Reduction of Population Levels of Some Indigenous Bacteria by Lactobacilli in the Gastrointestinal Tract of Gnotobiotic Rats

Microbiol. Immunol. Vol. 21 (9), 495-503, 1977 Reduction of Population Levels of Some Indigenous Bacteria by Lactobacilli in the Gastrointestinal Tract of Gnotobiotic Rats Tsugio WATANABE, Masami MOROTOMI, Yasuo KAWAI, and Masahiko MUTAI Department of Intestinal Microbiology, Yakult Institute for Microbiological Research, Kunitachi, Tokyo (Received for publication, January 10, 1977) Abstract Effects of each of three indigenous Lactobacillus groups on other bacterial populations were separately investigated in gnotobiotic rats. In the wall of the non-glandular part of the stomach, contents of the stomach and contents of the upper part of the small intestine, some pre-associated indigenous bacteria were reduced to conventional population levels by introducing three groups of lactobacilli : Group I (Lactobacillus acidophilus and related strains), Group II (L. fermentum) and the groups mixed. However, no obvious reduction in cell numbers of the pre-associated bacteria occurred in the case of Group III (L. murini). Lactobacilli are considered to be one of the most important members of the intestinal microflora of rats (6) and mice (2). The lactobacilli in conventional and gnotobiotic rats are most predominant in the stomach and the small intestine. In the cecum, rectum and feces, lactobacilli are also maintained at high population levels. We investigated localization of the indigenous lactobacilli at the species level in a previous report (11). In that report, it was revealed that the lactobacilli consisted of L. acidophilus and related strains, L. fermentum and L. murini, and furthermore these three groups of lactobacilli colonized differently in various parts of the gastrointestinal tract. On the other hand, populations of some indigenous bacteria such as E. coli, staphylococci, streptococci and bacteroides were suppressed in the stomach and the small intestine by the lactobacilli although mechanisms of this suppression are not yet known (6). Therefore, in this study, we investigated Lactobacillus suppression by preparing various gnotobiotic rats to which each of the Lactobacillus groups were separately administered in addition to pre-associated bacteria such as E. coli staphylococci, streptococci, bacteroides and fusiform bacteria. In this report, roles of each Lactobacillus group in the suppression are described by comparing bacterial flora of gnotobiotic rats containing lactobacilli with those of lactobacilli-free gnotobiotic and conventional rats. 495

496 T. WATANABE ET AL MATERIALS AND METHODS Bacteria used. Bacteria were isolated from normal conventional rats. The media for both isolation and growth, and cultural conditions are shown in Table 1. Of these strains, five (Escherichia coli N-1, Staphylococcus epidermidis N-1, Streptococcus fecalis var. liquefaciens Y-5, Bacteroides fragilis GAM-8 and Fusiform bacteria EG-1) were designated as GB 5 bacteria. Strains of Lactobacillus acidophilus and related strains, L. fermentum, and L. murini were designated as Group I, II and III respectively (11). Table I. Strains of bacteria used in contamination: their origin, media used, and cultural conditions Rats used. Conventional (Cony) rats, CDF strain, 7 weeks old and germfree rats, CDF strain, 4-5 weeks old were used. Diet and other experimental conditions were the same as in the previous report (6). Inoculation of animals. Five mixtures of bacterial strains were prepared in accordance with the methods described previously (6, 11). They were mixtures of GB5 bacteria, Group I, II, III and the three groups mixed, and the five experimental groups of gnotobiotic rats, which were prepared by associating each of the bacterial mixtures with stomach tubes into germfree animals in the schedule shown in Table 2, were designated as GB5, GB5 +GI, GB5 +GII, GB5 +GIII and GB5+ Mix respectively (Table 2). Preparation of gastrointestinal specimens. The animals were killed with CO2 gas at the same age (7 weeks old, refer Table 2). Sections of alimentary tract used were as follows : stomach, upper part and lower part of the small intestine (11), and cecum.

LACTOBACILLUS SUPPRESSION IN RAT INTESTINE 497 Table 2. Preparation of gnotobiotic animal groups and the experimental schedule Gnotobiotic animal groups were maintained in vinyl isolators during the experimental period. Arrows ( ) mean inoculation of a bacterial mixture indicated. Culture techniques. Dilution techniques of gut contents and the wall of the nonglandular part of the stomach, media used, and inoculation size were the same as in a previous report (11). Expression of data in figures and Lactobacillus suppression. The data in the figures indicate the range (mean + SD) and "range of mean" (6). Lactobacillus influences on population levels of GB5 bacteria were classified into three degrees : "suppression," "weak suppression" and "no suppression." If the population level of a GB5 bacterium was suppressed to the conventional level by contamination of lactobacilli, it was expressed as "suppression." "Weak suppression" was assumed if the population of a GB5 bacterium was higher than that of the conventional level although significant reduction was observed. If no change was observed or no change could be detected because the population of a GB5 bacterium in the GB5 animals was below 4 ~ 103 per ml, it was expressed as "no suppression." RESULTS Bacterial Flora on the Wall of the Non-Glandular Part of the Stomach (Fig. 1) i) Lactobacillus populations of experimental animal groups. In the GB5 +Mix, the Lactobacillus population level was higher than those of GB5 +GI, GB5 +GII and GB5 + GIII. ii) GB5 + GI animals. Population levels of E. coli, staphylococci and streptococci were reduced significantly (P<0.01) but were higher than those in conventional animals (weak suppression). iii) GB5 +GII animals. Numbers of staphylococci cells were suppressed to the conventional level, and those of E. coli and streptococci were suppressed weakly. iv) GB5 +GM animals. Group III lactobacilli did not reduce populations of the GB5 bacteria at all. v) GB5 + Mix animals. Population levels of E. coli, staphylococci and streptococci were suppressed to conventional levels. Because bacterial cell numbers of bacteroides and fusiform bacteria were below 4 ~ 103, the Lactobacillus suppression of these bacteria could not be detected in this area.

498 T. WATANABE ET AL Fig. 1. Population levels of indigenous bacteria on the wall of the non-glandular part of the stomach in gnotobiotic and conventional rats. Five animals were used in each experimental group. a, GB5 rats; b, GB5 +GI rats; c, GB5 +GII rats; d, GB5 +GIII rats; e, GB5 +Mix rats; f, Conventional rats. Solid lines, mean and standard deviation; broken lines, range of mean. Fig. 2. Population levels of indigenous bacteria in the contents of the stomach in gnotobiotic and conventional rats. See legend to Fig. 1.

LACTOBACILLUS SUPPRESSION IN RAT INTESTINE 499 Bacterial Flora in the Stomach Contents (Fig. 2) i) Lactobacillus populations of experimental animal groups. Lactobacillus populations of the three groups of gnotobiotic rats which were associated individually with a different single group of lactobacilli (GB5 +GI, GB5 +GII and GB5 +GM) were the same. However, Lactobacillus populations of GB5 +Mix and conventional animals were somewhat higher than those of GB5 + GI or GB5 +GIL ii) GB5 +GI animals. Only the bacteroides population was suppressed weakly. Reductions in other bacterial populations did not occur when the GB5 rats were associated with Group I lactobacilli. iii) GB5 +GII animals. The population level of bacteroides was suppressed to the conventional level by administration of Group II lactobacilli. Weak suppression in numbers of E. coli and staphylococci was also observed. iv) GB5 +GIII animals. No changes in numbers of any of the GB5 bacteria were detected when the GB5 rats were further associated with Group III lactobacilli. v) GB5 + Mix animals. In this animal group, population levels of all the GB5 bacteria except for fusiform bacteria were suppressed to conventional levels by administration of the mixed lactobacilli. Bacterial Flora in Contents of the Upper Part of the Small Intestine (Fig. 3) The Lactobacillus suppression of the numbers of staphylococci, bacteroides and fusiform bacteria cells could not be detected because the population levels of these bacteria were very low. Of the three single Lactobacillus groups, only Group II showed suppressive effects because the numbers of streptococci were suppressed to Fig. 3. Population levels of indigenous bacteria in the upper part of the small intestine in gnotobiotic and conventional rats. See legend to Fig. 1.

500 T. WATANABE ET AL the conventional level and those of E. coli were suppressed weakly by administration of this group of lactobacilil to lactobacilli-free animals, although population levels of lactobacilli in this animal group (GB5 +GII) were somewhat lower than those of other experimental animal groups. On the other hand, association of the mixed lactobacilli was more effective than Group II because the population level of E. coli was also suppressed to the conventional level in addition to that of streptococci. Bacterial Flora in Contents of the Lower Part of the Small Intestine (Fig. 4) Lactobacillus populations of GB5 +GIII and GB5 +Mix animals were higher than those of GB5 +GI, GB5 +GII and conventional rats. In the latter animal groups, viable counts of E. coli and streptococci were similar to those of lactobacilli which were most predominant in both the wall and contents of the stomach and the upper part of the small intestine. In this part, apparent Lactobacillus suppression was not observed in any experimental animal group, but there were four cases in which some of the pre-associated GB5 bacteria were suppressed weakly by lactobacilli. There were slight reductions in numbers of E. coli and streptococci in the GB5 + GI group, and of streptococci and bacteroides in the GB5 + Mix animals. Fig. 4. Population levels of indigenous bacteria in the lower part of the small intestine in gnotobiotic and conventional rats. See legend to Fig. 1. Bacterial Flora in Contents of the Cecum (Fig. 5) Population levels of lactobacilli in the gnotobiotic animal groups were almost the same as those for E. coli, streptococci, bacteroides and fusiform bacteria. In conventional rats, fusiform bacteria was the predominant bacterial species among the five bacteria examined.

LACTOBACILLUS SUPPRESSION IN RAT INTESTINE 501 Fig. 5. Population levels of indigenous bacteria in the cecum in gnotobiotic and conventional rats. See legend to Fig. 1. Although differences in the Lactobacillus populations were observed in the gnotobiotic rats, effects of the different lactobacilli were similar, i.e. numbers of E. coli and streptococci were suppressed weakly in all the gnotobiotes. DISCUSSION It is well known that bacterial flora are altered by many factors such as diet (1, 3, 10), animal strain (5, 8) and environmental conditions (10). Therefore, attention should be paid to assessment of changes produced by various experimental treatments. In this study, reductions in the population levels of the pre-associated GB5 bacteria were observed when lactobacilli-free gnotobiotic rats were associated with various kinds of Lactobacillus mixtures, and these reductions were classified into three degrees : "suppression," "weak suppression" and "no suppression" (see MATERIALS AND METHODS). However, reduction in a population level classified as "weak suppression" should be considered as no change because it was possible that such reductions might be produced by unknown factors other than Lactobacillus contamination although they were statistically significant. Therefore, we regarded only changes which were classified as "suppression" as obvious reductions produced by the lactobacilli introduced. Lactobacillus suppression. Results are summarized in Table 3 to show the relationships among factors such as post-associated lactobacilli, pre-associated GB5 bacteria, and the five gastrointestinal parts examined. Lactobacillus suppression was restricted within the wall of the non-glandular part

502 T. WATANABE ET AL Table 3. Antagonistic relationships between lactobacilli and GB5 bacteria in the five gastrointestinal parts (summary of the data illustrated in Figs. 1-5) GI, GII, GIII and Mix indicate Group I, Group II, Group III and the three groups mixed respectively. E, STA, STR, B and F indicate E. coli N-1, Staph. epidermidis N-1, Str. fecalis var. liquefaciens Y-5, B. fragilis GAM-8 and fusiform bacteria EG-1 respectively. of the stomach, contents of the stomach and the upper part of the small intestine. With respect to bacterial species suppressed and gastrointestinal parts where the Lactobacillus suppression was observed, these results were similar to those reported in a previous paper (6) in which it was stated that population levels of some indigenous bacteria were controlled by the presence of the lactobacilli in the gnotobiotic rats. Furthermore, it should be pointed out that in these parts lactobacilli are predominant in both the gnotobiotic and conventional rats. It is also important that in the cecum where lactobacilli are not predominant, there was no Lactobacillus suppression of the population levels of the GB5 bacteria. As shown in Table 3, Group II lactobacilli (L. fermentum) was more effective than Group I (L. acidophilus and related strains) or Group III (L. murini) in the suppression. From these results, we presume that reductions in cell numbers of some indigenous bacteria observed in GB5 + Mix and similar results reported previously (6) are produced mainly by Group II (L. fermentum). There are several reports concerning microbial interference between indigenous bacteria, or between indigenous and non-indigenous microbes in vivo (4, 6, 7). However, mechanisms involved in these microbial antagonisms including the Lactobacillus suppression described in this report have not been resolved perfectly.

LACTOBACILLUS SUPPRESSION IN RAT INTESTINE 503 Although little is known about Lactobacillus suppression, we consider that these mechanisms should be analyzed separately as two types of phenomena. One of these is a microbial interference on the wall of the stomach, and the other is a interference in luminal contents. It is considered that differences in bacterial ability to adhere to the epithelial cells of the wall may partly affect their suppressive influence in the former phenomenon. Results obtained from a different series of experiments seem to support the speculation mentioned above, i.e, lactobacilli suppressed markedly population levels of staphylococci in the wall of the non-glandular part of the stomach (6) and lactobacilli, especially Group II (L. fermentum) which was effective in the suppression observed in this study, showed a higher ability than that of staphylococci in adhesion to the keratinized cells of the non-glandular part of the stomach in vitro (9). The latter microbial interference observed in luminal contents is very complex because many factors may be involved in these mechanisms such as bacterial population levels, bacteria products, and physiological conditions of each gastrointestinal part. Therefore, further studies should be carried out in more simple gnotobiotic animals than those used in these experiments. The authors wish to thank the animal care staff in our institute for their cooperation, and also M. Miyazaki for her helpful assistance. REFERENCES 1) Brownlee, A., and Moss, W. 1961. The influence of diet on lactobacilli in the stomach of the rat. J. Path. Bact. 82: 513-516. 2) Dubos, R., Schaedler, R.W., Costello, R., and Hoet, P. 1965. Indigenous, normal, and autochthonous flora of the gastrointestinal tract. J. Exp. Med. 122: 67-76. 3) Dubos, R., and Schaedler, R.W. 1962. The effect of diet on the fecal flora of mice and on their resistance to infection. J. Exp. Med. 115: 1161-1171. 4) Freter, R. 1962. In vivo and in vitro antagonisms of intestinal bacteria against Shigella flexneri. II. The inhibitory mechanism. J. Infect. Dis. 110: 38-46. 5) Maejima, K., Maejima, F., and Tajima, Y. 1966. Viable count of various bacteria in the digestive tract of mice. Fecal count in different strains of mice. Exp. Anim. 15: 104-112. 6) Morotomi, M., Watanabe, T., Suegara, N., Kawai, Y., and Mutai, M. 1975. Distribution of indigenous bacteria in the digestive tract of conventional and gnotobiotic rats. Infect. Immun. 11: 962-968. 7) Savage, D.C. 1969. Microbial interference between indigenous yeast and lactobacilli in the rodent stomach. J. Bacteriol. 98: 1278-1283. 8) Schaedler, R.W., and Dubos, R. 1962. The fecal flora of various strains of mice. Its bearing on their susceptibility to endotoxin. J. Path. Bacteriol. 90: 495-513. 9) Suegara, N., Morotomi, M., Watanabe, T., Kawai, Y., and Mutai, M. 1975. Behavior of microflora in the rat stomach: adhesion of lactobacilli to the keratinized epithelial cells of the rat stomach in vitro. Infect. Immun. 12: 173-179. 10) Tannock, G.W., and Savage, D.C. 1974. Influences of dietary and environmental stress on microbial populations in the murine gastrointestinal tract. Infect. Immun. 9: 591-598. 11) Watanabe, T., Morotomi, M., Kawai, Y., and Mutai, M. 1977. Distribution of indigenous lactobacilli in the digestive tract of conventional and gnotobiotic rats. Microbiol. Immunol. 21: 183-191. Requests for reprints should be addressed to Dr. T. Watanabe, Department of Intestinal Microbiology, Yakult Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186, Japan.