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Antimicrobial Agents and Chemotherapy, March 2006, p. 1067-1070, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.1067-1070.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

David A. Boyd,1 Mark A. Miller,2 and Michael R. Mulvey1*

National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba,1 SMBD-Jewish General Hospital and McGill University, Montreal, Quebec, Canada2

Received 28 September 2005/ Returned for modification 20 October 2005/ Accepted 5 December 2005


   ABSTRACT
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Enterococcus gallinarum N04-0414 (MIC for vancomycin, 256 µg/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.


   TEXT
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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 (vanSD) or the vanRD 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 µg/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.


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  		TABLE 1. Primers used in this study

 

Figure 1
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  		FIG. 1. Schematic representation of the vanD (A) and vanC (B) gene clusters from E. gallinarum N04-0414. White arrows represent coding sequences and indicate direction of transcription. Primers used in PCRs are named and their locations indicated by black triangles. PCR products that were sequenced are indicated by horizontal black bars (A), and the vanC gene cluster and ddl2 gene region in E. gallinarum N04-0414 and ATCC 49573, respectively, that were sequenced are indicated by hatched boxes (B). Stem-loop structures between vanXD and orfS and between ddl2 and ntpD are indicated. The sequence of the direct repeats (DR) and the stem-loop structure flanking the ATCC 49573 ddl2 gene are shown (B). A schematic representation of the E. gallinarum BM4174 vanC gene cluster and the surrounding region is shown for comparative purposes, and the promoter (PC) of this region is indicated.

 
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). The E. gallinarum N04-0414 Van proteins displayed between 71 and 91% identity to the corresponding proteins from other vanD operons. The VanDN04-0414 ligase exhibited 81 to 82% identity to the VanD1, VanD2, and VanD3 ligases but only 78 to 79% identity to the VanD4 and VanD5 ligases.

VanD strains express vancomycin resistance constitutively due to defective regulatory proteins caused by residue substitutions affecting the function of VanSD or VanRD (11, 12), deletions or insertions in the vanSD gene causing frameshifts or truncated proteins (4, 6, 11), or interruption of vanSD by an insertion element (12). The E. gallinarum N04-0414 vanSD gene contains no insertions or deletions, and the five conserved blocks of amino acids (H, N, G1, F, and G2) found in the transmitter regions in histidine protein kinases are present (20). However, for the E. gallinarum N04-0414 VanSD protein, we note an Asn->Asp change at position 314 in the ATP-binding G1 box and an Ala->Ser change at position 338 in a hydrophilic region near the ATP-binding G2 box. The E. gallinarum N04-0414 VanRD protein contains the conserved Asp-19, Asp-53, and Lys-101 residues of response regulators as well as the other conserved residues in the effector domains of VanR-type regulators. Vancomycin resistance in E. gallinarum N04-0414 is constitutive (data not shown). If the E. gallinarum N04-0414 vanD operon is constitutively expressed, it is likely due to changes in the VanSD protein and not in the VanRD protein. Whether it is the substitutions in the N04-0414 VanSD protein noted above or other substitutions contributing to a constitutive resistance phenotype awaits further experiments.

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 vanXD1 and is transcribed as part of the operon (7). In E. faecium N97-0330, the vanXD3 gene is followed closely by a stem-loop structure and a putative transcriptional regulator gene (4). In E. gallinarum N04-0414, the vanXD 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). Thus, the three vanD operons mentioned above are found in unique genetic environments.

We characterized the E. gallinarum N04-0414 vanC1 operon and determined that it shares 99.5% nucleotide identity with the BM4174 vanC1 operon (Fig. 1B) (2). Identities between the corresponding proteins range from 98.9 to 100%. One difference between the VanSC proteins was at position 320, which is a Ser in E. gallinarum BM4174 but a Gly in N04-0414. Ser-320 has been postulated to affect VanSC function and lead to constitutive expression of the vanC1 operon (19). None of the other changes in VanSC from E. gallinarum strains exhibiting constitutive vancomycin resistance (Arg-200->Leu, Asp-312->Asn, or Asp-312->Ala) were found in the N04-0414 VanSC protein, and thus this protein resembles the VanSC proteins from strains with inducible resistance (19). These data indicate that the vanC1 operon is likely inducible in E. gallinarum N04-0414.

As mentioned above, VanD-type strains contain defective ddl genes (4, 5, 6, 11, 12, 21). E. gallinarum BM4174 expresses two ddl genes, ddl (14), which is in an unknown location in the chromosome, and ddl2, which is found 230 bp downstream of the vanC1 operon but in the opposite orientation (Fig. 1B) (1). The two proteins share 24% identity, and both genes are expressed (1). E. gallinarum N04-0414, ATCC 49573, and 19 routine strains from our collection were all found to posses a ddl gene by PCR. Sequence analysis showed that the E. gallinarum N04-0414, ATCC 45973, and BM4174 ddl genes shared 99% identity and the corresponding proteins 100% identity. The ddl2 gene was not found downstream of vanSC in E. gallinarum N04-0414; however, 58 bp downstream of the vanSc gene on the opposite strand, we did identify a partial ORF whose putative product exhibited 66% amino acid identity to the C-terminal end of the NtpD ATPase subunit D from Clostridium tetani (Fig. 1B) (GenBank accession no. AAO35588). PCR to detect the ddl2 gene elsewhere in the E. gallinarum N04-0414 chromosome was negative. In E. gallinarum ATCC 49573 and the 19 routine strains, the ddl2 gene was mapped between vanSC and ntpD. Sequence analysis of this region in E. gallinarum ATCC 49573 revealed a ddl2 gene 230 bp downstream of vanSC, as found in E. gallinarum BM4174 (Fig. 1B). However, a mutation in the middle position of codon 306 (TTA->TGA) introduces a stop codon and presumably a truncated Ddl2 protein. Interestingly, the E. gallinarum ATCC 49573 ddl2 gene is found on a 1,343-bp fragment that is flanked by a 9-bp direct repeat sequence, ACAAACGAA (positions 5824 to 5832 of GenBank accession no. DQ022190) (Fig. 1B). We note that, whereas the ddl genes and the vanC1 operons have a G+C content of 42% to 44%, the ddl2 genes have a 37% to 38% G+C content. It may be that the ddl2 gene was acquired by E. gallinarum from a foreign source by a site-specific mechanism involving the direct repeat sequence. In E. gallinarum N04-0414, a precise deletion of the ddl2 gene has occurred at the 9-bp sequence, presumably after the acquisition of the vanD operon, though this assumption is speculative. We could not transfer vancomycin resistance to E. faecium GE-1 or E. faecalis JH2-2 (Fusr Rifr) by conjugation. No other vanD-type operon has been successfully transferred in vivo.

The high-level vancomycin resistance of E. gallinarum N04-0414 is due to the presence of a vanD operon and perhaps achieved in concert with the deletion of ddl2. To our knowledge, this is the first report of VanD-type resistance in E. gallinarum.

Nucleotide sequence accession numbers. The sequences for the E. gallinarum N04-0414 vanD operon, vanC1 operon, and ddl gene were submitted to the GenBank database and assigned accession numbers DQ172830, DQ022190, and AY971750, respectively. The sequences for the E. gallinarum ATCC 49573 ddl gene and ddl2 gene were assigned accession numbers AY971751 and DQ015971, respectively.


   ACKNOWLEDGMENTS
 
We thank Romeo Hizon and the staff of the NML DNA Core Facility for expert technical assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba, Canada R3E 3R2. Phone: (204) 789-2133. Fax: (204) 789-5020. E-mail: Michael_mulvey{at}phac-aspc.gc.ca. Back


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Antimicrobial Agents and Chemotherapy, March 2006, p. 1067-1070, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.1067-1070.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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