Gnotobiotic Mice. Samples at different levels of the digestive tract. were obtained in the following fashion. The animals

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1 ANTIMIcRoBIAL AGENTS AND CHZMOTHZRAPNY, Jan. 1978, p Copyright 1978 American Society for Microbiology Vol. 13, No. 1 Printed in U.S.A. Production of an Antibiotic Substance by Bacillus licheniformis Within the Digestive Tract of Gnotobiotic Mice R. DUCLUZEAU,l F. DUBOS,l P. RAIBAUD,1 AND G.D. ABRAMS2 Laboratoire d'ecologie Microbienne, Institut National de la Recherche Agronomique, 7835 Jouy-en-Josas, France,' and Department ofpathology, University ofmichigan, Ann Arbor, Michigan Received for publication 1 May 1977 In monoxenic mice, vegetative cells and spores of Bacillus licheniformis were enumerated and in vivo antibiotic production was measured at various levels of the digestive tract and in the feces. The apparent independence of vegetative cell and spore populations in the cecum and feces, as well as the marked fluctuations of these two populations in the stomach and small intestine, suggested that sporulation ofb. licheniformis and production of antibiotic occur only in the upper levels of the digestive tract. Study of dixenic models wherein B. licheniformis was inoculated after Clostridium perfringens or Lactobacillus sp. or before Lactobacillus sp. revealed the simultaneous disappearance of B. licheniformis from the stomach and antibiotic from the feces. This strain persisted in the cecum, but only in the form of vegetative cells. These models demonstrate that the activity of the microflora in the upper segments can affect the equilibrium of the microflora throughout the digestive tract. In an earlier study (4) we observed that the establishment of a strain ofbacillus licheniformis in the digestive tract of axenic (germfree) mice prevents the subsequent establishment of Clostridium perfringens in these same animals. This antagonistic effect was shown to depend upon the in vivo production of an antibiotic substance similar to bacitracin by B. licheniformis. However, it was found that certain strains of bacteria, if established in the digestive tract before or at the same time as B. licheniformis, were able to prevent the production of antibiotic by the latter strain. In particular, the prior establishment of C. perfringens in gnotobiotic mice prevented both the production of antibiotic and the antagonistic effect of B. licheniformis when subsequently implanted. The purpose of the present work was to elucidate the factors which condition the production of antibiotic by B. licheniformis in vivo. MATERIALS AND METHODS Animals. The animals used in these studies were adult axenic (germfree) C3H mice. The methods of maintenance and inoculation of animals used in the present study, as well as the methods of fecal sampling, have been described previously (4). The mice were generally fed a commercial diet (Labena Special Duquesne Purina), designated as 97 "D.'+ In particular experiments, as indicated in the text, the animals were fed a semisynthetic diet, designated as "S." The composition of this diet has been given elsewhere (2). Samples at different levels of the digestive tract were obtained in the following fashion. The animals were killed by cervical elongation and immediately autopsied. The stomach, cecum, and colon were sampled, and the small intestine was divided into three equal segments designated, beginning proximally, as SIR, SI2, and SI3. These samples were weighed, placed in ten times their weight of sterile water, and homogenized with an Ultraturrax. The suspensions thus obtained were considered as 1-l dilutions of the samples. Bacterial strains, culture media, and enumeration. The origin of the strains of B. licheniformis and C. perfringens as well as the methods used in their culture and enumeration have been described previously (4). Strain 313 of Lactobacillus sp., isolated from the feces of conventional rats, was also used. This strain belongs to group Th 5, which has been described (8). It was grown on medium LAPT glo (8) and selectively enumerated on medium AGAT (7). Measurement of the oxidation-reduction potential of cecal contents. The method described by Wostmann and Bruckner-Kardoss for the rat was used (1). Morphological assessment of the association between bacteria and gastric mucosa. Samples of the gastric mucosa and wall were obtained from animals harboring B. licheniformis alone or along with other selected strains, gently washed by immersion in

2 98 DUCLUZEAU ET AL. saline to remove food particles, and fixed in neutral 1% Formalin. Samples were prepared for light microscopy by paraffin embedding, sectioning, and staining with hematoxylin and eosin. Samples intended for examination by scanning electron microscopy were dehydrated in ethanol, infiltrated with amyl acetate, and critical-point dried. After gold coating, the specimens were examined with the JS MU3 scanning electron microscope. Assay of antibiotic produced by B. licheniformis. Antibiotic content of the feces was assayed by the method previously described (4), which consists of measuring, in a test culture of C. perfringens, the radius of the inhibition zone formed around a fecal pellet pressed into the medium. To assess the content of antibiotic in the stomach, small intestine, or cecum, approximately 1 mg of contents of the organ in question were used in place of feces, and the test organism used was a strain of facultatively anaerobic Corynebacterium species. Measurement of transit through the digestive tract. The rate of transit through the digestive tract was measured by the method of Contrepois and Gouet (3) by using the spores of a strictly thermophilic B. subtilis as a marker. RESULTS Sporulation of B. licheniformis and production of antibiotic in the feces of monoxenic mice. B. licheniformis was implanted in a group of 12 axenic mice fed commercial diet D (group A), and the animals were held for 9 days after inoculation. During this period, feces were sampled 3 times at intervals of a few days beginning 8 days after inoculation. In each sample, consisting of a pool of 12 fecal pellets freshly passed by 12 mice, the number of spores and the number of vegetative cells of B. licheniformis were determined. The zones of inhibition produced by four randomly selected fecal pellets were also measured. It was determined (Fig. 1) that the number of vegetative cells in the feces and the radius of the inhibition zone varied only slightly during the period of observation. The number of spores, however, was much more variable. The same experiment was performed with two other groups of six mice each. Group B was fed commercial diet D, but the mice were given drinking water containing 1 g/liter of manganese sulfate, which produces sporulation of B. licheniformis in vitro. Group C received semisynthetic diet S and deionized water to drink. These two groups were held for 5 days, during which 2 fecal samples were secured. As shown in Fig. 1, the number of spores of B. licheniformis in the feces of group B was slightly lower than that observed in group A. However, the number of vegetative cells and the radius of the zones of inhibition were similar in the two groups. In the animals of group ANrIMICROB. AGENTS CHEMOTHER. 3 vegetative spore J rodius of ontibiotic T inhibition zone dietory regimen FIG. 1. Influence of dietary regimen on the number of spores and vegetative cells ofb. licheniformis and on the production of antibiotic in the feces of monoxenic mice. Group A, animals receiving diet D and deionized water; group B, animals receiving diet D and MnSO4 solution; group C, animals receiving diet S and deionized water. The broad bars represent the arithmetic means of the various values. The vertical lines at the ends of the bars indicate the ranges of values. The means in group A are derived from 3 pooled samplings; the means in groups B and C are derived from 2 pooled samplings. C, the number of vegetative cells was approximately 1-fold lower than in the other two groups, but the number of spores and the zones of inhibition were similar to those in group A. The same analyses were performed on the feces of infant mice suckled by mothers from group A. Vegetative cells of B. licheniformis were only irregularly present in these animals, always in low numbers (14 on the average). No spores could be detected, and zones of inhibition were never observed around the feces from suckling mice. Distribution of vegetative cells and spores of B. licheniformis and of antibiotic in different segments of the digestive tract of monoxenic mice. In 18 monoxenic mice inoculated at least 1 days earlier, six levels of the digestive tract were sampled. Vegetative cells and spores of B. licheniformis were enumerated, and the in vitro inhibition of bacterial growth by antibiotic was assessed. The results are shown in Fig. 2. The number of spores of B. licheniformis in the upper segments of the digestive tract was found to be low, extremely variable, and in the stomach, sometimes even zero. The number of cells counted in an unheated sample was in some instances greater than the number of spores recovered after heating, thus reflecting the number of vegetative cells in the original sample. In other instances, the numbers before and after heating were close, indicating that

3 VOL. 13, 1978, 1977 ANTIBIOTIC PRODUCTION IN DIGESTIVE TRACT 99 the number of vegetative cells was equal to or less than the number of spores in the sample. In these upper segments ofthe tract, production of antibiotic was observed only in rare samples, with no apparent correlation with the number ofb. licheniformis present. In the cecum and colon, the number of vegetative cells ofb. licheniformis was higher than distribution at vorious lveis of the digestive troct FIG. 2. Distribution of spores and vegetative cells of B. licheniformis and antibiotic produced at various levels in the digestive tract of monoxenic mice. S, Stomach; SI1, SI2, and SI3, proximal, middle, and distal thirds of small intestine, respectively; Cec, cecum; Col, colon. The broad bars represent the arithmetic means of the various values. The vertical lines at the ends of the bars indicate the ranges of values. L *6L C- -ọ 2 - a. E 8, c e 6 --4, 2 L t Si 1 in the upper segments of the tract, and was very constant from one animal to the next. In contrast, the number of spores remained extremely variable and at a level similar to that in the upper segments of the tract. The antibiotic was constantly present in cecum and colon, and the extent of inhibition was less variable from one animal to the next than in the upper segments of the digestive tract. Kinetics of establishment of B. licheniformis and production of antibiotic at different levels of the digestive tract in monoxenic mice. Seven axenic mice were given an intragastric inoculum containing, in.2 ml, 7 x 16 spores ofb. licheniformis and 2 x 16 spores of B. subtilis serving as a transit marker. At intervals between and 48 h after inoculation, one animal was autopsied, and in each segment of the digestive tract, the number of vegetative cells and spores of B. licheniformis and the number of spores of B. subtilis were determined. The presence of antibiotic was also assessed. The results are shown in Fig. 3. The spores of the transit marker disappeared rapidly in all segments of the digestive tract. Accordingly, the spores of B. licheniformis present at the second sampling and beyond represent spores newly formed in situ and not simply residual spores of the inoculum. It can be seen that in the first four levels studied, the numbers of vegetative cells and spores of B. licheniformis remained close to one another ~~~~~~~4' Iṫ.. ) SI 2 b 2' 35 4' 5' - SI3 1 CcC 1 Col.o ~ 4 E l hours.. vegetative cells of B. licheniformis spores of B.licheniformis o.-..-o thermophilic tronsit morker spores FIG. 3. Kinetics of the implantation ofb. licheniformis and the production of antibiotic at different levels of the digestive tract in monoxenic mice. S, Stomach; SI,, SI2, and SI3, proximal, middle, and distal thirds of small intestine, respectively; Cec, cecum; Col, colon. Ordinates represent either the log1, of the number of bacteria or the radius in millimeters of the antibiotic inhibition zones in individual mice.

4 1 DUCLUZEAU ET AL. but fluctuated widely. In the cecum and colon, on the other hand, the number of vegetative cells stabilized rapidly at a level clearly above that in the upper segments. The number of spores, however, remained variable and of the same order of magnitude as in the upper segments. The presence of antibiotic was detected only occasionally within the upper segments, but regularly within the cecum and colon beginning at hour 3. Four additional experiments of this type were performed, yielding equivalent results. Influence of the establishment of various bacteria in the digestive tract of gnotobiotic mice on the growth, sporulation, and antibiotic production in vivo of B. licheniformis. A group of 1 axenic mice was inoculated with a strain of C. perfringens and, 3 days later, with a strain of B. licheniformis. Ten days later, the mice were sacrificed, and one fecal pellet was obtained from each at the moment of sacrifice. There was no evidence of antibiotic in any of these fecal pellets (Table 1), confirming our previous results (4). There was a popu- ANTIMICROB. AGENTs CHEMOTHER. lation of C. perfringens in the stomach of these animals, but there were neither spores nor vegetative cells of B. licheniformis. In the cecum, the population of vegetative cells of B. licheniformis was as abundant as that of C. perfringens. This population of B. licheniformis was only slightly less than that seen in monoxenic mice harboring that strain. However, practically no spores of B. licheniformis were found in the cecum of dixenic animals, in contrast to the population of spores in monoxenic mice. Finally, it was found that the oxidation-reduction potential was approximately 2 mv lower in the cecum of animals harboring both C. perfringens and B. licheniformis than in animals carrying only the latter strain. In two additional groups of axenic mice, B. licheniformis and strain 313 of Lactobacillus sp. were established. In one group, B. licheniformis was the first to be implanted, and in the other it was the second. Both strains of bacteria were enumerated in stomach and cecum. Vegetative cells of B. licheniformis were present in the cecum in both groups at TABLZ 1. Influence ofc. perfringens and Lactobacillus sp. on the number ofspores and vegetative cells of B. licheniformis in stomach and cecum ofdixenic mice and on antibiotic content of the feces Radius (mm) of Bacteria inoculated Bacteria enumerated Stomacha Cecuma antibiotic inhi- No. of feces B. licheniformis B. licheniformis alone VCb (3.8-8.) ( ) (5-12) B. licheniformis TSc (<2-4.3) ( ) C. perfringens then B. licheniformis VC < B. licheniformis (<2-<2) ( ) (-) B. licheniformis TS <2 <2 (<2-<2) (<2-2.6) C. perfringens VC ( ) (8.-8.9) Lactobacillus 313 B. licheniformis VC < then B. lichenifor- (<2-2.7) (8.-9.3) (-4) mis B. licheniformis TS <2 <2 (<2-<2) (<2-2.3) Lactobacillus ( ) (9.-9.7) B. licheniformis B. licheniformis VC < then Lactobacillus (<2-2.6) (9.-9.5) (-) 313 B. licheniformis TS <2 <2 (<2-<2) (<2-2.5) Lactobacillus (6.-8.) (8.-9.9) a Arithmetic mean of the log1o of the number of bacteria per gram of fresh organ. Figures in parentheses indicate the range. b VC, Vegetative cells. c TS, Thermoresistant spores.

5 VOL. 13, 1978, 1977 levels equal to those in monoxenic animals, but spores were not found. In the stomach, neither vegetative cells nor spores ofb. licheniformis were found. In neither group was there evidence of a zone of inhibition around the feces. Morphological assessment of the association -between bacteria and gastric mucosa. Certain organisms, notably lactobacilli, are known to colonize portions of the gastric epithelium (5, 9). Since the preceding experiments pointed to the importance of the gastric population of B. licheniformis, the manner of association of this strain with the gastric mucosa was examined by light and scanning electron microscopy. In no instance was it possible to demonstrate attachment of B. licheniformis to the nonglandular epithelial surface of the stomach. This squamous epithelium retained the bare appearance of that in the axenic animal. Bacilli were readily visualized in the mucous blanket covering the glandular portion of the stomach in monoxenic mice (Fig. 4). It was not possible to determine whether the bacteria were present on the mucus in higher concentrations than throughout the gastric contents. Examination of the stomachs of dixenic animals revealed, as expected, attachment of lactobacilli to the nonglandular surface. tdc. ANTIBIOTIC PRODUCTION IN DIGESTIVE TRACT 11,,g DISCUSSION It is known that the production of antibiotic by B. licheniformis is linked to the process of sporulation (1). Therefore, in the design of experiments, attention was centered on this aspect of the population of B. licheniformis. Our attempts to manipulate the in vivo rate of sporulation by varying the diet of the animals did not produce the expected effects, however. When added to the diet of the animals, manganese sulfate, which increases the rate of sporulation in vitro, seemed instead to have an inhibitory effect on sporulation in vivo. Utilization of a semisynthetic diet led to a diminution in the number of vegetative cells in the feces, as we had previously observed with other strains of bacteria (2), but was without effect on the number of spores. The diet of suckling mice allowed only minimal growth ofb. licheniformis in the digestive tract, but inhibited sporulation. Nonetheless, the several experiments did yield data pointing to certain conclusions about the factors which condition in vivo antibiotic production. The observation (Fig. 2 and 3) of a different ratio of spores to vegetative cells of B. licheniformis in cecum and colon as compared with that in upper segments of the digestive tract I Ant "' MA-4 5N.L.. Jr4., - IIt -. ev -~~~ A. ~ d- Z i FIG. 4. Scanning electron micrograph ofglandular mucosa of stomach in a monoxenic mouse harboring B. licheniformis. The epithelium is covered by a mucous blanket which forms the background of the photo. Many bacilli and a few food particles adhere to the mucus (x7,).

6 12 DUCLUZEAU ET AL. suggests that sporulation probably does not occur in cecum, colon, or feces. Thus, despite a 1,-fold increase in the number of vegetative cells in the lower tract as compared with stomach and small intestine, there was no change in the number of spores present. It has been previously demonstrated that B. licheniformis, which is capable of multiplying under anaerobic conditions, sporulates only under aerobic conditions (6); we have verified this for the strain of B. licheniformis used in this work. It seems likely that cecal and colonic contents of monoxenic mice are too anaerobic to allow the sporulation of B. licheniformis, whereas conditions in the upper segments of the tract are more favorable. It also seems likely that sporulation and antibiotic production actually begin in the stomach, given the retention of contents at that level, followed by relatively rapid transit through the small intestine. In fact (Fig. 3), spores are regularly demonstrable in the stomach and, on occasion, so is antibiotic activity. However, in the upper segments of the digestive tract, particularly in the stomach, the populations of spores and vegetative cells fluctuate, seemingly independently. It is conceivable that, in the upper tract, physico-chemical conditions, such as average ph and relative aerobiasis, are transiently favorable to the multiplication of B. licheniformis and, then, at another time, are favorable to sporulation. These conditions vary with the feeding activity and digestive physiology of the animal. The spores produced at a given time in the stomach or small intestine move downward along the digestive tract. Greater or lesser numbers are recovered in the upper segments of the tract, depending on the interval between actual formation of the spores and sampling. In the lower segments of the tract, variation is reduced by relative stasis of the digestive contents in the distended cecum of the monoxenic mice and by slow passage through the colon. Coprophagy was not prevented in these experiments, but cannot be responsible by itself for the presence of vegetative cells and spores of B. licheniformis in the stomach. Indeed, in individual animals, the ratio between vegetative cells and spores was very different in the stomach than in the colonic contents. In certain gastric samples, for instance, vegetative cells were observed in the absence of spores; in others, only spores were seen. In each case, both spores and vegetative cells were present in the colonic contents. In addition, in the dixenic animals, a large population of vegetative cells of B. licheniformis was seen in the lower bowel, whereas B. licheniformis was absent from the stomach. ANTIMICROB. AGENTS CHEMOTHER. Production of antibiotic, like sporulation, is irregular in the upper segments of the digestive tract. It is likely that the antibiotic produced during intermittent cycles of sporulation is distributed along the entire digestive tract. Relative transit times and stasis of part of the digestive contents in the cecum would explain why antibiotic is found with great constancy in the feces after being produced in the upper tract. Our experiments with dixenic animals support this line of reasoning. It was found that the establishment of C. perfringens or Lactobacillus sp. resulted in the disappearance of vegetative cells and spores of B. licheniformis from the stomach. In the cecum, however, only the spores disappeared, suggesting that the spores are formed by vegetative cells in the stomach and possibly in the small intestine, but not in the cecum. The disappearance of spores from the digestive tract coincided with the disappearance of antibiotic from the feces. This indicates that the effect of C. perfringens and of Lactobacillus sp. is primarily on the production of antibiotic, and does not involve destruction or inhibition of antibiotic once formed. In fact, in our previous study (4), the antibiotic substance produced by B. licheniformis was found to be stable, at least as judged by resistance to heating and to changes in ph. The mechanism by which C. perfringens and Lactobacillus sp. suppress sporulation and antibiotic production by B. licheniformis is no doubt complex. Our results demonstrate that vegetative cells ofb. licheniformis cannot grow in the upper digestive tract when C. perfringens or Lactobacillus sp. are established, but continue to flourish in the lower bowel in the presence of these strains. The reasons for this are not apparent. Competition for attachment sites in the stomach does not seem to be a factor, since even in the monoxenic animal, B. licheniformis does not attach to the epithelium in the manner of lactobacilli. Alteration in oxidation-reduction potential likewise does not explain the difference between stomach and cecum, since B. licheniformis continues to grow even in the reduced conditions in the cecum associated with C. perfringens. In any event, the disappearance of antibiotic production could be explained simply as the elimination of B. licheniformis from those segments of the digestive tract in which sporulation and antibiotic production are possible. Alternatively, the antagonistic strains could also have an inhibitory effect on sporulation per se. The precise physico-chemical mechanisms of interaction between the three bacterial strains in the upper digestive tract were not investi-

7 VOL. 13, 1978, 1977 gated in these studies. Whatever these mechanisms, it is clear that the population of B. licheniformis is associated with a potent antagonistic effect on C. perfringens throughout the tract. Thus this model demonstrates, at least in the mouse, the potential impact of the bacterial population of the upper segments on the equilibrium of the microflora along the entire length of the digestive tract. ACKNOWLEDGMENT This study was supported by NATO research grant 689. LITERATURE CITED 1. Berniour, R. W., and G. D. Novelli Some characteristics ofbacitracin production by Bacillus licheniformis. Arch. Biochem. Biophys. 87: Chopin, A., R. Ducluzeau, and P. Raibaud Effet du regime alimentaire sur l'equilibre entre 14 souches microbiennes ensemencees dans le tube digestif de souris axeniques adultes et sur l'etablissement de ces souches chez leurs descendants entre la naissance et le sevrage. Can. J. Microbiol. 2: Contrepois, M., and P. Gouet Utilisation d'une technique microbiologique pour la mesure de la vitesse de transit des microparticules dans le tractus ANTIBIOTIC PRODUCTION IN DIGESTIVE TRACT 13 digestif des ruminants. C. R. Acad. Sci. (Paris) 268: Ducluzeau, R., F. Dubos, P. Raibaud, and G. D. Abrams Inhibition of Clostridium perfringens by an antibiotic substance produced by Bacillus licheniformis in the digestive tract of gnotobiotic mice: effect of other bacteria from the digestive tract. Antimicrob. Agents Chemother. 9: Fuller, R Ecological studies of the lactobacillus flora associated with the crop epithelium of the fowl. J. Appl. Bacteriol. 36: Haavik, H. I Studies on the formation of bacitracin by Bacillus licheniformis: effect of glucose. J. Gen. Microbiol. 81: Raibaud, P., M. Caulet, J. V. Galpin, and G. Mocquot Studies on the bacterial floral of the alimentary tract of pigs. II. Streptococci: selective enumeration and differentiation of the dominant group. J. Appl. Bacteriol. 24: Raibaud, P., J. V. Galpin, R. Ducluzeau, G. Mocquot, and G. Oliver Le genre Lactobacillus dans le tube digestif du rat. I. Caracteres des souches homofermentaires isolees de rats holo et gnotoxeniques. Ann. Inst. Pasteur 124A: Savage, D. C., R. Dubos, and R. W. Schaedler The gastrointestinal epithelium and its autochthonous bacterial flora. J. Exp. Med. 127: Wostmann, B. S., and E. Bruckner-Kardoss Oxidation-reduction potentials in cecal contents of germfree and conventional rats. Proc. Soc. Exp. Biol. Med. 121: Downloaded from on November 29, 218 by guest