Enterococcus gallinarum N04-0414 Harbors a VanD-Type Vancomycin Resistance Operon and Does Not Contain a D-Alanine:D-Alanine 2 (ddl2) Gene
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ABSTRACT
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
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.
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 AsnAsp change at position 314 in the ATP-binding G1 box and an AlaSer 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-200Leu, Asp-312Asn, or Asp-312Ala) 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 (TTATGA) 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.
EFERENCES
Ambúr, O.-H., P. E. Reynolds, and C. A. Arias. 2002. D-Ala:D-Ala ligase gene flanking the vanC cluster: evidence for presence of three ligase genes in vancomycin-resistant Enterococcus gallinarum BM4174. Antimicrob. Agents Chemother. 46:95-100.
Arias, C. A., P. Courvalin, and P. E. Reynolds. 2000. vanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174. Antimicrob. Agents Chemother. 44:1660-1666.
Arthur, M., P. Reynolds, and P. Courvalin. 1996. Glycopeptide resistance in enterococci. Trends Microbiol. 4:401-407.
Boyd, D. A., J. Conly, H. Dedier, G. Peters, L. Robertson, E. Slater, and M. R. Mulvey. 2000.
Molecular characterization of the vanD gene cluster and a novel insertion element in a vancomycin-resistant enterococcus isolated in Canada. J. Clin. Microbiol. 38:2392-2394.
Boyd, D. A., P. Kibsey, D. Roscoe, and M. R. Mulvey. 2004. Enterococcus faecium N03-0072 carries a new VanD-type vancomycin resistance determinant: characterization of the VanD5 operon. J. Antimicrob. Chemother. 54:680-683.
Casadewall, B., and P. Courvalin. 1999. Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339. J. Bacteriol. 181:3644-3648.
Casadewall, B., P. E. Reynolds, and P. Courvalin. 2001. Regulation of expression of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339. J. Bacteriol. 183:3436-3446.
Courvalin, P. 2005. Genetics of glycopeptide resistance in gram-positive pathogens. Int. J. Med. Microbiol. 294:479-486.
Dahl, K. H., G. S. Simonsen, . Olsvik, and A. Sundsfjord. 1999. Heterogeneity in the vanB gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci. Antimicrob. Agents Chemother. 43:1105-1110.
Dalla Costa, L. M., P. E. Reynolds, H. A. P. H. M. Souza, D. C. Souza, M.-F. I. Palepou, and N. Woodford. 2000. Characterization of a divergent vanD-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil. Antimicrob. Agents Chemother. 44:3444-3446.
Depardieu, F., M. Kolbert, H. Pruul, J. Bell, and P. Courvalin. 2004. VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis. Antimicrob. Agents Chemother. 48:3892-3904.
Depardieu, F., P. E. Reynolds, and P. Courvalin. 2003. VanD-type vancomycin-resistant Enterococcus faecium 10/96A. Antimicrob. Agents Chemother. 47:7-18.
Dutka-Malen, S., B. Blaimont, G. Wauters, and P. Courvalin. 1994. Emergence of high-level resistance to glycopeptides in Enterococcus gallinarum and Enterococcus casseliflavus. Antimicrob. Agents Chemother. 38:1675-1677.
Evers, S., B. Casadewall, M. Charles, S. Dutka-Malen, M. Galimand, and P. Courvalin. 1996. Evolution of structure and substrate specificity in D-alanine:D-alanine ligases and related enzymes. J. Mol. Evol. 42:706-712.
Gomez, A., M. Ladire, F. Marcille, and M. Fons. 2002. Trypsin mediates growth phase-dependent transcriptional regulation of genes involved in biosynthesis of ruminococcin A, a lantibiotic produced by a Ruminococcus gnavus strain from a human intestinal microbiota. J. Bacteriol. 184:18-28.
Liu, Y.-G., N. Mitsukawa, T. Oosumi, and R. F. Whittier. 1995. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8:457-463.
Liu, Y.-G., and R. F. Whittier. 1995. Thermal asymmetric PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674-681.
Ostrowsky, B. E., N. C. Clark, C. Thauvin-Eliopoulos, L. Venkataraman, M. H. Samore, F. C. Tenover, G. M. Eliopoulos, R. C. Moellering, Jr., and H. S. Gold. 1999. A cluster of VanD vancomycin-resistant Enterococcus faecium: molecular characterization and clinical epidemiology. J. Infect. Dis. 180:1177-1185.
Panesso, D., L. Abadía-Patio, N. Vanegas, P. E. Reynolds, P. Courvalin, and C. A. Arias. 2005.
Transcriptional analysis of the vanC cluster from Enterococcus gallinarum strains with constitutive and inducible vancomycin resistance. Antimicrob. Agents Chemother. 49:1060-1066.
Parkinson, J. S., and E. C. Kofoid. 1992. Communication modules in bacterial signalling proteins. Annu. Rev. Genet. 26:71-112.
Perichon, B., B. Casadewall, P. E. Reynolds, and P. Courvalin. 2000. Glycopeptide-resistant Enterococcus faecium BM4416 is a VanD-type strain with an impaired D-alanine:D-alanine ligase. Antimicrob. Agents Chemother. 44:1346-1348.
Perichon, B., P. Reynolds, and P. Courvalin. 1997. VanD-type glycopeptide resistant Enterococcus faecium BM4339. Antimicrob. Agents Chemother. 41:2016-2018.
eynolds, P. E., and P. Courvalin. 2005. Vancomycin resistance in enterococci due to synthesis of precursors terminating in D-alanyl-D-serine. Antimicrob. Agents Chemother. 49:21-25.
Woodford, N. 2001. Epidemiology of the genetic elements responsible for acquired glycopeptide resistance in enterococci. Microb. Drug Resist. 7:229-236.
National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba
1 SMBD-Jewish General Hospital and McGill University, Montreal, Quebec, Canada(David A. Boyd,Mark A. Mil)
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
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.
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 AsnAsp change at position 314 in the ATP-binding G1 box and an AlaSer 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-200Leu, Asp-312Asn, or Asp-312Ala) 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 (TTATGA) 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.
EFERENCES
Ambúr, O.-H., P. E. Reynolds, and C. A. Arias. 2002. D-Ala:D-Ala ligase gene flanking the vanC cluster: evidence for presence of three ligase genes in vancomycin-resistant Enterococcus gallinarum BM4174. Antimicrob. Agents Chemother. 46:95-100.
Arias, C. A., P. Courvalin, and P. E. Reynolds. 2000. vanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174. Antimicrob. Agents Chemother. 44:1660-1666.
Arthur, M., P. Reynolds, and P. Courvalin. 1996. Glycopeptide resistance in enterococci. Trends Microbiol. 4:401-407.
Boyd, D. A., J. Conly, H. Dedier, G. Peters, L. Robertson, E. Slater, and M. R. Mulvey. 2000.
Molecular characterization of the vanD gene cluster and a novel insertion element in a vancomycin-resistant enterococcus isolated in Canada. J. Clin. Microbiol. 38:2392-2394.
Boyd, D. A., P. Kibsey, D. Roscoe, and M. R. Mulvey. 2004. Enterococcus faecium N03-0072 carries a new VanD-type vancomycin resistance determinant: characterization of the VanD5 operon. J. Antimicrob. Chemother. 54:680-683.
Casadewall, B., and P. Courvalin. 1999. Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339. J. Bacteriol. 181:3644-3648.
Casadewall, B., P. E. Reynolds, and P. Courvalin. 2001. Regulation of expression of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339. J. Bacteriol. 183:3436-3446.
Courvalin, P. 2005. Genetics of glycopeptide resistance in gram-positive pathogens. Int. J. Med. Microbiol. 294:479-486.
Dahl, K. H., G. S. Simonsen, . Olsvik, and A. Sundsfjord. 1999. Heterogeneity in the vanB gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci. Antimicrob. Agents Chemother. 43:1105-1110.
Dalla Costa, L. M., P. E. Reynolds, H. A. P. H. M. Souza, D. C. Souza, M.-F. I. Palepou, and N. Woodford. 2000. Characterization of a divergent vanD-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil. Antimicrob. Agents Chemother. 44:3444-3446.
Depardieu, F., M. Kolbert, H. Pruul, J. Bell, and P. Courvalin. 2004. VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis. Antimicrob. Agents Chemother. 48:3892-3904.
Depardieu, F., P. E. Reynolds, and P. Courvalin. 2003. VanD-type vancomycin-resistant Enterococcus faecium 10/96A. Antimicrob. Agents Chemother. 47:7-18.
Dutka-Malen, S., B. Blaimont, G. Wauters, and P. Courvalin. 1994. Emergence of high-level resistance to glycopeptides in Enterococcus gallinarum and Enterococcus casseliflavus. Antimicrob. Agents Chemother. 38:1675-1677.
Evers, S., B. Casadewall, M. Charles, S. Dutka-Malen, M. Galimand, and P. Courvalin. 1996. Evolution of structure and substrate specificity in D-alanine:D-alanine ligases and related enzymes. J. Mol. Evol. 42:706-712.
Gomez, A., M. Ladire, F. Marcille, and M. Fons. 2002. Trypsin mediates growth phase-dependent transcriptional regulation of genes involved in biosynthesis of ruminococcin A, a lantibiotic produced by a Ruminococcus gnavus strain from a human intestinal microbiota. J. Bacteriol. 184:18-28.
Liu, Y.-G., N. Mitsukawa, T. Oosumi, and R. F. Whittier. 1995. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8:457-463.
Liu, Y.-G., and R. F. Whittier. 1995. Thermal asymmetric PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674-681.
Ostrowsky, B. E., N. C. Clark, C. Thauvin-Eliopoulos, L. Venkataraman, M. H. Samore, F. C. Tenover, G. M. Eliopoulos, R. C. Moellering, Jr., and H. S. Gold. 1999. A cluster of VanD vancomycin-resistant Enterococcus faecium: molecular characterization and clinical epidemiology. J. Infect. Dis. 180:1177-1185.
Panesso, D., L. Abadía-Patio, N. Vanegas, P. E. Reynolds, P. Courvalin, and C. A. Arias. 2005.
Transcriptional analysis of the vanC cluster from Enterococcus gallinarum strains with constitutive and inducible vancomycin resistance. Antimicrob. Agents Chemother. 49:1060-1066.
Parkinson, J. S., and E. C. Kofoid. 1992. Communication modules in bacterial signalling proteins. Annu. Rev. Genet. 26:71-112.
Perichon, B., B. Casadewall, P. E. Reynolds, and P. Courvalin. 2000. Glycopeptide-resistant Enterococcus faecium BM4416 is a VanD-type strain with an impaired D-alanine:D-alanine ligase. Antimicrob. Agents Chemother. 44:1346-1348.
Perichon, B., P. Reynolds, and P. Courvalin. 1997. VanD-type glycopeptide resistant Enterococcus faecium BM4339. Antimicrob. Agents Chemother. 41:2016-2018.
eynolds, P. E., and P. Courvalin. 2005. Vancomycin resistance in enterococci due to synthesis of precursors terminating in D-alanyl-D-serine. Antimicrob. Agents Chemother. 49:21-25.
Woodford, N. 2001. Epidemiology of the genetic elements responsible for acquired glycopeptide resistance in enterococci. Microb. Drug Resist. 7:229-236.
National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba
1 SMBD-Jewish General Hospital and McGill University, Montreal, Quebec, Canada(David A. Boyd,Mark A. Mil)