Effect of Bacterial Contamination on Cecal Size and Cecal Contents of Gnotobiotic Rodents

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1 JOURNAL OF BACTERIOLOGY, Aug. 1969, p American Society for Microbiology Vol. 99, No. 2 Printed in U.S.A. Effect of Bacterial Contamination on Cecal Size and Cecal Contents of Gnotobiotic Rodents WALTER J. LOESCHE Forsyth Dental Center, Boston, Massachusetts Received for publication 27 March 1969 In the present investigation the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. Of the species tested, Bacteroides oralis and Fusobacterium nucleatum did not establish in germ-free mice. Streptococcus mutans, Clostridium difficile, a Neisseria strain and two recent cecal isolates established, but failed to exert an effect upon the cecum of mice. A group K streptococcus and B. fragilis increased the cecal size apparently by increasing the levels of water-soluble protein, peptides, and carbohydrates in the cecal contents. Mixed ileal bacteria decreased the cecal size by preventing accumulation of soluble proteins and carbohydrates in the cecum. A Peptococcus strain caused a reduction by lowering the levels of insoluble material in the cecum. When this strain was combined with two Clostridium isolates and introduced into gnotobiotic rats, 50 to 65% cecal reduction was observed. This polycontamination did not decrease the per cent water of the cecal contents but caused lower levels of both soluble and insoluble material to accumulate in the cecum. No net nitrogen absorption from the distal small intestine occurred in either the germ-free or polycontaminated rats. The bacterial flora indigenous to the intestinal tract of rodents apparently influences the size of the cecum, for, in the germ free state, this organ enlarges and together with its contents can account for up to 25% of the body weight (9, 18). Contamination of germ-free animals with mixed fecal and intestinal bacteria obtained from conventional animals results in a return to normal cecal size (9). A degree of bacterial specificity is involved, since reduction of the cecum is not attained with most pure cultures of bacteria. However several investigators (11, 22, 23) have shown that certain bacteria alone, or in mixtures, will effect a reduction in cecal size. Little information is available as to how these bacteria effect this transition. In the present investigations, the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. In those contaminations in which cecal size was changed from germ-free values, the cecal contents were fractionated to determine whether the microbes were specificantly altering the soluble carbohydrates, soluble proteins, insoluble fraction, or any combination of these. The results obtained suggested that in these experiments there were several ways in which the microbes affected cecal size. A previous investigation had shown that, in germ-free rats, nitrogen accumulated as digesta passed from the distal small intestine into the cecum, whereas in conventional rats 90% of the nitrogen found in intestinal contents was reabsorbed prior to the cecum (15). It was therefore possible that a contamination which was successful in reducing cecal size might prevent the accumulation of endogenous nitrogen in the cecum. When a combination of microbes capable of reducing cecal size in both rats and mice was obtained, studies were performed to determine whether this combination would promote net nitrogen absorption from the distal half of the small intestine. 520 MATERIALS AND METHODS Germ-free rats strain CDF and mice strain CD-1 were obtained from the Charles River Breeding Laboratories (North Wilmington, Mass.). They were housed in plastic germ-free isolators and fed either autoclaved diet 5010C (Purina) or autoclaved diet L356 (General Biochemical Incorporation) ad lib. Conventional mice of the same strain were maintained under routine animal room conditions. For studies involving nitrogen absorption from the small intestine, a powdered diet containing chromic oxide was used. This diet was sterilized by irradiation and consisted of 30% milk powder, 63% dextrose, 3% liver powder, 2% vitamin mix (Gustafsson Formula, General Biochemical Incorporation), 1% Salt Mix W (Nutritional Biochemical Corp., Cleveland, Ohio), and 1% Cr2O3. Germ-free mice were contaminated with the follow-

2 VOL. 99, 1969 GERM-FREE RODENT CECUM AND CONTAMINATION 521 ing bacterial strains: Bacteroidesfragilis NTCC strain 9343, presumably the most numerous organism in the intestinal tract of mammals (21); Bacteroides oralis strain R42; Fusobacterium nucleatum strain 7CF, selected because it is an amino acid fermenter and amino acids are known to accumulate in the germ-free cecum (5); Clostridium difficile ATCC strain 9689, an organism which has been shown to reduce cecal size in the mouse (23); a group K streptococcus strain (courtesy of S. Hayano, Saitama Institute of Public Health, Saitama, Japan), selected because of its neuraminadase activity, which might have an effect on cecal size by hydrolyzing the sialoproteins which accumulate in the germ-free cecum (15); and Streptococcus mutans strain GS5 and an uncharacterized Neissera strain, both of which were under investigation for cariogenic activity in our laboratory. Intestinal and cecal isolates were obtained by high-dilution plating of intestinal and cecal contents on blood-agar plates and incubation under an 80% nitrogen, 10% hydrogen, and 10% carbon dioxide atmosphere. Representative colonies from the highest dilution plate were screened for growth in the presence of 10% bile and 1% gastric mucin. Because both bile (10) and mucins (13) are present in elevated amounts in germfree ceca, it was thought that organisms inhibited by these compounds would be ineffective in reducing cecal size. Those isolates capable of reducing cecal size were characterized by methods described in the Manual of Microbiological Methods (24) and elsewhere (16). Mice were killed by neck fracture 3 to 5 weeks after contamination, and the ceca were quickly removed and weighed, and their contents were expressed; soluble and insoluble fractions were obtained as described previously (14). With the rats, the procedure varied slightly in that the cecum and small intestine were removed under ether anesthesia. The soluble fraction was analyzed for soluble protein, soluble carbohydrate, 10% trichloroacetic acid-precipitable protein, 80% ethyl alcohol-precipitable protein and carbohydrate, and trichloroacetic acid and ethyl alcohol-soluble protein and carbohydrate. The insoluble fraction was analyzed for dry weight, carbohydrate, and nitrogen. Protein was measured by the procedure of Lowry et al. (17) with albumin as a TABLE 1. standard. Carbohydrate was measuied by the anthrone procedure (19) with galactose as a standard, and nitrogen was determined by the Conway micro diffusion method after micro-kjeldahl digestion (5). When ratios of nitrogen to chromium were to be determined, homogenized material from the distal half of the small intestine or from the cecum was dried to constant weight and the nitrogen and chromium present in a 50-mg sample were determined. Chromium was measured by the method of Kimura and Miller (12). RESULTS Effect of microbes on cecal size. Not all bacteria would establish in the germ-free animal. Of the species tested, B. oralis and F. nucleatum did not establish in germ-free mice. S. mutans strain GS5, the Neisseria strain, C. difficile, and two of the cecal isolates established but failed to exert an effect upon the cecum. Contents of the terminal ileum of conventional mice were capable of reducing cecal size (Table 1), but the five most numerous colonial types isolated from these conventionalized mice collectively had no effect on cecal size when introduced into germ-free mice. The group K streptococcus and B. fragilis caused an increase in cecal size (Table 1). An anaerobic gram-positive coccus (Peptococcus sp.) isolated from the cecum of conventional mice was capable of reducing the cecum by 40%, whereas two motile Clostridium strains (12C and 13X) isolated from conventionalized mice caused a 25% reduction. When these three strains were introduced together, a 50% reduction was observed (Table 1). This combination also proved effective in reducing the cecum in rats ingesting either diet L356 (47% reduction) or the powdered irradiated glucose chromium diet (64% reduction) (Table 3). If B. fragilis was added to this combination the coccus was found to be suppressed and to have eventually disappeared. Effect of microbes on cecal contents. The cecal Effect of various microbes on cecal size of gnotobiotic micea I MI ~~~~~~~~~Cecum Gnotobiotic state animals Body weight Weight of cecal cent of body animals contents ~~~~~~weight as per g g Germ-free b 2.4 i Conventional ~ i Mixed ileal bacteria : Group K streptococcus Bacteroidesfragilis i Peptococcus : Two Clostridium strains Peptococcus plus Clostridium strains : a Mice were fed autoclaved diet 5010C ad lib. b Expressed as value plus or minus standard error of the mean.

3 522 LOESCHE J. BACrERIOL. contents of animals in which contamination caused a change in the size of the cecum were fractionated (Table 2). The data are presented in milligrams per 100 g of adjusted body weight, i.e. body weight minus cecal weight, to eliminate the variable contribution the cecum was making to the body weight. Because the germ-free cecum was seven times larger than the cecum of the conventional animals, a six- to eightfold increase in soluble protein, carbohydrate, etc. over conventional values might be expected. In a similar manner, the B. fragilis and K streptococcus animals would have 10 times, and the animals contamninated with the ileal contents twice, the conventional levels. When cecal contents of germ-free mice were compared to those of conventional mice, there was, as expected, an absolute increase of each of the measured components (Table 2). However, the levels of soluble carbohydrate were increased 29 times, and those of soluble protein were increased 11 times over the conventional values. The dry weight of the insoluble fraction was six times the conventional value. Apparently the germ-free cecum can be characterized not only by its higher water content (9) but also by increased amounts of water-soluble protein and carbohydrate; 55% of the soluble protein and 36% of the soluble carbohydrate could not be precipitated by either 10% trichloroacetic acid or 80% ethyl alcohol, suggesting that they were of low molecular weight (Table 2). In contrast, in the conventional animals, almost all the soluble protein was of high molecular weight. Animals contaminated with either B. fragilis or the group K streptococcus also showed a disproportionate increase in soluble carbohydrates and proteins. The carbohydrate was approximately 50 times elevated over conventional values TABLE 2. in the B. fragilis animals and 70 times higher in the group K streptococcus contaminated animals (Table 2). Of this carbohydrate, 70 to 80% was soluble both in 10% trichloroacetic acid and 80% ethyl alcohol. Contamination with these bacteria and resultant increase in cecal size thus appeared to be associated with high levels of low molecular weight carbohydrates. The remaining contaminations described in this investigation deal with situations in which the cecal size was reduced by microbes. When animals were contaminated with ileal contents, a complex flora established which was effective in decreasing cecal size to 2% of the body weight. This reduction was associated with low levels of soluble protein and carbohydrate which resembled the conventional values, whereas, the dry weight of the insoluble fraction was almost three times greater than the amount found in the conventional animals. Apparently the bacteria introduced were capable of lowering the soluble components to a greater extent than the insoluble material. The opposite effect was observed in animals contaminated with an anaerobic gram-positive coccus (Peptococcus sp.), as the level of soluble protein was 11 times greater than that found in the conventional animal, but the insoluble fraction was only 2.4 times the amount found in the conventional mice. The decreased levels of insoluble material might reflect an ability of this organism to solubilize the large macromolecules and cellular debris which would be present in this fraction. Contamination with two Clostridium strains led to a slight reduction in cecal size, but the analysis of cecal contents did not provide any information as to how this was effected. The various cecal fractions were slightly lower than Effect of various microbes on the cecal contents of gnotobiotic mice, Soluble Gnotobiotic condition Total protein Trichloroacetic weight acid- and ethyl Total acid- and ethyl alcohol-soluble carbohydrate alcohol-soluble protein carbohydrate TrichoroaeticInsoluble dry Germ-free i : Conventional Mixed ileal bacteria : ± : Bacteroidesfragilis i i i K streptococcus i Peptococcus strain Two Clostridium strains : ± ±t Peptococcus plus Clostridium strain ± d : a Mice were fed autoclaved diet SO1OC and water ad lib. Values are expressed as milligrams per 100 g of adjusted body weight (body weight minus weight of cecum) A standard error of the mean.

4 VOL. 99, 1969 GERM-FREE RODENT CECUM AND CONTAMINATION 523 the germ- the corresponding amounts found in free controls (Table 2). When germ-free mice were contaminated with both the anaerobic coccus and Clostridium strains, cecal reduction was maximal. The insoluble dry weight decreased as was seen previously in the mice monocontaminated with the coccus. There were also lower levels of soluble carbohydrate and protein, with about 65% of this material being soluble in 10%, trichloroacetic acid or 80% ethyl alcohol or both (Table 2). This might reflect the contribution of the Clostridium strains to cecal reduction. This contamination of Peptococcus and Clostridium strains was then introduced into young 35- to 40-day-old female germ-free rats to observe whether their ability to reduce the cecum was unique for mice. These rats were fed either diet L356 or a powder diet containing chromium. After 3 weeks of contamination, representative animals, as well as germ-free controls of similar age, were killed. No reduction of the cecum was noted, because, in both groups of animals, the cecum accounted for about 5 to 6%7 of the body weight. High-dilution cecal cultures of the contaminated animals revealed that the anaerobic coccus was either absent or present in low numbers. The coccus was reintroduced into the contaminated isolator, and subsequent fecal culturing verified that it had become established. The contaminated and germ-free animals were maintained for another 5 weeks, and then all animals were killed. Contamination resulted in approximately a 50% reduction in cecal size in animals ingesting diet L356 and a 65 % reduction in animals ingesting the chromium diet (Table 3). Despite this reduction, the polycontamination TABLE 3. had no effect on the water concentration, because in all instances, water accounted for 82 to 85% of the wet weight of the cecal contents. When the contents were fractionated, ceca from contaminated rats showed a reduction in the soluble carbohydrates, soluble protein, and insoluble dry weight, regardless of which diet was being consumed, when they were compared to their suitable germ-free controls (Table 3). Thus the effect of this contamination in rats was almost a uniform decrease of all measured cecal components. The diet was not without some effect, because animals on diet L356 were 80 to 100 g heavier than animals of similar age ingesting the powdered diet. Also, on diet L356, the soluble protein was in excess of the soluble carbohydrate, whereas on the powdered diet the soluble carbohydrate outweighed the protein. Previous studies had shown that, in the germfree rat, nitrogen was accumulating as intestinal contents passed from the distal small intestine into the cecum, whereas, in the conventional rat, about 90%,0 of the intestinal nitrogen was reabsorbed prior to the cecum (15). In the present experiment, the effect of contamination with the anaerobic coccus and Clostridium strains on the recovery of endogenous nitrogen could be assessed in the animals ingesting the diet containing 1 % Cr2O3 (Table 4). Smaller amounts of chromium and nitrogen were found in the distal small intestine and cecum of the contaminated animals as compared to the germ-free controls. However, the nitrogen to chromium ratios increased by the same amount in passage from the small intestine to cecum, indicating that for both groups nitrogen was still accumulating in the cecum. Thus, this polycontamination did not have an effect on Effect of polycontamination on cecal size and cecal contents of gnotobiotic rats Gnotobiotic condition L356 diet Powdered chromium diet Germ-free Contaminateda Germ-free Contaminated Body wt (g) ± 11.1b d Cecal contents wt (g) i i: Water (%) in cecal contents Cecum as per cent of body wt Soluble fraction Soluble protein i 13.2c 131 i Soluble carbohydrate ± d h 3.3 Insoluble fraction Dry wt I a Polycontaminated with Peptococcus strain 11 and Clostridium strains 12C and 13X. b Values are expressed + standard error of the mean. c Values for soluble and insoluble fractions are expressed in milligrams per 100 g of adjusted body weight (body weight minus cecal weight).

5 524 LOESCHE J. BACTERIOL. one of the basic abnormalities of the germ-free animal, i.e. failure to absorb endogenous nitrogen from the terminal small intestine. Attempts were made to characterize the three bacteria capable of reducing cecal size. Strain 11 was an anaerobic gram-positive coccus, which did not ferment mannitol, was catalase positive, and reduced nitrates. As such it has been tentatively identified as Peptococcus saccharolyticus (3). Other characteristics are given in Table 5. Strains 12C and 13X were motile Clostridiwn species TABLE 4. Effect of polycontamination on the net absorption of nitrogen from small intestinal contents of gnotobiotic rats Determination Germ-f ree Contaminateda Small Cecum Small Cecum intestine intestine cm Nitrogen 5.8b Chromic oxide Nitrogen chromic oxide ratio a Contaminants included a Peptococcus strain and two Clostridium strains b Values are expressed in milligrams per 100 g of body weight. TABLE 5. whose biochemical characteristics are very similar (Table 5). They are listed separately only because they could be easily distinguished on the basis of colonial morphology. They could not be readily placed into any Clostridium species described by Bergey's Manual (3). They were able to grow in the absence of carbohydrates producing foulsmelling end products. None of the biochemical characteristics recorded was of value in explaining how these bacteria helped to reduce cecal size. DISCUSSION The role bacteria play in maintaining normal cecal size is not simple. Nor is it likely that only one specific bacterium is responsible, since several unique events are observed in the intestinal tract of germ-free rodents. The problem of cecal enlargement probably originates in the small intestine where, in the normal or conventional animal, endogenous proteins, glycoproteins, mucopolysaccharides, and other cellular remnants are almost quantitatively digested and the resulting monomers reabsorbed (25). In the absence of bacteria, this digestion does not occur or is greatly reduced, and, as a result, these endogenous materials pass through the intestine with some of them accumulating in the cecum. The loss of endogenous nitrogen and calories seriously upsets the animals' metabolic capacity, and conservative Biochemical characteristics of microbes capable of reducing cecum of gnotobiotic rodents Species Peptococcus strain 11 Clostridium strain 12C Clostridium strain 13X Gram stain +, Coccus +, Rod spores +, Rod spores Motility + + Carbohydrate fermentation Glucose + SI+, gas Galactose Mannose Fructose + SI+ +, gas Rhamnose _ Ribose _ SI+ S1+, gas Xylose _ Lactose _ Maltose Sl+a +, gas Cellobiose Sucrose SI+ Starch Hydrolyzed Mannitol Nitrate reduction + H2S production + + Gelatin liquefaction + + Catalase + Indole + + Litmus milk Acid reduced curd Acid reduced curd Acid reduced curd partially digested partially digested partially digested a Slightly positive.

6 VOL. 99, 1969 GERM-FREE RODENT CECUM AND CONTAMINATION 525 mechanisms such as lower cell renewal rates (1) and lower basal metabolic rates come into play (9) İn the germ-free cecum, the considerable amounts of trypsin, chymotrypsin (14), and other host enzymes (6, 20) which are present presumably continue their degradation of macromolecules, as suggested by the significant amounts of low molecular weight peptides and carbohydrates found in the present study and by the high levels of amino acids reported by Combe and Pion (4). If these low molecular weight compounds cannot pass through the cecal wall in appreciable amounts, then their collected effect will be one of water retention. Some of the peptides could exert pharmacological effects on the smooth muscles of the cecal wall, initiating or causing further distention of the cecum. Gordon (8) found considerable amounts of kallidin or bradykinin in cecal contents of germ-free rats, and this compound may be contributory to cecal enlargement. Some intestinal preparations such as the rat duodenum are relaxed by kinins (7). The normal intestinal flora, either directly or as a result of its waste products, apparently is responsible for normal peristaltic activity (2). In the absence of microbes, transit of material through the digestive tract is sluggish and pooling could easily occur in blind loops such as the cecum. Bacterial contamination of germ-free animals could interrupt or alter any or all of the above events. In the most successful contamination reported in the present investigation, i.e. the Peptococcus plus two Clostridium strains in germfree rats ingesting a powdered diet, a 65% reduction in cecal size was not associated with any reabsorption of nitrogen from the distal small intestine. Thus this contamination failed to reverse, perhaps, the primary event in the sequence leading to cecal enlargement, even though these bacteria established in high numbers in the distal small intestine, i.e. 101 per g (wet weight) of contents. Perhaps their presence in the intestinal tract of rats served to increase peristalsis, and, as a result, less material accumulated in the small intestine and cecum. The Peptococcus, either alone or in combination with the Clostridium strains, appeared to reduce the cecum in mice by decreasing the insoluble fraction with only slight effects on the soluble fraction. This mode of cecal reduction differed from the pattern observed in mice contaminated with ileal contents, since the undefined flora which established in these animals appeared to reduce the soluble carbohydrates and proteins to normal values without exerting an appreciable effect on the insoluble fraction. This reduction of water-soluble materials would provide conditions favoring inspissation of water and perhaps explains why the ceca in these animals were only 2 % of the body weight. The contaminations with B. fragilis and the group K streptococcus showed that certain bacteria can actually increase cecal size. In both instances, the increase was associated with elevated levels of soluble carbohydrate and protein, which would increase the number of osmotically active molecules in the cecum, thereby promoting water retention and cecal enlargement. None of the above contaminations resulted in the establishment of a normal cecal size in gnotobiotic animals. No bacteria were found which caused degradation and net reabsorption of nitrogen from the distal small intestine. Because of this, endogenous proteins, glycoproteins, and other cellular material continue to pass into the cecum, where they were acted upon in different ways by the various bacteria introduced into the gnotobiotic animal. The patterns of cecal reduction which occurred suggested that in the normal animal a concert of various microbes may be responsible for small cecal size. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant DE from the National Institute of Dental Research. The technical assistance of Suzanne Banghart and Ernest Smith is gratefully appreciated. LITERATURE CITED 1. Abrams, G. D., H. Bauer, and H. Sprinz Influence of the normal flora on mucosal morphology and cellular renewal in the ileum. A comparison of germfree and conventional mice. Lab. Invest. 12: Abrams, G. D., and J. E. Bishop Effect of the normal flora on gastrointestinal motility. Proc. Soc. Exp. Biol. Med. 126: Breed, R. S., E. G. D. Murray, and N. R. Smith Bergey's manual of determinative bacteriology. 7th ed. The Williams & Wilkins Co., Baltimore. 4. Combe, E., and R. Pion Note sur la composition en acides amines du contenu de caecum de rats axeniques et de rats temoins. Ann. Biol. Anim. Biochim. Biophys. 6: Conway, E. J Microdiffusion analysis and volumetric error. Crosby Lockwood and Sons, London. 6. Dahlquist, A., B. Bull, and B. E. Gustafsson Rat intestinal 6 bromo 2-naphthyl glycosidase and disaccharidase activities. I. Enzymatic properties and distribution in the digestive tract of conventional and germfree animals. Arch. Biochem. Biophys. 109: Erdos, E. G Hypotensive peptides: bradykinin, kallidin and eledoisin. Advan. Pharmacol. 4: Gordon, H. A A bioactive substance in the caecum of germfree animals. Nature (London) 205: Gordon, H. A Is the germfree animal normal? A review of its anomalies in young and old age, p In M. E. Coates (ed.), The germfree animnal in research. Academic Press, London.

7 526 LOESCHE J. BACTERIOL. 10. Gustafsson, B. E., and A. Norman Physical state of bile acids in intestinal contents of germfree and conventional rats. Scand. J. Gastroenterol. 3: li. Hudson, J. A., and T. D. Luckey Bacteria induced morphologic changes. Proc. Soc. Exp. Biol. Med. 116: Kimura, F. T., and V. L. Miller Improved determinations of chromic oxide in cow feed and feces. J. Agr. Food Chem. 5: Lindstedt, G., S. Lindstedt, and B. E. Gustafsson Mucus in intestinal contents of germfree rats. J. Exp. Med. 121: Loesche, W. J Protein and carbohydrate composition of cecal contents of gnotobiotic rats and mice. Proc. Soc. Exp. Biol. Med. 128: Loesche, W. J Accumulation of endogenous protein in the cecum of the germfree rat. Proc. Soc. Exp. Biol. Med. 129: Loesche, W. J., S. S. Socransky, and R. J. Gibbons Bacteroides oralis, proposed new species isolated from the oral cavity of man. J. Bacteriol. 88: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Luckey, T. D Germfree life and gnotobiology. Academic Press, Inc., New York. 19. Neish, A. C Analytical methods for bacterial fermentations. Report No , National Research Council of Canada. 20. Reddy, B. S., and B. S. Wostmann Intestinal disaccharidase activities in the growing germfree and conventional rats. Arch. Biochem. Biophys. 113: Rosebury, T Microorganisms indigenous to man. Mc- Graw-Hill Book Co., Inc., New York. 22. Schaedler, R. W., R. Dubois, and R. Costello Association of germfree mice with bacteria isolated from normal mice. J. Exp. Med. 122: Skelly, B. J., P. C. Trexler, and J. Tanami Effect of a Clostridium species upon cecal size of gnotobiotic mice. Proc. Soc. Exp. Biol. Med. 110: Society of American Bacteriologists Manual of microbiological methods. McGraw-Hill Book Co., Inc., New York. 25. Twombly, J., and J. H. Meyer Endogenous nitrogen secretions into the digestive tract. J. Nutr. 74: Downloaded from on April 9, 2018 by guest