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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
Species identification was carried out with the automated VITEK 2 system (bioMérieux, Marcy lÉtoile, France), used according to the manufacturers 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 IaId; 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).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 patients 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 |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Rice, L. B. (2001). Emergence of vancomycin-resistant enterococci. Emerging Infectious Diseases 7, 1837.[ISI][Medline]
3 . Hayden, M. K. (2000). Insights into the epidemiology and control of infection with vancomycin-resistant enterococci. Clinical Infectious Diseases 31, 105865.[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, 2317.[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, 45714.
6 . National Committee for Clinical Laboratory Standards. (2003). Performance Standards for Antimicrobial Susceptibility TestingThirteenth 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, 21614.
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, 108491.
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, 6814.[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, 57781.[ISI][Medline]