methylene blue milk, no coagulation; acid formation from glucose (final ph 4.7), STREPTOCOCCUS UBERIS

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1 THE PHYSIOLOGY AND NUTRITION OF STREPTOCOCCUS UBERIS H. W. SEELEY Laboratory of Bacteriology, College of Agriculture, Cornell University, Ithaca, New York Received for publication April 12, 1951 In about 1928 there appeared descriptions of a streptococcus, associated with disturbances of the bovine udder, the outstanding properties of which differed markedly from those of the common mastitis organism, Streptococcus agalactiae. In the interval from 1928 to 1932, reports appeared by Klimmer, Haupt, and Roots (1928) in Germany, by Diernhofer (1932) in Austria, and by S. J. Edwards (1932) in England, which succeeded largely in lifting the status of this organism to the level of a distinct species. The German workers described a collection of 18 cultures obtained from milk, which they called "Group II," and which reacted as follows: sucrose and salicin but not raffinose were fermented; litmus milk was reddened but neither reduced nor coagulated; growth occurred in litmus milk after 2 weeks at 1 C; methylene blue was "transitorily" reduced; a final ph of 5. to 5.3 in lactose broth was reached; and sodium hippurate was hydrolyzed. On blood agar plates the reaction was described as greeniing or indifferent. There is little doubt that the organisms studied by Edwards in England and reported in 1932 as "Group III" streptococci corresponded to those of Klimmer. In addition to having most of the characteristics described by Klimmer, the "Group III" strains were shown to ferment inulin and mannitol, to give a final ph of 4.5 to 4.8 in glucose broth, and to split esculin. A point of difference appeared, however, in the observations of these two investigators in regard to the behavior of these organisms in litmus milk, and this difference has persisted in succeeding reports by other workers. Edwards found that litmus milk was not only reddened, as the Germans had reported, but also was partially reduced and coagulated when large inocula were used. Reports of widely assorted reactions in lifmus milk appear in the literature. This subject is considered more fully under "Results." Diernhofer (1932) examined a number of strains of the English "Group III" organism and pronounced them to be identical with strains isolated in his own laboratory from the milk from infected udders. At this time he proposed for the organism the species name Streptococcus uberis. Diernhofer confirmed the majority of results reported by Edwards and applied additional tests. A brief summary of his description of the species follows. "Smooth round colonies on agar, turbidity in broth; diplococci; litmus milk gradually reddened, no coagulation. At 1 C (with a scanty inoculum) no change in litmus milk; transient reduction of methylene blue milk, no coagulation; acid formation from glucose (final ph 4.7), lactose, sucrose, mannitol, and salicin, but not from raffinose and glycerol. Hippurate and esculin were split; sugar-free blood plates showed greening in deep colonies...." Minett (1934) objected to the adoption of the species name pro- 17

2 18 H. W. SEELEY [VOL. 62 posed by Diernhofer on the grounds that an international agreement should be reached and that the naming of the species should be delayed until a more complete investigation of the older literature could be made. Plastridge (1939), working at the Storrs Agricultural Experiment Station, compared his group "Ba" streptococci with a strain of the Streptococcus uberis of Diermhofer and found them to be in agreement, and he adopted the name "Streptococcus uberis" for his group "Ba" cultures. Despite the lack of harmony with regard to a species name mutually acceptable, the use of the name proposed by Diernhofer has become common in this country. Efforts to place S. uberis in existing classifications of the streptococci on a physiological basis have resulted in differences of opinion. Plastridge and Williams (1939) expressed the view that S. uberis bears a closer physiological resemblance to Lancefield's group E than to any other group. Little et al. (1946) consider that "while the physiological properties of S. uberis are not identical with any of the species described by Sherman, they resemble the general properties of the Enterococcus division more closely than those of any of the other groups described by him." Hansen (1935) observed that S. uberis may form a transitional type between S. agalactiae and the fecal streptococci. The serology of S. uberis is not yet on a reliable footing. Reports on this subject were reviewed by Stableforth (1946). Previous to the time of this review, the existence of a group-specific or species-specific antigen was not demonstrated although many "type" reactions were recorded. Since then Jacob (1947) has reported a serological affinity of S. uberis to members of group E. Although many of the physiological reactions of the S. uberis have been proved beyond dispute, others remain in question. In the work reported in the present paper a fairly extensive review of the reactions of this organism was undertaken. The standard tests used for purposes of classification were combined with a study of the nutrition of S. uberis to try to establish a clearer definition of the species and to disclose outstanding disparities or likenesses between S. uberis and other members of the genus Streptococcus. EXPERIMENTAL PROCEDURES AND RESULTS Sources and selection of cultures. Eleven of the strains of S. uberis were procured from Dr. Plastridge and comprised part of a collection built up over a number of years. Included among these was the "Streptococcus uberis I" of Diernhofer. Forty-one additional strains were secured during the course of the study from a large number of cultures isolated from the milk of cows afflicted with udder infections. Reliable serological methods were unavailable for the examination of these cultures inasmuch as the existence of a group-specific antigen is questionable and a species-specific antigen in S. uberis is not readily shown. Notwithstanding the fact that fermentation reactions are in general among the most variable known characteristics of a given species, the combination of the ability to ferment inulin and the inability to ferment raffluose is rare among streptococci, and this relatively unique property was weighed heavily in the selection of S. uberis cul-

3 1951] PHYSIOLOGY AND NNUTRION OF S. UBER19 tures from the collection of cultures examined. Strains otherwise typical of S. uberis but which fail to ferment trehalose (Miller and Heishman, 194), which fail to ferment inulin (Little et al., 1946), and which fail to hydrolyze sodium hippurate (Slanetz and Naghski, 194), have been reported, and allowances were made for these and other possible variations. The ability to ferment mannitol seems to be an especially constant attribute of the species. In the original collection of isolations were several enterococci that subsequently proved to be Streptococcus faecalis. These were studied physiologically with the cultures of S. uberis for comparative purposes and provided a check on the media and methods, the reactions of this species being well-established. Physiological tests-methods. The procedures used in making the tests reported are sufficiently well-known so as to make another description redundant. Inocula were made by pipette, and the source was an 18 to 24 hour culture of active cells prepared from a previous serial transfer. The temperature of incubation for all cultures was 37 C unless otherwise stated. Physiological tests-results. A summary of the physiological reactions of S. uberis appears in table 1. In regard to the glycerol fermentation, positive tests Were found only after 1 days' incubation under aerobic conditions. Because these tests were weak, the fermentation was followed by potentiometric measurement rather than by the usual indicators. Several strains of S. faecalis in the same circumstances attacked glycerol both aerobically and anaerobically. It should be made clear that under ordinary test conditions, i.e., employing an indicator and incubating the culture for 7 days, this weak reaction might easily escape detection and that the glycerol fermentation is of no value in ordinary identification procedures for S. uberis. The justification for enlarging upon this particular test lies in the fact that the aerobic and anaerobic use of glycerol is a reaction of decided importance in the classification of the enterococci, and this point of similarity of S. uberis to this group is interesting. The slow aerobic use of glycerol by S. uberis occurred under conditions in which Streptococcus lactis was negative and S. faecalis was strongly positive. Reference has already been made to a report (Slanetz and Naghski, 194) of the failure of S. uberis to hydrolyze sodium hippurate. Whereas all of the strains tested in the present work were positive, about 1 per cent were very weakly so, and it seems likely that the ability strongly to hydrolyze sodium hippurate is a variable trait of the species. On the matter of temperature limits of growth, S. uberis gives variable results which appear to depend on the previous treatment of the culture. During the course of the study, the ability of S. uberis to grow at 45 C was reversed. In the early work that was done with cultures that had been stored at 4 C for several weeks but which were thoroughly active at the time of use, no growth occurred at 45 C. Later in the study after a lapse of several months during which cultures were exposed to room temperature and transferred a number of times, prompt growth, equal to the decisive growth of the enterococci, occurred when the cultures were placed at 45 C. Strains of S. faecalis held under identical conditions

4 H. W. SEELEY [VOL. 62 grew rapidly, both directly after refrigeration for several weeks and after standing at room temperature for several months. TABLE 1 Summary of physiological characteristics of Streptococcus uberis NUMBER OF cul- TURES TESTED NUMER OF TESTS POSITIVE Xylose... Arabinose... Glucose... Fructose... Maltose... Lactose... Sucrose... Trehalose... Melibiose... Raffinose... Inulin... Mannitol... Sorbitol... Dulcitol... Glycerol: aerobically... anaerobically... Salicin... Esculin... Starch hydrolyzed... Gelatin liquefied... Sodium hippurate hydrolyzed... Tyrosine decarboxylated... Growth at 1 C... Growth at 45 C... Survival at 6 C for 3 min... Tolerance of bile salts... 1%... Conc 2%... 3%... 14%... Resistance to.1% methylene blue... Growth in 6.5% NaCl... Growth in 4.% NaCl... Growth in litmus milk... reddening... reducing... coagulating... Polysaccharide formation from sucrose... Reaction on blood agar... Final ph in glucose broth (see text) (see text) 51 5 (see text) (see text) indifferent or slight greening range, At 1 C, S. uberis grew moderately and not as heavily as S. faecalis. Growth at this temperature occurred whether inoculation was made by pipette or needle. Unfortunately this test of tolerance of the low temperature was not repeated after

5 1951] PHYSIOLOGY AND NUTRITION OF S. UBERIS ill the cultures had stood at room temperature for several months. The ability of S. uberis to make a limited growth corresponds to the results of a series of tests in which S. uberis was inoculated in small numbers into sterile milk held at 1 C and plated at intervals; although no important increase in population was noted, about one-third of the cultures grew slightly (Seeley et al., 1944). The fact that Minett (1934) regarded acid production at 1 C in litmus milk an important characteristic of the "Group III" organisms, whereas Ferguson (1938) found no growth of S. uberis at 1 C attests further to the variability of response to this test. These results clearly demonstrate an unstableness of the species to tolerance tests and serve to illustrate the point that the reaction found will depend upon the previous manner of handling of the culture. A similar inconstancy of reaction is also notable when S. uberis is cultivated in litmus milk. At the time of isolation all of the cultures showed acid production and varying degrees of reduction, and many coagulated, some thus affecting the milk in a manner indistinguishable from S. faecalis. As in the case of the response to the 45 C temperature, the litmus milk reactions changed, so that after storage at 4 C for 3 months all of the cultures reddened litmus only and neither reduced nor coagulated. The reactions reported in table 1 are those found when the majority of cultures were freshly isolated. The striking variability of S. uberis in litmus milk shows up in the reports of various workers. Seelemann (1939) described the reaction in litmus milk as reddening with neither coagulation nor reduction. Ferguson (1938) reported that litmus milk was not reduced before curdling and that some strains curdled the milk whereas others did not. Slanetz and Naghski (194) found that acid and curd formation and variations of these reactions appeared. Hansen (1935) found only acid production with no curdling in the three cultures examined by him. Plastridge and Hartsell (1937) described acid production as the only reaction in litmus milk. In a later publication (1942) Plastridge et al. list acid alone, or acid, coagulation and partial reduction reactions. Minett (1934) called attention to the point that the reactions in litmus milk were variable depending upon the size of the inoculum. It has been the observation of the present author that this conclusion of Minett, along with the manner of testing and the history of the culture, largely explains the variation in the reports on this matter. A few cultures that produced only a reddening of litmus when inoculated by a needle in addition reduced and curdled litmus when the inoculation was made with one drop of the culture. A strain of S. lactis tested under parallel conditions reduced and curdled litmus milk within 24 hours regardless of the technique of inoculation. All of the cultures of S. uberis differed from the enterococci in that they failed to decarboxylate tyrosine. A further examination of the decarboxylating ability of a single strain revealed that it did not decarboxylate tryptophane, phenylalanine, leucine, valine, arginine, glutamic acid, tyrosine, methionine. isoleucine, glycine, histidine, alanine, or lysine. These tests were made on a Warburg respirometer employing substrates of L or DL configuration in concentrations ranging from M/5 to M/3. The tests were made at ph 5.5 under nitrogen. Nutrition-cultures. Nineteen strains of S. uberis were employed, comprising

6 2 H. W. SEELEY [VOL. 62 from the collection of Dr. Plastridge and 8 additional cultures, all of which were studied in the foregoing part of this investigation. The physiological reactions of the strains selected agree with those of the strain which originated from Diernhofer, except for one which did not ferment inulin. Nutrition-methods. The methods used in the study of the nutrition of S. uberis are essentially those outlined in previous papers from the Cornell laboratories dealing with nutrition of the streptococci, Smiley et al. (1943), Niven and Sherman (1944), and others. Two organisms, Streptococcus salivarius and S. faecalis, which have been studied in detail and reported in the two references just cited, were tested concurrently with S. uberis, and their reactions provided TABLE 2 Minimum synthetic medium (exclusive of amino acid requirements) for the growth of Streptococcus uberis CO"NIQON1 CONCENTRATION PER 1 ML Casein hydrolyzate (prepared from Labco "vitamin-free" casein)... 5 mg Glucose... 1 mg K2HPO mg Thioglycolate (Na)... 1 mg Salts: NaCl... 2 mg MgSO mg FeSO Ag Mg9l2*4H pg DL-Tryptophan mg Uracil....5 mg Nicotinic acid....5 mg Pyridoxine HCI mg ThiaminHHCI....1 mg Riboflavin....1 mg Pantothenic acid (Ca)....1 mg Biotin....1 pg Folic acid....1 pg a control on the media and methods. S. salivariu-s has been shown to require thiamine, nicotinic acid, riboflavin, biotin, and pantothenic acid and was used in experiments involving the omission of these vitamins. S. faecalis was used in experiments involving the omission of folic acid, pyridoxine, and tryptophane, all of which it requires. A third species, S. lactis, having need of one or the other of glutamine or asparagine (Niven, 1944) and differing in other particulars from the other two control species, was used here also. The incubation temperature in all nutrition experiments was 37 C. Cultures were examined after 24 and 48 hours, and the degree of growth was measured in terms of turbidity. Table 2 shows the constituents, in quantity, of the final medium, (synthetic excepting the casein hydrolyzate) which supported the growth of S. uberis in a manner equal to, and with some strains, better than beef infusion broth.

7 1951] PHYSIOLOGY AND NUTRITION OF S. UBERIS 3 Nutrition-results. Choline, inositol, and para-aminobenzoic acid, which were included in the original medium, were soon found to be unnecessary for growth and were excluded during most of the experiments. None of the 19 strains required glutamine or asparagine, and in this respect the species is similar to the enterococci. The presence of xanthine in the medium altered the rate of growth. In the absence of xanthine, growth was exceedingly rapid and complete in about 2 hours. When xanthine was present full growth was not attained for about 48 hours, at which time no appreciable difference could be detected from the total growth in the xanthine deficient medium. Guanine and adenine were without any noticeable effect. Uracil alone, of the purines and pyrimidines tested, exerted a pronounced positive influence on the growth and in its absence only one-half to one-third of the full growth occurred in 8 of 9 strains tested. Of the 19 cultures of S. uberis tested, all required nicotinic acid, pyridoxine, thiamine, riboflavin, pantothenic acid, and biotin. Fifteen of the 19 strains required folic acid. The amino acid requirements of a few strains of S. uberis revealed that the following were indispensable for the growth of the species: tryptophane (19 strains studied previously were tested and all required this amino acid), phenylalanine, arginine, valine, leucine, and glutamic acid. Other data secured on stimulatory and nonessential amino acids indicate a fairly complex amino acid requirement for S. uberis. Experiments with strepogenin failed to disclose any effect of this substance, the same manner of growth taking place in its presence or in its absence. DISCUSSION The problem of the classification of S. uberis is especially confusing as a result of the variability of its reactions to some tests that are important in separating the major divisions of the streptococci, and because of the fact that other of its reactions, although stable enough, overlap the divisions. If one is willing to accept the test of the growth at 45 C, the milk reactions and the aerobic use of glycerol as positive for S. uberis in spite of the qualifications explained earlier, a fair case can be made out for the conformation of S. uberis to the enterococcus division. Even so, the remaining points of distinction lie outside of the limits of variability encountered within the 4 "species" of the enterococcus division. In the ability to grow on 4 per cent bile blood agar and in the ability to survive a temperature of 6 C for 3 minutes, different strains of S. uberis are divided. In other tests as growth in 6.5 per cent NaCl, the decarboxylation of tyrosine, the requirement for thiamine (all strains of S. uberis tested needed this vitamin supplied in contrast to the 19 strains of enterococci tested by Niven and Sherman, none of which needed this vitamin), S. uberis is quite different from the enterococci. This breach is further widened by the fact that a group D antigenic relationship for S. uberis, although mentioned in some reports, has not been clearly demonstrated, and in fact has been contradicted (Jacob, 1947). The failure of many workers to identify a group antigen in S. uberis allies it

8 4 H. W. SEELEY [VOL. 62 with the streptococci of the "viridans" division in this respect. S. uberis, however, grows to some extent at 1 C and all strains tested hydrolyze arginine; these reactions are unlike those of the "viridans" division the defined members of which are largely negative on both counts. Further comparisons of S. uberis to recognized species, groups, or divisions are equally fruitless inasmuch as the reactions of the species are distributed across the lines of the general divisions. Although one can point to some undeniably important relationships in several directions, a conspicuous relationship, either in terms of physiology or nutritional requirements, does not exist. Whereas the author has no intention of discussing the serology of S. uberis, it is perhaps worthy of mention that a serum which appeared to be in the nature of a species-specific serum was prepared; it reacted with extracts of of the serological types of S. uberis as defined by Plastridge and Williams (1939) and with a number of other untyped strains of the organism, and at the same time did not react with extracts of several of the more prominent members of Lancefield's grouping or with extracts of several of the "viridans" streptococci. This work remains to be completed. SUMMARY The physiological reactions and nutritional requirements of a collection of strains of Streptococcus uberis have been studied, and the results are reported. The relationship of S. uberis to the recognized species and groups of streptococci is discussed. REFERENCES DIERNHOFER, K Asculinboullon als Hilfsmittel fur die Differenzierung von Euterund Milchstreptokokken bei Massenuntersuchungen. Milchw. Forsch., 13, EDWARDS, S. J Studies on bovine mastitis. VI. The nonhemolytic streptococci of bovine mastitis and their relationship to certain saprophytic streptococci from cattle. J. Comp. Path. and Therap., 45, FERGUSON, JEAN 1938 The distribution of the mastitis streptococci in dairy herds. Cornell Vet., 28, HANSEN, P. ARNE 1935 New York State Agr. Exp. Sta. Tech. Bul. #232. III. The identity of Streptococcus agalactiae. JACOB, M. M Investigations on streptococcal mastitis and the streptococci. II. A serological study of the haemolytic streptococci of group E (Lancefield) and of Streptococcus uberis. Univ. Reading: Thesis. KLIMMER, M., HAUPT, H., AND ROOTS, ELM Zur Trenung einiger in der Milch vorkommender Streptokokken mit besonderer Beruchsichtigung der Isolierung des Str. agalactiae Guillebeau. Zbl. Bakt., I (Orig.), 17, LITTLE, R. B., BROWN, J. HOWARD, AND PLASTRIDGE, W. N Bovine mastitis, a symposium (ed. by R. B. Little, W. N. Plastridge). McGraw-Hill. Chap. V. Bacteriology of mastitis. MILLER, W. T., AND HEISHMAN, J Bovine mastitis caused by unusual types of streptococci. Cornell Vet., 3, MINFTT, F. C Streptococcus mastitis in cattle; bacteriology and preventive medicine. Proc. 12th Int. Vet. Cong., 2, NIVEN, C. F., JR., AND SHEEMAN, J. M Nutrition of the enterococci. J. Bact., 47,

9 1951] PHYSIOLOGY AND NuTRITION OF S. UBERIS 5 NIVEN, C. F., JR Nutrition of Streptococcus lactis. J. Bact., 47, PLASTRIDGE, W. N Streptococcus agalactiae as an entity and definite etiological agent in bovine mastitis. Rep. Proc. 3d Int. Cong. for Microbiology, PLASTRIDGE, W. N., ANDERSON, E. O., AND WEIRETHER, F. J Storrs Agr. Exp. Sta. Bul. 24. Infectious bovine mastitis. 8. The control of Streptococcus agalactiae mastitis by a segregation program based on periodic laboratory tests. PLASTRIDGE, W. N., AND HARTSELL, S. E Biochemical and serological characteristics of streptococci of bovine origin. J. Infectious Diseases, 61, PLASTRIDGE, W. N., AND WILLIAMS, L. F Abst., J. Bact., 88, SEELEMANN, M Vorkommen und biologiches Verhalten von Tierund menschenpathogenen Streptokokken in der Milch. Zbl. Bakt., I. (Orig.), 144, SEELEY, H. W., ANDERSON, E. O., AND PLASTRIDGE, W. N Mastitis and the plate count of milk. V. The behavior of Streptococcus uberis in milk held at different temperatures. J. Milk Tech., 7, SLANETZ, L. W., AND NAGHSKI, J. 194 Studies on streptococci of bovine mastitis. J. Infectious Diseases, 66, SMILEY, K. L., NIVEN, C. F., JR., AND SHERMAN, J. M The nutrition of Streptococcus salivarius. J. Bact., 45, STABLEFORTH, A. W Bovine mastitis, a symposium (ed. by R. B. Little, W. N. Plastridge). McGraw-Hill. Chap. VI. Serological classification of the mastitis streptococci. Downloaded from on September 6, 218 by guest