(1946), and Elek (1948) have described different methods. Stuart, van Stratum, and Rustigian (1945) found the method of Rustigian

Transcription

1 A COMPARISON OF THE PHENYLPYRUVIC ACID REACTION AND THE UREASE TEST IN THE DIFFERENTIATION OF PROTEUS FROM OTHER ENTERIC ORGANISMS SVERRE DICK HENRIKSEN State Institute for Public Health, Bacteriological Department, 081o, Norway Received for publication June 6, 1950 It was shown by Henriksen and Closs (1938) and Closs and Henriksen (1938) that Proteus strains could be differentiated from other enteric organisms by their action on phenylalanine (PA). Whereas the former rapidly produced large quantities of phenylpyruvic acid (PPA), demonstrable by a color reaction with ferric ions, the latter produced only small quantities of PPA or none at all. A rapid test was described that seemed to be highly specific for Proteus. At that time no simple method was available for testing urease activity, and no comparison of the two reactions was made. In the last few years, however, several good methods for the study of urea decomposition have become available. Thus Rustigian and Stuart (1941), Ferguson and Hook (1943), Christensen (1946), and Elek (1948) have described different methods. Stuart, van Stratum, and Rustigian (1945) found the method of Rustigian and Stuart very specific. Out of 600 Proteus strains only 5 gave weak or negative reactions, whereas 1,000 cultures of other enteric organisms failed to give positive reactions within 48 hours. Christensen claimed that this medium would only allow the growth of organisms capable of utilizing urea as a sole source of nitrogen, and that organisms capable of hydrolyzing urea, but incapable of utilizing ammonia as a source of nitrogen, might escape detection. Consequently he devised a medium that is richer in nutritive substances (peptone and glucose). On this medium Proteus strains give a much more rapid and a much stronger reaction than other organisms, but paracolon, Aerobacter, and intermediate types might produce a weak positive reaction within 24 hours and a strong reaction after 3 to 5 days. Thus both media are well suited for the differentiation of Proteus from other organisms. That of Rustigian and Stuart seems to be more specific, whereas that of Christensen is better suited for the detection of weak urease activity. The results of a comparison of these reactions were reported by Cook (1948). METHODS PPA Reaction Before the standard method was chosen, several preliminary experiments were performed in order to study the influence of various factors on the results. The basic method in all these experiments was the following: 20- to 24-hour agar slant cultures were suspended in 0.5 ml 0.85 per cent saline and mixed with equal volumes of 0.2 per cent DL-phenylalanine in saline, in ordinary test tubes. The mixtures were shaken and placed nearly flat on a table, or in the incubator, in 225

2 2126 2VERRE DICK HENRISEN [vol. 60 order to provide a large surface area. After incubation the mixture was acidified with 10 per cent H2SO4 with phenol red as an indicator. Then 4 or 5 drops of half-saturated FeNH4(SO4)2 were added, the tubes shaken, and the results read as soon as the green color had reached maximal development (within about 1 minute). Experiment 1: Incubation temperature. Tubes incubated at room temperature gave weaker reactions than tubes incubated at 37 C after 30 minutes. After 2 hours the difference was only slight, and after 4 hours both sets of tubes gave equal reactions. Experiment 2: Incubation time. After 30 minutes the reactions were weak or moderately strong; after 2 hours they were nearly maximal, but a slight increase could be seen after 4 hours. No further increase occurred after 24 hours. Experiment 3: Influence of alkalinization. One drop of 0.01 per cent phenol red was added to all tubes. One set was made alkaline by dropwise addition of M/10 Na2CO3, whereas the reaction was not adjusted in a second set. The reactions were only very slightly stronger in the alkaline series, if at all. Experiment 4: Addition of (NH4)2SO4. After incubation, one set of tubes was saturated with (NH4)2S04 by the addition of a few crystals, whereas none were added to a second series. When the iron reagent was added, the former set of tubes showed a just perceptible increase in the strength of the reactions. Experiment 5: Addition of merthiolate. In one series of tubes the PA solution was preserved with 0.01 per cent methiolate, whereas no preservative was used in a second series. The reactions showed no difference. The strains used in these experiments were one of Proteus mirabilis and one X19 (Proteus vulgaris). Based on these experiments the following method was adopted: Inoculate the whole surface of an agar slant. Incubate overnight; if growth is good, suspend in 0.5 ml saline. Occasional strains (though rarely Proteus strains) give insufficient growth, in which case the growth from several slants should be used. The suspension should contain about 50,000 million organisms or more, but need not be standardized. Transfer the suspension to a test tube and add 0.5 ml of 0.2 per cent DL-PA in saline (or 0.1 per cent L-PA), preserved with 0.01 per cent merthiolate. Add 1 drop of 0.01 per cent phenol red and make alkaline with M/10 Na2CO3. Shake vigorously and place tubes nearly flat on a table. Leave for 4 hours and add 10 per cent H2SO4 dropwise until the color changes through yellow to pink. Add sufficient (NH4)2SO4 to saturate the solution and then 4 or 5 drops of half-saturated FeNH4(SO4)2. Shake well and read results when the green color is at a maximum or, if negative, after about 1 minute. Positive reactions are graded as + (faint greenish color), ++ (distinct, but weak or only moderately strong green color), and (dark-green to bluishgreen color). Urease Reaction Tubes of Christensen's medium are inoculated in the customary manner and incubated at 37 C; the results are read after 24 hours. Positive reactions are

3 1950] PHENYLPYRUVIC ACID REACTION AND UREASE TEST 227 graded similarly: + (pink to red color in a shallow layer of medium just below bacterial growth), ++ (purple color corresponding to slope but not reaching bottom of butt), and +++ (purple color reaching to bottom of butt). MATERIAL In all, 646 strains have been tested so far. The majority were freshly isolated strains from feces or from samples of water. A snall number of strains were laboratory stock cultures, representing ShigeUa, "Arizona," Proteus X strains, and Klebsiella (8 type strains received from Kauffmann, which, to simplify the table, have been included in the Aerobacter group). All strains had been tested for fermentation of lactose, mannitol, maltose, glucose, and sucrose and for indole production. Strains from drinking water had also been tested for the IMVIC reactions, likewise all fecal strains that gave a positive PPA or U (urease) test. On the other hand, it was impracticable to determine the IMVIC reactions of all TABLE 1 Phenylpyruvic acid (PPA) and urease (U) reactions of Proteus U j _ NUMBER 01 SNS PPA P. vulgaris P. mirabilis P. morganii Proteu8 ap 3 3 Total the negative fecal paracolon strains, and it is consequently not definitely known how many intermediate or Aerobacter strains are included in this group. It was felt to be of greater interest to make sure of a correct classification of all strains that gave positive reactions. RESULTS The reactions of the Proteus group are shown in table 1. All strains gave a 3+ PPA reaction and 128 out of 137 strains gave a 3+ U reaction. Four strains of Proteus morganii gave only a 2+ U reaction. This is not surprising as it has been shown previously that P. morganrii may attack urea at a slower rate than other Proteus species (Stuart, van Stratum, and Rustigian, 1945). One strain of P. morganii gave a negative U reaction. Unfortunately the strain was discarded before the test could be repeated, but, in view of the fact that another strain from the same patient gave a 3+ reaction, it is probable that the failure of the strain to split urea may have been due to a technical error or to a temporary suppression of the enzyme activity. A stock strain of Proteus X19 gave con-

4 228 SVERRE DICK HENRIKSEN [VOL;. 60 sistently negative U reactions in repeated tests, whereas the PPA reaction was 3+. It is worth noting that the PPA reaction was maintained in spite of the ab- TABLE 2 Reactions of Salmonella, Shigella, and "Arizona" group8 U - +~~~~~~~~~~~~~~~~~~~~~~~~~~ I - ~~-NUKMBR 07r STRAIN PPA S. typhi S. paratyphi B S. typhimurium.9 9 S. paradysenteriae S. sonnei Arizona group Total TABLE 3 Reactions of Escherichia-Aerobacter group and of various U other organisms - - t +, ,2+ 3+ NUMBER on PPA mans _ +, E. coli Intermediate coli Paracoli * * Intermediate paracoli...j Aerobacter Alcaligenes t Pseudomonas Unidentified gram-negative rods Total * The figures may include strains of both the colon and the intermediate groups, as IMVIC reactions of negative strains were not determined. t Two strains of Alcaligenes gave a 2+ PPA reaction; all other strains in this column gave only very weak reactions (+). sence of urease activity. Stuart, van Stratum, and Rustigian (1945) found that one of their X strains produced a U-negative variant. Finally 3 strains, designated Proteus sp. in table 1, deserve mention. These strains did not agree completely with any of the Proteu speciesbut showed marked

5 1950] PHENYLPYRUVIC ACID REACTION AND UREASE TEST 229 resemblance to P. morganii. They did not swarm on agar at 37 C or at 28 C; they produced acid and a small amount of gas from glucose after 24 hours, and from sucrose after 3 days. The H2S and Voges-Proskauer tests were negative and gelatin was not liquefied. The methyl red reaction was weakly positive (orange color), and no growth occurred in Koser's citrate medium. One of the strains was slightly motile; another one produced indole. Thus the strains differ from the typical P. morganii in the fermentation of sucrose, in lack of motility, or in indole production. It seems reasonable to consider them as atypical strains of P. morganii. The strains were consistently negative in the U test and gave 3+ reactions for PPA. Thus again the PPA test remained positive in spite of loss-or absence-of urease activity. The reactions of the Salmonella, Shigella, and "Arizona" groups are shown in table 2. With the exception of two strains of Salmonella paratyphi B, which showed weak PPA reactions, all strains were negative. The results obtained with strains of the Escherichia-Aerobacter group and various other organisms are shown in table 3. Discrepancies between the results of the two tests are more frequent in this group, but in most cases the discrepancies are only minor. Thus 11 strains gave negative U reactions and weakly positive PPA reactions, which in only two cases were strong enough to be considered as 2+ reactions. Thirty-four strains gave + or 2+ reactions (mostly only + reactions) in the U test and negative PPA reactions. Four Aerobacter strains gave 3+ U reactions and a + PPA reaction, and 4 strains gave weak reactions in both tests. In 22 cases the discrepancies were more marked, with 3+ U reactions and a negative PPA reaction. Such strains were found in all groups, but more frequently in the intermediate and Aerobacter groups. Thus, on the whole, there were more positive U reactions with negative PPA reactions than vice versa. DISCUSSION The faculty of rapid transformation of PA to PPA coincides with strong urease activity in the vast majority of cases. The association is close enough to make it tempting to assume that the two reactions might be different expressions of the activity of the same enzyme system. The fact that a number of strains showed minor discrepancies between the results of the two tests does not necessarily speak against this assumption. Both are crude qualitative reactions, and variations in the size of the inoculum, the incubation time, and other factors may explain such discrepancies. Thus, previous experience (Henriksen and Closs, 1938) has shown that a number of strains of the Escherichia-Aerobacter group may give positive PPA reactions if the reaction mixture is incubated for 24 hours. It has also been shown (Christensen, 1946) that such strains may give positive U reactions after several days. The fact that more strains gave a positive U test with a negative PPA test than vice versa might easily be explained as due to the longer incubation time, which would tend to favor the detection of weak and slow urease activity. It may be more difficult to reconcile the fact that some Proteus strains may give a strongly positive PPA test and a negative U test, with this assump-

6 230 SVERRE DICK HENRIKSEN [VOL. 60 tion. However that may be, the agreement between the two tests is close enough to make it permissible to use them alternatively. The specificity of the PPA reaction seems to be equal to, or occasionally to surpass, that of the U test, carried out by Christensen's method. As his medium is designed to be sensitive rather than specific, a comparison with Rustigian and Stuart's method might be more fitting. Judging from the results published by Stuart, van Stratum, and Rustigian (1945) and the results reported here, the PPA test would appear to be practically as specific, especially if very slight reactions are disregarded. In rare cases the PPA test may be superior, as shown by the fact that Proteus strains may show a strong positive reaction although the urease activity is subnormal or absent. No Proteus strain has yet been found that failed to give a strongly positive PPA reaction within 4 hours. Thus the PPA test might be a valuable supplement to the U test in such rare cases, or it might safely be used instead of the latter test. It seems unlikely that this will happen, as the U test is very simple and convenient and, as a rule, very reliable. Also the PPA test as used in this study may seem slightly more cumbersome. Further it may sometimes be difficult to get PA, as has been the case during certain stages of this study. The relatively high cost of the substance is not very important as 1 g is sufficient for as many as 1,000 tests, and by reducing the volumes it might go even further. For routine use the method could safely be simplified somewhat. Thus the following modification would be adequate according to previous experience: Suspend the growth from a 20- to 24-hour agar slant culture in 0.5 ml of saline and mix in a test tube with an equal volume of 0.2 per cent DL-PA and 1 drop of 0.01 per cent phenol red. Place nearly horizontally on the desk at room temperature after vigorous shaking and leave for 4 hours. Add 10 per cent H2S04 dropwise until the color changes through yellow to pink, and then 4 to 5 drops of half-saturated FeNH4(SO4)2. Shake and read results when the green color is at a maximum, or after 1 minute in negative cases. This reaction might be of value in laboratories that rarely need to make a urease test and would not want to prepare a special medium for the test. All the necessary reagents could be stored in dry form and solutions prepared as required, or a PA solution could be kept, preserved with merthiolate. BSUARY The faculty of rapid transformation of phenylalanine into phenylpyruvic acid nearly always coincides with strong urease activity and is practically limited to members of the Proteus group. A simple routine test for the detection of phenylpyruvic acid production is described. The specificity and sensitivity of the test are equal to, and in rare cases superior to, those of the urease test. REFERENCES CHRISTENSEN, W. BLAME 1946 Urea decomposition as a means of differentiating Proteus and paracolon cultures from each other and from Salmonella and Shigella types. J. Bact., 52,

7 1950] PHENYLPYRUVIC ACID REACTION AND UREASE TEST 231 CLOSS, K., AND HENRIKSEN, S. D tjber den Nachweis von d- und b-phenylalanin in biologischen Flhssigketien. Z. physiol. Chem., 254, COOK, G. T Urease and other biochemical reactions of the Proteus group. J. Path. Bact., 60, ELEK, STEPHEN D Rapid identification of Proteus. J. Path. Bact., 60, FERGUSON, W. W., AND HooK, A. E Urease activity of Proteus and Salmonella organisms. J. Lab. Clin. Med., 28, HENRIKSEN, S. D., AND CLOSS, K The production of phenylpyruvic acid by bacteria. Acta Path. Microbiol. Scand., 15, RUSTIGIAN, R., AND STUART, C. A Decomposition of urea by Proteus. Proc. Soc. Exptl. Biol. Med., 47, RUSTIGIAN, R., AND STUART, C. A The biochemical and serological relationships of the organisms of the genus Proteus. J. Bact., 49, STUART, C. A., VAN STRATUM, E., AND RUSTIGIAN, R Further studies on urease production by Proteus and related organisms. 3. Bact., 49, Downloaded from on October 2, 2018 by guest