1 Nosocomial Infections, National Microbiology Laboratory, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2; 2 Victoria General Hospital, Victoria, British Columbia; 3 Vancouver General Hospital, Vancouver, British Columbia, Canada
Received 4 June 2004; returned 24 June 2004; revised 29 June 2004; accepted 1 July 2004
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Abstract |
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Methods: PCR was used to amplify the entire vancomycin resistance operon and the complete nucleotide sequence was determined by dideoxy cycle sequencing.
Results: Analysis revealed a VanD-type operon with 94% nucleotide identity to the VanD4 operon and 90% nucleotide identity to the VanD1/D3 operons. A set of universal primers was designed in order to identify all current vanD variants by PCR.
Conclusions: E. faecium N03-0072 carries a new VanD-type operon, designated VanD5.
Keywords: glycopeptide resistance , van PCR , E. faecium
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Introduction |
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Materials and methods |
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A female patient with a urinary tract infection complicated with renal stones had received multiple courses of vancomycin to treat infections caused by vancomycin-susceptible E. faecium that had been isolated from urine, pre- and post-operatively, and from a post-operative flank wound infection, in January 2003. Further surgery to relieve complications was carried out in February 2003, at which time a nephrostomy was created. A vancomycin-resistant enterococcus (VRE) was isolated from nephrostomy urine on March 18, 2003, while a subsequent urine screen on March 23, 2003 was negative for VRE. A rectal/perineal screen on April 4, 2003 was positive for VRE and this isolate, labelled N03-0072, was sent to the National Microbiology Laboratory for further characterization as part of a national surveillance initiative conducted by the Canadian Nosocomial Infection Surveillance Program. Control strains used were E. faecium ATCC 51559 (vanA), E. faecalis ATCC 51299 (vanB), Enterococcus gallinarum ATCC 49573 (vanC1), Enterococcus casseliflavus (vanC2), Enterococcus flavescens (vanC3), E. faecium BM4339 (vanD1), E. faecium N97-330 (vanD3), E. faecium 10/96A (vanD4), E. faecalis N00-410 (vanE), and E. faecalis 24AE010247 (vanG1).2 Strain identification was conducted by standard biochemical tests and glycopeptide MICs were determined by Etest (AB Biodisk, Solna, Sweden).
DNA methodology
Primers used in this study are listed in Table 1. Multiplex PCR for vanA, vanB, ddlE.faecium and ddlE.faecalis was as follows: each 25 µL reaction contained 1x PCR Buffer II, 3 mM MgCl2, 0.2 mM dNTPs, 1.25 U AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA), 0.5 µM each of primers for the vanA, vanB and E. faecium ddl genes, and 0.75 µM of primers for the E. faecalis ddl gene. PCR was carried out with an initial denaturation at 94°C for 10 min followed by 30 cycles at 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min, followed by a 5 min hold at 72°C. PCR for vanD, vanE and vanG was carried out singly as above. The N03-0072 ddl gene was amplified and sequenced using primers ddlF-1 and ddlF-2. PCR with D-Ala-D-X degenerate primers V3 and V4 was done with initial denaturation at 94°C for 10 min followed by 15 cycles at 94°C for 30 s, 55°C for 1 min with decrease in temperature of 0.67° per cycle, and 72°C for 2.5 min, followed by 20 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 2.5 min, followed by a 5 min hold at 72°C. Long PCR was with the Expand Long Template PCR system as described by the manufacturer (Roche Diagnostics, Indianapolis, IN, USA). The entire VanD5 operon was isolated as two overlapping amplicons using long PCR with primers RDNH2 and HD-2 and with HD-1 and vanX3'. Cloning of PCR products was done using pPCR Script Cam Cloning Kit (Stratagene, La Jolla, CA, USA).
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The nucleotide sequences of the E. faecium N03-0072 VanD5 operon and of its ddl gene have been deposited in the GenBank Database under accession numbers AY489045 and AY489046, respectively.
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Results and discussion |
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The MICs of vancomycin and teicoplanin for E. faecium N03-0072 were 64 mg/mL and 1 mg/L, respectively. The isolate was negative in multiplex PCR for vanA, vanB and ddlE.faecalis, but, as expected, was positive for ddlE.faecium. PCR was also negative for vanD, vanE and vanG. PCR was therefore carried out with primers V3 and V4 and the 630 bp product was cloned and sequenced. Analysis revealed a product that exhibited 8991% nucleotide (nt) identity with the known vanD genes and hence, this variant was designated vanD5. PCR for vanD had apparently failed because of sequence divergence at the vanD-2 primer site as shown previously for vanD4.3 The entire VanD5 operon was characterized by completely sequencing two overlapping amplicons, one spanning vanRvanH, and one spanning vanHvanX. A total of 5653 bp of contiguous sequence was obtained and analysis revealed six open reading frames (ORFs) homologous to characterized VanD operon genes, vanRSYHDX. Overall the VanD5 operon shared 9091% nt identity with the VanD1 and VanD3 operons and the partially sequenced VanD2 operon, and 94% nt identity with the VanD4 operon (data not shown). In contrast, the VanD4 operon shares only 85% nt identity with the VanD1 and VanD3 operons, whereas the latter two share 97% nt identity. Table 2 shows a percentage identity matrix of the proteins of the VanD operon. For these comparisons, the putative full length non-mutated proteins of the truncated VanSD3, VanSD4, VanYD4 and VanYD5 proteins were used (see below and footnote a of Table 2). The vanYD5 gene has a mutation at codon 26 that causes a frameshift and subsequent truncation after amino acid 51. The vanYD4 gene contains a different frameshift mutation that presumably leads to a truncated non-functional protein.4 It is interesting to note that amongst the VanYD and VanD proteins, the ones from the VanD5 operon are more similar to their homologues from the VanD4 operon than to the ones from the VanD1/D2/D3 operons, whereas amongst VanHD and VanXD proteins, the opposite is true. This may reflect recruitment of genes from different VanD operons in the assembly of the VanD5 operon. The regulatory proteins VanR and VanS are highly conserved, exhibiting 9399% amino acid identity.
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The VanD1, VanD3 and VanD4 operons are expressed constitutively, presumably due to mutation in their vanSD genes; a Ser-166Pro substitution in vanSD1 which occurs near the site of autophosphorylation, a frameshift in vanSD3 that leads to a truncated protein, and an insertion element disrupting the vanSD4 gene.4,5 We could not detect any obvious mutation in the vanSD5 or vanRD5 genes even though growth experiments indicated constitutive expression of the VanD5 operon (data not shown). In contrast, the VanD2 operon was found to be inducible.6 In addition, the ddl gene (D-Ala-D-Ala ligase) in strains harbouring the VanD1, VanD3 and VanD4 operons have all been found to contain mutations that lead to a non-functional enzyme and hence a lack of peptidoglycan precursors terminating in D-Ala-D-Ala.4,5,7,8 The E. faecium BM4339 (VanD1) ddl gene contains a 5 bp insertion leading to a truncated protein, the E. faecium N97-330 (VanD3) ddl gene is interrupted by ISEfm1 (also called IS19), and the E. faecium 10/96A (VanD4) ddl gene contains a Gly-184
Ser mutation that leads to a non-functional protein.4,5,7,8 Thus, the constitutive expression of the VanD operons allows these strains to grow in the presence or absence of vancomycin. Analysis of the sequence of the ddl gene of E. faecium N03-0072 (accession no. AY489046) showed that the insertion of an additional C-residue at codon 322 introduces a frameshift that changes the amino acid sequence for the next 11 residues, followed by a stop codon. Hence, the last 25 amino acids of the presumptive wild-type Ddl protein are absent (data not shown). Though this does not directly involve residues critical for enzymic function, it presumably perturbs secondary and/or tertiary structure and probably affects enzyme function.9 An analysis of the E. faecium A902 (VanD2) ddl gene, which has been deposited in GenBank (accession no. AF215736), revealed a Glu-13
Gly mutation in a residue that is involved in substrate binding.9 A vancomycin-dependent strain, E. faecium BM4484, contains the Glu-13
Gly change and produces a non-functional Ddl protein.9 Thus, all VanD-containing strains isolated to date produce a non-functional D-Ala-D-Ala ligase enzyme. Even though vancomycin resistance in VanD2 strain A902 is reported to be inducible and the strain is not vancomycin dependent, the absence of a functional ddl enzyme leads one to speculate that there is, at the least, a low basal level of VanD2 produced constitutively in this strain.
Transfer experiment
Attempts to transfer vancomycin resistance from E. faecium N03-0072 to E. faecium GE-1 by conjugation were unsuccessful. No other VanD variant has been successfully transferred by conjugation in vitro.3,6,10
Design of a universal vanD primer set
The original vanD1/vanD2 primer set designed from the vanD1 sequence, also detects the vanD2 and vanD3 genes in PCR analysis.5,6,10 However, due to sequence divergence, these primers failed to detect the vanD4 and vanD5 variants (this study).3 Based on a sequence alignment, we designed a new primer set, vanD-U1/vanD-U2, for use in detection of all extant vanD genes in PCR analyses. A PCR experiment with this primer set showed that the expected 628 bp amplicon was detected in vanD1, vanD3, vanD4 and vanD5-containing strains, whereas no amplicons were produced in strains containing vanA (E. faecium), vanB (E. faecalis), vanC1 (E. gallinarum), vanC2 (E. casseliflavus), vanC3 (E. flavescens), vanE (E. faecalis), or vanG (E. faecalis). Since the primers were designed from areas in the vanD genes exhibiting 100% sequence identity, vanD2 would also be detected with the vanD-U1/vanD-U2 primer set.
Although all VanD-type strains to date have been readily identified as vancomycin-resistant using standard phenotypic susceptibility methods, the genetic variability identified between the vanD subtypes could cause difficulties in identification using published PCR protocols. We suggest laboratories adopt the vanD-U1/vanD-U2 primer set described above to detect all known VanD-type variants.
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Acknowledgements |
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Members of the Canadian Nosocomial Infection Surveillance Program (CNISP): Dr Elizabeth Bryce, Vancouver General Hospital, Vancouver, BC; Dr John Conly, Foothills Medical Centre, Calgary, Alta; Dr John Embil, Health Sciences Centre, Winnipeg, Man.; Dr Joanne Embree, Health Sciences Centre, Winnipeg, Man.; Dr Michael Gardam, University Health Network, Toronto, Ont.; Ms Denise Gravel, Centre for Infectious Disease Prevention and Control, Health Canada; Dr Elizabeth Henderson, Peter Lougheed Centre, Calgary, Alta; Dr James Hutchinson, Health Sciences Centre, St. John's, Nfld; Dr Michael John, London Health Sciences Centre, London, Ont.; Dr Lynn Johnston, Queen Elizabeth II Health Sciences Centre, Halifax, NS; Dr Pamela Kibsey, Victoria General Hospital, Victoria, BC; Dr Joanne Langley, I.W.K. Grace Health Science Centre, Halifax, NS; Dr Mark Loeb, Hamilton Health Sciences Corporation, Hamilton, Ont.; Dr Anne Matlow, Hospital for Sick Children, Toronto, Ont.; Dr Allison McGeer, Mount Sinai Hospital, Toronto, Ont.; Dr Sophie Michaud, CHUS-Hôpital Fleurimont, Sherbrooke, Que.; Dr Mark Miller, SMBD-Jewish General Hospital, Montreal, Que.; Dr Dorothy Moore, Montreal Children's Hospital, Montreal, Que.; Dr Michael Mulvey, Canadian Science Centre for Human and Animal Health, Health Canada; Ms Marianna Ofner-Agostini, Centre for Infectious Disease Prevention and Control, Health Canada; Ms Shirley Paton, Centre for Infectious Disease Prevention and Control, Health Canada; Dr Virginia Roth, The Ottawa Hospital, Ottawa, Ont.; Dr Andrew Simor, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ont.; Dr Geoffrey Taylor, University of Alberta Hospital, Edmonton, Alta; Dr Mary Vearncombe, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ont.; Dr Alice Wong, Royal University Hospital, Saskatoon, Sask.; Dr Dick Zoutman, Kingston General Hospital, Kingston, Ont.
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Footnotes |
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Members of the Canadian Nosocomial Infection Surveillance Program (CNISP) are listed in the Acknowledgements
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References |
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