VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis

Abstract

Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

FIG. 1.

Schematic representation of the vanD gene cluster from E. faecium strain BM4538 (ddl [G₉₅₆A] vanR_D [G₄₁₉A]) and a recombinant plasmid. Open arrows represent coding sequences and indicate the direction of transcription. The PCR fragment internal to the vanD gene used as a probe in the hybridization experiments is indicated above the corresponding region. Horizontal bars depict the PCR products corresponding to overlapping amplified fragments from strains A902 (ddl [A₃₈G] vanS_D [Δ1bp₆₅₇]), BM4538 (ddl [G₉₅₆A] vanR_D [G₄₁₉A]), BM4539 (ddl [::7bp₈₇₀] vanS_D [::7bp₇₅₃]), and BM4540 (ddl [::7bp₃₆₁] vanS_D [::7bp₇₅₃]). The positions of the 5′ ends of the primers complementary to the sequence of reference VanD-type strain BM4339 (ddl [::5bp₃₇] vanS_D [C₅₁₇A]) are in parentheses, with black arrowheads showing the direction of DNA synthesis. Numbering begins at the A residue of the ATG start codon of the vanR_D gene from BM4339 (ddl [::5bp₃₇] vanS_D [C₅₁₇A]). The sizes of the PCR products are indicated in boldface digits. The insert in plasmid pAT822 cloned under the control of the P₂ promoter is represented by a dashed line, and the vector is indicated in parentheses. For the recombinant plasmid, arrowheads represent the locations and the orientations of the oligodeoxynucleotides used for amplification of the insert.

FIG. 2.

Analysis of SmaI-digested genomic DNA of VanD-type clinical isolates by PFGE (left) and Southern hybridization (right) with a vanD-specific probe. Bacteriophage λ concatemers (lanes λ; Biolabs) were used as molecular size markers, and the sizes are indicated at the left. The sizes of the hybridizing fragments are indicated at the right.

FIG. 3.

Schematic representation of the genes for d-Ala:d-Ala ligases of enterococci. The positions of the amino acids implicated in the binding of d-Ala1, d-Ala2, and ATP and conserved in E. faecium and E. faecalis are indicated by dotted, hatched, and black bars, respectively (25, 46). In E. faecium strains A902 (ddl [A₃₈G] vanS_D[Δ1bp₆₅₇]) and BM4538 (ddl [G₉₅₆A] vanR_D [G₄₁₉A]), the single-base differences relative to the sequence of ddl from E. faecium BM4147, which lead to a Glu-to-Gly substitution at position 13 and a Ser-to-Asn substitution at position 319, respectively, are indicated in italics. In E. faecalis strains BM4539 (ddl [::7bp₈₇₀] vanS_D [::7bp₇₅₃]) and BM4540 (ddl [::7bp₃₆₁] vanS_D [::7bp₇₅₃]), a 7-bp insertion (italics) at the positions corresponding to amino acids 290 and 121, respectively, is responsible for a frameshift mutation that leads to the synthesis of 297- and 128-amino-acid peptides instead of the putative 348-amino acid Ddl.

FIG. 4.

Alignment of the deduced amino acid sequences of VanS_D sensors. Numbers at the left refer to the first amino acid in the corresponding sequence. Numbers at the right refer to the last amino acid in the corresponding line. Identical amino acids are indicated by asterisks below the alignment, and the isofunctional amino acids are indicated by dots below the alignment. Conserved motifs H, N, G1, F, and G2 are indicated above the alignment with dashed lines (38). The histidine residue in boldface is the putative autophosphorylation site. The proline at position 173, putatively responsible for constitutive expression of resistance in BM4339, is indicated in italics and boldface. The truncated amino acid sequences of VanS_D from A902 (ddl [A₃₈G] vanS_D [Δ1bp₆₅₇]), BM4539 (ddl [::7bp₈₇₀] vanS_D [::7bp₇₅₃]), and BM4540 (ddl [::7bp₃₆₁] vanS_D [::7bp₇₅₃]) lack the four conserved blocks (N, G1, F, and G2).

FIG. 5.

Alignment of the deduced amino acid sequences of the effector domains of various VanR-type regulators with those of E. coli PhoB and OmpR regulators. Numbering refers to the amino acid sequence of VanR_D. PhoB consists of 229 residues in two functional domains: a 124-amino-acid N-terminal receiver domain and a 99-amino-acid C-terminal effector domain with DNA binding and transactivation functions (14). Fully conserved residues are highlighted in gray, and residues belonging to the PhoB hydrophobic core are underlined. PhoB residues implicated in DNA binding and protein-protein interaction are indicated by black circles and asterisks, respectively. The glycine (G) which is conserved in the VanR-type regulators and in the regulators of the same family, such as PhoB and OmpR of E. coli, is boxed. The glutamate (E) at position 140 (numbering of VanR_D), indicated in boldface and italics, corresponds to the mutation in E. faecium BM4538 (ddl [G₉₅₆A] vanR_D [G₄₁₉A]).

FIG. 6.

Proportions of late soluble cytoplasmic peptidoglycan precursors (A) and VanY_D d,d-carboxypeptidase specific activity in membrane extracts (B) from BM4339 (ddl [::5bp₃₇] vanS_D [C₅₁₇A]) and derivatives of BM4458 (vanS_D [C₅₁₇A]) and BM4459 (vanS_D [C₅₁₇A]). The strains studied and the pAT392 [P₂ aac(6′)-aph(2′′)] and pAT822 [P₂ vanS_D aac(6′)-aph(2′′)] plasmids used for transformation are indicated at the bottoms of the charts. Induction was performed with 2 μg of vancomycin per ml. The levels of resistance to vancomycin (Vm) and teicoplanin (Te) are indicated under the panel displaying the peptidoglycan precursors. Specific activity was defined as the number of nanomoles of product formed at 37°C per minute per milligram of protein contained in the extracts.