5 7 7 . 15 : 5 7 6 . 8 5 1 . 2 5 2

STAPHYLOCOCCAL PHOSPHATASE, GLUCURONIDASE AND SULPHATASE

MARY BARBER, B. W. L. BROOKSBANK and S. W. A. KUPER From the Departments of Bacteriology and Chemical Pathology,

St Thomas’s Hospital Medical School, London

BARBER et al. (1948) carried out experiments to determine what organisms may be responsible for the destruction of “ pregnandiol-like glucuronide ” (Bisset et al., 1948) in unpreserved human urine. A number of organisms likely to be present in non-catheter specimens of urine, such as Bact. coli, Strep. fmal i s , and staphylococci were tested, and it was found that certain strains of Staph. albus destroyed both pregnandiol-like glucuronide and pregnandiol glucuronidate. Since a few strains of Staph. pyogenes tested at the same time were without apparent effect on these compounds, it was considered desirable to make a further study of the distribution of glucuronide-destroying enzymes among staphylococci. While studying glucuronidase pro- duction, the same organisms were tested for phosphatase and, at the suggestion of Professor L. Young, for sulphatase.

The use of phenolphthalein phosphate as a substrate in the detection of bacterial phosphatase was first recommended by Bray and King (1942). The advantage of this compound for such tests is obvious owing to the ease with which free phenolphthalein can be detected. We decided, therefore, to use phenolphthalein glucuronide, phenolphthalein phosphate and phenolphthalein sulphate as substrates for the present investigation.

METHODS

Substrates. Phenolphthalein diphosphoric acid was prepared from phenol- phthalein and phosphorus oxychloride in pyridine-chloroform according to the procedure of Fishman et al. (1948). Some of the gummy mass of phenolphthalein diphosphoric acid so obtained was dried in a vacuum desiccator over calcium chloride and used for the phosphatase tests. Phenolphthalein diphosphoric acid, unlike the calcium s a l t t h e preparation used by Bray and King (1942), is readily soluble in water. The solution is of course acid, but can be brought to a neutral or slightly alkaline p H by dilution with phosphate buffer or a bacteriological medium at p H 7.4. A sample of the solid was converted to the sodium salt and purified according to the procedure of Huggins and Talalay (1945). The pure sodium salt was analysed for total phosphorus and for sodium. Two analyses gave values for P of 10.61 and 10.42 per cent. ; the calculated P value for C,,H,,O,,P~a, was 10.24 per cent. The corresponding figures for Na were 11.9 and 11.4 per cent.; calculated value 19.0 per cent. The

J. PATH. BACT.-POL. lgIIr 57

58 11.1. BARBER, B. W . I,. BROORSBAXK AND S. W . A h7VPER

calculated values for the trisodium salt of the diphosphate, C,,H,,Ol1P,Na,, were P, 11.04 per cent. ; Na, 12.28 per cent. It is not possible to distinguish between the penta- and trisodium salts from the analytical figures for sodium, but it is unlikely that the compound is not a diphosphate. It will be noted that Huggins and Talalay also found anomalous figures, namely Na, 11 *68 per cent. ; P, 9.46 per cent.

The bulk of the phenolphthalein diphosphoric acid was made into a neutral, essentially pyridine-free solution. This was injected int,o rabbits a t first in two simultaneous daily injections of 5 ml., each containing approximately 500 mg., and later in two simultaneous daily doses of half this amount. The rabbits (8 in all) were kept in metabolism cages and about 60 g. of phenolphthalein phosphate administered to them. Two died under the large dosage and were replaced, but all survived the lesser dosage. They were fed on cabbage and carrot,s, with water ad libitum. The urine was collected under toluene and processed according to Fishman et al. for phenolphthalcin glucuronide. The yield of the cryst,alline cinchonidine salt of phenolphthalein glucuronide, twice recrystallised from hot methanol by the addition of hot ethyl acetate, was 10.1 g. An approximately equal quantity of cruder material was a.lso obtained. The purified cinchonidine salt gave 93.5 per cent. of the theoretical quantity of phenolphthalein for a, salt wit>h one molecule of methyl alcohol of crystallisa- tion when hydrolysed by boiling with 4NHC1. A nearly 0.01 M solution of sodium phenolphthalein glucuronidate was prepared from the crystalline cinchonidine salt by the procedure of Talalay et al. (1946). This was diluted as required for the glucuronidase tests.

Phenolphthalein sulphate is a new compound and was prepared by Professor

Preparation of medium. Stock solutions, approximately 0.01 M , of the three substrates were prepared in water, sterilised by Seitz filtration and stored in a refrigerator. For tests, these solutions were added separately, with aseptic precaut,ions, to broth consisting of Lab-Lemco 1 per cent., peptone (Evans) 1 per cent. and NaCl 0.5 per cent. a t pH 7.4-7.6. The final concentration of the substrate used was in most cases 1/50th of the stock solution (i.e. 0.0002 M , or approximately 0-01 per cent.). Higher concentrations increased the rate of liberation of free phenolphthalein ; lower concentrations gave fewer positive reactions.

It was found that the solut.ions were stahle at, a t,emperature of 100°C. when diluted 1 in 50 in the buffered medium a t p H 7.4 used for the tests. The substrates were hydrolysed, however, a t higher temperatures or if heated in acid solution.

Brot,h plus substrate was inoculated with the organism to be tested and incubated a t 37" C. At various intervals a sample was removed and made just alkaline by the ca.refd addition of N/10 NaOH, when a pink colour develops in the presence of free phenolpht,halein. (With excess of alkali the pink colour obtained with free phenolphthalein disappears.) Unless a positive result was obtained earlier, the cultures were kept in the incubator for from 7 to 14 days.

Sources of staphylococci. Tests were carried out on 160 coagulase-positive strains of staphylococci, hereafter referred to as Staph. pyogenes, and 75 coagulase- negative strains. The former were isolated from cases of staphylococcal infection or nasal carriers in seven different London hospitals, the Kent and Canterbury Hospital and St Mary's Hospital, Manchester. The date of isolation ranged from 1947 to the time of the present investigation; 80 of the strains were penicillin-sensitive and 80 penicillin-destroying. Of the coagulwse-negative staphylococci, 55 were recently isolated from the anterior nares of students and staff of this hospital and 20 from pathological specimens (12 nasal swabs and 8 miscellaneous) from four London hospitals.

L. Young.

Test f o r enzyme production.

THREE STAPHYLOCOCCUS ENZYMES 5 9

Coagulase-positive

160

0

RESULTS From table I it will be seen that Staphylococcus

pyogenes and coagulase-negative staphylococci differ significantly in their production of phosphatase. All the 160 strains of Staph. pyogenes

TABLE I

Phosphatase production by stuphylococci

Phosphatase.

Coagulase-negative

1

54

Phosphatase production

Gluouronidase-positive .

Glucuronidase-negative . .

Total . .

Positive within 24 hours * .

Negative at 24 hours . Positive in 2-7 days

Negative at 7 days .

Coagnlase-positive Coagulase-negative

6 (3.7 per cent.) 12 (16 per cent.)

154 63

160 75

Total .

I 2o 0

I

75 160 I

* All coagnlase-positive strains gave this reaction in 18 hours or less ; the single coagnlase-negative strain in this category gave a reaction between 18 and 24 hours.

liberated sufficient free phenolphthalein to give a deep red colour on the addition of alkali in 18 hours or less. Only 1 of the 75 coagulase- negative strains gave a comparable result, namely positive in 18- 24 hours. This strain was isolated in association with Staph. pyogenes from the anterior nares of a member of the staff of this hospital. Of the other coagulase-negative strains, 54 gave a positive reaction in from 2 to 7 days and 20 were negative at the end of a week.

The number of strains liberating phenolphthalein from phenolphthalein glucuronide is given in table 11. It will be

Clucuroniduse.

TABLE I1

Clucuroniduse production by staphylococci

60 M . BARBER, B. W . L. BROOKSBANR AND S. W . A KUPER

Of the 6 glucuronidase-producing strains of Staph. pyogenes, 3 were isolated from nasal swabs, 2 from ear swabs and 1 from the conjunctiva. Nine of the 12 coagulase-negative strains liberating phenolphthalein were from the anterior nares and 1 was from the air of a maternity department. The source of the remaining two is unknown.

Sulphatase. None of the strains of staphylococci tested was capable of liberating phenolphthalein from phenolphthalein sulphate to any considerable degree. Two strains of Staph. pyogenes, one isolated from the anterior nares and one from a specimen of faxes, gave a weakly positive reaction after 7-8 days’ incubation. None of the coagulase-negative strains gave a positive reaction (table IIT).

~ _ _ _ _ _ _ _ _ _ _ ~ ~~ ~

Sulphatase production Coagulase-positive

Sulphatase-positive . 2

TABLE I11

Sulphatase production by staphylococci

Coagulase-negative

0

Sulphatase-negative .

Total .

In contrast to this, an aerobic sporing bacillus, encountered as a contaminant, gave a positive reaction in 5 days and a strain of Salm. paratyphi B in 4 days.

188 73

160

Correlation of results and relation to other Jindings

Staph, pyogenes. Of the 2 sulphatase-positive strains, 1 also produced glucuronidase and 1 did not. As shown in table IV, there was no correlation between the production of these enzymes and of penicillinase.

TABLE IV

Coagulase-positive staphylococci

1 Glucuronidase-po8itive . Sulphatase-positive .

Penicillin-sensitive Penicillin-destroying

3 3

1 1

Coaguluse-negative staphylococci. Table V relates phosphatase and glucuronidase activity. It will be seen that the two enzymes do not go together. Of the 12 glucuronidase-positive strains, 5 also produced phosphatase, 7 did not.

THREE STAPHYLOCOCCUS E N 2 Y M E S 61

Glucuronidase-positive . .

Glucuronidase-negative . .

Phosphatase-positive Phosphatase-negative

I 5 7

50

Total . 55 1 20

Glucuronidrtse-positive . .

Glucuronidase-negative . .

Total . --

Phosphatase-positive .

Phosphatase-negative .

Total .

phatase are given in table VI. Again there is no apparent correlation between any of these activities.

TABLE VI

Coagulase-negative staphylococci

Msnnitol-positive 1 Mannitol-negative -

4 8

10 53

14 61

8 48

6 13

14 61

Effect of concentration of substrate

Two strains of coagulase-negative staphylococci showing glucu- ronidase activity were each inoculated into tubes of broth containing three different concentrations of phenolphthalein glucuronide, namely 0.0028 per cent. in tube 1, 0.01 per cent. in tube 2 (equalling the concentrations used in previous tests) and 0-1 per cent. in tube 3. After 48 hours at 37OC., the amount of free phenolphthalein was estimated. The results are shown in table VII. It will be seen that the fourfold increase in substrate between tubes 1 and 2 caused a considerable increase in the amount of phenolphthalein liberated, whereas the further tenfold increase in substrate between tubes 2 and 3 had comparatively little effect on the amount of phenolphthalein liberated. A third strain, recorded as negative in tahle 11, was found to liberate 2-3 pg./ml. of phenolphthalein when grown in broth containing 0.1 per cent. of substrate.

62 M . BARBER, B. W. L. BROOKSBANK A N D S. W . A . RUPER

Four coagulase-negative strains were tested qualitatively after 24 hours’ incubation in 3 different concentrations of phenolphthalein phosphate, namely 0.01, 0.05 and 0.1 per cent. Two of the strains

TABLE VII

Effect of concentration of substrate o n amount of phenolphthalein liberated

-

Phenolphthalein liherated

Concentration of substrate (g. per 100 nil.)

0.0025 .

0.01

0.1 . .

Strain P6009

6 6

18.0

18.4

Percentage of total present

54.9

30.0

3.1

Stmiri luS994

10.3

22.4

24.1

Percentage of total present

85.8

37.3

4.0

gave a negative result in all three tubes ; these two strains had both been negative after 7 days in the previous tests. Two strains gave a positive reaction in the tube containing the highest concentration of substrate (0.1 per cent.) but were negative in the other two tubes ; these strains gave a positive result in 2 and 4 days respectively in the previous tests, when the concentration of substrate was 0.01 per cent.

DISCUSSION In 1932 Gordon and Cooper demonstrated the presence of glycero-

phosphatase in chloroform-killed suspensions of Staph. aureus and Bact. coli, and in 1933 Boivin and Mesrobeanu also recorded phos- phatase activity of these organisms. With acid-insoluble phosphate substrates, the latter workers found greatest activity at p H 10, whereas with acid-soluble phosphates the optimum pH was 6. They favoured the view that these organisms had two distinct enzymes but could not rule out the possibility that the results were due to a single enzyme, the optimum p H varying with the substrate.

The introduction by Bray and King (1942) of phenolphthalein phosphate as a substrate has made the detection of bacterial phos- phatase relatively simple. These workers (Bray and King, 1943 ; King, 1943) studied a large number of micro-organisms belonging to a wide variety of species for phosphatase activity by incorporating phenolphthalein phosphate in an egg medium. The great majority of the organisms studied showed some activity, but 60 strains of Corynebacterium diphtheria? all gave negative results. Ten strains each of Staph. aureus and Staph. albus (coagulase test not recorded) all gave a strongly positive reaction. The amount of substrate used by these workers was about 40 times that used in the present investiga-

THREE STAPHYLOCOCCUS ENZYMES 6 3

tion, which may account for their finding no apparent difference in reaction between strains of Staph. aureus and Staph. albus. Bray and King (1943) found that reduction in the amount of phenolphthalein phosphate in the medium eliminated weak reactors. In the present investigation, it was found that an increase in the amount of substrate accelerated positive reactions in coagulase-negative staphylococci and the concentration used for the tests was that giving maximum differentiation.

Several studies of bacterial glucuronidase have appeared recently. From cultures of Buct. coli, Buehler et al. (1949) and Smith and Mills (1950) prepared cell-free filtrates which were able to liberate the aglycones from /3-D-glucuronides. The enzyme or enzymes responsible have been loosely classified as /3-glucuronidase, although no tests of their specificity or mode of action have been made. Karunairatnam and Levvy (1950) demonstrated an enzyme liberating free phenol and phenolphthalein from their respective glucuronides in cell-free preparations from medium-sized micro-organisms in the rumen of sheep. Buehler et al. showed that /3-glucuronidase production by Bact. coli was stimulated by the presence of menthol glucuronide in the medium. Smith and Mills (1950) found that glucuronidase was not produced by strains of Bact. coli grown in nutrient agar or peptone medium, but that transference to a medium containing 0.1 per cent. menthol glucuronide stimulated glucuronidase production within 24 hours. For maximal production of the enzyme these workers recommended 0.5 per cent. menthol glucuronide in the medium, although with this concentration the amount of free menthol liberated was sufficient to inhibit growth completely. It is possible, therefore, that in the present investigation more strains of staphylococci would have given positive results if a greater concentration of substrate had been used.

SUMMARY

We tested 160 strains of Staph. pyogenes and 75 strains of coagulase- negative staphylococci for their capacity to liberate free phenolph- thalein from phenolphthalein phosphate, phenolphthalein glucuronide and phenolphthalein sulphate in fluid medium.

All 160 strains of Staph. pyogenes but only 1 coagulase-negative strain liberated free phenolphthalein from phenolphthalein phosphate in 24 hours or less ; of the remaining coagulase-negative strains 54 gave a positive reaction with this substrate in 2-7 days and 20 were negative at the end of a week.

With phenolphthalein glucuronide 6 (3.7 per cent.) strains of Staph. pyogenes and 12 (16 per cent.) coagulase-negative strains gave a positive reaction.

Only 2 strains of Xtaph. pyogenes and none of the coagulase- negative staphylococci showed any sulphatase activity.

64 M . BARBER, B. W . L. BROOKSBANK AND S. W . A . KUPER

Our thanks are due to Professor L. Young for the supply of phenol- phthalein sulphate and for much valuable advice and criticism.

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