Growth and Invasiveness of Candida albicans in the Germ-Free and Conventional Mouse After Oral Challenge

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1 APPLIED MICROBIOLOGY, Sept., 1966 Copyright 1966 American Society for Microbiology Vol. 14, No. 5 Printed in U.S.A. Growth and Invasiveness of Candida albicans in the Germ-Free and Conventional Mouse After Oral Challenge A. W. PHILLIPS AND EDWARD BALISH' Department of Bacteriology and Botany, Biological Research Laboratories, Syracuse University, Syracuse, New York Received for publication 18 March 1966 ABSTRACT PHILLIPS, A. W. (Syracuse University, Syracuse, N.Y.), AND EDWARD BALISH. Growth and invasiveness of Candida albicans in the germ-free and conventional mouse after oral challenge. Appl. Microbiol. 14: Candida albicans was established in large numbers throughout the gut after one oral challenge in the germ-free and in the conventional mouse. Of the strains tested, only the germfree ND 1 mouse appeared to be susceptible to infection, and this was confined to the stomach mucosa; lesions contained large numbers of hyphal and mycelial forms with blastospores. These forms were also seen in the gut of resistant germ-free ND 4 mice after challenge. Only budding yeast forms were seen in the gut contents from conventional animals. The concentration of sulfhydryl-containing compounds was decreased in the stomach contents from germ-free mice. The stomach tissue of conventional animals seemed to be more acidic than that of germ-free animals, and association of C. albicans with conventional mice neutralized some of this acidity. Eh values of contents from the gut of unchallenged mice were usually higher in conventional than in germ-free animals; after challenge, the Eh in both groups decreased. Some reciprocal effects of intestinal microorganisms and host are discussed in relation to intestinal candidiasis. It has been reported that Candida albicans could not be isolated from the conventional laboratory mouse (5). Further, these authors and others could not infect mice by oral challenge with C. albicans (10, 14). Huppert and Cazin stated that, without dietary antibiotics, C. albicans could not survive in the alimentary tract. A recent review on C. albicans is that by Winner and Hurley (18). This investigation was undertaken to determine whether C. albicans could colonize the gut of the germ-free and the conventional mouse, and, if so, whether infection results. MATERIALS AND METHODS Culture. C. albicans ATCC was maintained on Sabouraud dextrose (Difco) slants. The virulence of this strain was determined in adult white mice (ND 4) of the Swiss Webster strain (Manor Farms, Inc., Staatsburg, N.Y.) and in ND 1 mice, by use of intraperitoneal injections. In both groups, the LD5o 'Present address: Argonne National Laboratory, Argonne, Ill. was found to be 106 viable cells of C. albicans. Isolates from animals were cultured on Corn Meal Agar, (Difco) to verify formation of mycelia, blastospores, and chlamydospores by C. albicans. Germ-free mice. Young adult germ-free and conventional mice of Lobund strains ND 1 and ND 4 were obtained from the Lobund Laboratory, University of Notre Dame, Notre Dame, Ind., and from Manor Farms, Inc., Staatsburg, N.Y., respectively; from the latter source, the MF-1 strain was purchased. Animals received either a purified diet A (1) or a crude diet B (no. 5010C from Ralston Purina Co., St. Louis, Mo.) which were sterilized by autoclaving for 25 min at 125 C. Microbiological and histological procedures. The methods and materials employed in the microbiological, histological, and related procedures were described previously (la). Eh and ph values of contents from the alimentary tract. Samples of contents from different segments of the gut were determined as described previously (la). Sulfhydryl determinations. The sulfhydryl (SH) concentration in contents from the gut was measured by the method of Klotz and Carver (7). A 1-g amount was suspended in 5 ml of distilled water and then 737

2 738 PHILLIPS AND BALISH APPL. MICROBIOL. centrifuged at 800 X g for 15 min. Supernatant fractions of all samples were held under nitrogen until assayed; the sensitivity of this method was 0.05,umole of SH per ml i 5%. Preparation of inoculum. C. albicans was grown in Sabouraud dextrose broth (Difco) for 18 hr at 37 C, and then mixed with the diet within an isolator (13) containing animals. RESULTS Results with ND 1 mice. These animals weighed about 40 g when received. All challenged mice were fed a single dose of 106 viable C. albicans cells per animal. After receiving the purified diet A for 4 months, the gnotobiotic animals weighed about 25 g, whereas conventional animals did not lose weight. Unchallenged germ-free control mice remained uncolonized and showed no weight loss. Within 48 hr of challenge, stool samples from the gnotobiotic group contained many hyphal forms of C. albicans, whereas only yeast forms were present in the conventional group. Lesions were found only in the stomachs of the gnotobiotic group and appeared to be confined to the mucosal area; large numbers of hyphae and mycelia with blastospores were present. The microscopic appearance of these lesions was similar to that found in crop infections of gnotobiotic chicks after feeding C. albicans (la); hyphae and mycelia were the predominant forms of C. albicans in these lesions. The lesions were established in some mice within 7 days of challenge, and, in the remaining animals, within 21 days of challenge; at this time, animals were autopsied. Microscopic examinations of tissues from challenged conventional mice showed no infections. Only yeast forms of C. albicans were found in contents from the alimentary tract of conventional animals. That C. albicans became established in all challenged animals was indicated by counts taken on contents from the small intestine. These colony counts were about 107 and 106 per gram (wet weight) in gnotobiotic and conventional mice, respectively. The hyphal form of C. albicans predominated in the gnotobiotic group. Results with ND 4 mice. Animals were 3 to 4 months of age when challenged and weighed about 35 g. Germ-free and conventional groups received autoclaved diet B. Neither group showed loss in body weight during the 21-day period of observation after challenge. Hyphal forms of C. albicans were found in stools from the gnotobiotic group only; the control animals showed only the yeastlike form. At autopsy, no macroscopic or microscopic evidence of infection could be found in any tissue examined from either group of animals. Examination of contents from the gut indicated that C. albicans became as well established in these animals as in the previous groups of mice. Hyphae were present in large numbers (about 50% of the total count) only in the gut of the gnotobiotic group, but these numbers were not as large as in the infected animals described above. Results with MF-I mice. Two breeding colonies of germ-free MF-1 mice receiving diet B were challenged and observed for a period of 18 months. C. albicans colonized (yeast form) the gut of these animals, but failed to infect the young or adults of either group. Similarly, conventional control mice were colonized but not infected. SH levels. The concentration of SH compounds in tissues and contents from different segments of the gut of germ-free and conventional ND 4 mice were determined at 21 days after oral challenge; in both groups, tissues from stomach, small intestine, and cecum had concentrations of 3, 6, and 5 Amoles of SH per g (wet weight), respectively. Differences were found in gut contents from corresponding sections of the tract of gnotobiotic and conventional mice (Table 1). The SH level appeared to be decreased in stomach contents from gnotobiotic mice. The SH concentration in contents from the small intestine and the cecum of both test and control animals appeared to be higher than in the stomach contents of gnotobiotic animals; the latter also had decreased SH levels in cecal contents. The predominance of hyphal forms of C. albicans in gnotobiotic animals is noted in Table 1. TABLE 1. Sulfhydryl levels in contents from the alimentary tract of germ-free and conventional ND] mice after oral challenge with Candida albicans Microflora before challenge Morphology of C. albicans in stomach contentsa Stomach SH in contents, pmoles/g (wet wt)b Small intestine Germ-free.40% H, 60% Y i 0.11 Conventional % Y 1.5 i a H = hyphal form; Y = yeast form. b The values shown are the mean i SD of five samples.

3 VOL. 14, 1966 GROWTH AND INVASIVENESS OF C. ALBICANS 739 TABLE 2. ph values in the alimentary tract of conventional and gnotobiotic mice (ND4 strain) Microflora of animals ph in tissuesa ph in contentsa Stomach intestine Cecum Stomach Small Cecum intestine ~~~~~intestine Ceu Germ-free Conventional Monoflora of Candida albicans Conventional + C. albicans a Mean of ph values from nine animals. TABLE 3. Eh values in the alimentary tract of conventional and gnotobiotic mice (ND4 strain) Microflora of animals Eh in tissues (mv)g Eh in contents (mv) Stomach Small intestine Cecum Stomach Small intestine Cecum Germ-free ± ± ± ± 3 Conventional i i ± ± 13 Monoflora of Can- +40 ±t ± 3-2 ± i ± 12-7 ± 8 dida albicans. Conventional + C. +39 ± ± 2 +3 i i ± 7-23 ± 11 albicans a Mean values 4 SD on samples from eight animals. TABLE 4. Comparative weights of intestinal tissues and their contents from gnotobiotic and conventional mice (ND 4 strain) Microflora of animals Tissue (g, wet wt)a Contents (g, wet Wt)a Stomach intestine Small Cecum Stomach intestine Small Cecum Germ-free ± ± ± 0.50 Conventional ± ± ± ± 0.30 Candida albicans monoflora ± ± ± ± 0.20 Conventional + C. albicans ± ± ± ± ± ± 0.20 a The values shown are the means ± SD on samples from nine animals. Eh and ph in the alimentary tract. In the stomach tissues of unchallenged animals, those of conventional mice were inclined to be acidic, whereas those of germ-free animals were close to neutral ph (Table 2). Challenge with C. albicans seemed to neutralize the acidic nature of conventional stomach tissue. No appreciable changes were seen in ph of intestinal contents from any group of animals (Table 2). Eh values on tissues of the alimentary tract seem to involve less variation than those in contents from the gut (Table 3). In unchallenged mice, stomach and cecal tissues usually were increased in Eh values among conventional animals, whereas the values appeared to be similar in tissue from the small intestine. Contents from the intestine of conventional mice had higher Eh values than those from germ-free animals, and, after challenge, these values were markedly decreased in both gut tissues and contents of different segments of the tract (Table 3). It appeared that, in gnotobiotic mice with stomach lesions containing hyphae, rather dense layers of PAS-positive material were present, as in germ-free ND1 mice. The comparative weights of intestinal tissues and their contents from gnotobiotic and conventional ND 4 mice are given in Table 4. The tissues of the small intestine appeared to be somewhat heavier in conventional than in gnotobiotic

4 740 PHILLIPS AND BALISH APPL. MICROBIOL. animals. The stomach and cecal tissues were comparable in weight in both groups. The most notable difference was the marked increase in cecal contents of gnotobiotic animals, which was not altered greatly by the challenge during 21 days [see Gordon (2)]. Attempts to isolate C. albicans and other Candida species from unchallenged conventional mice revealed none; this agrees with other data (5). DISCUSSION Apparently the environment of the alimentary tract of certain gnotobiotic and conventional mice is adequate to support the growth of large numbers of C. albicans. Thus, it may be surprising that others (5) and we could not isolate this organism from the conventional laboratory mouse. Perhaps this is owing simply to the absence of C. albicans in the environment of the laboratory animal, since we observed that C. albicans became established in unchallenged, germ-free mice housed in the same isolator with orally challenged germ-free animals (Phillips, unpublished data). These two groups of animals were kept in separate glass jars. Further evidence that the invasiveness of C. albicans is dependent upon conversion of yeast into hvphae is suggested by the observation that infection occurred only in those animals in which complete conversion occurred. Further, the stomach lesions contained predominately hyphae, or mycelia (with blastospores). The absence of infection in our ND 4 and MF-1 mice indicates either strain differences in susceptibility to infection, or diet received. The significance of the PAS-positive material in the stomach mucosa of germ-free ND 1 mice is not clear. Possibly this material enhanced their susceptibility to invasion by hyphae, since certain polysaccharides were shown to promote filament formation in C. albicans (12). Hog gastric mucin also enhanced the virulence of this organism (9, 15) Ȧnother observation concerns an apparent decrease in the concentration of SH groups in contents from the intestinal tract of germ-free mice (Table 1). This decrease was most marked in contents from the stomach of susceptible germ-free mice, suggesting that intestinal bacteria produce SH compounds in amounts sufficient to prevent formation of hyphae. Nickerson and associates showed that SH compounds prevented filament formation and promoted cell division in C. albicans (6, 11). Thus, an adequate intestinal SH level may be a basis for resistance to candidiasis. Possibly the different diets employed influenced levels of SH compounds and others which are believed to affect cell division. The decreased weight of tissue in the small intestine and the increased weight of cecal contents in germ-free mice confirm previous observations on a variety of germ-free mammals [reviewed by Luckey (8)]. Indeed, we have seen deaths from ruptured ceca, and one mouse had a cecum which was 50% of its body weight. Removing germ-free animals to a conventional environment brings the cecal weight to normal within 4 to 7 days (17). This cannot be achieved with several microbial species individually. Thus, Proteus vulgaris, Escherichia coli, and Lactobacillus acidophilus did not influence cecal size in gnotobiotic rats (4). Clostridium difficile, isolated from the ceca of conventional mice, reduced cecal size in gnotobiotic mice within 48 hr (B. Skelly, Bacteriol. Proc., p. 67, 1963). C. albicans in our gnotobiotic mice showed no marked effect on cecal weight during 18 months of association. Gordon (3) reperted the presence of a substance having bradykininlike properties in the ceca of germ-free mice and rats, but its relation to the cecal phenomenon is not clear. Further, Wiseman and Gordon (19) reported decreased levels of the above substance as well as decreased cecal weight in gnotobiotic rats with Salmonella typhimurium. Cecal enlargement in germ-free mice was corrected in striking fashion after association with bacterioides (16). ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant AM We thank D. N. Mardon, F. R. Hathaway, and Jose Capo for maintenance of germ-free animals, and Barbara Rauch for histological preparations. LITERATURE CITED 1. BAER, P. N., AND W. L. NEWTON Studies on peridontal disease in the mouse. III. The germfree mouse and its conventional control. Oral Surg. Oral Med. Oral Pathol. 13: la. BALISH, E., AND A. W. PHILLIPS Growth, morphogenesis, and virulence of Candida albicans after oral inoculation in the germ-free and conventional chick. J. Bacteriol. 91: GORDON, H. A Morphological and physiological characterization of germfree life. Ann. N.Y. Acad. Sci. 78: GORDON, H. A Demonstration of a bioactive substance in caecal contents of germ-free animals. Nature 205: GUSTAFSSON, B Germfree research at the Institute of Histology, University of Lund, p In G. Tunevall [ed.], Recent progress in microbiology. Charles C Thomas, Publisher, Springfield, Ill.

5 VOL. 14, 1966 GROWTH AND INVASIVENESS OF C. ALBICANS HUPPERT, M., AND J. CAZIN, JR Pathogenesis of Candida albicans infections following antibiotic therapy. II. Further studies of the effect of antibiotics on the in vitro growth of Candida albicans. J. Bacteriol. 70: JILLSON, 0. F., AND W. J. NICKERSON Mutual antagonism between pathogenic fungi. Inhibition of dimorphism in Candida albicans. Mycologia 40: KLOTZ, I. M., AND B. CARVER Aspectrophotometric titration for the determination of sulfhydryl groups. Arch. Biochem. Biophys. 95: LUCKEY, T. D Germfree life and gnotobiology. Academic Press, Inc., New York. 9. MANKOWSKI, Z The experimental pathogenicity of various species of Candida in Swiss mice. Trans. N.Y. Acad. Sci. 19: MOURAD, S., AND L. FRIEDMAN Active immunization of mice against Candida albicans. Proc. Soc. Exptl. Biol. Med. 106: NICKERSON, W. J., AND N. J. W. VAN RiJ The effect of sulfhydryl compounds, penicillin, and cobalt on the cell division mechanism of yeasts. Biochim. Biophys. Acta 3: NICKERSON, W. J., AND Z. MANKOWSKI Role of nutrition in the maintenance of yeast shape in Candida. Am. J. Botany 40: PHILLIPS, A. W., H. R. NEWCOMB, R. LACHA- PELLE, AND E. BALISH Rearing of germfree and monocontaminated chicks in rigid plastic isolators. Appl. Microbiol. 10: RAO, R. G., AND M. SIRSI The pathogenicity of Candida albicans and the effect of Nystatin on experimental candidiasis. Indian J. Med. Res. 50: SALVIN, S. B., J. C. CORY, AND M. K. BERG The enhancement of the virulence of Candida albicans in mice. J. Infect. Diseases 90: SCHAEDLER, R. W., R. DuBos, AND R. COSTELLO Association of germfree mice with bacteria isolated from normal mice. J. Exptl. Med. 122: WAGNER, M Serologic aspects of germfree life. Ann. N.Y. Acad. Sci. 78: WINNER, H. I., AND R. HURLEY Candida albicans. J. and A. Churchill Ltd., London. 19. WISEMAN, R. F., AND H. A. GORDON Reduced levels of a bioactive substance in the caecal content of gnotobiotic rats mono-associated with Salmonella typhimurium. Nature 205: Downloaded from on May 2, 2018 by guest