Influence of the Indigenous Gastrointestinal Microbial Flora on Duodenal Alkaline Phosphatase Activity in Mice

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1 INFECTION AND IMMUNITY, June 1971, p Copyright 1971 American Society for Microbiology Vol. 3, No. 6 Printed in U.S.A. Influence of the Indigenous Gastrointestinal Microbial Flora on Duodenal Alkaline Phosphatase Activity in Mice DIANE P. YOLTON, CAROL STANLEY,' AND DWAYNE C. SAVAGE Department of Microbiology, University of Texas, Austin, Texas Received for publication 28 January 1971 Alkaline phosphatase activity was assayed by two procedures in duodenal homogenates from specific pathogen-free (SPF) mice of various ages, adult germfree mice, adult ex-germ-free mice colonized with an indigenous microflora from the SPF mice, and adult ex-germ-free mice monocontaminated with a Lactobacillus sp. indigenous to the SPF mice. In the SPF neonates, the activity remained at low levels until at least 12 days of age, increased to high levels at 20 days of age, and then fell to adult levels between the early neonatal and later high levels. In the germ-free mice, the activity levels were significantly higher than the levels in SPF mice at any age. In contrast, in the ex-germ-free animals, colonized by an entire indigenous microflora, the values fell within the range for adult SPF animals. In the ex-germ-free mice colonized only by the Lactobacillus sp., the activity levels were intermediate between the values for germ-free and SPF mice. These findings show that the indigenous microbial flora influences considerably the intestinal alkaline phosphatase activity in the mouse. Alkaline phosphatase activity (EC ) in the mucosa of the mouse duodenum begins to increase at about 11 days after birth of the animals and reaches peak values 10 days later. Thereafter, the activity falls to adult levels intermediate between the neonatal and peak values (13). Three entities with differing mobilities in starch-gel electrophoresis have alkaline phosphatase activity in the duodenums of newborn mice up to 11 days of age. In contrast, an undetermined number of entities with graded small differences in electrophoretic mobility have phosphatase activity in the duodenums of 20-day-old and adult mice (6). The many entities in the weanling and adult animals may be different molecular forms of alkaline phosphatase that develop in epithelial cells as such cells migrate from the crypts up to the tips of the duodenal villi (6). This migration of epithelial cells from the crypts to the villous tips in the mouse duodenum is known to be influenced by the indigenous alimentary microbial flora (11). The major components of this alimentary microflora, the anaerobic bacteria, colonize the mouse gastrointestinal tract and reach high population levels during the second week after birth (17). During the same period, as well, the activity levels and molecular forms of Present address: 265 Southwest Allen, Beaverton, Ore alkaline phosphatase change from the neonatal to the adult condition (6). These facts suggested to us that the alimentary microflora influences in some way the molecular forms and activity levels of alkaline phosphatase in the mucosa of the mouse gastrointestinal canal. We tested this hypothesis by measuring directly the levels of alkaline phosphatase activity in the duodenal mucosa of specific pathogen-free (SPF) mice, germ-free mice, and ex-germ-free mice colonized with an indigenous microflora from the SPF mice. In addition, in an effort to establish a model for studying the influence of the microflora on these intestinal enzymes, we assayed the duodenal alkaline phosphatase activity of germfree mice monocontaminated with an indigenous Lactobacillus sp. MATERIALS AND METHODS Mice. Animals used were germ-free ARS/Ha/ICR mice and their conventional counterparts Ha/ICR SPF mice (A. R. Schmidt, Madison, Wis.). The SPF mice were housed in groups of eight adults or one mother with litter in plastic boxes with paper covers (Isocage, Carworth, New City, N.Y.) with either nonsterilized or autoclave-sterilized Ab-Sorb-Dri (Ab- Sorb-Dri, Inc., Garfield, N.J.) for bedding. They were maintained on either Wayne Lab-Blox or autoclavesterilized Sterilizable Wayne Lab-Blox (Allied Mills,

2 VOL. 3) MICROBIAL FLORA 769 Inc., Chicago, Ill.). Their drinking water was either tap water acidified with M HCl or autoclavesterilized water that was then acidified with M HCl. Sterile or nonsterile food, bedding, and water were used, depending upon the particular experiment. The number of mice per litter was reduced to eight at birth. The germ-free and monocontaminated mice were maintained in inflatable vinyl isolators (24), with autoclave-sterilized Ab-Sorb-Dri for bedding, autoclave-sterilized Sterilizable Wayne Lab-Blox for food, and sterile water. Sterility tests on the germ-free animals were performed by established procedures (25). Some of the germ-free mice were housed under the conditions described for the SPF mice with sterile bedding and water. They were given food contaminated with fecal pellets from SPF mice. These mice are referred to as conventionalized ex-germ-free. Monocontamination of germ-free mice. An indigenous Lactobacillus strain was used for monocontamination of germ-free mice. It had been isolated on a medium developed for selection of lactobacilli, 1OA (20), from a stomach homogenate from an Ha/ICR mouse kept in our colony. The Lactobacillus strain was partially characterized (reference 22; Table 1). The bacteria were introduced into the germ-free isolators as pure broth cultures. The cultures were poured on the food of mice to be monocontaminated. The following procedure was used to insure that the mice were only monocontaminated. For each 10 monocontaminated mice, cecal homogenates from two were plated onto Brain Heart Infusion Agar (BHI; Difco), Sabouraud's Dextrose Agar (Difco), T7T (20), methylene blue agar (20), 10A (20), a medium for selection of bacteroides and clostridia (-S; reference 20), and blood-agar (Fisher Scientific Co., Pittsburgh, Pa.). The BHI, Sabouraud's, and blood-agar plates were incubated at 37 C aerobically and anaerobically (20). The 1OA and - S plates were incubated at 37 C anaerobically. The T7T and methylene blue plates were incubated at 37 C aerobically. After 5 to 7 days of incubation, the mice were considered to be monocontaminated if only the Lactobacillus sp. grew on any of the media. Enzyme assay. Alkaline phosphatase activity was assayed by either one of two procedures. Procedure one, used early in the experimentation, was replaced by procedure two, a more rapid assay system. In procedure one, the mice were killed at the same time each day with chloroform. Lengths of intestine of 1 to 3 mm and 3 to 5 mm for young and adult mice, respectively, were cut just posterior to the pylorus and freed of surrounding connective tissue and lumenal contents. The intestinal segments from a litter of 8 baby mice or 10 adult mice of the same sex were pooled. Chilled deionized water (5 to 10 ml) was added to each pool. The pools were then homogenized in a tissue grinder. The alkaline phosphatase activity in the homogenates was determined by using the King- Armstrong method (8) as modified by Moog (14). In procedure two, the mice were killed at the same time each day by cervical separation. A 1-cm section of intestine just posterior to the pylorus was cut and TABLE 1. Properties of the organism used for monocontamination Test media Tests performed Results 1OA brotha Microscopy Gram-positive rods Catalase pro- b duction Spore production Turbidity Microaerophilic growth 10 A + 0.5% Motility agara Litmus milk, Acid produc- Acid, clot tion, coagulation of milk Phenol red broth Fermentation + 1% of: carbohydratec Glucose Acid, no gas Lactose NGd Triple sugar iron agarc Indole nitrate medium, Production of H2S Production of indole Nitrate reduction a Medium for selection of lactobacilli (20). b Negative results. c Difco. d No growth. e BioQuest. freed of surrounding connective tissue and lumenal contents. Each duodenum was homogenized in 1 ml of 0.05 M tris(hydroxymethyl)aminomethane (Tris), ph 7.0, in a tissue grinder. The homogenate was centrifuged at approximately 2,500 X g for 10 min, and the supernatant solution was removed. The alkaline phosphatase activity in the supernatant solution was determined by using p-nitrophenol phosphate as the substrate (1). The reaction mixture contained 0.1 ml of 0.5 M MgCl2, 0.1 ml of 0.04 M p-nitrophenol phosphate, 0.1 ml of enzyme in 0.05 M Tris at ph 7.0, and 1.7 ml of 0.1 M Tris at ph 9.0. The reaction was started with the addition of the enzyme. The mixture was incubated at 37 C. The results were read at 415 nm on a DU spectrophotometer. In both procedures, the protein concentration was determined by the method of Lowry et al. (12) by using bovine serum albumin as the standard. Enumeration of bacteria. Segments of the gastrointestinal tract, always with contents intact, were weighed and then homogenized with Teflon grinders in 5 ml of sterile charcoal water (20). The homoge-

3 770 YOLTON, STANLEY, AND SAVAGE INFEC. IMMUN. nates were diluted in charcoal water in 10-fold steps. Calibrated loopfuls of each dilution were then spread on the surface of 1OA agar medium. The plates were incubated anaerobically at 37 C (20). Tissue sections. Segments of stomach and duodenum with contents intact were frozen in a 2% solution of methyl cellulose (17) in 0.15 M saline on the freezing shelf of a microtome-cryostat (International Equipment Co., Needham Heights, Mass.). Sections of frozen tissues, cut at 4 /im, were fixed for 60 sec in absolute methyl alcohol and stained with a modified tissue Gram stain (3). RESULTS Alkaline phosphatase activity in SPF, germ-free, and conventionalized ex-germ-free mice (procedure one). In early experiments, the alkaline phosphatase activity was assayed by procedure one in homogenates of the duodenums of germ-free, conventionalized ex-germ-free, and SPF mice (Table 2). In SPF neonates, the activity remains at mean levels of 0.64 units up to at least 12 days of age. Thereafter, the activity increases; mean activity levels of 5.66 units are found in 20-day-old animals. After 20 days of age, however, the activity declines; in adults, the levels lie between TABLE 2. the early neonatal levels and those determined in 20-day-old animals. In contrast, in the germ-free adults the activity levels are significantly higher than the levels in SPF mice at any age. In conventionalized ex-germ-free mice, however, the activity is within the range of levels found in the adult SPF mice. Monocontamination of germ-free mice with lactobacilli. The results from these first experiments prompted us to establish an experimental model to study further the effect of the microflora on alkaline phosphatase activity. This model involved monocontaminating germ-free animals with a member of the indigenous microflora. A strain of indigenous lactobacilii isolated from the stomach of an SPF Ha/ICR mouse was used as the monocontaminating microorganism. The particular Lactobacillus sp. established throughout the gastrointestinal tracts of the monocontaminated ex-germ-free mice (Table 3). Population estimates were made 7 days after the animals were exposed to the bacteria. The numbers of lactobacilli recoverable from the tissues of the monocontaminated mice were essentially the same as could be recovered from correspond- Alkaline phosphatase activity in the duodenal mucosa ofspecific pathogenz-free (SPF), germ-free, and conventionalized ex-germ-free Ha/ICR mice AMice Specific activitya No. of assays Type Age Range Mean deviation SPF days 20b NCC SPF days 12d NC' SPF weeks 22d Germ-free weeks 5d Ex-germ-freee weeks 3d a Assayed by procedure one, micromoles of phenol produced per minute per milligram of protein at 37 C. b Each assay done with a pool of eight mice (1 litter)., Not calculated. d Each assay done with a pool of 10 mice, all males or all females. e Associated for at least 5 days with the indigenous microbial flora of SPF Ha/ICR mice. TABLE 3. Number of lactobacilli in the digestive tract of adult specific pathogeii-free (SPF) anzd monocontamintated Ha/lICR mice Mice Gastrointestinal segment cultured" Type No. Stomach Duodenum Ileum Cecum SPF (7-8)b 7 (5-8) 8 (7-8) 8 (7-8) Monocontaminated 10 8 (7-8) 6 (6-8) 8 (5-8) 7 (7-8) a Cultured anaerobically (20) at 37 C on 10A medium (20). bmedium and range (in parentheses) of logso of the numbers of bacteria per gram of fresh tissue. c Cultured at 7 days after the germ-free mice were exposed to the lactobacilli.

4 VOL. 3, 1971 MICROBIAL FLORA 771 FIG. 1. Photomicrographs of frozen stomach sections stained by the tissue-gram stain method. Numerous rod-shaped bacteria (darkest layer) can be seen in close association with the mucosa (bottom). X 1,200. (A) Specific pathogen-free mouse. (B) Ex-germ-free mouse associated for 7 days with a Lactobacillus sp. ing tissues of SPF mice. Apparently, the lactobacilli colonized the stomachs and guts of the monocontaminated mice in the same pattern as they colonize those areas in the SPF mice. In both cases, the number of bacteria per gram of gastrointestinal tissue ranged from 106 to 108. Lactobacilli, found normally in the stomachs of mice, form a layer on the stratified squamous epithelium of the nonsecreting portion of the stomach (references 17, 21; Fig. IA). A similar layer was found on the nonsecreting epithelium in the stomachs of the monocontaminated animals (Fig. ib). Such layers are not formed in the 'duodenums; in frozen sections lactobacilli could be seen only in the lumens of the small bowels of either SPF or the monocontaminated mice. Alkaline phosphatase activity in SPF, germ-free, and monocontaminated ex-germ-free mice (procedure two). Procedure two was used in assays of duodenal alkaline phosphatase activity in the ex-germ-free mice monocontaminated with lactobacilli and in animals from SPF and germ-free control groups. The monocontaminated mice were assayed for alkaline phosphatase activity 2, 4, or 7 days after they were exposed to the lactobacilli (Table 4). As previously established with the findings of assays made by procedure one, the activity levels in the germ-free mice were significantly higher than the levels in the SPF mice. The mean activity of 7.75 units in the germ-free animals contrasts sharply with a mean of 1.89 units found in the SPF animals. This high germfree activity was reduced to a mean of 3.79 units, however, when the germ-free mice were associated with lactobacilli. This level lies between the germ- TABLE 4. Alkaline phosphatase activity in the duodenums of adult Ha/ICR specific pathogenfree (SPF), germ-free mice, and ex-germ-free mice monocontaminated with lactobacilli Type of micea Specific activityb Range Mean deviation SPF Germ-free Monocontaminatedc a Individual mice were assayed; 20 animals per group; all males, 5 to 7 weeks old. b Assayed by procedure two; micromoles of p-nitrophenol produced per minute per milligram of protein at 37 C. c Assayed at 2, 4, and 7 days after the mice were exposed to lactobacilli. Enzyme activity was found to be the same at all three times, so the results were pooled for this table. free and SPF values, but closer to the SPF values. Therefore, this strain of lactobacillus reduces the germ-free levels of alkaline phosphatase activity but does not reduce them completely to the levels found in mice with a complete indigenous microflora. DISCUSSION The term "alkaline phosphatase" may actually describe a number of enzymes which in vitro can catalyze the same reactions, but which in vivo may have entirely different enzymatic capacities.

5 772 YOLTON, STANLEY, AND SAVAGE INFEC. IMMUN. Certainly differences in purified alkaline phosphatase preparations from different tissues have been recognized (10). However, even the same tissue may contain more than one kind of alkaline phosphatase. In particular, the small intestine of the mouse can contain at least two alkaline phosphatases distinguishable by their catalytic properties (14). This heterogeneity has hindered studies of the biological function of alkaline phosphatases. The enzymes may play a role, however, in the active transport function of the microvilli (2, 4), absorption of cholesterol and other lipids (15), and absorption of monosaccharides (9). Our findings on the development of alkaline phosphatase activity in mice from birth to adulthood confirm earlier work by Moog (13). She and her co-workers have studied duodenal alkaline phosphatases in mice of several conventional strains. Such animals usually have complex bacterial floras (19). The flora of the Ha/ICR SPF strain is less complex and better defined than that of conventional mice (18). In these SPF animals, however, the levels of duodenal alkaline phosphatase activity are similar to those found by Moog in the conventional mice. The activity levels in either the SPF or conventional mice are strikingly lower, however, than the levels in germ-free mice. Moreover, the high germ-free levels of alkaline phosphatase activity fall to the SPF values in ex-germ-free mice colonized by an indigenous microflora. As assessed histochemically, germ-free mice also have higher alkaline phosphatase activity in their cecums than do conventional mice (7). Clearly, the intestinal microbial flora can dramatically affect duodenal alkaline phosphatase activity in the mouse. Several factors had to be considered in the design of an experimental model to study the affects of the microflora on alkaline phosphatase activity. In any such model, a known change in the flora must produce a significant change in alkaline phosphatase activity. Our preliminary evidence suggested that oral antibiotic treatment of SPF mice did not affect the alkaline phosphatase activity. Therefore, monocontamination of germ-free mice was attempted (21). An indigenous Lactobacillus strain was selected for the monocontamination experiments. Lactobacilli inhabit the stomachs of mice (5) and consequently can be found normally in appreciable numbers in the duodenum (5). It seemed desirable to use in our model an organism of the type that normally reaches high population levels in the region of the tissue assayed for alkaline phosphatase activity. The lactobacillus strain used colonized the entire gastrointestinal tract of the gnotobiotic mice and formed a layer along the nonsecreting gastric epithelium of the stomachs of the animals. Most importantly, though, the lactobacilli were present in high numbers in the duodenal lumens. Two days of association with the lactobacilli is sufficient to reduce the alkaline phosphatase activity in the monocontaminated mice. Longer exposure up to 7 days does not further change the activity level which is intermediate between the germ-free and SPF values. It is not surprising that alkaline phosphatase activity of germ-free mice does not return completely to SPF values in the mice contaminated with only one indigenous microorganism. Germ-free mice must be associated with more than one microbial species to convert their cecums to conventional size and their intestinal mucosa to conventional histological form (21, 23). The influence of the intestinal bacteria on the mammalian host may be due not only to the presence of bacteria but also to interactions among the bacteria themselves. In conclusion, the alimentary microbial flora profoundly affects in some way the levels of alkaline phosphatase activity in the mouse intestinal mucosa. Clearly, the gastrointestinal microflora cannot be ignored in studies of intestinal alkaline phosphatases and other intestinal enzymes as well (16). The indigenous microbial flora produces profound effects upon the host's intestinal morphology (23) and physiology. Thus, this microflora must be considered a major component of the gastrointestinal canal. ACKNOWLEDGMENTS This investigation was funded by Public Health Service research grant AI and training grant AI from the National Institute of Allergy and Infectious Diseases and training grant GM from the National Institute of General Medical Sciences. LITERATURE CITED 1. Bessey, 0. A., 0. H. Lowry, and M. J. Brock A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem. 164: Boyd, C. A. R., D. S. Parsons, and A. V. Thomas The presence of K+-dependent phosphatase in intestinal epithelial cell brush borders isolated by a new method. Biochim. Biophys. Acta 150: Brown, J. H., and L. 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