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J Clin Microbiol. Apr 2006; 44(4): 1567–1570.
PMCID: PMC1448630

Rapid Identification of Enterococcus hirae and Enterococcus durans by PCR and Detection of a Homologue of the E. hirae mur-2 Gene in E. durans

Abstract

During an evaluation of PCR for identification of isolates of Enterococcus hirae, a homologue with 82% identity to E. hirae mur-2 was identified in Enterococcus durans and was named mur-2ed. PCR using primers for two genes (copY and murG) of E. hirae strains showed amplification with E. hirae strains only. PCR (under high-stringency conditions) with primers for the mur-2ed gene gave the expected amplification product only with E. durans strains. A combination of murG and mur-2ed primers in a multiplex PCR assay differentiated E. hirae from E. durans in all cases. PCR using these primers appears to be a rapid alternative for identification of E. hirae and E. durans isolates.

Phenotypic and biochemical similarities among many enterococcal species and the existence of strains with unusual or aberrant phenotypes make accurate species identification a challenge. PCR detection of the ddl genes (9, 10, 14), rRNA sequence-based testing (1, 6, 7, 21), direct sequencing of the groES gene (20), commercially available API 20 S strips (BioMerieux, SA, Plainview, N.Y.), and a nonradioactive DNA probe (AccuProbe culture identification tests; Gen-Probe, Inc., San Diego, CA) have all been used for identification of Enterococcus spp. (4), but these systems have focused mainly on identification of Enterococcus faecalis and Enterococcus faecium, which account for over 90% of clinical isolates belonging to this genus (16). Other species such as Enterococcus durans and Enterococcus hirae may not be recognized because many laboratories do not routinely identify enterococci to the species level. In addition, distinguishing some species such as E. hirae and E. durans poses difficulty due to phenotypically aberrant strains (21) and/or variation in the sugar fermentation profiles (5, 11, 12). Molecular methods like PCR directed to the ddl genes of E. durans/E. hirae (14), sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell protein, tRNA interpacer PCR, and arbitrarily primed PCR analysis (6) have successfully identified E. durans/E. hirae isolates. With the exception of PCR, these techniques are difficult to implement in the clinical laboratory.

In the present study, we explored the utility of primers designed from some of the gene sequences of E. hirae available in GenBank for PCR identification of this species. During the study, we also detected a homologue of a muramidase gene (mur-2) of E. hirae (accession no. M77639) in an E. durans isolate and evaluated the use of primers designed from this gene (designated mur-2ed) for PCR identification of E. durans isolates.

Bacteria used in the study were 15 isolates previously identified as E. durans (from Spain, the United States, and Canada) including ATCC 49479 (American Type Culture Collection [ATCC], Manassas, VA) and 5 as E. hirae (from Spain, the United States [including ATCC], Argentina, and Switzerland), some of which were provided by R. R. Facklam, Centers for Disease Control and Prevention, Atlanta, GA; C. Bantar, Buenos Aires, Argentina; N. Liassine, Geneva, Switzerland; and G. J. Tyrrell, Alberta, Canada. Other enterococci used in the study were 170 E. faecalis isolates, 181 E. faecium isolates, 1 E. avium isolate, 5 E. casseliflavus isolates, 5 E. gallinarum isolates, 5 E. mundtii isolates, 5 E. raffinosus isolates, and 2 E. solitarius isolates from our collection (15). The majority of these clinical isolates came from the United States, but some were from Thailand, Argentina, Colombia, Belgium, and Spain.

PCR amplification was carried out to determine the specificity of primers (Table (Table1)1) selected from the sequences of copY, murG, and mur-2 genes of E. hirae (2, 8, 13, 17) available in GenBank. Following extraction of genomic DNA (250 ng) (22), the PCR conditions used included 70 pmol of the above-mentioned primers; 200 μM each of dATP, dCTP, dGTP, and dTTP; 60 mM Tris-HCl (pH 8.5); 2 mM MgCl2; 15 mM ammonium sulfate; and 2 U Taq polymerase (Platinum Taq polymerase; Invitrogen). Initial (low-stringency PCR) cycling parameters were as follows: 1 cycle of a 2-min denaturation at 94°C; 35 cycles consisting of a 1-min denaturation at 94°C, a 2-min annealing at 55°C, and a 3-min extension at 72°C; and one final extension of 10 min at 72°C. The PCR protocol was also applied to fresh colonies from the agar plate containing a pure culture. The colony suspension was prepared by mixing several colonies in 100 μl of Tris-EDTA (pH 8.0), boiling 10 min, and vortexing vigorously. One microliter of the suspension was used as template for the PCR experiments.

TABLE 1.
Oligonucleotides used for PCR identification of E. durans and E. hirae isolates

In later experiments, high-stringency PCR (HS-PCR) conditions were used for E. hirae identification with mur-2 primers. The cycling conditions for the HS-PCR protocol were as follows: 1 cycle of a 2-min denaturation at 94°C; 30 cycles consisting of a 1-min denaturation at 94°C, a 15-second annealing at 60°C, and extension for 1 min 30 seconds at 72°C; and one final extension of 7 min at 72°C. For E. durans identification with mur-2ed primers, the HS-PCR conditions were similar to PCR with E. hirae except that the annealing temperature was raised to 61°C. The PCR products were analyzed by automated DNA sequencing using a model 377 DNA sequencer (ABI, Foster City, CA). Sequence analysis was done using the BLAST network service of the NCBI and the GCG software package (Genetics Computer Group, Madison, WI). The PCR product amplified from the genomic DNA of E. durans using mur-2 primers by decreasing the annealing temperature to 47°C for 2 min was cloned into the pCR2.1 vector of the TA cloning kit (Invitrogen, San Diego, CA) and subsequently sequenced. The PCR product amplified from E. durans using mur-2ed primers and a previously cloned mur-2 gene of E. hirae (2) were also used as DNA probes along with efaA and aac-(6′)-Ii for E. faecalis and E. faecium, respectively (3, 19), to hybridize to DNA from 393 enterococci lysed on a nylon membrane using the conditions published previously (19). Results from PCR identification of a subset of 13 enterococci including seven E. durans isolates, one alleged E. durans isolate (ATCC 49479), one E. durans isolate (W185) with an aberrant phenotype (21), and four E. hirae isolates were also compared with results from phenotypic identification to evaluate the specificity of primers for accurate identification. Six additional previously identified E. durans isolates, one E. hirae isolate, and eight other species of enterococci were also included in low-stringency PCR and HS-PCR identification tests. For phenotypic identification of E. hirae and E. durans, previously published criteria were used (11, 12).

PCR with primers for copY, murG, and mur-2 genes of E. hirae resulted in amplification of products from all E. hirae isolates (total DNA and colony suspension), the sequences of which showed 99% identity by GAP analysis to their respective genes in GenBank. Of the other enterococci, only E. durans showed any amplification product with mur-2 primers, while use of copY and murG did not result in any amplification product. The nucleotide sequence from the E. durans mur-2 PCR product (521 bp) showed 84% identity to nucleotides 544 to 799 of mur-2 (accession no. M73369) of E. hirae. The predicted amino acid sequence of Mur-2ed showed 86% similarity and identity to the Mur-2 protein of E. hirae by GAP analysis. Based on this similarity to the mur-2 sequence of E. hirae, this gene was designated mur-2ed. Colony lysate hybridization with 15 E. durans isolates, 5 E. hirae isolates, 170 E. faecalis isolates, 181 E. faecium isolates, 5 E. casseliflavus isolates, 5 E. raffinosus isolates, 4 E. gallinarum isolates, 1 E. avium isolate, 5 E. mundtii isolates, and 2 E. solitarius isolates demonstrated the species specificity of the mur-2ed and E. hirae mur-2 (2) probes, as they specifically hybridized to their respective species but not to other enterococcal species tested.

Based on the specificity of gene probes for mur-2ed and mur-2, we retested all the E. durans isolates including ATCC 49479 using HS-PCR and mur-2ed primers and identified 14/15 isolates as E. durans, 8 of which are shown in Table Table2.2. Isolate ATCC 49479, which was sent to us as an E. durans isolate, was identified as E. hirae by PCR, as it showed amplification with E. hirae mur-2 primers and no amplification with mur-2ed primers; gene probe hybridization and phenotypic identification results (Table (Table2)2) also identified ATCC 49479 as E. hirae, supporting the utility of mur-2ed primers for accurate and rapid identification. Both mur-2ed primers of E. durans and mur-2 primers of E. hirae, by HS-PCR, specifically amplified PCR products from their respective species (Table (Table2)2) but not from other species, demonstrating their usefulness for rapid identification if used for HS-PCR as described above. Among the other 14 isolates previously identified as E. durans, W185 was a known phenotypically aberrant strain (21) and, based on phenotypic tests, would have been identified as E. raffinosus. Our phenotypic tests on W185 were the same as those described earlier (21), while the PCR and gene probe hybridization results using mur-2ed and mur-2 primers for PCR and the respective PCR products as DNA probes identified it as E. durans, in agreement with its previous identification using internally transcribed spacer PCR (21).

TABLE 2.
Results of E. durans and E. hirae identification by phenotypic tests and/or HS-PCR

A multiplex PCR assay (using purified total DNA as the template) under low-stringency conditions using primers (70 pmol) targeting the murG and mur-2ed genes was designed to determine if it could discriminate between E. durans and E. hirae isolates. The PCR yielded the corresponding amplification products of 521 bp only for E. hirae isolates (including ATCC 49479) and of 177 bp only for E. durans isolates (Fig. (Fig.1).1). To test the specificity of the multiplex PCR assay, it was also performed in a mixture containing equal amounts of purified total DNA (ca. 250 ng) from E. hirae TX2817, E. durans ATCC 6056, E. faecalis OG1RF, E. faecium TX2466, and an E. gallinarum clinical isolate. Amplification of both 521-bp and 177-bp bands was obtained with no additional bands observed, confirming the specificity of the murG and mur-2ed primers for E. hirae and E. durans.

FIG. 1.
Multiplex PCR of Enterococcus isolates using murG and mur2ed primers under low-stringency conditions with total DNA as the template. Lane 1, molecular weight marker; lane 2, E. durans ATCC 49479 (identified as E. hirae in this work); lanes 3 and 4, E. ...

In conclusion, E. durans and E. hirae have previously been considered closely related species belonging to the same species group (5, 12, 18), and the sequence similarities between these two species in the mur-2 gene region support this relationship. Hybridization results under high-stringency conditions using gene probes for mur-2ed and mur-2 showed their species specificity for E. durans and E. hirae, respectively. The use of copY, murG, and mur-2 primers for E. hirae and mur-2ed primers for E. durans and HS-PCR and/or multiplex PCR (murG and mur2ed primers) appears to provide a rapid and accurate identification alternative to biochemical testing.

Nucleotide sequence accession number.

The mur-2ed gene sequence has been entered in GenBank under accession no. AF46783.

Acknowledgments

We thank Richard R. Facklam, Centers for Disease Control and Prevention, Atlanta, Ga., and Gregory J. Tyrrell, University of Alberta Hospital, Alberta, Canada, for providing some of the isolates.

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