Evaluation of Two Fluorogenic Assays for Identification of Streptococcus Species Isolated from Bovine Mammary Glands and S. H. KING Department of Animal Science Institute of Agriculture University of Tennessee Knoxville 37901-1071
K. R. MATTHEWS, S. P. OLIVER,'
ABSTRACT
INTRODUCTION
Liquid and agar assays that utilized 4-methylumbelliferyl-conjugated glucuronide, PD-galactoside, or Nacetyl-P-D-glucosaminide,and agglutination by Dolichos bijlorus lectin were evaluated for identification of Streptococcus species isolated from bovine mammary glands. A greater number of Streptococcus uberis isolates were negative for N-acetyl-P-D-glucosaminidaseby the liquid assay compared with the agar assay. Enzyme profiles for Streptococcus dysgalactiae were similar by both assays. Streptococcus Efysgalactiae was the only species that agglutinated when mixed with lectin from D. bigorus. Most Streptococcus agalactiae isolates were positive for P-D-glucuronidase and P-D-galactosidase by both assays. Two Streptococcus equinus strains had negative enzyme profiles by the liquid assay; however, both strains had enzyme profiles consistent for S. equinus by the agar method. Incorporation of 4-methylumbelliferyl-conjugated substrates into trypticase soy agar did not appear to alter agar characteristics and eliminated aliquoting substrates and inoculating tubes. More than one enzyme profile was produced per Streptococcus species or serogroup by both methods. However, some profiles were similar between species, which hindered accurate identification of Streptococcus species. (Key words: streptococci, bovine, identification)
PD-
Received July 13, 1990. Accepted September 17, 1990. 'Send reprint request to S. P. Oliver. 1991 J Dairy Sci 74:421425
Streptococcus species encompass a large heterogeneous group of bacteria. Predominant species involved in bovine mastitis are Streptococcus agalactiae, Streptococcus dysgalacthe. and Streptococcus uberis (11, 17). Other species involved less frequently include Streptococcus equinus, Streptococcus equi, Streptococcus alactolyticus, Streptococcus saccharolyticus, Streptococcus acidimonius, Enterococcus faecalis, and Enterococcus faecium (17). Identification and characterization of Streptococcus species of bovine origin have been based primarily on a few biochemical tests including: CAMP test, esculin and hippurate hydrolysis, reduction of .l% methylene blue milk, and inulin and raffinose fermentation (2). These tests are adequate for identification of S. agalactiae but less accurate for differentiation of other Streptococcus species. The most reliable procedures for identification of Streptococcus species include a battery of conventional biochemical tests in combination with serotyping. Watts (17) proposed a scheme to identify streptococci isolated from bovine mammary glands: it utilized commercial serogroup reagents, rapid commercial tests, and conventional carbohydrate utilization tests. This system is laborious, time consuming, expensive, and may not be practical for use in all laboratories. Schaufuss et al. (14) described a more rapid and convenient method for identification of streptococci of bovine origin. The procedure utilized 4-methylumbellife~l-wnjugatedsubstrates for detection of bacterial enzymes. This method was based on generation of fluorescence when free 4-methylumbelliferone was released by enzyme hydrolysis of substrates (1). Various 4-methylumbelliferylconjugated substrates have been used for rapid detection of enzyme activities of a variety of microorgan421
422
h4A'ITHEwS ET AL.
isms (5, 7, 8, 9, 13, 16). Additionally, use of (14). The 4-methylumbelliferylconjugated sublectins that unnbine specifically with defined strates p-D-glucuronide, p-D-galactoside, and sugar structures has been studied for detecting N-acetyl-p-D-glucosaminide(Sigma Chemical Group C streptococci (12, 14). The purpose of Co.,St. Louis,MO)were prepared as described this study was to compare the liquid assay by Slifkin and Gil (15). Briefly, p-Ddescribed previously by Schaufuss et al. (14) glucuronide (15 pnol/L) and p-D-galactoside with a method that employs incorporation of (5 p o l / L ) stock solutions were dissolved in .2 4-methylumbelliferyl substrates into agar base ml of dimethyl sulfoxide, and .2 ml of Nmedium to conventional methods for differenti- acetyl-&D-glucosaminide (15 pnol/L) was disation of streptococci isolated from bovine milk. solved in N, Ndimethylfoxmamide. AU substrate solutions were further diluted to 10 ml in MATERIALS AND METHODS acetate buffer, pH 5.2. Substrate solutions were filter sterilized using .45 pn filters (Aerodisc, Streptococcal isolates (n = 96) of bovine Gelman Sciences Inc., AM Arbor, MI) and origin were evaluated. Most strains (n = 78) stored up to 2 wk at -2o'C. were isolated from bovine milk samples colColonies of each streptococcal isolate were lected from the University of Tennessee dajr suspended in the respective 4-methylumbelresearch herds. Remaining strains (n = 14) were Werylconjugated substrate solution (14). Reacsupplied by J. L. Watts, H ill Farm Research tants were incubated for 1 to 2 h at 37'C, and Station, Homer, LA. The following American Type Culture Collection (ATCC, Rockville, 20 pl of .lN NaOH or saturated NaHCO3 were MD) reference strains were included: S. agdac- added to each inoculated substrate. Slifkin and tiae, ATCC 27956; S. dysgalactiae, ATCC Gil(15) reported that the addition of NaOH or 27957; S. uberis, ATCC 27958; and S. equinus, NaHC03 enhanced fluorogenic response when visualized by W light (366 nm). A positive ATCC 27960. reaction was indicated by development of a distinct light blue fluorescence. Conventional Biochemical Tests Bacteria were stored at -8o'C in 10% skim milk (BBL, Cockeymille, MD). Prior to testing, isolates were subcultured twice in -ti-
case soy broth (BBL), streaked for isolation on trypticase soy agar medium supplemented with 5% sheep blood (BBL), and incubated 18 h at 37'C. Streptococcal isolates were identified on the basis of hemolytic characteristics; growth in 6.5% NaCI; hydrolysis of sodium hippurate, esculin, and arginine; and CAMP reaction (2). In addition, reduction of .l% methylene blue milk,production of acetyhethyl carbinol, and growth in 10 and 40% bile were determined (4). Acid production from carbohydrates was detected using an agar plate method (3). C a d x ~ hydrates tested were mannitol, arabinose, raffinose, ribose, sorbitol, inuljn, trehalose, glycerol, xylose, salicin, lactose, and sucrose (6). Presence of Lancefield group antigens was determined with a latex agglutination system (Streptex, Wellcome Diagnostics, Research Triangle Park, NC). Liquid Assay
The presence of thee constitutive enzymes was evaluated as described by Schaufuss et al. Journal of Dairy Science Vol. 74, No. 2, 1991
Agar Assay
Trypticase soy agar (BBL) was prepared according to manufacturer's directions and supplemented with 150 mg/L of the respective 4-methylumbelliferyl-conjugated substrates (i.e., B-D-glucuronide, N-acetyl-6-D-glucosaminide, or PD-galactoside). Medium was dissolved by mixing for 30 min at 1 W C and sterilized by autoclaving for 15 min. Medium (pH 7.2) was tempered at 45°C for 20 min and poured (20 ml) into 100 mm diameter petri plates. Plates were stored at 4'C not longer than 2 wk Plates were streaked radially with 10 j.~l of a 24-h broth culture and incubated for 18 to 24 h at 37'C. No more than six streptococcal c u l m s were streaked per plate. plates were read under an W lamp at 366 nm without addition of .lN NaOH or saturated NaHCO3. Lectin Agglutination
Lectin (1 mg) from Dulichus bifurus (Sigma) was suspended in 1 ml phosphate-buffered saline, pH 7.2 @ifm Laboratories, Detroit, MI) (14). Lectin suspension (20 pL) was added to
423
IDENTfFICATION OF STREeTocOCCI
TABLE 1. ~etstimof species-specific enzyme activity of streptococci isolated from bovine milk using the liquid assay.' NUIllbeX
organism
tested
Streptococcus agclrcrctioe
A
&curonidme
d 27956 5 1
Streptococcus dysgalactiae
Streptococcus uberis
Streptococcus equinus
Enterococcus s p p 3
eD
ATCC 27957 23 4 ATCC 27958 23 21 Am27960 4 2 2 5 2
Enyme activity N-acetvl-EDRDg l m a d a s ~ ;Talactosidase
+ +
+
+ -
+
-
+ + + + + +
+ +
+ + +
+ + -
-
-
-
-
-
-
c
+ + -
+ + +
-
-
+ +
+
-
Agglutination by Dolichos bi~omlectin
+ +4
+5
-
-
-
-
'Liqaid assay utdizing 4-mthylumbellifcryl-conjugatcd substrates. ?4merican Type culture collection.
3Enterococcusfaecdis and Enterococcus faecium. and 3 negative reactions. 5 ~ 0 t a lof 3 positive and 1 negative reactions.
4Tota1 of 20 positive
20 Cl.1 of a buffered streptococcal suspension and placed on a microscope slide. As a control, 20 pl of bacterial suspension were added to 20 p.l of buffer. Slides were rotated by hand for 1 min. A distinct agglutination of bacteria and no auto-agglutination of the buffer control indicated a positive reaction.
ilar. For all isolates tested, no differences between NaOH or NaHCO3 in fluorogenic inten-
sity were observed. Identification of streptococcal isolates using 4-methylumbelliferyl-conjugatedsubstrate agar base medium produced more consistent results than the liquid assay uable 2). All S. agulactiae isolates were positive for PD-glucuronidase and f&D-galactosidase. Most strains of S. RESULTS dysguluctiue (n = 24) produced only &DDetection of Streptococcus species specific glucuronidase, with the exception of four enzymes using 4-methylumbelljferyl substrates, strains that utilized all three substrates. A p p x &D-glucuronide, N-acetyl-&D-glucosaminide, imately 86% of S. dysgaluctiue strains agglutior p-D-galactoside by the liquid assay is in nated when mixed with D. biflorus lectin. None Table 1. All Streprococcus species exhibited of the other streptococcal species produced an two or more enzyme profiles. However, each agglutination reaction. About 82% (n = 37) of species had one predominant profile, with the S. uberis strains utilized all three substrates. exception of S. uberis, which had approxi- Streptococcus equinus and enterococci were mately equal number of strains positive and negative for P-D-glucuronidase. Most strains of negative for N-acetyl-&D-glucosaminidase. S. equinus (7 of 9) and enterococci (5 of 7) Only S. dysgalactiue strains agglutinated when hydrolyzed only N-acetyl-&D-glucosaminidase mixed with the lectin of D. biflorus. However, and j3-D-galactosidase. four S. dysgalactiue strains failed to agglutinate at lectin concentration as high as 1 mg/ml. DISCUSSION Only 2 of 96 strains (S. equinus) produced In previous studies (10, 14, 15). assays to negative enzyme pfiles. Enzyme p f j l e s for Enterococcus f'calis and E. faecium were sim- identify streptococci using 4-methylumbelJournal of Dairy Science VoL 74, No. 2, 1991
424
MATLlIEwS ET AL.
TABLE 2. Differentiation of Streptococcus species of bovine origin utilizing 4-metbylumbeUiferyl-conjugatedsubstrate agar medim. NUmk
BB
Enyme activity N-acelyl-fbD-
BD-
organism
tested
glucuronidast glacosaminidase galactosidase
Streptococcus agalacricle
ATCC' 27956
+
4
Streptococcus dysgalactiae
2 ATCC 27957 23 4
Streptococcus uberis
ATCC 27958 36
Streptococcus eguinus
ATCC27960
8 6
Enterococcus spp2
2 5 2
+
+ +
+
+ +
+ + -
-
+w5
+ -
+ + + -
+ + + + +
+ + +
-
-
+ + + + + + -4.
-
Agglutination by Dolicbs bifloms lectin
-
+ +3 +4
-
-
-
~Anleri- Type culm Collection. 2Enterococcus faecah and Enterococcus faecium. 3Total of 21 positive mi 2 negative reactions. %otal of 2 positive and 2 negative reactions. 5(w) = weak reaction.
liferyl-conjugated substrates were either carried out in test tubes or on filter paper. However, Trepeta and Edberg (16) reported on incorporation of 4-methylumbelliferyl-p-D-glucuronide into MacConkey agar base as a rapid method for isolation and identification of Escherichia coli. No changes in selectivity or lactose fermentation characteristics of MacConkey agar were observed when the umbelliferyl reagent was incorporated into the medium. In the present study, incorporation of 4-methylumbelliferyl-conjugated substrates into qpticase soy agar did not appear to alter agar characteristics and eliminated aliquoting substrates and inoculating tubes. Small volume (20 pl) of substrate solution, coupled with small test tubes, made it difficult to suspend streptococcal colonies, detracting from the utility of the liquid assay. Differences in enzyme profiles may be pronounced between human and bovine isolates. Lammler et al. (9) attempted to differentiate group B streptococci of human and bovine origin using the liquid assay. Results showed that neither human nor bovine isolates produced N-acetyl-P-D-glucosiuninidase. HowJournal of Dairy Science Vol. 74, No. 2, 1991
ever, 74 of 77 bovine isolates and only 10 of 82 isolates from humans produced p-D-galactosidase. All human and bovine isolates evaluated produced p-D-glucuronidase. Slifkin and Gil (15) employed a filter paper assay and screened 180 streptococcal isolates from human throat cultures. The enzyme profile for group B streptococci was the same as that for human isolates reported by Lammler et al. (9). Slifkin and Gil (15) reported also that all three fluorogenic enzyme substrates were hydrolyzed by all strains of group C streptococci. In addition, those group C streptococci were all agglutinated when mixed with the lectin of D. bifzorus. In the present study, 86% of S. dysgalactiue (group C) hydrolyzed only p-Dglucuronide and approximately 92% of those strains agglutinated when mixed with the lectin of D. biforus (Table 2). Schaufuss et al. (14) reported on rapid differentiation of streptococci isolated from cows with mastitis. The liquid assay was used, but pD-mannosidase activity was assayed, not p-Dgalactosidase. In contrast to results of the present study for S. dysgalactiae, they (14)reported 77% of S. dysgalactiae isolates hydrolyzed N-
425
IDENTIPICATION OF STREPTOCOCCI
acetyl-P-D-glucosaminide.Variability reported by Schaufuss et al. (14) in hydrolysis of Nacetyl-p-D-glucosaminideby S. uberis isolates was similar to that in Table 1. Therefore, differences in enzyme profiles for streptococcal spe cies may depend on origin. The agar assay was simple to perform compared with the liquid assay. However, species identification of streptococci was not enhanced. More than one enzyme profile was produced per Streptococcus species or serogroup, and some profiles were similar between species. Effective use of either assay would q u k additional tests, such as plating isolates on blood esculin agar, to detect esculin hydrolysis and hemolytic patterns from individual colonies. Use of D. bzjZorus lectin alone was effective in identifying S. dysgalactiae (86%) correctly. The test was simple, rapid, and may be recommended as an additional test when identifying S. dysgalactiae. Streptococcus equinus and Enterococcus species can be identifed readily because they were the only species tested which did not hydrolyze p-Dglucuronide. However, differentiation of group D nonenterocaxi from Enterococcus species was not possible with either 4methylumbelliferyl assay. Results of this study indicate that neither the liquid nor agar assays should be recommended exclusively for identification of bovine strep tococci. A disadvantage of both fluorogenic assays was the number of enzyme profiles compiled for each Streptococcus species. The necessity of additional biochemical tests, length of time from isolation of the organism to assay completion, and variability of enzyme profiles for each species preclude rapid identification of streptococci. ACKNOWLEDGMENTS
This investigation was supported by the Tennessee Agricultural Experiment Station and the University of Tennessee College of Veterinary Medicine Center of Excellence Research Program in Livestock Diseases and Human Health. Authors express their appreciation to Arlene Stewart for excellent clerical assistance.
REFERENCES
lBradtnuy, J. M. 1977. Rapid biochemical tests for characterization of W mycoplasnetales. J. Clin. Microbiol. 5531. ZBrown, R. W.. D. A. Bamum, D. E. Jasper, J. S. McDonald, and W. D. Schultze. 1981. Page 16 in Microbiological procedures for use in the diagnosis of bovine mastitis. Natl. Mastitis Counc., Arlington, VA. 3 Colman, G., and R E O . Williams. 1972. Taxonomy of some human viridans streptococci. Page 218 in Seep tococci and streptococcal diseases, recognition,understaudiug, and management. L.W.Wannamaker and I. Matsen, ed. Academic Ress. Inc., New York, NY. 4Cowan. S. T., and K. J. Steel. 1977. Mauual for identifcation of medical bacteria. 2nd ed. Cambridge University Press, Cambridge, Engl. 5 Facklam, R. R. 1976. A review of the microbiological techuiques for the isolation and identification of strep tococci. Crit. Rev. Clia Lab. Sci. 6287. 6Facklam, R R 1977. physiological di€fexentiation of viridaw sireptococci J. Clin. MicrobioL 5:184. 7Facklam, R R., and R. B. Carey. 1985. Streptococci and aerococci. Page 154 in Manual of cliuical microbiology. Am. Soc. Microbiol., Washington, DC. 8 Grange, J. M., and K. Clark. 1977. Use of umbelliferone derivatives in the study of enzyme activities of mycobacteria. J. clin Path. 30:151. 9 Lammler, C., P. Schaufuss,and H. Blobel. 1986. galactosidase activity of serological Group B. Zentralbl. Bakteriol. Hyg. A261, 167. lOMaddoch, J. L., and M. J. Greenau 1975. A rapid method for idenbacterial enzymes. J. Clin pathol. 28:686. 11 McDonald, T. J., and J. S. McDonald. 1976. Strep tococci isolated from bovine int~amammtayinfections. Am. J. Vet. Res. 37377. 120ttensoone1, F.,Y.Nakamizo, M. Sam, Y. Miyamoto, and K. Takkawa. 1974. Lectins detectiug group C streptococci. Infect.. Immuu 9:971. 13 Robison, B. J. 1984. Evaluation of a fluomgenic assay for detection of Escherichia coli in foods. Appl. Envimn. Microbiol. a 2 8 5 14 Schaufuss, P., C. Lammler, and H.Blobel. 1986. Rapid differentiation of streptococci isolated from cows with mastitis. I. Clin. Microbiol. 24A098. 15SlifLin, M., and G. M Gil. 1983. Rapid biochemical tests for the identification of groups A, B, C, F, and G streptococci from throat cultures. J. Clin. Microbial. 1827. 16 Trepeta, R W., and S. C. Edberg. 1984. Methylumbellifeayl-J%D-glucuronide-basedmedium for rapid isolation and identification of Escherichia coli. J. Clin Mimbiol. 19:172. 17Wam, J. L. 1988. Characterization and identification of streptococci isolated from bovine mammary glands. J. Dairy Sci. 71:1616.
PD-
Journal of Dairy Science Vol. 74, No. 2, 1991
K. R. MATTHEWS, S. P. OLIVER,'
ABSTRACT
INTRODUCTION
Liquid and agar assays that utilized 4-methylumbelliferyl-conjugated glucuronide, PD-galactoside, or Nacetyl-P-D-glucosaminide,and agglutination by Dolichos bijlorus lectin were evaluated for identification of Streptococcus species isolated from bovine mammary glands. A greater number of Streptococcus uberis isolates were negative for N-acetyl-P-D-glucosaminidaseby the liquid assay compared with the agar assay. Enzyme profiles for Streptococcus dysgalactiae were similar by both assays. Streptococcus Efysgalactiae was the only species that agglutinated when mixed with lectin from D. bigorus. Most Streptococcus agalactiae isolates were positive for P-D-glucuronidase and P-D-galactosidase by both assays. Two Streptococcus equinus strains had negative enzyme profiles by the liquid assay; however, both strains had enzyme profiles consistent for S. equinus by the agar method. Incorporation of 4-methylumbelliferyl-conjugated substrates into trypticase soy agar did not appear to alter agar characteristics and eliminated aliquoting substrates and inoculating tubes. More than one enzyme profile was produced per Streptococcus species or serogroup by both methods. However, some profiles were similar between species, which hindered accurate identification of Streptococcus species. (Key words: streptococci, bovine, identification)
PD-
Received July 13, 1990. Accepted September 17, 1990. 'Send reprint request to S. P. Oliver. 1991 J Dairy Sci 74:421425
Streptococcus species encompass a large heterogeneous group of bacteria. Predominant species involved in bovine mastitis are Streptococcus agalactiae, Streptococcus dysgalacthe. and Streptococcus uberis (11, 17). Other species involved less frequently include Streptococcus equinus, Streptococcus equi, Streptococcus alactolyticus, Streptococcus saccharolyticus, Streptococcus acidimonius, Enterococcus faecalis, and Enterococcus faecium (17). Identification and characterization of Streptococcus species of bovine origin have been based primarily on a few biochemical tests including: CAMP test, esculin and hippurate hydrolysis, reduction of .l% methylene blue milk, and inulin and raffinose fermentation (2). These tests are adequate for identification of S. agalactiae but less accurate for differentiation of other Streptococcus species. The most reliable procedures for identification of Streptococcus species include a battery of conventional biochemical tests in combination with serotyping. Watts (17) proposed a scheme to identify streptococci isolated from bovine mammary glands: it utilized commercial serogroup reagents, rapid commercial tests, and conventional carbohydrate utilization tests. This system is laborious, time consuming, expensive, and may not be practical for use in all laboratories. Schaufuss et al. (14) described a more rapid and convenient method for identification of streptococci of bovine origin. The procedure utilized 4-methylumbellife~l-wnjugatedsubstrates for detection of bacterial enzymes. This method was based on generation of fluorescence when free 4-methylumbelliferone was released by enzyme hydrolysis of substrates (1). Various 4-methylumbelliferylconjugated substrates have been used for rapid detection of enzyme activities of a variety of microorgan421
422
h4A'ITHEwS ET AL.
isms (5, 7, 8, 9, 13, 16). Additionally, use of (14). The 4-methylumbelliferylconjugated sublectins that unnbine specifically with defined strates p-D-glucuronide, p-D-galactoside, and sugar structures has been studied for detecting N-acetyl-p-D-glucosaminide(Sigma Chemical Group C streptococci (12, 14). The purpose of Co.,St. Louis,MO)were prepared as described this study was to compare the liquid assay by Slifkin and Gil (15). Briefly, p-Ddescribed previously by Schaufuss et al. (14) glucuronide (15 pnol/L) and p-D-galactoside with a method that employs incorporation of (5 p o l / L ) stock solutions were dissolved in .2 4-methylumbelliferyl substrates into agar base ml of dimethyl sulfoxide, and .2 ml of Nmedium to conventional methods for differenti- acetyl-&D-glucosaminide (15 pnol/L) was disation of streptococci isolated from bovine milk. solved in N, Ndimethylfoxmamide. AU substrate solutions were further diluted to 10 ml in MATERIALS AND METHODS acetate buffer, pH 5.2. Substrate solutions were filter sterilized using .45 pn filters (Aerodisc, Streptococcal isolates (n = 96) of bovine Gelman Sciences Inc., AM Arbor, MI) and origin were evaluated. Most strains (n = 78) stored up to 2 wk at -2o'C. were isolated from bovine milk samples colColonies of each streptococcal isolate were lected from the University of Tennessee dajr suspended in the respective 4-methylumbelresearch herds. Remaining strains (n = 14) were Werylconjugated substrate solution (14). Reacsupplied by J. L. Watts, H ill Farm Research tants were incubated for 1 to 2 h at 37'C, and Station, Homer, LA. The following American Type Culture Collection (ATCC, Rockville, 20 pl of .lN NaOH or saturated NaHCO3 were MD) reference strains were included: S. agdac- added to each inoculated substrate. Slifkin and tiae, ATCC 27956; S. dysgalactiae, ATCC Gil(15) reported that the addition of NaOH or 27957; S. uberis, ATCC 27958; and S. equinus, NaHC03 enhanced fluorogenic response when visualized by W light (366 nm). A positive ATCC 27960. reaction was indicated by development of a distinct light blue fluorescence. Conventional Biochemical Tests Bacteria were stored at -8o'C in 10% skim milk (BBL, Cockeymille, MD). Prior to testing, isolates were subcultured twice in -ti-
case soy broth (BBL), streaked for isolation on trypticase soy agar medium supplemented with 5% sheep blood (BBL), and incubated 18 h at 37'C. Streptococcal isolates were identified on the basis of hemolytic characteristics; growth in 6.5% NaCI; hydrolysis of sodium hippurate, esculin, and arginine; and CAMP reaction (2). In addition, reduction of .l% methylene blue milk,production of acetyhethyl carbinol, and growth in 10 and 40% bile were determined (4). Acid production from carbohydrates was detected using an agar plate method (3). C a d x ~ hydrates tested were mannitol, arabinose, raffinose, ribose, sorbitol, inuljn, trehalose, glycerol, xylose, salicin, lactose, and sucrose (6). Presence of Lancefield group antigens was determined with a latex agglutination system (Streptex, Wellcome Diagnostics, Research Triangle Park, NC). Liquid Assay
The presence of thee constitutive enzymes was evaluated as described by Schaufuss et al. Journal of Dairy Science Vol. 74, No. 2, 1991
Agar Assay
Trypticase soy agar (BBL) was prepared according to manufacturer's directions and supplemented with 150 mg/L of the respective 4-methylumbelliferyl-conjugated substrates (i.e., B-D-glucuronide, N-acetyl-6-D-glucosaminide, or PD-galactoside). Medium was dissolved by mixing for 30 min at 1 W C and sterilized by autoclaving for 15 min. Medium (pH 7.2) was tempered at 45°C for 20 min and poured (20 ml) into 100 mm diameter petri plates. Plates were stored at 4'C not longer than 2 wk Plates were streaked radially with 10 j.~l of a 24-h broth culture and incubated for 18 to 24 h at 37'C. No more than six streptococcal c u l m s were streaked per plate. plates were read under an W lamp at 366 nm without addition of .lN NaOH or saturated NaHCO3. Lectin Agglutination
Lectin (1 mg) from Dulichus bifurus (Sigma) was suspended in 1 ml phosphate-buffered saline, pH 7.2 @ifm Laboratories, Detroit, MI) (14). Lectin suspension (20 pL) was added to
423
IDENTfFICATION OF STREeTocOCCI
TABLE 1. ~etstimof species-specific enzyme activity of streptococci isolated from bovine milk using the liquid assay.' NUIllbeX
organism
tested
Streptococcus agclrcrctioe
A
&curonidme
d 27956 5 1
Streptococcus dysgalactiae
Streptococcus uberis
Streptococcus equinus
Enterococcus s p p 3
eD
ATCC 27957 23 4 ATCC 27958 23 21 Am27960 4 2 2 5 2
Enyme activity N-acetvl-EDRDg l m a d a s ~ ;Talactosidase
+ +
+
+ -
+
-
+ + + + + +
+ +
+ + +
+ + -
-
-
-
-
-
-
c
+ + -
+ + +
-
-
+ +
+
-
Agglutination by Dolichos bi~omlectin
+ +4
+5
-
-
-
-
'Liqaid assay utdizing 4-mthylumbellifcryl-conjugatcd substrates. ?4merican Type culture collection.
3Enterococcusfaecdis and Enterococcus faecium. and 3 negative reactions. 5 ~ 0 t a lof 3 positive and 1 negative reactions.
4Tota1 of 20 positive
20 Cl.1 of a buffered streptococcal suspension and placed on a microscope slide. As a control, 20 pl of bacterial suspension were added to 20 p.l of buffer. Slides were rotated by hand for 1 min. A distinct agglutination of bacteria and no auto-agglutination of the buffer control indicated a positive reaction.
ilar. For all isolates tested, no differences between NaOH or NaHCO3 in fluorogenic inten-
sity were observed. Identification of streptococcal isolates using 4-methylumbelliferyl-conjugatedsubstrate agar base medium produced more consistent results than the liquid assay uable 2). All S. agulactiae isolates were positive for PD-glucuronidase and f&D-galactosidase. Most strains of S. RESULTS dysguluctiue (n = 24) produced only &DDetection of Streptococcus species specific glucuronidase, with the exception of four enzymes using 4-methylumbelljferyl substrates, strains that utilized all three substrates. A p p x &D-glucuronide, N-acetyl-&D-glucosaminide, imately 86% of S. dysgaluctiue strains agglutior p-D-galactoside by the liquid assay is in nated when mixed with D. biflorus lectin. None Table 1. All Streprococcus species exhibited of the other streptococcal species produced an two or more enzyme profiles. However, each agglutination reaction. About 82% (n = 37) of species had one predominant profile, with the S. uberis strains utilized all three substrates. exception of S. uberis, which had approxi- Streptococcus equinus and enterococci were mately equal number of strains positive and negative for P-D-glucuronidase. Most strains of negative for N-acetyl-&D-glucosaminidase. S. equinus (7 of 9) and enterococci (5 of 7) Only S. dysgalactiue strains agglutinated when hydrolyzed only N-acetyl-&D-glucosaminidase mixed with the lectin of D. biflorus. However, and j3-D-galactosidase. four S. dysgalactiue strains failed to agglutinate at lectin concentration as high as 1 mg/ml. DISCUSSION Only 2 of 96 strains (S. equinus) produced In previous studies (10, 14, 15). assays to negative enzyme pfiles. Enzyme p f j l e s for Enterococcus f'calis and E. faecium were sim- identify streptococci using 4-methylumbelJournal of Dairy Science VoL 74, No. 2, 1991
424
MATLlIEwS ET AL.
TABLE 2. Differentiation of Streptococcus species of bovine origin utilizing 4-metbylumbeUiferyl-conjugatedsubstrate agar medim. NUmk
BB
Enyme activity N-acelyl-fbD-
BD-
organism
tested
glucuronidast glacosaminidase galactosidase
Streptococcus agalacricle
ATCC' 27956
+
4
Streptococcus dysgalactiae
2 ATCC 27957 23 4
Streptococcus uberis
ATCC 27958 36
Streptococcus eguinus
ATCC27960
8 6
Enterococcus spp2
2 5 2
+
+ +
+
+ +
+ + -
-
+w5
+ -
+ + + -
+ + + + +
+ + +
-
-
+ + + + + + -4.
-
Agglutination by Dolicbs bifloms lectin
-
+ +3 +4
-
-
-
~Anleri- Type culm Collection. 2Enterococcus faecah and Enterococcus faecium. 3Total of 21 positive mi 2 negative reactions. %otal of 2 positive and 2 negative reactions. 5(w) = weak reaction.
liferyl-conjugated substrates were either carried out in test tubes or on filter paper. However, Trepeta and Edberg (16) reported on incorporation of 4-methylumbelliferyl-p-D-glucuronide into MacConkey agar base as a rapid method for isolation and identification of Escherichia coli. No changes in selectivity or lactose fermentation characteristics of MacConkey agar were observed when the umbelliferyl reagent was incorporated into the medium. In the present study, incorporation of 4-methylumbelliferyl-conjugated substrates into qpticase soy agar did not appear to alter agar characteristics and eliminated aliquoting substrates and inoculating tubes. Small volume (20 pl) of substrate solution, coupled with small test tubes, made it difficult to suspend streptococcal colonies, detracting from the utility of the liquid assay. Differences in enzyme profiles may be pronounced between human and bovine isolates. Lammler et al. (9) attempted to differentiate group B streptococci of human and bovine origin using the liquid assay. Results showed that neither human nor bovine isolates produced N-acetyl-P-D-glucosiuninidase. HowJournal of Dairy Science Vol. 74, No. 2, 1991
ever, 74 of 77 bovine isolates and only 10 of 82 isolates from humans produced p-D-galactosidase. All human and bovine isolates evaluated produced p-D-glucuronidase. Slifkin and Gil (15) employed a filter paper assay and screened 180 streptococcal isolates from human throat cultures. The enzyme profile for group B streptococci was the same as that for human isolates reported by Lammler et al. (9). Slifkin and Gil (15) reported also that all three fluorogenic enzyme substrates were hydrolyzed by all strains of group C streptococci. In addition, those group C streptococci were all agglutinated when mixed with the lectin of D. bifzorus. In the present study, 86% of S. dysgalactiue (group C) hydrolyzed only p-Dglucuronide and approximately 92% of those strains agglutinated when mixed with the lectin of D. biforus (Table 2). Schaufuss et al. (14) reported on rapid differentiation of streptococci isolated from cows with mastitis. The liquid assay was used, but pD-mannosidase activity was assayed, not p-Dgalactosidase. In contrast to results of the present study for S. dysgalactiae, they (14)reported 77% of S. dysgalactiae isolates hydrolyzed N-
425
IDENTIPICATION OF STREPTOCOCCI
acetyl-P-D-glucosaminide.Variability reported by Schaufuss et al. (14) in hydrolysis of Nacetyl-p-D-glucosaminideby S. uberis isolates was similar to that in Table 1. Therefore, differences in enzyme profiles for streptococcal spe cies may depend on origin. The agar assay was simple to perform compared with the liquid assay. However, species identification of streptococci was not enhanced. More than one enzyme profile was produced per Streptococcus species or serogroup, and some profiles were similar between species. Effective use of either assay would q u k additional tests, such as plating isolates on blood esculin agar, to detect esculin hydrolysis and hemolytic patterns from individual colonies. Use of D. bzjZorus lectin alone was effective in identifying S. dysgalactiae (86%) correctly. The test was simple, rapid, and may be recommended as an additional test when identifying S. dysgalactiae. Streptococcus equinus and Enterococcus species can be identifed readily because they were the only species tested which did not hydrolyze p-Dglucuronide. However, differentiation of group D nonenterocaxi from Enterococcus species was not possible with either 4methylumbelliferyl assay. Results of this study indicate that neither the liquid nor agar assays should be recommended exclusively for identification of bovine strep tococci. A disadvantage of both fluorogenic assays was the number of enzyme profiles compiled for each Streptococcus species. The necessity of additional biochemical tests, length of time from isolation of the organism to assay completion, and variability of enzyme profiles for each species preclude rapid identification of streptococci. ACKNOWLEDGMENTS
This investigation was supported by the Tennessee Agricultural Experiment Station and the University of Tennessee College of Veterinary Medicine Center of Excellence Research Program in Livestock Diseases and Human Health. Authors express their appreciation to Arlene Stewart for excellent clerical assistance.
REFERENCES
lBradtnuy, J. M. 1977. Rapid biochemical tests for characterization of W mycoplasnetales. J. Clin. Microbiol. 5531. ZBrown, R. W.. D. A. Bamum, D. E. Jasper, J. S. McDonald, and W. D. Schultze. 1981. Page 16 in Microbiological procedures for use in the diagnosis of bovine mastitis. Natl. Mastitis Counc., Arlington, VA. 3 Colman, G., and R E O . Williams. 1972. Taxonomy of some human viridans streptococci. Page 218 in Seep tococci and streptococcal diseases, recognition,understaudiug, and management. L.W.Wannamaker and I. Matsen, ed. Academic Ress. Inc., New York, NY. 4Cowan. S. T., and K. J. Steel. 1977. Mauual for identifcation of medical bacteria. 2nd ed. Cambridge University Press, Cambridge, Engl. 5 Facklam, R. R. 1976. A review of the microbiological techuiques for the isolation and identification of strep tococci. Crit. Rev. Clia Lab. Sci. 6287. 6Facklam, R R 1977. physiological di€fexentiation of viridaw sireptococci J. Clin. MicrobioL 5:184. 7Facklam, R R., and R. B. Carey. 1985. Streptococci and aerococci. Page 154 in Manual of cliuical microbiology. Am. Soc. Microbiol., Washington, DC. 8 Grange, J. M., and K. Clark. 1977. Use of umbelliferone derivatives in the study of enzyme activities of mycobacteria. J. clin Path. 30:151. 9 Lammler, C., P. Schaufuss,and H. Blobel. 1986. galactosidase activity of serological Group B. Zentralbl. Bakteriol. Hyg. A261, 167. lOMaddoch, J. L., and M. J. Greenau 1975. A rapid method for idenbacterial enzymes. J. Clin pathol. 28:686. 11 McDonald, T. J., and J. S. McDonald. 1976. Strep tococci isolated from bovine int~amammtayinfections. Am. J. Vet. Res. 37377. 120ttensoone1, F.,Y.Nakamizo, M. Sam, Y. Miyamoto, and K. Takkawa. 1974. Lectins detectiug group C streptococci. Infect.. Immuu 9:971. 13 Robison, B. J. 1984. Evaluation of a fluomgenic assay for detection of Escherichia coli in foods. Appl. Envimn. Microbiol. a 2 8 5 14 Schaufuss, P., C. Lammler, and H.Blobel. 1986. Rapid differentiation of streptococci isolated from cows with mastitis. I. Clin. Microbiol. 24A098. 15SlifLin, M., and G. M Gil. 1983. Rapid biochemical tests for the identification of groups A, B, C, F, and G streptococci from throat cultures. J. Clin. Microbial. 1827. 16 Trepeta, R W., and S. C. Edberg. 1984. Methylumbellifeayl-J%D-glucuronide-basedmedium for rapid isolation and identification of Escherichia coli. J. Clin Mimbiol. 19:172. 17Wam, J. L. 1988. Characterization and identification of streptococci isolated from bovine mammary glands. J. Dairy Sci. 71:1616.
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