A SYSTEMATIC STUDY OF MICROORGANISMS WHICH DECOMPOSE THE SPECIFIC CARBOHYDRATES OF THE PNEUMOCOCCUS1 GRACE M. SICKLES AND MYRTLE SHAW Division of Laboratories and Research, New York State Department of Health, Albany The soil bacteria which split the specific carbohydrates of the pneumococci differ in form and in cultural characters. The results of experimental studies on their immunological significance have already been reported (Sickles and Shaw, 1933, 1934). A systematic study of the different strains has been made in order to determine their relationships to each other and to known groups of soil organisms; this report records the results of that study. The cultures described are of four distinct types: 1. Large spore-bearing rods which decompose type III pneumococcus specific carbohydrate, similar to those described by Dubos and Avery (1931); and, in addition, a strain which also utili,zes agar. 2. Very small non-sporulating rods which attack the non-typespecific carbohydrate obtained from a degraded type I pneumococcus and the so-called "C" substance from typical strains. 3. Bacteria, oval in form, which decompose the specific carbohydrate of type II pneumococcus. 4. Slender rods with tapering ends, which utilize the specific carbohydrate of type I pneumococcus. SOURCE OF STRAINS AND METHODS OF ISOLATION With the exception of the very small non-sporulating rod which acts on the atypical I carbohydrate, of which we have only one culture, several strains of each of the above types were obtained I Presented before the Society Pennsylvania, December 29, 1933. of American Bacteriologists, Philadelphia, 415 JOURNAL OF BACTERIOLOGY, VOL. 28. NO. 4
416 GRACE M. SICKLES AND MYRTLE SHAW from swamps and other uncultivated soils in different localities. One strain which decomposes the carbohydrate of pneumococcus type III was isolated from manure. Two kinds of mineral media were employed in the isolation and cultivation of these bacteria. Medium A was that described by Dubos and Avery (1931) in their report on the isolation of an organism which decomposed the capsular polysaccharide of type III pneumococcus. This consists of: (NH4)2S04.....1.0 gram K2HP4.2.0 grams Tap water.1000.0 cc. Reaction not adjusted-usually about ph 7.4. Medium S has the following composition: MgSO47 H20...0.2 gram NHIH2PO4... 1.5 grams CaCl2... 0.1 gram KCl... 0.1gram FeCls... Trace Distilled water.1000.0 cc. 10 cc. N/i NaOH added per liter; ph from 7.2 to 7.4 To these mineral media were added the specific pneumococcus carbohydrates as a source of carbon, in concentrations varying from 0.002 to 0.01 per cent. The soil samples were suspended in sterile water and inoculations made into flasks of mineral media containing the specific carbohydrates. These were incubated at different temperatures and under aerobic and anaerobic conditions. Since it was soon evident that no growth of the carbohydrate-decomposing organisms occurred under anaerobic conditions, later cultures were limited to those grown aerobically. At intervals during the incubation, the cultures were tested for precipitation with the specific antipneumococcus serum. An absence of precipitation or a diminution in amount was the criterion for the presence in the mixed culture of an organism capable of decomposing the carbohydrate. As soon as precipitation was no longer obtained, the culture was transferred to fresh me-
MICROORGANISMS WHICH DECOMPOSE CARBOHYDRATES 417 dium containing the specific carbohydrate. This served as a means of enrichment and to some extent eliminated extraneous forms unable to grow in these simple media. After a number of transfers in liquid medium, the carbohydrate of the medium was utilized in a shorter time, either because of the increased activity of the organisms or because of the continual elimination of contaminants. Blood-agar plates were streaked from these liquid cultures. Since the cultures which decomposed the type III pneumococcus polysaccharide were spore-bearers, heating to 80 C. before plating greatly facilitated their isolation. Colonies were fished into mineral medium containing the carbohydrate and the active fishings selected by the precipitation test. The cultures were then replated several times to assure their purity. When the mixed cultures which decomposed the type II carbohydrate were plated on blood-agar or any agar containing beefextract or peptone, all the fishings from these plates were inactive, indicating that the active organism failed to grow on these solid media. It was necessary to continue the process of enrichment and dilution until the variety of contaminants was so reduced that only a few types appeared when plated on beef-extract peptone agar. At this stage the enrichment cultures were plated on a semisolid agar made of mineral medium A, 0.8 per cent agar, and 0.01 per cent type II pneumococcus carbohydrate. Characteristic pink colonies appeared, which proved to be active organisms. The mixed cultures, which utilized type I pneumococcus carbohydrate, contained, even after considerable enrichment, contaminants which overgrew the active organisms on all the solid media. The pure culture was finally isolated by continuing the dilution and enrichment procedures until the active liquid culture gave no growth when plated on beef-extract agar. This apparently pure culture was then plated on mineral medium S containiing 0.8 per cent agar and 0.005 per cent type I pneumococcus carbohydrate. The small, pale yellow colonies developing slowly on this medium were fished and replated. The cultural and biochemical properties were then studied.
418 GRACE M. SICKLES AND MYRTLE SHAW METHODS OF IDENTIFICATION Standard media were prepared in accordance with formulae given in the Manual of Methods of the Society of American Bacteriologists (1932, 1933). Gram stains were made according to the Kopeloff and Beerman modification, and flagella were stained by Shunk's method, all as given in the Manual (1932). In the study of the fermentation reactions of those strains which failed to grow on standard media, the test substances were added in 1 per cent concentration to mineral media. For the other strains, the action on carbohydrates was tested in the mineral media, also in Hiss serum water and in 1 per cent peptone solution. For the determination of the optimum temperature, resistance to heat, and the limiting hydrogen-ion concentration, a medium previously found to be most favorable to the growth of the organism was used. This medium varied with the type of organism. Results were verified by using the mineral medium containing the specific carbohydrate. Growth of the organism under any given circumstance could then be determined not only by turbidity in the tube but also by decomposition of the specific polysaccharide. In the case of the strains which decomposed the type III or atypical I pneumococcus carbohydrate, inoculations were made from colonies grown on medium free from pneumococcus carbohydrate to avoid introducing any specific enzyme with the inoculum. DESCRIPTIVE CHARACTERS Bacteria which decompose the carbohydrate of pneumococcus type III Morphology. Straight rods with rounded ends, occurring singly or in pairs. Vegetative cells 2.5 to 3.5j in length by 0.6 to 0.8us in width. Spores 1.5 by 2.5uA. Motile, possessing from four to six peritrichous flagella. Gram-negative. Figure 1 illustrates the vegetative cells and spores. Colonies on blood agar or beef-extract peptone agar. Round, entire; usually about 2 mm. in diameter. Two types of colonies appear on plates-one white and opaque, the other gray and
MICROORGANISMS WHICH DECOMPOSE CARBOHYDRATES 419 translucent. The white colonies produce spores; the gray ones are non-sporulating. This dissociation appears to be characteristic of all the strains studied, although it is more marked in some strains than in others. One strain utilizes agar, and colonies of this strain, while identical in appearance with those of the other strains, lie in depressions formed in the agar because of the softening effect on the gel. No liquid occurs in these depressions. Figures 2 and 3 illustrate the structure and type of colonies. X4 if4 FIG. 1. Bacillus palustris var. gelaticus: Bacteria from blood-agar plate after twenty-eight hours' incubation. Gram. 1lagnification, X 1000. Agar slants. Moderate growth. Usually made up of colonies of both above types. Potato slants. Growth heavy, v-iscid, yellowish, moist, and glistening. Nutrient gelatin. No liquefaction. Beef-extract peptone broth. Moderate even turbidity in twentyfour hours. Beef-infusion broth. A less favorable medium than the beefextract broth, at least forty-eight hours' incubation being neces- i OW 1 I
420 GRACE M. SICKLES AND MYRTLE SHAW sary to promote growth. In old cultures, a heavy viscid sediment occurs. Litmus milk. Litmus reduced. No digestion. No change in reaction. Starch agar. Starch hydrolyzed weakly by two strains, strongly by the two other strains. FIG. 2. Bacillus palustris var. gelaticus: Ninety-six-hour growth on blood-agar plate, showing colonial structure of sporulating type. Natural size. Utilization of carbohydrates. Acid but no gas produced from glucose, lactose, sucrose, maltose, dextrin, mannitol, xylose, galactose, inulin, and salicin by all strains. Although glucose is utilized by the organisms, this carbohydrate has an inhibiting effect on the growth of the cultures if added to an otherwise favorable medium. Agar was utilized by only one strain: neither acids nor reducing sugars could be determined in the decomposition products, although the gel properties were obviously changed.
MICROORGANISMS WHICH DECOMPOSE CARBOHYDRATES 421 Relation to oxygen. Aerobic. Temperature relations. Growth was obtained at from 22 to 42 C. -Optimum temperature from 35 to 37 C. Effect of reaction of medium. ph limits of growth from 5.5 to 8.5. Optimum not definite as the organism grew well over a considerable range. Usually grown at ph 7.2 to 7.4. FIG. 3. Bacillus palustris: Differences in appearance of sporulating and nonsporulating colonies on blood agar. Natural size. Bacteria which decompose the non-type-specific carbohydrate Morphology. Small slender rods, occurring singly and in pairs, 0.7 to 1.0, long. Width less than 0.51A. No spores. Non-motile. Gram-negative. Agar colonies. On blood agar, barely visible in twenty-four hours; after four days, dull rust-colored colonies about 1 mm. in diameter. Round, entire, umbilicate, and rather dry. On beef-
422 GRACE M. SICKLES AND MYRTLE SHAW extract agar, colonies are similar except that color is yellowish gray. Agar slants. On beef-extract agar, very slight, thin, yellowish gray growth. On blood-agar slant, moderate growth; same color and texture as in colonies on blood agar. Potato slants. Moderate growth, bright orange in color. Potato darkened. Nutrient gelatin. At 370C., gelatin cultures liquefied in one week. At room temperature, liquefaction slower, napiform; yellow sediment along line of pumiiture. Beef-extract peptone broth. Moderate even turbidity. If nontype-specific carbohydrate is added to broth, a heavier growth occurs with yellow sediment. Beef-infusion broth. No growth. Litmus milk. After two weeks' incubation, a soft curd. Slight reduction of litmus. No change in reaction indicated. Starch agar. Very active hydrolysis. Utilization of carbohydrates. Acid but no gas produced from glucose, lactose, sucrose, maltose, dextrin, inulin; very slight action on mannitol; salicin not fermented. Relation to oxygen. Facultative. Temperature relations. Limits of growth from 22 to 39 C. No growth at 41 C. Optimum temperature from 35 to 37 C. Cells killed by heating for ten minutes at 52 C. Enzyme produced by strain against pneumococcus carbohydrate withstands 56 C. for ten minutes. Effect of reaction of medium. ph limits of growth from 6.5 to 9.0. Optimum between 7.0 and 7.5. Microdrganisms which decompose the carbohydrate of pneumococcus type II Morphology. Extremely pleomorphic. Young cells are oval in form, 1.5 by 2.0 ua, usually in pairs, and contain granules which stain deeply with basic dyes. Older cultures contain cells which may be from 12 to 15 u in length. The granules in these occur in many different forms and arrangements. Nonmotile. Gramnegative. No spores noted. Figures 4, 5, and 6 illustrate some of the forms in which these organisms occur.
MICRO6RGANISMS WHICH DECOMPOSE CARBOHYDRATEs 423 4% Jib a~~~~~~~ Fic4.Sacharbateriu ovl-yugclsgono urs eioi agaėtylneblu. agifcaio,p*t000 /4 as40-t dil- N. #10 0 V.-:- t A* ILA V -' FIG. 5. Saccharobacterium ovate: Seven-day culture grown on sucrose semisolid agar. Methylene blue. Magnification, X 1000.
424 GRACE M. SICKLES AND MYRTLE SHAW Agar colonies. On beef-extract agar, no growth. On blood agar, no growth. Medium A + pneumococcus II carbohydrate + 0.8 per cent agar. Very small round pink colonies pinpoint in size after about five days' incubation. After two weeks' incubation, they increase in size to 1 mm. in diameter. They are quite coherent so that, in fishing, whole colonies are removed from the agar plate. FIG. 6. Saccharobacterium ovale: Seven-day culture grown in beef-extract peptone broth containing 1 per cent sucrose. Methylene blue. Magnification, X1000. Beef-extract-agar slants. No growth. Potato slants. No growth. Nutrient gelatin. No growth. Beef-extract peptone broth. No growth. Beef-extract peptone + 1 per cent sucrose. Moderate turbidity. Yellowish sediment. Beef-infusion broth. No growth. Beef-infusion broth + 1 per cent sucrose. No growth. Litmus milk. No growth. &w_..l 4w 6
MICROORGANISMS WHICH DECOMPOSE CARBOHYDRATES 425 Starch. No growth on nutrient agar containing starch. On agar plates containing mineral medium A and starch, there was a definite zone of hydrolysis. Utilization of carbohydrates. In mineral medium A containing 1 per cent of the test carbohydrate, lactose and sucrose were utilized by all the strains, while maltose, xylose, and dextrin were decomposed by some of the strains. There was only a slight change in reaction. No growth occurred in the tubes containing inulin, mannitol, salicin, or 1 per cent glucose. When the concentration of glucose was reduced to 0.1 per cent, a slight amount of growth was apparent. Experiments on the addition of glucose to a favorable medium, such as the mineral base plus pneumococcus II carbohydrate, showed that glucose, even in 0.1 per cent concentration, had a marked inhibitory effect. Relation to oxygen. Aerobic. In deep tubes of a semisolid medium, the growth of the culture is sharply delimited at 4 or 5 mm. from the surface. Temperature relations. Limits of growth from 20 to 37 C. Optimum temperature from 34 to 35 C. Organisms are killed by heating for ten minutes at 54 C. Effect of reaction of medium. ph limits of growth from 6.4 to 7.8. No growth at 6.0. Optimum ph from 7.0 to 7.4. Effect of sodium chloride on growth. The addition of 0.5 per cent sodium chloride to any favorable medium completely prevents growth of the organism. Microorganisms which decompose the carbohydrate of pneumococcus type I Morphology. Extremely pleomorphic. Young organisms are pointed, often curved rods, 2 by 0.5 I,u having a densely staining granule in the central portion of the cell. The tapering pointed ends remain unstained. As the cultures become older, the ends of the cells become rounded, and in old cultures the organism may be a long oval or its form may be spherical or pear shaped. This change in form is probably associated with autolysis, as at this stage the cells stain weakly and subcultures usually fail to grow. The organisms are nonmotile and Gram-negative. No spores
426 GRACE M. SICKLES AND MYRTLE SHAW wo -W -0 f Do * ar IVS t a r I_ 'I t t V. FIG. 7. Saccharobacterium acuminatum: Forty-eight-hour culture in medium S containing 1 per cent sucrose. Crystal violet. 'Magnification, X 1000. 7. Downloaded from http://jb.asm.org/ on September 3, 2018 by guest FIG. 8. Saccharobacterium acuminatum: Six-day culture on agar medium S containing 1 per cent sucrose. Crystal violet. Magnification, X 1000.
MICROORGANISMS WHICH DECOMPOSE CARBOHYDRATES 427 have been noted. Figures 7 and 8 illustrate some of the forms in which these organisms occur. Agar colonies. On beef-extract agar, no growth. On blood agar, no growth. Medium S + pneumococcus I carbohydrate + 0.8 per cent agar. Very tiny pale yellow colonies. Even after several weeks of incubation, they remain less than 0.5 mm. in diameter. On medium S containing 1 per cent sucrose and agar, the colonies develop more rapidly to 1 mm. in diameter, are pale yellow in color, and have the same texture as the colonies of the micro6rganisms which decompose the type II pneumococcus carbohydrate. Beef-extract-agar slants. No growth. Nutrient gelatin. No growth. Potato slants. No growth. Beef-extract peptone broth. No growth. Beef-infusion broth. No growth. Litmus milk. No growth. Starch. Not hydrolyzed. Utilization of carbohydrates. In mineral medium S containing 1 per cent of the test carbohydrate, the sucrose culture was the only one showing growth. Glucose, lactose, maltose, dextrin, inulin, mannitol, and salicin were not utilized. Further experiments indicated that glucose in 0.1 per cent concentration would support growth, but is somewhat inhibitory if added to an otherwise favorable medium. Relation to oxygen. Aerobic. Temperature relations. Limits of growth from 20 to 340C. No growth at 370C. Optimum temperature from 28 to 32 C. Organisms are killed by heating for ten minutes at 480C. Effect of reaction of medium. ph limits of growth from 6.0 to 7.8. Optimum ph from 6.6 to 7.2. Effect of sodium chloride on growth. The addition of 0.7 per cent sodium chloride to any favorable medium completely prevents growth of the culture. Effect of peptone and beef extract. The addition of 0.3 per cent beef extract or 0.5 per cent peptone completely inhibits growth.
428 GRACE M. SICKLES AND MYRTLE SHAW Peptone in concentrations as low as 0.1 per cent has a deleterious effect on the development of the organism. SPECIFICITY OF ACTION ON PNEUMOCOCCUS CARBOHYDRATES With the exception of the strain which utilizes agar, the action of these organisms is quite specific when the pneumococcus carbohydrates are used as a sole source of carbon in a mineral medium. For example, the cultures which split the type II carbohydrate are unable to utilize the carbohydrates of pneumococci types I and III or the atypical I strain. It has been found, however, that, when additional sources of energy are furnished, the specificity is much less marked. Certain of the strains which split the type III carbohydrate may then utilize atypical I carbohydrate; most of those which split the type II carbohydrate will decompose the type III carbohydrate, but no strain except the pointed rods will attack the type I carbohydrates. It may thus be possible to broaden the activity of the other strains by cultivation in a suitable medium, and this point is now under investigation. DISSOCIATION An outstanding character of the strains which decompose the type III pneumococcus carbohydrate is their tendency to dissociate into sporulating and nonsporulating strains which produce characteristic colonies on beef-extract peptone agar or blood agar. This dissociation is spontaneous and is not permanent although subcultures will remain true to type for a short time before they in turn produce the two types of colonies. This example of dissociation in a spore-forming rod seemed of especial interest because such distinctive characters as the decomposition of pneumococcus carbohydrate and the digestion of agar make it easy to determine that both types of colonies are variants of the same strain. Gee (1927) has4described a similar type of dissociation in an organism resembling B. vulgatws, isolated from spoiled haddock. CLASSIFICATION The bacteria which decompose the carbohydrate of pneumococcus type III belong in the genus Bacillus but do not agree with
MICROORGANISMS WHICH DECOMPOSE CARBOHYDRATES 429 the description of any of the species listed in that genus in Bergey's Manual of Determinative Bacteriology (1934). Although only a limited number of the more common forms of the genus Bacillus have been tested by us as to their ability to decompose pneumococcus carbohydrate, the evidence indicates that this particular character is unusual. We propose the name Bacillus palustris for these organisms. A study of the descriptions of the species that digest agar, as given by Gran (1902), Biernacki (1911), Gray and Chalmers (1924), Aoi and Orikura (1928), Lundestad (1928), Angst (1929), Waksman and Bavendamm (1931), Goresline (1933), and Nichols (1933), indicates that the bacillus that acts on pneumococcus carbohydrate and agar differs from all the above in that it forms spores. Panek (1905) has described an organism which he named Bacterium betaevtscosum. Since the original of this paper was not seen, we do not know whether a definite statement was made concerning sporulation, but Panek's strain differs in another character. It is a facultative anaerobe. The digestion of agar is relatively rare among the bacteria, but it is a question whether this character is of sufficient value to warrant placing in a different species the one strain which splits pneumococcus carbohydrate and also utilizes agar. It seems logical to consider this strain a variety rather than a species and to name it, therefore, B. palustris var. gelaticus. Although we have not been able to identify it with any species described in Bergey's Manual (1934), the very small rod which specifically utilizes the atypical I carbohydrate may be classified in the genus Flavobacterium. We suggest the name Flavobacterium ferrugineum for this organism. The relationship of the microorganisms that decompose the carbohydrate of pneumococcus types I and II to those organisms that digest cellulose and which are classed in the genera Cytophaga, Cellfalcicula, and Cellvibrio in the family Mycobacteriaceae is suggested by such characters as their form and pleomorphism, their failure to grow on standard media, and the inhibiting effects on growth of glucose, sodium chloride, and peptone. The toxic action of reducing sugars and of peptone on Cytophaga were noted by Hutchinson and Clayton (1919) and on a similar cellulose-di-
430 GRACE M. SICKTEB AND MYRTLE SHAW gesting organism by Dubos (1928). Moreover, the picture presented by the cultures which act on the type I carbohydrate as they change from the pointed rods of young cultures to the slightly rounded form, similar in appearance to the young cells of the strains which act on the type II carbohydrate, and finally to the coccoid form of old cultures, resembles the series of changes recorded by Winogradsky (1929) and Krzemieniewska (1930, 1933) in the microorganisms which decompose cellulose. However, since no growth has been obtained with our cultures in liquid or on solid media with cellulose as a source of carbon, they obviously cannot be classed in the above genera. Because of their apparent relationship to other genera in the family Mycobacteriaceae, we have classified them in this group. A new genus, Saccharobacterium, established on the basis of the utilization of the bacterial polysaccharides is proposed in which both strains would be placed because of their similarity in metabolic activities. The name Saccharobacterium ovale is suggested for the strains which split the carbohydrate of pneumococcus type II and Saccharobacterium acuminatum for the pointed rods which decompose the specific polysaccharide of pneumococcus type I. SUMMARY Four types of microbrganisms which decompose the specific and non-type-specific carbohydrates of the pneumococcus were isolated from soils and studied in detail. Methods for the isolation and purification of the cultures are given. The organisms are considered to be new species. The name Bacillus palustris is suggested for those which decompose the specific carbohydrate of pneumococcus type III. The strain which also attacks the gel properties of agar is considered to be a variety, gelaticus, of the above species. The name Flavobacterium ferruginreum is given to that microorganism which utilizes the non-type-specific carbohydrate obtained from a degraded type I pneumococcus. A new genus, Saccharobacterium, in the family Mycobacteri-
MICROORGANISMS WHICH DECOMPOSE CARBOHYDRATES 431 aceae has been proposed for the microbrganisms which decompose the specific carbohydrates of pneumococcus types I and II. The name S. ovale is suggested for the strains which act on the carbohydrate of pneumococcus type II and S. acuminatum for those which decompose the polysaccharide of pneumococcus type I. REFERENCES ANGST, E. G. 1929 Washington (State) Univ. Puget Sound, Biol. Sta. Pub., 7, 49. AoI, K., AND ORIKURA, J. 1928 Centralbl. f. Bakt., II Abt., 74, 321. BERGEY, D. H., AND OTHERS 1934 Bergey's Manual of Determinative Bacteriology. Fourth Edition. The Williams & Wilkins Company, Baltimore. BIERNACKI, W. 1911 Centralbl. f. Bakt., II Abt., 29, 166. Dumos, R. J. 1928 Jour. Bact., 15, 223. DuBos, RENA, AND AVERY, 0. T. 1931 Jour. Exper. Med., 54, 51. GEE, A. H. 1927 Jour. Infect. Dis., 41, 355. GORESLINE, H. E. 1933 Jour. Bact., 26, 435. GRAN, H. H. 1902 Bergens Museums Aarbog., No. 2, 1. (Cited in BERGBY, D. H., AND OTHERS 1934 Bergey's Manual of Determinative Bacteriology. Fourth Edition, 187. The Williams & Wilkins Company, Baltimore.) GRAY, P. H. H., AND CHALMERS, C. H. 1924 Ann. Applied Biol., 11, 324. HuTcHINsON, H. B., AND CLAYTON, JAMES 1919 Jour. Agr. Sc., 9, 143. KRZEMIENIEWSKA, H. 1930 Acta Societatis Botanicorun Polonice, 7, 507. KRZEMIENIEWSKA, H. 1933 Arch. f. Mikrobiol., 3, 394. LUNDESTAD, JON 1928 Centralbl. f. Bakt., II Abt., 75, 321. NICHOLS, A. A. 1933 Zentralbl. f. Bakt., II Abt., 88, 177. PANEK, K. 1905 Bull. Ac. SC. de Cracovie, January. (Abstracted in 1905 Bull. de l'inst. Pasteur, 3, 592.) SICKLES, G. M., AND SHAW, MYRTLE 1933 Jour. Infect. Dis., 53, 38. SICKLES, G. M., AND SHAW, MYRTLES 1934 Proc. Soc. Exper. Biol. & Med., 31, 443. Society of American Bacteriologists. Manual of methods for pure culture study of bacteria. 1932, Leaflet 4 and 6. 1933, Leaflet 2. Soc. Amer. Bacts., Geneva, N. Y. WAKSMAN, S. A., AND BAVENDAMM, W. 1931 Jour. Bact., 22, 91. WINOGRADSKY, S. 1929 Ann. de I'Inst. Pasteur, 43, 549.