ass J 95:1349-1355, 1966 Dixon JM: The fate of bacteria in the small intestine. J Path Bact 79:131-140, 1960 Drasar BS, Shiner M: Studies on the intestinal flora II Bacterial flora of the small intestine in patients with gastrointestinal disorders. Gut 10:812819, 1969 Delliapiani AW, Girdwood RH: Bacterial changes in small intestine in malabsorptive states and in pernicious anemia. Clin Sci 26:359-374, 1964 Tabaqchali S, Hatzioannov J, Booth CC: Bile salt deconjugation and steatorrhea in patients with a stagnant-loop syndrome. Lancet 2:10-13, 1968 Donaldson RM: Role of intestinal microorganisms in malabsorption. Fed Proc 26:1426-1429, 1967 Wirts CW, Goldstein F: Studies of the mechanism of post gastrectomy steatorrhea. Ann Intern Med 58:25-36, 1963 Goldsworthy NE, Florey H: Some properties of mucus, with special reference to its antibacterial functions. Brit J Exp Path 11:192- 208, 1930 Wilson GS, Miles AA: Principles of bacteriology and immunity. Baltimore, Williams & Wilkins Co, 1964, p 1253-1254 Halbert SP: Antagonism of coliform bacteria against Shigellae. J Immun 58:153-157, 1948 Nieman C: Influence of trace amounts of fatty acids on the growth of microorganisms. Bact Rev 18:147-163, 1954 Fuller R, Moore JH: The inhibition of the growth of Clostridium welchii by lipids isolated from the contents of the small intestine of the pig. J Gen Microbiol 46:23-41, 1967