GROWTH AND MANOMETRIC STUDIES ON CARBOHYDRATE UTILIZATION

GROWTH AND MANOMETRIC STUDIES ON CARBOHYDRATE UTILIZATION BY SHIGELLA FLEXNERI' ARVID L. ERLANDSON, JR.,2 AND WILLIAM H. MACKEY Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland The conventional biochemical tests of sugar fermentations have proved especially important in the identification of Shigella and other enteric organisms. These biochemical reactions are, in general, consistent and duplicable (Edwards and Ewing, 1955; Madsen, 1949) and are the common laboratory procedure in identification of enteric unknowns. Despite their importance as diagnostic tools, data are lacking on the biological significance, if any, of these biochemical properties. It must be acknowledged that little is known today of the biological significance of most of the metabolic properties used in such classification. The present study was designed to determine the utilization of 14 carbohydrates by 18 strains of the 6 serotypes of Shigellaflexsneri, as indicated by (1) their ability to support growth when used as the sole carbohydrate source in a synthetic medium, -(Erlandson and Mackey, 1957), (2) their ability to stimulate the oxygen uptake of nonproliferating cells, and (3) their ability to ferment these carbohydrates. EXPERIMENTAL METHODS Culture. Three strains from each of the 6 serotypes of S. flexneri were selected at random from the Naval Medical Department Reference Collection of Enterobacteriaceae (Babcock et al., 1951). The 18 strains selected were biochemically and serologically characteristic of their respective serotypes and were maintained in a lyophilized state. Carbohydrates. Ten per cent stock solutions were prepared by dissolving the requisite amount of carbohydrate in the appropriate diluent; i.e., distilled water for fermentation analyses, phosphate buffer for manometric studies, or basal 1 The opinions or assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. 2 Present address: Research Division, Parke, Davis, and Company, Detroit, Michigan. Received for publication November 18, 1957 medium for growth studies. The following carbohydrates were utilized: D-glucose, D-mannose, D-fructose, D-galactose, sucrose, trehalose, raffinose, maltose, D-arabinose, D-ribose, L-sorbose, D-xylose, i-rhamnose, and lactose. The stock solutions were sterilized by Seitz filtration, transferred aseptically to sterile screw-capped test tubes and stored in the refrigerator. Fermentation studies. Conventional laboratory procedures were followed. Duplicate tubes of Andrade's broth containing.5 per cent carbohydrate were inoculated with each of the strains and incubated at 37 C for 3 days. The tubes were checked daily for acid and gas production. Growth studie. The basal medium (Erlandson and Mackey, 1957) was composed of phosphate buffer,.67 M; sodium chloride,.85 per cent; magnesium sulfate,.5 per cent; thiamin hydrochloride,.3 per cent; niacin,.1 per cent; and aspartic acid,.25 per cent. This was made up in redistilled, deionized water, adjusted to ph 7., and autoclaved at 15 lb pressure for 15 min. The carbohydrates, previously sterilized by Seitz filtration, were added aseptically to the basal medium to yield a final concentration of 1 per cent. Inocula preparation was the same as in previous experiments (Erlandson and Mackey, 1957). Growth was measured turbidimetrically using a Coleman Universal spectrophotometer, with a 6 m,u filter, using the basal medium as a control. Maximal growth was usually obtained in 48 hr, but all cultures were incubated 1 days before discarding. Manometric studies. Resting cell suspensions were prepared as previously described (Erlandson and Ruhl, 1956). Approximately.4 mg of cellular nitrogen was used per Warburg vessel and all reactions were run aerobically at ph 7. and 37 C. Carbohydrate substrates were dissolved in.67 M phosphate buffer and the ph adjusted to 7.. The rate of oxygen consurmption was 53 Downloaded from http://jb.asm.org/ on July 6, 218 by guest

19581 CARBOHYDRATE UTILIZATION BY S. FLEXNERI 531 expressed as QO,(N) with endogenous respiration subtracted. RESULTS The ability of 18 strains re- Fermentation studies. of S. Jlexneri to ferment 14 carbohydrates is corded in table 1. The data indicate that glucose, mannose, fructose, and galactose were fermented by all the strains tested within 24 hr. Trehalose also was fermented by all strains, but a delayed fermentation occurred in some cases. Each of the 3 strains of S. flexneri 1, 3, and 5 showed a delayed fermentation of sucrose, whereas no fermentation was evident with the strains of S. flexneri 2, 4, and 6 tested. Raffinose and maltose were fermented by several strains but followed no definite pattern as to strain or serotype. The remaining carbohydrates were not fermented by any of the 18 strains. Growth studies. Brain-heart infusion broth and agar (Difco) supported excellent growth of all Organism TABLE 1 Biochemical reactions of Shigella flexneri strains from the initial inoculum. Tubes containing synthetic medium with the appropriate carbohydrates, supplied singly, developed visible turbidity at various time intervals, depending both on the strain employed and on the individual carbohydrate (table 2). As a rule, maximal growth was obtained in 24 to 48 hr. In the second and succeeding subcultures, maximal growth was attained within 24 hr. This growth sequence appeared consistent for all 6 serotypes. As indicated in table 2, the combination of 18 strains and 14 carbohydrates yielded 87 instances of successful growth. Glucose, mannose, fructose, and galactose supported growth of all 18 strains. Trehalose supported the growth of 12 strains, sucrose supported growth of 5 strains, and the remaining carbohydrates were incapable of functioning as the carbon and energy source for growth of S. flexneri. Cultures were incubated Carbohydrate* Glucose Man- Fruc- Galac- Trehalose Sucrose Raffiinose Maltose Othert nose tose tose strain 848 A A A A A (A)ls strain 829 A A A A A (A)7 (A) R2 A R2 strain 846 A A A A A (A)7 A A R2 strain 97-5 A A A A A (A)56 strain 938-2 A A A A A (A)2R7 A strain 956-2 A A A A A _ (A)2 A strain 113 A A A A (A)2 (A)18 I strain 157-3 A A A A (A)2 (A)7 A A R3 strain 12-3 A A A A A (A)7 A A R2 strain 124 A A A A A - - _ strain 1234 A A A A A - A R2 strain 1255 A A A A A A R2 strain 1393 A A A A A (A)18 _ strain 1323 A A A A A (A)4 A A R3 strain 1391 A A A A A (A)3 R7 A A strain 1443 A A A A (A)4 _ strain 1427 A A A A (A)8 _ A strain 146 A A A A A _ A * A = acid; G = gas; ( ) = delayed reaction; and R = reverted. Subscript indicates day biochem- (no subscript indicates 24 hr). t Other sugars were arabinose, ribose, sorbose, xylose, rhamnose, and lactose. ical reaction was discernible Downloaded from http://jb.asm.org/ on July 6, 218 by guest

532 ERLANDSON AND MACKEY [VOL. 75 1 days with no change in growth results. Increasing the inoculum from 16 to 18 organisms failed to produce growth in these cases. The basal medium supplemented with glucose, mannose, fructose, or galactose supported growth serially through 1 subcultures, which indicated their sufficiency as carbohydrate sources. Serial transfers in the trehalose-supplemented media also were successful with the exception of S. flexneri 1 strain 829, which grew initially only after a delay of 6 days and S. flexneri 3 strain 113, with which very little initial growth was evident. Serial transfers in the sucrose-supplemented medium yielded inconsistent results. Postincubation characterization of the cells by means of cellular morphology and serology was made and in all cases the cells were found to conform to the serotype characteristics possessed by the strains at the beginning of the growth studies. Manometric studies. The effect of 14 carbohydrates on oxygen uptake of 18 strains of S. flexneri is recorded in table 3. As evidenced in the fermentation and growth studies, glucose, mannose, fructose, and galactose were oxidized by all 18 strains tested. From these data, it may be seen that there is considerable strain variation in regard to the influence of a carbohydrate substrate on oxygen uptake by resting cells. Oxygen uptake with both glucose and mannose appears to be relatively consistent between strains and for the most part, within serotypes. Such is not evident concerning the other carbohydrates, where both strain and serotype variations were noted. In all strains tested, glucose and mannose TABLE 2 Growth of Shigella flexneri in a synthetic medium with a single carbohydrate source Percentage Light Transmittance (24 to 48 hr) Organism Supplement to basal medium (1%) Glucose Mannose Fructose Galactose Trehalose Sucrose Other* strain 848 31 45 29 44 37 64 1 strain 829 25 28 25 48 32 (6) 1 1 strain 846 49 43 46 52 39 1 1 strain 97-5 5 6 67 59 1 1 1 strain 938-2 26 55 24 63 29 1 1 strain 956-2 52 55 44 65 1 1 1 strain 113 37 6 59 52 87 72 1 strain 157-3 25 39 29 3 1 1 1 strain 12-3 3 4 4 31 1 1 1 strain 124 39 41 39 45 38 1 1 strain 1234 28 36 21 5 1 1 1 strain 1255 29 48 38 41 46 1 1 strain 1393 34 48 41 56 42 5 1 strain 1323 4 7 33 45 39 75 1 strain 1391 38 43 61 37 43 41 1 strain 1443 47 5 52 52 1 1 1 strain 1427 32 42 28 34 1 1 1 strain 146 49 39 49 46 1 1 1 * Other sugars were raffinose, maltose, rhamnose, sorbose, lactose, arabinose, ribose, and xylose. Endogenous (basal without carbohydrate source) subtracted. Maximal drop of 2 units with endogenous. Inoculum 1 X 16 cells. Downloaded from http://jb.asm.org/ on July 6, 218 by guest

1958] CARBOHYDRATE UTILIZATION BY S. FLEXNERI 533 provided superior activity, followed by fructose and galactose. Among these substrates oxidized, trehalose activity was intermediate betweeni mannose and fructose or between fructose and galactose. Sucrose activity, in those strains utilizing sucrose, was least of the carbohydrates utilized. DISCUSSION In the growth studies reported here with the strains employed, glucose, mannose, fructose, and galactose were capable of functioning as the sole carbohydrate source for all strains tested. These 4 carbohydrates were also fermented within 24 hr and oxidatively dissimilated at a rapid rate by nonproliferating cells. While trehalose was fermented by all strains tested, Organism strain 848 strain 829 strain 846 strain 97-5 strain 938-2 strain 956-2 strain 113 strain 157-3 strain 12-3 strain 124 strain 1234 strain 1255 strain 1393 strain 1323 strain 1391 strain 1443 strain 1427 strain 146 some reactions being delayed, not all the strains tested were capable of utilizing this carbohydrate as the carbon and energy source. However, a relationship did exist between those strains which were able to utilize this compound for growth and those capable of oxidative dissimilation. This observation is essentially true of sucrose also. Although 2 strains were able to dissimilate sucrose and yet not utilize it nutritionally, it should be noted that the extent of oxidative dissimilation was comparatively small. This is also true of the oxidation of maltose by 3 strains. A number of strains were capable of fermenting maltose. Several of these were weak, however, and reverted to alkaline. Eight strains were capable of fermenting raffinose but none could TABLE 3 Oxidative dissimilation of carbohydrates by Shigella flexneri Qo2(N) Substrate Glucose Mannose Fructose IGalactose Trehalose Sucrose Maltose 158 873 889 846 1187 813 1175 664 126 866 834 746 985 1321 734 823 15 976 899 859 93 787 1 791 116 659 1146 866 825 737 952 1232 682 823 9 843 * Other sugars were raffinose, rhamnose, sorbose, lactose, ribose, xylose, and arabinose. Each Warburg flask contained: 1 ml (approximately.4 mg N) of cell suspension; 1.5 ml.67 M phosphate buffer ph 6.8;.5 ml substrate (1 Mmoles);.1 ml 1 per cent KOH (center well). Control endogenous rates subtracted (average 25 IAL per hr). All rates were linear for at least 9 min. 433 639 712 545 723 583 86 391 133 333 634 328 849 14 317 199 95 75 397 338 449 515 332 265 585 273 478 269 162 191 195 326 129 173 223 12 423 127 67 1 48 889 127 822 522 684 259 72 212 86 13 228 124 77 8 47 Other* Downloaded from http://jb.asm.org/ on July 6, 218 by guest

534 ERLANDSON AND MACKEY [VOL. 75 utilize raffinose as the carbohydrate source and none was capable of dissimilating raffinose. None of the other sugars tested was either fermented, oxidatively dissimilated, or capable of functioning as the carbon and energy source for growth of the test organism. Our studies indicate, therefore, that there is a definite relationship between the ability of nonproliferating cells of S. flexneri to oxidatively dissimilate certain carbohydrates and the ability of the organism to utilize the same carbohydrate as the sole carbohydrate source for growth in a chemically defined medium. Further, these strains of Shigella can be carried in a basal medium with a variety of carbohydrates, singly supplied, and after numerous transfers still retain the characteristics present prior to growth in this simple medium. The ability of Shigella to ferment glucose, mannose, fructose, and galactose is compatible with the previous observations on oxidation and utilization for growth. This relationship is not evident with trehalose, sucrose, raffinose, and maltose, however. ACKNOWLEDGMENT The authors wish to express their gratitude to E. L. Lampley, HMC, USN, and R. J. Madden, HM3, USN, for technical assistance. SuA RY Eighteen strains of Shigella flexneri (3 each of the 6 serotypes) were studied for their ability to grow in a simple basal medium containing 1 of 14 carbohydrates as the sole carbon and energy source. Glucose, mannose, fructose, and galactose supported growth of all strains tested. Trehalose and sucrose supported the growth of only certain strains and none of the other carbohydrates were capable of supporting growth. Manometric studies with the same strains and carbohydrates indicate that a relationship exists between the ability of nonproliferating cells to oxidatively dissimilate the individual carbohydrates and the ability of the carbohydrates to support growth in a synthetic medium. Attempts to determine a relationship between the diagnostic criterion of carbohydrate fermentation with the other two properties proved only partly successful. REFERENCES BABCOCK, M. C., GILMORE, J. D., AND BARNES, L. A. 1951 Naval medical department reference collection of Enterobacteriaceae. Reports of the Naval Medical Research Institute. EDWARDS, P. R. AND EWING, W. H. 1955 Identification of Enterobacteriaceae Burgess Publishing Co., Minneapolis. ERLANDSON, A. L., JR., AND RUHL, R. F. 1956 Oxidative dissimilation of amino acids and related compounds by. J. Bacteriol., 72, 78-712. ERLANDSON, A. L., JR., AND MACKEY, W. H. 1957 Nutrition of Shigella: Growth of Shigella fiexneri in a simple, chemically defined medium. J. Bacteriol., 75, 253-257. MADSEN, S. 1949 On the classification of the Flexner types. Einar Munksgaard, Copenhagen. Downloaded from http://jb.asm.org/ on July 6, 218 by guest