tional method of identifying the enterococci, using bile-esculin and 6.5% NaCl tolerance, is accurate butrequires upto a 48-h incubation period insome cases (1).
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1983, p. 1275-1277 0095-1137/83/111275-03$02.00/0
Vol. 18, No. 5
Rapid Identification of Enterococci G. S. BOSLEY,'* R. R. FACKLAM,1 AND D. GROSSMAN2 Centers for Disease Control, Atlanta, Georgia 303331; and Pharmacia Diagnostics, Piscataway, New Jersey
088542 Received 3 June 1983/Accepted 15 August 1983
A 4-h method was devised to differentiate the non-beta-hemolytic streptococci into three categories: enterococci, group D nonenterococci, and viridans streptococci. All of the Streptococcus faecalis, 90% of the Streptococcus faecium (enterococci), and 96% of the Streptococcus bovis biotype I (group D nonenterococci) cultures were correctly identified by the 4-h method. The less commonly isolated group D cultures had lower rates of correct identification by this method. None of the viridans streptococci was identified incorrectly. The enterococcal strains of streptococci are taken from tryptic soy sheep blood agar plates more resistant to antimicrobial agents than are that had been inoculated with streptococcal culthe nonenterococcal strains; therefore, it is im- tures and incubated overnight at 35°C. The PYR portant that the enterococcal strains be rapidly broth was inoculated with a loopful of colonies and accurately identified (5, 7, 8). The conven- and incubated for 4 h at 35°C, after which 1 drop tional method of identifying the enterococci, of PYR reagent (N,N-dimethylaminocinnamalusing bile-esculin and 6.5% NaCl tolerance, is dehyde) was added to the broth. After 1 min, a accurate but requires up to a 48-h incubation deep red color was read as positive, and all other period in some cases (1). Recently, Schierl and colors, including orange, were interpreted as Blazevic reported a rapid test for identification negative reactions. of enterococci, but only 83% of the enterococci The group D streptococcal coagglutination were positive after 4 h (6). The Phadebact group test was performed as indicated by the manufacD coagglutination test (Pharmacia Fine Chemi- turer. A direct colony method was tested by cals, Piscataway, N.J.) has been reported to be a smearing a loopful of colonies onto the card rapid and specific test for identifying the group provided in the kit. The cells were then emulsiD streptococci; however, a second test (6.5% fied with the reagent, and the suspension was NaCl) is required to specifically identify the agitated for 1 min and read for agglutination. enterococcal group D strains (4). Cultures that failed to give positive group D L-Pyrrolidonyl-,-naphthlamide (PYR) is a reactions were inoculated into 2-ml of Toddsubstrate which is hydrolyzed by 100% of the Hewitt broth (BBL Microbiology Systems, group A Streptococcus pyogenes and the group Cockeysville, Md.) and retested for group D D enterococci (J. Godsey, R. Schulman, and L. reactions after incubation for 4 h at 35°C. Again, Eriquez, Abstr. Annu. Meet. Am. Soc. Microbi- the broth cultures were retested after overnight ol. 1981, C84, p. 276). An agar medium contain- incubation when the 4-h test was negative. Intering PYR and requiring overnight incubation has pretations of the results were as follows: posibeen described for presumptive identification of tive group D and PYR broth reactions identified group A streptococci and for the separation of the group D enterococci; positive group D with the enterococci from the nonenterococci (3). negative PYR reactions identified the group D The group A streptococci and group D entero- nonenterococci; and a positive reaction in PYR cocci were found to give positive reactions to alone indicated an unidentified group containing PYR, whereas all other streptococci did not. aerococci and some strains of enterococci. This study describes our evaluation of a modiAll streptococcal and aerococcal strains used fication of the PYR test when interpreted with were from human specimens submitted to the the Phadebact group D test for use as a rapid (4- Streptococcus Laboratory, Centers for Disease h) identification procedure for the enterococci. Control, Atlanta, Ga., by federal, state, and city The PYR broth, obtained from Carr-Scarbor- health departments. All of the cultures were ough Microbiologicals, Inc., Stone Mountain, grouped by the Lancefield extraction method Ga., is composed of Todd-Hewitt broth with and identified by conventional testing (1). 0.01% PYR and is received in 0.2-ml amounts in Table 1 lists the results of testing 238 cultures screw cap tubes. Inoculum for both the PYR for PYR hydrolysis and the results for the Phabroth and group D coagglutination test was debact D serogrouping. All of 78 enterococcal 1275
1 276
J. CLIN. MICROBIOL.
NOTES
TABLE 1. Streptococcal serogroup D reactions (Phadebact group D) and hydrolysis of PYR Organism
No.
tested
Organism
No. of positive Phadebact D reactions
No. of
No.iiv ofR reactions
Direct
4-h broth
Ovemight
S. faecalis S.faecium S. avium S. durans
32 28 13 5
32 28 13 5
30 24 8 4
2 1 0 0
0 3 0 0
S. bovis I S. bovis II S. equinus
24 12 2
0 0 0
23 5 0
0 0 0
0 0 0
5 103 4 10
2 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
Aerococci Viridans streptococci Group B streptococci Other streptococci Groups H, K, N, and 0
cultures (Streptococcus faecalis, Streptococcus faecium, Streptococcus avium, and Streptococcus durans) gave positive hydrolysis reactions on PYR. None of the 38 nonenterococcal group D cultures (Streptococcus bovis I, S. bovis II, and Streptococcus equinus) gave positive reactions. All of 32 cultures of S. faecalis and 90% (25 of 28) of S. faecium cultures were identified as enterococci with positive PYR and Phadebact D grouping reactions after 4 h. The S. faecium cultures that failed to react with the group D reagent in the direct or 4-h broth test reacted with the group D reagent after the broth had been incubated overnight. Sixty-two percent (8 of 13) of S. avium cultures were identified after 4 h. None of the remaining five strains reacted with the group D reagent even after testing the broth that had been incubated overnight. Eighty percent (4 of 5) of the S. durans cultures were identified by the 4-h method. The unidentified strain also failed to react with the group D reagent after the overnight incubation period. Ninety-six percent (23 of 24) of S. bovis I strains were identified as group D nonenterococci at the 4-h incubation period. Forty-two percent (5 of 12) of S. bovis II strains were also identified as group D nonenterococci at the 4-h incubation period. None of the strains of S. bovis (I or II) that failed to react with the direct test or 4-h broth test was grouped by the overnight culture test. Two S. equinus cultures were tested, but neither was grouped after any time period. None of the aerococci, viridans streptococci, or the nonhemolytic group B or groups H, K, N, and 0 streptococci gave group D coagglutination reactions. The six viridans streptococci that we reported as reacting with the Phadebact group D reagent were found to be mixed cultures upon
reexamination (Bosley and Facklam, Abst. Annu. Meet. Am. Soc. Microbiol. 1983, C290, p. 360). Purified cultures of these strains failed to react with the group D reagent. Only two cultures of aerococci among this latter group of cultures gave positive PYR reactions. Inconsistent PYR reactions prevented an accurate prediction of the identification of the aerococci. Ninety-five percent of the more frequently occurring strains of enterococci (S. faecalis and S. faecium) were accurately identified. Since it is the S. faecalis and S. faecium strains that are more resistant to antimicrobial agents (5, 7, 8) and more frequently occurring, the failure to identify the remaining enterococcal strains is less critical. The failure to identify all of the S. bovis cultures as group D nonenterococci was disappointing, but 96% of the S. bovis biotype I strains, which are associated with colonic cancer (3), were accurately identified. The fact that none of the viridans or other non-group D streptococci, other than aerococci, was identified as enterococci was encouraging. We believe that this rapid procedure will be an important tool for the clinical laboratory which has determined the need to separate enterococci from the other streptococci in a short period of time. LITERATURE CITED 1. Facklam, R. R. 1980. Streptococci and aerococci, p. 88110. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed.
American Society for Microbiology, Washington, D.C. 2. Facklam, R. R., L. G. Thacker, B. Fox, and L. Eriquez. 1982. Presumptive identification of streptococci with a new test system. J. Clin. Microbiol. 15:987-990. 3. Klein, R. S., R. A. Recco, M. T. Catalana, S. C. Edberg, J. I. Casey, and N. H. Steilbigel. 1977. Association of Streptococcus bovis with carcinoma of the colon. N. EngI. J. Med. 297:525-544.
VOL. 18, 1983 4. Lim, D. V., and M. H. Marnell. 1980. Confirmatory identification of group D streptococci by slide coagglutination. Curr. Microbiol. 4:151-154. 5. Moellering, R. C., Jr., 0. M. Koreniowski, M. A. Sande, and C. B. Wennersten. 1971. Species-specific resistance to antimicrobial synergism in Streptococcus faecium and Streptococcusfaecalis. J. Infect. Dis. 140:202-208. 6. Schierl, S. A., and D. J. Blazevic. 1981. Rapid identification
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of enterococci by reduction of litmus milk. J. Clin. Microbiol. 14:227-228. 7. Thornsberry, C., C. N. Baker, and R. R. Facklam. 1974. Antibiotic susceptibility of Streptococcus bovis and other group D streptococci causing endocarditis. Antimicrob. Agents Chemother. 5:228-233. 8. Watanakunakorn, C. 1971. Penicillin combined with gentamicin or streptomycin: synergism against enterococci. J. Infect. Dis. 124:581-586.
