D-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium

Abstract

Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.

Fig. 1.

Phylogenetic analysis of vancomycin resistance ligases. Branch support values are indicated on the phylogram. The neighbor-joining method (34) was used for sequence comparison. Phylogenetic analysis was conducted using MEGA 4 (35).

Fig. 2.

Schematic representation of the vanN operon. Identified −35 and −10 regions are underlined in the P_N promoter region. The transcription start site is indicated in boldface.

Fig. 3.

Alignment of deduced amino acid sequences of VanS_N (E. faecium UCN71)_, VanS_L (E. faecalis N06-0364), VanS_C (E. gallinarum BM4174), VanS_G (E. faecalis BM4518), and VanS_E (E. faecalis N00-410). Identical residues are marked in black, conserved amino acid motifs of histidine kinases (H, N, G1, F, and G2) are indicated by solid lines, and VanS_N predicted hydrophobic transmembrane domains are indicated by dotted lines_. The P₁₅₆S and R₂₀₄Q substitutions in VanS_N are indicated by asterisks.