STREPTOCOCCI' ANIMAL STRAINS OF HEMOLYTIC. hydrolyze sodium hippurate. Avery (1929) also demonstrated

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1 THE BIOCHEMIICAL CHARACTERS OF HUMAN AND ANIMAL STRAINS OF HEMOLYTIC STREPTOCOCCI' PHILIP R. EDWARDS Department of Animal Pathology, Kentucky Agricultural Lexington, Kentucky Experiment Station, Received for publication, July 29, 1931 There have been numerous efforts to discover a biochemical method of differentiating streptococci of human and animal origin. In part, these have been successful. The work of Ayers (1916), Ayers, Johnson and Davis (1918), and Avery and Cullen (1919) has demonstrated that certain hemolytic streptococci of bovine origin can be differentiated from human streptococci by the final hydrogen-ion concentration produced when they are grown in glucose broth. Working with the same group of bovine streptococci, Ayers and Rupp (1922) were able to separate them from streptococci of human origin by their ability to hydrolyze sodium hippurate. Avery (1929) also demonstrated that the members of this particular group of bovine streptococci were tolerant to concentrations of methylene blue which inhibited streptococci of human origin. Frost and his associates (1926, 1927, 1929) have carried out extensive studies of streptococci of bovine origin and classified a large number of strains. There remains however a large group of hemolytic streptococci derived from bovines and other domestic animals which has not been successfully differentiated from human streptococci. This group of streptococci is characterized by low acid production in glucose broth, inability to hydrolyze sodium hippurate, lack of tolerance to methylene blue, and active hemolysis of blood cells 1 The investigation reported in this paper was carried out in connection with a project of the Kentucky Agricultural Experiment Station, and is published by the permission of the director. 259

2 260 PHILIP R. EDWARDS in a fluid medium. These characters are likewise possessed by streptococci of human origin. It is with this group of undifferentiated animal strains that the present paper is concerned. The cultures studied consisted of a group of 90 strains of streptococci isolated from horses, cattle, swine and chickens, together with 36 strains of human origin used for comparison. These cultures all produced a relatively low acidity in glucose broth, the final hydrogen ion concentration never exceeding ph 4.9. TABLE 1 Source of cultures Animal strains: Equine, os uteri Equine, aborted fetuses... 3 Equine, septicemia and arthritis Equine, pneumonia... 2 Bovine, septicemia... 3 Bovine, placenta... 1 Bovine, milk... 1 Chicken, femur... 9 Swine, septicemia... 2 Swine, abortion... 4 Human strains: Throat cultures, sore throat and scarlet fever suspects Broncho-pneumonia... 4 Scarlet fever, stock cultures... 7 Mastoiditis... 3 Meningitis... 1 Erysipelas... 1 Smallpox... 1 In contrast to this, the so-called high acid producing strains have, in our hands, produced acidity ranging from ph 4.4 to 4..0 None of the cultures hydrolyzed sodium hippurate and all were markedly hemolytic in a fluid medium. The question of hemolysis deserves some consideration. While high acid-producing, sodium hippurate-hydrolyzing streptococci may be hemolytic to a certain degree and produce fairly well-defined zones of clearing on a blood plate, they are not, in our experience, nearly so markedly hemolytic as the group under consideration. The

3 HUMAN AND ANI'MAL STRAINS OF STREPTOCOCCI hemolytic activity of all the strains studied here has been roughly titrated. As a result it has been found that 0.05 cc. of the supernatant fluid of centrifuged broth cultures of these streptococci is able completely to hemolyze 0.2 cc. of a 5 per cent suspension of washed sheep cells. The high acid-producing cultures of bovine origin have not proved nearly so active against red cells, the majority of them not producing complete hemolysis in amounts of 0.5 cc. The source of the strains studied is given in table 1. ACID PRODUCTION While it has been stated above that all the cultures reported upon were of the low acid-producing type, different degrees of acid production from glucose have been noted in the human and TABLE 2 Final hydrogen ion concentration in glucose meat infusion broth cultures of streptococci 3 Animal strains...3* Human strains... ph * Figures indicate number of strains at various hydrogen ion concentrations. 261 animal strains. In determining the final hydrogen ion concentration of the cultures, meat infusion broth, adjusted to ph 7.8 and containing 1 per cent glucose, was inoculated with the strains to be tested and incubated at 37 C. for ninety-six hours. The hydrogen ion concentration was then determined by comparison with standard buffer solutions. It was found that the average hydrogen ion concentration reached by the animal strains was ph 5.1 while that attained by the human strains was only ph The reaction of the individual strains is given in table 2. With the exception of seven cultures the difference observed between the human and animal strains is distinct and consistent and seems to indicate a difference in the metabolism of the cultures. It is of interest in this connection that Jones (1919) noted that strains of hemolytic streptococci of equine origin JOURNAL OF BACTERIOLOGY, VOL. XXIII, NO. 3

4 262 PHILIP R. EDWARDS produced a higher titrable acidity in carbohydrate mediums than did human streptococci. FERMENTATIVE REACTIONS The fermentative reactions of streptococci have been studied extensively and in general the results have been discouraging. This method of study, once regarded as being of great promnise, has proved disappointing. There are a few notable exceptions, however, such as the studies of Holth (quoted by Adsersen (1915)), Adsersen (1915) and Ogura (1929) on the fermentative clharacters of Sir. eqti. These workers have examined more than 250 cultures of this species and found their action on fermentable substances absolutely constant. Not only did all strains of the micro6rganism exhibit the same fermentative reactions but these reactions differed from those of human streptococci and of other strains of equine streptococci. These observers found that Str. equi failed to ferment lactose, sorbitol and trehalose. All the other cultures examined fermented one or more of these substances. In addition Ogura demonstrated that the majority of equine strains fermented sorbitol and failed to ferment trehalose, while the few human strains which he examined fermented trehalose and failed to ferment sorbitol. Since these results offered promise of a method of differentiation, Ogura's studies have been extended to a larger number of strains from a greater variety of animal species. In the present investigation the 90 strains of animal origin and the 36 human strains have been tested for their ability to ferment sucrose, salicin, lactose, sorbitol, arabinose, xylose, sorbose, trehalose, mannitol, raffinose, rhamnose, dulcitol, glycerol, and inulin. In performing the tests the following basic medium was used: Mleat infusion cc. Casein digest cc. Andrades indicator... 1 per cent The casein digest was prepared according to the method of Kulp and Rettger (1924). The reaction was adjusted to ph 7.6 and

5 HUMAN AND ANIMAL STRAINS OF STREPTOCOCCI 263 the medium sterilized. The fermentable substances were sterilized by filtration and added to the previously sterilized broth. After inoculation the tubes were incubated ten days before final recording of results. Of all the substances tested, only lactose, trehalose, sorbitol and glycerol proved to be of any value for differentiation. The results obtained with these substances are given in table 3. It can be seen that the 36 human strains without exception formed acid from trehalose but did not attack sorbitol. On the contrary, 85, or 94 per cent, of the animal strains failed to ferment trehalose but produced acid from sorbitol. The remaining five animal strains reacted as did the human strains in regard to these two substances. It will be noted, however, that the percentage of TABLE 3 Fermentation reactions LACTOSE SORBI- TREHA- GLYCEROL TOL LOSE Animal strains...{ , Human strains , ±, indicates acid production; - indicates no acid production. lactose fermenters in this group is much lower than in the human cultures. Only one of these five animal strains fermented lactose, while 34 of the 36 human cultures produced acid from this sugar. Also the percentage of glycerol fermenters is higher, 100 per cent of the atypical animal strains attacking glycerol, whereas acid was formed from this substance by only 50 per cent of the human cultures. It is of interest that no streptococcus has been encountered which ferments both trehalose and sorbitol. With the exception of Str. equi, which is not considered in this paper, we have not found a strain which fails to ferment one of these two substances, one or the other always being attacked. We have confirmed the statements of Holth, Adsersen and Ogura that Str. equi ferments neither lactose, sorbitol nor trehalose.

6 264 PHILIP R. EDWARDS DISCUSSION It has been shown above that the animal strains examined in this work produced a higher acidity in glucose broth than did the human strains. However, this method may not be of practical value since some of the human strains produce almost as great a degree of acidity as do the animal strains. The final hydrogen ion concentration produced by the 36 human strains included in this group is lower than that reported by many workers for human streptococci. Numerous references may be found in the literature to human streptococci which produce a final reaction of ph 5.0 to 5.2 in glucose broth. We are unable to explain the lower acidity produced by our series of human cultures. The work has been repeated several times and the results confirmed by other persons in the laboratory. The rather constant differences observed are indicative of a basic difference in the metabolism of the organisms. In the great majority of instances the results of the fermentation tests were sharp and distinct. The only doubtful or delayed fermentations obtained were in the tests with glycerol. In testing lactose, trehalose, and sorbitol positive reactions were nearly always apparent in twenty-four hours and were never delayed beyond forty-eight hours. Gly-cerol was fermented very slowly in these tests and in some instances the production of acid was very slight. Positive tests with this sugar were rarely apparent before the fourth or fifth day. It should be stated that the most vigorous glycerol fermenters in this series were the five atypical animal strains. They produced acid earlier and in greater amount than any of the human cultures. The question naturally arises as to whether the five atypical strains of animal origin may be in reality human strains. It is the writer's opinion that they are not. Three of these atypical strains were recovered from the os uteri of mares, one from a case of septicemia in a foal, and the fifth from an aborted swine fetus. These five cultures grow much more vigorously in artificial media than the other animal strains and somewhat more vigorously than the human cultures. They produce higher acidity in glucose broth than the human strains. As pointed out above the

7 HUMAN AND ANIMAL STRAINS OF STREPTOCOCCI percentage of lactose fermenters is much lower and the percentage of glycerol fermenters higher in these five cultures than in the human strains. These facts indicate that they are not human strains of streptococci which have been transferred to animals. If the results obtained in this study can be confirmed with a larger number of cultures it may be said with certainty that any hemolytic streptococcus producing acid from sorbitol and failing to ferment trehalose is of animal origin. On the other hand if a strain produces acid from trehalose and fails to ferment sorbitol, it is highly probable that the organism is of human origin. The study of this series of cultures is being continued in the hope that additional differences may be established between the human and animal cultures and an exact method found of differentiating the atypical animal strains from the human type. In conclusion it should be said that the results obtained here apply only to the group under consideration and the method is not applicable to other groups of streptococci. The few high acid-producing, sodium hippurate hydrolyzing strains which have been tested do not give concordant results. SUMMARY In a series of 126 strains of hemolytic streptococci, 90 of animal and 36 of human origin, it has been demonstrated that the animal cultures produce a higher acidity in glucose broth than do the human cultures. It has also been demonstrated that 94 per cent of the animal strains can be clearly differentiated from the human cultures by the fact that they produce acid from sorbitol and fail to ferment trehalose. The streptococci of human origin all ferment trehalose and fail to attack sorbitol. The writer wishes to take this opportunity to express his grateful appreciation to Dr. W. W. Dimock and Dr. D. J. Healy for their interest, co6peration and helpful suggestions throughout this work. REFERENCES ADSERSEN 1915 Centralbl. Bakt., I, 0., 76, 111. AVERY 1929 Jour. Exp. Med., 50,

8 266 PHILIP R. EDWARDS AVERY AND CULLEN 1919 Jour. Exp. Med., 29, 215. AYERS 1916 Jour. Bact., 1, 84. AYERS, JOHNSON AND DAVIS 1918 Jour. Infect. Dis., 23, 290. AYERS AND RuPP 1922 Jour. Infect. Dis., 30, 388. BROWN, FROST AND SHAW 1926 Jour. Infect. Dis., 38, 381. FROST, GuMM AND THOMAS 1927 Jour. Infect. Dis., 40, 698. HAYNES 1929 Jour. Infect. Dis., 45, 316. JONES 1919 Jour. Exp. Med., 30, 159. OGURA 1929 Jour. Jap. Soc. Vet. Sci., 8, 174. KULP AND RETTGER 1924 Jour. Bact., 9, 357.