Enterococcus gallinarum N04-0414 Harbors a VanD-Type Vancomycin Resistance Operon and Does Not Contain a d -Alanine: d -Alanine 2 ( ddl2 ) Gene

Abstract

Enterococcus gallinarum N04-0414 (MIC for vancomycin, 256 g/ml) harbored a vanD-type vancomycin resistance operon as well as the intrinsic vanC1 operon. The D-Ala:D-Ala ligase 2 gene (ddl2) was not present in the strain, though it is found downstream of the vanS gene from the vanC operon in E. gallinarum ATCC 49573 and 19 other E. gallinarum strains tested.Acquired glycopeptide resistance in enterococci can be mediated through three types of D-Ala:D-Lac ligases, VanA, VanB, and VanD, and two types of D-Ala:D-Ser ligases, VanE and VanG (3,8,23,24). Low-level intrinsic resistance by VanC-type D-Ala:D-Ser ligases is found in Enterococcus gallinarum (VanC1), Enterococcus casseliflavus (VanC2), and Enterococcus flavescens (VanC3) (23). E. gallinarum has been previously associated with both VanA (13) and VanB (9). VanD has been found in relatively few strains of Enterococcus faecium and Enterococcus faecalis (4,5,10,11,18,21,22). All VanD-type strains characterized to date contain a mutated D-Ala:D-Ala ligase gene (ddl), and thus expression of the vanD operon is necessary for peptidoglycan synthesis and growth of the organism (4,5,6,7,11,12,21). Constitutive expression of the vanD operon in these strains is achieved by mutations in either the vanS gene from the vanD operon (vanS D ) or the vanR D gene (4,11,12).E. gallinarum N04-0414 was isolated from the rectal swab specimen of a male patient who had received a course of vancomycin for a methicillin-resistant Staphylococcus aureus infection. Vancomycin and teicoplanin MICs were determined by Etest (AB Biodisk, Solna, Sweden) and shown to be 256 and 12 g/ml, respectively. Standard PCRs were carried out as previously described (5). Thermal asymmetric interlaced PCR (16, 17) was carried out with either AmpliTaq Gold or a Herculase-Taq mix (Stratagene, La Jolla, CA). Primers used in this study are listed in Table 1. Primer locations and PCR products sequenced are shown in Fig. 1.PCRs for vanD-type genes with universal vanD primers (5) and with primers HD-3 and vanX-U1 were successful. Analysis of the product of the latter PCR revealed an open reading frame (ORF) of 1,032 bp that coded for a protein of 343 amino acids with 81% identity to the VanD1 protein from E. faecium BM4339 (6,22). The rest of the vanD operon was obtained by standard and thermal asymmetric interlaced PCR, and a total of 6,424 bp of the sequence was characterized (Fig. 1A) There are few data available on the regions flanking the characterized vanD operons. In E. faecium BM4339, an integrase gene, intD, is found downstream of vanX D1 and is transcribed as part of the operon (7). In E. faecium N97-0330, the vanX D3 gene is followed closely by a stem-loop structure and a putative transcriptional regulator gene (4). In E. gallinarum N04-0414, the vanX D gene is followed by a stem-loop structure, after which is found, on the opposite strand, an ORF (orfS) coding for a putative protein that exhibits 40% identity to the RumK histidine kinase of the Ruminococcus gnavus ruminococcin A synthesis operon (15). T...

Characterization of VanYn, a novel D,D‐peptidase/D,D‐carboxypeptidase involved in glycopeptide antibiotic resistance in Nonomuraea sp. ATCC 39727

“…Despite the high identity of the conserved sequences belonging to the active sites, the overall identity between VanY n and previously described VanY and VanX enzymes in enterococci is moderate: 46% with VanX D from Enterococcus gallinarum [27], 40% with VanX A from E. faecium [28], 39% with VanX B from E. faecium (accession no. http://www.ncbi.nlm.nih.gov/protein/ADA62242.1), 34% with VanX B from Enterococcus faecalis [29], 38% with VanXY C from E. gallinarum [30], 27% with VanY B from E. faecalis [29], VanY G from E. faecalis [31] and VanY B from E. faecium [32], and 25% with VanY A from E. faecium [28].…”

“…Alignment of Zn 2+ ‐binding and active site sequences shows that VanY n shares well‐conserved motifs with VanY, VanX and VanXY enzymes from different strains of glycopeptide‐resistant enterococci with VanA [28], VanB [29,32], VanC [15,30], VanD [27,35] and VanG [31] phenotypes. It is worth noting that VanY n possesses an Asp active site residue that is involved in recognition of the α‐NH 3 + of d ‐Ala‐ d ‐Ala in VanX and that is not present in known VanY sequences.…”

vanD and vanG -Like Gene Clusters in a Ruminococcus Species Isolated from Human Bowel Flora

“…strain CCRI-16110 carries a vanD gene cluster closely related to the vanD1 gene cluster in enterococci. So far, the vanD gene cluster has been described only for enterococci (4,10,33), and the present report is the first descriptive study of the vanD gene cluster in a nonenterococcal species.…”

Current Trends of Emergence and Spread of Vancomycin-Resistant Enterococci

“…Due to different strategies in establishing those complex and highly regulated networks independently and via different routes five different vanD cluster types had arranged and were characterized so far (Boyd et al, 2000;Depardieu et al, 2004). Up to now, VanD-type resistance still is a rare van resistance type among enterococci but has been described in a VanC type E. gallinarum N04-0414, too (Boyd et al, 2006b). In this strain the vancomycin resistance phenotype is constitutive but typical VanD strain features are lacking (mutations in vanS D linked to constitutive expression; shut-down of housekeeping D-Ala-D-Ala ligase activity, etc.).…”