First VanD-Type Vancomycin-Resistant Enterococcus raffinosus Isolate

The vancomycin-resistant Enterococcus raffinosus strain GV5 was resistant to vancomycin (MIC, 1,024 μg/ml) and teicoplanin (MIC, 256 μg/ml). The species of strain GV5 was determined by sequencing of a specific PCR product for the 16S rRNA gene of E. raffinosus. GV5 was isolated from a stool specimen and from a bedsore on the necrotic inferior limb of a diabetic 73-year-old man in Japan.

DNA sequence analysis of the vanD operon was performed by sequencing the PCR products with primers specific for each gene in the vanD4 operon of E. faecium 10/96A (8); it showed that GV5 encodes a 5,654-bp vanD gene cluster consisting of vanR_DS_DY_DH_DDX_D, which is homologous to the corresponding genes in the reported VanD-type strains and is located on the chromosome (accession no. AB242319) (3, 6, 7, 9). The vanD gene cluster was compared with that of the corresponding genes of the vanD4 gene cluster of E. faecium 10/96A (Fig. 1) (8). vanR_D and vanX_D were completely identical to the equivalent genes in 10/96A. There was one amino acid substitution in both VanH_D and VanD, where Ile₁₆₉ was converted to Phe and Gly₁₂₁ was converted to Val, respectively. The reported VanS_D contains five blocks of the conserved sequences H, N, G1, F, and G2 (2, 4, 8), which are contained in phosphate transmitters of two-component regulator systems (1, 11). Block H sequences consist of the residues L₁₆₄AHDLKTPLS₁₇₃, including a putative autophosphorylation site, His₁₆₆ (14). The Thr₁₇₀ residue in the block H sequence has been replaced by Ile in VanS_D of GV5, suggesting that this mutation might result in the constitutive expression of resistance due to impaired VanS_D function to dephosphorylate phosphorylated VanR_D. vanY_D of GV5, which has a molecular size of 1,068 bp, is completely identical to that of 10/96A with the exception of an additional adenosine insertion in vanY_D of 10/96A (8). The nucleotide sequence from position 346 to position 354 of GV5 vanY_D is C₃₄₆AAAAAAAC₃₅₄, and the sequence from position 346 to position 355 of 10/96A vanY_D is C₃₄₆AAAAAAAAC₃₅₅. If an adenosine residue were inserted within the seven adenosines located between nucleotides 346 and 354 of GV5 vanY_D, the codon sequence at positions 415 to 417 of the resulting gene would become the TGA translation stop codon as a result of the frameshift mutation, and translation would be terminated prematurely after amino acid 138, as in the VanY_D protein of 10/96A (8).

FIG. 1.

Schematic representation of the vanD gene cluster from E. raffinosus strain GV5 and Northern blot analysis of the vanD cluster. (A) Open arrows represent coding sequences and indicate the direction of transcription. The PCR fragments internal to the vanS_D, vanY_D, and vanD genes used in the hybridization experiments are indicated below the corresponding regions. Amino acids with arrows within parentheses indicate substitutions compared with the reported sequence of the vanD4 operon of E. faecium 10/96A (8). (B) Northern hybridization was performed according to a protocol described previously (13). RNAs were prepared from strains cultured with 6 μg/ml of vancomycin (+VCM) or without vancomycin (−VCM) for 2 h. Thirty micrograms of RNA was used in each lane. The sizes of the RNAs were determined by using the sizes of RNA molecular weight markers (Invitrogen, Inc.), and the arrows and the numbers on the left indicate the positions and sizes of the largest bands in each experiment.

In Northern hybridizations with vanY_D and vanD probes, identical bands of about 3.7 kb in size, which correspond to the transcript of vanY_DH_DDX_D (5), were observed in both the absence and the presence of vancomycin (Fig. 1). The vanS_D probe detected an approximately 1.9-kb band, which corresponds to the size of the transcript of vanR_DS_D, in the absence and presence of vancomycin (Fig. 1). These results indicate that the vanD4 cluster in GV5 is expressed constitutively (2, 3, 6, 9, 12).

Analysis of the d-Ala:d-Ala ligase gene (ddl) on the chromosome of strain GV5 (accession no. AB242318) revealed that there are two amino acid substitutions—Asn₂₇₁ is converted to Asp, and Gly₃₁₉ is converted to Asp—compared to the wild-type DDL of E. raffinosus JCM8733 (accession no. AB242317), which implies that the amino acid substitutions might result in impaired function of GV5 DDL (10).

Several VanD-type vancomycin-resistant enterococci have been identified among E. faecium and E. faecalis (3, 6, 7, 9). We have described the first VanD-type E. raffinosus strain and showed evidence that there is species divergence in enterococci that encode VanD resistance as well as nucleotide divergence between the VanD determinants (3, 6, 7, 9).