A COMPARATIVE STUDY OF KNOWVN FOOD-POISONING STAPHYLOCOCCI AND RELATED VARIETIES JAMES B. EVANS AND C. F. NIVEN, JR. Division of Bacteriology, American Meat Institute Foundation, and the Department of Bacteriology and Parasitology, The University of Chicago, 939 East 67th Street, Chicago, Illinois Received for publication January 20, 1950 The question as to whether the enterotoxigenic staphylococci represent a homogeneous group, based upon their physiological characteristics, yet remains open. Various studies reported in the literature have indicated that such cultures may be relatively heterogeneous in their cultural characteristics. However, many of these studies would appear to be invalidated by the lack of an adequate test for the enterotoxin. In a number of reports the sole criterion for the toxigenic capacity of the cultures investigated was the fact that they had been isolated from foods incriminated in food-poisoning outbreaks. Also, the physiological characteristics that have been considered most frequently have proved to be variable when applied to staphylococci, as well as to other groups of microorganisms. Stritar and Jordan (1935), after studying pigmentation, gelatin liquefaction, hemolysin production, and the fermentation of lactose and mannitol, concluded that "the power to provoke food poisoning is not limited to any recognizable variety of Staphylococcus." They found no evidence of homogeneity in biochemical, hemolytic, or agglutinative characters. The special gelatin medium devised by Stone (1935) for the identification of food-poisoning staphylococci has been found to be of little value. Chapman, Lieb, and Curcio (1937) proposed a series of six tests for the purpose of identifying food-poisoning staphylococci, but these investigators indicated that a considerable amount of variation among these tests arose when cultures were carried in laboratory media over a period of time. They referred to these strains as "degenerate variants." Hussemann and Tanner (1949) reported the coagulase reaction to be a promising test for detecting foodpoisoning staphylococci. They found that of 28 strains of staphylococci from foods incriminated in food-poisoning outbreaks 27 gave a positive coagulase test, and of 40 strains from normal foods only 1 was coagulase-positive. They also reported that the classical tests of pigmentation, hemolysis, mannitol fermentation, bromthymol blue agar, growth in the presence of 7.5 per cent sodium chloride, and Stone's reaction were of little differential value. In the present work a collection of 114 strains of staphylococci has been studied. The majority of these were isolated from foods examined as possible causative agents in food poisonings; the others were isolated from apparently normal foods and from various clinical sources. Some of the cultures studied have been maintained on laboratory media for varying periods of time up to 19 years, but others were isolated during the course of this investigation and studied immediately after isolation. The ability of 31 of these strains to produce enterotoxin had been 545
546 JAMES B. EVANS AND C. F. NIVEN, JR. [vol. 59 tested by controlled feeding tests with'young rhesus monkeys (Macaca mulatta) by Dr. G. M. Dack, Dr. M. J. Surgalla, and their associates. METHODS For the coagulase test only young, vigorous cultures were used. Stock cultures were transferred daily in broth for at least 3 days before testing. This procedure, however, was omitted for those strains that had recently been isolated from foods. For the test 2 drops of a 24-hour culture were mixed with 0.5 ml of citrated rabbit plasma in a 9-by-75-mm vial and incubated for 3 hours at 30 C. Lyophilized plasma was reconstituted with distilled water for the test. When unknown cultures were tested, a known coagulase-positive strain was included for control purposes. The basal medium used for the fermentation tests contained 1.0 per cent tryptone, 0.5 per cent yeast extract, 0.5 per cent sodium chloride, and 0.004 per cent bromeresol purple. The sugars and alcohols were sterilized separately by autoclaving 5 per cent aqueous solutions, and were added aseptically to the basal medium to a final concentration of 0.5 per cent. Arginine hydrolysis was detected by growing the culture for 3 days at 30 C in 1.0 per cent tryptone, 0.5 per cent yeast extract, 0.5 per cent sodium chloride, and 0.3 per cent arginine, and testing qualitatively for ammonia production with Nessler's reagent. RESULTS AND DISCUSSION Among the strains in the collection studied 22 were known to produce enterotoxin as determined by animal feeding tests. Without exception, all of these known food-poisoning strains proved to be coagulase-positive when subsequently tested. Additional physiological studies on these strains, as shown in table 1, indicate that they comprise a very homogeneous group of microorganisms. For example, all cultures reduced nitrate, were capable of initiating growth at 10 C and 45 C and in a 20 per cent sodium chloride broth, hydrolyzed arginine, and produced an orange or cream pigment on an agar medium containing 7.5 per cent sodium chloride (Chapman, 1945, 1946). All cultures were capable of growing anaerobically in a glucose broth. In addition, all strains fermented mannitol anaerobically. With the exception of glycerol these enterotoxin-producing cultures were also homogeneous in their fermentative characteristics. Of the 9 cultures tested that did not yield any evidence of enterotoxin production, 8 proved to be coagulase-positive. With minor exceptions, these 8 cultures appeared to be identical in all respects to the enterotoxigenic staphylococci. For comparative purposes, 41 additional strains of coagulase-positive staphylococci were included in this study. None of these cultures have been tested for their enterotoxigenic capacities, although a large percentage of them had been isolated from foods incriminated in food-poisoning outbreaks. As shown in table 1, these cultures are also identical with the known enterotoxigenic staphylococci. The most striking features of these data are the extreme homogeneity of the coagulase-positive group considered as a whole and the lack of any distinction between enterotoxigenic strains and those coagulase-positive microorganisms
1950] STUDY OF FOOD-POISONING STAPHYLOCOCCI 547 that appear to lack the capacity to produce the toxin. This is particularly striking when it is considered that these cultures have been isolated over a span of TABLE 1 Physiological characteristics of known toxigenic staphylococci as compared to other cultures studied COAGULASZ-POSITIVE COAGULASZ- Enterotoxinpositive Enterotoxinnegative Enterotoxin not tested NEGATIVE No. of cultures tested 22 1 8 1 41 1 43 Catalase production... 100 100 100 100 Nitrate reduction... 100 100 100 63 Acetoin production... 100 100 100 47 Pigmentation: White... 0 12 2 37 Cream... 5 0 5 33 Orange... 95 88 93 30 Growth: 10C... 100 88 98 91 45 C... 100 100 100 28 15% NaCl... 100 100 100 88 20% NaCl... 100 100 98 88 Hydrolysis: Stone's gelatin... 55 50 51 44 Starch... 0 0 0 5 Sodium hippurate... 82 100 85 65 Esculin... 0 0 0 40 Arginine... 100 75 100 23 Fermentation: Mannitol, aerobic... 100 100 100 81 Mannitol, anaerobic... 100 100 100 5 Glucose, anaerobic... 100 100 100 70 Xylose... 0 0 0 33 Arabinose... 0 0 0 33 Mannose...... 100 100 100 58 Fructose...... 100 100 100 95 Galactose... 95 100 98 42 Maltose... 100 100 100 95 Lactose... 100 100 100 65 Sucrose... 100 100 100 91 Trehalose... 100 100 100 86 Raffinose... 0 0 0 9 Inulin...0 0 0 12 Dextrin... 100 100 95 12 Glycerol... 68 100 78 77 Sorbitol... 0 0 0 33 nearly 20 years. These studies would indicate that the variation and degeneration of stock cultures is much less than has been implied.
58JAME B. EVANS AN) C. F. NIVEN, JR. 548 [VOL. 59 Furthermore, it would seem that the coagulase-positive staphylococci constitute a clearly definable species, as was proposed by Cowan and Shaw (1949) and others. The fermentation pattern of the strains in this study is in close agreement with the collection of 94 coagulase-positive strains studied by Christie and Keogh (1940). According to the present system of classification used in Bergey's Manual (Breed, Murray, and Hitchens, 1948) these organisms would be divided among Micrococcus pyogenes var. aureus, M. pyogenes var. albus, and M. aurantiacus, based upon their ability to liquefy gelatin and to produce a pigment. However, since gelatin hydrolysis was the most variable characteristic noted in this otherwise homogeneous group it would appear taxonomically unsound to use this test as a primary means of separation. Of the 43 coagulase-negative staphylococci in this study, animal feeding tests had been conducted on only one culture. No evidence of enterotoxin production was detected. Many of these cultures have been isolated from foods implicated in food-poisoning outbreaks. Moreover, a number of these cultures might be considered as food-poisoning varieties based upon the other tests that have been used for such purposes, such as pigmentation, gelatin liquefaction, aerobic mannitol fermentation, and lactose fermentation. These cultures comprised an extremely heterogeneous group, no well-defined subgroups being apparent. Owing to the heterogeneity of these microorgisms no single physiological test was found that would separate all strains from the coagulase-positive group. The most useful test noted was the anaerobic fermentation of mannitol (Evans, 1947). The two coagulase-negative strains that have been found positive thus far on this test differed from the coagulase-positive group in at least 11 of the characteristics listed in table 1. They were also unusual in their vitamin requirements (Evans, 1948). When the complete physiological pattern of the individual microorganisms was studied, all coagulase-negative strains could be clearly set apart from the coagulase-positive group. Every strain differed from the typical coagulase-positive culture in at least five of the tests listed in table 1. When tested in combination, characteristics such as orange pigmentation on an agar medium containing 7.5 per cent sodium chloride, acetoin production, nitrate reduction, esculin hydrolysis, growth at 45 C, and fermentation of xylose, arabinose, mannose, galactose, lactose, and sorbitol assumed importance for differential purposes. The fermentation of dextrin would appear to be a fairly useful test, but the reaction is frequently slow and weak. The production of ammonia in arginine broth also seems to be of value under the conditions used in this survey. In most cases the coagulase-positive strains gave a strong test for ammonia, whereas the majority of the coagulase-negative strains failed to hydrolyze arginine. However, when other basal media and conditions were employed for this test, the results were often anomalous. Also, many of the cultures produced considerable amounts of ammonia from other nitrogenous constituents of the basal medium. These studies would imply that most, if not all, food-poisoning staphylococci are members of the coagulase-positive group. Whether any of the coagulasenegative cultures are enterotoxigenic must await further animal feeding tests.
1950] STUDY OF FOOD-POISONING STAPHYLOCOCCI 549 As yet, however, there appears to be no recorded case of a coagulase-negative culture having produced enterotoxin under experimental conditions. Chapman, Lieb, and Curcio (1937) pointed out that, with the few coagulase-negative cultures that were sent to them as food-poisoning strains, in no case was there good circumstantial evidence to warrant such a label. The single coagulase-negative strain of Hussemann and Tanner (1949) that had been isolated from a foodpoisoning outbreak has been lost, but there was reason to question the designation of this strain as a food-poisoning organism (Hussemann, 1949). If the hypothesis is accepted that all food-poisoning staphylococci are members of the coagulase-positive group, the question arises as to what percentage of such cultures can produce enterotoxin. The present study throws little light on this problem. Little is known concerning the optimum conditions necessary for the production of enterotoxin by a growing culture of Staphylococcus. The inherent weakness and difficulty in conducting anaimal or human feeding tests are well recognized by those engaged in such studies. From a practical point of view, however, it would seem advisable to view with extreme suspicion any food containing coagulase-positive staphylococci in large numbers. ACKNOWLEDGMENT The authors wish to express their deep appreciation to Dr. G. M. Dack and Dr. M. J. Surgalla of the Food Research Institute, the University of Chicago, for many of the cultures used in this study and for making available the results of the feeding tests conducted in their laboratories, which were the criteria for enterotoxin production used in this study. SUMMARY A physiological study has been conducted on 114 staphylococci, including 22 strains that were known to produce enterotoxin as evidenced by feeding tests with rhesus monkeys. Based upon the 29 physiological tests employed, the enterotoxigenic strains comprised an extremely homogeneous group. All toxigenic cultures were coagulase-positive and appeared to be identical with all of the other coagulase-positive strains, which included 8 cultures that failed to give evidence of enterotoxin production. These studies indicate that most, if not all, enterotoxigenic staphylococci are members of the coagulase-positive group, but they do not preclude the possibility that some varieties of coagulase-negative staphylococci might also be associated with food poisoning. REFERENCES BREED, R. S., MURRAY, E. G. D., AND HITCHENS, A. P. 1948 Bergey's manual of determinative bacteriology. 6th ed. Williams & Wilkins Co., Baltimore. CHAPMAN, G. H. 1945 The significance of sodium chloride in studies of staphylococci. J. Bact., 50, 201-203. CHAPMAN, G. H. 1946 A single culture medium for selective isolation of plasma-coagulating staphylococci and for improved testing of chromogenesis, plasma coagulation, mannitol fermentation, and the Stone reaction. J. Bact., 51, 409-410. CHAPMAN, G. H., LIEB, C. W., AND CuRcio, L. G. 1937 Isolation and cultural differentiation of food-poisoning staphylococci. Food Research, 2, 349-367.
550 JAMES B. EVANS AND C. F. NIVEN, JR. [VOL. 59 CHRISTIE, R., AND KEOGH, E. V. 1940 Physiological and serological characteristics of staphylococci of human origin. J. Path. Bact., 51, 189-197. COWAN, S. T., AND SHAW, C. 1949 Outline classification of Bacterium and Staphylococcus. J. Gen. Microbiol., 3, 69-73. EVANS, J. B. 1947 Anaerobic fermentation of mannitol by staphylococci. J. Bact., 54, 266. EVANS, J. B. 1948 Studies of staphylococci with special reference to the coagulase-positive types. J. Bact., 55, 793-800. HUSSEMANN, D. L. 1949 Personal communication. HU5SEMANN, D. L., AND TANNER, F. W. 1949 A comparison of strains of staphylococci isolated from foods. Food Research, 14, 91-97. STONE, R. V. 1935 A cultural method for classifying staphylococci as of the "food poisoning" type. Proc. Soc. Exptl. Biol. Med., 33, 185-187. STRITAR, J., AND JORDAN, E. 0. 1935 Is a special variety of Staphylococcus concerned in food poisoning? J. Infectious Diseases, 56, 1-7. Downloaded from http://jb.asm.org/ on March 11, 2019 by guest