Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Boyd, D. A. Search for Related Content PubMed PubMed Citation Articles by Boyd, D. A.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, June 2006, p. 2217-2221, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01541-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

VanG-Type Vancomycin-Resistant Enterococcus faecalis Strains Isolated in Canada

David A. Boyd,¹ Tim Du,¹ Romeo Hizon,¹ Brynn Kaplen,¹ Travis Murphy,¹ Shaun Tyler,¹ Shirley Brown,² Frances Jamieson,² Karl Weiss,³ Michael R. Mulvey,^1* and the Canadian Nosocomial Infection Surveillance Program

National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada,¹ Central Public Health Laboratory, Ontario Ministry of Health, Toronto, Ontario, Canada,² Hôpital Maisonneuve-Rosemont, Montreal, Quebec, Canada³

Received 1 December 2005/ Returned for modification 2 January 2006/ Accepted 26 March 2006

Top Abstract Text References

 
Enterococcus faecalis G1-0247 (vancomycin MIC, 16 µg/ml) was found to harbor a vanG operon 99% identical to the vanG operon in E. faecalis BM4518. E. faecalis N03-0233 (vancomycin MIC, 16 µg/ml) was found to harbor a novel vanG operon, vanG2, on an element in a different chromosomal location than the vanG-harboring elements in G1-0247 and BM4518.

Top Abstract Text References

 
The vanG operon provides low-level resistance to vancomycin through the action of the D-Ala-D-Ser VanG ligase (9). The operon consists of three regulatory genes, vanU_GR_GS_G, which are cotranscribed from the P_UG promoter, followed by the resistance region consisting of five genes, vanY_GW_GGXY_GT_G, which are cotranscribed from the P_YG promoter (1). To date the vanG operon has been found only in a few related strains of Enterococcus faecalis isolated in Australia (1, 7). The transfer of VanG resistance was via ca.-240-kb elements to the chromosome of susceptible E. faecalis strains (1). The vanG operon and the chromosomal insertion site of the element harboring it have been characterized in E. faecalis BM4518 (vancomycin MIC, 16 µg/ml; teicoplanin MIC, 0.5 µg/ml) (1).

This report describes the characterization of two E. faecalis strains exhibiting low-level vancomycin resistance that were isolated in Canada.

Genomic libraries were constructed with partial Sau3A fragments using Lambda ZAP (Stratagene, La Jolla, CA). Primers used in PCRs are listed in Table 1. Vectorette PCR, inverse PCR, and thermal asymmetric interlaced (TAIL)-PCR (5, 6, 10) were used to isolate genomic regions that could not be isolated from the genomic libraries.

View this table: [in this window] [in a new window]

TABLE 1. Primers used in this study

E. faecalis G1-0247 (vancomycin MIC, 16 µg/ml, and teicoplanin MIC, 0.75 µg/ml by Etest; AB Biodisk, Solna, Sweden) was isolated at Hôpital Maisonneuve-Rosemont, Montreal, Quebec, in December 2001 from a rectal swab from a patient with type 1 diabetes with complications and chronic renal failure. The patient had several hospitalizations since March 2000 and received vancomycin on several occasions during this period. Further analysis of E. faecalis G1-0247 by PCR gave a positive reaction for the vanG gene. Using a combination of cloning, PCR techniques, and sequencing, we characterized 14,301 bp encompassing the vanG operon (accession no. DQ212986) (Fig. 1). Analysis revealed that the E. faecalis BM4518 and G1-0247 vanG operon regions shared >99% nucleotide identity, with there being only three differences. One was a T at position 1950 (accession no. DQ212986) in the orfG23-vanU_G intergenic region, which was a C in BM4518 (position 17015 of accession no. AY271782), and one was a G at position 5930 (accession no. DQ212986) in the first position of codon 265 of the vanW_G gene (GAAGlu-265), which was an A in BM4518 (AAALys-265; position 20994 in accession no. AY271782). The third difference was a T at position 359 of the E. faecalis G1-0247 vanY_G gene (position 4581 in accession no. DQ212986) which was absent in the BM4518 vanY_G gene. Hence, whereas in E. faecalis BM4518 the resulting frameshift leads to the introduction of a stop codon 6 bp further downstream (1), G1-0247 contains a full-length vanY_G gene. However, E. faecalis BM4518 produces another inducible D,D-carboxypeptidase activity presumably located elsewhere in the chromosome, and so VanY_G activity is presumably not essential for resistance (1). Whether the E. faecalis G1-0247 VanY_G protein is active and whether another D,D-carboxypeptidase activity is present await biochemical evidence. Downstream of the vanG operon were two open reading frames (ORFs) located between orfG24 and orfG25 that were not annotated for E. faecalis BM4518 (1). The first ORF, orfG26, is 429 bp (positions 10874 to 11302 in accession no. DQ212986) and codes for a putative protein of 142 amino acids exhibiting 50% identity to a sigma 24 homolog identified from the Streptococcus suis 89/1591 genome (GenBank locus tag Ssui801000309). The second ORF, orfG27, is 372 bp (positions 11889 to 12260 of accession no. DQ212986) and codes for a putative protein of 123 amino acids exhibiting 63% identity to the PemK-like MazF protein of Thermoanaerobacter tengcongensis MB4 (accession no. AE008691, locus tag TTE2166). Analysis of orfG25 and downstream flanking DNA showed 100% identity between E. faecalis BM4518 and G1-0247 in this region, indicating that an element harboring vanG in G1-0247 is inserted in the identical location, near the 3' end of a gene labeled orfG1 in BM4518 (Fig. 1). In the fully sequenced E. faecalis V583 genome this gene (locus tag EF0728) is located at positions 686280 to 687653 (8). The left junction region of the E. faecalis G1-0247 vanG element (accession no. DQ212987) was isolated by TAIL-PCR, and analysis showed that, whereas the locus EF0728 sequences were 100% identical, the next 343 bp, which define the 3' end of orfG1, exhibited only 86% identity, including differences leading to five amino acid changes (data not shown). Further, after the orfG1 stop codon the sequences diverged even more significantly, exhibiting only 43% identity. In the five VanG-type clinical isolates studied, the element harboring the resistance genes was located on chromosomal SmaI fragments sized from 400 to 530 kb (1). It was postulated that each strain acquired different elements independently or that the same transconjugant underwent DNA rearrangements upon, or following, integration. The differences in vanG elements may occur near the left end, whereas the right end containing the vancomycin resistance genes and the putative site-specific OrfG25 recombinase is conserved.

View larger version (26K): [in this window] [in a new window]

FIG. 1. Schematic of left junction regions and right junction regions from the vanG and vanG2 elements from E. faecalis G1-0247 and N03-0233, respectively, compared to the corresponding regions from E. faecalis BM4518. The regions cloned or characterized by PCR are indicated by two-headed arrows. Regions missing in the vanG2 operon compared to the corresponding regions in the vanG operon are indicated by dashed lines. Percent identities between the corresponding proteins encoded by the vanG and vanG2 regions are shown. The sequences surrounding the left and right genomic junctions of the vanG and vanG2 elements are shown at the bottom; nucleotides shown above the E. faecalis G1-0247 IR-L indicate differences from the BM4518 sequence. DR-L, direct repeat left; DR-R, direct repeat right; IR-L, inverted repeat left; IR-R, inverted repeat right.

E. faecalis N03-0233 (vancomycin MIC, 16 µg/ml; teicoplanin MIC, 0.5 µg/ml), isolated in 2003 from a rectal swab of a 55-year-old patient hospitalized in southern Ontario, was negative by PCR for vanA, vanB, vanD, vanE, and vanG. Analysis of a 600-bp product obtained with the V1/V2 degenerate primer pair for D-Ala-D-Xxx ligase genes (3) revealed a partial gene, labeled vanG2, with 87% nucleotide identity to the vanG gene from E. faecalis BM4518. Using a combination of cloning, PCR, and sequencing, a total of 12,547 bp of the region containing the vanG2 operon was determined (accession no. DQ222944) (Fig. 1). The vanG2 operon was found to consist of seven ORFs, the regulatory region consisting of vanU_G2R_G2S_G2, and the resistance region consisting of vanW_G2GXY_G2T_G2. Analysis revealed that the failure of the vanG1/vanG2 primer pair to amplify the vanG2 gene was due to sequence differences in the primer binding sites (data not shown). There was no vanY_G2 gene found between vanS_G2 and vanW_G2. The proteins encoded by the vanG2 operon share between 74 and 96% identity to the corresponding proteins encoded by the vanG operon (Fig. 1). The region downstream of vanT_G2 is similar to the corresponding region in the vanG operon, though the OrfG25 and OrfG₂25 proteins exhibit only 21% identity. There was no orfG₂23 homolog present in the vanG2 operon upstream of vanU_G2. Analysis downstream of orfG₂25 showed that this region was 100% identical to the dctP gene of E. faecalis V583, which encodes the TRAP dicarboxylate transporter DctP subunit (locus tag EF0429) (8). Thus, the element containing the vanG2 operon was found in a different chromosomal location than the element harboring the vanG operon. The left junction of the vanG2 element was isolated by TAIL-PCR (accession no. DQ222945). Analysis revealed the target site of integration was the 2-bp sequence CA equivalent to positions 400063 and 400064 of the E. faecalis V583 genome (8). In E. faecalis N03-0233 this 2-bp sequence flanks imperfect 8-bp inverted repeats that define the ends of the vanG2 element (left end, positions 142 to 149 of accession no. DQ222945; right end, positions 12210 to 12217 of accession no. DQ222944) (Fig. 1). We detected a 300-bp ORF, labeled orfG₂1, starting 155 bp downstream of the left junction integration site, whose putative product showed no significant homology to extant proteins in the GenBank database.

Vancomycin resistance was inducible in both E. faecalis G1-0247 and N03-0233 (data not shown) as it was in BM4518 (1). Given the near identity of the vanG operons, regulation of resistance in E. faecalis G1-0247 is likely the same as in BM4518. An alignment of the regions upstream of vanU_G2 and vanW_G2 with the P_UG and P_YG promoters indicated that similar promoter regions exist in the vanG2 operon (P_UG2 and P_WG2 in Fig. 2A and B, respectively). Further, putative VanR_G/P-VanR_G binding regions are conserved upstream of the –35 box in the P_WG2 region (Fig. 2B and C). It was postulated for the E. faecalis BM4518 vanG operon that one or more of these 12-bp regions are the core binding sites for VanR_G/P-VanR_G, allowing for inducible positive control of the resistance genes (1). It was noted (1) that these regions are highly conserved relative to the VanR_A/P-VanR_A consensus binding region in VanA-type Enterococcus faecium (Fig. 2C) (4).

View larger version (47K): [in this window] [in a new window]

FIG. 2. Alignments of the promoter regions from the vanG and vanG2 operons. Identical nucleotides are blocked. A) Alignment of the regulatory gene promoters PUG and PUG2; B) alignment of the resistance gene promoters PYG and PWG2; C) alignment of the putative binding sites for VanRG/P-VanRG and VanRG2/P-VanRG2 (a, b, and c in panel B) and the consensus binding site for VanRA/P-VanRA (4). An overall consensus binding site is shown. D is A, G, or T; R is A or G; W is A or T; and Y is C or T.

In E. faecalis BM4518, vancomycin resistance was cotransferred with ermB at a low frequency (2 x 10^–9 copies per donor) but only with selection on erythromycin (1). Both E. faecalis G1-0247 and N03-0233 harbored ermB, and transconjugants of E. faecalis JH2-2 carrying ermB could easily be selected on erythromycin after mating with either strain by cross-streaking on solid media. However, analysis of six transconjugants of each type showed that none harbored either vanG or vanG2. As with BM4518, no transconjugants could be selected on vancomycin. As we only analyzed a total of 12 transconjugants, it may be that ermB vanG transconjugants did exist but weren't characterized. Alternatively, ermB may be found on a mobile genetic element either separate from or embedded within the elements harboring vanG or vanG2 and more readily transferred. Also, the functions necessary for vanG transfer may not be functional in E. faecalis G1-0247 or N03-0233, or the conditions necessary for transfer were not met with the mating method used.

Enterococci harboring vanG-type genes are extremely rare; to date only a few strains from Australia and Canada have been isolated. In a previous study, vanG genes exhibiting 77% to 100% identity to the E. faecalis BM4518 vanG gene have been found in the fecal flora of humans, though the organisms harboring these genes could not be identified (2). The similarity of the regions characterized here and in E. faecalis BM4518 indicate that the vanG and vanG2 operons reside on elements that belong to a family of putatively conjugative related genetic elements. The difference in the chromosomal locations of the elements is most likely due to the action of the diverse OrfG25 site-specific recombinases. The absence of a vanY_G2 gene in the vanG2 operon is likely not detrimental to resistance expression, as the VanY_G activity may be redundant in enterococci if the strain contains another D,D-carboxypeptidase activity, as found for E. faecalis BM4518 (1).

Nucleotide sequence accession numbers. The vanG operon region and left junction region of the vanG element from E. faecalis G1-0247 were assigned accession no. DQ212986 and DQ212987, respectively, and the vanG2 operon region and left junction region of the vanG2 element from E. faecalis N03-0233 were assigned accession no. DQ222944 and DQ222945, respectively, in the GenBank database.

 
We thank Patrice Courvalin (Institut Pasteur) for providing us with sequence data before its publication.

Members of the Canadian Nosocomial Infection Surveillance Program are as follows: Elizabeth Bryce, Vancouver General Hospital, Vancouver, BC; John Conly, Foothills Medical Centre, Calgary, Alta.; Gordon Dow, The Moncton Hospital, Moncton, NB; John Embil, Health Sciences Centre, Winnipeg, Man.; Joanne Embree, Health Sciences Centre, Winnipeg, Man.; Michael Gardam, University Health Network, Toronto, Ont.; Denise Gravel, Centre for Infectious Disease Prevention and Control, Public Health Agency of Canada; Elizabeth Henderson, Peter Lougheed Centre, Calgary, Alta.; James Hutchinson, Health Sciences Centre, St. John's, Nfld.; Michael John, London Health Sciences Centre, London, Ont.; Lynn Johnston, Queen Elizabeth II Health Sciences Centre, Halifax, NS; Pamela Kibsey, Victoria General Hospital, Victoria, BC; Joanne Langley, I. W. K. Grace Health Science Centre, Halifax, NS; Mark Loeb, Hamilton Health Sciences Corporation, Hamilton, Ont.; Anne Matlow, Hospital for Sick Children, Toronto, Ont.; Allison McGeer, Mount Sinai Hospital, Toronto, Ont.; Sophie Michaud, CHUS-Hôpital Fleurimont, Sherbrooke, Que.; Mark Miller, SMBD-Jewish General Hospital, Montreal, Que.; Dorothy Moore, Montreal Children's Hospital, Montreal, Que.; Michael Mulvey, National Microbiology Laboratory, Public Health Agency of Canada; Marianna Ofner-Agostini, Centre for Infectious Disease Prevention and Control, Public Health Agency of Canada; Shirley Paton, Centre for Infectious Disease Prevention and Control, Public Health Agency of Canada; Virginia Roth, The Ottawa Hospital, Ottawa, Ont.; Andrew Simor, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ont.; Geoffrey Taylor, University of Alberta Hospital, Edmonton, Alta.; Monali Varia, Centre for Infectious Disease Prevention and Control, Public Health Agency of Canada; Mary Vearncombe, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ont.; Alice Wong, Royal University Hospital, Saskatoon, Sask.; and Dick Zoutman, Kingston General Hospital, Kingston, Ont.

 
* 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.

Top Abstract Text References

 

1
1. Depardieu, F., M. G. Bonora, P. E. Reynolds, and P. Courvalin. 2003. The vanG glycopeptide resistance operon from Enterococcus faecalis revisited. Mol. Microbiol. 50:931-948.[CrossRef][Medline]
2
2. Domingo, M.-C., A. Huletsky, R. Giroux, K. Boissinot, F. J. Picard, P. Lebel, M. J. Ferraro, and M. G. Bergeron. 2005. High prevalence of glycopeptide resistance genes vanB, vanD, and vanG not associated with enterococci in human fecal flora. Antimicrob. Agents Chemother. 49:4784-4786.[Abstract/Free Full Text]
3
3. Dukta-Malen, S., C. Molinas, M. Arthur, and P. Courvalin. 1992. Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine: D-alanine ligase-related protein necessary for vancomycin resistance. Gene 112:53-58.[CrossRef][Medline]
4
4. Holman, T. R., Z. Wu, B. L. Wanner, and C. T. Walsh. 1994. Identification of the DNA-binding site for the phosphorylated VanR protein required for vancomycin resistance in Enterococcus faecium. Biochemistry 33:4625-4631.[CrossRef][Medline]
5
5. 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.[CrossRef][Medline]
6
6. Liu, Y.-G., and R. F. Whittier. 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674-681.[CrossRef][Medline]
7
7. McKessar, S. J., A. M. Berry, J. M. Bell, J. D. Turnidge, and J. C. Paton. 2000. Genetic characterization of vanG, a novel vancomycin resistance locus of Enterococcus faecalis. Antimicrob. Agents Chemother. 44:3224-3228.[Abstract/Free Full Text]
8
8. Paulsen, I. T., L. Banerjei, G. S. A. Myers, K. E. Nelson, R. Seshadri, T. D. Read, D. E. Fouts, J. A. Eisen, S. R. Gill, J. F. Heidelberg, H. Tettelin, R. J. Dodson, L. Umayam, L. Brinkac, M. Beanan, S. Daugherty, R. T. DeBoy, S. Durkin, J. Kolonay, R. Madupu, W. Nelson, J. Vamathevan, B. Tran, J. Upton, T. Hansen, J. Shetty, H. Khouri, T. Utterback, D. Radune, K. A. Ketchum, B. A. Dougherty, and C. M. Fraser. 2003. Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 299:2071-2074.[Abstract/Free Full Text]
9
9. Reynolds, 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.[Free Full Text]
10
10. Singer, T., and E. Burke. 2003. High-throughput TAIL-PCR as a tool to identify DNA flanking insertions. Methods Mol. Biol. 236:241-271.[Medline]

---

Antimicrobial Agents and Chemotherapy, June 2006, p. 2217-2221, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01541-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Boyd, D. A., Willey, B. M., Fawcett, D., Gillani, N., Mulvey, M. R. (2008). Molecular Characterization of Enterococcus faecalis N06-0364 with Low-Level Vancomycin Resistance Harboring a Novel D-Ala-D-Ser Gene Cluster, vanL. Antimicrob. Agents Chemother. 52: 2667-2672 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Boyd, D. A. Search for Related Content PubMed PubMed Citation Articles by Boyd, D. A.