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Journal of Clinical Microbiology, November 2005, p. 5796-5799, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5796-5799.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Epidemiology and Molecular Analysis of Intestinal Colonization by Vancomycin-Resistant Enterococci in Greek Hospitals

Achilleas Gikas,1* Athanasia Christidou,1 Efstathia Scoulica,1 Paulos Nikolaidis,3 Athanasios Skoutelis,2 Stamatina Levidiotou,4 Sofia Kartali,5 Efstratios Maltezos,5 Simeon Metalidis,3 John Kioumis,6 George Haliotis,7 Sofia Dima,8 Maria Roumbelaki,1 Nikoleta Papageorgiou,1 Evangelos I. Kritsotakis,1 and Yannis Tselentis1

Department of Clinical Bacteriology, Parasitology, Zoonosis and Geographical Medicine, University Hospital of Heraklion, Crete, Greece,1 Department of Infectious Diseases, University Hospital of Patras, Patras, Greece,2 Department of Internal Medicine, "AHEPA" General Hospital, Thessaloniki, Greece,3 Department of Microbiology-Infectious Diseases, University Hospital of Ioannina, Ioannina, Greece,4 Department of Microbiology-Infectious Diseases, University Hospital of Alexandroupolis, Alexandroupolis, Greece,5 Department of Intensive Care Unit, "Papanikolaou" General Hospital, Thessaloniki, Greece,6 Department of Internal Medicine, General Hospital of Chalkis, Euboea, Greece,7 Patient Care Area of Internal Medicine, General Hospital of Chania, Chania, Greece8

Received 3 June 2005/ Returned for modification 20 July 2005/ Accepted 5 August 2005


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ABSTRACT
 
From 1,246 specimens collected from 13 Greek hospitals, 266 vancomycin-resistant enterococci strains were isolated from 255 patients (20.5%). The VanA phenotype was present in 82 (30.8%) strains, the VanB phenotype in 17 (6.4%) strains, the VanC1 phenotype in 152 (57.1%) strains, and the VanC2/C3 phenotypes in 15 (5.6%) strains. When only VanA and VanB phenotypes were considered, the overall prevalence was 7.5%. Eighty-six isolates exhibiting the VanA or VanB phenotype were analyzed by pulsed-field gel electrophoresis (PFGE), and 46 PFGE groups were found.


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TEXT
 
In Greece, the first clinical isolate of Enterococcus faecium with the VanA phenotype was reported in 2000 (10). Introduction and dissemination of vancomycin-resistant enterococcus (VRE) strains in two tertiary hospitals were also reported in the same year (3, 9). At the end of 2000, a cluster of VRE cases was investigated at the University Hospital of Heraklion (1). New cases of VRE infection were detected during 2002 in the same hospital. In order to estimate the rates of intestinal colonization, characterize the isolates, and investigate the molecular epidemiology of VRE in Greek hospitals, a point-prevalence study was undertaken.

On 1 November 2002, 13 hospitals (5 university and 8 district general [DG]) scattered throughout the country participated in the study (Fig. 1). Through one-in-four systematic sampling, patients from every patient-care unit were randomly selected.



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  		FIG. 1. Distribution of participating hospitals in the Greek territory.

Patients' selection, specimen collection and culture, and identification, and determination of resistance to vancomycin were performed in every hospital by the same methodology. Isolates were sent to a reference center for further investigation with molecular methods.

Fecal samples or rectal swabs were obtained and cultured in Enterococcus broth and bile esculine azide agar, both supplemented with 6 mg/liter of vancomycin (1). The isolates were primarily identified as enterococci, subsequently tested for resistance to glycopeptides by the disk diffusion agar method, and further confirmed by E-test according to the manufacturer's guidelines (AB Biodisk, Solua, Sweden). Species level identification was performed by conventional methods (biochemical characteristics, motility, and pigment production).

Species identification and resistance phenotypes were confirmed by a multiplex PCR assay as described elsewhere (1, 8).

Clonal distribution of strains harboring either the vanA or the vanB locus was examined by pulsed-field gel electrophoresis (PFGE) of SmaI chromosomal macrorestriction digest (11, 15).

A total of 1,246 specimens were tested and 266 VRE strains were isolated from 255 (20.5%) patients. Species distribution was as follows: E. faecium, 79 strains (29.7%); Enterococcus faecalis, 12 (4.5%); Enterococcus gallinarum, 153 (57.5%); Enterococcus casseliflavus/flavescens, 16 (6.0%); Enterococcus avium, 4 (1.5%); and Enterococcus hirae, 2 (0.8%). The highest prevalence was detected in renal dialysis units (28.9%) followed by surgical and medical wards (22.6% and 19.0%, respectively). Adult intensive care units (ICUs) had the lowest prevalence (14.9%). No VRE was obtained from neonatal ICUs (Table 1).


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  		TABLE 1. VRE prevalence by hospital and ward type

No significant difference of overall VRE frequencies was observed between university and DG hospitals ({chi}2 test, P = 0.531). However, stratification by patient-care area showed that university hospitals had higher rates for ICUs and surgical wards ({chi}2 test, P = 0.015 and P < 0.001, respectively). DG hospitals had higher rates in renal dialysis units ({chi}2 test, P = 0.008).

Analysis of VRE phenotypes showed significantly different frequencies between the university and DG hospitals ({chi}2 test, P = 0.001). VanA and VanB rates were higher in the university hospitals than in DG hospitals (39.6% and 8.1% versus 19.7% and 4.3%, respectively), whereas rates of isolates with VanC1 and VanC2/C3 phenotypes were higher in DG hospitals than in university hospitals (68.4% and 7.7% versus 48.3% and 4%, respectively).

When the isolates with the VanA and VanB phenotypes were considered as a group separately from the isolates with the VanC phenotypes, different results were obtained. Ninety-nine such strains were isolated from 93/1,246 patients, giving a prevalence of VRE with the VanA/B phenotypes of 7.5% (Table 2).


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  		TABLE 2. VRE prevalence (isolates with VanA/VanB phenotypes) by hospital and ward type

The highest prevalence of VRE with the VanA/B phenotypes was detected in medical wards (9.4%), followed by surgical wards (8.6%), adult ICUs (6.0%), and renal dialysis units (5.4%).

At the ward level, university hospitals had higher frequencies regarding VanA and VanB phenotypes for renal dialysis units and surgical wards than DG hospitals ({chi}2 test, P = 0.002 and P = 0.017, respectively), whereas for ICUs and medical wards, no difference was observed ({chi}2 test, P = 0.903 and P = 0.165, respectively).

PCR results were in agreement with the identification and resistance phenotypes of all isolates. For the four E. avium and two E. hirae isolates, PCR amplification yielded only the vanA resistance element. One E. gallinarum isolate and one E. casseliflavus isolate generated products corresponding to the presence of vanA gene along with the vanC1 and vanC2/C3 genes, respectively.

Eighty-six isolates exhibiting the VanA or VanB phenotype were analyzed by PFGE, and 46 distinct patterns are depicted (Fig. 2) (13). The 65 E. faecium isolates with the VanA phenotype formed 32 patterns, whereas the 4 E. faecalis isolates with the VanA phenotype formed 4 patterns. Ten E. faecium isolates with the VanB phenotype formed four patterns, and four E. avium isolates with the VanA phenotype formed three patterns. Three different VanA phenotype patterns were formed from E. casseliflavus, E. gallinarum, and E. hirae isolates, respectively.



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  		FIG. 2. The distinct PFGE patterns of E. faecium with the VanA phenotype (panel A), E. faecium with the VanB phenotype (panel B), and E. faecalis with the VanA phenotype (panel C). All the other VanA-resistant enterococci are shown in panel D.

The patterns of the isolates collected, in all except two hospitals, were clearly unrelated. In hospital HER-1, 17 different patterns were found; in PT-1, 19; in HER-2, 5; in THES-1, 2; in THES-2, 3; and in IO-1, 2.

In hospital HER-2, 10 strains with the VanA phenotype out of 15 (66.7%) exhibited a distinct PFGE pattern. This pattern was also found in three isolates in the neighboring hospital (HER-1). In HER-1, three groups of seven, three, and six strains with identical PFGE patterns together accounted for 51.6% (16/31) of the tested strains. These three clusters showed two different phenotypic profiles for E. faecium with the VanA phenotype and one phenotypic profile for E. faecium with the VanB phenotype.

Our study provides the first estimation of the intestinal colonization with VRE in Greek hospitals. These rates are among the highest reported in Europe (6, 12).

In this study, no VRE colonization was found in the neonatal units, which is consistent with the literature (14).

When the isolates with the VanC phenotypes, having a low clinical impact, were excluded and rates of isolates with the VanA/B phenotypes were considered separately, the overall rate was much lower (7.5%) and a different distribution by ward and hospital type was revealed. Thus, the inclusion of the isolates with VanC phenotypes may lead to an overestimation of the clinical problem and to the detection of completely different areas of concern for epidemiological intervention or prevention actions.

One E. gallinarum vanA-harboring strain and one E. casseliflavus vanA-harboring strain were isolated in the present study. This type of resistance was first described by Dutka-Malen et al. in 1994 (5). E. gallinarum and E. caseliflavus vanA-harboring strains (0.7% of vanA isolates each) were detected in renal dialysis units in Belgium (4). Recently, a dissemination of E. gallinarum with the VanA phenotype within an ICU was reported in Argentina (2).

It has been suggested that if VRE are not controlled soon after introduction into a hospital, clonal outbreaks may evolve, subsequently leading to polyclonal endemicity (7). Accordingly, strains coming from hospital HER-1 may have been recently introduced in the neighboring hospital, HER-2, since 10 out of 15 strains exhibited the same PFGE profile of E. faecium with the VanA phenotype. Moreover, our findings indicate an endemic situation already installed within HER-1 and PT-1 hospitals. In the remaining hospitals, the numbers of isolates are too small to draw conclusions on the PFGE patterns. However, one could suggest that the introduction of VRE is recent, as such isolates had not been previously detected.

Surveillance for VRE is not routinely performed in Greece, and isolation procedures are sporadically employed. Thus, prompt attention for the detection of new cases of VRE colonization and disease and employment of infection control policies are mandatory, so as to control this pathogen and prevent emergence on a wide scale. The use of molecular methods is especially important in identifying breakdown in infection control measures.


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ACKNOWLEDGMENTS
 
We thank Peggy Kanellos for her assistance in editing and literature research concerning this study.


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FOOTNOTES
 
* Corresponding author. Mailing address: University Hospital of Heraklion-1352/71110, Crete, Greece. Phone: 00.30.2810.375050. Fax: 00.30.2810.392847. E-mail: gikas{at}med.uoc.gr. Back


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Journal of Clinical Microbiology, November 2005, p. 5796-5799, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5796-5799.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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