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Laboratoire Universitaire d'Antibiologie, Faculte de Medecine, Avenue Kennedy, 30900 Nmes, France
Laboratoire de Bacteriologie, Virologie, Parasitologie, CHU de Nmes, Groupe Hospitalo-Universitaire de Caremeau, Place du Professeur Robert Debre, 30029 Nmes Cedex 9, France
Laboratoire de Bacteriologie, CHU Arnaud de Villeneuve, 371, avenue du Doyen Gaston Giraud, 34295 Montpellier cedex 5, France
Service de Medecine Interne B, CHU de Nmes, Groupe Hospitalo-Universitaire de Caremeau, Place du Professeur Robert Debre, 30029 Nmes Cedex 9, France
ABSTRACT
We report the first strain of glycopeptide-resistant Enterococcus faecium from Europe that contains a vanD allele isolated from blood cultures of an immunocompromised patient hospitalized in a French university hospital. Based on phenotypic results, PCR sequencing, pulsed-field gel electrophoresis, and Southern blotting, the isolate was assigned to E. faecium with a chromosomally located VanD allele most closely related to the VanD1 allele.
TEXT
Glycopeptides inhibit cell wall synthesis through the interaction of their N termini with the D-alanyl-D-alanine termini of peptidoglycan precursors. The resulting complexes prevent the transglycosylases from incorporating disaccharide pentapeptide subunits into the growing peptidoglycan chain, thereby inhibiting cell wall synthesis (15). Since 1988, many reports described the increase of vancomycin resistance in enterococci from Europe and the United States (6, 13). This resistance to glycopeptides occurs via a common mechanism involving modification of the peptidoglycan biosynthetic pathway (8). Five phenotypes of acquired glycopeptide resistance have been identified in enterococci. VanA and VanB are the most common types. They are caused by complex gene clusters that may be plasmid and/or transposon carried (8). VanE and VanG types have each been identified in single strains of Enterococcus faecalis (11, 15). Finally, to date, five Enterococcus faecium strains with VanD resistance have been reported in the United States, in Canada, and in Brazil (4, 7, 16, 18, 19). These isolates were recovered from stool cultures, urine, and blood cultures from immunocompromised patients. Each contained a distinct vanD allele, vanD1 to vanD5 (4, 7, 16, 18, 19).
Since November 2003, a 56-year-old man had been regularly admitted to the hematology and oncology unit of Nmes University Hospital for the treatment of an acute myeloid leukemia. He was hospitalized on February 2004 for febrile neutropenia. Physical examination did not reveal a focal site of infection. E. faecium was isolated from three blood cultures. Despite an intravenous antibiotic therapy using levofloxacin and quinupristin/dalfopristin, the patient died 5 days after his admission.
E. faecium was determined biochemically using the Vitek 2-AST N017 identification card (Biomerieux, Marcy-l'Etoile, France). Susceptibility to antibiotics was determined with the E-test method (AB Biodisk, Solna, Sweden) on blood agar plates, and antibiotic MICs were interpreted according to the recommendations of the Antibiotic Susceptibility Testing Committee of the French Society for Microbiology with Enterococcus faecalis ATCC 29212, E. faecium B91 (VanA+), and E. faecium B132 (VanB+) as reference strains (20). The three isolates from the blood cultures showed the same resistant pattern. They were resistant to penicillin G (MIC, 16 μg/ml), gentamicin (MIC, >2,000 μg/ml), streptomycin (MIC, >2,000 μg/ml), tetracycline (MIC, 16 μg/ml), linezolid (MIC, >4 μg/ml), daptomycin (MIC, 16 μg/ml), and macrolide-lincosamide-streptogramin B-type antibiotics (MIC, >8 μg/ml). They were also resistant to vancomycin (MIC, >256 μg/ml) and teicoplanin (MIC, >256 μg/ml). They were susceptible only to quinupristin/dalfopristin (MIC, 0.5 μg/ml) and intermediate to levofloxacin (MIC, 2 μg/ml). Levels of growth of these strains in the absence of and following the addition of vancomycin (8 μg/ml) were identical (data not shown), indicating that resistance was expressed constitutively. One of the three isolates was genotypically studied and named E. faecium NEF1.
To determine the genotype responsible for the unusual resistance of this strain, we used a multiplex PCR assay previously described by Dutka-Malen et al., which allows simultaneous detection of the glycopeptide resistance genotypes vanA and vanB, which encode D-alanine-D-lactate (D-Ala-D-Lac) ligases, and vanC-1 and vanC-2/3, which encode D-Ala-D-Ser ligases (9). At the same time, multiplex PCR allows the identification to the species level of the following clinically relevant enterococci: E. faecium, E. faecalis, E. gallinarum, and E. casseliflavus/E. flavescens. In this assay, PCR amplicons are discriminated from each other based on their molecular sizes. This protocol was modified according to Patel et al. in order to bypass the DNA extraction procedure and to process the PCR assay directly on an isolated colony from blood agar or Mueller-Hinton plates (17). Four previously characterized vancomycin-resistant Enterococcus strains, which carry either vanA, vanB, vanC-1, or vanC-2/3 genes, were used as controls. Multiplex PCR performed on the enterococcal isolate resistant to vancomycin and teicoplanin yielded a 550-bp amplified fragment specific for an internal fragment of the gene encoding D-Ala-D-Ala ligase of E. faecium. However, despite the high level of resistance to glycopeptides of this E. faecium isolate, none of the vanA, vanB, vanC1, or vanC2/3 resistance determinants could be amplified. Using degenerate V3 (5'-GAR GAT GGI TSC ATM CAR GGW-3') and V4 (5'-MGT RAA ICC IGG CAK RGT RTT-3') primers, which allow amplification of fragments internal to genes encoding related ligases (12), we obtained a 630-bp fragment. This fragment was cloned into pGEMT (Promega, Madison, WI) to yield the pGEMT-Van plasmid and introduced into Escherichia coli via transformation. The insert was sequenced. The deduced 223-amino-acid partial peptide showed approximately 90% identity with five VanD ligases listed in the GenBank database. Primer walk sequencing was carried out to amplify the entire D-Ala-D-Lac ligase (MWG-Biotech, Ebersberg, Germany). Deduced amino acid sequences were aligned using ClustalW (http://www.ebi.ac.uk/clustalw/). The complete region detected was predicted to encode a ligase of 343 amino acids (Fig. 1). This VanD peptide showed 85 to 95% amino acid identity to the VanD2 (AF153050 [16]), VanD3 (AF175293 [3]), VanD4 (AF277571 [7]), and VanD5 (AAR37060 [2]) ligases and 68% and 67% identity with, respectively, the VanA and VanB (B1 and B2) ligases (M97297 [1], U35369 [10], and AY145441 [14]) (Table 1). It shared a maximum of 98% amino acid identity with the VanD1 ligase (AF130997 [5]).
To search for a possible chromosomal location of the vanD allele, we used pulsed-field gel electrophoresis of I-CeuI-restricted DNA as previously described (2). A transfer of DNA from electrophoresis gel to a nylon membrane was accomplished by capillary transfer. For DNA probing of the Southern blot, we used an amplification product obtained by PCR of E. faecium NEF1 using the vanD1 primers previously described (18). This product was labeled with digoxigenin-dUTP following the manufacturer's instructions (Boehringer Mannheim, Indianapolis, Ind.) and hybridized on I-CeuI-restricted patterns (Fig. 2). The vanD1 probe hybridized with E. faecium NEF1 DNA, confirming the chromosomal localization of this resistance gene as previously described for the vanD1 gene (18). All these results point to the fact that the resistance gene in E. faecium NEF1 is most closely related to the vanD1 allele.
In this study, we reported to the best of our knowledge the first strain of vancomycin-resistant E. faecium from Europe that contains a vanD allele. Since this episode of bacteremia, no further strains belonging to this genotype have been isolated in our hospital. Similar to previous patients reported with VanD-type glycopeptide-resistant Enterococcus, this patient had multiple medical problems, i.e., underlying medical illness (diabetes mellitus), immunocompromised status (neutropenic leukemia or lymphoma), prolonged hospital stays, multiple courses of antibiotics (vancomycin, expanded-spectrum cephalosporins), and instrumentation (central venous catheter) (16). For a few months, our patient had been treated several times with vancomycin, but, during this period, the patient had no enterococcal infections. He received also -lactams (amoxicillin plus clavulanic acid, imipenem, and cephalosporins), fluoroquinolones, aminoglycosides, and pristinamycin. However, he did not have any links with the United States, Canada, or Brazil, the countries where the five VanD-type glycopeptide-resistant E. faecium strains have been previously described. Interestingly, the five E. faecium strains with VanD-type resistance reported before were isolated in patients with multiple medical problems: diabetes mellitus, aplastic anemia, and, above all, three cases of orthoptic liver transplantation.
Phenotypically, this E. faecium presents a very high level of resistance against teicoplanin compared with the other described vanD phenotypes. It may represent constitutive high-level expression of the vanD gene cluster. The resistance gene in E. faecium NEF1 is most closely related to the VanD1 allele. The E. faecium NEF1 peptide showed 98% amino acid identity to the VanD1 ligases and a vanD1 probe hybridized with the E. faecium NEF1 DNA. The residues believed to comprise the active site of VanA are conserved in all enterococcal D-Ala-D-Ala ligases, including our isolate (7) (Fig. 1). It will be interesting to characterize the entire vanD operon in E. faecium NEF1 to detect potential variations in its organization compared to vanD1 to vanD5 operons.
We showed that the vanD gene of our isolate was chromosomally located as previously described for the other VanD isolates (8). This distribution may contribute to the paucity of VanD-type resistant strains in contrast to the widespread and increasing prevalence of vancomycin-resistant enterococci carrying vanA or vanB genes in France.
(This work was presented in part in 6th European Congress of Chemotherapy and Infections, Paris, France, December 2004, abstr. 348/78P.)
ACKNOWLEDGMENTS
We are very grateful to Josiane Campos for performing the enterococcal multiplex PCR assay.
This work was supported by Universite de Montpellier 1 (BQR, BQ 68).
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