INTRODUCTION

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The VanC phenotype, as found in Enterococcus gallinarum, E. casseliflavus, and E. flavescens, is characterized by intrinsic low-level resistance to vancomycin. The nucleotide sequences of the vanC-1 gene in E. gallinarum, the vanC-2 gene in E. casseliflavus, and the vanC-3 gene in E. flavescens have been reported, although there is some disagreement as to whether E. flavescens is a legitimate enterococcal species (3, 7, 10, 18). Descheemaeker et al. were unable to discriminate between E. casseliflavus and E. flavescens using pulsed-field gel electrophoresis (PFGE) or oligonucleotide D11344-primed PCR (3). Previous attempts to differentiate the vanC-2 and vanC-3 genes by PCR analysis have also been unsuccessful (4, 11). Although these organisms are not as clinically significant as the other members of the group II enterococci, i.e., E. faecalis and E. faecium, clusters of infection have previously been reported (5, 6). In 1991, Pompei et al. reported four strains of yellow-pigmented enterococci resembling E. casseliflavus, all of which were isolated from surgical patients (13). These four strains were further characterized by DNA-DNA hybridization and biochemical tests, and the differences were considered significant enough for the strains to be designated a new species, E. flavescens, with the type strain CCM 439 (12). E. casseliflavus was incriminated in a series of bloodstream infections in a hemodialysis unit over an 8-month period (9). Pigment production and the motility test can usually differentiate E. faecium from the three enterococcal species carrying vanC genes, while acid production from ribose is the only biochemical test capable of differentiating E. casseliflavus and E. flavescens. The variable nature of these characteristics, however, complicates the proper identification of these species necessary for epidemiologic studies (5, 6). The recent report of vanC-1-containing E. faecalis and E. faecium isolates also emphasizes the importance of differentiation of the vanC genes for epidemiologic studies (11).

The purposes of this study were (i) to detect and differentiate the vanC genotypes, if possible, and (ii) to study the distribution of the respective vanC genes among a collection of typical and atypical enterococcal strains.

	MATERIALS AND METHODS

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Bacterial isolates. The 30 isolates of enterococci with the VanC phenotype characterized in this study, including 21 E. casseliflavus or E. casseliflavus-E. flavescens isolates and 9 E. gallinarum isolates, are listed in Table 1. All of the enterococcal isolates were identified at the Centers for Disease Control and Prevention by standard methods (5, 6, 19). The control strains included E. faecalis A256 (vanA genotype [16]), V583 (vanB genotype [14]), and ATCC 19433 (type strain); E. faecium ATCC 19434 (type strain), and E. gallinarum VR-42 (vanC-1 genotype [2]). E. gallinarum SS-1228 (ATCC 49573 [type strain]) (vanC-1 genotype), E. casseliflavus SS-1229 (ATCC 25788 [type strain]) (vanC-2 genotype [10]), and E. flavescens SS-1317 (CCM-439 [type strain]) (vanC-3 genotype [10]) strains were included in the study. E. flavescens SS-1317 (CCM-439), SS-1318 (CCM-440), and SS-1319 (CCM-441) were received from Raffaello Pompei, Rome, Italy (12, 13).

Antimicrobial susceptibility testing. All isolates were tested by the broth microdilution method as described by the National Committee for Clinical Laboratory Standards (8) with cation-adjusted Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.). The antimicrobial agents tested were ampicillin, ciprofloxacin, gentamicin, penicillin, rifampin, streptomycin, teicoplanin, and vancomycin. E. faecalis ATCC 29212 was the quality control strain used in the susceptibility testing.

PCR amplification of antimicrobial resistance genes. The vanC genes that encode intrinsic low-level glycopeptide resistance in the enterococci were amplified by PCR with oligonucleotide primers selected from published sequences (4, 7, 10) with the assistance of the OLIGO software (National Biosciences, Hamel, Maine). The PCR reaction mix and conditions for amplification of the 796-bp vanC-1 gene product of E. gallinarum (2) and the 484-bp vanC-2 product of E. casseliflavus (15) have been described previously. The primers used for detection of the above genes were (5'3') (+) GAA AGA CAA CAG GAA GAC CGC and () ATC GCA TCA CAA GCA CCA ATC for vanC-1 (2) and (+) CGG GGA AGA TGG CAG TAT and () CGC AGG GAC GGT GAT TTT for vanC-2 (15).

Analysis of genomic DNA restriction patterns by PFGE. The isolates were grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) overnight at 37°C with shaking. A modification of the procedure described by Smith and Cantor (17) was used for preparation of the agarose plugs. Briefly, the pellet from 300 µl of broth was resuspended in 300 µl of TE buffer (10 mM Tris, 0.1 mM EDTA [pH 8.0]), mixed with an equal volume of 1.8% agarose (65 to 68°C; Bio-Rad Chromosomal Grade Agarose; Bio-Rad Chemical Co., Hercules, Calif.) in TE buffer, and poured into a plug mold. Mutanolysin (20 U/ml), instead of lysozyme, and 1 to 2 µg of RNase per ml was added to the EC lysis buffer (6 mM Tris [pH 7.5], 1 M NaCl, 100 mM EDTA [pH 7.5], 0.5% Brij-58, 0.2% sodium deoxycholate, 0.5% Sarkosyl) used for treatment of the plugs overnight at 37°C. This solution was replaced by ESP solution (0.5 M EDTA [pH 8.0], 1% Sarkosyl, and 1 mg of proteinase K per ml), and the plugs were incubated overnight at 50°C with gentle shaking. The plugs were washed with TE buffer and then stored at 4°C in TE buffer until needed. After digestion with 20 U of SmaI (New England BioLabs, Bedford, Maine), the plugs were placed in the wells of a 1% FastLane agarose (FMC BioProducts, Bedford, Maine) in 0.5× TBE and were electrophoresed at 200 V with a 0.1-s20-s pulse time for 20 h at 14°C in the Bio-Rad CHEF-DRII system. The gels were stained with ethidium bromide, the restricted DNA was visualized with UV light, and the images were photographed with Polaroid type 55 film (Polaroid Corporation, Cambridge, Mass.) or were saved with the Foto Analyst Archiver System (Fotodyne, Inc., Hartland, Wis.) to floppy disks. The data were analyzed, and a dendrogram was generated with the Advanced Quantifier 1-D Match version 2.1 for Windows software (BioImage, Ann Arbor, Mich.).

	RESULTS AND DISCUSSION

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The goal of this study was to detect and differentiate the three vanC determinants and to examine the distribution of these genes in a collection of both typical and atypical enterococci. The biochemical characteristics and identification results of the typical and atypical VanC enterococci are given in Table 1. Although members of the group II enterococci with VanC phenotypes normally are arginine positive, motile, pigmented (except for E. gallinarum), and ribose positive (except for E. flavescens), the collection of strains selected for the present study included strains with variable results in these reactions that would, in most cases, prevent their proper identification. Precise identification of such strains was previously obtained by using analysis of whole-cell protein profiles and DNA-DNA reassociation experiments (18, 19).

Phenotypically, E. gallinarum is distinguished from the E. faecium, E. casseliflavus, and E. flavescens strains by its motility and lack of pigmentation (5, 6). However, three of the E. gallinarum strains included in our study were nonmotile, and two E. casseliflavus strains were nonmotile and nonpigmented. On the other hand, E. flavescens has been differentiated from E. casseliflavus by its lack of acid production from ribose. Five of the E. casseliflavus-E. flavescens isolates included in the present study were negative for acid production from ribose after conventional incubation for 7 days, suggesting that they were E. flavescens. When the period of incubation was extended to 1 month, all were positive for acid production from ribose, thus eliminating the only phenotypic species distinction. Atypical isolates, such as strains 1673-93 and 1703-93, as well as 616-93, 2628-95, and 2629-95, would appear to be E. faecium by conventional tests, because they are nonmotile and nonpigmented, but PCR with the F1 and F2 primers as described by Dutka-Malen et al. (4) failed to amplify the 550-bp ddl_{E. faecium} gene product in strains 1673-93 and 1703-93, suggesting that they are not E. faecium. Such strains would not be adequately identified on the basis of phenotypic characteristics alone but could be identified only by additional tests such as acidification of methyl--D-glucopyranoside (MGP) and susceptibility to efrotomycin as recently proposed (1).

The 796-bp vanC-1 PCR product was amplified only from the E. gallinarum isolates and not from the E. casseliflavus or E. casseliflavus-E. flavescens isolates (Table 1 and Fig. 1A). As expected, due to the extensive homology (98.3%) shared in the vanC-2 and vanC-3 gene sequences, all of the E. casseliflavus or E. casseliflavus-E. flavescens isolates produced the 484-bp vanC-2 PCR product; all E. gallinarum isolates were negative (Table 1 and Fig. 1A). Only the E. casseliflavus-E. flavescens isolates produced the 224-bp vanC-3 product (Table 1 and Fig. 1B). Organisms yielding a vanC-1 gene product were reported as E. gallinarum, those yielding the vanC-2 product only were reported as E. casseliflavus, and those yielding both the vanC-2 and the vanC-3 products were reported as E. casseliflavus-E. flavescens.

View larger version (54K): [in this window] [in a new window]   FIG. 1.   Amplification of the vanC-1, vanC-2, and vanC-3 enterococcal genes by PCR. The standard in lane 1 is the 100-bp DNA ladder (Gibco/BRL, Gaithersburg, Md.). (A) vanC-1 (796-bp) and vanC-2 (484-bp) products amplified from selected study and control isolates with vanC-1 and vanC-2 primers in the reaction mix. Lanes 2 to 7 contain E. casseliflavus and E. casseliflavus-E. flavescens strains, and A256 and V583 are E. faecalis strains. (B) The vanC-3 (224-bp) products amplified from E. casseliflavus-E. flavescens strains are seen in lanes 4 to 6.

We also varied the concentrations of the primers in our multiplex PCR mixture containing the vanC-1 (0.1 µM), vanC-2 (0.1 µM), and vanC-3 (0.2 µM) primers in an attempt to eliminate the production of the vanC-2 product by the vanC-3-containing isolates (Fig. 2). We were not successful in eliminating the product, but only the vanC-3 isolates produced both the 484-bp (vanC-2) and the 224-bp (vanC-3) products. In the multiplex PCR, a 525-bp product was also amplified with both the vanC-2 and vanC-3 genes resulting from the combination of the vanC2(+) and vanC3() primers. The 525-bp product appeared to predominate in isolates with the vanC-3 gene, while the 484-bp product predominated with the vanC-2 gene. Only the E. gallinarum (vanC-1) isolate produced the expected 796-bp product.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Multiplex PCR of the vanC genes of enterococcal strains with the VanC phenotype. The reaction mix contained the vanC-1, vanC-2, and vanC-3 primers for amplification of the respective genes. Lanes 1 to 6, E. casseliflavus and E. casseliflavus-E. flavescens isolates; lanes 3 to 5, the 224-bp vanC-3 product specific for E. casseliflavus-E. flavescens isolates. Also, the 525-bp product that appears to be more prominent in E. casseliflavus-E. flavescens isolates is seen. The 796-bp vanC-1 gene product amplified in E. gallinarum strains is in lanes 10 and 12. The molecular size standard is the 100-bp DNA ladder (Gibco/BRL Life Technologies, Inc.).

The MICs for the VanC study isolates ranged from 2 to 8 µg/ml for vancomycin; most E. gallinarum isolates were intermediate (8 µg/ml), and all were susceptible to teicoplanin and did not show high-level resistance to gentamicin or streptomycin (Table 1). None of the antimicrobials tested distinguished among the species.

As seen in the dendrogram (Fig. 3), PFGE was not useful for differentiating the E. casseliflavus and E. flavescens isolates. In all E. gallinarum strains except for SS-1228 (ATCC 49573 [type strain]) and 616-93, only smaller molecular fragments were seen (<150 kb) in the PFGE patterns.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Dendrogram derived from analysis of the PFGE patterns of SmaI-digested genomic DNA of enterococcal strains with the VanC phenotype. E. casseliflavus-E. flavescens strains are labeled E. flavescens in the figure because of space limitations.

Antimicrobial susceptibility testing and PFGE were not able to distinguish among strains possessing the three vanC gene genotypes. In contrast, the use of the three sets of PCR primers allowed us to detect and differentiate the vanC-1 (E. gallinarum), vanC-2 (E. casseliflavus), and vanC-3 (E. casseliflavus-E. flavescens) genes from typical and atypical enterococcal strains. In all of the isolates characterized as E. gallinarum, the 796-bp vanC-1 gene product was amplified. Of the 21 E. casseliflavus or E. casseliflavus-E. flavescens isolates, all were shown to carry the vanC-2 gene, and 10 of them also possessed the vanC-3 gene. Therefore, the presence of the vanC genes may indicate a marker of species identification, and their detection may be an adjunct tool to assist in the precise identification of atypical strains. In a recent investigation testing some of the strains included in the present study, E. casseliflavus and E. flavescens were found to be a single species, and the term E. casseliflavus was recommended to be retained as the species denomination (18, 19). Taking this into account and considering our results, the presence of vanC-3 genes in some of the E. casseliflavus strains indicates that strains possessing the vanC-3 gene may constitute genotypic variants of this species. According to our data, such variants seem to be more frequently associated with ribose-negative variants, including those with a delayed reaction, of E. casseliflavus, which were denominated E. casseliflavus-E. flavescens, since the scope of the present study would not allow us to speculate on the validity of the designation E. flavescens as a separate species.