Negative Staphylococci with the MS-2 System ROY J

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Rapid Determination of Novobiocin Resistance of Coagulase- Negative Staphylococci with the MS-2 System ROY J. ALMEIDA1 AND JAMES H. JORGENSEN2* Departments of Microbiology1 and Pathology,2 The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 Received 31 August 1982/Accepted 20 October 1982

Special novobiocin elution disks were prepared for testing of coagulase- negative staphylococci in the MS-2 system (Abbott Laboratories, Diagnostics Div., Irving, Tex.). The MS-2 system correctly classified 91.5% of 82 isolates as either susceptible or resistant to novobiocin in an average time of 5.8 h.

Since 1975, when Kloos and Schleifer pub- The MS-2 system has been shown to provide lished their simplified scheme for routine identi- rapid, accurate antimicrobial susceptibility re- fication of human Staphylococcus species (4), sults in previous studies (8): however, the means interest in the coagulase-negative staphylococci for determining novobiocin resistance are not (C-NS) has grown. These organisms now have presently available from the manufacturer. been shown to cause a variety of infections, A group of 82 C-NS isolates was used in this including prosthetic valve endocarditis (7). One study. These urinary tract isolates were classi- species, Staphylococcus saprophyticus, recent- fied according to the Kloos simplified scheme ly has been shown to be a prominent cause of and were tested for novobiocin resistance by urinary tract infections in young women (3) and conventional means (4). Of these 82 isolates, 62 nongonococcal urethritis in men (2). A key char- were S. saprophyticus (novobiocin resistant) acteristic for recognition of S. saprophyticus in and 20 were S. epidermidis (novobiocin suscep- the Kloos simplified scheme is the determination tible). Novobiocin elution disks for use in the of susceptibility or resistance to 1.6 p.g of novo- MS-2 system were prepared as follows. Novobi- biocin per ml. Although three human C-NS ocin (Sigma Chemical Co., St. Louis, Mo.) was species (S. saprophyticus, S. cohnii, and S. carefully weighed and added to sterile water to xylosus) are resistant to novobiocin, S. sapro- achieve a concentration of 68 jig/ml. This solu- phyticus is the most frequently isolated novobio- tion was filter sterilized with a 0.22-nm-pore-size cin-resistant C-NS. Anderson et al. (1) conclud- filter (Nalgene Labware Div., Nalge/Sybron ed that novobiocin-resistant urinary tract Corp., Rochester, N.Y.), and 20 ,ul of the 68-,ug/ isolates may be reported as presumptive S. ml solution was applied to 6-mm-diameter absor- saprophyticus, and, more recently, Marrie et al. bent paper disks (Schleicher & Schuell Co., (6) determined that a test for resistance to the 5- Keene, N.H.). Disks were allowed to dry at ,ug novobiocin disk has a 93% positive predictive room temperature for 4 to 6 h and then were accuracy as a test for presumptive identification stored at 4°C in a dessicator until used. Novobi- of S. saprophyticus. Moreover, Kloos and Wolf- ocin elution disks were manually inserted into shohl recently concluded (5) that a test for standard MS-2 biphasic susceptibility cartridges novobiocin susceptibility was an important ad- for testing. During the test, these disks provided junctive test for accurate identification of C-NS a novobiocin concentration of 1.6 ,ug/ml in the by a commercially available biochemical test lower reaction chamber, after transfer of0.85 ml system. The Kloos simplified method for deter- of inoculated MS-2 broth. mining novobiocin resistance involves the use of A pilot study was carried out to determine a a 5-,ug novobiocin disk and P agar as the test means for interpreting novobiocin susceptibility medium. This method requires overnight incu- tests in the MS-2 system. For this purpose, two bation followed by measurement of the zone of isolates of S. epidermidis (ES0451 and EE0470) inhibition. Isolates which are inhibited "from 1 and two isolates of S. saprophyticus (SE1912 to 5 mm from the edge of the disk," are classi- and SE2160) were grown overnight on Trypti- fied as novobiocin resistant (4). The purpose of case soy agar plates with 5% sheep blood (BBL this study was to determine if the MS-2 system Microbiology Systems, Cockeysville, Md.). A (Abbott Laboratories, Diagnostics Div., Irving, sterile inoculating needle was touched to four or Tex.) could be used to provide a rapid, same-day five well-isolated, morphologically identical col- indication of novobiocin resistance of C-NS. onies on each plate and inoculated into screw- 558 VOL. 17, 1983 NOTES 559 Vertical Scale is 1.6 OD

Time (each mark is 30 minutes) Vertical Scale is 1.6 OD B

Time (each mark is 30 minutes) FIG. 1. The MS-2 system growth curves of S. epidermidis EE0470 (A) and S. saprophyticus SE1912 (B) tested against 10 antimicrobial agents. CTL, Growth control; 10, trimethoprim-sulfamethoxazole (25 ,ug); 9, cephalothin (10 ,ug); 8, ampicillin (2.5 ,ug); 7, nitrofurantoin (30 p.g); 6, erythromycin (3 ,ug); 5, gentamicin (4 ,ug); 4, clindamycin (0.5 pLg); 3, methicillin (5 ,ug); 2, penicillin (1 U); and 1, novobiocin (1.4 p.g). OD, Optical density. capped glass test tubes (16 by 150 mm) contain- (0.5 pLg); gentamicin (4 ,ug); erythromycin (3 jig); ing 4 ml of 0.85% sterile saline solution. The nitrofurantoin (30 pug); ampicillin (2.5 ,ug); ceph- contents of each tube were mixed vigorously to alothin (10 ,ug); trimethoprim-sulfamethoxazole yield a uniform cell suspension, and the turbidity (25 Fg). was adjusted to a density of a 0.5 to 1 McFarland A 400-,lI portion of the prepared saline sus- opacity standard. The MS-2 disk loader-sealer pension of each organism was pipetted into an was used to load and seal the disks of the MS-2 transfer cartridge containing 15 ml of MS-2 following antimicrobial agents into each of four culture medium. Cartridges then were inserted MS-2 transfer cartridges: novobiocin (1.4 ,ug); into an MS-2 analysis module, and the research penicillin (1 U); methicillin (5 ,ug); clindamycin system version of the MS-2 was used for the 560 NOTES J. CLIN. MICROBIOL. determinations. The MS-2 system was instruct- cin in these bacteria were similar to those of all ed to transfer the bacteria-medium mixture from of the other antimicrobial agents tested by us. the upper growth chamber to the lower reaction Thus, similar results probably could have been chambers of the transfer cartridges when the obtained with interpretive programming for any change in the optical density reached 0.008 (this of the other drugs. The average time of incuba- is a routine procedure when the clinical system tion necessary to produce results for both the S. program for susceptibility testing is used). The epidermidis and S. saprophyticus strains was 5.8 cartridges were incubated and monitored over- h (range 4.8 to 6.1 h). night in the MS-2 analysis module before the These experiments indicate a high degree of growth curves were examined. agreement between results obtained by novobio- The growth curves of one S. epidermidis cin susceptibility testing by the Kloos simplified (EE0470) isolate and one S. saprophyticus method and those obtained with the MS-2 sys- (SE1912) isolate are shown in Fig. 1. S. epider- tem. Although novobiocin disks and MS-2 pro- midis EE0470 showed uninhibited growth in the gramming for their interpretation are not cur- control position of the cuvette and in position 2 rently available from Abbott Laboratories, the (penicillin disk). Growth was inhibited, howev- MS-2 system can be used to determine novobio- er, in position 1 (novobiocin disk). S saprophyti- cin resistance in clinical isolates by interpreta- cus SE 1912 showed growth in the control tion with another antibiotic algorithm. We chose position and in position 1 (novobiocin disk). The amikacin for this purpose, although the novobio- results were identical to results of novobiocin cin curves closely resembled those of the other susceptibility testing of these strains by the antibiotics included in our testing. Even greater Kloos simplified method. They also were identi- accuracy could be achieved if novobiocin kinetic cal to the Bauer-Kirby disk diffusion test results data were employed to develop a unique inter- for the nine other antimicrobial agents included pretive program, as was done by Abbott Labora- in the MS-2 system. tories for the currently available antimicrobial The clinical system program of the MS-2 agents. Thus, it is possible to identify gram- system then was instructed to interpret results of positive, catalase-positive, coagulase-negative, novobiocin testing with a novobiocin disk occu- novobiocin-resistant urinary tract staphylococ- pying a position in the test cartridge that was cal isolates presumptively as S. saprophyticus programmed to contain amikacin. This was done by same-day testing in the MS-2 system. because novobiocin was not included in the We thank the Medical Center Hospital microbial pathology antimicrobial agent battery list of the MS-2 laboratory, San Antonio, Tex., and Virginia Thomas of the system. Thus, the system was instructed to Department of Microbiology at The University of Texas interpret the growth kinetics in position 1, con- Health Science Center at San Antonio, San Antonio, for the taining novobiocin, as if an amikacin disk had clinical isolates they generously provided for this study. been placed there. A total of 20 isolates of S. epidermidis and 62 isolates of S. saprophyticus LITERATURE CITED manner. were tested in this 1. Anderson, J. D., A. M. Clarke, M. E. Anderson, J. L. Of the 20 S. epidermidis strains tested, 18 Isaac-Renton, and M. G. McLoughlin. 1981. Urinary tract were interpreted by the MS-2 system as being infections due to Staphylococcus saprophyticus biotype 3. susceptible to novobiocin when programmed Can. Med. Assoc. J. 124:415-418. 2. Hovelius, B., I. Thelin, and P.-A. Mardh. 1979. Staphylo- interpretive criteria for amikacin were used. The coccus saprophyticus in the aetiology of nongonococcal remaining two isolates were reported a having urethritis. Br. J. Vener. Dis. 55:369-374. an intermediate reaction to novobioir (amika- 3. Jordon, P. A., A. Irvani, G. Richard, and H. Baer. 1980. cin). Of the 62 S. saprophyticus strains tested, Urinary tract infection caused by Staphylococcus sapro- were system as being phyticus. J. Infect. Dis. 142:510-515. 57 interpreted by the MS-2 4. Kloos, W. E., and K. H. Schleifer. 1975. Simplified scheme resistant to novobiocin (amikacin). Two isolates for routine identification of human Staphylococcus species. were interpreted as having an intermediate reac- J. Clin. Microbiol. 1:82-88. tion, and three isolates failed to grow adequately 5. Kloos, W. E., and J. F. Wolfshohl. 1982. Identification of Staphylococcus species with the API STAPH-IDENT Sys- for testing in the MS-2 medium. Therefore, in tem. J. Clin. Microbiol. 16:509-516. comparison to the Kloos simplified method for 6. Marrie, T. J., C. Kwan, M. A. Noble, A. West, and L. determining novobiocin resistance, the MS-2 Duffield. 1982. Staphylococcus saprophyticus as a cause of system was able to correctly classify 90 and urinary tract infections. J. Clin. Microbiol. 16:427-431. of S. epidermidis and S. 7. Speller, D. C., and R. G. Mitchell. 1973. Coagulase-nega- 92%, respectively, tive staphylococci causing endocarditis after cardiac sur- saprophyticus strair'- with amikacin susceptibil- gery. J. Clin. Pathol. 26:517-522. ity criteria. 8. Thornsberry, C., J. P. Anhalt, J. A. Washington II, L. R. Although the program for interpretation of McCarthy, F. D. Schoenknecht, J. C. Sherris, and H. J. amikacin was used in these experi- Spencer. 1980. Clinical laboratory evaluation of the Abbott kinetics MS-2 automated antimicrobial susceptibility testing sys- ments, close inspection of Fig. 1A and 1B sug- tem: report of a collaborative study. J. Clin. Microbiol. gests that the kinetics of inhibition by novobio- 12:375-390.