THE CHARACTERISTICS OF STRAINS OF SELENOMONAS ISOLATED FROM BOVINE RUMEN CONTENTS

THE CHARACTERISTICS OF STRAINS OF SELENOMONAS ISOLATED FROM BOVINE RUMEN CONTENTS MARVIN P. BRYANT Dairy Husbandry Research Branch, U.S.D.A., Beltsville, Maryland The genus Selenomonas Boskamp (1922) includes large crescent-shaped bacteria with a characteristic tuft of flagella attached to the middle of the concave side of the cell. These bacteria have been divided into three species mainly on the basis of habitat; namely, S. palpitans from the intestinal tract of guinea pigs, S. sputigena from the human mouth, and S. ruminantium from the rumen contents of herbivorous animals (Lessel and Breed, 1954). Although S. ruminantium was observed in rumen contents by Certes in 1889 and more recently by many other workers, no reports have appeared on the cultivation or physiology of this species. MacDonald (1953) described the cultural and some physiological characteristics of S. sputigena (Spirillum sputigenum). Studies on the characteristics of bacteria isolated from high dilutions of bovine rumen contents have shown that at least four different genera of anaerobic, motile, curved, rod-shaped bacteria occur (Bryant and Small, 1956a, b). One of these organisms has been identified as Selenomonas ruminantium (Certes, 1889; Wenyon, 1926) solely on the basis of the characteristic morphology, arrangement of flagella, and habitat. Also, it has been found that the bacteria designated as RO-HD by Huhtanen and Gall (1953) belong to this species. The present work was carried out to determine the morphological, cultural, and physiological characteristics of pure cultures of S. ruminantium. Received for publication December 27, 1955 grass pasture plus grain mixture, and strain D47 was isolated from a mature cow on a ration of alfalfa hay plus grain mixture. Strains B358 and B438 were isolated from two calves at 13 weeks of age on a ration of alfalfa hay plus grain mixture. Strain B385 was isolated from a 3-weekold calf on a ration of whole milk, alfalfa hay, and grain mixture. Strains HD,, HD4, and HDL were obtained through the courtesy of Dr. Lorraine Gall. Cultures for determining fermentation products of strains GA192, PC18, and HDI were prepared as described by Bryant and Doetsch (1954) except that glucose was used in the medium in place of cellulose. In this procedure the cultural vessels were evacuated so that initial partial pressure of carbon dioxide was low and analyses of fermentation gases could be made. Cultures for determination of fermentation acids of strains GA31, B358, B385, B438, HD4, and HDL were grown in rumen fluidglucose liquid medium containing a carbonic acid-bicarbonate buffer (Bryant and Small, 1956a). Analyses of fermentation products were carried out as described for strain 28 of Butyrivibrio (Bryant and Small, 1956a). To determine if strains fermented lactate, they were inoculated into carbohydrate basal medium containing rumen fluid (Bryant and Small, 1956a) with 1.0 per cent of lactic acid added. The lactic acid was neutralized to ph 7.0 with NaOH before it was added to the medium. A control medium was the same but minus the lactate. The cultures and controls METHODS were observed for growth (visible turbidity) Methods for isolation, maintenance of cultures, for one week at 37 C. None of the controls and study of morphological and physiological showed an appreciable amount of growth. The characteristics were the same as previously described (Bryant and Small, 1956a). lated lactate medium were analyzed for organic lactate cultures showing growth and uninocu- Strains GA31 and GA192 were isolated from acids. The differences in amounts of acids between rumen contents of two mature cows maintained the inoculated and uninoculated media were on a ration of fresh alfalfa. Strains PC3 and PC18 assumed to be the amounts of acids produced were isolated from a heifer on a ration of blue from lactate. Actually, a small amount of acid 162

19561 CHARACTERISTICS OF SELENOMONAS STRAINS FROM RUMEN 163 probably was derived from cysteine-hcl. This was added to the media as reducing agent and was known to be catabolized by most strains (see Results). RESULTS The organisms were rather large, curved, crescent-shaped rods usually 0.8 to 1 /L in width and 2 to 7 js long with bluntly tapered ends. Occasionally a strain would show most cells from 2 to 5,I in width. The reason for this change in size was not established. They were usually arranged as singles with occasional diplo-rods and short chains. A few helicoidal filaments up to about 20, in length were occasionally present. Strain D49 was smaller (about 0.6,z in diameter) and most cells of strain B385 were only slightly curved. The strains were gram negative though a few cells in a smear often appeared to be gram positive. The majority of cells of the strains, with the exception of D49 and B385, had a single tuft of a few to 12 or more flagella. The point of attachment of the tuft was often difficult to observe but usually it appeared to originate on the concave side of the cell at a central position or near the end. Cells of strain D49 and B385 often had two tufts of flagella which appeared to be attached at random over the surface of the cell. An attempt was made to clarify the picture of the arrangement of flagella by preparing stains of older cultures which were past the period of active growth (5 days of incubation). However, there was no more regularity in the point of attachment of the tufts. Surface colonies were entire, slightly convex, and translucent, and were light tan in color. They were from 2 to 6 mm in diameter. Deep colonies were thin and lens-shaped. Growth in glucose liquid medium was heavily turbid but some lightly flocculent sediment was often present. Growth occurred in glucose medium in which yeast extract and trypticase were added in place of rumen fluid; but, with the exception of strains HD,, HD4, and HDL, growth was somewhat better in the rumen fluid medium. All strains grew well in glucose medium with a N2 gaseous phase and without bicarbonate added. All strains grew well at 30 and 37 C. None grew at 22 or 50 C but strains varied in their ability to grow at 45 C. All strains were strictly anaerobic. They would not grow in media in which the resazurin was not reduced. Physiological characteristics are shown in table 1. H2S production was very rapid in all strains that were positive. The medium became quite black after only 24 hr of incubation. NO3 reduction occurred only in the strains obtained from Dr. Gall. The minimum ph in the glucose medium of low buffer capacity was the lowest obtained with any of the anaerobic rumen bacteria that have been studied to date. The Voges-Proskauer test was negative on all strains except PC-3, B438, GA31, and PC18 which were weakly positive in one run. When the strains were tested a second time, all were negative. Because of the copious production of H2S, an experiment was set up to determine its source. Possible sources in the medium used to detect H2S production were trypticase, S04, and cysteine-hcl. Five media were prepared with the following basal ingredients in common: ferric ammonium citrate, 0.05 per cent; yeast extract, 0.2 per cent; glucose, 0.1 per cent; agar, 0.5 per cent; resazurin, 0.0001 per cent; Na2CO3, 0.4 per cent; and equilibration with 100 per cent CO2. Medium 1 contained the basal incredients plus 1.5 per cent of trypticase and 0.05 per cent of cysteine-hcl; medium 2 contained the basal plus 1.5 per cent of trypticase and 0.05 per cent of ascorbic acid; medium 3 contained the basal plus 0.05 per cent of cysteine- HCI; medium 4 contained the basal plus 0.05 per cent of ascorbic acid, and medium 5 contained the basal plus 0.05 per cent of ascorbic acid and 0.1 per cent of Na2SO4. When the H2S positive strains were stabinoculated into these media, the only media showing a rapid production of H25 were those containing cysteine-hcl (media 1 and 3). Strain HD4 showed a very small amount of H2S in medium 2 after 18 days. No evidence of H2S production was obtained from medium 4 or 5. The results indicate that these bacteria have a very active cysteinase. It can be seen in table 2 that the principal acids produced by these organisms grown in rumen fluid-glucose medium varied considerably. Strain GA192 produced mainly lactic acid. Strains HD1 and HD4 produced mainly pro-

164 BRYANT [vol. 72 TABLE 1 Some physiological characteristics of eleven strains of Selenomonas isolated from rumen fluid* Characteristic Strain GA192 GA31 PC3 PC18 D47 B358 B385 B438 HDi HD- EHIDL H2S production... + + + + + + _ + + + + NO, reduction... + + + Casein digestion. + - - Growth at 45 C... - - + - + + + - Minimum ph in glucose... 4.4 4.4 4.4 4.3 4.4 4.3 5.2 4.4 4.3 4.4 4.3 Acid from sucrose... + + + + - + + + + + + Acid from trehalose... + + + - _ - - + - w Acid from glycerol... - + + + w - - + Acid from mannitol... + + + + + + - + + + Acid from dextrin... + - + - - + + + + + + Acid from inulin... + + + - + + + + - w Acid from salicin... + + + + w + - + + + + Acid from esculin... + + + + + + - + + + + Acid from xylan... + Hydrolysis of starch...+ - + - + + + + + Fermentation of lactate - w + _ - + * None of the strains produced catalase or indole, liquefied gelatin, produced acid from gum arabic or pectin, or hydrolyzed cellulose. All strains produced acid from xylose, arabinose, galactose, fructose, lactose, maltose, and cellobiose. "w" refers to a weak reaction. TABLE 2 Some products produced in rumen fluid-glucose medium by strains of Selenomonas isolated from rumen contents* Product GA192 GA31 PC18 B358 B385 B438 HD)a HD, HDL Combustible gas... 0 0 0 Carbon dioxide... 0.79 1.44 2.62 Butyric acid...0 0.18 0.12 0.16 2.38 0.24 0.39 0.18 0 Propionic acid... 0.12 0.51 1.38 1.26 0.27 0.65 2.31 3.60 0.61 Acetic acid. 0.12 0.83 0.89 1.33-0.95 0.86 1.67 2.50 0.50 Formic acid.0...o 0.16 0.30 0.13 0.92 0.22 0.35 0 0 Succinic acid.0 0.54 0.09 0.18 0 0.33 0.35 0 0 Lactic acid... 2.89 4.69 2.05 4.32 1.88 5.49 0 0.09 3.77 Volatile alcohol... 0 0 0 * Products calculated as mm per 100 ml of medium. pionic and acetic acids. The products produced by other strains, with the exception of B385, lay between the two extremes. Some butyric, formic, and succinic acids were detectable in most cultures. Strain B385 differed markedly in products from the others. The chief acids produced were butyric, lactic, and formic. It showed a considerable uptake of acetic acid. Strains PC18 and EID) produced a rapid fermentation of lactate with dense growth showing in two days. Strain GA31 did not show an appreciable amount of growth until four days after inoculation. Data in table 3 show that the lactate-fermenting strains produced propionic and acetic acids from lactate with a ratio of propionic to acetic of 1.8, 1.7, and 1.9, respectively. As none of the strains tested produced hydrogen gas from glucose, it seems probable that the strains of Selenomon carry on a lactate fermentation similar to that of the genus Propionibacterium.

19561 CHARACTERISTICS OF SELENOMONAS STRAINS FROM RUMEN TABLE 3 Acids produced in the lactate fermentation by three 8train8 of Selenomonas isolated from rumen contents* mm Per 100 MI of Medium Strain Strain Strain PC18 HD4 GA31 Propionic acid.6.... 5.44 5.70 1.17 Acetic acid.3.03 3.35 0.62 Lactic acid... -9.77-10.84-2.51 * No appreciable amounts of butyric, formic or succinic acids were produced. DISCUSSION The strains of bacteria included in this study appear to form a rather well defined group with the exception of strain B385 which differed from all other strains studied in not producing HzS, in having a higher minimum ph of growth, in fermenting not fermenting salicin or esculin, in xylan, and in producing different fermentation products. Also it appeared to be somewhat different morphologically because it had less curved cells and, although the flagella occurred in tufts, the tufts seldom appeared to arise from the middle of the concave side of the cell. The difference in type of flagellation appears to be only a difference of degree and may not be of importance. The detailed studies of MacDonald and Madlener (1955) on Selenomonas sputigena, using the dark field, flagella stains, and the electron microscope, suggested that the flagella may be randomly distributed about the circumference of the cell. Because of the large differences in physiological characteristics between strain B385 and all other strains, it seems logical to exclude this strain from the species Selenomonas ruminantium. Strain B385 has characteristics very similar to Butyrivibrio fibradissolvens (Bryant and Small, 1956a) except that the latter species is monotrichous, usually smaller in size, and ferments esculin and salicin. The HD strains obtained from Dr. Gall were similar to the strains isolated by the author except that the former strains reduced NO3 and the latter did not. Huhtanen and Gall (1953) reported that RO-HD1 and RO-HD4 types differed in the fermentation of dextrin, starch, 165 sucrose, and lactose. In the present study both of the types fermented these carbohydrates. The strains in the present study, excluding B385, show some physiological differences. The question arises whether or not these differences are valid criteria for separating the strains into more than one species. The only differences in characteristics which appeared to correlate were the fermentation of glycerol and lactate. As the lactate fermentation may assume considerable importance in the rumen, it is recommended that the lactate-fermenting strains be given variety status and named Selenomnm ruminantium var. lactilyticas. The differences in the fermentation products recovered from the glucose medium do not appear to be a good basis for sub-dividing the species as the various strains showed a more or less continual variation from those producing mainly lactic acid to those producing mainly propionic and acetic acids. The present strains of Selenomonas ruminantium have some physiological similarities to S. sputigena studied by MacDonald (1953). These include temperature range; the non-requirement of substantial amounts of carbon dioxide for growth, fermentation of glucose, sucrose, and mannitol; and failure to produce indole. S. sputigena reduced nitrate as did the HD cultures of the present study. The minimum ph of 5.0 for growth of S. sputigena was quite low as compared to other species of anaerobic rods included in MacDonald's study but was somewhat higher than the minimum ph obtained in the present study. This apparent difference may have been due to the differences in media and methods used. The only known characteristic of S. sputigena which appears to separate it from S. ruminantium is the failure of the former to produce H2S. The criteria presently used to differentiate species of the genus Seknomona are based almost entirely on the habitat (Lessel and Breed, 1954). This criterion appears rather superficial, but must be used until detailed descriptions of the species based on comparable cultural methods are available. Organisms morphologically similar to Selenomonas have been observed repeatedly during microscopic examination of rumen contents of cows and sheep on a wide variety of rations.

166 BRYANT [VOL. 72 However, when large numbers of colonies were picked from rumen fluid-glucose-cellobiose (RGCA) medium inoculated with rumen contents from cows on a variety of rations (Bryant and Burkey, 1953a, b, c), organisms morphologically and physiologically similar were infrequently isolated. They accounted for 4, 4, 3, 2, 1, 0.5, 0, 0, and 0 per cent of total isolates from animals fed soy bean hay plus grain, clover pasture, bluegrass pasture plus grain, fresh alfalfa, grain mixture, alfalfa hay plus grain, alfalfa hay, alfalfa silage, and wheat straw, respectively. These data suggest that Selenomas may contribute more to the rumen fermentation in animals fed rations containing more soluble carbohydrate as opposed to rations such as silage or straw which contain little soluble carbohydrate. The minimum ph tolerated and the fact that some strains of Selenomonas ferment lactate suggest that these organisms might become of increased importance in rumens of animals on rations high in the more readily available carbohydrates. These types of rations often cause a drop in the ph and a substantial increase in the amount of lactic acid in the rumen (Hungate et al., 1952; Phillipson, 1952). Although very few samples of rumen contents of low ph have been studied, one case was found which tends to substantiate the above reasoning (Bryant and Small, 1955). A 3-week-old calf which received an inoculum of fresh rumen contents at 1 week of age was fed a ration of alfalfa hay-grain mixture, ad libitum, plus whole milk. The calf ate unusually large quantities of the grain mixture (over 1 lb per day) during the third week of life. When rumen contents were studied, the ph was found to be 5.5, the number of lactate fermenters was high (600,000,000 per ml), and 2 of 20 colonies picked from the RGCA medium were presumptively identified as Selenomonas. Two of the six colonies picked from a tube of the lactate medium (Gutierriez, 1953) were presumptively identified as Selenomonas. Examination of a gram-stained smear of rumen contents from this animal showed organisms of the shape and size of Selenomonas as one of the most numerous types. Selenomonas is one of only a few kinds of glycerol-fermenting organisms found in large numbers in rumen contents of adult animals. Johns (1953) reported that glycerol was fermented mainly to propionic acid in the rumen of sheep. One can theorize that Selenomonas was one of the main organisms responsible for this glycerol fermentation. ACKNOWLEDGMENT The author wishes to acknowledge the assistance of Mrs. Nola Small in the determination of many of the physiological characteristics and in the maintenance of cultures during the course of this work. SUMMARY A study was made of the characteristics of 10 strains of Selenomonas ruminantium and 1 strain of a morphologically similar organism. Eight of the strains were isolated from the rumens of adult cows, heifers or calves. Three strains were obtained from Dr. Lorraine Gall. The strains of Selenomonas were anaerobic, large, curved, usually crescent-shaped rods with tufts of flagella often attached to the middle of the concave side of the cell. These bacteria produced H2S from cysteine and a ph of 4.3 to 4.4 in glucose medium. NO3 reduction and the Voges-Proskauer reaction varied. All strains produced acid from glucose, xylose, arabinose, galactose, fructose, lactose, maltose, cellobiose, esculin, and salicin. Most strains produced acid from sucrose and mannitol, and some strains produced acid from trehalose, glycerol, dextrin, and inulin. Strains varied in ability to hydrolyze starch and ferment lactate. Acids produced during growth on rumen fluidglucose medium varied. Some strains produced mainly lactic acid and small amounts of propionic and acetic acids. Some strains produced chiefly propionic and acetic acids, while acids produced by other strains lay between the two extremes. Small amounts of butyric, formic, and succinic acids were detectable in most cultures. Analysis of gas produced by three strains indicated carbon dioxide and no combustible gas. The lactate-fermenting strains produced propionic and acetic acids from lactate. It was suggested that these strains be given variety status and called S. ruminantium var. lactilyticas. A strain of bacterium morphologically similar to Selenomonas differed from it in not producing H2S, in not fermenting salicin or esculin, in fermenting xylan, in having a higher minimum ph of growth, and in producing mainly butyric, formic, and lactic acids with an uptake of acetate in rumen fluid-glucose medium.

19561 CHARACTERISTICS OF SELENOMONAS STRAINS FROM RUMEN 167 REFERENCES BOSKAMP, E. 1922 Ueber Bau, Lebensweise und systematische Stellung von Selenomonas palpitans (Simons). Centr. Bakteriol. Parasitenk., Abt. I, Orig., 88, 58-73. BRYANT, M. P. AND BURKEY, L. A. 1953a Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J. Dairy Sci., 36, 205-217. BRYANT, M. P. AND BURKEY, L. A. 1953b Numbers and some predominant groups of bacteria in the rumen of cows fed different rations. J. Dairy Sci., 36, 218-224. BRYANT, M. P. AND BURKEY, L. A. 1953c The bacterial flora in the rumen of heifers fed a ration of alfalfa silage. Bureau Dairy Industry-Information-151. BRYANT, M. P. AND DOETSCH, R. N. 1954 A study of actively cellulolytic rod-shaped bacteria of the bovine rumen. J. Dairy Sci., 37, 1176-1183. BRYANT, M. P. AND SMALL, N. 1955 The development of the flora and fauna in the rumen of growing calves. J. Dairy Sci., 38, 607. BRYANT, M. P. AND SMALL, N. 1956a The anaerobic monotrichous butyric acid-producing rod-shaped bacteria of the rumen. J. Bacteriol., 72, 16-21. BRYANT, M. P. AND SMALL, N. 1956b Characteristics of two new genera of anaerobic curved rods isolated from the rumen of cattle. J. Bacteriol., 72, 22-26. CERTES, A. 1889 Note sur les micro-organismes de la panse des ruminants. Bull. soc. zool. France, 14, 70-73. GUTIERRIEZ, J. 1953 Numbers and characteristics of lactate utilizing organisms in the rumen of cattle. J. Bacteriol., 66, 123-128. HUHTANEN, C. N. AND GALL, L. S. 1953 Rumen organisms I. Curved rods and a related rod type. J. Bacteriol., 65, 548-553. HUNGATE, R. E., DOUGHERTY, R. W., BRYANT, M. P., AND CELLO, R. M. 1952 Microbiological and physiological changes associated with acute indigestion in sheep. Cornell Vet., 42, 423-449. JOHNS, A. T. 1953 Fermentation of glycerol in the rumen of sheep. New Zealand J. Sci. Technol. 35, 262-269. LESSEL, E. F., JR. AND BREED, R. S. 1954 Selenomonas Boskamp, 1922-A genus that includes species showing an unusual type of flagellation. Bacteriol. Revs., 18, 165-169. MACDONALD, J. B. 1953 The motile non-sporulating anaerobic rods of the oral cavity. Thesis, University of Toronto, 95 pp. MACDONALD, J. B. AND MADLENER, E. M. 1955 Studies on Spirillum sputigenum. Abst. 33rd Gen. Meeting Intern. Assoc. Dental Research, paper 169. PHILLIPSON, A. T. 1952 The fatty acids present in the rumen of lambs fed on a flaked maize ration. Brit. J. Nutrition, 6, 190-198. WENYON, C. M. 1926 Protozoology, Vol. I. Bailliere, Tindall and Cox, London.