A Convenient Method for Differentiation of Coagulase-Negative Staphylococci Isolated from Bovine Mammary Glands1

A Convenient Method for Differentiation of Coagulase-Negative Staphylococci Isolated from Bovine Mammary Glands' J. L. WAlTSF C. H. RAY, and P. J. WASHBURN Mastitis Research Laboratory Hill Farm Research Station Louisiana Agricultural Experiment Station Louisiana State University Agricultural Center Route 1, Box 10 Homer 71040 ABSTRACT

Abbreviation key: AC = arabinosecellobiose, TM = trehalose-mannitol.

The utility of trehalosemannitol broth and arabinosecellobiose broth for identification of Staphylococcus epidermidis and novobiocin-resistant staphylococci was determined using 236 coagulasenegative staphylococci isolated from bovine mammary glands. None of the 49 S. epidermidis strains was positive in &alose-mannitol broth; whereas, all strains of Staphylococcus hyicus, Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus horninis, Staphylococcus warneri, and Staphylococcus simulans were positive. Of the novobiocin-resistant staphylococcal species, only Staphylococcus saprophyticus was negative in arabinosecellobiose broth. Except for one strain of Staphylococcus sciuri and one strain of Staphylococcus kloosii, all remaining strains of novobiocin-resistant staphylococcal species were positive in arabinosecellobiose broth. Results indicate that trehalose-mannitol broth is an acceptable method for identification of S. epidennidis isolated fmm bovine mammary glands. Furthermore, arabinose-cellobiose broth is a useful method of screening for novobiocin-resistant staphylococci. (Key words: coagulase-negative staphylococci, identification, bovine mastitis)

INTRODUCTION

Coagulase-negative staphylococci are frequently isolated from bovine teat skin, teat canals, and milk (I, 2, 3, 5, 10). These organisms, previously considered nonpathogenic, are now recognized to cause elevated SCC and reduced milJt production (1,2).Recent studies (5, 10) have demonstrated that the distribution of coagulase-negative staphylococci may be indicative of specific management practices such as postmiIking teat antisepsis. Thus, accurate identitication of these organisms is needed for development of improved mastitis control methods and epidemiological studies. Conventional microtube methods for identification of coagulase-negative staphylococci are tedious and timeconsuming (9). Commercial microbial identification systems offer the advantage of being rapid, accurate, and convenient (7, 8, 9, 11, 12, 13). Several systems have yielded acceptable accuracy with staphylococci isolated from bovine mammary glands (8, 9, 11, 12). However, these systems are expensive; costs range from $2.50 to $4.00 per isolate. An inexpensive identification method is needed that would reduce reliance on commercial systems for identification of coagulase-negative staphylococci isolated from bovine mammary glands. Recently, Knapp and Washington (6) reposed that trehalosemannitol both could be used to differentiate Staphylococcus epidermidis from other staphylococcal species. However, no veterinary isolates were included in their study. The purpose of this study was to determine the utility of TM broth for differentiation of S. epidermidis isolated from bovine mammary glands. In addition, the utility of

o

Received July 2, 1990. Accepted Septembe~17, 1990. 'Approved for publication by the director of the Louisiana Asrjcultural J?xp&ncnt Station as h3anuscript Number W804368. komspmiing au&or. msent address: ~nimal ~ealth 'Lherapeatics Research, 7923-190-MR, The Upjohn CWpany. Knlnma.mo, MI 49002. 1991 I Dairy Sci 74426428

426

427

DIFFERENTIAnON OF STAFWYLOCOCCI

arabinose-cellobiose (AC) broth to separate broth were dispensed into 12 x 75 mm sterile novobiocin-resistant organisms such as Staphy- disposable plastic tubes. A single, well-isolated lococcus xylosus was also determined, colony removed from the surface of a blood agar plate was used to inoculate each medium. Inoculated tubes were incubated for 24 h at MATERIALS AND METHODS 37'C and observed for color change. A change in color from purple to yellow was considered Bacterla to be positive. A total of 236 coagulase-negative staphylococci isolated from bovine mammary RESULTS AND DISCUSSION glands and stored in the Mastitis Research LabThe coagulase-negative staphylococci are oratory culture collection were studied. Isolates from each species were selected to represent a divided into two groups based upon novobiocin wide variety of herds and geographic regions. SusceptibiJity (2, 4). Of the novobiocin-suscep All isolates had been identified using a previ- tible, coagulase-negative staphylococci, S. hyiously described conventional method (9) and cus, S. chromogenes, and S. epidermidis are the the API Staph-Trac system (Analytab Products, most frequently isolated from bovine mammary Plainview, NY). The following reference glands (1, 3, 5, 10). Differentiation of these strains were included in the study: Sraphylmoc- species from other novobiocin-susceptible cus hyicus ATCC 11249; Staphylococcus chro- coagulase-negative staphylococci by convenmgenes ATCC 43764, Staphylococcus simu- tional methods is tedious and time
TrehaloseMannltol and Arablnose-Celloblose Broth

Trehalose-mannitol broth was prepared as described by Knapp and Washington (6) by adding 2% D-trehalose and 2% D-mannitol to purple broth base (BBL Microbiology Systems). Arabinosecellobiose broth was prepared by adding L-arabinose and D-cellobiose to purple broth base (BBL Microbiology Systems) to achieve a final concentration of 2% for each carbohydrate. Two milliliters of TM and AC

s.

epi&ds

S. haemolyticus horninis warneri

s. s. s.

simulans

S. sapmphyticus

s. xyrosus s. sciwi S. S. S. S.

equonun gdliMm Moosii

arleme

56 49 1 15 23 5 3

17 6 3 1

4 4

100 0

0 0

100 100 100 100 100 100 100 100

0 0 0

100 100

100

0

0 100 83 100 100 75 100 ~~

staphylococcus

species.

Journal of Dairy Science Vol. 74, No. 2, 1991

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WATlS ET AL.

Knapp and Washington (6) for coagulase-negative staphylococci other than S. epidermidis. Furthermore, these workers (6) determined that using .2 ml of TM broth allowed differentiation of 94% of S, epidermidis isolates at 6 h. Thus, isolates yielding positive reactions at incubation times of less than 24 h would not be considered S. epidermidis. These isolates could be further differentiated using additional tests such as DNase (S. hyicus-positive) (4) or a commercial identifkation system. Of the novobiocin-resistant, coagulase-negative staphylococcal species tested, only StaphyZocuccw suproplryticw was negative in AC broth. All 17 S. xylosus and five of six (83%) S. sciuri strains were positive in AC broth. Of the remaining four novobiocin-resistant species tested, all were positive in AC broth except one isolate of S. kloosii. However, these four species recently have been described and have not been isolated from bovine mammary glands. Thus, AC broth is an acceptable method of screening for novobiocin-resistant, coagulasenegative staphylococci such as s. xylosus. Furthermore, AC broth could be inoculated at the same time as TM broth, permitting preliminary screening of isolates prior to use of commercial identification systems. Separation of staphylococci based upon novobiocin susceptibility can be performed using either antibioticimpregnated media or disk diffusion techniques (2, 4, 9). However, the user must be aware of interpretive criteria for each technique. Use of AC broth would provide a simpler technique for separation of coagulase-negative staphylococci. However, further identification would be needed to place positive in AC broth strains into an exact species. In summary, TM broth is an acceptable method for differentiation of coagulase-negative staphylococci isolated from bovine mammary glands. Furthermore, AC broth is a useful screening method for separation of novobiocinresistant, coagulase-negative staphylococci. These tests are extremely cost-effective with cost per isolate placed at $.23. Use of TM and

J o d of Dairy Science Vol. 74. No. 2, 1991

AC broth would reduce the need for costly commercial systems in the routine identification of coagulase-negative staphylococci isolated from bovine mammary glands.

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