Female Rats. Iowa Received for publication 26 April lactate by degradation of the tissue glycogen (1,

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1977, p Copyright 1977 American Society for Microbiology Vol. 34, No. 5 Printed in U.S.A. Role of Estrogen in Controlling the Genital Microflora of Female Rats BRYAN LARSEN,12* A. J. MARKOVETZ,l AND R. P. GALASK"2 Departments ofmicrobiology' and Obstetrics and Gynecology,2 The University of Iowa, Iowa City, Iowa Received for publication 26 April 1977 Plate counts of viable bacteria recovered by lavage from rat vaginae demonstrated that the number of bacteria associated with the vaginal epithelium varied cyclically and that this pattern was abolished by ovariectomy. After ovariectomy, vaginal bacterial counts remained relatively stable at low levels. The estrogen 17f,-estradiol (1,3,5(10)-estratriene-3,17,B-diol cypionate) administered to ovariectomized rats caused a significant increase in vaginal bacterial counts on day 3 post-treatment. A similar effect was seen in non-ovariectomized rats, but a larger dose of estrogen was necessary to obtain such a response, suggesting that an endogenous estrogen antagonist may have been present in non-ovariectomized animals. Progesterone (4-pregnene-3,20-dione) given with estradiol diminished the effect of the estrogen on vaginal bacterial counts, but did not abolish it. Progesterone administered without estradiol had no detectable effect on vaginal bacterial counts. These findings suggested that the cyclic variation in bacterial content of rat vaginae could be explained primarily as the effect of the secretory pattern of ovarian estrogen. Numerous investigations have revealed that steroid hormones incubated with certain (primarily gram-positive) bacterial species in vitro inhibit these microorganisms (3, 6, 12, 14, 18, 25, 26, 28, 29). Further, animal studies showed that exogenously administered steroid (norethindrone) or a steroid-like compound (diethylstilbestrol) may decrease the severity of experimental staphylococcal infections in rabbits (25, 27). However, little is known about the microbiological implications of endogenously secreted steroid hormones. The reproductive hormones produce numerous physiological changes within the host, including enhancement of phagocytic activity (15-17), which might influence host response to infection. In addition, the well-known changes in carbohydrate and protein metabolism could result in alterations of the host's indigenous microflora by altering properties of the colonized tissue. However, little is known about this possibility. It is suggested by the earlier literature that an association exists between the presence or absence of ovarian function in primates and the types of bacteria present in the vaginal tract, but the reason for this relationship is not clear (12, 19, 20). The traditional view states that glycogen is deposited in the vaginal epithelium in response to estrogenic hormones, and this glycogen provides a substrate that promotes growth of lactobacilli, which in turn produce 534 lactate by degradation of the tissue glycogen (1, 2). The acid thus produced is believed to restrict vaginal flora to "aciduric" species (1, 2). Some evidence, however, indicates that the effects of estrogen on vaginal epithelium result in acidification of the vaginal environment independent of the presence of lactobacilli (13, 21-23). Because of controversy in the older literature and because contemporary methods have not been applied to gain an understanding of the microecology of the vaginal tract, we have been examining the microflora of female rat genital tracts (7-10). This system is especially interesting because the vaginal tract of rats is free of appreciable glycogen (H. Miura, 1928, cited in references 1 and 2) and, as demonstrated previously (7), has a ph near neutrality, yet vaginal bacterial counts have been shown to vary with the estrous cycle stage (8). The highest bacterial counts were consistently observed during estrus phase of the cycle, and miniimal counts occurred during the diestrus phase (8). Further, the relationship between vaginal bacterial counts and the predominant cell types of the vaginal epithelium have also been demonstrated both by viable bacterial counts (9) and by scanning electron microscopy (10). Because the cytological characteristics of the vaginal epithelium reflect the response to ovarian hormones, it is reasonable to suspect that the demonstrated fluctuations in vaginal bacterial counts are due to effects of

2 VOL. 34, 1977 ovarian hormone secretion on the vagina, but this concept lacks experimental support. Therefore, the following study was undertaken to investigate the effects of ovarian function and exogenously administered ovarian hormones on the bacterial content of rat vaginae. MATERIALS AND METHODS Animals. Experimental animals used in this study were virgin, female, randomly bred, albino rats obtained from Small Animal Supply Company (Omaha, Neb.). Rats were housed in stainless-steel cages in a temperature- and light-controlled room (12-h light, 12-h dark) and were provided food ad libitum. No vaginal procedures were performed on rats of less than 2 months or more than 12 months of age. Enumeration of vaginal bacteria was accomplished by vaginal lavage and plate count. The material obtained, using a standardized volume of sterile saline for lavage, was diluted, plated on blood agar, and incubated at 37 C in an anaerobic chamber for 24 h before counting. The reliability of this technique has been evaluated previously (8). It was demonstrated that the standard error of colony counts of multiple platings of material obtained by vaginal lavage was about 10%. In addition, we found that vaginal lavage recovered at least 90% of culturable bacteria and, therefore, provided a reliable indication of the number of bacteria on the vaginal epithelium (8, 9). Bacterial counts. In the present study, viable counts are expressed as colony-forming units per rat vagina. Where appropriate, counts are combined and expressed as a geometric mean (1g). Culture methods. Culture of genital tissue was accomplished by sacrificing a rat, wetting the fur with 70% ethanol, and exposing the viscera with a midline incision. The pubic symphysis was divided to further expose the internal genitalia. Vagina and cervix were removed and divided longitudinally, and one half of the tissue was placed immediately in chopped meatglucose medium and subsequently transferred to an anaerobic chamber. After 4 days of anaerobic incubation at 37 C, the chopped meat medium was subcultured onto sheep blood agar, and anaerobic species were identified by criteria of the Virginia Polytechnic Institute Anaerobe Manual (4). Facultative species were isolated by grinding the other half of the genital tissue in saline and sea sand with mortar and pestle. The mascerated tissue was streaked on sheep blood agar and incubated in a candle jar, a representative of each colonial type was isolated and identified by accepted techniques (11). Pasteurella pneumotropica was characterized as a grayish, weakly hemolytic colony on blood agar. It was a highly pleiomorphic, gramnegative rod. A weak A/A reaction was observed on triple sugar iron agar (TSI), and urea was slowly hydrolized. Catalase and cytochrome oxidase were both positive. The organism was nonmotile and failed to grow on Maconkey agar. Identification of this organism was verified by the State Reference Laboratory, Iowa State Hygienic Laboratory, Iowa City, Iowa. Ovariectomy was performed by dorsal approach on etherized rats as described in detail elsewhere (5). Control animals were subjected to sham operation in HORMONAL REGULATION OF RAT GENITAL MICROFLORA which the ovaries were brought through the skin incision and then replaced in the body cavity. Unless otherwise indicated, a 2-week recovery period was observed before animals were used in the experiments. Hormones. Steroid hormones included the estrogen, 1,3,5(10)-estratriene-3,17,8-diol cypionate (Upjohn Company, Kalamazoo, Mich.), hereafter referred to as estradiol; and the progestin, progesterone (4- pregnene-3,20-dione) purchased from Eli Lilly Co., Indianapolis, Ind. The hormones were diluted in peanut oil before intraperitoneal injection. Purity of hormone preparations used conformed to USP criteria. RESULTS To determine if the cyclic variations of female rat genital microflora, which had been observed previously (8, 9), were related to ovarian function, the effect of surgical ablation of the ovaries on the quantitative vaginal flora was studied. Twenty rats of the same age were placed into two groups, and ovariectomy was performed on the first group. A sham operation was performed on animals of the second group, which served as controls. A postoperative resting period was not observed, and vaginal counts were done on the day of surgery and on each of 12 subsequent days. Figure 1 compares the result of an ovariectomy to a sham operation on vaginal bacterial counts in two age-matched rats. After ovariectomy, bacterial counts diminished and remained within approximately 1 logarithm of 5 x 104, whereas in the sham-operated (control) rat, cyclic variation in bacterial counts continued. The data in Fig. 1 are exemplary of data obtained in an additional 9 ovariectomized or 9 a1o0 'a 106 U0 104 Ovariectomized Postoperative Day Sham Operated Control 535 FIG. 1. Effect ofovariectomy on cyclicity ofvaginal bacterial counts. Vaginal bacterial counts are expressed as colony-forming units (CFU) during the first 12 postoperative days in ovariectomized and sham-operated control rats. Nine additional ovariectomized and nine control rats were also studied and gave results similar to those shown.

3 536 LARSEN, MARKOVETZ, AND GALASK control rats, although counts from the 10 ovariectomized rats as a group tended to cluster about 1 x 104 rather than 5 x 104 as shown for the ovariectomized rat in Fig. 1. It should be noted that some daily variation in vaginal bacterial counts in ovariectomized animals occurred, but, in contrast to variation observed in control animals, the daily fluctuation in ovariectomized animals did not follow any identifiable pattern, nor were the daily changes of the large magnitude characteristic of counts obtained from control rats. This is further illustrated by Table 1, in which the magnitude of daily fluctuation in vaginal bacterial counts is reflected by comparison of average minimal and maximal counts in the two groups. Data obtained before the second postoperative day were excluded from this comparison so that the differences in the data would better reflect the postoperative rather than the preoperative hormonal condition of the animals. Peak bacterial counts in control rats reached levels approximately 100 times average peak levels seen in ovariectomized rats, whereas minimal counts were comparable in the two groups. It was concluded that substantial quantitative differences in the bacterial content of vaginal tracts of ovariectomized as compared to control rats exists, but it was not known whether a corresponding change in the types of organisms present in genital tracts of these two groups of animals could be demonstrated. To answer this question, six ovariectomized and six control rats were sacrificed 30 days after surgical procedure. The stage of the estrous cycle was not determined in the control rats at the time of sacrifice since we wished not to perform any manipulations on these animals that might alter the bacterial content of the genital tract. Vaginal and cervical tissue were excised and cultured aerobically and anaerobically. As shown in Table 2, ovariectomized rats had a microflora that appeared less diverse than TABLE 1. Comparison of minimal and maximal vaginal bacterial counts on postoperative days 2 through 12 Gg minimal Group ig maximal countsa counts Control (non-ovariecto- 1.5 x x 107 mized) rats' Ovariectomized ratsb 2.8 x x 105 athe minimal count was recorded for each rat during the period of postoperative days 2 through 12, and mean count was computed for 10 rats. A similar procedure was followed for computation of maximal counts. hn= 10. APPL. ENVIRON. MICROBIOL. TABLE 2. Comparative bacteriology of rat genital tissue from six ovariectomized and six control rats No. of isolatesa Organism Ovariecto mized Controls a-hemolytic streptococcus 0 (1)h 7 (1)' Pasteurella pneumotropica 0 (0) 5 (0) Nonhemolytic streptococcus 0 (0) 3 (0) Lactobacillus species 2 (0) 3 (0) Diphtheroids 2 (0) 2 (0) Proteus mirabilis 1 (1) 2 (0) Staphylococcus epidermidis 3 (2) 1 (0) Group D streptococcus 0 (1) 0 (0) Bacteroides fragilis od 4 Bacteroides corrodens 0 1 Fusobacterium species 0 2 Eubacterium species 0 1 Peptostreptococcus anaerobius 0 1 Propionibacterium species 0 1 Veillonella alcalescens 0 1 a Mean number of isolates per rat: ovariectomized, 1.33 (0.83); controls, 3.83 (2.00). See bforexplanationof parentheses. I Parentheses indicate isolates recovered only by subculture from chopped meat-glucose medium. ' Two rats each had two strains of a-hemolytic streptococci. With this exception, the number of isolates corresponds to the number of animals positive for the listed organisms. e Anaerobes were isolated only by subculture from chopped meat-glucose medium. No attempt was made to recover anaerobic species from aerobically ground tissue. that of control rats. The most striking difference was the failure to isolate any anaerobic bacterial species from ovariectomized rats. Likewise, with the exception of Staphylococcus epidermnidis, facultative species were most frequently isolated from control rats. Additionally, five facultative isolates from ovariectomized rats could only be isolated after subculture from enrichment medium, whereas only one isolate from the control group required subculture for isolation. The above data suggested to us that ovarian activity may have influenced colonization of the rat vaginal tract. Estrogen is the ovarian hormone secreted before estrus, and we demonstrated previously that estrus marks the time when vaginal bacterial counts are highest (8, 9). Therefore, the effect of estrogen upon the number of bacteria recoverable from vaginal tracts of rats was studied. Preliminary studies suggested that estrogen may be the compound responsible for periodic expansion of the genital bacterial population seen in control rats. To verify this concept and to simultaneously determine if a dose-response relationship exists between estrogen and bacterial population increases, four groups of ovariectomized rats with three rats in each group were given estradiol (5, 2.5, and 1.25 Ag as single doses) or a control injection of peanut oil. Vaginal tract bacterial counts were measured for 15 days. As shown in Fig. 2, estradiol administra-

4 VOL. 34, 1977 ~1O (cf) to e e a rats. Each group was composed of three rats and received 5, 2.5, 1.25, or 0 4g of estradiol. Data presentation is similar to the previous figure. HORMONAL REGULATION OF RAT GENITAL MICROFLORA 537 tion elevated bacterial counts in all cases. As in the previous study, increase in bacterial counts was delayed until post-treatment day 3. Differences in dosage appeared unrelated to the magnitude of the increase in bacterial counts but may have influenced the duration ofthe elevated counts. The control injection of peanut oil did not have any detectable influence upon bacterial counts. The results of similar experiments performed with 12 control rats are presented in Fig. 3. Although bacterial counts of ovariectomized rats were affected by as little as 1.25 ilg of estradiol (Fig. 2), the same dosage failed to elicit a similar response in control rats. Similarly, 2.5 gg of estradiol failed to cause a sustained elevation in bacterial counts, and cyclicity of counts was only slightly, if at all, altered. After treatment with 5,tg of estradiol bacterial counts in control rats 10~ 106 -AJ -j z a w LuA DAY OF STUDY FIG. 3. Response of vaginal tract bacterial counts (CFU) of individual control rats to 0 or 5 lug of estradiol. Additional control rats were treated with 1.25 or 2.5 mg of estradiol; results obtained are not identifiably different from data for the untreated rats and are, therefore, not shown. This study and presentation of data are similar to the previous figure. Data from each rat are plotted on a separate graph.

5 538 LARSEN, MARKOVETZ, AND GALASK increased and generally remained above 1 x 106 for 9 to 11 days; however, even at this dosage a tendency toward cyclicity in bacterial counts was evident (Fig. 3). Apparently, vaginal bacterial counts in control rats were not as sensitive to the effect of estradiol as were those in ovariectomized rats. This apparent refractoriness of control rats to estrogen suggested to us the presence of an endogenous estrogen antagonist that was not present in ovariectomized rats. Because progesterone is the natural antagonist or modifier of many biological effects of estrogen in vivo, we studied its effect on vaginal bacterial counts in ovariectomized rats. As a preliminary study, 12 ovariectomized rats were placed into four groups, three rats per group. The first group received a control injection of peanut oil. The second group received 5,ug of estradiol. The third group was treated with 5 jig of estradiol and 100,ug of progesterone, followed by two additional 100-,ug doses of progesterone on 2 subsequent days. The last group received 100,ug of progesterone on each of the first 3 days of the study. Vaginal bacterial counts were measured daily for 15 days. As expected, counts in rats receiving control injections of oil were fairly constant for the duration of the study and remained below 1 x 105. Rats treated with progesterone alone showed a pattern of counts similar to that of animals receiving the control injection. The pattern of counts in estradioltreated rats was consistent with the pattern established earlier (Fig. 2). In rats treated with both estradiol and progesterone, bacterial counts were increased on post-treatment day 3 but began to decline more rapidly than counts in rats receiving only estrogen. However, this difference was not large, and, therefore, the possibility of a true progesterone effect on bacterial counts required further verification. Twelve additional ovariectomized rats were studied in an experiment similar to the one just described, with the exception that a larger dose of progesterone was used. Three rats received 5,Ag of estradiol. Three rats received 500,ug of progesterone on each of the first 3 days of the study. Three animals received a combined estradiol-progesterone regimen consisting of 5,ug of estradiol and 500 ug of progesterone on day 1 of the study and 500,g of progesterone on each of the following 2 days (Fig. 4). These results are similar to those obtained in the previous study, which used a lower progesterone dose; however, the decline in bacterial counts after the initial rise was more pronounced. It appears from these data that progesterone antagonizes estrogen action in sustaining high-bacterial counts, but under the conditions of this study it did not prevent transient elevation of counts. APPL. ENVIRON. MICROBIOL. DISCUSSION In this study virgin female rats were used as experimental models to determine if the bacterial population colonizing a vaginal tract is responsive to the hormonal milieu within the host as predicted by previous studies (8, 9). The results suggest three conclusions. First, the ovaries represent the pacemakers for the cyclicity of bacterial counts, as evidenced by cessation of regular cyclic patterns in bacterial counts after ovariectomy. In addition to the effect of ovariectomy on the quantitative microflora, a qualitative effect was observed. A second conclusion is that the periodic expansion of vaginal bacterial population in nonovariectomized rats may be accounted for by estrogenic hormone. Third, progesterone may modify the effect of estrogen on bacterial counts in vaginal tracts. In nonovariectomized rats, it can be postulated that secretion ofprogesterone, which follows estrogen secretion, accounts for the rapid decline of bacterial counts after estrus (9), although the decrease in counts may simply reflect the cessation of estrogen secretion. Since during the normal estrous cycle progesterone secretion follows estrogen secretion and is ephemeral, lasting approximately one half of 1 day, it would seem that cyclic changes are most likely explained by the dynamics of estrogen secretion. A challenge to the afore-stated conclusion that ovarian function is responsible for changes in bacterial counts throughout the estrous cycle may be raised since cytological verification of the presence of ovarian cycles in intact rats was not performed. However, the absence of the ovaries was the only difference between the two groups (ovariectomized versus control) of rats studied. In view of this and the demonstrated effects of exogenously administered hormones on vaginal bacterial counts, the conclusion that cyclic fluctuations in vaginal counts are due to ovarian function is reasonable. Perhaps the greater question is whether cyclicity was truly abolished in ovariectomized rats. We noted that a certain fluctuation in vaginal counts occurred after ovariectomy. However, as stated above, the range of these fluctuations was substantially smaller than the range of fluctuations in control rats. In addition, fluctuations occurred randomly in ovariectomized rats but with regularity in control animals. Therefore, the conclusion that cyclic variation in bacterial counts was present in control rats but not in ovariectomized rats is warranted. It should be mentioned that ovariectomy does not absolutely deprive the rat of estrogen, since the adrenal may synthesize some estrogen. This amount, however, is small and does not produce enough estrogen to cause the

6 VOL. 34, 1977 HORMONAL REGULATION OF RAT GENITAL MICROFLORA 539 rvyi 1o7- Controls Estrogen J c cn > 1031 iu L > 0 Progesterone : 11 Xr Day Of Study Response of vaginal bacterial counts (CFU) in ovariectomized rats receiving estrogen and/or FIG. 4. progesterone. Four groups of ovariectomized rats (three per group) were given a control injection of oil or 5,g of estradiol, 1.5 mg ofprogesterone (in fractionated dosage 50() 1ug/day for 3 days), or both estrogen and progesterone. Presentation of data is the same as in the preceding figures. cytological changes in the vagina associated with enhancement of vaginal colonization. Perhaps the low levels of adrenal estrogen may have caused some of the low-level fluctuations in vaginal counts observed in castrate rats. The mechanism whereby estrogen and possibly progesterone alter the pattern of vaginal colonization in rats has not yet been established. Several effects of estrogen are known that could indirectly influence the vaginal microflora, including effects on vaginal cytology, mucus secretion, and vascularity. Alternatively, the hormones could act directly on bacteria; however, the low in vivo concentrations of the hormones in intact animals and the time lag between steroid administration and effect in ovariectomized rats suggest that the effect of the hormones on bacterial counts is mediated rather than direct. The time lag between estrogen administration and increase in bacterial counts conresponds

7 540 LARSEN, MARKOVETZ, AND GALASK with the time required to produce vaginal cornification. Further, estrogen secretion in intact rats substantially precedes vaginal cornification by about 1 day (24). We have shown that vaginal cornification is associated with peak vaginal bacterial counts (9). These points serve to emphasize that the effect of estradiol on bacterial counts is probably indirect. Studies are continuing to attempt to elucidate the mechanism of hormone action on genital microflora. LITERATURE CITED 1. Cruickshank, R., and A. Sharman The biology of the vagina in the human subject. I. Glycogen in the vaginal epitheliem and its relation to ovarian activity. J. Obstet. Gynaecol. Br. Emp. 41: Cruickshank, R., and A. Sharman The biology of the vagina in the human subject. II. The bacterial flora and secretion of the vagina in relation to glycogen in the vaginal epithelium. J. Obstet. Gynaecol. Br. Emp. 41: Fitzgerald, T., and W. W. Yotis Interference with cellular incorporation of substrates into Staphylococcus aureus by hormones. J. Med. Microbiol. 4: Holdeman, L V., and W. E. C. Moore (ed.) Anaerobe Laboratory Manual. Virginia Polytechnic Institute and State University, Blacksburg. 5. Ingle, J., and J. Q. Griffith Surgery of the rat, p In E. J. Farris and J. Q. Griffith (ed.), The rat in laboratory investigation. Hafner Publishing Company, New York. 6. Koch, M. L The bactericidal action of beta progesterone. Am. J. Obstet. Gynecol. 59: Larsen, B., A. J. Markovetz, and R. P. Galask The indigenous microflora of the female rat genital tract. Proc. Soc. Exp. Biol. Med. 151: Larsen, B., A. J. Markovetz, and R. P. Galask Quantitative alterations in the genital microflora of female rats in relation to the estrous cycle. J. Infect. Dis. 134: Larsen, B., A. J. Markovetz, and R. P. Galask The relationship of vaginal cytology to alterations of the genital microflora of female rats with respect to the estrous cycle. Appl. Environ. Microbiol. 33: Larsen, B., A. J. Markovetz, and R. P. Galask Scanning electron microscopy of vaginal colonization. Appl. Environ. Microbiol. 33: Lennette, E. H., E. H. Spaulding, and J. P. Truant (ed.) Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 12. Lester, G., and 0. Hetcher Effect of deoxycorticosterone on growth microorganisms. J. Bacteriol. 93:627. APPL. ENVIRON. MICROBIOL. 13. Lewis, R. M., and L. Weinstein The production of vaginal acidity by estrin, its importance in the treatment of gonorrheal vaginitis. Surg. Obstet. Gynecol. 63: Morse, S. A., and T. J. Fitzgerald Effect of progesterone on Neisseria gonorrhoea. Infect. Immun. 10: Nicol, T., D. L. J. Bilbey, L M. Charles, J. L Cordingly, and B. Vernon-Roberts Oestrogen: the natural stimulant of body defence. J. Endocrinol. 30: Nicol, T., and I. D. Helmy Influence of oestrogenic hormones on the reticulo-endothelial system in the guinea pig. Nature (London) 167: Nicol, T., B. Vernon-Roberts, and D. C. Quantock The influence of various hormones on the reticuloendothelial system: endocrine control of body defence. J. Endocrinol. 331: Varricchio, F., N. J. Doorrenbos, and A. Stevens Effect of asasteroids on gram-positive bacteria. J. Bacteriol. 93: Von Haam, R., and L. Rosenfield The effect of various sex steroids upon experimental pneumococcus infections in mice. J. Infect. Dis. 70: Weinstein, L, M. Bogin, J. H. Howard, and B. B. Finkelstone A survey of the vaginal flora at various ages with special reference to the Doderlein bacillus. Am. J. Obstet. Gynecol. 32: Weinstein, L, and J. H. Howard The incidence of the Doderlein vaginal bacillus during the postclimacterium. Yale J. Biol. Med. 10: Weinstein, L., and J. H. Howard The effect of estrogenic hormone on the H-ion concentration and the bacteriologic content of the human vagina with special reference to the Doderlein bacillus. Am. J. Obstet. Gynecol. 37: Weinstein, L., N. W. Walvro, D. V. Worthington, and E. Allen The influence of estrogenic hormone on the H-ion concentration of and bacterial flora of the vagina of the immature monkey. Yale J. Biol. Med. 11: Yoshinaga, K., R. A. Hawkins, and J. F. Stocker Estrogen secretion by the rat ovary in vivo during the estrous cycle and pregnancy. Endocrinology 85: Yotis, W. W In vivo and in vitro action of norethindrone on staphylococci. J. Bacteriol. 94: Yotis, W. W., and S. I. Baman An evaluation of diethylstilbestrol as an inhibitor of the growth of staphylococci. Yale J. Biol. Med. 41: Yotis, W. W., and S. L. Baman Diethylstilbestrol: a suppressor of induced furunculosis in rabbits. Yale J. Biol. Med. 41: Yotis, W. W., and J. M. Cummings Alteration in the multiplication of Staphylococcus aureus by Enovid. Can. J. Microbiol. 15: Yotis, W. W., and J. Waner Antimicrobial properties of testosterone and its intermediates. Antonie Van Leeuwenhoek J. Microbiol. Serol. 34: