Abstract
A total of 107 Enterococcus strains, 10 Vagococcus fluvialis strains, and 8 Lactococcus garvieae strains were tested for acidification of methyl-α-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.
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-α-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.
(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].)
MATERIALS AND METHODS
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).
TABLE 1.
Strains of Enterococcus and related genera used in this study
| Species | Strain(s)a | No. of strains |
|---|---|---|
| E. faecium | Human strains | 23b |
| Type strain ATCC 19434 | 1 | |
| Reference strains SS 442 and SS 960 | 2 | |
| E. gallinarum | Human strains | 21 |
| Type strain ATCC 49573 | 1 | |
| E. casseliflavus | Human strains | 20 |
| Type strain ATCC 25788 | 1 | |
| Reference strain SS 937 | 1 | |
| “E. flavescens” | Food strain | 1 |
| Type strain ATCC 49996 | 1 | |
| Reference strains SS 1318 and SS 1319 | 2 | |
| E. faecalis | Human strains | 10 |
| Type strain ATCC 19433 | 1 | |
| Reference strain SS 499 | 1 | |
| E. mundtii | Human strains | 4 |
| Type strain ATCC 43186 | 1 | |
| Reference strains SS 1233 and SS 1234 | 2 | |
| E. dispar | Human strains | 2 |
| Type strain ATCC 51266 | 1 | |
| E. durans | Type strain ATCC 19432 | 1 |
| Reference strain SS 497 | 1 | |
| E. avium | Type strain ATCC 14025 | 1 |
| E. malodoratus | Type strain ATCC 43197 | 1 |
| E. hirae | Reference strain SS 1226 | 1 |
| E. pseudoavium | Type strain ATCC 49372 | 1 |
| E. raffinosus | Reference strain SS 1278 | 1 |
| E. cecorum | Type strain ATCC 43198 | 1 |
| E. saccharolyticus | Type strain ATCC 43076 | 1 |
| E. columbae | Reference strain SS 1310 | 1 |
| E. sulfureus | Type strain ATCC 49903 | 1 |
| L. garvieae | Human strains | 5 |
| Type strain ATCC 43921 | 1 | |
| Reference strains SS 1290 and SS 1311 | 2 | |
| V. fluvialis | Human strains | 9 |
| Type strain ATCC 94515 | 1 | |
| Total | 125 |
ATCC, American Type Culture Collection; SS, standard strain from the culture collection of the Streptococcus Laboratory, Centers for Disease Control and Prevention.
Includes eight vancomycin-resistant strains.
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
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 2.
Results of MGP acidification and EFRO susceptibility tests for the identification of Enterococcus species and some related genera
| Species | Phenotypic characteristica
|
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MAN | SOR | ARG | ARA | SBL | RAF | TEL | MOT | PIG | SUC | PYU | MGP | EFRO (n) | |
| Group I | |||||||||||||
| E. avium | + | + | − | + | + | − | − | − | − | + | + | + | R (1) |
| E. malodoratus | + | + | − | − | + | + | − | − | − | + | + | − | S (1) |
| E. raffinosus | + | + | − | + | + | + | − | − | − | + | + | + | R (1) |
| E. pseudoavium | + | + | − | − | + | − | − | − | − | + | + | + | R (1) |
| E. saccharolyticus | + | + | − | − | + | + | − | − | − | + | − | + | R (1) |
| Group II | |||||||||||||
| E. faecalisb | +* | − | +* | − | + | − | + | − | − | +* | + | − | R (12) |
| L. garvieae | + | − | + | − | − | − | − | − | − | v | − | − | S (8) |
| E. faeciumc | +* | − | + | + | v | v | − | − | − | +* | − | − | S (26) |
| E. casseliflavusd | + | − | +* | + | v | + | −* | +* | +* | + | v | + | R (26) |
| E. mundtii | + | − | + | + | v | + | − | − | + | + | − | − | S (7) |
| E. gallinarume | + | − | +* | + | − | + | − | +* | − | + | − | + | R (22) |
| Group III | |||||||||||||
| E. durans | − | − | + | − | − | − | − | − | − | − | − | − | S (2) |
| E. hirae | − | − | + | − | − | v | − | − | − | + | − | − | S (1) |
| E. dispar | − | − | + | − | − | + | − | − | − | + | + | + | R (3) |
| Group IV | |||||||||||||
| E. sulfureus | − | − | − | − | − | + | − | − | + | + | − | + | R (1) |
| E. cecorum | − | − | − | − | + | + | − | − | − | + | + | − | R (1) |
| Group V | |||||||||||||
| E. columbae | + | − | − | + | + | + | − | − | − | + | + | − | R (1) |
| V. fluvialis | + | − | − | − | + | − | − | + | − | + | − | + | R (10) |
MAN, mannitol; SOR, sorbose; ARG, arginine; ARA, arabinose; SBL, sorbitol; RAF, raffinose; TEL, 0.04% tellurite; MOT, motility; PIG, pigment; SUC, sucrose; PYU, pyruvate; +, >90% positive; −, <10% positive; ∗, occasional exceptions (<3% of strains showed aberrant reactions); v, variable; R, resistant; S, susceptible.
Included one sorbitol-negative, four mannitol-negative, and one arginine-negative strain.
Included four mannitol-negative strains.
Included three nonmotile, eight arginine-negative, and three nonpigmented strains.
Included five nonmotile and four arginine-negative strains.
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).
TABLE 3.
Key tests, including MGP acidification and EFRO susceptibility tests, for the identification of the group II enterococcal species
| Species | Result fora:
|
|||
|---|---|---|---|---|
| MOT | PIG | MGP | EFRO | |
| E. faecalis | − | − | − | R |
| E. faecium | − | − | − | S |
| E. casseliflavus | +b | +b | + | R |
| E. mundtii | − | + | − | S |
| E. gallinarum | +b | − | + | R |
Abbreviations and symbols are as in Table 1.
Occasional variant reaction.
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.
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