Japan. J. Microbiol. Vol. 14 (4), 279-284, 1970 Determination of Pseudomonas aeruginosa II. Acylamidase by Biochemical Test Methods the Identification Test, a Modified Biochemical Test for of Pseudomonas aeruginosa Taketoshi ARAI, Masako OTAKE, Seiji ENOMOTO, Sachiko GOTO, and Shogo KUWAHARA Department of Microbiology, Showa Pharmaceutical College, Tokyo, and Department of Microbiology, Toho University School of Medicine, Tokyo (Received for publication, January 9, 1970) ABSTRACT A rapid and simple test method for the detection of acylamidase activity of Pseudomonas aeruginosa was devised. One loopful of a nutrient agar overnight culture of a test organism was inoculated into 1 ml of a test medium consisting of 0.2% KH2PO4, 0.01% MgSO4 7H2O, 0.5% NaCl and 0.1% acetamide (final ph 6.8). After aerobic incubation at 37C for 6 hr, one drop of Nessler's reagent was dropped into the test medium. A reddish-brown sediment appeared immediately if results were positive. Of 40 test strains of P. aeruginosa 39 gave strongly positive results. A strain showed a weakly positive result after 6 hr incubation, but the reaction became stronger after 18 hr culture. Other species of Pseudomonas, and various species of bacteria such as genera Vibrio and Aeromonas, and family Enterobacteriaceae were negative in this test. From these experimental results, the acylamidase test was considered to be highly specific for strains of P. aeruginosa, and therefore useful as a reliable method for the identification of this species. In a previous paper [1] an improved technique was reported for microbial gluconate oxidation. Although this reaction was considered to be one of the most reliable tests for the speciation of P. aeruginosa, there exist strains of this species which, though rare, are negative for gluconate, oxidation. Further some other species of Pseudomonas and Enterobacteriaceae gave positive results in this reaction. In 1960, Kelly and Clarke [4] reported the presence of an aliphatic amidase which was capable of hydrolysing either acetamide or propionamicle. It appeared likely, from our experiences, that this enzymatic activity was fairly specific for P. aeruginosa. Consequently, attempts were made to devise a simple and reproducible method for the detection of acylamidase activity, and this was accomplished by using a synthetic medium containing acetamide as the substrate for enzymatic action, and Nessler's reagent was used as the test reagent. The present paper reports our experience with this procedure, its specificity, and our results. MATERIALS AND METHODS Basal medium. An inorganic salts solution of the following composition was used 279
280 T. ARAI, M. OTAKE, S. ENOMOTO, S. GOTO AND S. KUWAHARA as the basal media. Table 1. List of the test strain KH2PO4 2.0 g NaGl 5.0 g MgSO4 7H2O 0.1 g Redistilled water 1000 ml ph 6.8 To this basal solution acetamide was added in various concentrations, the reaction was again adjusted to 6.8, and each solution dispensed into 15 ~170 mm test tubes in 1 ml amounts, and then autoclaved at 121 C for 15 min. Strains used. A total of 78 strains of 18 species as listed in Table 1 were used. Thirteen strains of P. aeruginosa were isolated from patients at the Central Laboratory of Toho University Hospital. Aeromonas and V. parahaemolyticus strains were kindly supplied by the Yokohama Quarantine Station, and some strains of enterobacteria by Tokyo-to Laboratories for Medical Sciences. All strains were subcultured on nutrient agar slants wiht successive transfers at one month intervals. Strains of V. parahaemolyticus were cultured in a nutrient broth or agar medium containing 3% NaCl. Method for detection of acylamidase activity. One loopful of a nutrient agar overnight culture of the test organism was inoculated into 1 ml of the acetamide medium,and after incubation at 37 C, aerobically for 6-18 hr, one drop of Nessler's reagent was added to the culture. A reddishbrown sediment was immediately produced if a positive result appeared. The intensity of the Nessler reaction was expressed as -, }, +, ++, and +++. Nessler's reagent was prepared as follows: One gram of HgC12 was added to 6 ml of distilled water and heated to dissolve completely. Separately, 2.5 g of KI was dissolved in 6 ml of distilled water, and this was mixed with the HgCl2, solution. Then 6 g of KOH was dissolved in 6 ml of distilled water, and added to the above solution. After stirring well, 13 ml more of distilled water was added, and the total solution filtered before use. RESULTS Determination of the Composition of the Basal Inorganic Salts Solution a) Examination for the presence of acylamidase activity with aqueous acetamide solution. A 1% aqueous acetamide solution without any additional salt component was autoclaved, and then 1 ml of this solution was inoculated with one loopful of an overnight culture of P. aeruginosa, incubated at 37 C for various incubation times and the presence of ammonia produced by acylamidase activity was checked with Nessler's reagent. As shown in Table 2, Nessler reaction was weakly positive at 6 hr, and strongly positive at 24 hr, although no bacterial growth was observed. b) Effect of the addition of MgSO4 to the test medium. To the basal composition mentioned above and to the one lacking MgSO4 0.1% acetamide was added and, after sterilization, they were inoculated in
MODIFIED BIOCHEMICAL TEST FOR IDENTIFICATION OF P. AERUGINOSA 281 the same way as above and incubated to investigate the effect of magnesium ion on growth and acylamidase activity. As seen in Table 3, the addition of MgSO4 to 0.01% caused only slight differences in Nessler's reaction, although a definite increase in bacterial growth was noted with the addition of MgSO4. c) Effect of the addition of sodium acetate to the test medium. To the basal composition sodium acetate was incorporated to 0.1 and 0.5% respectively besides 0.1% acetamide, and the Nessler's reaction was compared with that lacking acetate. As shown in Table 4, the addition of sodium acetate did not exert any affect on the acylamidase activity. Table 2. Acylamidase test using 0.1% aqueous acetamide solution - `+++ indicates intensity of the Nessler reaction Table 3. Effect of the addition of magnesium sulfate on the acylamidase test. Relation between the Amount o f Acetamide and the Acylamidase Activity Acetamide was added to the basal composition to various concentrations, and the effect of the concentration of acetamide on intensity of Nessler reaction was examined. The results are given in Table 5. Nessler reaction tended to be weaker when the concentration of acetamide was below 0.02%. When it was 0.05%, the results varied depending on the strain used. When it was above 0.1%, all the test strains of P. aeruginosa gave strongly positive reaction. Consequently, 0.1% was considered the optimal concentration of acetamide for the test medium. Relation between the Size of the Inoculum and Intensity of Nessler Reaction Cells of P. aeruginosa culture on nutrient agar slant were suspended in sterile saline so as to contain 109 viable cells per ml, and serial ten-fold dilution were prepared from this original suspension. Each dilution, in an amount of 0.05 ml, was inoculated into 1 ml of acetamide medium, Table 4. Effect of the addition of sodium acetate on the acylamidase test Tests were performed after 24 hr incubation at 37C. Table 5. Relation between the concentration of acetamide and the intensity of Nessler reaction Tests were performed after 24 hr incubation at 37C.
282 T. ARAI, M. OTAKE, S. ENOMOTO, S. GOTO AND S. KUWAHARA Table 6. Relation between the size of inoculum and the intensity of Nessler reaction Table 7. Relation between the time of incubation and intensity of Nessler reaction and Nessler reaction was examined. The results are shown in Table 6. As seen in this table, the effect of the inoculum size was quite negligible after 24 hr culture, but at 6 hr, an inoculum size of below 106 cells per ml of test medium gave a negative reaction, 107 cells per ml of test medium gave varying results depending on strains, and above 108 viable cells per ml of test medium was required to produce the positive results. According to our experimental results, one loopful from a nutrient agar slant with a standard platinum loop contained approximately 109 viable cells, and therefore one loopful inoculum to 1 ml of acetamide medium was quite sufficient to give a positive result after only 6 hr incubation. Relation between Time of Incubation and Intensity o f Nessler Reaction One loopful of an overnight culture on nutrient agar of the test strain of P. aeruginosa was inoculated into 1 ml of acetamide medium, and incubated at 37C for various times of incubation to examine the effect of incubation time on the acylamidase activity. The results are shown in Table 7. After incubation for more than 5 hr, the reaction was always strongly positive regardless of the incubation time. Results of Acylamidase Test of 40 Clinical Isolates of P. aeruginosa and Related Species Fourty fresh clinical isolates of P. aeruginosa were investigated for their acylamidase activity by the test method mentioned above. After 6 hr incubation all the test strains showed strongly positive results except for one strain, which gave weakly positive reaction at 6 hr, but turned more strongly positive after 18 hr incubation. Seven strains of P. fluorescens, and each one strain of P. fragi, P. chlororaphis and P. putrefaciens were cultured at 18C for various times of incubation, and tested for their acylamidase activity. As shown in Table 8, the reaction was negative in all the test strains after 48 hr incubation, and even after 72 hr of incubation only one strain gave a weakly positive reaction, and 3 strains showed doubtful results. Results of acylamidase test with related
MODIFIED BIOCHEMICAL TEST FOR IDENTIFICATION OF P. AERUGINOSA 283 Table 8. Acylamidase activities of various species of Pseudomonas other than Pseudomonas aeruginosa Table 9. Acylamidase activities of related gram-negative bacteria bacterial species, such as Aeromonas spp. V. parahaemolyticus, Achromobacter, E. coli, Klebsiella, Enterobacter, Citrobacter and Proteus group are given in Table 9. All of the test strains gave negative results not only at 6 hr but also even after 18 hr incubation. Thus, acylamidase activity appeared to be highly specific to P. aeruginosa, and the test highly discriminatory. DISCUSSION Buhlmann and his coworkers [2] described a test method for the deamination of acetamide by P. aeruginosa using a modification of Christensen's urea broth [3]. However, this method required at least 12 hr incubation to give clear-cut results. The use of an inorganic salts solution containing acetamide as a sole source of carbon and nitrogen in our present investigation resulted in the more rapid detection of acylamidase activity. We used Nessler's reagent as the test reagent to detect the presence of ammonia resulting from enzymatic action. Kelly and Kornberg [6] detected amidase activity from cells grown in a synthetic medium containing acetate as a sole carbon source. In our present experiments, how- Tests were performed after 6 hr incubation at 37C. ever, we could not confirm any affect of the addition of acetate to the acetamide medium. Although acylamidase activity could be detected by inoculating a large amount of resting cells into a 1% aqueous solution of acetamide, more distinct and reproducible results were obtained when a test strain was inoculated into the above described medium and incubated at 37C for more than 6 hr. Since it is well known that alkaline earth metals cause sediment production by Nessl-
284 T. ARAI, M. OTAKE, S. ENOMOTO, S. GOTO AND S. KUWAHARA er's reagent, it was of considerable importance to determine if the addition of Mg ion exerted any effect on the test. Actually, a slight pale yellowish turbidity appeared in the control medium several minutes after the addition of Nessler's reagent. When the reaction was negative, however, the medium was colorless and transparent immediately after the addition of the reagent, and, if positive, red-brownish sediment was produced rapidly so that the determination could be made quite easily. In the Buhlmann's method, acetamide was added to a final concentration of 1% to the test medium. However, in our present test method, 0.1% was sufficient for successful result, presumably because a synthetic medium containing acetamide as a sole nutrient source was used as test medium and method to detect ammonia directly was employed. Kelly and Kornberg [6] recommended the use of Seitz filtration of the acetamide solution as a method of sterilization. But, in our present experiments, the compositions containing acetamide was sterilized by autoclaving at 121C for 15 min, enabling the preparation of the test medium to be done easily. Since the Nessler reaction was confirmed to be negative in the control medium, the effect of sterilization by heating seemed negligible for the present object. Buhlmann and his coworkers reported that out of 49 strains of P. aeruginosa 45 were strongly positive and 4 strains were weakly positive after incubation at 37C for 12 hr. In our experiments, all except one of the 40 test strains gave strongly positive results after 6 hr incubation at 37C when a large size of inoculum was used. If the Nessler reaction was performed after 24 hr incubation, the results were invariably positive with the strains of P. aeruginosa regardless of the size of inoculum. According to Buhlmann's paper 12 strains of other species of Pseudomonas were all negative in acylamidase test. In our experiments, all the test strains of P. fluorescens, P. fragi and P. chlororaphis were negative in this reaction even after 48 hr incubation, and related genera such as Aeromonas, Achromobacter, and V. parahaemolyticus were also all negative after incubation at 37C for 24 hr. Thus the acylamidase test devised by the authors was found to be highly specific to P. aeruginosa, and is therefore considered to be useful as a means of the identification of this species. ACKNOWLEDGEMENT We want to express our deep thanks for the supply of test strains to Dr. K. Shimizu of the Central Laboratory of Tokyo University Hospital, Dr. H. Abe of Yokohama Quarantine Station and Mr. Kudoh of Tokyo-to Laboratories for Medical Sciences. REFERENCES [1] Arai, T., Enomoto, S., and Kuwahara, S. 1970. Determination of Pseudomonas aeruginosa by biochemical test methods I. An improved method for gluconate oxidation test. Japan. J. Microbiol. 14: 49-56. [2] Buhlmann, X., Vischer, W. A., and Bruhin, H. 1961. Identification of apyocyagenic strains of Pseudomonas aeruginosa. J. Bacteriol. 82: 787-788. [3] Christensen, W. B. 1946. Urea decomposition as a means of differentiating Proteus and Paracolon cultures from each other and from Salmonella and Shigella types. J. Bacteriol. 52: 461-466. [4] Kelly, M., and Clarke, P. H. 1960. Amidase production by Pseudomonas aeruginosa. Biochem. J. 74: 21. [5] Kelly, M., and Clarke, P. H. 1962. An inducible amidase produced by a strain of Pseudomonas aeruginosa. J. Gen. Microbiol. 27: 305-316. [6] Kelly, M., and Kornberg, H. L. 1962. Discontinuity of amidase formation by Pseudomonas aeruginosa. Biochim. Biophys. Acta 59: 517-519.