Comparison of methods for determining DNase and phosphatase

JOURNAL OF CLINICAL MICROBIOLOGY, May 1989, p. 1127-1129 0095-1137/89/051127-03$02.00/0 Copyright C) 1989, American Society for Microbiology

Vol. 27, No. 5

Comparison of Methods for Determining DNase and Phosphatase Activities of Staphylococcit BRUCE E. LANGLOIS,* ROBERT J. HARMON, KATHERINE AKERS, AND DEBRA K. AARON Department of Animal Sciences, University of Kentucky, Lexington, Kentucky 40546-0215 Received 17 August 1988/Accepted 9 February 1989

A greater percentage of DNase-positive strains was detected with DNase test agar than with DNase test agar containing 0.005% methyl green or 0.005% toluidine blue (P < 0.01). No significant differences were obtained in the percentage of phosphatase-positive strains with the four methods compared. On the basis of ease of use, P agar containing para-nitrophenylphosphate disodium (0.495 mg/ml) would be the preferred method for determining phosphatase activity of staphylococci.

Little information is available concerning the best method for determining DNase and phosphatase activities of staphylococci, especially for newly recognized species and species isolated from nonhuman sources. Therefore, this study was undertaken to compare methods using agar media for determining DNase and phosphatase activities of reference, type, and bovine strains of staphylococci. This study used 30 reference and type strains, representing 23 Staphylococcus species (1). Each strain was tested a minimum of three times on each medium. In addition, 274 isolates from bovine milk which had been identified in our laboratory as staphylococci by the API STAPH Trac (API Analytab Products, Plainview, N.Y.) rapid identification system were used. Three media supplemented with DNA were used to determine production of DNase by staphylococci: (i) DNase test agar (DTA) (Difco Laboratories, Detroit, Mich.), (ii) DTA containing 0.005% methyl green (DMG) (Difco), and (iii) DTA containing 0.005% toluidine blue (DTB) (4). Duplicate DTA plates and single DMG and DTB plates were inoculated per culture by radially streaking (3 cm) up to six cultures on each medium. After 24 and 48 h of incubation at 35°C, the DTA plates were flooded with 1 N HCI and the plates were observed for clear zones surrounding growth on the plates. After 24 and 48 h, the DMG plates were examined for a clearing around the streaks and the DTB plates were examined for a bright pink zone surrounding growth on a royal blue background. Four methods were used to determine phosphatase activity of the staphylococci: (i) P agar (3) containing 0.495 mg of para-nitrophenylphosphate disodium per ml (PNP) (Sigma 104 phosphatase substrate; Sigma Chemical Co., St. Louis, Mo.), (ii) P agar containing 0.023 mg of phenolphthalein monophosphate per ml (PMP; Sigma), (iii) P agar containing 0.067 mg of phenolphthalein diphosphate per ml (PDP; Sigma), and (iv) the phosphatase test from API STAPH Trac kit (PAL; Analytab). Duplicate PMP and PDP plates and single PNP plates were prepared per culture by radially streaking (3 cm) up to six cultures on each medium. After 24 and 48 h of incubation at 35°C, 1 ml of concentrated ammonium hydroxide was added to the lids of the PMP and PDP plates, and the bottoms were inverted Corresponding author. t Journal paper 88-5-159 of the Kentucky Agricultural Experiment Station. *

and placed over the lids. Development of red colonies indicated free phenolphthalein and thus phosphatase activity. Yellow color surrounding growth on PNP agar indicated phosphatase activity. The PAL results were obtained from the STAPH Trac kits used to identify each isolate used in this study. Chi-square analyses (2) were used to examine the differences between the proportions of positive isolates obtained for the different media. All reference and type strains tested grew on DTA within 24 h; however, 74.2 and 29% of the strains failed to grow on DMG or DTB, respectively (Table 1). The variable results obtained with some strains may be due to differences in the amount of inoculum streaked on the agar. In another study (data not presented), inhibition on DMG or DTB was less when colonies were streaked directly on the agar than when a loop was used to streak broth cultures. S. aureus gave strong positive DNase reactions with the three media, and it was the only DNase-positive species tested which did not give variable results. S. chromogenes, S. hyicus, and S. intermedius showed strong positive reactions with DTA and DTB but variable results with DMG. Variable results were obtained for S. carnosus and S. lentus after 24 h with DTA; however, all results were positive after 48 h. Weak reactions were obtained for S. capitis, S. gallinarum, and S. simulans only after 48 h of incubation with DTA. Results obtained with bovine strains of staphylococci were similar to those obtained for the reference and type strains (Table 2). A greater percentage of DNase-positive strains was detected with DTA than with DMG or DTB (P < 0.01). The percentage of positive strains was greater with DTB than with DMG (P < 0.05). Greater percentages of DNase-positive strains were obtained after incubation for 48 h than with 24 h of incubation (P < 0.05). In general, some bovine strains of each DNase-positive species were inhibited by DMG and DTB. Strains of S. chromogenes were inhibited the most. Less than 3% of bovine S. chromogenes strains showed production of DNase on DMG after 24 h compared with 97.3% on DTA and 36.4% on DTB. DTB was more inhibitory to bovine S. aureus strains than was DMG. Approximately 23% of bovine S. epidermidis strains and 11.1% of S. hominis strains showed production of DNase with DTA after 24 h. None of these strains was positive with DMG or DTB after 24 h. Incubation 1127

1128

J. CLIN. MICROBIOL.

NOTES

TABLE 1. Comparison of methods for determining production of DNase by reference and type staphylococcal strains

Response" at indicated time on: DTA"

Staphylococcus species

-

NG

NG

+ +

+ + -

+ + NG NG NG

+

NG, w NG, w NG, w

+

w, w, -

w w + + +

+ + NG NG NG

NG, w

+

+

NG -, w NG

NG -, w NG

-

NG, NG, NG, -

NG, -

NG,-

-

w

NG NG. -

-

-

-

w, -

+ + +

+ +

w, -

+ -

w

-

+

+ w -

+ +

+ +

NG,-

NG,NG, NG,-

NG NG -

-,

w

-, w

NG, NG NG, -

NG,NG, -

NG, -

-, +

+

NG NG -

NG NG

NG,-

NG,-

+ +

+ + +

w

NG, -

NG, NG -. + -, + NG, + NG NG NG NG, w NG,-

-

48h

24h

48h

24h

48h

24h

S. arlettae BP36 S. aureus ATCC 12600 S. aureus ATCC 25923 S. auricularis ATCC 33753 S. capitis ATCC 27840 S. capitis CE2 S. caprae ATCC 35538 S. carnosus MA S. carnosius F61 S. caseolyticius ATCC 13548 S. chromogenes May 1 S. cohnii ATCC 29974 S. cohnii JL143 S. cohnii DBM388 S. epidermnidis ATCC 14990 S. equoruin PA218 S. gallinarum ATCC 35539 S. gallinarum VIII S. haemolylicus ATCC 29970 S. hominis ATCC 27844 S. hyicus (coagulase negative) BG1222 S. hyicus (coagulase positive) BG1479 S. intermedijus ATCC 29633 S. kloosii DM56 S. lentus ATCC 29070 S. saprophyticus ATCC 15035 S. sciairi ATCC 29062 S. simulans ATCC 27848 S. warneri ATCC 27836 S. xylosus ATCC 29971

DTB

DMG

NG - + - +

NG, + NG NG, w NG NG, w W, w-

+

-

NG,-

NG, -

-

-, Negative reaction; +, positive reaction; NG, no growth, w, weak reaction. "After the addition of HCI to DTA following incubation.

TABLE 2. Comparison of methods for determining production of DNase by staphylococci of bovine origin for 48 h was required for detection of DNase production by S. simulans with DTA. Over 18% of S. simulans strains showed production of DNase with DMG after 24 h. The false-positive results obtained for some DNase-nega-

tive strains (S. haemolyticus and S. saprophyticus) with DMG apparently were due to changes in the color of the medium surrounding their growth that were not related to depolymerization of DNA by DNase. Results of this study indicate that many bovine strains and reference and type strains of newly described and recognized Staphylococcus species do not grow on DMG or DTB. Therefore, these two media are not recommended for use to detect the production of DNase by staphylococci of bovine origin or for use with many of the newly defined Staphylo-

species. All reference and type strains grew on the media used to determine phosphatase activity. In general, similar results were obtained with the four methods used to determine phosphatase activity of the type and reference strains (Table 3). More variable results were observed with PMP than with the other methods. S. kloosii gave variable results with all methods except PNP, while S. equorum, S. lentus, and S. saprophyticus gave variable results with PMP. Variable results were obtained for S. caprae and S. carnosus F61 with coccus

PAL. No significant differences were obtained in the percentages of phosphatase-positive bovine strains within a species for the four methods (Table 4).

% Positive reactions

Staphylococcus species

S. S. S. S. S. S. S.

aureus caseolyticus chromogenes epidermidis

haemnolyticis

hominis hyicus (coagulase negative) S. hvicus (coagulase positive) S. intermedius S. lentus S. sapprophyticus S. Scilluri S. simulans S. warneri S. xyloss S. hominisl S. warneri

St'aphvlococcuis spp.

No. of iso-

DTA"

plates

DMG

DTB

48 h

24 h

48 h

24 h

75

100

100

96.0 98.7

1

0

0

110 22 2 9 5

100

100

2

50

1 1

0 0

S

0

97.3 99.1 2.7 32.8 22.7 50.0 0 31.8 50 100 0 0 11.1 22.2 0 11.2 40 100 60.0 100

1

2 il 5 4 18

0

48 h

85.3

88.0

0

0

36.4 87.3 0 0 0 0 0 0 60 0

0

100

100

100

100

0

100

50

100

0 0

0 0

0 100

0 0

0 0

0 0 0 0 0

0 0 0 0 0

O

O

100 100 100 100 36.4 18.2 54.5 0 20 0 0 0 0 0 0 0 11.1 16.7 33.3 0 O

24 h

O

O

O

72.3 79.6 29.6 50.0 38.7 61.3 Mean"' "After the addition of HCI to DTA following incubation. ">Chi-square analysis; DTA > DMG, DTB (P < 0.01); DTB > DMB (P < 0.05); DTA 48 h > DTA 24 h (P < 0.05); DMG 48 h > DMG 24 h. DTB 24 h (P < 0.01).

VOL. 27, 1989

NOTES

TABLE 3. Comparison of methods for determining phosphatase activity of reference and type staphylococcal strains

24 h

arlettae BP36 aureus ATCC 12600 aureus ATCC 25923 auricldaris ATCC 33753 S. capitis ATCC 27840 S. capitis CE2 S. caprae ATCC 35538 S. carnosus MA S. carnosus F61 S. caseolyticus ATCC 13548 S. chromogenes May 1 S. cohnii ATCC 29974 S. cohnii JL143 S. cohnii DBM388 S. epidermidis ATCC 14990 S. equoruin PA218 S. gallinarurn ATCC 35539 S. gallinarum VIII S. haemolvticus ATCC 29970 S. hominis ATCC 27844 S. hyicus (coagulase negative) BG1222 S. hyicus (coagulase positive) BG1479 S. intermedius ATCC 29633 S. kloosii DM56 S. lentus ATCC 29070 S. saprophyticus ATCC 15035 S. sciuri ATCC 29062 S. simulans ATCC 27848 S. warneri ATCC 27836 S. xylosus ATCC 29971

Staphvlococcus species

PNP______ PAL,____

species

S. S. S. S.

TABLE 4. Comparison of methods for determining phosphatase activity of staphylococci of bovine origin

Response" at indicated time on: PMP PDP PNP PAL

Staphylococcus

48 h

24 h

48 h

24 h

24 h

w + + -

w

-

+ + -

+ + -

+ + -

+ + + -

+ + + -

-

-

-

-

-

-

+ +

w, -

S. (aur1elus S. caseoNlticls S. chrormogenes

+ + + + +

+

+

+

+

+

+ +

+ + +

w, - w

-

-

w

+

+

+

+

+

+

StaphYlococcils

-

-

-

-

+ +

+ +

+ +

+ +

+ + + + +

+

+ + + + +

+ + + + +

No. of

S. S. S. S.

%` Positive reactions PDP PNP, 48 h 24 h 48 h 24 h

PMP

isolates

24 h

epiderimidis haemoNlvtics hominis hvicius (coagulase negative) S. hvicius (coagulase positive) S. intrrmnediius S. lentus S. saprophvticuls S. sciluli S. simrulans S. warneri S. xlosuls S. hoilninisl S. wvar-neri

-

1129

PAL, 24 h

2 9 5

100 100 100 100 100 100 0 0 0 0 0 0 96.4 98.2 98.2 98.2 98.2 98.2 90.9 95.5 90.9 90.9 90.9 100 0 0 0 0 0 0 0 0 0 0 0 0 80 80 80 80 80 100

1

1(0

100

100

100

100

100

2 1 1

100

100

100

100

100

100

0 0

0 0

0 0

0 0

0 0

0 0

75 1 110 22

2 il 5 4 18

5

100 100 45.5 63.6 O

O

75 100 11.1 16.7

50 45.5 O

75 5.6

50 100 54.5 45.5 O

O

50 36.4 O

75 100 100 5.6 5.6 5.6

O

O

O

O

O

O

80.3

82.8

80.3

80.7

81.0

81.4

spp.

+

+

+

+

+

+

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Mean' "

Chi-square analysis found no significant differences.

-

staphylococci. However, on the basis of ease of use, PNP would be the preferred method of determining phosphatase activity of staphylococci.

-,+ -,w -+ -,w -,+ w -, w -,w -, w - w - w

The technical assistance of Sara Jenkins is gratefully acknowledged.

+ +

+ +

+ +

+ +

+ +

+

+

+

+

+

+

+

LITERATURE CITED 1. Langlois, B. E., R. J. Harmon, and K. Akers. 1988. U se of lysostaphin and bacitracin susceptibility for routine presumptive identification of staphylococci of bovine origin. J. Food Prot. 51:24-28. 2. Matthews, D. E., and V. T. Farewell. 1985. Using and understanding medical statistics. S. Karger, New York. 3. Phillips, E., and P. Nash. 1985. Culture media, p. 1051-1092. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.). Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 4. Waller, J. R., S. L. Hodel, and R. N. Nuti. 1985. Improvement of two toluidine blue O-mediated techniques for DNase detection. J. Clin. Microbiol. 21:195-199.

-, Negative reaction; +, positive reaction; w. weak reaction.

Except for S. simulans, 75% of the strains within phosphatase-positive species were positive for phosphatase. Approximately 46% of bovine strains identified as S. simulans were positive for phosphatase by all methods except PAL. Results of this study indicate that any of the four methods would be suitable for determining phosphatase activity of