JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1984, p. 279-280 Vol. 19, No. 2 0095-1137/84/020279-02$02.00/0 Copyright © 1984, American Society for Microbiology NOTES Five-Hour Novobiocin Test for Differentiation of Coagulase- Negative Staphylococci BRIAN J. HARRINGTONl* AND J. MICHAEL GAYDOS2 Mercy Hospital, Toledo, Ohio 43624,1 and The Toledo Hospital, Toledo, Ohio 436062 Received 22 August 1983/Accepted 20 October 1983

A 5-h broth disk test, read visually for growth or no growth to determine resistance of coagulase-negative staphylococci to 1.6 ,ug of novobiocin per ml, was evaluated as a rapid test for the presumptive identification of Staphylococcus saprophyticus. The correlation with an overnight disk diffusion test was 100%.

Recently, there has been an increase in the awareness that 29060, and S. simulans ATCC 27851. All 35 of the S. coagulase-negative staphylococci may be a cause of urinary saprophyticus isolates and 20 of the S. epidermidis isolates tract infections (4, 8, 13, 18). In particular, Staphylococcus were from urine. The other eight S. epidermidis isolates and saprophyticus has been shown tô be a common cause of the four S. hemolyticus isolates were from wound or blood urinary tract infections in young, sexually active female specimens. Coagulase testing was performed by both slide outpatients (4, 9, 19). Kloos and Schliefer showed that S. and tube tests, and only coagulase-negative isolates were saprophyticus, unlike the other commonly occurring coagu- tested for novobiocin resistance. Species identification was lase-negative staphylococci in humans, is resistant to novo- made by the API Staph-Ident system (Analytab Products, biocin at 1.6 ,ug/ml (10). Hovelius and Mardh found that their Plainview, N.Y.) or the AutoMicrobic system Gram-Posi- S. saprophyticus isolates had novobiocin minimal inhibitory tive Identification Card (Vitek Systems, Inc., Hazelwood, concentrations of between 128 and 512 ,ug/ml (7). Marrie and Mo.) or both. Disk diffusion susceptibility testing was per- Kwan reported that the minimal inhibitory concentrations of formed on Mueller-Hinton agar (BBL Microbiology Sys- novobiocin for S. saprophyticus and Staphylococcus cohnii tems) by the National Committee for Clinical Laboratory were in the range of 16 to 32 p.g/ml, whereas for Staphylo- Standards Bauer-Kirby method (17), except a 5-ktg novobio- coccus epidermidis and other novobiocin-susceptible, coag- cin disk was used and the susceptible breakpoint diameter of ulase-negative staphylococci, the minimal inhibitory concen- inhibition was >16 mm. Because it was known from disk trations of novobiocin were in the range of 0. 125 to 0.5 ,ug/ml diffusion testing that these organisms grow in Trypticase soy (14). Kloos and Smith, in the Manual ofClinical Microbiolo- broth at 37°C to a distinct turbidity (at least to a 0.5 gy (11), recommend the use of a 5-,ug novobiocin disk on P McFarland standard) within 5 h, this medium was used for agar as an overnight disk diffusion technique (10). P agar is a the broth disk test. The control tube of broth for this test was peptone-yeast extract-glucose medium which was not avail- the Bauer-Kirby inoculum broth. The test procedure consist- able commerically until recently. Several investigators have ed of very lightly inoculating two tubes each containing 3.0 reported the use of other, more readily available media for ml of Trypticase soy broth with the organism to be tested, as the disk diffusion test. Almeida and Jorgensen (1) compared though both tubes were to be used for the disk diffusion the results obtained with Mueller-Hinton agar with those inoculum (17). Immediately after inoculation, one 5-,ug no- obtained with P agar and concluded that Mueller-Hinton vobiocin disk (BBL Microbiology Systems) was added to agar is a suitable alternative to P agar. Goldstein et al. (6) one ofthe tubes; the tube then was gently shaken for 5 to 10 s compared the results obtained with Mueller-Hinton agar, to aid elution of the drug from the disk to give a nominal Trypticase soy agar (BBL Microbiology Systems, Cockeys- novobiocin concentration of 1.6 ,ug/ml of broth. Both tubes ville, Md.) with sheep blood, and P agar and found that the were then placed in a 37°C heat block or water bath. The test results were similar. In addition, other media have been used was read when the control tube showed turbidity compara- (19) or not specified (5, 7, 15, 16, 18) by European workers to ble to that of a 0.5 McFarland standard. test for novobiocin resistance. In our study, we compared There was 100% correlation between these two tests with the results obtained with an overnight disk diffusion tech- the 70 strains tested. For the 35 S. saprophyticus isolates, nique with those obtained with a broth disk procedure read the diameters of the zones of inhibition around the 5-,ug after 5 h. novobiocin disk ranged from 7.5 to 10.1 mm. For the 28 (This work was presented at the 83rd Annual Meeting of clinical isolates of S. epidermidis, the diameters ranged from the American Society for Microbiology, New Orleans, La., 6 23.1 to 30.3 mm, and for the S. hemolyticus isolates, the to 11 March 1983 [J. M. Gaydos and B. J. Harrington, Abstr. diameters ranged from 20.3 to 28.2 mm. The stock strains of Annu. Meet. Am. Soc. Microbiol. 1983, C372, p. 373].) Staphylococcus sciuri, Staphylococcus simulans, and S. All isolates tested were fresh clinical isolates from urine, epidermidis gave zone diameters of 8.1, 25.8, and 30.5 mm, blood, or wound cultures, apart from the three stock strains respectively. In the broth disk test, all strains which were Staphylococcus epidermidis ATCC 14990, S. sciuri ATCC resistant by the disk diffusion method were visibly turbid within 5 h, whereas strains which were susceptible by the * Corresponding author. disk diffusion method showed no visible growth or turbidity 279 280 NOTES J. CLIN. MICROBIOL. after 5 h. After further incubation of these strains overnight, Pathol. 31:348-350. there still was no growth in the presence of 1.6 i.g of 6. Goldstein, J., R. Schulman, 1:. Kelly, G. McKinley, and J. Fung. novobiocin per ml. 1983. Effect of different media on determination of novobiocin From the results of this study, it appears that not only is resistance for differentiation of coagulase-negative staphylo- cocci. J. Clin. Microbiol. 18:592-595. testing these organisms in Trypticase soy broth an accept- 7. Hovelius, B., and P. Mardh. 1977. On the diagnosis of coagu- able alterative to disk diffusion, but the broth disk testing has lase-negative staphylococci with emphasis on Staphylococcus the advantage that the results are available within 5 h. There saprophyticus. Acta Pathol. Microbiol. Scand. Sect. B 85:427- is at least one commerically available same-day biochemical 434. test identification system for staphylococci which often calls 8. John, J. F., Jr., P. K. Gramling, and N. M. O'Dell. 1978. for the detetmination of resistance and susceptibility to Species identification of coagulase-negative staphylococci from novobiocin as a confirmatory test; thin is the API Staph- urinary tract isolates. J. Clin. Microbiol. 8:435-437. Ident system. If the broth disk test is set up at the same time 9. Jordan, P. A., A. Iravani, G. A. Richard, and H. Baer. 1980. as the API Staph-Ident system, the novobiocin result, if Urinary tract infection caused by Staphylococcus saprophyti- that the cus. J. Infect. Dis. 142:510-515. needed, would be available at the time biochemical 10. Kloos, W. E., and K. H. Schleifer. 1975. Simplified scheme for system identification is able to be read. If biochemical routine identification of human Staphylococcus species. J. Clin. testing for identification is not performed, a presumptive Microbiol. 1:82-88. identification of the organisms as S. saprophyticus can be 11. Kloos, W. E., and P. B. Smith. 1980. Staphylococci, p. 83-87. In made on the basis of novobiocin resistance at 1.6 ,ug/ml (3, E. H. Lenette, A. Balows, W. J. Hausler, Jr., and J. P. Truant 11, 19). The use of this broth disk test enables the laboratory (ed.), Manual of clinical microbiology, 3rd ed. American Socie- to report a presumptive identification of S. saprophyticus ty for Microbiology, Washington, D.C. within 5 h rather than after 18 to 24 h. Two recent reports of 12. Leighton, P. M., and M. White. 1983. Rapid determination of rapid novobiocin susceptibility testing in broth (2, 12) have novobiocin susceptibility for the identification of Staphylococ- cus saprophyticus. Diagn. Microbiol. Infect. Dis. 1:261-264. required the use of automated instruments; however, the 13. Lewis, J. F., S. R. Brake, D. J. Anderson, and G. N. Vredeveld. method we describe here can be performed without such 1982. Urinary tract infection due to coagulase-negative staphy- equipment. lococcus. Am. J. Clin. Pathol. 77:736-739. 14. Marrie, T. J., and C. Kwan. 1982. Antimicrobial susceptibility of Staphylococcus saprophyticus and urethral staphylococci. LITERATU1E CITED Àntimicrob. Agents Chemother. 22:395-397. 1. Almeida, R. J., and J. H. Jorgensen. 1982. Use of Mueller- 15. Mitchell, R. G. 1968. 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