1 Department of Microbiology, Hippokration University Hospital, Thessaloniki; 2 Department of Medical Microbiology, University of Thessalia, Larissa; 3 Department of Microbiology, Faculty of Nursing, School of Health Sciences, University of Athens, 123 Papadiamantopoulou Street, 11527 Athens, Greece
Received 27 February 2004; returned 14 April 2004; revised 19 April 2004; accepted 21 April 2004
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Abstract |
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Methods: Specimens for the surveillance were collected from separate patients in two periods (JanuaryMay 1999 and JanuaryMay 2003); 258 specimens were submitted during the first period and 149 during the second period.
Results: Three patients (1.2%) were colonized with VRE during the first period, whereas 52 (34.9%) were colonized during the second period. Two VRE isolates of the first period were Enterococcus faecalis and one Enterococcus faecium, whereas those of the second period were E. faecium except for three E. faecalis and two Enterococcus gallinarum. All VRE isolates apart from the two E. gallinarum isolates were positive for the vanA gene. The 48 vancomycin-resistant E. faecium were classified into eight clonal types, one of those predominating with 29 isolates; the remaining included one to nine isolates. The five vancomycin-resistant E. faecalis formed four distinct clonal types.
Conclusions: The study reports a substantially higher prevalence of VRE carriage when the surveillance was repeated after a 4 year time interval. Urgent infection control measures are needed to prevent emergence of VRE outbreaks in our hospital setting.
Keywords: VRE , surveillance , genotyping
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Introduction |
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In Greek hospitals, unlike others in the United States and Europe, VRE causing clinical infections have only recently been recognized and outbreaks of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis have been reported from hospitals in the region of Athens.5,6 However, no systematic study has been done to estimate the prevalence of VRE colonization in hospitalized patients. In that respect, a cross-sectional survey was conducted in a tertiary Greek hospital with no documented VRE infections, in order to determine the degree of VRE colonization among hospitalized patients.
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Materials and methods |
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The study was conducted at Hippokration University Hospital, Thessaloniki, Greece. This is the largest tertiary referral hospital in Northern Greece having 1012 beds (51 with acute facilities) and more than 55 000 admissions each year. Specimens for the study were collected in two periods. The first period was from January through May 1999 and the second period from January through May 2003. In the study were included adult patients randomly chosen from those hospitalized for more than 48 h and less than 5 days in eight high-risk units (renal, transplant, oncology and intensive care units).
Culture and identification
Faecal samples were diluted with sterile saline and plated onto enterococcosel agar (BBL Microbiology Systems, Cockeysville, MD, USA) with and without 6 mg/L of vancomycin. Plates were incubated at 35°C and read after 24 and 48 h of incubation. From each sample, colonies showing macroscopically morphological differences and whose colony morphology was consistent with that of enterococci were subcultured and characterized as enterococci by additional tests (salt tolerance, growth on bile-aesculin agar, catalase activity). Identification to species level was carried out with the automated Vitek system (bioMérieux, Marcy l'Étoile, France). Identification of VRE was also confirmed by PCR analysis using specific primers described by Dutka-Malen et al.7
Susceptibility testing
For all distinct enterococcal isolates that grew on the screening agar supplemented with 6 mg/L of vancomycin, MICs of vancomycin and teicoplanin were determined by an agar dilution method.8 Interpretative criteria for susceptibility status were those of the NCCLS.8 The Vitek system was used in addition to determine susceptibility to a range of antimicrobials (ampicillin, ciprofloxacin, erythromycin, gentamicin, streptomycin and tetracycline). E. faecalis ATCC 29212 was used for quality control.
Genotyping testing
Isolates for which the vancomycin MIC was >4 mg/L were analysed for the presence of the vanA, vanB, vanC1, vanC2 and vanE gene by PCR using primers and conditions that were described previously.7,9 Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA was carried out with a contour-clamped homogeneous electric field apparatus (CHEF DRIII apparatus; Bio-Rad Laboratories, Hemel Hempstead, UK) and banding patterns of the strains were compared visually.10 Ten glycopeptide-sensitive E. faecium isolates, which were recovered during the study periods, were also chosen at random and used as controls.
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Results |
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Three of the 258 patients (1.2%) were colonized with VRE during the first period, whereas 52 (34.9%) were colonized during the second period. Two VRE isolates of the first period were identified as E. faecalis and one as E. faecium whereas 47 VRE that were recovered during the second period were identified as E. faecium isolates; of the remaining isolates, three were E. faecalis and two were Enterococcus gallinarum. In the selective medium containing vancomycin, all VRE isolates were recovered and in addition 13 enterococcal isolates that were found intermediate (MIC 816 mg/L) or susceptible (MIC <8 mg/L) to vancomycin by the agar dilution method. The MICs of vancomycin for the VRE ranged from 32 to >256 mg/L and those of teicoplanin ranged from 4 to 128 mg/L. Most of the vancomycin-resistant E. faecium exhibited resistance to ampicillin (95.8%) and high levels of streptomycin (87.5%) and gentamicin (60.4%) (Table 1). In addition, seven enterococci that were identified as E. gallinarum (six isolates) or Enterococcus casseliflavus (one isolate) were vancomycin intermediate.
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Discussion |
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The prevalence of VRE during the second period of the study seems closest to the levels that were observed recently among high-risk patients in the United States but higher than that reported among patients admitted to at-risk wards of European hospitals.12 However, comparison of the data is very difficult and should be done cautiously since the populations studied differ not only in age and sex but also in many clinical parameters. The design of a study (only specimens from high-risk units) may also overestimate the overall prevalence of VRE in the hospital setting. In addition, several reports count as VRE enterococcal isolates that exhibit intermediate-level resistance to vancomycin.13 It must also be mentioned that detection methods are not the same and in several studies, enrichment methods have been used increasing rates of VRE isolation.13,14
It has been suggested that if VRE are not controlled soon after introduction into a hospital, the first sporadic cases of colonization may evolve into hospital outbreaks, which can be especially difficult to control.4 Therefore, a reduction in the clinical use of vancomycin as well as other antibiotics (metronidazole, clindamycin and cephalosporins) that can increase rate of VRE carriage has been impressed upon our clinicians. In addition, the observation that several VRE were clonal in PFGE analysis, emphasizes the need for urgent infection control measures in order to prevent the emergence of clinical infections due to VRE in our hospital. Further studies to determine the presence of putative virulence properties such as the enterococcal surface protein Esp in the identified VRE clones will be important in continuing to monitor this situation.
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Footnotes |
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References |
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2 . Low, D. E., Keller, N., Barth, A. et al. (2001). Clinical prevalence, antimicrobial susceptibility, and geographic resistance patterns of enterococci: results from the SENTRY antimicrobial surveillance program, 19971999. Clinical Infectious Diseases 32, Suppl. 2, 13345.[CrossRef]
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