Nov 2, 1972 - h before acetoin can be detected (1). In Enterobacter aerogenes, acetoin and 2,3- butanediol are formedby three enzymes (Fig. 1): the pH 6 ...
APPLID MICROBIOLOGY, Mar. 1973, p. 511-512 Copyright 0 1973 American Society for Microbiology
Vol. 25, No. 3 Printed in U.S.A.
Effect of Acetate upon the Formation of Acetoin in Klebsiella and Enterobacter and its Possible Practical Application in a Rapid Voges-Proskauer Test KLAUS BRYN1, JAN C. ULSTRUP, AND FREDRIK C. STbRMER Department of Microbiology, Ullevdl Hospital, and Department of Biochemistry, University of Oslo,
Blindern, Norway Received for publication 2 November 1972
Acetate stimulates the formation of acetoin during 1-h incubation of Voges-Proskauer-positive strains of Klebsiella and Enterobacter. Of these organisms, 124 of 126 strains were recognized as positive in the presence of acetate, and 106 were recognized as positive in its absence.
The Voges-Proskauer (VP) test has found wide acceptance in clinical laboratories as a means of classifying strains of Enterobacteriaceae, based on acetoin production. Much effort has been exerted to reduce the time necessary for a positive test, and this has been extensively reviewed (5, 7, 8, 12). Acetoin has been detected directly in moisture from incubated slants of triple sugar iron (TSI) medium (2), provided the slants are acid. Strains producing alkaline slants when grown on TSI medium have to be incubated for 4 to 6 h before acetoin can be detected (1). In Enterobacter aerogenes, acetoin and 2,3butanediol are formed by three enzymes (Fig. 1): the pH 6 acetolactate-forming enzyme (E1), acetolactate decarboxylase (E,) (EC 4.1.1.5), and diacetyl(acetoin) reductase (Es) (3). Acetate has multiple effects on this pathway. It induces the three enzymes (3, 10), activates El (11), and regulates the balance between acetoin and 2, 3-butanediol (S. H. Larsen and F. C. Stormer, Eur. J. Biochem., in press). The aim of this report was to investigate whether the effects of acetate could be taken advantage of to reduce the time necessary for detection of VP-positive organisms. Bacteria were obtained from clinical material, and from water samples (kindly supplied by V. Gaustad, Oslo Board of Health). They were identified after growth overnight at 37 C on slants of double sugar iron (DSI) medium I Present address: Methodology Department, National Institute of Public Health, Oslo, Norway.
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which is identical to TSI medium with sucrose omitted, mannitol motility tubes (0.2% mannitol, 0.25% Oxoid agar no. 3), and fluid indole urea medium (0.3% L-tryptophan and 2% urea in saline phosphate buffer with phenol red). The organisms were classified as VP positive or negative by incubation for 48 h at 37 C in 6 ml of 1% glucose broth (standard VP test). The ability of bacterial strains to produce acetoin was investigated by transferring one loopful of cells, grown overnight on DSI medium, to tubes containing 0.2 ml of sterile Koser's citrate medium (9, page 56). The latter was modified by adjustment of pH, and addition of various levels of glucose and sodium acetate, where indicated. The tubes were incubated for 60 min in a water bath at 37 C and tested for acetoin (1). Preliminary experiments were performed with E. aerogenes strain 1033. Bacteria were transferred from DSI medium to tubes of Koser citrate medium that contained various quantities of glucose and sodium acetate. These were then adjusted to different pH values. Acetoin production was optimal at a glucose concentration of 1%. Equivalent amounts of acetoin were formed at pH 6.0 in the presence of 10 to 25 mM acetate, and at pH 7.0 in the presence of 0.1 to 0.2 M acetate. At pH 6.0, 0.1 M acetate was completely inhibitory. Further experiments were performed with 1% glucose and 0.1 M sodium acetate in Koser medium (pH 6.8). Isolated strains were classified as VP positive or negative according to the standard VP test.
NOTES
512 Pyruvate
E'
acetolactate E acetoin
E
2
3-butanediol
NADH NAD FIG. 1. Formation of acetoin and 2,3-butanediol in Enterobacter aerogenes. E1 pH 6 acetolactateforming enzyme, E2 acetolactate decarboxylase, and Es diacetyl(acetoin) reductase. (The reduction of diacetyl to acetoin is omitted.) =
=
=
Inocula from a slant of DSI medium were incubated for 1 h in the presence and absence of acetate and tested for acetoin. As expected, none of the 94 VP-negative strains produced detectable amounts of acetoin regardless of whether acetate was present. The effect of acetate on 131 VP-positive strains was investigated. In Klebsiella, 97/98 produced acetoin in the presence of acetate as compared to 90/98 when acetate was omitted from the medium. In Enterobacter, the effect was even more pronounced: 27/28 produced acetoin in the presence of acetate, and only 16/28 produced acetoin in the absence of acetate. The VP reaction was much stronger when acetate was present in the incubation medium. The two strains which were not recognized as acetoin producers in our test (1 Klebsiella and 1 Enterobacter) were investigated further. Their ability to produce acetoin during 1 h of incubation was established when 0.1 M acetate was incorporated in the DSI slant. In this way, induction by acetate of E,, E2, and E3 probably was enhanced during growth on DSI slants. Unfortunately, acetate seriously inhibited growth and acid production on the slant (used as a criterion in identification of bacteria), and it could not be used routinely. Various compounds have been incorporated in incubation media to speed up the VP test (7). Weak buffers were recommended (4), and a positive effect of pyruvate has been reported (M. J. Pickett and M. L. Scott, Bacteriol. Proc., p. 110, 1955) which can be explained by a conversion of pyruvate to acetate during the incubation period. Pyruvate caused some induction, compared to the effect of acetate, of the acetoin-producing enzymes in E. aerogenes (10). Agreement between the classical VP test and
APPL. MICROBIOL.
our 1 h "acetate test" was not found for Proteus. Five strains, VP positive by the standard test, did not produce detectable amounts of acetoin, regardless of whether acetate was present. Incorporation of acetate in the slants did not promote the acetoin-producing capacity. It has been noted by others (12) that acetoin formation in Proteus is variable and strongly dependent on conditions of culture, making the VP test of little taxonomic value for the Proteus group (6). The lack of response to acetate in the five VP-positive strains of Proteus may indicate a different mechanism of acetoin formation. Organisms, possessing the mechanism of acetate-stimulated acetoin production which is found in E. aerogenes, can be recognized as VP positive more rapidly when acetate is incorporated in test media. We, therefore, want to focus the attention on the potential value of this compound in the differentiation of Enterobacteriaceae. LITERATURE CITED 1. Barry, A. L., and K. L. Feeney. 1967. Two quick methods for Voges-Proskauer test. Appl. Microbiol. 15:1138-1141. 2. Benjaminson, M. A., B. C. de Guzman, and A. J. Weil. 1964. Voges-Proskauer test: expeditious techniques for routine use. J. Bacteriol. 87:234-235. 3. Bryn, K., 0. Hetland, and F. C. Stormer. 1971. The reduction of diacetyl and acetoin in Aerobacter aerogenes. Evidence for one enzyme catalyzing both reactions. Eur. J. Biochem. 18:116-119. 4. Clarke, P. H., and S. T. Cowan. 1952. Biochemical methods for bacteriology. J. Gen. Microbiol. 6:187-197. 5. Eddy, B. P. 1961. The Voges-Proskauer reaction and its significance: a review. J. Appl. Bacteriol. 24:27-41. 6. Edwards, P. R., and W. H. Ewing. 1972. Identification of
7. 8. 9. 10. 11. 12.
Enterobacteriaceae. Burgess Publishing Company, Minneapolis, Minn. Hartman, P. A. 1968. Miniaturized microbial methods. Adv. Appl. Microbiol. Suppl. 1:14-16. Ljutov, V. 1963. Technique of Voges-Proskauer test. Acta Pathol. Microbiol. Scand. 58:325-335. Society of American Bacteriologists. 1957. Manual of microbiological methods. McGraw-Hill Book Company, Inc. New York. Stormer, F. C. 1968. Evidence for induction of the 2, 3-butanediol-forming enzymes in Aerobacter aerogenes. FEBS Lett. 2:36-38. Stormer, F. C. 1968. The pH 6 acetolactate-forming enzyme from Aerobacter aerogenes. I. Kinetic studies. J. Biol. Chem. 243:3735-3739. Suassuna, I., U. R. Suassuna, and W. H. Ewing. 1961. The methyl red and Voges-Proskauer reactions of Enterobacteriaceae. Public Health Lab. Bull. Conf. State Prov. Public Health Lab. Dir. 19:67-75.
