First Characterization of a Cluster of VanA-Type Glycopeptide-Resistant Enterococcus faecium, Colombia

Diana Panesso, Sigifredo Ospina, Jaime Robledo, María Claudia Vela, Julieta Peña, Orville Hernández, Jinnethe Reyes, and César A. Arias

Disclosures

Emerging Infectious Diseases. 2002;8(9) 

In This Article

Abstract and Introduction

From August 1998 to October 1999, glycopeptide-resistant enterococci (GRE) were isolated from 23 infected patients at a teaching hospital in Medellín, Colombia. Identification at the species level and by multiplex polymerase chain reaction assay indicated that all isolates were Enterococcus faecium. The isolates were highly resistant to ampicillin, ciprofloxacin, gentamicin, penicillin, streptomycin, teicoplanin, and vancomycin; they were susceptible only to chloramphenicol, linezolid, and nitrofurantoin. Determination of glycopeptide genotype indicated the presence of the vanA gene in all isolates. Molecular typing by pulsed field gel electrophoresis showed that all isolates were closely related. This study is the first molecular characterization of GRE in Colombia.

Enterococci normally colonize the intestinal tract of humans and other animals, with urinary tract infection being the most common enterococcal infection reported in humans.[1] In recent years, enterococci have become important nosocomial pathogens: the organisms have been reported as the second leading cause of urinary tract infections and the third leading cause of nosocomial bacteremia in hospitalized patients.[2] The most commonly identified species is Enterococcus faecalis, followed by E. faecium.[3]E. gallinarum, E. casseliflavus, and E. durans have been reported less often.[4,5]

The most important characteristics of these organisms include their inherent resistance to several antimicrobial agents and their ability to acquire resistance determinants. Resistance against such diverse groups of drugs as β-lactams, macrolides, aminoglycosides, and glycopeptides continues to evolve. The ability to grow in the presence of glycopeptides results from the change of the C-terminal residue of peptidoglycan precursors (D-Ala) to D-lactate (VanA, VanB, and VanD phenotypes)[6,7] or D-serine (VanC, VanE, and VanG phenotypes).[8,9,10] The change alters the affinity of the glycopeptide for its natural target.[6] Six different gene clusters have been described (vanA-B-C-D-E-G).[6,10,11,12] The most predominant phenotype in E. faecium is VanA; VanA strains are highly resistant to both vancomycin and teicoplanin. The vanA gene cluster is located on transposons or related elements[6] and has also been found in nonenterococcal species such as Arcanobacterium (Corynebacterium) haemolyticum, Oerskovia turbata, Bacillus circulans, and Streptococcus gallolyticus.[13,14,15,16] A van cluster with a high degree of homology to the vanA cluster (designated vanF) has been found in the biopesticide organism Paenibacillus popilliae.[17]

Since the initial discovery of glycopeptide-resistant enterococci (GRE) in the United Kingdom,[18] nosocomial isolates of GRE have been reported from around the world;[14] these isolates have also been found in healthy people in the community outside the hospital.[19] In Latin America, GRE have been reported in Argentina[20] and Brazil.[21] We report here the first isolation and characterization of a cluster of VanA-type glycopeptide-resistant E. faecium in a teaching hospital in Colombia.

Hospital San Vicente de Paul is a 650-bed teaching hospital providing tertiary care for Medellín, Colombia, and neighboring towns, an area with a population of 1.5 million inhabitants. From August 1998 to October 1999, we collected organisms from 23 patients. Enterococci were isolated from infected patients by classical microbiologic techniques.[3] Identification at the species level was performed by the Vitek gram-positive card (bioMérieux SA, Marcy l'Etoile, France), according to the manufacturer's recommendations.

Initial identification of resistance to vancomycin was performed by the Vitek system (bioMérieux SA). We confirmed resistance to vancomycin, determining MICs by an agar dilution method as recommended by the National Committee for Clinical Laboratory Standards[22] on Mueller-Hinton agar plates (ICN Pharmaceuticals Inc., Madison, WI). MICs were performed in duplicate. The following antimicrobial agents were obtained as reference powders of known potency and tested: ampicillin, ciprofloxacin, chloramphenicol, gentamicin, penicillin, streptomycin, teicoplanin, vancomycin (ICN Pharmaceuticals, Inc.), and linezolid (Pharmacia Corp., Peapack, NJ). Susceptibility to nitrofurantoin (MIC <32 µg/mL) was determined by the Vitek system (bioMérieux SA). In addition to determining MICs, high-level resistance to streptomycin was tested at concentrations of 2 mg/mL; E. faecalis ATCC 29212 was used as control strain. Three well-characterized strains of enterococci belonging to the genotypes vanA (E. faecium BM4147), vanB (E. faecalis V583), and vanC (E. gallinarum BM4174) were included as GRE control strains.

van
Genes

For species identification of enterococcal isolates, the genes encoding D-alanine-D-alanine ligases specific for E. faecium (ddlE. faecium ), E. faecalis (ddlE. faecalis ), vanC-1 (E. gallinarum), and vanC-2 (E. casseliflavus) were detected by a multiplex PCR assay, as described by Dutka-Malen et al..[23] Primers D1 (5´ GCTTCTTCCTTTACGACC) and D2 (GTTCCAGTCCTAAAAAAC) for the ddl gene of E. avium were included in the multiplex mixture. A similar multiplex PCR protocol was performed separately for detection of van genes by using specific primers for vanA, vanB, vanC-1, and vanC-2 genes.[23] E. faecium BM4147 (vanA), E. faecalis V583 (vanB), and E. gallinarum BM4174 (vanC-1) were used as control strains.

Molecular typing was performed by pulsed-field gel electrophoresis (PFGE). Chromosomal DNA was obtained by the procedure of Antonishyn et al.:[24] a loopful of bacterial colonies from a 24-h isolate was grown until A 600 was 0.1 in brain heart infusion broth at 37°C. Bacteria were harvested by centrifugation at 4°C, and the pellet was resuspended in cell suspension buffer (1M NaCl, 10 mM Tris-HCl, pH 8.0). The suspension was embedded in 1.5% agarose and disks were made. Disks were placed in lysis buffer (6mM Tris-HCl, pH 8, 1 M NaCl, 100 mM EDTA, 0.5% Brij-58, 0.2% Na deoxycholate, and 0.5% N-lauroyl sarcosine) with additional RNase (20 µg/mL) and lysozyme (1 mg/mL) and incubated for 4 h at 37°C. The disks were washed with EDTA-sarcosine buffer (0.5 M EDTA, pH 8, and 0.1% N-lauroyl sarcosine), placed in proteinase K solution (100 µg/mL), and incubated overnight at 50°C with mild agitation. Disks were washed four times with Tris-EDTA buffer (Tris 10 mM, pH 7.5, and 1mM EDTA) for 30-60 min at room temperature on a rocker.

DNA was digested as described.[25] Briefly, DNA fixed in the agarose disks was preincubated in 1 mL of buffer E (6 mM Tris, pH 8, 20 mM KCl, 6 mM MgCl2, and 6 mM 2-mercaptoethanol) at 25°C for 30 min. Restriction was performed for 17 h in 60 µL of restriction buffer containing SmaI (20 U) at 25°C. The reaction was stopped by addition of 10 µL of sterile loading buffer. Gels were prepared with 1% agarose in 0.5x TBE buffer (50 mM Tris, pH 8, 50 mM boric acid, 0.2 mM EDTA). A DNA ladder (50-1000 kb) was used as the molecular size marker. Fragments were separated by electrophoresis (CHEF-DR II system, Bio-Rad Laboratories, Inc., Richmond, CA) at 6 V/cm, with switch times ramped from 1 s to 35 s over 23 h at 14°C. After staining with ethidium bromide, the restricted DNA fragments were viewed under UV light and photographed. A vancomycin-susceptible strain of E. faecium isolated in the same hospital was included in the PFGE protocol as the control. We interpreted the band patterns by the criteria of Tenover et al..[26]

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