Arai Et Al-1970-Microbiology and Immunology

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Japan. J. Microbiol.

Vol. 14 (4), 279-284, 1970

Determination of Pseudomonas aeruginosa

by Biochemical Test Methods

II. Acylamidase Test, a Modified Biochemical Test for

the Identification of Pseudomonas aeruginosa

Taketoshi ARAI, Masako OTAKE, Seiji ENOMOTO, Sachiko GOTO,

nd Shogo KUWAHARA

Departmentof Microbiology, howaPharmaceutical College,Tokyo, and Department

of Microbiology, oho UniversitySchoolof Medicine,Tokyo

(Received or publication, anuary 9, 1970)

ABSTRACT

rapid and simple test method for the detection of acylamidase activity of Pseudo-

monas 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,

.01 MgSO4.7H2O, 0.5 NaCl and 0.1 acetamide (final pH 6.8). After aerobic incuba-

tion 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,

nd 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 glu-

conate oxidation. Although this reaction

was considered to be one of the most reli-

able tests for the speciation of P. aerugi-

nosa, there exist strains of this species

which, though rare, are negative for gluco-

nate, oxidation. Further some other species

of Pseudomonas and Enterobacteriaceae

gave positive results in this reaction.

n 1960, Kelly and Clarke [4] reported

the presence of an aliphatic amidase which

was capable of hydrolysing either acet-

amide or propionamide. It appeared likely,

from our experiences, that this enzymatic

activity was fairly specific for P. aeruginosa.

Consequently, attempts were made to de-

vise a simple and reproducible method for

the detection of acylamidase activity, and

this was accomplished by using a synthetic

medium containing acetamide as the sub-

strate for enzymatic action, and Nessler's

reagent was used as the test reagent.

he present paper reports our experi-

ence with this procedure, its specificity, and

our results.

MATERIALS AND METHODS

asal medium. An inorganic salts solu-

tion of the following composition was used

279



280

T. ARAI, M. OTAKE, S. ENOMOTO, S. GOTO AND S. KUWAHARA

as the basal media.

KH2PO4 2.0g

NaC1 5.0 g

MgSO4.7H20 0.1 g

Redistilledwater 1000ml 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 x 170 mm test tubes in

1 ml amounts, and then autoclaved at

121 C for 15 min.

trains used. A total of 78 strains of 18

species as listed in Table 1 were used.

Thirteen strains of P. aeruginosa were iso-

lated from patients at the Central Labo-

ratory of Toho University Hospital. Aero-

monas 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 sub-

cultured on nutrient agar slants wiht suc-

cessive transfers at one month intervals.

Strains of V. parahaemolyticus were cul-

tured in a nutrient broth or agar medium

containing 3 NaCl.

ethod for detection of acylamidase ac-

tivity. One loopful of a nutrient agar over-

night culture of the test organism was in-

oculated into 1 ml of the acetamide medi-

um,and after incubation at 37 C, aerobical-

ly for 6-18 hr, one drop of Nessler's rea-

gent was added to the culture. A reddish-

brown sediment was immediately produced

if a positive result appeared.

he intensity of the Nessler reaction

was expressed as - +, ++, and +

essler's reagent was prepared as fol-

lows: One gram of HgC12 was added to 6

ml of distilled water and heated to dissolve

completely. Separately, 2.5 g of KI was dis-

solved in 6 ml of distilled water, and this

was mixed with the HgC12 solution. Then

6 g of KOH was dissolved in 6 ml of dis-

tilled water, and added to the above solu-

tion. After stirring well, 13 ml more of dis-

tilled water was added, and the total solu-

tion filtered before use.

RESULTS

Determination of the Composition of the

asal Inorganic Salts Solution

a) Examination for the presence of acyl-

amidase activity with aqueous acetamide

solution. A 1 aqueous acetamide solu-

tion without any additional salt compo-

nent 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 pro-

duced by acylamidase activity was checked

with Nessler's reagent.

s 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

gSO4 0.1 acetamide was added and,

after sterilization, they were inoculated in

Table 1. List of the test strain



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

.01 caused only slight differences in

Nessler's reaction, although a definite in-

crease 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 composi-

tion 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.

Relation between the Amount of Aceta-

mide and the Acylamidase Activity

Acetamide was added to the basal com-

position 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.

essler reaction tended to be weaker

when the concentration of acetamide was

below 0.02 . When it was 0.05 , the re-

sults varied depending on the strain used.

When it was above 0.1 , all the test strains

of P. aeruginosa gave strongly positive re-

action. 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 nutri-

ent agar slant were suspended in sterile

saline so as to contain 109 viable cells per

ml, and serial ten-fold dilution were pre-

pared from this original suspension. Each

dilution, in an amount of 0.05 ml, was in-

oculated into 1 ml of acetamide medium,

Table 2. Acylamidase test using 0.1

aqueous acetamide solution

Table 3. Effect of the addition of magnesium

ulfate on the acylamidase test

Table 4. Effect of the addition of sodium

acetate on the acylamidase test

Table 5. Relation between the concentration of acetamide

nd the intensity of Nessler reaction



282 T. ARAI M. OTAKE S. ENOMOTO S. GOTO AND S. KUWAHARA

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 posi-

tive results. According to our experimental

results one loopful from a nutrient agar

slant with a standard platinum loop con-

tained 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 of Nessler Reaction

ne loopful of an overnight culture on

nutrient agar of the test strain of P. aeru-

ginosa was inoculated into 1 ml of aceta-

mide medium and incubated at 37C for

various times of incubation to examine the

effect of incubation time on the acylami-

dase activity. The results are shown in

Table 7. After incubation for more than

5 hr the reaction was always strongly posi-

tive regardless of the incubation time.

Results of Acylamidase Test of 40 Clinical

Isolates of P. aeruginosa and Related

Species

ourty fresh clinical isolates of P. aeru-

ginosa were investigated for their acyla-

midase activity by the test method men-

tioned 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.

even strains of P. flu orescens 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.

esults of acylamidase test with related

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



MODIFIED BIOCHEMICAL TEST FOR IDENTIFICATION OF P. AERUGINOSA 283

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

Biihlmann and his coworkers [2] de-

scribed 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 con-

taining acetamide as a sole source of car-

bon and nitrogen in our present investi-

gation 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 enzy-

matic action.

elly and Kornberg [6] detected amidase

activity from cells grown in a synthetic

edium containing acetate as a sole carbon

source. In our present experiments, how-

ever, we could not confirm any affect of the

addition of acetate to the acetamide medi-

um. Although acylamidase activity could

be detected by inoculating a large amount

of resting cells into a 1 aqueous solution

of acetamide, more distinct and repro-

ducible results were obtained when a test

strain was inoculated into the above de-

scribed medium and incubated at 37C for

more than 6 hr.

ince it is well known that alkaline earth

metals cause sediment production by Nessl-

Table 8. Acylamidase activities of various

species of Pseudomonas other than

Pseudomonas aeruginosa

Table 9. Acylamidase activities of related

gram-negative bacteria



284 T. ARAI, M. OTAKE, S. ENOMOTO, S. GOTO AND S. KUWAHARA

er's reagent, it was of considerable im-

portance 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, how-

ever, the medium was colorless and trans-

parent 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 Biihlmann'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 composi-

tions 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 heat-

ing 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 invaria-

bly positive with the strains of P. aeru-

ginosa regardless of the size of inoculum.

ccording to Bahlmann'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

fter incubation at 37C for 24 hr. Thus the

acylamidase test devised by the authors

was found to be highly specific to P. aeru-

ginosa, 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 UniversityHospital,

Dr. H. Abe of YokohamaQuarantine Station and

Mr. Kudoh of Tokyo-to Laboratories or Medical

Sciences.

REFERENCES

[ 1 Arai, T., Enomoto,S., and Kuwahara,S. 1970.

Determinationof Pseudoinonas eruginosa

by biochemicalest methods . An improved

methodfor gluconateoxidation est.Japan.

J. Microbiol.14: 49-56.

[ 2 Bilhlmann, X., Vischer,W. A., and Bruhin,

H. 1961. Identification of apyocyagenic

strains of Pseudoinonas eruginosa. . Bac-

teriol. 82: 787-788.

[ 3 Christensen,W. B. 1946.Urea decomposition

as a means of differentiatingProteus and

Paracolon cultures from each other and

from Salmonella and Shigella types. J.

Bacteriol. 2: 461-466.

[ 4 Kelly, M., and Clarke, P. H. 1960.Amidase

production by Pseudoinonas eruginosa.

Biochem. . 74: 21.

[ 5 Kelly, M., and Clarke,P. H. 1962.An induci-

ble amidaseproduced by a strain of Pseu-

domonasaeruginosa. . Gen. Microbiol. 7:

305-316.

[ 6 Kelly,M., and Kornberg,H. L. 1962. Discon-

tinuity of amidase formation by Pseudo-

monas aeruginosa.Biochim.Biophys.Acta

59: 517-519.

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Arai Et Al-1970-Microbiology and Immunology