Persistence of a clone of glycopeptide-resistant Enterococcus faecalis among patients in an intensive care unit of a Greek hospital

Spyros Pournaras1,*, Helen Malamou-Lada2, Maria Maniati1, Dimitra Mylona-Petropoulou2, Helen Vagiakou-Voudris2, Athanassios Tsakris3 and Antonios N. Maniatis1

1 Department of Medical Microbiology, University of Thessalia, Larissa; 2 Department of Clinical Microbiology, G. Gennimatas General Hospital, Athens; 3 Department of Microbiology, Faculty of Nursing, School of Health Sciences, University of Athens, Athens, Greece

Received 21 August 2003; returned 12 October 2003; revised 16 October 2003; accepted 23 October 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To investigate an outbreak of glycopeptide-resistant Enterococcus faecalis (GREF) in the intensive care unit (ICU) of ‘G. Gennimatas’ General Hospital, Athens, Greece.

Materials and methods: Between August 2000 and November 2001, 20 highly GREF isolates were recovered from severe infections of separate patients in the ICU. The isolates were tested by PCR, PFGE, mating experiments and plasmid analysis.

Results: All isolates carried the vanA gene. Nineteen isolates fitted to one clone by macrorestriction analysis with four subclones being consecutively detected. Each subclone seemed to predominate for a specific time period. Additionally, four GREF isolates related to the ICU clone were recovered from other wards of the hospital.

Conclusions: Our findings indicate that a monoclonal GREF outbreak persisted for more than 1 year in a large Greek hospital. The rate of GREF isolation declined after the application of infection control measures.

Keywords: glycopeptide resistance, Enterococcus faecalis clone, outbreak


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enterococci are opportunist nosocomial pathogens and have the capacity to develop and transfer antimicrobial resistance. During recent years, glycopeptide-resistant enterococci (GRE) have emerged in several hospitals and national surveillance surveys have documented an increase in their frequency of isolation.1 GRE have a broad geographical distribution probably as a result of an animal reservoir, at least in Europe.2 In the United States, GRE are a particularly serious problem and it is thought that they have been disseminated in the hospital environment mainly by cross-contamination.1 Among common enterococcal species, glycopeptide-resistance is usually disseminated among Enterococcus faecium isolates and glycopeptide-resistant strains of this species have been implicated in several outbreaks of hospital infections.3 Glycopeptide-resistant Enterococcus faecalis (GREF) strains have also been detected; their prevalence is generally low2 and hospital outbreaks as a result of such organisms have occasionally been described.4 In Greece, glycopeptide-resistant enterococci still represent an uncommon and occasional experience for most clinical laboratories and previous studies have reported only the dissemination of glycopeptide-resistant E. faecium strains in Greek hospitals.5

In one of the largest general hospitals in Athens, Greece, glycopeptide-resistant E. faecium were rarely isolated up to the year 2000, with no reports of GREF. However, in August 2000 a GREF isolate was recovered from a severe clinical infection sample of an intensive care unit (ICU) patient. Thereafter, several GREF isolates were recovered from patients in the ICU. The similar antimicrobial susceptibility patterns of these isolates prompted an investigation to determine whether limited spread of a single clone had occurred.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

During the study period (August 2000 to November 2001), 20 GREF isolates were consecutively collected from severe clinical infections of 20 separate patients in the ICU of ‘G. Gennimatas’ General Hospital. Four GREF isolates that were recovered during the same period from clinical specimens of four separate patients in other departments of the hospital were also included (Table 1). Most isolates were recovered from blood and intravenous catheters of patients with bloodstream infections. In four patients, GREF were isolated from aspirated pus samples of severe purulent wound infections. All patients were hospitalized for more than 5 days and most of them (20 of 24) had been treated with multiple antimicrobials that included glycopeptides (either vancomycin or teicoplanin), before the isolation of these organisms.


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Table 1. Date, clinical source of isolation, resistance phenotype and PFGE profile for 24 vanA positive E. faecalis isolates
 
Species identification and susceptibility testing

Species identification was carried out with the automated VITEK 2 system (bioMérieux, Marcy l’Étoile, France), used according to the manufacturer’s instructions. The VITEK 2 system was also used to determine susceptibility to a range of antimicrobials (penicillin, ampicillin, gentamicin, streptomycin, ciprofloxacin, tetracycline and vancomycin). Susceptibility status was defined according to the NCCLS guidelines.6 When GREF isolates were recovered, MICs of vancomycin and teicoplanin were also determined by an agar dilution method.6

PCR analysis and PFGE analysis

A multiplex PCR assay for the detection of vanA, vanB and vanE genes was carried out using primers and conditions that were described previously.7 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.8 Seven E. faecalis isolates, susceptible to glycopeptides, which were recovered during the study period, were also compared to the resistant ones by PFGE.

Mating experiments

The GREF isolates were used as donors in filter mating experiments, with E. faecalis JH2-2 (Fusr and Rifr) as the recipient strain. Transconjugant colonies were selected on BHI agar plates containing rifampicin (100 mg/L), fusidic acid (25 mg/L) and vancomycin (10 mg/L). The VITEK 2 system was used to determine the antimicrobial susceptibility pattern and the resistance that was transferred to the transconjugants. MICs for transconjugants of vancomycin and teicoplanin were determined by an agar dilution method.6 Plasmid DNA was extracted by an alkaline lysis procedure9 and separated by electrophoresis in 0.8% (w/v) agarose gels. The plasmid DNA band was extracted from the agarose gel with the QIAquick Gel Extraction kit (QIAGEN) and was used as template DNA in a PCR reaction for the detection of the vanA gene.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antibiotic resistance phenotypes of the 24 GREF isolates are shown in Table 1. All isolates had similar phenotypes, being resistant to erythromycin, ciprofloxacin, vancomycin and teicoplanin, but remaining susceptible to penicillin, ampicillin and tetracycline. In addition, all GREF isolates exhibited high-level resistance to streptomycin and 23 of them to gentamicin also. The MICs for the isolates of vancomycin ranged from 64 to 128 mg/L and of teicoplanin from 16 to 32 mg/L.

PCR analysis revealed that all the isolates were positive for the vanA gene. According to their macrorestriction profiles, all isolates but one, that belonged to the unrelated PFGE type II, were classified into a major clone that was subdivided into four subclones (subtypes Ia–Id; Table 1 and Figure 1). The isolates within each subtype were indistinguishable. The index GREF isolate belonged to the PFGE subtype Ia; it was identified in August 2000 from the blood cultures of a septicaemic patient in the ICU. Thereafter, GREF isolates of the subtype Ib were recovered in September 2000, of subtype Ic in November 2000 and of subtype Id in February 2001. GREF isolates that belonged to the subtype of the index GREF isolate were detected from July 2001 up to the end of the outbreak. The four GREF isolates that were collected from other departments of the hospital also belonged to subtypes Ib, Ic and Id (Table 1 and Figure 1). Macrorestriction profiles of seven vancomycin-susceptible E. faecalis, collected randomly during the same period from inpatients in the ICU (five isolates) and other departments (two isolates) were also examined; they yielded totally different profiles from those of the GREF isolates (data not shown).



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Figure 1. PFGE of SmaI-restricted genomic DNA of 20 glycopeptide-resistant E. faecalis isolates tested in this study. The origin and the PFGE type of each isolate are shown in Table 1. Lane M, molecular mass marker (kb).

 
Plasmid analysis of the clinical isolates revealed the presence in all isolates of a plasmid ranging in size from 60 to 70 kb; 19 isolates of subtypes Ia, Ib and Id harboured the ~70 kb plasmid, whereas four isolates of the subtype Ic carried the slightly smaller (~60 kb) plasmid. A plasmid was not visualized in the strain of the PFGE type II. It was found by PCR, using the plasmid DNA as template, that these plasmids contained the vanA gene. The glycopeptide-resistant phenotype was transferable from 16 isolates. Transfer frequencies varied between 5.2 x 10–5 and 4.5 x 10–7 transconjugants per donor cell. In most donor isolates, resistance to erythromycin, gentamicin and streptomycin was co-transferred with glycopeptide resistance. A plasmid of size similar to that of the respective donor was visualized in the transconjugants.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Studies on glycopeptide-resistant enterococci are usually limited to E. faecium isolates while GREF isolates have been only sporadically recovered with clonal outbreaks more limited worldwide.4 The findings of the study demonstrate that this GREF clone persisted in our ICU for more than 1 year. Four subtypes of the major clone were consecutively detected, and each of them seemed to predominate for a specific time period. This observation could suggest that each subtype possibly resulted from the previous one after genetic events resulting in the changes in PFGE profiles that were observed. Thus, it could be postulated that the index GREF strain in the ICU might have been the ancestor of the remaining subtypes and the genetic events possibly occurred within the hospital.

The GREF clone in this report predominated in a hospital with a prevalence of methicillin-resistant Staphylococcus aureus (MRSA) of 60%. Thus, MRSA infections that are usually treated with vancomycin would have increased the selective pressure for this multiresistant GREF clone, which subsequently spread to other departments of the hospital. GREF have a prolonged survival on environmental surfaces but also on gloved and ungloved fingertips.10 Therefore, poor compliance with hand washing procedures by health care workers might have resulted in their rapid spread in our hospital setting. Although potential reservoirs were not detected in environmental samples from the ICU, the incidence of isolation declined significantly after the study period. The infection control team of the hospital implemented weekly surveillance cultures and strict antiseptic techniques that included the rigorous use of alcohol-chlorhexidine solutions by visitors and staff before touching any patient and before leaving the unit. Preferably, alcohol-chlorhexidine nebulizers were put at each patient’s bedside. Education programmes of the nursing as well as the medical staff of the ICU and the other wards of the hospital were undertaken. Also, a reduction in the clinical use of glycopeptides as well as other antibiotics (metronidazole, clindamycin and cephalosporins) that can increase the rate of colonization with GREF possibly reduced the further spread of GREF. Indeed, although isolation cubicles with dedicated equipment and staff were not used, there were only three episodes of infections as a result of GREF in the ICU from November 2001 through July 2003.

Sporadic cases as a result of glycopeptide-resistant enterococci can easily evolve into monoclonal and then polyclonal outbreaks, which can be especially difficult to control.3 In our hospital, it seemed that the monoclonal outbreak of GREF was apparently not followed by horizontal spread of glycopeptide resistance to unrelated E. faecalis isolates. Since only clinical isolates were collected during this study, the rate of colonization with the GREF clonal strain or other GREF strains has not been estimated. Further studies are needed to determine whether similar GREF strains exist in other Greek hospitals.


    Acknowledgements
 
We are grateful to R. V. Goering for critical review of the manuscript before submission.


    Footnotes
 
* Corresponding author. Tel: +30-2410-682509; Fax: +30-2410-682508; E-mail: pournaras{at}med.uth.gr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . 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, 1997–1999. Clinical Infectious Diseases 32, Suppl. 2, 133–45.[CrossRef]

2 . Rice, L. B. (2001). Emergence of vancomycin-resistant enterococci. Emerging Infectious Diseases 7, 183–7.[ISI][Medline]

3 . Hayden, M. K. (2000). Insights into the epidemiology and control of infection with vancomycin-resistant enterococci. Clinical Infectious Diseases 31, 1058–65.[CrossRef][ISI][Medline]

4 . Biavasco, F., Miele, A., Vignaroli, C. et al. (1996). Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis. Microbial Drug Resistance 2, 231–7.[ISI][Medline]

5 . Maniatis, A. N., Pournaras, S., Kanellopoulou, M. et al. (2001). Dissemination of clonally unrelated erythromycin- and glycopeptide-resistant Enterococcus faecium isolates in a tertiary Greek hospital. Journal of Clinical Microbiology 39, 4571–4.[Abstract/Free Full Text]

6 . National Committee for Clinical Laboratory Standards. (2003). Performance Standards for Antimicrobial Susceptibility Testing—Thirteenth Informational Supplement M100-S13, Table 2D. NCCLS, Wayne, PA, USA.

7 . Fines, M., Perichon, B., Reynolds, P. et al. (1999). VanE, a new type of acquired glycopeptide resistance in Enterococcus faecalis BM4405. Antimicrobial Agents and Chemotherapy 43, 2161–4.[Abstract/Free Full Text]

8 . Morrison, D., Woodford, N., Barrett, S. P. et al. (1999). DNA banding pattern polymorphism in vancomycin-resistant Enterococcus faecium and criteria for defining strains. Journal of Clinical Microbiology 37, 1084–91.[Abstract/Free Full Text]

9 . Woodford, N., Morrison, D., Cookson, B. et al. (1993). Comparison of high-level gentamicin-resistant Enterococcus faecium isolates from different continents. Antimicrobial Agents and Chemotherapy 37, 681–4.[Abstract]

10 . Noskin, G. A., Stosor, V., Cooper, I. et al. (1995). Recovery of vancomycin-resistant enterococci on fingertips and environmental surfaces. Infection Control and Hospital Epidemiology 16, 577–81.[ISI][Medline]