INFECTION AND IMMUNITY, Jan. 1972, p. 1-7Copyright © 1972 American Society for Microbiology

Vol. 5, No. IPrinted in U.S.A.

Changes in the Mouse Intestinal Microflora DuringWeaning: Role of Volatile Fatty Acids

ADRIAN LEE AND ERICA GEMMELLDepartment of Medical Microbiology, University ofNew South Wales, Sydney, Australia

Received for publication 5 August 1971

The influence of volatile fatty acids on the ecology of the bacterial flora of themouse intestinal tract has been studied in three situations where large fluctuationsin the composition of the microflora have been observed. Young mice were shownto ingest solid food particles when 11 days old; this correlated with the appearanceof strictly anaerobic fusiform bacilli in the intestinal lumen and a 10,000-fold de-crease in numbers of coliform bacilli. Over the same period, volatile fatty acids wereshown by gas-liquid chromatography to appear in the intestinal content. It is sug-gested that the fusiform bacilli are responsible for the presence of the volatile acids(especially butyric acid) which exert an inhibitory effect on the coliform bacteria,resulting in the decline in numbers. When germ-free mice are placed in a specificpathogen-free mouse colony, changes in the intestinal flora occurred which weresimilar to those observed in the young mice approaching weaning. Once again, thedecline in the coliform population correlated with the appearance of significantlevels of butyric acid in the large intestine. In a further series of experiments, micewere fed penicillin and levels of the intestinal fatty acids were measured. The anti-biotic eliminated the anaerobic fusiforms from the intestine, resulting in the disap-pearance of significant levels of butyric acid and a million-fold increase in thenumbers of coliform bacilli.

Shortly after birth, the gastrointestinal tracts ofrodents are colonized with large numbers ofaerobic bacteria belonging to the family Entero-bacteriaceae (the coliforms). From the 12th dayof life, the numbers of these organisms declineand reach minimum levels at about day 21. Theselevels are maintained for the rest of the animal'slife unless it is placed under stress or administeredantibiotics. Previous studies have shown a corre-lation between the fall in coliform numbers andappearance of strictly anaerobic bacteria whichrapidly become the dominant members of the in-testinal flora of the adult animal; no explanationfor the bacterial antagonism was proposed, how-ever (10, 14).

In other contexts, several ways whereby thepresence of one bacterial species may influencethe behavior of another have been suggested,e.g., changes in redox potential (11) and competi-tion for fermentable carbon sources in a reducedenvironment (5). An alternative mechanism, sup-ported by some experimental evidence, has impli-cated short-chain volatile fatty acids, especiallybutyric acid (3, 11). Thus, Bohnoff and Miller (3)demonstrated that adult mice, fed previously withantibiotics, became highly susceptible to oralSalmonella infection; they believed antibiotics

eliminated members of the normal flora whichwere capable of forming volatile fatty acids invivo.

In the present communication, the influence ofthe volatile fatty acids on the ecology of the mi-croflora of the mouse intestinal tract is consideredin greater detail. To do this, three rather differentmodels have been used, namely, (i) neonatal mice,(ii) germ-free mice, and (iii) adult specific patho-gen-free (SPF) mice treated orally with penicillin.In each case, the intestinal microflora is vastlydifferent from that of the adult or normal animaland thus provides a means of correlating the pro-duction of the fatty acids with the presence orabsence of the strictly anaerobic autochthonousorganisms.

MATERIALS AND METHODS

Animals. Mice were obtained from the SPF colonymaintained in the Department of Medical Microbiol-ogy at the University of New South Wales (UNSW).This colony originated from germ-free animal$(Charles River Breeding Laboratories, Inc., NorthWilmington, Mass.) and has been maintained underconditions appropriate to retain the SPF state. Forone series of experiments, germ-free mice were kindlysupplied by Margaret Holmes from the C3H colony

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maintained at the Walter and Eliza Hall ResearchInstitute, Melbourne.

Extraction of volatile fatty acids. Approximately 1 gof cecum and large intestine was accurately weighedand homogenized in 5 ml of distilled water with a

Teflon grinder. The homogenate was centrifuged toremove debris; the supernatant fluid was made alka-line (ca. pH 9.0) with 0.1 N NaOH and stored, untildistillation, in a deep freezer. The thawed alkalinemixture was adjusted to pH 2.8 to 3.2 with 1 M phos-phate buffer (equal volumes of 1 M NaH2PO4 and 1 MNa2HPO4). This solution was immediately transferredto a Markam still, and 1 g of MgSO4 was added. Thevolatile fatty acids were steam-distilled into 10 ml of0.1 N NaOH until 300 ml of distillate had been col-lected. The distillate was reduced at 100 C in a hot-airoven and finally evaporated to dryness at 58 C. Thevolatile acids were liberated, when required for analy-sis, by 0.8 ml of 30% phosphoric acid (9).Gas chromatographic separation of the volatile fatty

acids. The volatile fatty acids were routinely quanti-tated on a column of 15% Carbowax 20M (Analabs,Inc.) on ChromoSorb W DMCS (60 to 80 mesh ob-tained from Johns-Manville Corp.) packed in stain-less-steel tubing (internal diameter of 3 mm). Thecolumn was maintained isothermally at 147 C; thenitrogen carrier gas and hydrogen flow rates were 15cc/min.

Individual volatile acids were determined on anAerograph 1520 gas-liquid chromatograph with out-put from a flame ionization detector coupled to a 1 mvLeeds and Northrup Speedomax Recorder with chartintegrater. Peaks were identified and quantitated byreference to chromatograms of 0.1% standards ofvolatile fatty acids which had been distilled in anidentical manner to the intestinal samples.

Enumeration of intestinal bacteria. Coliform bacte-ria (lactose- and late lactose-fermenting organisms)were counted as follows. Samples of intestinal ho-mogenate were cultured on tergitol 7 agar (Difco)with added tetrazolium by using the loop dilutiontechnique described previously (15). For countinganaerobic fusiform bacteria, smears of intestinal ho-mogenate were prepared and stained by Gram'smethod. The proportion of tapering fusiform rodswas estimated as described in a previous publication(10).

RESULTS

Changes in the intestinal microflora of theUNSW SPF mouse during weaning. Previousstudies (6, 10, 13-15) were carried out withAmerican strains of SPF mice. It was important,therefore, to determine whether a similar patternof bacterial colonization was found in the miceused in this study. Figure 1 shows the develop-ment of the flora in a representative litter ofUNSW mice. Measurements were carried out ex-

actly as in a previous paper (10). The predictedchanges in the flora as the mice approach weaningwere seen. Coliforms were present in very largenumbers until about day 14 when they began to

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decline to levels found in the litter mother. Thisrepresents a drop in bacterial count of 10,000organisms per g of large bowel. The strictlyanaerobic tapered-fusiform rods first appeared atday 14 and increased in number until day 22 whenthey became the dominant members of the bacte-rial flora.Time of weaning of the UNSW mouse. In pre-

vious studies, the period of weaning had been es-timated by casual observation of the baby mice.It seemed important for subsequent studies to de-termine the weaning time of the UNSW mousemore accurately. To do this, the normal diet pro-vided to the litter mothers was ground, mixedwith 10% activated charcoal, and then repelleted.It was assumed that as soon as the baby mouse

began to ingest the solid diet the black particlesof food would clearly be visible in the previouslymilk white stomach. Figure 2 shows the stomachsof young mice of four litters which were killed atdifferent days after birth. Black food particleswere first seen at day 11, whereas it was obviousthat the stomachs became totally black at aboutday 14. Presumably, about this time, the youngmice were taking considerable quantities of solidfood although weaning was not necessarily com-

plete. From the above results, it is clear that thisjust preceded the establishment of the anaerobesin the intestinal tract and the decline in coliformnumbers.Appearance of volatile fatty acids in the large

intestine of baby mice. The ceca- and large intes-tines of baby mice of differing ages were removedand homogenized in 5 ml of charcoal water. Vola-tile acids were extracted, and samples were in-

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jected into a gas-liquid chromatograph. Quantita-tive estimates of butyric acid concentrations areshown in Fig. 3. No butyric acid was found in theintestine of very young mice; it was present insignificant amounts when the animals were 12 to14 days old. The concentration of the acid rapidlyincreased, reaching a maximum at about 18 daysof age; this correlates well with the appearance ofthe fusiform bacteria in high numbers. The othermajor acid, acetic acid, was detectable muchearlier, significant amounts being present at 7days. The concentration gradually increased to amaximum at about day 16. The concentrations ofthe acid shown in Fig. 3 cannot be compared withother fatty acid levels described in the literature.In the present studies, the homogenate containedboth intestinal contents and tissue. As a result,the values shown are presumably less than theabsolute concentrations of acid present in the in-testinal lumen of these baby mice.

Propionic, isobutyric, isovaleric and valericacids were in lower concentrations than aceticand butyric acids in the adult animal. These acidsall appeared at about day 16 in the baby mice andreached maximum concentration in a very shorttime; i.e., at less than day 15 none was detectablebut all were present at maximum concentrationat 16 days.Development of the intestinal microflora in germ-

free mice after removal from a sterile environment.When germ-free mice are placed in an SPF rodentcolony, the gastrointestinal tract is colonized withbacteria in a similar fashion to that of newbornmice. Coliform bacteria establish in very highnumbers initially but then decline as strictlyanaerobic bacteria appear in the intestinal con-tent. This was illustrated in the following experi-ment. Germ-free mice were taken from their iso-lators and placed in the breeding room of theSPF colony. At different periods, randomly se-

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lected animals were removed and their ceca andlarge intestines were homogenized. The homoge-nates were cultured for coliforms and examinedmicroscopically for the presence of fusiform bac-teria. Although only small numbers of animalswere available, it is suggested (Table 1) that coli-forms reached peak concentrations 4 days afterremoval from the isolators and then declined tothe low levels characteristic of the SPF animal.

Because of the results reported above on babymice, it was of interest to determine the volatilefatty acid pattern in the intestines of these germ-free mice after removal from a sterile environ-ment. The results in Fig. 4 are the chromatographtracings of the gut content of the animals de-scribed in Table 1. The identity of the variouspeaks was ascertained by reference to tracings ofpure standards of volatile fatty acids. Only a traceof acetic acid was found in the intestine of thegerm-free animal; it possibly originated frommouse tissue as the whole homogenized gut wasextracted. However, after removal from the germ-free environment, other acids progressively ap-peared. Significant quantities of acetic and butyricacids were seen after 7 days; this period coincidedwith the decline in coliform numbers (see above).

Volatile fatty acids in the intestinal tract of miceafter oral administration of penicillin. Oral admin-istration of penicillin to mice eliminates the nor-mal anaerobic flora and is followed by an increasein the number of coliform bacteria in the intes-tine. In the following experiment, adult UNSWmice were given penicillin in their drinking water(0.3 g per liter) and killed after 3 days. The ceca

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TABLE 1. Levels of coliform bacteria in theintestinal content of germ-free mice placed in aspecific pathogen-free (SPF) mouse colony

TimeinSPF ~~~~~~~Presence ofTime inday

Coliform levela (logso per g) fusiformbacteria

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0 FIG. 4. Gas chromatographic separation of volatile, fatty acids in the large intestines ofgerm-free mice afterb. differing periods ofassociation with a specific pathogen-

free mouse colony. Peaks: 1, acetic acid; 2, propionicacid; 3, isobutyric acid; 4, butyric acid; 5, isovalericacid.

of these animals were removed, homogenized, andcultured for coliform organisms. The homogenatewas steam-distilled, and the volatile fatty acidswere collected. Samples of the acids were injectedinto a gas-liquid chromatograph; two representa-tive tracings are shown in Fig. 5.The level of coliforms in the control animal was

low (104 per g); as anticipated, it reached highlevels in the treated mice (ca. 1010 per g). Acetic,propionic, isobutyric, butyric, isovaleric, andvaleric acids were all present in the control ani-mal; acetic and butyric acid were in highest con-

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PENICILLIN TREATED MOUSE

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FIG. 5. Gas chromatographic separation of volatilefatty acids in the large intestine ofa normal adult mouseand a mouse treated with penicillin. Peaks: 1, aceticacid; 2, propionic acid; 3, isobutyric acid; 4, butyricacid; 5, isovaleric acid; 6, valeric acid.

centrations. This contrasted sharply from thefatty acid profile of the antibiotic-treated animals.In these, only acetic acid was present in concen-trations comparable to the control. A smallamount of propionic acid was detected as weretrace amounts of isobutyric and isovaleric acids.However neither butyric nor valeric acids werefound. Microscopic examination of the intestinalcontents of these animals indicated that, althoughlarge numbers of fusiform-shaped bacteria werepresent in normal untreated mice, these organismshad been eliminated from the penicillin-treatedanimals.

DISCUSSION

The mouse gastrointestinal tract serves as anexcellent model for the study of interactions be-tween bacterial populations within a normallystable ecosystem. Many species of organism arepresent, some in exceedingly high numbers, whileinbalances in these populations may have dele-terious effects on the host. Thus, as others haveshown (3), the animals may become highly sus-ceptible to oral infection by intestinal bacterialpathogens. The bacteria which appear to domi-nate the ecology of the large bowel in rodents area group of strictly anaerobic tapered-fusiformrods which have recently been cultured by specialtechniques; although no detailed taxonomicstudies have been reported, it appears they maybelong to the genera Eubacterium, Fusobacterium,or, indeed, may be a new species (6).

Conditions which do not permit the growth of

these fusiform bacilli lead to great fluctuations inthe flora of the gut and allow establishment ofvast numbers of members of the Enterobacteri-aceae. Thus, treatment of mice with streptomycinresults in the elimination of fusiform bacteria andlowers the lethal dose of Salmonella typhimuriwnfrom 108 to ca. 1 organism (3). Again, when miceare fed with a chemically defined liquid diet, thethick layers of fusiforms in the mucosal epithe-lium are eliminated. This leads to an increase innumbers of lactose-fermenting coliforms from 104to 109 per g (T. D. Wilkins and W. R. Long,Bacteriol. Proc., p. 113, 1971).

In the present study, similar fluctuations incoliform numbers have been demonstrated in thegastrointestinal tracts of young mice as they ap-proach weaning. This has been reported previ-ously in a different SPF mouse colony when itwas suggested that the precipitating factor was theingestion of solid food by the young animals (10).Our experiments now seem to confirm that thechange in the intestinal microflora is just precededby ingestion of food particles. Presumably thesolid food has a profound effect on the local en-vironment, making it possible for the strict an-aerobes to establish in large numbers. This couldbe due to provision of new nutrients or, morelikely, the creation of highly reduced conditions.The main purpose of this investigation was to

demonstrate that certain changes of the intestinalmilieu, probably involving the fusiform bacilli,were associated with the spontaneous decline ofcoliforms in the natural situation. The volatileacids were examined primarily, as other workershave suggested that these agents may play an ex-tremely important role in the intestinal lumen(2, 3, 11). For reasons mentioned below, the ma-terial of most interest was butyric acid. This com-pound was absent in the very young animal andonly appeared as the fusiforms established. Bycomparing Fig. 1 and 3, it will be obvious thatthis acid was first detected a short time before thebacteria were found in significant numbers. How-ever, it must be stressed that the fusiform popula-tion is represented in terms of a percentage-of theintestinal flora, assessed microscopically; underthese circumstances it is clear, because of the vastnumbers of organisms present, a figure of only1 %o for the fusiforms could still represent an ab-solute concentration of 108 per g. The only othervolatile acid present in high concentration, aceticacid, approaches its maximum a significant timebefore any drop in coliform number is observed.The other acids are all present in much lowerlevels than acetic and butyric and appear atabout the same time as the latter.

It appears, therefore, that certain of the volatileacids, especially butyric, are produced by fusiform

1 2 3 4 5 6

NORMAL MOUSE

104Coliforms per gram

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bacteria and may influence the concentrations ofother bacteria in the intestine. This is, of course,only circumstantial evidence; in an attempt toconfirm it, two other models in which changes innumbers of coliforms could be correlated withthe fusiform organisms were examined. In thefirst, adult germ-free mice were placed in a roomwith mice which had a fully developed intestinalmicroflora. The changes in the flora in theseanimals exactly paralleled those of the youngmice approaching weaning. At first, the intestinewas colonized by miscellaneous gram-positiverods and cocci as well as very high levels of coli-forms. However, after a short interval, the intes-tine was colonized by anaerobes, presumablyderived from neighboring animals. This led tothe expected reduction in coliform numbers tothe low level characteristic of SPF mice. Thechromatographic traces of intestinal contents ofanimals killed at differing periods after removalfrom the sterile environment dramatically indi-cate that the coliform level only drops when sig-nificant quantities of butyric acid are present.The final experiments questioned the fatty

acid patterns of normal adult mice and micewhich had been administered penicillin orally.Under these conditions, it has been shownthat the fusiform flora is completely eliminatedand coliforms invade the large bowel in highnumbers (14). Once again butyric acid wasimplicated in this imbalance in the flora. Al-though virtually no butyric acid was detectedin animals given penicillin (and containingexceedingly large numbers of coliforms), thelevel of acetic acid was only minimally decreased.This suggests that the latter acid plays only aminor role in the control of coliform levels.The above experiments confirm the impor-

tance of certain volatile fatty acids as a majorfactor controlling bacterial populations withinthe intestinal tract (3). How they act is not yetfully resolved. It is interesting to record thatover 30 years ago Bergeim (1, 2) investigatedmechanisms involved in the destruction of micro-organisms in the gastrointestinal tract; in these,he demonstrated that acetic and butyric acidshad marked effects on the in vitro growth ofEscherichia coli and a yeast in concentrationsthat might be expected in the intestinal tract.Furthermore, he showed that by varying thediet the concentrations of acids could be altered,leading to significant changes in the intestinalflora. It is also pertinent to record that the highlevels of volatile fatty acids present in the rumenfluid of cattle and sheep may have antibacterialactivity. Thus, starvation of both species ofanimals leads to reduction of the acid leveland has been shown to allow the growth of

salmonellae and E. coli in the rumen. It hasbeen suggested that this may explain why theholding of cattle in yards before slaughter pre-disposes these animals to Salmonella and E. coliinfection (4, 7).

Experiments on the growth of Shigella speciesin vitro with cultures of human Bacteroidesstrains suggest that acetic and propionic acidsmay be important in restricting the former or-ganism; however, it is unlikely that conditionsin these cultures resemble conditions withinthe intestinal lumen (8). Nonetheless, this re-port again stresses the importance of the en-vironmental pH in determining the antibacterialactivity of short-chain fatty acids. As othershave suggested (11), butyric acid exerts a greaterantibacterial effect than acetic acid at the normalpH of the intestine (6.0 to 7.0) because moreacid is in the undissociated state. Furthermore,there is evidence to suggest that toxicity ofacids also tends to increase with the length ofthe carbon chain. Although little is known ofthe identity of these organisms, similar morpho-logical types are classified on their ability toproduce butyric acid as a major product (12).As culture techniques have been developed forthe strictly anaerobic fusiform bacteria, it isnow intended to monocontaminate germ-freemice to determine the number of species of bac-teria involved in the production of the fattyacids described above. Levels of volatile fattyacids in the human intestinal content are alsobeing investigated to determine whether theyare present in sufficient quantities to exert anti-bacterial effects.

ACKNOWLEDGMENTS

We aire grateful to W. R. McManus of the School of Wooland Pastoral Sciences, University of New South Wales, for theuse of the gas chromatograph. We also thank H. Lahoud and G.Edwards for helpful advice on the fatty acid analysis.

These studies were supported by a grant froni the NationalHealth and Medical Research Council of Australia.

LITERATURE CITED

1. Bergeim, 0. 1940. Toxicity of intestinal volatile fatty acidsfor yeaists and E. coli. J. Infec. Dis. 66:222-234.

2. Bergeim, O., A. H. Hanszen, L. Pincussen, and E. Weiss.1941. Relation of volatile fatty acids and hydrogen sulphideto the intestinal flora. J. Infec. Dis. 69:155-166.

3. Bohnoff, M., and C. P. Miller. 1962. Enhanced suscepti-bility to Salmonella infection in streptomycin-treated mice.J. Infec. Dis. 111:117-127.

4. Brownlie, L. E., and F. H. Grau. 1967. Effect of food intakeon growth and survival of salmonellas and Escherichia coliin the bovine rumen. J. Gen. Microbiol. 46:125-134.

5. Freter, R. 1962. In vivo and in, vitro antagonism of intestinalbacteria against Shigella flexnteri. J. Infec. Dis. 110:38-46.

6. Gordon, J., and R. Dubos. 1970. The anaerobic bacter ialflora of the mouse cecumn. J. Exp. Med. 132:251-260.

7. Grau, F. H., L. E. Brownlie, and M. G. Smith. 1969. Ef-

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fects of food intake on number of salmonellae an Escheri-chia coli in rumen and faeces of sheep. J. AppI. Bac-teriol. 32:112-117.

8. Hentges, D. J., and B. R. Maier. 1970. Inhibition of Shigellaflexnieri by the normal intestinal flora. Infec. Immun.2:364-370.

9. Lahoud, H. R., K. Prichard, W. R. McManus, and P. J.Schofield. 1971. Volatile fatty acid production by the adultliver fluke Fasciola hepatica. Comp. Biochem. Physiol.38:379-391.

10. Lee, A., J. Gordon, C. J. Lee, and R. Dubos. 1971. The mouse

intestinal flora with emphasis on the strict anaerobes. J.Exp. Med. 133:339-352.

11. Meynell, G. G. 1963. Antibacterial mechanisms of the mouse

gut. II. The role of Eh and volatile fatty acids in the normalgut. Brit. J. Exp. Pathol. 44:209-219.

12. Moore, W. E. C. 1970. Relationships of metabolic productsto taxonomy of anaerobic bacteria. Int. J. Syst. Bacteriol.20:535-538.

13. Savage, D. C., R. Dubos, and R. W. Schaedler. 1968. Thegastrointestinal epithelium and its autochthonous bacterialflora. J. Exp. Med. 127:67-76.

14. Savage, D. C., and R. Dubos. 1968. Alterations in the mousececum and its flora produced by antibacterial drugs. J.Exp. Med. 128:97-110.

15. Schaedler, R. W., R. Dubos, and R. Costello. 1965. Thedevelopment of the bacterial flora in the gastrointestinaltract of mice. J. Exp. Med. 122:59-66.

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