streptococci (S. viridans). It is the object of this paper to show

Transcription

1 VARIATIONS OF STREPTOCOCCI WITH A HEMOLYSIN PRODUCTION' NOTE ON M. FROBISHER, JR., AND E. R. DENNY2 From,.the Department of Pathology and Bacteriology, Johns Hopkins University, and the Medical Clinic, Johns Hopkins University and Hospital, Baltimore, Maryland Received for publication, March 9, 1928 Instances have been cited by Beitzke and Rosenthal (1906) Anthony (1909), Zoppritz (1909), Jaffe (1912), Rosenow (1914, A), and (1914, B), Hintze and Kuhne (1922), Pruska (1924), Morgenroth, Schnitzer and Berger (1925), Buerger and Ryttenberg (1907), Ludke and Polano (1909), Much (quoted by Hoessli, 1910), Jungman (1921), and others in which hemolytic streptococci (S. hemolyticus) have changed either spontaneously, or as a result of certain definite cultural procedures, into green producing streptococci (S. viridans). It is the object of this paper to show how error may arise from failure to recognize the true nature of a strain of streptococcus at the start of such investigations. Strains of Streptococcus viridans are frequently found which, in rabbit-blood agar, pour plates, incubated aerobically for twentyfour to forty-eight hours, at 37 C., produce zones of hemolysis 2 to 4 mm. in width about the deep colonies. A photograph of such a blood agar culture is reproduced in figure 1. Such a strain of streptococci might be reported as Streptococcus hemolyticus. Certainly, if one regards as S. hemolyticus any strain showing a wide zone of hemolysis in blood agar pour plates (and it appears likely that this has been done in the past) then this strain would be regarded as of that type. Production of a zone of hemolysis of any width, however, is not the differential point. Streptococci are of three main types as regards action in 5 per i Aided by a grant from the Ella Sachs Plotz Foundation. 2 Jacques Loeb Fellow in Medicine. 109

2 110 M. FROBISHER, JR., AND E. R. DENNY cent rabbit-blood, infusion-agar, pour plates incubated aerobically at 37 C. for twenty-four to forty-eight hours. Two of these are hemolytic. The third (Gamma type-brown, 1919) produces no change whatever and will not be discussed further in Downloaded from FIG. 1. BLOOD AGAR PLATE CONTAINING A MIXED CULTURE OF BETA TYPE AND ALPHA TYPE STREPTOCOCCI OF THE EXTREMELY HEMOLYTIC SORT MENTIONED IN THE TEXT The alpha type colony with the ring around it is the one shown in figure 2 this paper. Of the two hemolytic types, one produces about its colonies a zone of hemolysis devoid of visible blood corpuscles. This is the beta type of Smith and Brown (1915); Brown (1919). (Most strains of the so-called S. hemolyticus reported in the litera- on May 10, 2016 by PENN STATE UNIV

3 VARIATIONS OF STREPTOCOCCI ture are probably of this type but it is also probable that some of the more hemolytic alpha type strains have been mistaken for the beta type.) The other type also produces a zone of more or less hemolysis about its colonies. The production of the hemolytic zone is preceded by the production of a zone, close around the colony, of methemoglobinized corpuscles. This is the alpha type of Smith and Brown (1915); Brown (1919). S. viridans belongs to this type. There are also strains producing no grossly visible greenish color which belong to the alpha type. The corpuscles in this inner zone, once methemoglobinized, are not subject to hemolysis. 'Consequently, when hemolysin is produced later in the life of the colony, the blood cells remain around the colony as a more or less greenish or brownish zone. The clear zone appears only when, and if, the hemolysin is produced in sufficient ainount to diffuse beyond the zone of discolored corpuscles. For brevity these zones are here referred to as the "green" and "clear" zones respectively. The relative and absolute widths of the green and clear zones are prone to vary, sometimes within wide limits, as described in this paper. One zone may at times be so exaggerated as completely to mask the other. The literature contains a large number of reports of such variations. The hemolytic character of S. tiriidans is easily demonstrated by the anaerobic cultivation of the pour-plate cultures (Brown, 1919). Here the oxidation processes which are partly responsible (Hagan, 1925, and Brown, 1919) for the green zone are inhibited and the colonies so cultivated are indistinguishable from those of hemolytic (beta type) streptococci. In some cases, hemolysin production by alpha type streptococci occurs so early and so extensively as to make the green zone very narrow both absolutely and relatively. The green zone in such cases escapes notice unless the low power of the microscope be focussed upon the deep colony, when the true nature of the growth is made clearly visible. As a further check, recourse may be had to the effect of 1 cc. of a young, (eight to twelve-hour) well grown, 20 per cent horse serum, broth culture (de Kruif and Ireland, 1920) upon 1 cc. of a 5 per cent suspension of washed rabbit erythrocytes. The mixture is held in the water bath at ill

4 112 M. FROBISHER, JR., AND E. R. DENNY 370C. for one or two hours. Under such conditions, cultures of alpha type streptococci produce no trace of hemolysis. Methemoglobin production, with the development of brownish, greenish or purplish tints may be pronounced. By the routine use of the microscope, supplemented, in cases of doubt, by the test tube hemolysin test just described, one avoids error in the designation of streptococci as alpha or -beta types. I I FIG. 2. APPEARANCE OF THE ALPHA TYPE COLONY MARKED WITH A RING IN FIGURE 1, WHEN VIEWED WITH THE Low POWER OF THE MICROSCOPE The zone of discolored corpuscles close around the colony is clearly visible The tube hemolysin test must not be used as a substitute for microscopic examination of deep colonies in blood agar, because there are streptococci which, with the microscope, are clearly seen to be of the beta type, yet yield no test tube hemolysis. EXPERIMENTAL Fifty strains of streptococci producing wide zones of hemolysis, thirteen of which were considered, by gross inspection of the plate, as S. hemolyticus (beta type), and thirty-seven of which produced

5 VARIATIONS OF STREPTOCOCCI no grossly visible methemoglobinized zone, were subjected to the tests described above. Figure 1 shows the gross appearance of one of the pour plates and figure 2 shows the microscopic appearance of one of the colonies, with the zone of methemoglobinized cells clearly visible. The test-tube hemolysin te-st was absolutely negative in every case. The possibility of error through failure to use microscopic examination of colonies and the tube hemolysin test in the study of streptococci is quite evident. It seems desirable to point out in this connection, particularly in view of a statement by Holman (1916) and the proposed classification of pseudo-hemolytic streptococci by Cumming (1927), that colonies of either alpha or beta type streptococci may show little or no heinolysis or green production when growing on the surface of blood agar plates. It is extremely difficult in some cases (not all) to be certain of the nature of a surface colony. Holman's reason for grouping the alpha and gamma (indifferent) streptococci together is based on surface streak methods. Cumming's proposed classification is similarly based on this technique. In the latter study the tube hemolysis tests were made using 0.2 cc. of a twenty-four-hour culture plus 1 cc. of 3 per cent corpuscle suspension. Many workers have shown that hemolysin is extremely labile and reaches a maximum concentration early in the life of the culture, often disappearing after fourteen hours. Tests made after fourteen hours, therefore, may often give falsely negative results. It has also been observed by many workers that true, beta type hemolytic streptococci vary greatly in their hemolytic powers. The use of 0.2 cc. of a broth culture might therefore exclude from consideration many weakly hemolytic, but genuine, beta type streptococci. According to the proposed classification, streptococci failing in these tube hemolysin tests but giving wide zones of hemolysis on the surface of blood agar plates were classed as pseudo-hemolytic streptococci. It appears, then that such a group would include (a) slightly hemolytic beta type streptococci, (b) strongly hemolytic alpha type streptococci, and (c) strongly hemolytic pneumococci.3 This grouping would offer no advantages over the present systems. 3 Pneumococci are exactly like alpha type streptococci in respect to blood agar and test-tube hemolysin tests. 113

6 114 M. FROBISHER, JR., AND E. R. DENNY VARIATIONS The fifty strains referred to above were first kept for varying periods upon blood agar slants with monthly renewals. In May 1927 they were dried on pieces of sterile filter paper in vacuum jars (Brown, 1925, 1926) and held in the refrigerator until Febru- FIG. 3. BLOOD AGAR PLATE CULTURE OF THE SAME ALPHA TYPE STREPTOCOCCUS AS THaAT SHOWN IN FIGURE 1 This culture was made nearly two years later, after the organism had lost its exaggerated hemolytic power. The arrow almost touches a colony. Due to the very small zones, a good picture could not be made. The contrast between this plate and figure 1, however, is marked, and the plate is included as a matter of record. ary, At this time-all were plated out in the same manner as previously described. All were found to have lost their exaggerated hemolytic properties and to have reverted to the ordinary alpha type usually recognized as such without difficulty. Figure 3 shows the appearance under these conditions, of a blood agar plate culture of the same streptococcus as that shown in figure 1.

7 VARIATIONS OF STREPTOCOCCI Without microscopic examination and tube hemolysin test of the streptococcus under its original conditions, one might have concluded that a beta type streptococcus had mutated to an alpha type streptococcus. It is suggested, in the light of these illustrations, that many of the cases reported as mutations of streptococci are due to faulty observation and failure to recognize the type of streptococcus in the first place coupled with a diminution (not loss) of hemolytic power in a genuine alpha type streptococcus. SUMMARY AND CONCLUSIONS 1. For the study and proper classification of streptococci in blood agar the following are essential: a. Differentiation of types based on low power microscopic observation of deep and not of surface colonies in blood agar plates. b. Use of tube hemolysin tests with properly prepared cultures to supplement plate observations. 2. Genuine, alpha type streptococci have frequently been encountered which possess very marked hemolytic powers, producing hemolytic zones 2 to 4 mm. wide and appearing, to the naked eye, to be beta type (hemolyticus) streptococci. These strains never caused hemolysis when their broth cultures were mixed with red cell suspensions. When the deep colonies of such streptococci in blood agar pour plates were viewed with the low power microscope, they were seen to have about them, inside the hemolytic zone, the zone of methemogobinized cells common to all alpha type streptococci. 3. A number of such streptococci have been found to lose a large part (not all) of their hemolytic powers after being kept for some time under various artificial conditions and to appear like the ordinary and easily recognized alpha type of streptococcus. The diminution of hemolytic properties may be due to a change in the organisms or to some obscure variation in technique. 4. It is suggested that some of the reported mutations in which a beta tvpe streptococcus has been said to change into an alpha type streptococcus may be duq to failure to recognize the true character of the organism in the first place, accompanied by a diminution of the original hemolytic properties. 115

8 116 M. FROBISHER, JR., AND E. R. DENNY 5. It is possible that many of the anomalous results of many sorts reported by investigators of streptococci may be due to confusion in identification of alpha and beta types. REFERENCES ANTHONY, B. VAN H Some characteristics of the streptococci found in scarlet fever. Jour. Inf. Dis., 6, 332. BEITZKE, H., AND ROSENTHAL, Zur Unterscheidung der Streptokokken mittels Blutnahrboden. Ark. pathol. Inst. Berlin, 349. BROWN, J. H Blood agar for the study of streptococci. Monographs of The Rockefeller Institute, Number 9. BROWN, J. H The preservation of bacteria in vacuo. Abs. Bact., 9, 8. BROWN, J. H Vacuum tubes for the storage and shipment of bacteria. Science, 64, 429. CUMMING, W. M On the pseudohemolytic streptococci isolated from the sputum in pulmonary tuberculosis. Jour. Pathol. and Bact., 30, 279. DEKRUIF, P. H., AND IRELAND, P. M Streptolysin. Jour. Inf. Dis., 26, 285. HAGAN, W. A The green coloration produced by certain streptococci on blood-agar plates. Jour. Inf. Dis., 37, 1. HINTZE, K., AND KUHNE, R Zur Frage Umwandung haemolytischer Streptokokken in die Griun-Wachsende Form. Zentralbl. f. Bakt., 1 abt., Orig., 88, 352. HOLMAN, W. L The classification of streptococci. Jour. Med. Res., 34, 377. JAFFE, R Beobachtungen bei blutlosenden und bei Gramnegativen Streptokokken. Arch. f. Hyg., 76, 137. JOPPRITZ, B Ueber Streptokokken versuche. Med. Klin., 5, JUNGMAN, P Zur Klinik und Pathogenese der Streptokokken. Deut. Med. Woch., 47, 496. LUDKE AND POLANO 1909 Ueber Hemolyse der Streptokokken. Munch. Med. Woch., 56, 7. MORGENROTH, J., SCHNITZER, R., AND BERGER, E Ueber die Einheit der Streptokokken und Pneumokokken. 1 Mitteilung. Ztschr. f. Immunitatsforsch. u. exp. Therap. Teil 1, Orig., 43, 169. MUCH, H. (Quoted by Hoessli) Cited from Brown, J. H., loc. cit. PRUSKA 1924 Die Umwandlung des haemolysierenden in einem Nicht-haemolysierenden Streptokokkus. Centralbl. f. Bakt., 1 abt., Orig., Ref. 77, 208. RoSENOW, E. C. 1914a Transmutations within the streptococcus-pneumococcus group. Jour. Inf. Dis., 14, 1. RoSENOW, E. C. 1914b The etiology of acute rheumatism, articular and muscular. Jour. Inf. Dis., 14, 61. SMITH, T., AND BROWN, J. H A study of streptococci isolated from certain presumably milk-bone epidemics of tonsillitis occurring in Massachusetts in 1913 and Jour. Med. Res., 31, 455.