Bacterial Degradation of Gastrointestinal Mucins

Vo]' 54, No.2

GASTROENTEROLOGY

Copyright © 1968 by The Williams & Wilkins Co.

Printed in U.S.A.

BACTERIAL DEGRADATION OF GASTROINTESTINAL MUCINS II. Bacterial origin of fecal ABH(O) blood group antigen-destroying enzyllles LANSING

C.

HOSKINS,

M.D.

Gastroenterology Service, Veterans Administration Hospital, and Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio

The preceding papert furnished evidence that intestinal bacteria degrade gastrointestinal mucins and especially the carbohydrate moieties of these mucins. Since glycoproteins possessing ABH (0) blood group antigen activity are one part of the epithelial mucins secreted in the proximal gastrointestinal tract of man,2-4 degradation of ABH antigen activity can be an indicator of mucin degradation. The extensive decrease of blood group A antigen titer that was noted during incubation of the nondialyzable fraction of germ-free rat stools with fecal bacteria in vitrol suggested that intestinal microorganisms degraded mucin glycoproteins having blood group antigen activity. How this occurred was not clarified. However, it is known that feces of man, dogs, and rats contain enzymes capable of degrading the waterReceived July 7, 1967. Accepted August 26, 1967. Presented in part at the Fifty-First Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, Illinois, April 17,1967. Address request for reprints to: Dr. Lansing C. Hoskins, Veterans Administration Hospital, Cleveland, Ohio 44106. This investigation was supported by a research grant from the Veterans Administration. The author gratefully acknowledges the technical assistance of Margaret J. Woods and Lillian Hejduk. The experiment describing the effect of oral neomycin on fecal blood groupdestroying enzyme activity mentioned in the discussion section was performed in Dr. Norman Zamcheck's laboratory at The Mallory Institute of Pathology, Boston City Hospital, Boston, Massachusetts. The author gratefully acknowledges Dr. Zamcheck's advice and encouragement in t IlPse studies.

soluble ABH(O) blood group antigens. 5 - s Previous studies of these enzymes did not clearly establish whether they were produced solely by intestinal bacteria or were also produced by the host's intestinal mucosa or digestive organs. To define further the role of intestinal microorganisms in gut mucus degradation, I performed studies whose purpose was to establish the origin of fecal blood group antigen-destroying enzymes. This paper presents evidence that fecal blood groupdestroying (BGD) enzymes are produced by intestinal microorganisms and not by the host's tissues. The experimental evidence has three parts: (1) comparison of BGD enzyme activity in the stools of germ-free and conventional rats, (2) comparison of BGD enzyme activity in stools and gut tissue homogenates from conventional rats, and (3) demonstration that BGD enzyme activity forms in anaerobic culture media inoculated with conventional rat feces. Jejunal mucosa from one human subject whose stools consistently contain BGD enzyme activity was also assayed for this activity.

Materials and Methods Stools were collected from unfasted germfree and conventional rats. (The stools from germ-free rats were obtained through the courtesy of the Charles River Breeding Laboratories, Inc., Wilmington, Mass.). Blood agar and EMB plate cultures of the germ-free rat stools showed no growth after 72 hr at 37 C. The stools from both types of rats were weighed and homogenized separa tely for 90 sec in a small homogenizer with enough phosphate-buffered 0.15 M NaCl, pH 6.4, to make a 10% wlv suspension. Successive centrifugations at 700 X g and 20,000 X g removed 218

February 1968

BACTERIAL DEGRADATION OF MUCINS. II

particulate matter. The clear supernatant fluid, hereafter called "stool extract," was assayed for BGD activity. The protein concentration in these extracts was determined following dialysis of a measured volume of each extract against five 2-liter volumes of distilled water at 4 C. Tissue homogenates were prepared from unfasted male conventional rats anesthetized with sodium amy tal, 0.09 mg per g of body wt ip. The stomach and intestines were opened and flushed clean of mucus and contents with ice-cold saline. The mucosae of small intestine, cecum, and colon were removed by gently scraping the surface with a blunt spatula. Ten per cent wlv homogenates of whole stomach, liver, pancreas, small intestinal mucosa, and combined mucosa of cecum and colon were prepared in ice-cold 0.15 M NaCl by 4 min of homogenization in a chilled homogenizer. Following centrifugation for 20 min at 700 X (j, the opalescent supernatant fractions of these tissue homogenates were assayed for enzyme activities and protein concentration. Blood group-destroying activity in the fecal or tissue extracts was measured by determining the decrease of blood group antigen substrate titer during incubation with the extract. Equal volumes of supernatant fluid from fecal or tissue homogenate and blood group antigen substrate were incubated at 37 C under toluene. Aliquots were removed immediately after mixing and at various intervals thereafter were heated for 10 min in a boiling water bath to inactivate BGD enzyme activity, and the titer of antigen remaining in each aliquot was determined by hemagglutination inhibition. A decreasing titer of antigen during incubation indicated the presence of BGD activity in the extract. No change in antigen substrate titer during incubation indicated absence of BGD activity. BGD activity is expressed as An, the dpcrease in antigen titer, during the period of incubation. The blood group antigen substrates were (1) saliva from a blood group B secretor containing a high titer of B antigen, heated for 10 min at 100 C, and dialyzed against 0.02 ~I phosphate buffer, pH 6.4, containing 0.01 I\I l\lgCI 2 , and (2) a 1 mg per ml solution in the same buffer of hog gastric mucin, N.N.R. (Nutritional Biochemicals, Inc., Cleveland, Ohio, containing high titers of A and H antigens. Blood group antigen titer was determined hy a hemagglutination inhibition procedure. To serial 2-fold dilutions of sample was added 0.1 ml (20 hemagglutinating units) of

219

standardized commercial human immune antiA or anti-B sera, or Ulex europeus seed extract (anti-H). After 30 min at 37 C, 0.1 ml of a 4% wIv suspension of human A, B, or 0 red cells prewashed four times in saline was added and the presence or absence of hemagglutination was determined after 60min incubation at 37 C and 1- to 2-min centrifugation at 1200 rpm. The antigen titer in the sample is defined as the highest dilution of sample capable of inhibiting hemagglutination and is expressed as the exponent, n, of serial Y2" dilutions of sample at the hemagglutination end point. The end point in replicate tests using 2-fold dilutions rarely varied more than ± 1 dilution. In two experiments the ,B-galactosidase and maltase activities in the supernatant fraction from gut tissue and fecal homogenates of conventional rats were also measured. ,B-Galactosidase was assayed by the method of Lederberg9 on aliquots preadjusted to pH 5.0 with acetate buffer,'o and maltase was assayed by the method of Dahlqvist" on aliquots preadjusted to pH 6.5 with phosphate buffer." Protein concentration was measured by Eggstein and Kreutz's" modification of the method of Lowry et aL" using bovine serum albumin (Cohn fraction IV) (Nutritional Biochemicals, Inc., Cleveland, Ohio) as reference standard. To determine whether fecal bacteria produced BGD enzymes, 0.5-g portions of fresh stool obtained just prior to anesthesia and cecal contents from conventional rats were each suspended in 100 ml of sterile thioglycollate broth containing 0.1% D-glucose and incubated at 37 C. Aliquots of these cultures were removed at various times during 4 days of incubation and centrifuged at 10,000 X g. The supernatant fluid was then filtered through a 0.45 f.' Millipore filter to remove bacteria and was dialyzed against distilled water. Protein and BGD assays were then performed on the dialyzed supernatant fraction.

Results Comparison of BGD enzyme activity in the stools of germ-free and conventional rats. Two experiments similar in over-all design were performed. In the first study incubation of stool extracts with blood group antigen substrates was carried out for 4 hr; in the second study incubation was extended to 24 hr. The results, summarized for the latter experiment in figure

Vol.

HOSKINS

220 A ANTIGEN

B ANTI GEN

1

0-0

lsi

8

8 c

c

(\J

(\J

~

w

n 10

0

0

c

z

(\J

~

12

w

\X

i=

\

Z

« 8 « u. 0

a: ~ 4


i=

z

«4

~X

zS

~

I--

z

U.

0

152

t:

«4

~

I--

0

0 0

IS

8

24

\

w

It--X

al

I--

~

\

zS

X


H ANTIGEN

n 10

n 20

54, No.2

:I: U.

0

~

24 0 IS 8 INCUBATION TI ME, HOURS

X~

152 I-I--

0 0

IS

8

24

FIG. 1. Comparison of ABH(O) blood group antigen-destroying activity in extracts of stools from germ-free rats (0) and from 2 conventional rats (., X). The protein concentration of each extract in the incubation mixtures is 0.33 mg per ml, pH 6.4, temp 37C.

=

1. ABH(O) blood group-destroying (BGD) and glycosidase activities of tissue homogenates and stool extracts of a conventional rat

TABLE

Tissue

BGD activProtein ity" (L\.n/24 p- I hr) vs. Galac- Maltconen of homogase tosienate dase A B H ---

mg/ml

Whole stomach ..... Whole pancreas .....

- -

5.6 9.8

0 0 0 0

7.0 5.8 2.3

0 0 0 0

-- - -b units units

0 7.9 0

C

2.1 22

~Iucosa

Small intestine ... Cecum-colon. .... Stool extract ......

038 339 8.1 035 14 14 ?,1O 2.0 7.3

"BGD activity = An, the decrease in antigen titer, from beginning to end (24 hr) of incubation at pH 6.4, 37 C. Antigen titer is expressed as the exponent, n, of that serial 72" dilution of incubation mixture which was the highest dilution capable of inhibiting hemagglutination. b Millimicromoles of o-nitrophenyl-J3-D-galactoside hydrolyzed per minute per milligram of protein at pH 5.0, 37 C. C Millimicromoles of maltose hydrolyzed per minute per milligram of protein at pH 6.5, 37 C.

1, were similar in both experiments. Stool extracts of germ-free animals failed to diminish the titer of A, B, or H antigen substrate after 24 hr of incubation, indicat-

=

ing absence of BGD enzyme activity in these extracts, whereas extensive loss of A, B, and H antigens occurred during incubation with conventional rat stool extracts. Lack of BGD activity in the germ-free stool extract might have been due to enzyme inhibitors which were not present in the conventional rat stool extracts. A test for enzyme inhibitors was done as follows. BGD assays were performed on conventional rat stool extracts (protein concn = 0.68 mg per ml) with and without added germ-free stool extract (protein concn = 0.67 mg per m!). The rate at which the conventional stool extract destroyed A or H antigen at 2, 4, and 6 hr was no different in the presence of germ-free stool extract than in its absence, indicating that inhibitors of A- and H-destroying activity were not present in the germ-free stool extract. Comparison of BGD enzyme activity in stools, gut tissue homogenates, and cell-free fecal bacterial cultures from conventional rats. Table 1 shows the results observed when gut tissue homogenates and stool extracts from 1 conventional rat were assayed for BGD enzyme and disaccharidase activities. The tissue homogenates showed no BGD activity against A, B, or H anti-

Februa,·y 1968

221

BACTERIAL DEGRADATION OF MUCINS. II

gen after 24 hr of incubation with antigen substrate. In contrast, the stool extract had potent BGD activity. The presence of the two disaccharidases in the homogenates indicat es that the method of preparation was adequate to preserve BGD enzyme activity had it existed in these tissues. Table 2 shows the results observed when mucosal homogenates, dialyzed stool extract, and anaerobic cultures of stool and cecal contents from another conventional rat were assayed for BGD enzyme activity. Again, none was demonstrated in the intestinal homogenates, but considerable activity was present in the dialyzed stool extract. In addition, BGD enzyme activity developed in the bacterial cultures of stool and cecal contents to levels approaching that found in the stool extract. No BGD activity was detected in intestinal mucosal homogenates from 4 rats despite its presence in their stools. Even after 7 days of incubation at 37 C with mucosal homogenates of small intestine or cecum-colon, no decrease in the titer of A antigen substrate was noted. Liver homogenate of 1 animal (protein concn = 13 mg per ml) had no demonstrable BGD activity against A or H antigen, and liver homogenate from a 2nd animal contained no demonstrable BGD activity against B antigen during 72 hr of incubation. Although BGD activity enzyme was present in the stools from each of 6 conventional rats, it was not demonstrable in homogenates of cecal contents from 2 of these animals although it developed in the anaerobic culture of cecal contents from 2 others. Mucosal homogenates of small intestine and cecum-colon were tested for presence of BGD enzyme inhibitors in two separate experiments by the same method used for the germ-free stool extracts. Small intestinal mucosal homogenate added with conventional stool extract to A antigen substrate did not inhibit the rate of A antigen destruction. Although added cecum-colon homogenate did not inhibit the initial rate at which stool extract degraded A antigen, less degradation of A antigen was noted after 1 hr of incubation in the pres-

2. BGD activity of tissue homogenates , stool extract, and cell-free anaerobic cultures of stool and cecal contents of a conventional rat

TABLE

Sample

Protein conen of sample

BGD activity" (ll.n/24 hr) vs.

A ~~

mg/m/

Mucosa Small intestine. .. Cecum-colon . . . . . Dialyzed stool extract ........... Stool culture o timeb . . . . . .. . .. 24 hr. .... . . ... ... ... 96 hr ...... Cecal contents culture 24 hr . . ... . . .. ... . 96 hr ... ........ . .

B

H

----

5.6 4.6

0 0

0 0

0 .46

18

8

0.50 0.48 0.32

0 14 16

0 4 6

0 4 6

0.45 0.43

8 16

0 8

4 6

a BGD activity = ton, the decrease in antigen titer, from beginning to end (24 hr) of incubation at pH 6.4, 37 C. Ant igen titer is expressed as the exponent, n, of that serial ~n dilution of incubation mixture which was the highest dilution capable of inhibiting hemagglutination. b Aliquot obtained immediately after stool inoculum was thoroughly suspended in culture medium.

ence of cecum-colon homogenate than was noted in its absence. Therefore, while it is unlikely that small intestinal mucosa contained a BGD enzyme inhibitor, the possibility of partial inhibition by mucosal homogenates of cecum-colon cannot be excluded by the methods used in these studies. BGD enzyme activity in human jejunal mucosa. A peroral mucosal biopsy was obtained from the author's jejunum at the ligament of Treitz. The mucosal homogenate was prepared and assayed for blood group B-destroying enzyme activity by the same methods that were used for the rat tissues. Incubation with B antigen substrate for 24 hr revealed no evidence of Bdestroying enzyme activity in the mucosal homogenate, whereas the author's stool extracts and fecal anaerobic bacterial cultures consistently showed high B antigendestroying enzyme activity.

222

HOSKINS

Discussion

Thirty-six years ago, Schiff and his coworkers discovered that human saliva and stool contained enzymes capable of destroying water-soluble blood group antigens. 5 - 7 Workers disagreed concerning the source of this enzymatic activity. Some held that it was solely bacterial in origin l5-l6 whereas Schiff and co-workers maintained that it was derived at least in part from body secretions.5 - 6 Witebsky and Neter 17 assayed group A antigen-destroying enzyme activity at various levels of the human intestinal tract and found it absent in the jejunum, present in the terminal ileum, and strongest in colonic contents. Its distribution, therefore, paralleled that of the human intestinal fiora. ls There is now abundant evidence that certain microorganisms elaborate glycosidases capable of destroying ABH (0) antigens,19-23 and at least one strain of Clostridium welchi haying this property was isolated from human stool. 24 However, the question whether enteric microorganisms are the sole source of fecal blood group-destroying activity was neither pursued nor settled. The experiments reported here indicate that the fecal blood group-destroying activity is derived from intestinal bacteria. This actiyity develops in bacterial cultures of both stool and cecal contents, is not detectable in homogenates of gut tissues from the same rat, and is absent in the ~too ls of the germ-free rat. Additional, less direct, evidence was obtained in another experiment in which levels of fecal BGD actiyity were measured in the stools of each of 2 rats before, during, and after administration of neomycin sulfate in their drinking water. When the oral dose of antibiotic wa s sufficient to decrease the fecal Gram-negative aerobe count nearly to zero, BGD activity diminished, but it retul'llcd to pretreatm ent levels after neomycin was discontinued and the aerobe count had returned to pretreatment levels. It is likely that the observations reported here from studies on rats and one human jejunal mucosal biopsy are applicable to man. Although Schiff et al. 25 and Hartmalln and Hartmann 26 reported weak ac-

Vol. 54, No.2

tivity in various human tissues obtained at autopsy, they did not test gastrointestinal tissues, and maintenance of sterility in their studies is open to question. The microorganisms (s) responsible for fecal BGD activity has not been clearly identified. Strains of several species of the genus Clostridium elaborate exocellular blood group-destroying enzymes. Our review of the literature reveals two reports of microorgansims isolated from human feces having BGD activity, one being the strain of C. welchi isolated by Schiff,24 and other being Lactobacillus bifidus val'. Penn isolated by Gyorgy et al. l9 from breast-fed infants' stool. Despite the ubiquity of clostridia and lactobacilli in mammalian stools, preliminary efforts in this laboratory to identify the responsible organism (s) have not been successful. In culture studies of the stools of 1 human subject with potent fecal B antigen-destroying activity the responsible organism appears to be one or more heat-sensitive obligate anaerobes. BGD enzymes are consistently produced in anaerobic thioglycollate liquid cultures inoculated with feces from this individual. Well aerated cultures and cultures heated 30 min at 60 C after inoculation with the same stool have not produced BGD enzymes. Both C. welchi and an anaerobe morphologically resembling L. bifidus isolated from this subject's stools failed to produce B antigen-destroying activity when grown in anaerobic liquid media. On the basis of limited but similar studies, the organism(s) producing BGD enzymes in rat stools also appears to be an obligate anaerobe. Although not yet established, it is reasonable to infer from their bacterial origin and the known activity of bacterial BGD enzymes 19 - 22 that the fecal BGD enzymes are glycosidases. Use has been made of BGD enzymes from microorganisms to split the terminal sugars which confer ABH antigen specificity off from blood group substances. It is noteworthy that pancreas and intestinal mucosa lack BGD activity despite the abundance of amylase in the former and a variety of disaccharidaseR and other glycosidases in the latter.l2 The terminal glycosides conferring A, B, and

February 1968

BACTERIAL DEGRADATION OF MUCINS. II

H specificity in human blood group substances are a - N(acetyl) - n - galactosaminoyl-(1 ~ 3)-, a-n-galactosyl-(l ~ 3)-, and a-L-fucosyl- (1 ~ 2) -, respectively.4 Inability of pancreas and intestinal mucosa to cleave these glycoside bonds, despite the presence of glycosidases capable of cleaving a-n-glucosyl-(l ~ 4)- and ,B-n-galactosyl- (1 ~ 4) -glycosides as demonstrated in table 1, underscores the high specificity of individual glycosidases and strongly suggests that the carbohydrate moieties of gut mucins are not degraded by host enzymes secreted into the gut lumen. Rat fecal BGD enzymes show no apparent specificity toward A, B, or H antigen (fig. 1) even though A and H antigens are present in high titer and B in very low titer in their gut mucous secretions. In contrast, human fecal BGD enzymes excreted by group A or group B secretors show marked specificity toward whichever of these antigens, A or B, is secreted by the individual in his gut mucous secretions. 27 It is probable that fecal BGD enzymes constitute one group of perhaps a number of bacterial glycosidases active in the stepwise degradation of oligosaccharide moieties of mucus, releasing carbohydrates utilized by the bacteri·a for their nutrition.l Summary

The source of ABO blood group antigendestroying enzymes in mammalian stools was investigated. 1. The stools of conventional rats but not those of germ-free rats contained blood group-destroying (BGD) enzyme activity. 2. BGD enzyme activity was present in fecal homogenates of conventional rats and developed in liquid anaerobic bacterial cultures of stool and cecal contents during their incubation at 37 C. Homogenates of stomach, pancreas, liver, small intestinal mucosa, and mucosa of cecum and colon possessed no demonstrable BGD enzyme activity. 3. Jejunal mucosa from one human subject whose stools consistently contain BGD enzyme activity had no demonstrable BGD enzyme activity.

223

It is concluded that fecal blood groupdestroying enzyme activity arises from intestinal microorganisms. REFERENCES 1. Hoskins, L. C., and N. Zamcheck. 1968. Bacterial degradation of gastrointestinal mucins. 1. Comparison of mucus constituents in the stools of germ-free and conventional rats. Gastroenterology 54: 210-217. 2. Szulman, A. E. 1960. The histological distribution of blood group substances A and B in man. J. Exp. Med. 111: 785-800. 3. Szulman, A. E . 1962. The histological distribution of the blood group substances in man as disclosed by immunofluorescence. II. The H antigen and its relation to A and B antigens. J. Exp. Med. 115: 977-996. 4. Hoskins, L. C. 1967. The ABO blood group antigens and their secretion by healthy and diseased gastric mucosa. Ann. N. Y. Acad. Sci. 140 CArt. 2): 848-860. 5. Schiff, F ., and M. Akune. 1931. Blutgruppen und Physiologie. Munchen. Med. Wschr. 7'8: 657-660. 6. Schiff, F., and G. Weiler. 1931. Fermente und Blutgruppen 1. Biochem. Z. 235: 454-465. 7. Schiff, F., and G. Weiler. 1931. Fermente lind Blutgruppen II. Biochem. Z. 239: 489-492. 8. Eisler, M . 1932. Weitere Versuche uber das Verhalten von Antigenen gegenober verscheiden Stuhlextrakten. Z. Immunitaetsforsch. 77 : 472-476. 9. Lederberg, J. 1950. Beta-D-galactosidase of Escherichia coli, strain K-12. J. Bact. 60: 381-392. 10. HSll, L., and A. L. Tappel. 1965. Lysosomal enzymes and mucopolysaccharides in the gastrointestinal tract of the rat and pig. Biochim. Biophys. Acta 101: 83-89. 11. Dahlqvist, A. 1964. Method for assay of intestinal disaccharidases. Anal. Biochem. 'I: 18-25. 12. Dahlqvist, A. 1963. Rat-intestinal dextranase. Localization and relation to the other carbohydrases of the digestiye tract. Biochem. J. S6: 72-76. 13. Eggstein, M., and F. H. Kreutz. 1955. Vergleichende Untersuchungen zur quantitativen Eiweissbestimmung im Liquor lind eiweissarmen Nosungen. Klin. Wschr. 33: 879-884. 14. Lowry, O. H ., N. J. Rosebrough, A. L . Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. BioI. Chem. 193: 265-275. 15. Witebsky, E., and T. Satoh. 1933. Zur Frage des Blu tgruppenferments und del' Auschei-

HOSKINS

224

dung von Blutgruppensubstanz. Klin. Wschr. 1 : 948-949. 16. Siewrs, O. 1935. Beitrage zur Kenntnis der

17.

18.

19.

20.

Wirkung des sogenannten "Blutgruppenferment s" im Speichel. Z. Immunitaetsforsch . 85: 163-180. Witebsky, E., and E. Neter. 1935. Distribution of blood group properties and blood group property destroying factors in the intestinal tract of man. J. Exp. Med. 62 : 589-598. Donaldson, R. M. 1964. Normal bacterial populat ions of the intestine and their relation to intestinal function. New Eng. J . Med. 270: 938-945. GYOl'g~·. P., C. S. Rose, and G. F. Springer. 1954. Enzymatic inactivation of bifidus factor and blood group substances. J. Lab. Clin. Med. 43 : 543-552. Buchanan, D. J ., M. J . Crumpton, and W . T. J. Morgan. 1957. Studies in immunochemistry. 17. The decomposition of serologically specific H substance isolated from pig gastric mucin and human ovarian cyst fluids by an enzyme preparation obtained from CI. welchii (Type B). Biochem. J.

65: 186-196. 21. Naylor, I., and H. Baer. 1959. Studies of an

enzyme

produced by Bacillus

f1tlminans

Vol. 54, No.2

that inactivates blood group 0 substance. J . Bact. 77: 771-775. 22. Watkins, W. M. 1959. Enzymes of Trichomonas foetus. The action of cell-free extracts on blood group substances and low-molecular weight glycosides. Biochem. J. 71: 261-274. 23. Marcus, D . M., E. A. Kabat, and G. Schiffman. 1964. Immunochemical studies on

blood groups. XXXI. Destruction of blood group A activity by an enzyme from Clostridium tertium which deacetylates N-acetylgalactosamine in intact blood group substances. Biochemistry (Wash.) 3: 437-

443. 24. Schiff, F. 1935. Uber den Abbau gruppenspezi-

fischer Substanzen durch Bacterien . Klin. Wschr.1: 750-751. 25. Schiff, F., M. Cahen, and A. Stimpfi. 1932. Uber Blutgruppenfermente. Zbl. Bakt. (Orig.) Abt. I 106: 333-334. 26. Hartmann, G., and J . Hartmann. 1941. Destruction of group antigens by enzymes. Acta Path. Microbiol. Scand. 18: 367-388. 27. Hoskins, L. C. 1967. Specificity of fecal blood group-destroying enzymes from intestinal bacteria towards t heir host's blood group antigens (abstr.) . Clin. Res. 25: 234.