- Email: [email protected]
Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a German University Pediatric Hospital
Int. J. Hyg. Environ. Health, 147-152 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/intjhyg
International Journal of Hygiene and Environmental Health
Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a German University Pediatric Hospital Holger-Andreas Elsner1, 2, Ingo Sobottka2, Heinz-Hubert Feucht2, Egmont Harps3, Christoph Haun4, Dietrich Mack2, Rainer Ganschow3, Rainer Laufs2, Paul-Michael Kaulfers2 1 2 3 4
Medizinische Hochschule Hannover, Abteilung für Transfusionsmedizin, Hannover, Germany Universitäts-Krankenhaus Eppendorf, Institut für Medizinische Mikrobiologie und Immunologie, Hamburg, Germany Universitäts-Krankenhaus Eppendorf, Klinik und Poliklinik für Kinder- und Jugendmedizin, Kernklinik, Hamburg, Germany Universitäts-Krankenhaus Eppendorf, Klinik und Poliklinik für Kinder- und Jugendmedizin, Abteilung für Kinderkardiologie, Hamburg, Germany
Received November 18, 1999 · Accepted February 7, 2000
Abstract Nosocomial Infections caused by vancomycin-resistant enterococci (VRE) are an emerging threat to critically ill patients. At the University Hospital Eppendorf, VRE were isolated from 38 patients between August 1993 and April 1997, of whom 32 were hospitalized at the Department of Pediatrics. Pulsed-field gel electrophoresis revealed that 26 Enterococcus faecium isolates from patients of the Department of Pediatrics were identical or closely related, and that isolates from three additional patients of the same department were possibly related. All of these isolates were of vanA genotype. They were resistant to glycopeptides, ampicillin, ciprofloxacin, clindamycin, and erythromycin. Most isolates displayed high-level resistance to gentamicin, but all remained susceptible to quinupristin/dalfopristin. Implementation of stringent hand disinfection and environmental disinfection policies, as well as measures for patient isolation contained this first outbreak of VRE at a German Children’s hospital, which emphasizes the importance of hygienic measures for the control of nosocomial spread of these organisms. Key words: Enterococcus faecium – vancomycin-resistance – nosocomial outbreak – infection – peritonitis – liver transplantation – pediatry
Introduction Enterococci are important nosocomial pathogens. Infections in humans are mainly caused by Enterococcus faecalis and E. faecium, and common manifestations comprise urinary tract infections, sepsis, and intraabdominal and pelvic infections, and endocarditis (Murray, 1990 a). Enterococci are found in the normal
intestinal flora of most healthy adults. According to the Centers for Disease Control and Prevention (CDC), from 1989 through 1993 the percentage of nosocomial infections due to vancomycin-resistant enterococci (VRE) in USA has increased from 0.3 % to 7.9 % (CDC, 1993). Infections due to VRE were found to be associated with increased mortality, which was partially related to
Corresponding author: Dr. H.-A. Elsner, Medizinische Hochschule Hannover, Abteilung für Transfusionsmedizin, CarlNeuberg-Straße 1, D-30625 Hannover, Germany, Phone: +49 51 15 32 67 01, Fax: +49 51 15 32 20 79, E-mail: [email protected] 1438-4639/00/203/2-147 $ 15.00/0
148
H.-A. Elsner et al.
the lack of effective antimicrobial therapy (Linden et al., 1996; Stosor et al., 1998). An unusual accumulation of VRE infections was observed at the Department of Pediatrics, University Hospital Eppendorf, from December 1995 to April 1997. To prove or exclude a clonal spread, VRE isolates were retrospectively analysed by pulsed-field gel electrophoresis, antimicrobial susceptibility testings, colony blot hybridization, and PCR.
Materials and methods Hospital setting The University Hospital Eppendorf, Hamburg, Germany, is a 1567-bed acute care hospital. The wards of the Department of Pediatrics are located in several buildings: Wards at which VRE infections or colonizations were noted are the pediatric intensive care unit (ICU), the ward for pediatric cardiology, the ward for pediatric hepatology and nephrology, two rooms at the cardiosurgical ICU, and the ward for pediatric hematology and oncology. All wards are located in different buildings. The Department of Surgery and the Department of Internal Medicine, at which patients with VRE were also hospitalized, are located in the same building as the cardiosurgical ICU, but these departments belong to different parts of the building. Patients Between August 1993 and April 1997 VRE were isolated from 38 patients of the University Hospital Eppendorf of whom 35 (92.1 %) were detected between April 1996 and April 1997. Thirty-two patients (84.2 %) were hospitalized at the Department of Pediatrics, including the rooms of the cardiosurgical ICU, 4 (10.5 %) at the Department of Surgery, including surgical ICU, and 2 (5.3 %) at the ICUs of the Department of Internal Medicine. The criteria for nosocomial infections employed in our study corresponded to the CDC definitions of nosocomial infections, which were in part supplemented by the Robert Koch-Institute, Germany, in cooperation with the German National Reference Center for Hospital Hygiene (Robert Koch-Institut, 1998). Bacterial isolates Thirty-one of 32 isolates from pediatric patients and four of six isolates from patients of other departments were available for further testings. In addition, two environmental isolates from the Department of Pediatrics, and two VRE strains from peripheral hospitals were examined. Enterococcal isolates were identified due to the criteria published by Facklam and Collins, 1989, and minimum inhibitory concentrations (MICs) of 9 antibiotics were determined. Antibiotic Susceptibility Testings Enterococci were tested for antibiotic susceptibility by microbroth dilution in cation-adjusted Mueller-Hinton broth (Barry et al., 1997; National Committee for Clinical Labo-
ratory Standards, 1993) using commercial test kits (Merlin, Germany, kindly donated by Rhône-Poulenc Rorer, Germany). All quality control results were within expected ranges. In addition, isolates were tested for high-level resistance to gentamicin (MIC 500 µg/ml) and streptomycin (MIC 2000 µg/ml) by agar dilution on Brain Heart Infusion agar (Difco) (Swenson et al., 1995). Gentamicin and streptomycin were purchased from Fluka, Switzerland. E. faecalis ATCC 29 212, and Staphylococcus aureus ATCC 29 213 were used for quality control of antibiotic susceptibility testings (microbroth dilution). For testing of highlevel resistance to streptomycin (agar dilution), E. faecalis CCUG 34 289 (Culture Collection, University of Göteborg, Sweden) (= ATCC 51 299) was used as positive control, and E. faecalis ATCC 29 212 as negative control (Swenson et al., 1995). Beta-lactamase acticity was tested using Cefinase test disks (Becton Dickinson Europe, France). S. aureus ATCC 29 213 was used as positive control for testing of beta-lactamase acticity. Environmental investigations Environmental investigations were performed at the pediatric ICU in November 1996, December 1996, and January 1997; and at the cardiosurgical ICU in April 1997. At each investigation, 15–30 inanimate objects and the hands of the competent nurse were examined by direct plating on RODAC plates consisting of tryptone soya agar (Oxoid, GB) supplemented with 3 % saponine (Fluka, Buchs, Switzerland). Pulsed-field gel electrophoresis Pulsed-field gel electrophoresis was performed by a modification of the method described by Murray et al. (1990 b), using a contour-clamped homogenous electric field (CHEF DR III, BioRad, USA). Restrictions were performed with the endonucleases SmaI and ApaI, respectively. Pulse time was 5.0–25.0 s for 32.0 h. Lambda ladder DNA (BioRad) was used as size standard. DNA restriction patterns obtained by PFGE were interpreted according to the criteria published by Tenover et al., 1995. Detection of glycopeptide resistance by colony blot hybridisation Hybridization and PCR resistance gene amplification were performed with the same isolates as described for PFGE. In addition, one Enterococcus gallinarum strain isolated from stool of a pediatric patient was used. Colony blots were prepared according to the method of Moseley et al., 1980. VanA- (CCC CTT TAA CGC TAA TAC GAT CAA), vanB- (CCG CCA TCC TCC TGC AAA AAA), and vanC-probes (ATC GCA TCA CAA GCA CCA ATC) that have been originally described as primers for PCR (Clark et al., 1993), were used for detection of glycopeptide resistance. The five strains E. faecium ATCC 19 434 (vancomycin susceptible), E. faecalis A256 (VanA phenotype), E. faecium 228 (VanA phenotype), E. faecalis V583 (VanB phenotype), and E. faecium D366 (VanB phenotype) were used as control strains for colony blot hybridization and PCR analysis.
Nosocomial outbreak of VRE
149
Amplification of vanA, vanB and vanC genes by PCR PCR was performed as described in the study by Clark et al., 1993. All isolates were analyzed for the three genes. Positive and negative controls were included in each amplification run.
Results Patient characteristics Underlying diseases and conditions of the 32 pediatric patients were: Cardiovascular diseases, including malformation, endocarditis, and valvular heart defects: 11 patients (34,4 %); liver transplantation: 5 patients (15,6 %); dialysis: 4 patients (12,5 %); premature delivery: 4 patients (12,5 %); other diseases: 8 patients (25 %). The monthly incidence of new detection of VRE at the University Hospital Eppendorf between December 1995 and April 1997 is shown in Figure 1. In a retrospective clinical evaluation of all patients one adult and four pediatric patients were assumed to be infected with VRE: Patient A (2 months, male) developed peritonitis associated with isolation of a possibly related VRE strain from intraabdominal secretions. Other microorganisms were not isolated. He was successfully treated with the combination of vancomycin and imipenem/ cilastatin (Zienam®, MSD, Germany). Patient B (5 years, female) also suffered from peritonitis associated with isolation of VRE from intraabdominal secretions. One month later VRE and additionally Enterobacter cloacae were isolated. Treatment with the combination of fosfomycin and amikacin (Biklin®, Bristol-Myers Squibb, Germany), followed by the combination of imipenem/cilastatin and teicoplanin (Targocid®, Hoechst, Germany), and finally with the combination of ciprofloxacin (Ciprobay®, Bayer, Germany), clindamycin (Sobelin®, Pharmacia & Upjohn, Germany), and amikacin led to her recovery. Patient C (9 months, male) developed peritonitis associated with isolation of the VRE outbreak strain and of Staphylococcus epidermidis from intraabdominal secretions. Treatment was successfully performed with quinupristin/dalfopristin (Synercid®, RhônePoulenc Rorer, Germany), which renders an infection likely. However, 10 weeks later he developed a relapse, again with isolation of VRE, S. epidermidis and Citrobacter freundii from intraabdominal secretions. Patient D (8 months, male) suffered from partial atrioventricular septal defect. It was unclear whether the septicemia and endocarditis he developed was caused only by S. aureus or whether there was a co-
Fig. 1. Incidence of patients with VRE infection or colonisation between December 1995 and April 1997. Pediatry, other depts.
infection with VRE. S. aureus was isolated from five blood cultures and from the mitral valve, which had to be replaced by a mechanical valve, whereas VRE were isolated from only one blood culture. Under therapy with antistaphylococcal agents clinical signs of septicemia disappeared. Because of an early postoperative paravalvular leakage the patient was treated with quinupristin/dalfopristin and later in the course there were no signs of infection due to VRE. Patient E (49 y, male), an adult liver transplant recipient from the department of surgery developed VRE bacteremia and died, probably related to infection. Clinical data about antibiotic therapy before and after diagnosis of VRE bacteremia were not available. In the other patients there were no signs of infection, and colonization was most likely. Environmental investigations During the environmental investigations at the pediatric ICU in November 1996, December 1996, and January 1997 VRE were not detected. In April 1997, at the investigation of the pediatric rooms of the cardiosurgical ICU, VRE were detected on a toy that had been put down in a clean area, and on a flask with oily lotion. Species identification of enterococcal isolates Of the 31 clinical isolates from pediatric patients 29 were identified as E. faecium, one as E. faecalis, and one as E. gallinarum. Both environmental isolates, the four isolates from other departments and both VRE strains from other Hamburg hospitals were identified as E. faecium.
150
H.-A. Elsner et al.
Typing of enterococcal isolates PFGE patterns of the clinical E. faecium isolates of 26 pediatric patients and the two environmental isolates were indistinguishable or closely related. In the following these isolates will be referred to as PFGE group 1 isolates. Three additional clinical E. faecium isolates from pediatric patients showed four (restriction with SmaI) fragment differences, and were regarded to be possibly related (= PFGE group 2 isolates). Three of the four enterococcal strains isolated from pediatric patients who were likely to be infected by VRE belonged to PFGE group 1, and one to PFGE group 2, whereas the isolate from the adult liver transplant recipient showed a completely different fragment pattern. Fragment patterns of isolates of both groups and of another isolate that was not related are shown in Figure 2. The four vancomycin-resistant E. faecium isolates from other departments, and the two VRE control strains from other hospitals showed more than 7 fragment differences compared to the outbreak pattern indicating that there was no association with the outbreak (Figures not shown). Categorisation of all isolates was identical with respect to SmaI and ApaI restriction patterns. Antimicrobial Susceptibility Testing One E. faecalis isolate from a pediatric patient, and one E. faecium isolate from a peripheral hospital were
identified as VanB phenotype whereas the other 36 E. faecium isolates were identified as VanA. All 31 E. faecium PFGE group 1 and 2 isolates were resistant to ampicillin, clindamycin, erythromycin, vancomycin (MIC90 128 µg/ml), and teicoplanin (MIC90 256 µg/ml), and susceptible to quinupristin/dalfopristin (MIC90 = 1 µg/ml). Thirty isolates of them were resistant to ciprofloxacin, and one isolate was intermediate. Nineteen of them showed high-level resistance against gentamicin. High-level resistance to streptomycin and beta-lactamase-activity was not detected in any isolate. The E. gallinarum isolate was intermediate to vancomycin and susceptible to teicoplanin. Hybridization and PCR gene amplification studies Of the 36 isolates with VanA phenotype, 35 were identified as the corresponding genotype both by PCR and by colony blot hybridization with oligonucleotides. One E. faecium isolate, that was identified as vanA genotype by PCR hybridized with the vanA, vanB, and vanC probe. Another E. faecium isolate that was identified as vanA genotype by colony blot hybridization was negative by PCR. Genotypes of the 2 VanB phenotype isolates were identical to their phenotypes both by colony blot hybridization and PCR. The E. gallinarum isolate was identified by colony blot hybridization and by PCR as genotype vanC. The reference strains used in our study also displayed the expected genotypes both by colony hybridization and PCR analysis.
Discussion
Fig. 2. Restriction fragments of 8 VRE isolates after digestion with SmaI. Isolates no. 4, 6, 7 and 8 belong to PFGE group 1, whereas isolates no. 1, 3 and 5 belong to PFGE group 2. In contrast, isolate no. 2, which had been isolated from a surgical patient, was interpreted not to be part of the outbreak. Isolate no. 5 was isolated from wound secretions of a pediatric liver transplant recipients suffering from peritonitis, whereas the other isolates are likely to represent colonization flora.
At the University Hospital Eppendorf between August 1993 and April 1997, VRE were isolated from 38 patients, of whom 32 were hospitalized at the Department of Pediatrics. Pulsed-field gel electrophoresis revealed that 26 clinical and two environmental isolates from the Department of Pediatrics were identical or closely related (PFGE group 1), and that three additional clinical isolates from the same hospital were possibly related (PFGE group 2), which proves the nosocomial transmission of a vancomycin-resistant E. faecium outbreak strain. A number of nosocomial outbreaks due to VRE have been reported (Boyce et al., 1994; Boyle et al., 1993; Frieden et al., 1993; Handwerger et al., 1993; Karanfil et al., 1992; Livornese et al., 1992; Nourse et al., 1998; Uttley et al., 1998) to date. In Germany, preliminary data on an outbreak of vancomycin-
Nosocomial outbreak of VRE
resistant E. faecium mainly in nephrologic patients have been reported from the City Hospital of Nürnberg (Reinhardt et al., 1996). There has also been a preliminary report on the transmission of 9 vancomycin-resistant E. faecium at another specialized German hospital (Eckmanns et al., 1998). Our report is the first report of an outbreak of VRE at a Children’s Hospital in Germany, involving at least 32 patients. According to CDC criteria four pediatric patients had infections due to VRE. Three of them were liver transplant recipients. Infections in liver transplant recipients due to VRE have been previously described (Linden et al., 1996; Papanicolaou et al., 1996; Wade, 1995) and intraabdominal surgical procedures have been discussed as risk factors for infection/colonization with VRE (Boyle et al., 1993). Furthermore, it must be pointed out that four patients colonized with VRE underwent dialysis. Renal insufficiency has been previously described as a risk factor for infection/colonization with VRE (Handwerger et al., 1993) and was also present in most patients of the other German outbreak (Reinhardt et al., 1996). During the outbreak period, observation of nursing practices at the pediatric wards revealed that hand disinfection was not consistently performed between contacts to different patients. Antibiotic selection pressure, and frequent transfer of patients to other wards may have additionally favoured the spread of VRE. In order to prevent further spread of VRE via the hands of the staff, hygienic hand rub with alcoholbased disinfectants was consistently performed before and after contact with patients. The effectiveness of inoculated fingers with 60 % isopropyl alcohol has been demonstrated in a study which found a 4 log10 reduction of vancomycin-resistant Enterococcus faecium and gentamicin-resistant Enterobacter cloacae (Wade et al., 1991). In case of visible contamination with patients’ secretions hygienic handwash with disinfectant-detergents was followed by a hygienic hand rub. This procedure corresponds to recommendations in the German speaking countries (Rotter, 1996). In addition, colonized and infected patients were isolated, and toys and other materials were disinfected with alcohol or benzalkonium chloride based solutions after contact with patients. Although there was no change of antibiotic therapy schemes, implementation of measures for patient isolation, stringent hand disinfection and environmental disinfection policies finally brought the outbreak to an end, which emphasizes the importance of hand and environmental disinfection for the control of nosocomial transmission of VRE.
151
Acknowledgement. Four vancomycin-resistant enterococcal control strains used as controls in our study were a kind gift from Nancye C. Clark, Centers for Disease Control and Prevention (CDC), Atlanta, USA.
References Barry, A. L., Fuchs, P. C., Brown, S. D.: Provisional interpretive criteria for quinupristin/dalfopristin susceptibility tests. J. Antimicrob. Chemother. 39: Supplement A: 87–92 (1997). Boyce, J. M., Opal, S. M., Chow, J. W., Zervos, M. J., PotterBynoe, G., Sherman, C. B., Romulo, R. L. C, Fortna, S., Medeiros, A. A.: Outbreak of multidrug-resistant Enterococcus faecium with transferable vanB class vancomycin resistance. J. Clin. Microbiol. 32, 1148–1153 (1994). Boyle, J. F., Soumakis, S. A., Rendo, A., Herrington, J. A., Gianarkis, D. G., Thurberg, B. E., Painter, B. G.: Epidemiologic analysis and genotypic characterization of a nosocomial outbreak of vancomycin-resistant enterococci. J. Clin. Microbiol. 31, 1280–1285 (1993). CDC.: Nosocomial enterococci resistant to vancomycinUnited States, 1989–1993. Morbidity and mortality weekly report 42, 597–599 (1993). Clark, N. C., Cooksey, R. C., Hill, B. C., Swenson, J. M., Tenover, F. C.: Characterization of glycopeptide-resistant enterococci from U. S. hospitals. Antimicrob. Agents Chemother. 37, 2311–2317 (1993). Eckmanns, T., Huß, R., Rose, G., Rüden, H.: Bericht über einen krankenhausweiten Ausbruch von vancomycinresistenten Enterococcus faecium (VREF). Abstract, p. 44, 50. Tagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie und 25. Jahrestagung der Deutschen Gesellschaft für Infektiologie, Berlin, Germany, 4.–9. Oktober 1998. Facklam, R. R., Collins, M. D.: Identification of Enterococcus species isolated from human infections by a conventional test scheme. J. Clin. Microbiol. 27, 731–734 (1989). Frieden, T. R., Munsiff, S. S., Low, D. E., Willey, B. M., Williams, G., Faur, Y., Eisner, W., Warren, S., Kreiswirth, B.: Emergence of vancomycin-resistant enterococci in New York City. Lancet 342, 76–79 (1993). Handwerger, S., Raucher, B., Altarac, D., Monka, J., Marchione, S., Singh, K. V., Murray, B. E., Wolff, J., Walters, B.: Nosocomial outbreak due to Enterococcus faecium highly resistant to vancomycin, penicillin, and gentamicin. Clin. Infect. Dis. 16, 750–755 (1993). Karanfil, L. V., Murphy, M., Josephson, A., Gaynes, R., Mandel, L., Hill, B. C., Swenson, J. M.: A cluster of vancomycin-resistant Enterococcus faecium in an intensive care unit. Infect. Control. Hosp. Epidemiol. 13, 195– 200 (1992). Linden, P. K., Pasculle, A. W., Manez, R., Kramer, D. J., Fung, J. J., Pinna, A. D., Kusne, S.: Differences in outcomes for patients with bacteremia due to vancomycin-resistant Enterococcus faecium or vancomycin-susceptible E.faecium. Clin. Infect. Dis. 22, 663–670 (1996). Livornese, L. L., Dias, S., Samel, C., Romanowski, B., Taylor, S., May, P., Pitsakis, P., Woods, G., Kaye, D., Levison
152
H.-A. Elsner et al.
M. E., Johnson, C. C.: Hospital-acquired infection with vancomycin resistant Enterococcus faecium transmitted by electronic thermometers. Ann. Intern. Med. 117, 112–116 (1992). Moseley, S. L., Huq, I., Alim, ARMA, So, M., SamapourMotalebi, M., Falkow, S.: Detection of enterotoxinogenic Escherichia coli by DNA colony hybridisation. J. Infect. Dis. 142, 892:898 (1980). Murray, B. E.: The life and times of the enterococcus. Clin. Microbiol. Rev. 3, 46–65 (1990 a). Murray, B. E., Singh, K. V., Heath, J. D., Sharma, B. R., Weinstock, G.: Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. J. Clin. Microbiol. 28, 2059– 2063 (1990 b). National Committee for Clinical Laboratory Standards. 1993. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved standard M7-A3. NCCLS, Villanova, Pa. Nourse, C., Murphy, H., Byrne, C., O’Meara, A., Breatnach, F., Kaufmann, M., Clarke, A., Butler, K.: Control of a nosocomial outbreak of vancomycin resistant Enterococcus faecium in a pediatric oncology unit: risk factors for colonisation. Eur. J. Pediatr. 157, 20–27 (1998). Papanicolaou, G. A., Meyers, B. R., Meyers, J., Mendelson, M. H., Lou, W., Emre, S., Sheiner, P., Miller, C.: Nosocomial infections with vancomycin-resistant Enterococcus faecium in liver transplant recipients: risk factors for acquisition and mortality. Clin. Infect. Dis. 23, 760–766 (1996). Reinhardt, A., Klare, I., Just, H. M., Witte, W.: First report of a hospital outbreak of vancomycin-resistant Enterococcus faecium (VRE) of VAN-A type in Germany. Abstract J36, 36th ICAAC New Orleans 1996. Robert Koch-Institut in Zusammenarbeit mit dem Nationalen Referenzzentrum für Krankenhaushygiene. Definitionen nosokomialer Infektionen (CDC-Definitionen, zum Teil ergänzt), 1998.
Rotter, M.: Verfahren zur Händehygiene in deutschsprachigen Ländern. Zbl. Hyg. Umweltmed. 1996, 199: 334– 349. Stosor, V., Peterson, L. R., Postelnick, M., Noskin, G. A.: Enterococcus faecium becteremia: Does vancomycin resistance make a difference? Arch. Intern. Med. 158, 522– 527 (1998). Swenson, J. M., Ferraro, M. J., Sahm, D. F., Clark, N. C., Culver, D. H., Tenover, F. C., and The National Committee for Clinical Laboratory Standards Study Group on Enterococci: Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. J. Clin. Microbiol. 33, 3008–3018 (1995). Swenson, J. M., Clark, N. C., Sahm, D. F., Ferraro, M. J., Doern, G., Hindler, J., Jorgensen, J. H., Pfaller, M. A., Reller, L. B., Weinstein, M. P., Zabransky, R. J., Tenover, F. C.: Molecular characterization and multilaboratory evaluation of Enterococcus faecalis ATCC 51 299 for quality control of screening tests for vancomycin and high-level aminoglycoside resistance in enterococci. J. Clin. Microbiol. 33, 3019–3021 (1995). Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H., Swaminathan, B.: Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239 (1995). Uttley, A. H. C., Collins, C. H., Naidoo, J., George, R. C.: Vancomycin-resistant enterococci. Lancet 1, 57–58 (1988). Wade, J. J., Desai, N., Casewell, M. W.: Hygienic hand disinfection for the removal of epidemic vancomycin-resistant Enterococcus faecium and gentamicin-resistant Enterobacter cloacae. J. Hosp. Infect. 18, 211–218 (1991). Wade, J. J.: The ermergence of Enterococcus faecium resistant to glycopeptides and other standard agents – a preliminary report. J. Hosp. Infect. 30 (Supplement) 483–493 (1995).
International Journal of Hygiene and Environmental Health
Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a German University Pediatric Hospital Holger-Andreas Elsner1, 2, Ingo Sobottka2, Heinz-Hubert Feucht2, Egmont Harps3, Christoph Haun4, Dietrich Mack2, Rainer Ganschow3, Rainer Laufs2, Paul-Michael Kaulfers2 1 2 3 4
Medizinische Hochschule Hannover, Abteilung für Transfusionsmedizin, Hannover, Germany Universitäts-Krankenhaus Eppendorf, Institut für Medizinische Mikrobiologie und Immunologie, Hamburg, Germany Universitäts-Krankenhaus Eppendorf, Klinik und Poliklinik für Kinder- und Jugendmedizin, Kernklinik, Hamburg, Germany Universitäts-Krankenhaus Eppendorf, Klinik und Poliklinik für Kinder- und Jugendmedizin, Abteilung für Kinderkardiologie, Hamburg, Germany
Received November 18, 1999 · Accepted February 7, 2000
Abstract Nosocomial Infections caused by vancomycin-resistant enterococci (VRE) are an emerging threat to critically ill patients. At the University Hospital Eppendorf, VRE were isolated from 38 patients between August 1993 and April 1997, of whom 32 were hospitalized at the Department of Pediatrics. Pulsed-field gel electrophoresis revealed that 26 Enterococcus faecium isolates from patients of the Department of Pediatrics were identical or closely related, and that isolates from three additional patients of the same department were possibly related. All of these isolates were of vanA genotype. They were resistant to glycopeptides, ampicillin, ciprofloxacin, clindamycin, and erythromycin. Most isolates displayed high-level resistance to gentamicin, but all remained susceptible to quinupristin/dalfopristin. Implementation of stringent hand disinfection and environmental disinfection policies, as well as measures for patient isolation contained this first outbreak of VRE at a German Children’s hospital, which emphasizes the importance of hygienic measures for the control of nosocomial spread of these organisms. Key words: Enterococcus faecium – vancomycin-resistance – nosocomial outbreak – infection – peritonitis – liver transplantation – pediatry
Introduction Enterococci are important nosocomial pathogens. Infections in humans are mainly caused by Enterococcus faecalis and E. faecium, and common manifestations comprise urinary tract infections, sepsis, and intraabdominal and pelvic infections, and endocarditis (Murray, 1990 a). Enterococci are found in the normal
intestinal flora of most healthy adults. According to the Centers for Disease Control and Prevention (CDC), from 1989 through 1993 the percentage of nosocomial infections due to vancomycin-resistant enterococci (VRE) in USA has increased from 0.3 % to 7.9 % (CDC, 1993). Infections due to VRE were found to be associated with increased mortality, which was partially related to
Corresponding author: Dr. H.-A. Elsner, Medizinische Hochschule Hannover, Abteilung für Transfusionsmedizin, CarlNeuberg-Straße 1, D-30625 Hannover, Germany, Phone: +49 51 15 32 67 01, Fax: +49 51 15 32 20 79, E-mail: [email protected] 1438-4639/00/203/2-147 $ 15.00/0
148
H.-A. Elsner et al.
the lack of effective antimicrobial therapy (Linden et al., 1996; Stosor et al., 1998). An unusual accumulation of VRE infections was observed at the Department of Pediatrics, University Hospital Eppendorf, from December 1995 to April 1997. To prove or exclude a clonal spread, VRE isolates were retrospectively analysed by pulsed-field gel electrophoresis, antimicrobial susceptibility testings, colony blot hybridization, and PCR.
Materials and methods Hospital setting The University Hospital Eppendorf, Hamburg, Germany, is a 1567-bed acute care hospital. The wards of the Department of Pediatrics are located in several buildings: Wards at which VRE infections or colonizations were noted are the pediatric intensive care unit (ICU), the ward for pediatric cardiology, the ward for pediatric hepatology and nephrology, two rooms at the cardiosurgical ICU, and the ward for pediatric hematology and oncology. All wards are located in different buildings. The Department of Surgery and the Department of Internal Medicine, at which patients with VRE were also hospitalized, are located in the same building as the cardiosurgical ICU, but these departments belong to different parts of the building. Patients Between August 1993 and April 1997 VRE were isolated from 38 patients of the University Hospital Eppendorf of whom 35 (92.1 %) were detected between April 1996 and April 1997. Thirty-two patients (84.2 %) were hospitalized at the Department of Pediatrics, including the rooms of the cardiosurgical ICU, 4 (10.5 %) at the Department of Surgery, including surgical ICU, and 2 (5.3 %) at the ICUs of the Department of Internal Medicine. The criteria for nosocomial infections employed in our study corresponded to the CDC definitions of nosocomial infections, which were in part supplemented by the Robert Koch-Institute, Germany, in cooperation with the German National Reference Center for Hospital Hygiene (Robert Koch-Institut, 1998). Bacterial isolates Thirty-one of 32 isolates from pediatric patients and four of six isolates from patients of other departments were available for further testings. In addition, two environmental isolates from the Department of Pediatrics, and two VRE strains from peripheral hospitals were examined. Enterococcal isolates were identified due to the criteria published by Facklam and Collins, 1989, and minimum inhibitory concentrations (MICs) of 9 antibiotics were determined. Antibiotic Susceptibility Testings Enterococci were tested for antibiotic susceptibility by microbroth dilution in cation-adjusted Mueller-Hinton broth (Barry et al., 1997; National Committee for Clinical Labo-
ratory Standards, 1993) using commercial test kits (Merlin, Germany, kindly donated by Rhône-Poulenc Rorer, Germany). All quality control results were within expected ranges. In addition, isolates were tested for high-level resistance to gentamicin (MIC 500 µg/ml) and streptomycin (MIC 2000 µg/ml) by agar dilution on Brain Heart Infusion agar (Difco) (Swenson et al., 1995). Gentamicin and streptomycin were purchased from Fluka, Switzerland. E. faecalis ATCC 29 212, and Staphylococcus aureus ATCC 29 213 were used for quality control of antibiotic susceptibility testings (microbroth dilution). For testing of highlevel resistance to streptomycin (agar dilution), E. faecalis CCUG 34 289 (Culture Collection, University of Göteborg, Sweden) (= ATCC 51 299) was used as positive control, and E. faecalis ATCC 29 212 as negative control (Swenson et al., 1995). Beta-lactamase acticity was tested using Cefinase test disks (Becton Dickinson Europe, France). S. aureus ATCC 29 213 was used as positive control for testing of beta-lactamase acticity. Environmental investigations Environmental investigations were performed at the pediatric ICU in November 1996, December 1996, and January 1997; and at the cardiosurgical ICU in April 1997. At each investigation, 15–30 inanimate objects and the hands of the competent nurse were examined by direct plating on RODAC plates consisting of tryptone soya agar (Oxoid, GB) supplemented with 3 % saponine (Fluka, Buchs, Switzerland). Pulsed-field gel electrophoresis Pulsed-field gel electrophoresis was performed by a modification of the method described by Murray et al. (1990 b), using a contour-clamped homogenous electric field (CHEF DR III, BioRad, USA). Restrictions were performed with the endonucleases SmaI and ApaI, respectively. Pulse time was 5.0–25.0 s for 32.0 h. Lambda ladder DNA (BioRad) was used as size standard. DNA restriction patterns obtained by PFGE were interpreted according to the criteria published by Tenover et al., 1995. Detection of glycopeptide resistance by colony blot hybridisation Hybridization and PCR resistance gene amplification were performed with the same isolates as described for PFGE. In addition, one Enterococcus gallinarum strain isolated from stool of a pediatric patient was used. Colony blots were prepared according to the method of Moseley et al., 1980. VanA- (CCC CTT TAA CGC TAA TAC GAT CAA), vanB- (CCG CCA TCC TCC TGC AAA AAA), and vanC-probes (ATC GCA TCA CAA GCA CCA ATC) that have been originally described as primers for PCR (Clark et al., 1993), were used for detection of glycopeptide resistance. The five strains E. faecium ATCC 19 434 (vancomycin susceptible), E. faecalis A256 (VanA phenotype), E. faecium 228 (VanA phenotype), E. faecalis V583 (VanB phenotype), and E. faecium D366 (VanB phenotype) were used as control strains for colony blot hybridization and PCR analysis.
Nosocomial outbreak of VRE
149
Amplification of vanA, vanB and vanC genes by PCR PCR was performed as described in the study by Clark et al., 1993. All isolates were analyzed for the three genes. Positive and negative controls were included in each amplification run.
Results Patient characteristics Underlying diseases and conditions of the 32 pediatric patients were: Cardiovascular diseases, including malformation, endocarditis, and valvular heart defects: 11 patients (34,4 %); liver transplantation: 5 patients (15,6 %); dialysis: 4 patients (12,5 %); premature delivery: 4 patients (12,5 %); other diseases: 8 patients (25 %). The monthly incidence of new detection of VRE at the University Hospital Eppendorf between December 1995 and April 1997 is shown in Figure 1. In a retrospective clinical evaluation of all patients one adult and four pediatric patients were assumed to be infected with VRE: Patient A (2 months, male) developed peritonitis associated with isolation of a possibly related VRE strain from intraabdominal secretions. Other microorganisms were not isolated. He was successfully treated with the combination of vancomycin and imipenem/ cilastatin (Zienam®, MSD, Germany). Patient B (5 years, female) also suffered from peritonitis associated with isolation of VRE from intraabdominal secretions. One month later VRE and additionally Enterobacter cloacae were isolated. Treatment with the combination of fosfomycin and amikacin (Biklin®, Bristol-Myers Squibb, Germany), followed by the combination of imipenem/cilastatin and teicoplanin (Targocid®, Hoechst, Germany), and finally with the combination of ciprofloxacin (Ciprobay®, Bayer, Germany), clindamycin (Sobelin®, Pharmacia & Upjohn, Germany), and amikacin led to her recovery. Patient C (9 months, male) developed peritonitis associated with isolation of the VRE outbreak strain and of Staphylococcus epidermidis from intraabdominal secretions. Treatment was successfully performed with quinupristin/dalfopristin (Synercid®, RhônePoulenc Rorer, Germany), which renders an infection likely. However, 10 weeks later he developed a relapse, again with isolation of VRE, S. epidermidis and Citrobacter freundii from intraabdominal secretions. Patient D (8 months, male) suffered from partial atrioventricular septal defect. It was unclear whether the septicemia and endocarditis he developed was caused only by S. aureus or whether there was a co-
Fig. 1. Incidence of patients with VRE infection or colonisation between December 1995 and April 1997. Pediatry, other depts.
infection with VRE. S. aureus was isolated from five blood cultures and from the mitral valve, which had to be replaced by a mechanical valve, whereas VRE were isolated from only one blood culture. Under therapy with antistaphylococcal agents clinical signs of septicemia disappeared. Because of an early postoperative paravalvular leakage the patient was treated with quinupristin/dalfopristin and later in the course there were no signs of infection due to VRE. Patient E (49 y, male), an adult liver transplant recipient from the department of surgery developed VRE bacteremia and died, probably related to infection. Clinical data about antibiotic therapy before and after diagnosis of VRE bacteremia were not available. In the other patients there were no signs of infection, and colonization was most likely. Environmental investigations During the environmental investigations at the pediatric ICU in November 1996, December 1996, and January 1997 VRE were not detected. In April 1997, at the investigation of the pediatric rooms of the cardiosurgical ICU, VRE were detected on a toy that had been put down in a clean area, and on a flask with oily lotion. Species identification of enterococcal isolates Of the 31 clinical isolates from pediatric patients 29 were identified as E. faecium, one as E. faecalis, and one as E. gallinarum. Both environmental isolates, the four isolates from other departments and both VRE strains from other Hamburg hospitals were identified as E. faecium.
150
H.-A. Elsner et al.
Typing of enterococcal isolates PFGE patterns of the clinical E. faecium isolates of 26 pediatric patients and the two environmental isolates were indistinguishable or closely related. In the following these isolates will be referred to as PFGE group 1 isolates. Three additional clinical E. faecium isolates from pediatric patients showed four (restriction with SmaI) fragment differences, and were regarded to be possibly related (= PFGE group 2 isolates). Three of the four enterococcal strains isolated from pediatric patients who were likely to be infected by VRE belonged to PFGE group 1, and one to PFGE group 2, whereas the isolate from the adult liver transplant recipient showed a completely different fragment pattern. Fragment patterns of isolates of both groups and of another isolate that was not related are shown in Figure 2. The four vancomycin-resistant E. faecium isolates from other departments, and the two VRE control strains from other hospitals showed more than 7 fragment differences compared to the outbreak pattern indicating that there was no association with the outbreak (Figures not shown). Categorisation of all isolates was identical with respect to SmaI and ApaI restriction patterns. Antimicrobial Susceptibility Testing One E. faecalis isolate from a pediatric patient, and one E. faecium isolate from a peripheral hospital were
identified as VanB phenotype whereas the other 36 E. faecium isolates were identified as VanA. All 31 E. faecium PFGE group 1 and 2 isolates were resistant to ampicillin, clindamycin, erythromycin, vancomycin (MIC90 128 µg/ml), and teicoplanin (MIC90 256 µg/ml), and susceptible to quinupristin/dalfopristin (MIC90 = 1 µg/ml). Thirty isolates of them were resistant to ciprofloxacin, and one isolate was intermediate. Nineteen of them showed high-level resistance against gentamicin. High-level resistance to streptomycin and beta-lactamase-activity was not detected in any isolate. The E. gallinarum isolate was intermediate to vancomycin and susceptible to teicoplanin. Hybridization and PCR gene amplification studies Of the 36 isolates with VanA phenotype, 35 were identified as the corresponding genotype both by PCR and by colony blot hybridization with oligonucleotides. One E. faecium isolate, that was identified as vanA genotype by PCR hybridized with the vanA, vanB, and vanC probe. Another E. faecium isolate that was identified as vanA genotype by colony blot hybridization was negative by PCR. Genotypes of the 2 VanB phenotype isolates were identical to their phenotypes both by colony blot hybridization and PCR. The E. gallinarum isolate was identified by colony blot hybridization and by PCR as genotype vanC. The reference strains used in our study also displayed the expected genotypes both by colony hybridization and PCR analysis.
Discussion
Fig. 2. Restriction fragments of 8 VRE isolates after digestion with SmaI. Isolates no. 4, 6, 7 and 8 belong to PFGE group 1, whereas isolates no. 1, 3 and 5 belong to PFGE group 2. In contrast, isolate no. 2, which had been isolated from a surgical patient, was interpreted not to be part of the outbreak. Isolate no. 5 was isolated from wound secretions of a pediatric liver transplant recipients suffering from peritonitis, whereas the other isolates are likely to represent colonization flora.
At the University Hospital Eppendorf between August 1993 and April 1997, VRE were isolated from 38 patients, of whom 32 were hospitalized at the Department of Pediatrics. Pulsed-field gel electrophoresis revealed that 26 clinical and two environmental isolates from the Department of Pediatrics were identical or closely related (PFGE group 1), and that three additional clinical isolates from the same hospital were possibly related (PFGE group 2), which proves the nosocomial transmission of a vancomycin-resistant E. faecium outbreak strain. A number of nosocomial outbreaks due to VRE have been reported (Boyce et al., 1994; Boyle et al., 1993; Frieden et al., 1993; Handwerger et al., 1993; Karanfil et al., 1992; Livornese et al., 1992; Nourse et al., 1998; Uttley et al., 1998) to date. In Germany, preliminary data on an outbreak of vancomycin-
Nosocomial outbreak of VRE
resistant E. faecium mainly in nephrologic patients have been reported from the City Hospital of Nürnberg (Reinhardt et al., 1996). There has also been a preliminary report on the transmission of 9 vancomycin-resistant E. faecium at another specialized German hospital (Eckmanns et al., 1998). Our report is the first report of an outbreak of VRE at a Children’s Hospital in Germany, involving at least 32 patients. According to CDC criteria four pediatric patients had infections due to VRE. Three of them were liver transplant recipients. Infections in liver transplant recipients due to VRE have been previously described (Linden et al., 1996; Papanicolaou et al., 1996; Wade, 1995) and intraabdominal surgical procedures have been discussed as risk factors for infection/colonization with VRE (Boyle et al., 1993). Furthermore, it must be pointed out that four patients colonized with VRE underwent dialysis. Renal insufficiency has been previously described as a risk factor for infection/colonization with VRE (Handwerger et al., 1993) and was also present in most patients of the other German outbreak (Reinhardt et al., 1996). During the outbreak period, observation of nursing practices at the pediatric wards revealed that hand disinfection was not consistently performed between contacts to different patients. Antibiotic selection pressure, and frequent transfer of patients to other wards may have additionally favoured the spread of VRE. In order to prevent further spread of VRE via the hands of the staff, hygienic hand rub with alcoholbased disinfectants was consistently performed before and after contact with patients. The effectiveness of inoculated fingers with 60 % isopropyl alcohol has been demonstrated in a study which found a 4 log10 reduction of vancomycin-resistant Enterococcus faecium and gentamicin-resistant Enterobacter cloacae (Wade et al., 1991). In case of visible contamination with patients’ secretions hygienic handwash with disinfectant-detergents was followed by a hygienic hand rub. This procedure corresponds to recommendations in the German speaking countries (Rotter, 1996). In addition, colonized and infected patients were isolated, and toys and other materials were disinfected with alcohol or benzalkonium chloride based solutions after contact with patients. Although there was no change of antibiotic therapy schemes, implementation of measures for patient isolation, stringent hand disinfection and environmental disinfection policies finally brought the outbreak to an end, which emphasizes the importance of hand and environmental disinfection for the control of nosocomial transmission of VRE.
151
Acknowledgement. Four vancomycin-resistant enterococcal control strains used as controls in our study were a kind gift from Nancye C. Clark, Centers for Disease Control and Prevention (CDC), Atlanta, USA.
References Barry, A. L., Fuchs, P. C., Brown, S. D.: Provisional interpretive criteria for quinupristin/dalfopristin susceptibility tests. J. Antimicrob. Chemother. 39: Supplement A: 87–92 (1997). Boyce, J. M., Opal, S. M., Chow, J. W., Zervos, M. J., PotterBynoe, G., Sherman, C. B., Romulo, R. L. C, Fortna, S., Medeiros, A. A.: Outbreak of multidrug-resistant Enterococcus faecium with transferable vanB class vancomycin resistance. J. Clin. Microbiol. 32, 1148–1153 (1994). Boyle, J. F., Soumakis, S. A., Rendo, A., Herrington, J. A., Gianarkis, D. G., Thurberg, B. E., Painter, B. G.: Epidemiologic analysis and genotypic characterization of a nosocomial outbreak of vancomycin-resistant enterococci. J. Clin. Microbiol. 31, 1280–1285 (1993). CDC.: Nosocomial enterococci resistant to vancomycinUnited States, 1989–1993. Morbidity and mortality weekly report 42, 597–599 (1993). Clark, N. C., Cooksey, R. C., Hill, B. C., Swenson, J. M., Tenover, F. C.: Characterization of glycopeptide-resistant enterococci from U. S. hospitals. Antimicrob. Agents Chemother. 37, 2311–2317 (1993). Eckmanns, T., Huß, R., Rose, G., Rüden, H.: Bericht über einen krankenhausweiten Ausbruch von vancomycinresistenten Enterococcus faecium (VREF). Abstract, p. 44, 50. Tagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie und 25. Jahrestagung der Deutschen Gesellschaft für Infektiologie, Berlin, Germany, 4.–9. Oktober 1998. Facklam, R. R., Collins, M. D.: Identification of Enterococcus species isolated from human infections by a conventional test scheme. J. Clin. Microbiol. 27, 731–734 (1989). Frieden, T. R., Munsiff, S. S., Low, D. E., Willey, B. M., Williams, G., Faur, Y., Eisner, W., Warren, S., Kreiswirth, B.: Emergence of vancomycin-resistant enterococci in New York City. Lancet 342, 76–79 (1993). Handwerger, S., Raucher, B., Altarac, D., Monka, J., Marchione, S., Singh, K. V., Murray, B. E., Wolff, J., Walters, B.: Nosocomial outbreak due to Enterococcus faecium highly resistant to vancomycin, penicillin, and gentamicin. Clin. Infect. Dis. 16, 750–755 (1993). Karanfil, L. V., Murphy, M., Josephson, A., Gaynes, R., Mandel, L., Hill, B. C., Swenson, J. M.: A cluster of vancomycin-resistant Enterococcus faecium in an intensive care unit. Infect. Control. Hosp. Epidemiol. 13, 195– 200 (1992). Linden, P. K., Pasculle, A. W., Manez, R., Kramer, D. J., Fung, J. J., Pinna, A. D., Kusne, S.: Differences in outcomes for patients with bacteremia due to vancomycin-resistant Enterococcus faecium or vancomycin-susceptible E.faecium. Clin. Infect. Dis. 22, 663–670 (1996). Livornese, L. L., Dias, S., Samel, C., Romanowski, B., Taylor, S., May, P., Pitsakis, P., Woods, G., Kaye, D., Levison
152
H.-A. Elsner et al.
M. E., Johnson, C. C.: Hospital-acquired infection with vancomycin resistant Enterococcus faecium transmitted by electronic thermometers. Ann. Intern. Med. 117, 112–116 (1992). Moseley, S. L., Huq, I., Alim, ARMA, So, M., SamapourMotalebi, M., Falkow, S.: Detection of enterotoxinogenic Escherichia coli by DNA colony hybridisation. J. Infect. Dis. 142, 892:898 (1980). Murray, B. E.: The life and times of the enterococcus. Clin. Microbiol. Rev. 3, 46–65 (1990 a). Murray, B. E., Singh, K. V., Heath, J. D., Sharma, B. R., Weinstock, G.: Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. J. Clin. Microbiol. 28, 2059– 2063 (1990 b). National Committee for Clinical Laboratory Standards. 1993. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved standard M7-A3. NCCLS, Villanova, Pa. Nourse, C., Murphy, H., Byrne, C., O’Meara, A., Breatnach, F., Kaufmann, M., Clarke, A., Butler, K.: Control of a nosocomial outbreak of vancomycin resistant Enterococcus faecium in a pediatric oncology unit: risk factors for colonisation. Eur. J. Pediatr. 157, 20–27 (1998). Papanicolaou, G. A., Meyers, B. R., Meyers, J., Mendelson, M. H., Lou, W., Emre, S., Sheiner, P., Miller, C.: Nosocomial infections with vancomycin-resistant Enterococcus faecium in liver transplant recipients: risk factors for acquisition and mortality. Clin. Infect. Dis. 23, 760–766 (1996). Reinhardt, A., Klare, I., Just, H. M., Witte, W.: First report of a hospital outbreak of vancomycin-resistant Enterococcus faecium (VRE) of VAN-A type in Germany. Abstract J36, 36th ICAAC New Orleans 1996. Robert Koch-Institut in Zusammenarbeit mit dem Nationalen Referenzzentrum für Krankenhaushygiene. Definitionen nosokomialer Infektionen (CDC-Definitionen, zum Teil ergänzt), 1998.
Rotter, M.: Verfahren zur Händehygiene in deutschsprachigen Ländern. Zbl. Hyg. Umweltmed. 1996, 199: 334– 349. Stosor, V., Peterson, L. R., Postelnick, M., Noskin, G. A.: Enterococcus faecium becteremia: Does vancomycin resistance make a difference? Arch. Intern. Med. 158, 522– 527 (1998). Swenson, J. M., Ferraro, M. J., Sahm, D. F., Clark, N. C., Culver, D. H., Tenover, F. C., and The National Committee for Clinical Laboratory Standards Study Group on Enterococci: Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. J. Clin. Microbiol. 33, 3008–3018 (1995). Swenson, J. M., Clark, N. C., Sahm, D. F., Ferraro, M. J., Doern, G., Hindler, J., Jorgensen, J. H., Pfaller, M. A., Reller, L. B., Weinstein, M. P., Zabransky, R. J., Tenover, F. C.: Molecular characterization and multilaboratory evaluation of Enterococcus faecalis ATCC 51 299 for quality control of screening tests for vancomycin and high-level aminoglycoside resistance in enterococci. J. Clin. Microbiol. 33, 3019–3021 (1995). Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H., Swaminathan, B.: Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239 (1995). Uttley, A. H. C., Collins, C. H., Naidoo, J., George, R. C.: Vancomycin-resistant enterococci. Lancet 1, 57–58 (1988). Wade, J. J., Desai, N., Casewell, M. W.: Hygienic hand disinfection for the removal of epidemic vancomycin-resistant Enterococcus faecium and gentamicin-resistant Enterobacter cloacae. J. Hosp. Infect. 18, 211–218 (1991). Wade, J. J.: The ermergence of Enterococcus faecium resistant to glycopeptides and other standard agents – a preliminary report. J. Hosp. Infect. 30 (Supplement) 483–493 (1995).