Previous Article | Next Article 
Journal of Clinical Microbiology, June 1998, p. 1584-1587, Vol. 36, No. 6
Instituto de Microbiologia, Universidade
Federal do Rio de Janeiro, Rio de Janeiro 21941, Brazil,1 and
Division of Bacterial and
Mycotic Diseases, National Center for Infectious Diseases, Centers
for Disease Control and Prevention, Atlanta, Georgia
303332
Received 10 November 1997/Returned for modification 20 January
1998/Accepted 24 February 1998
A total of 107 Enterococcus strains, 10 Vagococcus fluvialis strains, and 8 Lactococcus
garvieae strains were tested for acidification of
methyl- The precise identification of
Enterococcus species such as Enterococcus
gallinarum and Enterococcus casseliflavus has
assumed additional importance in clinical microbiology because of the intrinsic low-level resistance to glycopeptides presented by these species and the difficulty in differentiating them from
Enterococcus faecium, which is frequently found to be a
cause of outbreaks of disease caused by vancomycin-resistant
enterococci (1, 5, 9, 14). These difficulties in the
identification of enterococcal species are further complicated by the
occurrence of "atypical" strains, such as nonmotile E. gallinarum and E. casseliflavus, which are
incorrectly identified as E. faecium and
Enterococcus mundtii, respectively, by the currently
recommended identification procedures (6). In addition,
we have found that E. faecium strains have very diverse
phenotypic characteristics (12), which presents a problem
for the differentiation of this species from atypical strains of
E. casseliflavus and E. gallinarum. In 1994, Miele et al. (8) described a test
based on susceptibility to elfamycin drugs for the rapid
differentiation of Enterococcus faecalis and E. faecium. More recently, Devriese et al. (3) demonstrated that a test based on the acidification of
methyl- (This study was presented in part at the 97th General Meeting of the
American Society for Microbiology, Miami Beach, Fla., 4 to 8 May 1997 [1a].)
Bacterial strains.
A total of 125 strains were
studied, including 26 E. faecium (8 vancomycin-resistant strains with atypical phenotypes previously studied by Teixeira et al. [12]), 22 E. casseliflavus, 22 E. gallinarum, 12 E. faecalis, 7 E. mundtii, 4 "Enterococcus flavescens," 3 Enterococcus
dispar, 2 Enterococcus durans, 8 Lactococcus garvieae, and 10 Vagococcus
fluvialis strains, and 1 type strain and/or reference strain of
each additional species of the genus Enterococcus (Table
1).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Use of Tests for Acidification of
Methyl-
-D-Glucopyranoside and Susceptibility to
Efrotomycin for Differentiation of Strains of
Enterococcus and Some Related Genera
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-D-glucopyranoside (MGP) and susceptibility to
100-µg efrotomycin (EFRO) disks. All 26 strains of
Enterococcus casseliflavus, including 3 nonmotile and 2 nonpigmented strains, acidified MGP and were resistant to EFRO. All 22 strains of Enterococcus gallinarum, including 5 nonmotile
strains, also acidified MGP and were resistant to EFRO. None of the 26 strains of Enterococcus faecium acidified MGP, and all were
susceptible to EFRO. Although all 12 Enterococcus faecalis
strains were also negative in the MGP test, they were resistant to
EFRO. Other enterococcal strains gave variable results. All 10 strains
of V. fluvialis and all 8 strains of L. garvieae gave positive and negative results, respectively, in the
MGP test and were, respectively, resistant and susceptible to
EFRO. These results indicate that tests of the production of acid from
MGP and susceptibility to EFRO can be used as adjunct tests in the identification of typical and atypical strains of enterococci in the
clinical microbiology laboratory.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-D-glucopyranoside (MGP) could be used to
differentiate E. casseliflavus and
E. gallinarum from E. faecium and
E. faecalis. The aim of the present study was to
evaluate the tests for acidification of MGP and susceptibility to
efrotomycin (EFRO) for the differentiation of typical and atypical phenotypes of enterococci and related microorganisms.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
TABLE 1.
Strains of Enterococcus and related genera
used in this study
Characterization of strains. Strains were tested for their phenotypic characteristics with a conventional biochemical test as recommended by Facklam and Teixeira (6). All atypical strains were also submitted to tests for reactivity with the AccuProbe Enterococcus culture confirmation test (Gen-Probe, Inc., San Diego, Calif.), analysis of whole-cell protein profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and DNA-DNA reassociation experiments as described by Teixeira et al. (13).
MGP test. The carbohydrate MGP was obtained from Sigma Chemicals, St. Louis, Mo. Heart infusion broth (Difco, Detroit, Mich.) containing 1% MGP and 0.006% bromocresol purple indicator was prepared, distributed in 2-ml aliquots (13- by 100-mm tubes), and autoclaved for 10 min at 121°C. The broth was inoculated with a drop or a loopful of an overnight broth culture or with several colonies taken from a blood agar plate. The carbohydrate broth was then incubated at 35°C for 7 days. A positive reaction was recorded when the broth indicator turned yellow.
EFRO susceptibility test. Disks containing 100 µg of EFRO each (kindly provided by Merck Sharpe & Dohme, Rahway, N.J.) were prepared by dissolving 1,000 mg of EFRO in 0.1 ml of dimethyl sulfoxide and diluting in 9.9 ml of sterile distilled water. Ten microliters of this solution was dispensed onto each filter paper disk. The disks were allowed to dry at room temperature for 5 to 6 h in the dark. A heavy inoculum was spread with a loop or a swab over one half of a Trypticase soy agar plate containing 5% sheep blood. The EFRO disk was placed in the heavy part of the streak, and the plate was incubated for 18 to 24 h at 35°C. The presence of a growth inhibition zone of any size was interpreted as indicative of susceptibility.
| |
RESULTS AND DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
|
|---|
The inoculated MGP broth media were incubated for up to 7 days, but most of the positive reactions were observed after 24 h of incubation. The only exceptions occurred for a few E. faecalis strains, which became positive after 2 weeks of incubation. However, only the results obtained up to 7 days of incubation were considered.
All 26 E. casseliflavus strains, including 3 nonmotile (2 arginine-negative), 2 nonpigmented (1 arginine-negative), and 8 arginine-negative strains, acidified the MGP broth and presented no zone of growth inhibition around the EFRO disks. The "E. flavescens" strains were considered E. casseliflavus strains because of recent evidence that the two are a single species (13). All 22 E. gallinarum strains, including 5 nonmotile and 1 arginine-negative strain, also acidified MGP broth and were resistant to EFRO. All the atypical E. gallinarum and E. casseliflavus strains were confirmed for species status by whole-cell protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DNA-DNA reassociation experiments as described by Teixeira et al. (13).
The 26 E. faecium strains tested, including mannitol-negative and vancomycin-resistant variants previously studied by Teixeira et al. (12), did not acidify MGP broth and were susceptible to EFRO. The 12 E. faecalis strains tested, which included 7 atypical variants (1 raffinose-positive, 1 raffinose-positive and sorbitol-negative, 1 arginine-negative, and 4 asaccharolytic strains), as well as Enterococcus cecorum and Enterococcus columbae strains, also did not acidify MGP, but they all were resistant to EFRO. Other enterococci gave variable results for the MGP and EFRO tests. Strains of V. fluvialis, a motile gram-positive coccus that has been isolated from human clinical sources and is related to the genus Enterococcus in its phenotypic characteristics and positive reaction in the AccuProbe Enterococcus culture confirmation test (11), acidified MGP broth and were resistant to EFRO. These results are similar to those obtained with motile Enterococcus species E. gallinarum and E. casseliflavus. However, these two species can be differentiated from V. fluvialis on the basis of other physiological tests such as arginine and arabinose (11). All eight strains of L. garvieae, which phenotypically resembles E. faecalis, were negative in the MGP broth test and were susceptible to EFRO. On the basis of these results, we recommend that the MGP and EFRO tests be added to the modified conventional test scheme proposed by Facklam and Teixeira (6) as shown in Table 2.
|
Table 3 shows the key reactions that can aid in the identification of phenotypically atypical enterococcal strains, especially of the group comprising nonmotile E. gallinarum, nonmotile or nonpigmented E. casseliflavus, and E. faecium strains. Both MGP broth and EFRO susceptibility tests yielded consistent results for these species, indicating that they can be used as adjunct tests in the identification of atypical enterococcal strains. These tests could be helpful not only as part of the conventional procedures for the identification of enterococci but also with automated systems, such as Vitek and MicroScan. The reliability of the combination of these commercial test systems with motility and pigmentation tests for differentiation of E. gallinarum and E. casseliflavus from E. faecium is still controversial (1, 7, 9, 10, 14). These tests would also be less expensive, faster, and easier to perform than elaborate molecular tests like PCR or pulsed-field gel electrophoresis (1, 2, 4, 5).
|
| |
ACKNOWLEDGMENTS |
|---|
This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Ministério da Ciência e Tecnologia (MCT/PRONEX) Brazil.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Childhood and Respiratory Diseases Branch MS-C02, Centers for Disease Control and Prevention, Atlanta, GA 30333. Phone: (404) 639-1379. Fax: (404) 639-3123. E-mail: rrf2{at}cdc.gov.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
|
|---|
| 1. | Cartwright, C. P., F. Stock, G. A. Fahle, and V. J. Gill. 1995. Comparison of pigment production and motility tests with PCR for reliable identification of intrinsically vancomycin-resistant enterococci. J. Clin. Microbiol. 33:1931-1933[Abstract]. |
| 1a. |
Carvalho, M. G. S.,
L. M. Teixeira, and R. R. Facklam.
1997.
Methyl--D-glucopyranoside for identification of atypical strains of Enterococcus gallinarum and Enterococcus casseliflavus, poster C-457, p. 200.
In
Abstracts of the 97th General Meeting of the American Society for Microbiology. American Society for Microbiology, Washington, D.C.
|
| 2. | Coque, M. T., and B. E. Murray. 1995. Identification of Enterococcus faecalis strains by DNA hybridization and pulsed-field gel electrophoresis. J. Clin. Microbiol. 33:3368-3369[Medline]. |
| 3. |
Devriese, L. A.,
B. Pot,
K. Kersters,
S. Lauwers, and F. Haesebrouck.
1996.
Acidification of methyl--D-glucopyranoside: a useful test to differentiate Enterococcus casseliflavus and Enterococcus gallinarum from Enterococcus faecium species group and from Enterococcus faecalis.
J. Clin. Microbiol.
34:2607-2608[Abstract].
|
| 4. | Donabedian, S., J. W. Chow, D. M. Shlaes, M. Green, and M. J. Zervos. 1995. DNA hybridization and contour-clamped homogeneous electric field electrophoresis for identification of enterococci to the species level. J. Clin. Microbiol. 33:141-145[Abstract]. |
| 5. | Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33:24-27[Abstract]. |
| 6. | Facklam, R. R., and L. M. Teixeira. 1997. Enterococcus, p. 669-682. In L. Collier, A. Balows, and M. Sussman (ed.), Topley & Wilson's microbiology and microbial infections, 9th ed. Edward Arnold, London, United Kingdom. |
| 7. | Iwen, P. C., D. M. Kelly, J. Linder, and S. H. Hinrichs. 1996. Revised approach for identification and detection of ampicillin and vancomycin resistance in Enterococcus species by using MicroScan panels. J. Clin. Microbiol. 34:1779-1783[Abstract]. |
| 8. |
Miele, A.,
B. P. Goldstein,
M. Bandera,
C. Jarvis,
A. Resconi, and R. J. Williams.
1994.
Differential susceptibilities of enterococcal species to elfamycin antibiotics.
J. Clin. Microbiol.
32:2016-2018 |
| 9. | Rosenberg, J., F. C. Tenover, J. Wong, W. Jarvis, and D. J. Vugia. 1997. Are clinical laboratories in California accurately reporting vancomycin-resistant enterococci? J. Clin. Microbiol. 35:2526-2530[Abstract]. |
| 10. | Sader, H. S., D. Biedenbach, and R. N. Jones. 1995. Evaluation of Vitek and API 20S for species identification of enterococci. Diagn. Microbiol. Infect. Dis. 22:315-319[Medline]. |
| 11. | Teixeira, L. M., M. D. G. S. Carvalho, V. L. C. Merquior, A. G. Steigerwalt, D. J. Brenner, and R. R. Facklam. 1997. Phenotypic and genotypic characterization of Vagococcus fluvialis, including strains isolated from human sources. J. Clin. Microbiol. 35:2778-2781[Abstract]. |
| 12. | Teixeira, L. M., R. R. Facklam, A. G. Steigerwalt, N. E. Pigott, V. L. C. Merquior, and D. J. Brenner. 1995. Correlation between phenotypic characteristics and DNA relatedness within Enterococcus faecium strains. J. Clin. Microbiol. 33:1520-1523[Abstract]. |
| 13. | Teixeira, L. M., M. G. S. Carvalho, V. L. C. Merquior, A. G. Steigerwalt, M. G. M. Teixeira, D. J. Brenner, and R. R. Facklam. 1997. Recent approaches on the taxonomy of the enterococci and some related microorganisms. Adv. Exp. Med. Biol. 418:387-391[Medline]. |
| 14. |
Vincent, S.,
R. G. Knight,
M. Green,
D. F. Sahm, and D. M. Shlaes.
1991.
Vancomycin susceptibility and identification of motile enterococci.
J. Clin. Microbiol.
29:2335-2337 |
Journal of Clinical Microbiology, June 1998, p. 1584-1587, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Shewmaker, P. L., Steigerwalt, A. G., Morey, R. E., Carvalho, M. d. G. S., Elliott, J. A., Joyce, K., Barrett, T. J., Teixeira, L. M., Facklam, R. R.
(2004). Vagococcus carniphilus sp. nov., isolated from ground beef. Int. J. Syst. Evol. Microbiol.
54: 1505-1510
[Abstract] [Full Text] -
Miller, M. B., Allen, S. L., Mangum, M. E., Doutova, A., Gilligan, P. H.
(2004). Prevalence of Vancomycin-Resistant Enterococcus in Prenatal Screening Cultures. J. Clin. Microbiol.
42: 855-857
[Abstract] [Full Text] -
Hayes, J. R., English, L. L., Carter, P. J., Proescholdt, T., Lee, K. Y., Wagner, D. D., White, D. G.
(2003). Prevalence and Antimicrobial Resistance of Enterococcus Species Isolated from Retail Meats. Appl. Environ. Microbiol.
69: 7153-7160
[Abstract] [Full Text] -
Tyrrell, G. J., Turnbull, L., Teixeira, L. M., Lefebvre, J., Carvalho, M. d. G. S., Facklam, R. R., Lovgren, M.
(2002). Enterococcus gilvus sp. nov. and Enterococcus pallens sp. nov. Isolated from Human Clinical Specimens. J. Clin. Microbiol.
40: 1140-1145
[Abstract] [Full Text] -
Angeletti, S., Lorino, G., Gherardi, G., Battistoni, F., De Cesaris, M., Dicuonzo, G.
(2001). Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing. J. Clin. Microbiol.
39: 794-797
[Abstract] [Full Text] -
Chen, D. K., Pearce, L., McGeer, A., Low, D. E., Willey, B. M.
(2000). Evaluation of D-Xylose and 1% Methyl-alpha -D-Glucopyranoside Fermentation Tests for Distinguishing Enterococcus gallinarum from Enterococcus faecium. J. Clin. Microbiol.
38: 3652-3655
[Abstract] [Full Text] -
von Gottberg, A., van Nierop, W., Dusé, A., Kassel, M., McCarthy, K., Brink, A., Meyers, M., Smego, R., Koornhof, H.
(2000). Epidemiology of Glycopeptide-Resistant Enterococci Colonizing High-Risk Patients in Hospitals in Johannesburg, Republic of South Africa. J. Clin. Microbiol.
38: 905-909
[Abstract] [Full Text] -
Iwen, P. C., Rupp, M. E., Schreckenberger, P. C., Hinrichs, S. H.
(1999). Evaluation of the Revised MicroScan Dried Overnight Gram-Positive Identification Panel To Identify Enterococcus Species. J. Clin. Microbiol.
37: 3756-3758
[Abstract] [Full Text] -
Hanson, K. L., Cartwright, C. P.
(1999). Comparison of Simple and Rapid Methods for Identifying Enterococci Intrinsically Resistant to Vancomycin. J. Clin. Microbiol.
37: 815-817
[Abstract] [Full Text] -
Clark, N. C., Teixeira, L. M., Facklam, R. R., Tenover, F. C.
(1998). Detection and Differentiation of vanC-1, vanC-2, and vanC-3 Glycopeptide Resistance Genes in Enterococci. J. Clin. Microbiol.
36: 2294-2297
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»