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Mucin degradation in human colon ecosystems
GASTROENTEROLOGY
1981;81:759-65
Mucin Degradation in Human Colon Ecosystems Fecal Population Densities of Mucin-Degrading Bacteria Estimated by a “Most Probable Number” Method RONALD
S. MILLER and LANSING
C. HOSKINS
Division of Gastroenterology, Veterans Administration Medical Center and Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio
Bacteria in anaerobic human fecal cultures degrade the oligosaccharide and protein moieties of gut mutin glycoproteins, but most bacterial isolates from feces are unable to degrade mucin. In order to define better the bacterial populations that degrade gut mutins we have estimated their population densities in feces of 11 healthy subjects by a “most probable number” method. Tenfold serial dilutions of fresh feces were prepared, and triplicate cultures were inoculated with from 10s6 g to lo-” g feces into an anaerobic medium containing 2 mg/mJ hog gastric mutin. The most probable number per gram dry fecal weight of (a) total bacteria, (b) facultative anaerobes, (c) mucin oligosaccharide-degrading bacteria, and (d) mucin protein-degrading bacteria was estimated from the frequency of bacterial growth and mucin degradation among the triplicate cultures at each level of fecal inoculum. Mucin-degrading bacteria were present in feces from every subject. The mean + SD Jog,, most probable number of both mutin oligosaccharide-degrading and of mucin proteindegrading bacteria was 8.7+ 0.8,whereas the Jog,, most probable numbers of total bacteria and of facultative anaerobes were 10.7+ 0.5and 7.82 1.1,respectively. There was no relationship between the presence of facultative anaerobes and mucin degradation. The most probable number of mucin-degrading bacteria was generally stable over periods of up Received July 30, 1980. Accepted March 20, 1981. Address requests for reprints to: Lansing C. Hoskins, M.D., Veterans Administration Medical Center, 10701 East Boulevard, Cleveland, Ohio 44106. This work was supported by a research grant from the Veterans Administration and by the Office of Naval Research, Contract NOOO14-79-C-0034. The authors thank E. T. Boulding for his technical assistance. 0 1981 by the American Gastroenterological Association OOlS-5085/81/100759-07$02.50
to 29 mo in 6 subjects, but occasional fluctuations of up to lOOO-fold were observed. We conclude that mucin glycoproteins in the human gut are degraded by bacterial subpopulations that average 1% of total fecal bacteria in healthy subjects. Thus, they may be regarded as one or more functionally distinct subsets of the normal fecal micropora. There is substantial evidence suggesting that the association between humans and their enteric microflora affects health. Due to the wide variety of metabolic reactions performed by enteric bacteria (1) fluctuations in the population densities of some bacterial species may influence health in subtle ways. But investigators wishing to study how host-microbial associations affect health are confronted with the facts that the human colonic microflora is a highly diverse community numbering on the order of 1O1*viable organisms per gram of colonic contents and that these organisms comprise up to 500 taxonomically distinct species whose rank order of abundance varies among individuals (23). Furthermore, biochemical reactions used for taxonomic classification of enteric species in cultures do not necessarily relate to their biologically important functions in the colon; hence it is difficult to assess the functional significance of numeric differences in fecal species when species are defined by current taxonomic methods. In the face of such complexity an alternative approach is: (a) to define the gut microflora in terms of subpopulations with functional activities that are postulated to affect health; (b) to develop methods for estimating the population densities of these subpopulations based on their functional activities; and (c) to determine if their population densities in subjects at risk for a given illness differ from healthy
760
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subjects. One of these functional activities is degradation of the host’s structural macromolecules, i.e., the glycoproteins and glycolipids of gut mucin and gut cell membranes. Bacterial enzymes are present in fecal extracts and in the cell-free supernates of anaerobic fecal cultures that degrade the oligosaccharide side chains of mucin glycoproteins (4-9) and, to a lesser extent, the mucin polypeptide core (4,9). These include glycosidases that cleave the alinked terminal glycosides conferring ABH(0) blood-group antigen specificity from mucin oligosaccharides (5-7) as well as from the cell walls of Escherichia coli 086 (10). Although the bacteria responsible for mucin degradation are poorly characterized taxonomically, we recently reported that those that degrade mucin oligosaccharides can be distinguished from other enteric bacteria by their ability to produce glycosidases as extracellular enzymes (8). Their fecal population densities in 7 healthy subjects ranged from 10e to lO”‘/g dry fecal weight. Here we report a procedure for estimating the fecal population densities of mucin-degrading bacteria by a “most probable number” (MPN) method and the findings in 11 healthy subjects. Most probable number estimates were obtained from the frequency of degradation of hexoses and proteins from hog gastric mucin (HGM) in triplicate anaerobic cultures of serially diluted feces. Hog gastric mutin was used because it is structurally man gastric and ovarian cyst mucins commercially available.
similar to hu(11) and it is
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cooled ethanol was added to a final concentration of 60% vol/vol. The resulting precipitate was dissolved in 0.1 M NaCl and again precipitated with ethanol to 60% vol/vol. This precipitate of purified mucin was dissolved in and dialyzed extensively against distilled water, and was then lyophilized. This gave a yield of 41% by weight. The dry
weight composition was: protein (modified method of Lowry et al. described below) 21%; hexoses [anthrone method (13) corrected for L-fucose] 22%; hexosamines (14), expressed as N-acetylhexosamines after hydrolysis in 2 N HCI 16 h at IOO’C, 30%; L-fucose (15) 9.5%; deoxyribonucleic acids (16) 3.6%; sialic acid (17) (after hydrolysis in 0.5 N H,SO, 60 min at SOY& 0.5%. Minimal hemagglutination inhibitory concentrations of A and H antigens were 60 and 320 ng/ml, respectively.
Methods Preparation, dispensing, storage, and inoculation of the prereduced anaerobic medium were performed under 95% Nz-5% CO, that had been passed over copper at 45O’C to remove traces of oxygen. A weighed fecal sample was dried in a vacuum oven, cooled over P,O,, and then reweighed to determine its dry weight. A second l-g fecal sample was thoroughly suspended in 10 ml anaerobic medium. From this initial suspension serial tenfold, thoroughly mixed dilutions were made in anaerobic medium to lo-” g/ml. One-milliliter aliquots from the lo-’ g/ml to the lo-” g/ml dilutions were inoculated into each of 3 tubes containing anaerobic medium. The resulting set of 16 tubes inoculated with 10-*-10-l’ g feces were incubated along with a control tube containing uninoculated medium for 48 h at W’C with occasional agitation. This incubation period was sufficient to permit the cultures inoculated with 1O-9-1O-” g
Materials and Methods Materials Stools from 11 healthy biomedical colleagues were collected in polyethylene bags, and aliquots were inoculated from 10 min to 2 h after collection. Subjects had not taken antibiotics in the previous 6 mo; they were 9 males and 2 females whose ages ranged from 28 to 57 yr (median, 45 yr). The anaerobic culture medium (“medium 75,” Table 1) contained defined, dialyzable constituents and acid hydrolyzed casein. Bacterial growth in this medium is limited by monosaccharide concentration. Hog gastric mucin was dissolved in this medium at a concentration of 2 mg/ml. For the present work, sterile vitamin K, and hemin solutions (12) were added to the autoclaved medium at final concentrations of 0.5 pg/ml and 5 pg/ml, respectively. Hog gastric mucin was partially purified from a commercial source (hog gastric mucin, Lot 6901, ICN Nutritional Biochemicals, Cleveland, Ohio). Twenty-five-gram batches were stirred for 20 h at 22°C in 1 L of 0.1 M NaCl containing 0.02 M phosphate buffer, pH 7.6, and a few drops of toluene. After the first hour the pH was readjusted to 7.0-7.4 with 2 N NaOH. After centrifugation at 10,000 g the supernate was cooled to 0” f 2°C and pre-
Table
1.
Composition
of Medium
75
Constituent
Amount per liter
Casamino acids” D-glucose KZHPO, KH,PO, (NH&SO, L-cystine D, L-tryptophan MgSO, 7H,O Resazurin Stock solution Ah Stock solution B’ Stock solution Cd Distilled water
15 g 1 g 14.3 g 2.4 g 1.0 g 0.4 g 0.4 g 0.1 g lmg 10 ml 10 ml 10 ml 1L
Boil in flask with reflux condenser until reduced; then add 2 g Sodium thioglycollate. pH after autoclaving = 6.9. a Difco Laboratories, Detroit, Michigan. b Stock solution A: 0.2 g NaCI, 0.2 g F&O,, 0.2 g MnSO,, 100 ml water. c Stock solution B: 0.2 g adenine, 0.2 g guanine, 0.2 g xanthine, 0.2 g uracil, 100 ml water. d Stock solution C: 40 mg pyridoxine, 20 mg niacin, 20 mg p-aminobenzoic acid, 10 mg calcium pantothenate, 10 mg riboflavin, 10 mg thiamine, 2 mg folic acid, 0.1 mg biotin, 0.1 mg vitamin B,,. 100 ml water.
October
MUCIN DEGRADATION
1961
feces, which had a longer lag-growth phase, to reach maximum optical turbidity. Preliminary studies showed that mucin degradation occurred during log-phase growth and was complete by the end of 48 h. Presence or absence of visible growth was recorded for each culture, and a loopful of each was inoculated onto blood agar and eosinmethylene-blue agar plates and incubated aerobically for 72 h to record the presence or absence of facultative anaerobes. Each culture was then centrifuged for 30 min at 12,000 g to sediment bacteria. A 2.0-ml aliquot of the supernate was removed and was heated 2 min at 100°C to inactivate enzymatic activity; it was then cooled in an ice bath, and 3.0 ml chilled 100% ethanol was added to a final concentration of 80% vol/vol. The mixture was maintained in the ice bath for at least 30 min and was then centrifuged at 1400 g. The precipitated residual mucin adhered to the glass tube and was drained free of supernate by inverting the tube. The precipitated mucin was then dissolved in either 5.0 ml or 10.0 ml 0.1 M NaCl for determination of mucin hexose and mucin protein concentrations. Mucin hexose concentration was determined by the anthrone-sulfuric acid method of Mokrasch (13), which uses D-galactose as standard. The decrease in mucin hexose concentration was used as an indicator of degradation of the mucin oligosaccharide side chains because: (a) the anthrone-sulfuric acid reagent reacts with L-fucose as well as with D-galactose; and (b) during degradation of mucin oligosaccharides by glycosidases in fecal cultures, L-fucose, hexoses, and hexosamines are split off at nearly comparable rates (9). Mucin protein concentration was determined by the following modification of the method of Lowry et al. (18): Full-color development of the Folin copper reagent with the protein moiety of mucin glycoproteins was impaired unless the oligosaccharide moieties were first degraded. This was accomplished by heating 1.0 volumes of sample in 5.0 volumes of reagent A (18) at 70°C for 45 min and, after cooling, adding 0.1 volume reagent B, and then proceeding with the conventional procedure. Standards of human serum albumin, included in each assay, were treated similarly. Percent degradation of mucin hexoses and mucin protein in each culture was determined from the formula:
% Degradation = l1
culture concentration in inoculated -.-. __ concentration in uninoculated medium
x 100 i
Several control experiments indicated that degradation was the major cause of loss of thanol-precipitable mucin in the fecal cultures. First, there was no evidence for mucin binding to bacteria to account for its loss from culture supernates: In one culture with 95% degradation of mucin hexoses after a 41-h incubation, the combined recovery of anthrone-reacting hexoses from the bacterial pellet and the ethanol precipitate fraction of the culture supernate was only 11% of the mucin hexoses originally in the medium. Second, there was extensive bacterial use of the degraded mucin oligosaccharides: In three cultures
IN HUMAN
COLON ECOSYSTEMS
761
with >90% degradation of mucin hexoses the combined recovery of anthrone-reacting hexoses in the 80% ethanolprecipitable and ethanol-soluble fractions of the culture supernate was (10% of the total hexoses initially in the medium. In addition, solublized glycoconjugates released from bacterial cell walls during bacterial growth were a minor contribution to the ethanol-precipitable hexoses in the culture supernate: in two studies, ethanol-precipitable hexoses released during growth in cultures containing no mucin amounted to 4% and 12%, respectively, of the initial amount of mucin hexoses in media containing mucin. Blood-group A and H antigen titers in the redissolved mucin precipitates were determined by using twofold serial dilutions in a previously described microtiter hemagglutination-inhibition method (7).
Definition
of Mucin Degradation
In order to establish whether degradation of mucin hexoses or mucin protein had occurred in a culture it was necessary to define the limits of variation of mucin hexose and protein concentrations recovered in cultures with no bacterial growth. We determined the coefficients of variation (CV) of the percent of mucin hexoses and protein recovered in 23 cultures exhibiting no growth that were compiled from all the studies. The coefficients of variation for percent recovery of mucin hexoses and mucin protein were *12.3% and fi15.2%, respectively. As 95% of the cultures with no mucin degradation would be expected to have recovery percentages within the limits defined by 100% f 2 CV, the lower limit for recovery of mucin hexoses and protein in these cultures would be 75% and i’O%, respectively, and any degradation of mucin hexoses >25% or of mucin protein >30% was defined as actual degradation.
Estimation of Bacteria
of the Fecal Population by the “Most Probable
Densities
Number”
Method This is a standard dilution-series method for estimating population densities of bacteria in water, soil, and other habitats, and counts obtained by this method correlate well with counts obtained by plating techniques (19,20). It provides a statistical estimate, the “most probable number,” of the population density of bacteria in a sample (21.22). Most probable number estimates are derived from the proportion of positive cultures among replicate cultures over the transition between those dilutions in which all replicates are positive and those in which none are positive; they are obtained by solving an equation either by the method of successive approximations or from published tables (23,24). In this work we obtained MPN estimates for total bacteria, facultative anaerobes, and bacteria that degraded mucin hexoses and mucin protein; the MPN estimates were derived from the frequency of bacte-. rial growth and mucin degradation among the three cultures of each serial dilution. Results are expressed as the log,, MPN/g dry fecal weight. For a tenfold dilution series with three replicate cultures per dilution, the 95% confidence interval is log,, MPN f 0.870 (21,22).
October 1981
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IN HUMAN COLON ECOSYSTEMS
763
suggest that the fecal population densities of mucindegrading bacteria are generally stable over time, but temporary fluctuations of up to 1000-fold may occur. Other Features Use of the MPN method for estimating fecal population densities of mucin-degrading bacteria assumes that the frequencies of mucin degradation among serially diluted replicate cultures follow a Poisson distribution. We tested this for mucin hexose-degrading bacteria by determining the frequency of mucin hexose-degradation in replicate cultures inoculated with feces from 1 subject. This was done by employing threefold serial dilutions and eight replicates per dilution; the results are shown in Figure 4. Computation from the observed frequency of mucin hexose-degradation gave a value of 13 for the MPN of mucin-degrading bacteria in the initial l.O-ml inoculum, which contained 1.1 x 10e8 g feces. The dashed line is the expected number of cultures with mucin hexose-degradation for a Poisson distribution when there is a mean of 13 mucin hexose-degrading bacteria in the initial dilution. There is fair agreement between the observed and the expected number. A 2 test for goodness of fit (26) showed that the differences between the observed and the expected frequencies were most likely due to chance (0.25 > p > 0.10). Degradation of mucin hexoses in individual cultures was generally greater than degradation of mutin protein. Thus, mucin hexose-degradation exceeded 90% in 57 (61%) of 93 cultures while the
VOLUME, 6,
OF l/WT/AL INOCUUhW,m/
Figure 4. Correspondence between observed number (dots) had expected number (dashed line) for a Poisson distribution of cultures degrading mucin hexoses among eight replicate cultures at each three fold serial dilution of fecal inoculum from 1.1 X lo-" g to 4.6 X 10-l’ g in fecal cultures from a single sample. The differences between observed and expected values are likely due to chance (y for goodness of fit = 2.926,2 degrees of freedom, 0.25 > p > 0.10).
median percent degradation of mucin protein in 90 cultures was 65%, and exceeded 70% in only 39. The glucose and high amino-acid content of “medium 75” did not appear to inhibit mucin degradation: In preliminary experiments measurements of glucose concentration using glucose oxidase showed that glucose rapidly decreased to undetectable levels during early bacterial growth, and that the extent of mucin protein degradation did not differ significantly among triplicate sets of cultures containing 15, 7.5, or 3 mg/ml Casamino acids (Table 1). Omission of Casamino acids from the medium resulted in diminished degradation of mucin hexoses and protein. As many of the oligosaccharide side chains of HGM have blood-group A and H antigenic-determinant glycosides on their nonreducing ends, we determined the relationship between the loss of A- and H-antigen titers and the percent hexose degradation in the ethanol-precipitable mucin recovered from 97 cultures. Complete loss of both antigens occurred only in cultures with extensive (76%-100%) mucin hexose degradation. In 10 of 17 cultures with moderate (5l%-75%) degradation, the titer of one of the antigens was unchanged while the titer of the other was either partially or completely lost, suggesting that lack of either one of the antigen-specific A- or H-degrading glycosidases in these cultures prevented the stepwise degradation of their side chains. Discussion
Figure 3. Variations of the estimated fecal population density of mucin protein-degrading bacteria over 6-19 mo in 6 healthy subjects.
It is well established by studies of germ-free animals (4,27), fecal cultures (4,9), and analysis of in-
764
GASTROENTEROLOGY
MILLER AND HOSKINS
testinal contents (28,29) that the enteric microflora degrades gut mucin glycoproteins. In anaerobic fecal cultures containing mucin, the degradation of the oligosaccharide side chains generally exceeds 90% by the time maximum optical turbidity is attained, while degradation of the polypeptide core ranges from 20%-80%. Yet the ability to degrade the oligosaccharide chains is not a feature of the most abundant bacterial species in feces. Thus, Salyers et al. (30) found that none of 188 strains of Bacteroides isolated from human colonic contents fermented porcine gastric mucin and that only three fermented bovine submaxillary mucin. The same workers tested 154 strains of seven other genera of fecal bacteria that, together with Bacteroides, comprise about 70% of the total fecal flora. Only eight strains from two species fermented porcine gastric mucin, and none degraded bovine submaxillary mucin. (31). The work reported here indicates that bacteria that do degrade mucin oligosaccharides can be regarded as a functionally distinct subset of normal fecal flora that in most subjects numbers on the order of 10B-lO’o/g dry fecal weight and comprises about 1% of the total bacterial population. We have recently found evidence that one basis for their ability to degrade mucin oligosaccharides is their capacity to release glycosidases as extracellular enzymes (8). In both respects mucin oligosaccharide-degrading bacteria are comparable to fecal bacteria that produce blood-group ABH antigen-degrading glycosidases. The latter are also subpopulations of human fecal bacteria (32); their blood-group-degrading glycosidases are extracellular enzymes adapted to the ABH-antigenic determinant glycosides on the oligosaccharides of their host’s gut mucins (8,7). It is yet to be determined whether complete degradation of the oligosaccharide side chains is accomplished by one strain producing all the requisite glycosidases, including blood-group-degrading enzymes, or by the cooperation of several strains that individually produce one or two of the requisite glycosidases. We are currently attempting to clarify this point by isolating and characterizing fecal strains of mucin oligosaccharide-degrading bacteria. Our MPN estimates of mucin protein-degrading bacteria may have been affected by the presence of oligosaccharide side chains on HGM in the culture medium. Thus, there is evidence that the oligosaccharide side chains of hog gastric mucin protect the underlying polypeptide core from degradation by pancreatic proteases (33). If this observation pertains in anaerobic fecal cultures, then detectable mutin protein degradation would be dependent upon prior degradation of the oligosaccharide side chains. Hence, in fecal samples where the true population density of mucin protein-degrading bacteria exceeds
Vol. 81, No. 4
that of mucin oligosaccharide-degrading bacteria, cultures at sample dilutions containing only the former may not show detectable degradation of mutin protein, and the derived MPN values may underestimate their true population density. Our results suggest that in most subjects the fecal population densities of mucin protein-degrading bacteria equal or exceed those of mucin oligosaccharide-degrading bacteria. Albeit crude, our MPN estimates of mucin-degrading bacteria permit defining them as one or more functionally distinct subsets of normal human fecal bacteria with fecal population densities that are generally stable and similar among healthy subjects but that may be altered in association with gastrointestinal disorders (34). More precise definition of the types and general characteristics of the bacteria comprising the subsets requires further study.
References Scheline RR. Metabolism of foreign compounds by gastrointestinal microorganisms. Pharmacol Rev 1973;25:451-523. 2. Moore WEC, Holdeman LV. Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbial 1974;27:96179. 3. Holdeman LV, Good IJ, Moore WEC. Human fecal flora; variation in bacterial composition within individuals and a possible effect of emotional stress. Appl Environ Microbial 1976;31:359-75. 4. Hoskins LC, Zamcheck N. Bacterial degradation of gastrointestinal mucins. I. Comparison of mucus constituents in the stools of germ-free and conventional rats. Gastroenterology 1968;54:210-7. 5. Hoskins LC. Bacterial degradation of gastrointestinal mucins. II. Bacterial origin of fecal ABH(0) blood group antigen-destroying enzymes. Gastroenterology 1968;54:218-24. 8. Hoskins LC. Ecological studies of intestinal bacteria. Relation between the specificity of fecal ABO blood group antigen-degrading enzymes from enteric bacteria and the ABO blood group of the human host. J Clin Invest 1969;48:664-73. 7. Hoskins LC, Boulding ET. Degradation of blood group antigens in human colon ecosystems. I. In vitro production of ABH blood group-degrading enzymes by enteric bacteria. J Clin Invest 1978;57:63-73. 8. Hoskins LC, Boulding ET. Mucin degradation in human colon exosystems. Evidence for the existence and role of bacterial subpopulations that produce glycosidases as extracellular enzymes. J Clin Invest 1981; (in press). 9. Variyam EP, Hoskins LC. Mucin degradation in human colon ecosystems. Degradation of hog gastric mucin by fecal extracts and fecal cultures. Gastroenterology 1981;81,741-8. 10. Cromwell CL, Hoskins LC. Antigen degradation in human colon ecosystems. Host’s ABO blood type influences enteric bacterial degradation of a cell surface antigen on Escherichi coli 086. Gastroenterology 1977;73:37-41. mucus glycoproteins 11. Allen A. Structure of gastrointestinal and the viscous and gel-forming properties of mucus. Br Med Bull 1978;34:28-33. 12. Holdeman LV, Cato EP, Moore WEC, eds. Anaerobe laboratory manual. 4th ed. Blacksburg, Va. Virginia Polytechnic Institute Anaerobe Laboratory, 1977. 1.
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13. Mokrasch LC. Analysis of sugar phosphates and sugar mixtures with the anthrone reagent. J Biol Chem 1954;208:55-9. 14. Boas NF. Method for the determination of hexosamines in tissues. J Biol Chem 1953$04:553-63. 15. Dische Z, Shettles LB. A specific color reaction for methylpentoses and a spectrophotometric micromethod for their determination. J Biol Chem 1948;175:595-603. 16. Burton K. A study of the conditions and the mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315-23. 17. Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959;234:1971-75. 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193265-75. 19. Ziegler NR, Halvorson HO. Application of statistics to problems in bacteriology. IV. Experimental comparison of the dilution method, the plate count, and the direct count for the determination of bacterial populations. J Bacterial 1935;29: 609-34. 20. Harris RF. Sommers LE. Plate-dilution technique for assay of microbial ecology. Appl Microbial 1968:16:330-34. 21. Cochran WG. Estimation of bacterial densities by means of “the most probable number.” Biometrics 1950;4:105-116. 22. Meynell GA, Meynell E. Theory and practice in experimental bacteriology. 2nd ed. Cambridge: Cambridge University Press, 1970:185-96. 23. deMan JC. The probability of most probable numbers. Eur J Appl Microbial 1975;1:67-78. 24. Hoskins JK. Most probable numbers for evaluation of coliaerogenes tests by fermentation tube method. U S Public
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Health Rep 1934;49:393-405. 25. Finegold SM, Sutter VL, Sugihara PT, et al. Fecal microbial flora in Seventh Day Adventist populations and control subjects. Am J Clin Nutr 1977:30:1781-92. 26. Snedecor GW, Cochran WG. Statistical methods. 6th ed Ames, Iowa: Iowa State University Press, 1967. 27. Lindstedt G, Lindstedt S, Gustaffson BE. Mucus in intestinal contents of germfree rats. J Exp Med 1965;121:201-13. 28. Vercellotti JR, Salyers AA, Bullard WS, and Wilkins TD Breakdown of mucin and plants polysaccharides in the human colon. Can J Biochem 1977;55:1190-6. 29. Ofosu F, Forstner J, Forstner G. Mucin degradation in the intestine. Biochem Biophys Acta 1978;543:476-83. 30. Salyers AA, Vercellotti JR, West SEH, Wilkins TD. Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon. Appl Environ Microbial 1977;33:319-22. 31. Salyers AA, West SEH, Vercellotti Jr, Wilkins TD. Fermentation of mucins and plant polysaccharides by anaerobic bacteria from the human colon. Appl Environ Microbial 1977: 34:529-33. 32. Hoskins LC, Boulding ET. Degradation of blood group antigens in human colon ecosystems. II. A gene interaction in man that affects the fecal population density of certain enteric bacteria. J Clin Invest 1976;57:74-82. 33. Variyam EP, Hoskins LC. Degradation of gut mucins. Car.bohydrate side chains protect the polypeptide core from pancreatic elastase (abstr). Gastroenterology 1978;74:1108. 34. Braverman A, Miller RS, Hoskins LC. Fecal population densities of coliforms and mucin-degrading bacteria in health and diarrhea1 illness (abstr). Gastroenterology 1980;78:1145.
1981;81:759-65
Mucin Degradation in Human Colon Ecosystems Fecal Population Densities of Mucin-Degrading Bacteria Estimated by a “Most Probable Number” Method RONALD
S. MILLER and LANSING
C. HOSKINS
Division of Gastroenterology, Veterans Administration Medical Center and Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio
Bacteria in anaerobic human fecal cultures degrade the oligosaccharide and protein moieties of gut mutin glycoproteins, but most bacterial isolates from feces are unable to degrade mucin. In order to define better the bacterial populations that degrade gut mutins we have estimated their population densities in feces of 11 healthy subjects by a “most probable number” method. Tenfold serial dilutions of fresh feces were prepared, and triplicate cultures were inoculated with from 10s6 g to lo-” g feces into an anaerobic medium containing 2 mg/mJ hog gastric mutin. The most probable number per gram dry fecal weight of (a) total bacteria, (b) facultative anaerobes, (c) mucin oligosaccharide-degrading bacteria, and (d) mucin protein-degrading bacteria was estimated from the frequency of bacterial growth and mucin degradation among the triplicate cultures at each level of fecal inoculum. Mucin-degrading bacteria were present in feces from every subject. The mean + SD Jog,, most probable number of both mutin oligosaccharide-degrading and of mucin proteindegrading bacteria was 8.7+ 0.8,whereas the Jog,, most probable numbers of total bacteria and of facultative anaerobes were 10.7+ 0.5and 7.82 1.1,respectively. There was no relationship between the presence of facultative anaerobes and mucin degradation. The most probable number of mucin-degrading bacteria was generally stable over periods of up Received July 30, 1980. Accepted March 20, 1981. Address requests for reprints to: Lansing C. Hoskins, M.D., Veterans Administration Medical Center, 10701 East Boulevard, Cleveland, Ohio 44106. This work was supported by a research grant from the Veterans Administration and by the Office of Naval Research, Contract NOOO14-79-C-0034. The authors thank E. T. Boulding for his technical assistance. 0 1981 by the American Gastroenterological Association OOlS-5085/81/100759-07$02.50
to 29 mo in 6 subjects, but occasional fluctuations of up to lOOO-fold were observed. We conclude that mucin glycoproteins in the human gut are degraded by bacterial subpopulations that average 1% of total fecal bacteria in healthy subjects. Thus, they may be regarded as one or more functionally distinct subsets of the normal fecal micropora. There is substantial evidence suggesting that the association between humans and their enteric microflora affects health. Due to the wide variety of metabolic reactions performed by enteric bacteria (1) fluctuations in the population densities of some bacterial species may influence health in subtle ways. But investigators wishing to study how host-microbial associations affect health are confronted with the facts that the human colonic microflora is a highly diverse community numbering on the order of 1O1*viable organisms per gram of colonic contents and that these organisms comprise up to 500 taxonomically distinct species whose rank order of abundance varies among individuals (23). Furthermore, biochemical reactions used for taxonomic classification of enteric species in cultures do not necessarily relate to their biologically important functions in the colon; hence it is difficult to assess the functional significance of numeric differences in fecal species when species are defined by current taxonomic methods. In the face of such complexity an alternative approach is: (a) to define the gut microflora in terms of subpopulations with functional activities that are postulated to affect health; (b) to develop methods for estimating the population densities of these subpopulations based on their functional activities; and (c) to determine if their population densities in subjects at risk for a given illness differ from healthy
760
GASTROENTEROLOGY
MILLER AND HOSKINS
subjects. One of these functional activities is degradation of the host’s structural macromolecules, i.e., the glycoproteins and glycolipids of gut mucin and gut cell membranes. Bacterial enzymes are present in fecal extracts and in the cell-free supernates of anaerobic fecal cultures that degrade the oligosaccharide side chains of mucin glycoproteins (4-9) and, to a lesser extent, the mucin polypeptide core (4,9). These include glycosidases that cleave the alinked terminal glycosides conferring ABH(0) blood-group antigen specificity from mucin oligosaccharides (5-7) as well as from the cell walls of Escherichia coli 086 (10). Although the bacteria responsible for mucin degradation are poorly characterized taxonomically, we recently reported that those that degrade mucin oligosaccharides can be distinguished from other enteric bacteria by their ability to produce glycosidases as extracellular enzymes (8). Their fecal population densities in 7 healthy subjects ranged from 10e to lO”‘/g dry fecal weight. Here we report a procedure for estimating the fecal population densities of mucin-degrading bacteria by a “most probable number” (MPN) method and the findings in 11 healthy subjects. Most probable number estimates were obtained from the frequency of degradation of hexoses and proteins from hog gastric mucin (HGM) in triplicate anaerobic cultures of serially diluted feces. Hog gastric mutin was used because it is structurally man gastric and ovarian cyst mucins commercially available.
similar to hu(11) and it is
Vol. 81, No. 4
cooled ethanol was added to a final concentration of 60% vol/vol. The resulting precipitate was dissolved in 0.1 M NaCl and again precipitated with ethanol to 60% vol/vol. This precipitate of purified mucin was dissolved in and dialyzed extensively against distilled water, and was then lyophilized. This gave a yield of 41% by weight. The dry
weight composition was: protein (modified method of Lowry et al. described below) 21%; hexoses [anthrone method (13) corrected for L-fucose] 22%; hexosamines (14), expressed as N-acetylhexosamines after hydrolysis in 2 N HCI 16 h at IOO’C, 30%; L-fucose (15) 9.5%; deoxyribonucleic acids (16) 3.6%; sialic acid (17) (after hydrolysis in 0.5 N H,SO, 60 min at SOY& 0.5%. Minimal hemagglutination inhibitory concentrations of A and H antigens were 60 and 320 ng/ml, respectively.
Methods Preparation, dispensing, storage, and inoculation of the prereduced anaerobic medium were performed under 95% Nz-5% CO, that had been passed over copper at 45O’C to remove traces of oxygen. A weighed fecal sample was dried in a vacuum oven, cooled over P,O,, and then reweighed to determine its dry weight. A second l-g fecal sample was thoroughly suspended in 10 ml anaerobic medium. From this initial suspension serial tenfold, thoroughly mixed dilutions were made in anaerobic medium to lo-” g/ml. One-milliliter aliquots from the lo-’ g/ml to the lo-” g/ml dilutions were inoculated into each of 3 tubes containing anaerobic medium. The resulting set of 16 tubes inoculated with 10-*-10-l’ g feces were incubated along with a control tube containing uninoculated medium for 48 h at W’C with occasional agitation. This incubation period was sufficient to permit the cultures inoculated with 1O-9-1O-” g
Materials and Methods Materials Stools from 11 healthy biomedical colleagues were collected in polyethylene bags, and aliquots were inoculated from 10 min to 2 h after collection. Subjects had not taken antibiotics in the previous 6 mo; they were 9 males and 2 females whose ages ranged from 28 to 57 yr (median, 45 yr). The anaerobic culture medium (“medium 75,” Table 1) contained defined, dialyzable constituents and acid hydrolyzed casein. Bacterial growth in this medium is limited by monosaccharide concentration. Hog gastric mucin was dissolved in this medium at a concentration of 2 mg/ml. For the present work, sterile vitamin K, and hemin solutions (12) were added to the autoclaved medium at final concentrations of 0.5 pg/ml and 5 pg/ml, respectively. Hog gastric mucin was partially purified from a commercial source (hog gastric mucin, Lot 6901, ICN Nutritional Biochemicals, Cleveland, Ohio). Twenty-five-gram batches were stirred for 20 h at 22°C in 1 L of 0.1 M NaCl containing 0.02 M phosphate buffer, pH 7.6, and a few drops of toluene. After the first hour the pH was readjusted to 7.0-7.4 with 2 N NaOH. After centrifugation at 10,000 g the supernate was cooled to 0” f 2°C and pre-
Table
1.
Composition
of Medium
75
Constituent
Amount per liter
Casamino acids” D-glucose KZHPO, KH,PO, (NH&SO, L-cystine D, L-tryptophan MgSO, 7H,O Resazurin Stock solution Ah Stock solution B’ Stock solution Cd Distilled water
15 g 1 g 14.3 g 2.4 g 1.0 g 0.4 g 0.4 g 0.1 g lmg 10 ml 10 ml 10 ml 1L
Boil in flask with reflux condenser until reduced; then add 2 g Sodium thioglycollate. pH after autoclaving = 6.9. a Difco Laboratories, Detroit, Michigan. b Stock solution A: 0.2 g NaCI, 0.2 g F&O,, 0.2 g MnSO,, 100 ml water. c Stock solution B: 0.2 g adenine, 0.2 g guanine, 0.2 g xanthine, 0.2 g uracil, 100 ml water. d Stock solution C: 40 mg pyridoxine, 20 mg niacin, 20 mg p-aminobenzoic acid, 10 mg calcium pantothenate, 10 mg riboflavin, 10 mg thiamine, 2 mg folic acid, 0.1 mg biotin, 0.1 mg vitamin B,,. 100 ml water.
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MUCIN DEGRADATION
1961
feces, which had a longer lag-growth phase, to reach maximum optical turbidity. Preliminary studies showed that mucin degradation occurred during log-phase growth and was complete by the end of 48 h. Presence or absence of visible growth was recorded for each culture, and a loopful of each was inoculated onto blood agar and eosinmethylene-blue agar plates and incubated aerobically for 72 h to record the presence or absence of facultative anaerobes. Each culture was then centrifuged for 30 min at 12,000 g to sediment bacteria. A 2.0-ml aliquot of the supernate was removed and was heated 2 min at 100°C to inactivate enzymatic activity; it was then cooled in an ice bath, and 3.0 ml chilled 100% ethanol was added to a final concentration of 80% vol/vol. The mixture was maintained in the ice bath for at least 30 min and was then centrifuged at 1400 g. The precipitated residual mucin adhered to the glass tube and was drained free of supernate by inverting the tube. The precipitated mucin was then dissolved in either 5.0 ml or 10.0 ml 0.1 M NaCl for determination of mucin hexose and mucin protein concentrations. Mucin hexose concentration was determined by the anthrone-sulfuric acid method of Mokrasch (13), which uses D-galactose as standard. The decrease in mucin hexose concentration was used as an indicator of degradation of the mucin oligosaccharide side chains because: (a) the anthrone-sulfuric acid reagent reacts with L-fucose as well as with D-galactose; and (b) during degradation of mucin oligosaccharides by glycosidases in fecal cultures, L-fucose, hexoses, and hexosamines are split off at nearly comparable rates (9). Mucin protein concentration was determined by the following modification of the method of Lowry et al. (18): Full-color development of the Folin copper reagent with the protein moiety of mucin glycoproteins was impaired unless the oligosaccharide moieties were first degraded. This was accomplished by heating 1.0 volumes of sample in 5.0 volumes of reagent A (18) at 70°C for 45 min and, after cooling, adding 0.1 volume reagent B, and then proceeding with the conventional procedure. Standards of human serum albumin, included in each assay, were treated similarly. Percent degradation of mucin hexoses and mucin protein in each culture was determined from the formula:
% Degradation = l1
culture concentration in inoculated -.-. __ concentration in uninoculated medium
x 100 i
Several control experiments indicated that degradation was the major cause of loss of thanol-precipitable mucin in the fecal cultures. First, there was no evidence for mucin binding to bacteria to account for its loss from culture supernates: In one culture with 95% degradation of mucin hexoses after a 41-h incubation, the combined recovery of anthrone-reacting hexoses from the bacterial pellet and the ethanol precipitate fraction of the culture supernate was only 11% of the mucin hexoses originally in the medium. Second, there was extensive bacterial use of the degraded mucin oligosaccharides: In three cultures
IN HUMAN
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761
with >90% degradation of mucin hexoses the combined recovery of anthrone-reacting hexoses in the 80% ethanolprecipitable and ethanol-soluble fractions of the culture supernate was (10% of the total hexoses initially in the medium. In addition, solublized glycoconjugates released from bacterial cell walls during bacterial growth were a minor contribution to the ethanol-precipitable hexoses in the culture supernate: in two studies, ethanol-precipitable hexoses released during growth in cultures containing no mucin amounted to 4% and 12%, respectively, of the initial amount of mucin hexoses in media containing mucin. Blood-group A and H antigen titers in the redissolved mucin precipitates were determined by using twofold serial dilutions in a previously described microtiter hemagglutination-inhibition method (7).
Definition
of Mucin Degradation
In order to establish whether degradation of mucin hexoses or mucin protein had occurred in a culture it was necessary to define the limits of variation of mucin hexose and protein concentrations recovered in cultures with no bacterial growth. We determined the coefficients of variation (CV) of the percent of mucin hexoses and protein recovered in 23 cultures exhibiting no growth that were compiled from all the studies. The coefficients of variation for percent recovery of mucin hexoses and mucin protein were *12.3% and fi15.2%, respectively. As 95% of the cultures with no mucin degradation would be expected to have recovery percentages within the limits defined by 100% f 2 CV, the lower limit for recovery of mucin hexoses and protein in these cultures would be 75% and i’O%, respectively, and any degradation of mucin hexoses >25% or of mucin protein >30% was defined as actual degradation.
Estimation of Bacteria
of the Fecal Population by the “Most Probable
Densities
Number”
Method This is a standard dilution-series method for estimating population densities of bacteria in water, soil, and other habitats, and counts obtained by this method correlate well with counts obtained by plating techniques (19,20). It provides a statistical estimate, the “most probable number,” of the population density of bacteria in a sample (21.22). Most probable number estimates are derived from the proportion of positive cultures among replicate cultures over the transition between those dilutions in which all replicates are positive and those in which none are positive; they are obtained by solving an equation either by the method of successive approximations or from published tables (23,24). In this work we obtained MPN estimates for total bacteria, facultative anaerobes, and bacteria that degraded mucin hexoses and mucin protein; the MPN estimates were derived from the frequency of bacte-. rial growth and mucin degradation among the three cultures of each serial dilution. Results are expressed as the log,, MPN/g dry fecal weight. For a tenfold dilution series with three replicate cultures per dilution, the 95% confidence interval is log,, MPN f 0.870 (21,22).
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MUCIN DEGRADATION
IN HUMAN COLON ECOSYSTEMS
763
suggest that the fecal population densities of mucindegrading bacteria are generally stable over time, but temporary fluctuations of up to 1000-fold may occur. Other Features Use of the MPN method for estimating fecal population densities of mucin-degrading bacteria assumes that the frequencies of mucin degradation among serially diluted replicate cultures follow a Poisson distribution. We tested this for mucin hexose-degrading bacteria by determining the frequency of mucin hexose-degradation in replicate cultures inoculated with feces from 1 subject. This was done by employing threefold serial dilutions and eight replicates per dilution; the results are shown in Figure 4. Computation from the observed frequency of mucin hexose-degradation gave a value of 13 for the MPN of mucin-degrading bacteria in the initial l.O-ml inoculum, which contained 1.1 x 10e8 g feces. The dashed line is the expected number of cultures with mucin hexose-degradation for a Poisson distribution when there is a mean of 13 mucin hexose-degrading bacteria in the initial dilution. There is fair agreement between the observed and the expected number. A 2 test for goodness of fit (26) showed that the differences between the observed and the expected frequencies were most likely due to chance (0.25 > p > 0.10). Degradation of mucin hexoses in individual cultures was generally greater than degradation of mutin protein. Thus, mucin hexose-degradation exceeded 90% in 57 (61%) of 93 cultures while the
VOLUME, 6,
OF l/WT/AL INOCUUhW,m/
Figure 4. Correspondence between observed number (dots) had expected number (dashed line) for a Poisson distribution of cultures degrading mucin hexoses among eight replicate cultures at each three fold serial dilution of fecal inoculum from 1.1 X lo-" g to 4.6 X 10-l’ g in fecal cultures from a single sample. The differences between observed and expected values are likely due to chance (y for goodness of fit = 2.926,2 degrees of freedom, 0.25 > p > 0.10).
median percent degradation of mucin protein in 90 cultures was 65%, and exceeded 70% in only 39. The glucose and high amino-acid content of “medium 75” did not appear to inhibit mucin degradation: In preliminary experiments measurements of glucose concentration using glucose oxidase showed that glucose rapidly decreased to undetectable levels during early bacterial growth, and that the extent of mucin protein degradation did not differ significantly among triplicate sets of cultures containing 15, 7.5, or 3 mg/ml Casamino acids (Table 1). Omission of Casamino acids from the medium resulted in diminished degradation of mucin hexoses and protein. As many of the oligosaccharide side chains of HGM have blood-group A and H antigenic-determinant glycosides on their nonreducing ends, we determined the relationship between the loss of A- and H-antigen titers and the percent hexose degradation in the ethanol-precipitable mucin recovered from 97 cultures. Complete loss of both antigens occurred only in cultures with extensive (76%-100%) mucin hexose degradation. In 10 of 17 cultures with moderate (5l%-75%) degradation, the titer of one of the antigens was unchanged while the titer of the other was either partially or completely lost, suggesting that lack of either one of the antigen-specific A- or H-degrading glycosidases in these cultures prevented the stepwise degradation of their side chains. Discussion
Figure 3. Variations of the estimated fecal population density of mucin protein-degrading bacteria over 6-19 mo in 6 healthy subjects.
It is well established by studies of germ-free animals (4,27), fecal cultures (4,9), and analysis of in-
764
GASTROENTEROLOGY
MILLER AND HOSKINS
testinal contents (28,29) that the enteric microflora degrades gut mucin glycoproteins. In anaerobic fecal cultures containing mucin, the degradation of the oligosaccharide side chains generally exceeds 90% by the time maximum optical turbidity is attained, while degradation of the polypeptide core ranges from 20%-80%. Yet the ability to degrade the oligosaccharide chains is not a feature of the most abundant bacterial species in feces. Thus, Salyers et al. (30) found that none of 188 strains of Bacteroides isolated from human colonic contents fermented porcine gastric mucin and that only three fermented bovine submaxillary mucin. The same workers tested 154 strains of seven other genera of fecal bacteria that, together with Bacteroides, comprise about 70% of the total fecal flora. Only eight strains from two species fermented porcine gastric mucin, and none degraded bovine submaxillary mucin. (31). The work reported here indicates that bacteria that do degrade mucin oligosaccharides can be regarded as a functionally distinct subset of normal fecal flora that in most subjects numbers on the order of 10B-lO’o/g dry fecal weight and comprises about 1% of the total bacterial population. We have recently found evidence that one basis for their ability to degrade mucin oligosaccharides is their capacity to release glycosidases as extracellular enzymes (8). In both respects mucin oligosaccharide-degrading bacteria are comparable to fecal bacteria that produce blood-group ABH antigen-degrading glycosidases. The latter are also subpopulations of human fecal bacteria (32); their blood-group-degrading glycosidases are extracellular enzymes adapted to the ABH-antigenic determinant glycosides on the oligosaccharides of their host’s gut mucins (8,7). It is yet to be determined whether complete degradation of the oligosaccharide side chains is accomplished by one strain producing all the requisite glycosidases, including blood-group-degrading enzymes, or by the cooperation of several strains that individually produce one or two of the requisite glycosidases. We are currently attempting to clarify this point by isolating and characterizing fecal strains of mucin oligosaccharide-degrading bacteria. Our MPN estimates of mucin protein-degrading bacteria may have been affected by the presence of oligosaccharide side chains on HGM in the culture medium. Thus, there is evidence that the oligosaccharide side chains of hog gastric mucin protect the underlying polypeptide core from degradation by pancreatic proteases (33). If this observation pertains in anaerobic fecal cultures, then detectable mutin protein degradation would be dependent upon prior degradation of the oligosaccharide side chains. Hence, in fecal samples where the true population density of mucin protein-degrading bacteria exceeds
Vol. 81, No. 4
that of mucin oligosaccharide-degrading bacteria, cultures at sample dilutions containing only the former may not show detectable degradation of mutin protein, and the derived MPN values may underestimate their true population density. Our results suggest that in most subjects the fecal population densities of mucin protein-degrading bacteria equal or exceed those of mucin oligosaccharide-degrading bacteria. Albeit crude, our MPN estimates of mucin-degrading bacteria permit defining them as one or more functionally distinct subsets of normal human fecal bacteria with fecal population densities that are generally stable and similar among healthy subjects but that may be altered in association with gastrointestinal disorders (34). More precise definition of the types and general characteristics of the bacteria comprising the subsets requires further study.
References Scheline RR. Metabolism of foreign compounds by gastrointestinal microorganisms. Pharmacol Rev 1973;25:451-523. 2. Moore WEC, Holdeman LV. Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbial 1974;27:96179. 3. Holdeman LV, Good IJ, Moore WEC. Human fecal flora; variation in bacterial composition within individuals and a possible effect of emotional stress. Appl Environ Microbial 1976;31:359-75. 4. Hoskins LC, Zamcheck N. Bacterial degradation of gastrointestinal mucins. I. Comparison of mucus constituents in the stools of germ-free and conventional rats. Gastroenterology 1968;54:210-7. 5. Hoskins LC. Bacterial degradation of gastrointestinal mucins. II. Bacterial origin of fecal ABH(0) blood group antigen-destroying enzymes. Gastroenterology 1968;54:218-24. 8. Hoskins LC. Ecological studies of intestinal bacteria. Relation between the specificity of fecal ABO blood group antigen-degrading enzymes from enteric bacteria and the ABO blood group of the human host. J Clin Invest 1969;48:664-73. 7. Hoskins LC, Boulding ET. Degradation of blood group antigens in human colon ecosystems. I. In vitro production of ABH blood group-degrading enzymes by enteric bacteria. J Clin Invest 1978;57:63-73. 8. Hoskins LC, Boulding ET. Mucin degradation in human colon exosystems. Evidence for the existence and role of bacterial subpopulations that produce glycosidases as extracellular enzymes. J Clin Invest 1981; (in press). 9. Variyam EP, Hoskins LC. Mucin degradation in human colon ecosystems. Degradation of hog gastric mucin by fecal extracts and fecal cultures. Gastroenterology 1981;81,741-8. 10. Cromwell CL, Hoskins LC. Antigen degradation in human colon ecosystems. Host’s ABO blood type influences enteric bacterial degradation of a cell surface antigen on Escherichi coli 086. Gastroenterology 1977;73:37-41. mucus glycoproteins 11. Allen A. Structure of gastrointestinal and the viscous and gel-forming properties of mucus. Br Med Bull 1978;34:28-33. 12. Holdeman LV, Cato EP, Moore WEC, eds. Anaerobe laboratory manual. 4th ed. Blacksburg, Va. Virginia Polytechnic Institute Anaerobe Laboratory, 1977. 1.
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MUCIN DEGRADATION
13. Mokrasch LC. Analysis of sugar phosphates and sugar mixtures with the anthrone reagent. J Biol Chem 1954;208:55-9. 14. Boas NF. Method for the determination of hexosamines in tissues. J Biol Chem 1953$04:553-63. 15. Dische Z, Shettles LB. A specific color reaction for methylpentoses and a spectrophotometric micromethod for their determination. J Biol Chem 1948;175:595-603. 16. Burton K. A study of the conditions and the mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315-23. 17. Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959;234:1971-75. 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193265-75. 19. Ziegler NR, Halvorson HO. Application of statistics to problems in bacteriology. IV. Experimental comparison of the dilution method, the plate count, and the direct count for the determination of bacterial populations. J Bacterial 1935;29: 609-34. 20. Harris RF. Sommers LE. Plate-dilution technique for assay of microbial ecology. Appl Microbial 1968:16:330-34. 21. Cochran WG. Estimation of bacterial densities by means of “the most probable number.” Biometrics 1950;4:105-116. 22. Meynell GA, Meynell E. Theory and practice in experimental bacteriology. 2nd ed. Cambridge: Cambridge University Press, 1970:185-96. 23. deMan JC. The probability of most probable numbers. Eur J Appl Microbial 1975;1:67-78. 24. Hoskins JK. Most probable numbers for evaluation of coliaerogenes tests by fermentation tube method. U S Public
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Health Rep 1934;49:393-405. 25. Finegold SM, Sutter VL, Sugihara PT, et al. Fecal microbial flora in Seventh Day Adventist populations and control subjects. Am J Clin Nutr 1977:30:1781-92. 26. Snedecor GW, Cochran WG. Statistical methods. 6th ed Ames, Iowa: Iowa State University Press, 1967. 27. Lindstedt G, Lindstedt S, Gustaffson BE. Mucus in intestinal contents of germfree rats. J Exp Med 1965;121:201-13. 28. Vercellotti JR, Salyers AA, Bullard WS, and Wilkins TD Breakdown of mucin and plants polysaccharides in the human colon. Can J Biochem 1977;55:1190-6. 29. Ofosu F, Forstner J, Forstner G. Mucin degradation in the intestine. Biochem Biophys Acta 1978;543:476-83. 30. Salyers AA, Vercellotti JR, West SEH, Wilkins TD. Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon. Appl Environ Microbial 1977;33:319-22. 31. Salyers AA, West SEH, Vercellotti Jr, Wilkins TD. Fermentation of mucins and plant polysaccharides by anaerobic bacteria from the human colon. Appl Environ Microbial 1977: 34:529-33. 32. Hoskins LC, Boulding ET. Degradation of blood group antigens in human colon ecosystems. II. A gene interaction in man that affects the fecal population density of certain enteric bacteria. J Clin Invest 1976;57:74-82. 33. Variyam EP, Hoskins LC. Degradation of gut mucins. Car.bohydrate side chains protect the polypeptide core from pancreatic elastase (abstr). Gastroenterology 1978;74:1108. 34. Braverman A, Miller RS, Hoskins LC. Fecal population densities of coliforms and mucin-degrading bacteria in health and diarrhea1 illness (abstr). Gastroenterology 1980;78:1145.