Identification of Enterococcus faecalis Strains by DNA Hybridization ...

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Departments of Medicine and Clinical Pathology. William Beaumont Hospital. Royal Oak, Michigan 48073. VOL. 33, 1995. LETTERS TO THE EDITOR. 3369.

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1995, p. 3368–3369 0095-1137/95/$04.0010

Vol. 33, No. 12

Identification of Enterococcus faecalis Strains by DNA Hybridization and Pulsed-Field Gel Electrophoresis bp) and for aac(69)-Ii (323 bp) were generated by PCR from E. faecalis OG1RF and from E. faecium GE1, respectively. We used the following oligonucleotides: 59-CGGGATGAACGA ATTGGGTGTGA-39 and 59-AATTTTACTCATACGTGCT TCGG-39 (for gyrA) and 59-GCGGTAGCAGCGGTAGAC CAAG-39 and 59-CGTAGGTGTCTTACCAAATGC-39 [for aac(69)-Ii], selected from published sequences (1, 4). Different enterococcal species were included as controls. Colony lysates of the isolates in question hybridized to the gyrA probe but not to the aac(69)-Ii probe. None of the non-E. faecalis strains hybridized to the gyrA probe, and none of the non-E. faecium strains hybridized to the aac (69)-Ii probe (Table 1).

We read with interest the recently published article by Donabedian et al. reporting the use of contour-clamped homogeneous electric field (CHEF) electrophoresis for species differentiation of enterococci (2). At that time, we agreed with the authors’ statement that ‘‘Enterococcus faecalis differed from all other species in always having a largest fragment of .400 kb,’’ as these findings were similar to our experience. However, we recently recovered several E. faecalis isolates that showed a pattern of SmaI-digested genomic DNA more similar to that of Enterococcus faecium than that of E. faecalis (all fragments less than 350 kb) (Fig. 1).

With the emergence of vancomycin-resistant isolates, the identification to the species level of enterococci has been performed more often. Unfortunately, a recent report describes vancomycin-resistant strains of E. faecium with atypical biochemical characteristics, which makes the identification of

TABLE 1. Hybridization to probes for gyrA and aac(69)-Ii Species

E. faecalis E. faecium E. casseliflavus E. gallinarum E. solitarius E. hirae E. mundtii E. raffinosus Atypical E. faecalis isolates

FIG. 1. CHEF electrophoresis of digested genomic DNA from enterococcal strains. Lanes: 1, E. faecalis OG1RF; 2, E. faecalis TX2527; 3, E. faecalis TX2528; 4, E. faecalis OG1RF; 5, E. faecium GE1; 6, lambda DNA ladder standard. Lanes 1, 2, 3, and 5 show SmaI-digested genomic DNA from the specified strains; lane 4 shows NotI-digested genomic DNA from OG1RF. The size of NotI-digested genomic DNA fragments from OG1RF (6) and the size of the smallest band of lambda DNA ladder standard are indicated on the left and on the right of the figure, respectively.

No. of strains studies b

2 5c 2 2 2 2 1 1 7d

Hybridzation to the following probea: gyrA


1 2 2 2 2 2 2 2 1

2 1 2 2 2 2 2 2 2

1, hybridization; 2, no hybridization. Including OG1RF (ATCC 47077); DNA sequence analysis of eight additional E. faecalis isolates also showed the presence of this gene (4). c Including GE1 (ATCC 51558). d All isolates appeared to represent a single clone. a

The seven isolates (from five different patients) studied were recovered during a study on vancomycin-resistant enterococci in Houston, Texas, and were identifed by API 20STREP (BioMerieux, Plainview, N.Y.) and standard biochemical tests (3) as E. faecalis. These strains were resistant to vancomycin and susceptible to teicoplanin and hybridized to an intragenic vanB probe; they were also highly resistant to gentamicin and streptomycin. Genomic DNA preparation and conditions for pulsed-field gel electrophoresis were performed as described by Murray et al. (5). E. faecalis OG1RF (ATCC 47077), E. faecium GE1 (ATCC 51558), and lambda concatamers (FMC Corp., Rockland, Maine) were used as molecular size markers (Fig. 1). All seven E. faecalis isolates studies appeared to represent a single clone; two (TX2527 and TX2528) are shown in Fig. 1. The largest fragment of SmaI-digested genomic DNA from these isolates was approximately 309 kb, which is almost 100 kb smaller than the largest fragment for E. faecalis published by Donabedian et al. (2). To corroborate the identification to the species level, the isolates were tested for the presence of genes coding for GyrA and AAC(69)-Ii, which are derived from E. faecalis and E. faecium, respectively, by colony lysis hybridization under highstringency conditions (1, 4, 7). Intragenic probes for gyrA (220


these strains more difficult (8). Some authors have used other tools for characterization of enterococci (whole-cell protein profiles, pulsed-field electrophoresis gel, and gene probes) (2, 8); however, these techniques usually have been performed with typical strains. Our results widen the definition of atypical vancomycin-resistant enterococci, in this case, E. faecalis isolates which were ‘‘atypical’’ on the basis of the SmaI-digestion patterns, and suggest that the use of DNA banding patterns for identification purposes must be undertaken with caution. Identification by hybridization with specific probes may prove helpful, and this method should be tested with additional strains. REFERENCES 1. Costa, Y., M. Galimand, R. Leclercq, J. Duval, and P. Courvalin. 1993. Characterization of the chromosomal aac(69)-Ii gene specific for Enterococcus faecium. Antimicrob. Agents Chemother. 37:1896–1903. 2. Donabedian, S., J. W. Chow, D. M. Shlaes, M. Green, and M. J. Zervos. 1995.


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3. 4. 5.


7. 8.


DNA hybridization and contour-clamped homogeneous electric field electrophoresis for identification of enterococci to the species level. J. Clin. Microbiol. 33:141–145. Facklam, R. R., and M. D. Collins. 1989. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J. Clin. Microbiol. 27:731–734. Korten, V., W. M. Huang, and B. E. Murray. 1994. Analysis by PCR and direct DNA sequencing of gyrA mutations associated with fluoroquinolone resistance in Enterococcus faecalis. Antimicrob. Agents Chemother. 38:2091–2094. Murray, B. E., K. V. Singh, J. D. Heath, B. R. Sharma, and G. M. Weinstock. 1990. Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. J. Clin. Microbiol. 28:2059–2063. Murray, B. E., K. V. Singh, R. P. Ross, J. D. Heath, G. M. Dunny, and G. M. Weinstock. 1993. Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function. J. Bacteriol. 175:5216–5223. Sambrook. J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Teixeira, L. M., R. R. Facklam, A. G. Steigerwalt, N. E. Pigott, V. C. Merquior, and D. J. Brenner. 1995. Correlation between phenotypic characteristics and DNA relatedness within Enterococcus faecium strains. J. Clin. Microbiol. 33:1520–1523.

Teresa M. Coque, Ph.D. Division of Infectious Diseases Department of Internal Medicine Barbara E. Murray, M.D. Division of Infectious Diseases Department of Internal Medicine Department of Microbiology and Molecular Genetics The University of Texas Medical School Houston, Texas 77030

Authors’ Reply We agree with Drs. Coque and Murray that the use of DNA patterns by pulsed-field electrophoresis for specific identification of enterococci should be done with caution. The SmaI


digestion patterns of the seven E. faecalis isolates studied by Coque and Murray were remarkably similar to patterns generally seen for E. faecium. Pulsed-field electrophoresis has been used extensively in epidemiologic investigations of enterococci. We found DNA probes and SmaI digestion patterns obtained by CHEF to be a reliable tool for identification of enterococci to the species level among the sample of isolates we studied (1). This has remained our experience. We found CHEF to be most useful in the differentiation of E. faecium from E. gallinarum. Our study included isolates from epidemiologically related and unrelated backgrounds and isolates with atypical characteristics. The study of additional isolates may provide important information on the interpretation of banding patterns. We do not suggest that CHEF information be used alone when enterococci are identified to the species level. As stated in our article, DNA probes and CHEF electrophoresis will not replace standard biochemical identification of enterococci, but they may provide information which can be of use in identifying some isolates to the species level. REFERENCE 1. 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.

Susan Donabedian, M.P.H. Division of Infectious Diseases Marcus J. Zervos, M.D. Departments of Medicine and Clinical Pathology William Beaumont Hospital Royal Oak, Michigan 48073

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