Rapid Diagnostic Test That Uses Isocitrate Lyase ... - Europe PMC

Vol. 13, No. 4

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1981, p. 661-665 0095-1137/81/040661-05$02.00/0

Rapid Diagnostic Test That Uses Isocitrate Lyase Activity for Identification of Yersinia pestis SHARON L. HILLIER AND W. T. CHARNETZKY* Department of Bacteriology and Public Health, Washington State University, Pullman, Washington 99164 Received 1 October 1980/Accepted 19 January 1981

The presence of high levels of isocitrate lyase activity in Yersinia pestis grown on blood agar base medium, as compared with low levels of this enzyme in Yersinia pseudotuberculosis and Yersinia enterocolitica, suggested that the differences in the levels of this enzyme could be used for the presumptive identification of Y. pestis. A modified, semiquantitative assay for isocitrate lyase activity is described which requires no expensive instrumentation, utilizes readily available chemicals and substrates, and requires only 20 min for completion. This test yielded positive results with all 108 isolates of Y. pestis tested and negative results with all strains of Y. pseudotuberculosis (68 isolates) and Y. enterocolitica (202 isolates) tested. Less than 2% of the approximately 1,300 non- Yersinia isolates from the family Enterobacteriaceae and none of the 93 isolates from the family Pseudomonadaceae yielded positive results. We conclude that this test provides for rapid identification of Y. pestis and should be useful in the initial screening of isolates from rodent and flea populations and in the presumptive identification of this organism from suspected cases of human plague. Isocitrate lyase (EC 4.1.3.1), one of two enzymes which comprise the glyoxylate bypass of the tricarboxylic acid cycle, catalyzes the aldol cleavage of isocitric acid to yield one molecule each of glyoxylic and succinic acids. This enzyme, as part of an anaplerotic sequence, is induced in most microorganisms only during growth on two-carbon compounds such as acetate or ethanol and is repressed during growth on carbohydrates (6). Yersinia pestis is unlike Yersinia pseudotuberculosis, Yersinia enterocolitica, and most other members of the family Enterobacteriaceae in that it possesses two forms of isocitrate lyase (4). One form, like that of the other members of the family Enterobacteriaceae, is produced only during growth on two-carbon compounds. The second form of isocitrate lyase is produced during growth on a wide variety of media, including complex media such as blood agar base. We believe that the presence of the latter form of isocitrate lyase during growth on complex medium could provide a useful criterion for the presumptive identification of Y. pestis, since under these conditions most bacteria do not maintain high levels of isocitrate lyase. Assays for isocitrate lyase activity are available but are normally applied to cell extracts made from large quantities of cells (8). These tests provide accurate quantification of isocitrate lyase activity but are more sensitive and more

quantitative than required to distinguish between the high levels present in Y. pestis and the comparatively low levels present in most other bacteria grown on complex medium. Since these tests are too time consuming for routine diagnostic application and require equipment unavailable in most diagnostic laboratories, we have designed a test based on a colorimetric assay for glyoxylic acid, a product of the enzymatic cleavage of isocitric acid. This test, which requires only common laboratory equipment and readily available chemicals which can be frozen until needed, takes only 20 min. We show here that this test reliably distinguished Y. pestis from Y. pseudotuberculosis and Y. enterocolitica. In addition, false-positive results were obtained with less than 2% of the non-Yersinia clinical isolates of the family Enterobacteriaceae. MATERIALS AND METHODS Bacterial cultures. The sources of the strains used are provided in the appropriate tables. Ail bacterial strains examined by the rapid test were grown on slants of blood agar base medium (BBL Microbiology Systems) at either 26 or 37°C. Cells were routinely transferred twice. The second transfer was tested after 15 to 18 h of incubation. Y. pestis, Y. enterocolitica, Y. pseudotuberculosis, and Escherichia coli isolates to be assayed for isocitrate lyase by a conventional assay were.cultured in the liquid, chemically-defined complex medium of Higuchi et al. (3) as modified by 661

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Brubaker (1). This medium, which contains 1% xylose, high levels of several amino acids, vitamins, and salts, did not induce high levels of isocitrate lyase in the other bacterial strains tested. Liquid cultures were grown in Erlenmeyer flasks with a nominal volume 10x that of the culture and aerated at 150 rpm. Preparation of crude extracts. Cells in the exponential stage of growth were collected by centrifugation at 12,000 x g for 15 min, washed twice, and suspended in 0.1 M morpholinepropanesulfonic acid (MOPS) buffer (pH 7.7) containing 5 mM MgCl2 and 1 mM ethylenediaminetetraacetate. After disruption in a French pressure cell at 20,000 lb/in2, unbroken cells and cellular debris were removed by centrifugation at 20,000 x g for 15 min at 4°C. The supernatant was used as the crude extract. Assays. Protein concentrations were determined by the Bio-Rad assay which is based on the binding of Coomassie brilliant blue as described by Bradford (2). Isocitrate lyase activity was determined by the method of McFadden and Howes (8). Crude extracts, diluted in MOPS buffer to appropriate protein concentrations, were incubated with DL-isocitric acid at a final concentration of 2.2 mM for 30 min at 30°C. The concentration of the glyoxylate phenythydrazone formed during the colorimetric reaction was determined by measuring the absorbance at 520 nm. Linearity of this assay was established between 5 and 60 min. One unit of isocitrate lyase activity is defined as the amount of enzyme which catalyzed the formation of 1 nmol of glyoxylate per minute. Specific activity is expressed as nanomoles of glyoxylate produced per minute per milligram of protein.

RESULTS Aceceptable medium for growth of ceils tested. The basis of this test is the difference in the levels of isocitrate lyase activity in cells grown in medium containing acetate and ethanol at levels below those required for induction of isocitrate lyase. Most common laboratory media satisfy this criterion. Blood agar base was chosen because it is generally available and readily prepared. Microorganisms of interest usually can be grown on this medium, although blood may be added to the medium to facilitate the growth of more fastidious organisms. The addition of blood to this medium does not interfere with the test described in this report. Levels of isocitrate lyase activities differentiated by this test. To determine the levels of activity to be compared, we determined the

specific activities of isocitrate lyase in cell-free extracts of Y. pestis, Y. enterocolitica, Y. pseudotuberculosis, and E. coli (Table 1). Consistent with earlier results (4), the lowest specific activity of isocitrate lyase detected in a Y. pestis extract was 35, whereas the highest specific activity in an extract from any of the other organisms tested was less than 2. We felt that determination of specific activities in cell-free extracts

TABLE 1. Isocitrate lyase activities of selected Yersinia species and E. coli grown on a liquid complex medium

Organism

No. tested

Avg sp

Range

actb5

Y. pestis 7 51 35-68 Y. pseudotuberculosis 5 1.7 NDC-1.9 Y. enterocolitica 4 1.3 ND-1.4 E. coli 5 0.8 ND-0.8 a Five independent determinations were made for each strain as described in the text. b Specific activities are expressed as nanomoles of glyoxylate formed per milligram of protein per minute. C ND, No detectable activity.

should not be required to differentiate between the high levels found in Y. pestis and the comparatively low levels found in the other bacteria tested. Since most bacteriologists should be able to readily adjust bacterial suspensions to within a twofold difference in optical density, we felt that a test on whole cells, visually adjusted to roughly equal optical densities would be preferable to an assay based on cell-free extracts. In general, bacterial suspensions having an absorbance of 0.4 to 0.6 at 620 nm were used in the following experiments. Initial tests on whole cells were unsuccessful since the levels of glyoxylate obtained when Y. pestis cells were used were too low to be reliably detected by the colorimetric assay. The addition of Triton X-100 to increase cell permeability, however, resulted in high levels of glyoxylate from Y. pestis cells when sufficient isocitrate was provided as substrate. For optimal detection of glyoxylate in the colorimetric assay, MOPS buffer was chosen. Considerably less glyoxylate phenythydrazone was detected when Tris or phosphate buffer was used. The formation of glyoxylate phenylhydrazone was the same as that described by McFadden and Howes (8). Attempts to eliminate the boiling step, or to substitute 2,4-dinitrophenylhydrazine for phenylhydrazine hydrochloride, resulted in unacceptable reductions in the subsequent color formation. The glyoxylate phenylhydrazone formed is temperature sensitive; therefore, rapid chilling after the boiling step was used to assure more consistent results. Less than 1.5 nmol of DL-isocitrate per assay mixture yielded less than optimal levels o- f glyoxylate, although higher concentrations of DLisocitrate did not significantly increase the subsequent glyoxylate levels. The optimum incubation period was determined by varying the time of incubation from 5 to 30 min. By 5 min, low but detectable levels of glyoxylate were produced by Y. pestis, and by 30 min some strains of Y. pseudotuberculosis also yielded detectable

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levels of glyoxylate. When cells of the strains used in Table 1 were incubated with isocitrate for 15 min and assayed for glyoxylate, these strains were clearly separated into two classes. All Y. pestis strains yielded high levels of glyoxylate, whereas ail of the other strains yielded levels at or below the lower limits of detection of the colorimetric assay, which is consistent with previously determined differences in the specific activity of isocitrate lyase. Based on these observations, we chose the following procedure for subsequent tests. Freshly grown cells were suspended in 200 p1 of MOPS buffer (pH 7.8) to an approximate absorbance of 0.4 to 0.6 at 620 nm. Two drops of 1% Triton X-100 and 20 1l of 80 mM DL-isocitric acid were added. The suspension was agitated and incubated at ambient temperature. After 15 min, 20 pd of 5% phenylhydrazine hydrochloride was added, and the suspension was heated to boiling and immediately cooled in an ice bath. 10 N HC1 (100,l) and 25% K3Fe(CN)6 (10 Pl) were added, and the suspension was agitated. Cultures were scored as positive if an intense red color, indicative of the glyoxylate phenylhydrazone, was evident within 2 min. Isocitrate lyase activity in Yersinia species. Each of the 108 isolates of Y. pestis subsequently tested by the rapid assay were positive for isocitrate lyase activity. Of these, 76 were human wild-type isolates, 14 were isolated from flea pools, and 18 were avirulent mutants. A1though the virulent wild-type strains produced more glyoxylate, all plague strains tested were clearly positive. Negative results were obtained with all 68 strains of Y. pseudotuberculosis tested, and with 202 isolates of Y. enterocolitica, 52 of which were human isolates (Table 2). This single enzymatic activity, then, readily distinguished Y. pestis from the two other most closely related Yersinia species. Isocitrate lyase activities of the other Enterobacteriaceae Of the 682 human isolates of E. coli tested, 12 were positive for isocitrate

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lyase activity. Similarly, few isocitrate lyase-positive isolates were found in the other genera of the family Enterobacteriaceae tested. Overall, less than 2% of approximately 1,300 non- Yersinia isolates of the family Enterobacteriaceae tested were positive for isocitrate lyase by the rapid test (Table 3). Other gram-negative organisms tested. Human isolates from 5 Pseudomonas species were tested; only 1 of 93 was positive for isocitrate lyase by this test. From the family Neisseriaceae, 57 isolates, including isolates of Neisseria, Moraxella, and Acinetobacter species, were tested. Of the 48 human isolates of Acinetobacter calcoaceticus tested over 50% were positive, with the positive isolates being distributed equally between A. calcoaceticus biotypes anitratus and lwoffii. Isolates from the other Neisseriaceae species tested were negative. DISCUSSION Y. pestis, unlike other members of the family Enterobacteriaceae, possesses high levels of isocitrate lyase activity when grown on blood agar base medium. The difference in levels of activity observed are great enough to allow ready distinction without precise measurement of cell numbers or quantitative determination of enzymatic activity. Accordingly, a simple and rapid test was designed which separates gram-negative bacteria solely on their level of isocitrate lyase activity. All Y. pestis isolates were clearly positive in this test, whereas all isolates of the two other major human pathogens in this genus, Y. pseudotuberculosis and Y. enterocolitica, were negative. This test, then, provides a better separation of Y. pestis and Y. pseudotuberculosis than do the conventional biochemical tests which rely heavily on urease and motility reactions. Some freshly isolated strains of Y. pestis, like Y. pseudotuberculosis, are urease positive (9). The separation of Y. pestis and Y. pseudotuberculosis by the motility reaction is possible only after 48 h of incubation, since approxi-

TABLE 2. Yersinia species tested for isocitrate lyase activity by the rapid test after growth on blood agar base medium Total

No.

% Posi-

tested

positive

tive

Y. pests Humana virulent lab strainsb 108 100 108 Y. pseudotuberculosis 68 Human,a, avirulent lab strainsb 0 0 Y. enterocolitica 202 Human, mammal, food, water,c lab strain8b 0 0 a Human wild-type strains were provided by T. J. Quan, Plague Branch, Centers for Disease Control, Vectorborne Diseases Division, Fort Collins, Co. b Strains were provided by R. R. Brubaker, Department of Microbiology, Michigan State University, East Lansing, Mich. c Strains were provided by T. F. Wetzler, Department of Environmental Health, University of Washington, Seattle, Wash.

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TABLE 3. Members of the family Enterobacteriaceae tested for isocitrate lyase activity by the rapid test after growth on blood agar base medium Organism

Source

Total tested

No. positive

% Positive

Escherichia coli 682 Humana 12 1.8 Citrobacter diversus Human 2 0 0 Citrobacter freundii Human 92 1 1.1 Salmonella arizonae 32 Human, sewage 0 0 Salmonella enteritidis 6 Human, sewage 0 0 Salmonella paratyphi Human 9 0 0 Salmonella typhimurium 5 Human, sewage 0 0 Shigella dysenteriae Human 2 0 0 Klebsiella ozaenae Human 2 0 0 Klebsiella oxytoca Human 61 1 1.6 Klebsiellapneumoniae Human 127 0 0 Enterobacter aerogenes Human 21 0 0 Enterobacter cloacae Human 75 2 2.7 Enterobacter agglomerans 'Human 16 0 0 Proteus mirabilis Human 114 3 2.6 Proteus rettgeri Human 2 0 0 Proteus vulgaris Human 3 0 0 Serratia liquefaciens Human 6 0 0 Serratia marcescens 37 Humanc 1 2.7 Serratia rubidaea Human 3 0 0 Morganella morganii Human 25 0 0 Providencia alcalfaciens Humand 8 0 0 Providencia stuartii Humand 10 0 0 a Ail human isolates were from Northwest Regional Laboratory, Lewiston, Idaho. b Ail sewage isolates were provided by R. J. Seidler, Oregon State University, Corvallis, Oreg. c Isolate was provided by A. W. Pasculle, Presbyterian-University Hospital, Pittsburgh, Pa. d Isolates were provided by H. Canawati, Department of Pathology, University of Southern California Liver Unit, Ranchos Las Amigos Hospital, Downey, Calif.

mately 50% of the Y. pseudotuberculosis, like Y. pestis, appear to be nonmotile after 24 h (7). Other biochemical characteristics such as acid production on carbohydrates and H2S production apparently vary widely between strains. In fact, it was difficult for us to find two references listing the same biochemical reactions for these organisms. In addition to its value in the distinction of Y. pestis from other Yersinia species, this characteristic also appears to offer a method for the rapid presumptive distinction between Y. pestis and other members of the family Enterobacteriaceae which, as a group, yielded only 2% positive reactions. However, isolates representative of all genera and species of the family Enterobacteriaceae were not tested. Expansion of this preliminary screening to include more aerobic, gram-negative rods may also prove valuable since positive reactions in those genera tested to date are also rare. High constitutive levels of isocitrate lyase have been reported in Pseudomonas indigofera (5). It is reasonable to assume that this will not be a unique finding, but rather that loosely controlled isocitrate lyases will be found in individual strains from other genera. Expansion of the base of organisms tested may yield other species in which significant numbers

of strains maintain high isocitrate lyase activity. In our initial screening of human isolates only one such organism was found, A. calcoaceticus. Although the incidence of human plague in the United States is quite low, each case is a lifethreatening situation in which prompt diagnosis and treatment are essential. The time required for this test compares well with that required for a Gram stain, determination of catalase and oxidase activities, and fluorescent-antibody tests. Addition of this test to initial screening procedures would facilitate presumptive identification of Y. pestis. We believe that this test should be of particular value in underdeveloped countries where diagnostic laboratory facilities are limited, in independent laboratories where bacteriophage typing and fluorescent-antibody testing for Y. pestis are impractical, and in laboratories involved in screening of wild rodent populations. It has been demonstrated that isocitrate lyase activity is absent from human tissues. It is an intriguing possibility that examination of infected tissues, bubo aspirates, or blood samples might provide an even more rapid diagnosis of cases of human plague based simply on biological stains, serological tests, and isocitrate lyase determinations. However, it has not been estab-

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lished that Y. pestis would reliably maintain high isocitrate levels in an infected host or that other microbes would not induce this enzyme during an infection. This test provides a mechanism for testing this possibility. (The test described here has been used by T. J. Quan from the Bureau of Laboratories, Vectorborne Diseases Division, Fort Colfins, Co., to screen additional isolates of Y. pestis [88], Pasteurella multocida [70], Francisella tularensis [43] and Y. enterocolitica [72]. The test was positive for ail plague isolates tested and negative for ail

nonplague isolates.)

pestis. III. Effects of calcium ions

4.

5.

6.

7.

LITERATURE CITD 1. Brubaker, R. R. 1970. Interconversion of purine mononucleotides in Pasteurellapestis. Infect. Immun. 1:446454. 2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of micrograms quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 3. Higuchi, K., L. L. Kupferberg, and J. L. Smith. 1959. Studies on the nutrition and physiology of Pasteurella

8. 9.

on the growth of virulent and avirulent strains of Pasteurella pestis. J. Bacteriol. 77:317-321. Huiler, S. L, and W. T. Charnetzky. 1980. Glyoxylate bypass enzymes in Yersinia species and multiple formal of isocitrate lyase in Yerstinic pestis. J. Bacteriol. 145: 452-458. Howes, W. V., and B. A. McFadden. 1962. Isocitrate lysse and malate synthase in Pseudomonas indigofera. II. Enzyme changes during the phase of adjustment and the early exponential phase. J. Bacteriol. 84:1222-1227. Kornberg, H. L, and S. R. Eldsen. 1963. The metabolism of 2-carbon compounds by microorganisms, p. 401464. In F. F. Nord (ed.), Advances in enzymology, vol. 23. Interscience Publishers, Inc., New York. Martin, W. J., and J. A. Washington. 1980. Enterobacteriaceae, p. 195-219. In E. H. Lennette, A. Balows, W. J. Hausler, and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. McFadden, B. A., and W. V. Howes. 1960. The determmnation of glyoxylic acid in biological systems. Anal. Biochem. 1:240-248. Moilaret, H. H., and E. Thal. 1974. Genus XI. Yersinia van Loghem, p. 330. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Willkins Co., Baltimore.