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Occurrence of Glycopeptide-Resistant Enterococci in Transplant Medicine Internal Wards in 2001–2005
Occurrence of Glycopeptide-Resistant Enterococci in Transplant Medicine Internal Wards in 2001–2005 G. Młynarczyk, W. Grzybowska, A. Młynarczyk, S. Tyski, D. Kawecki, M. Łuczak, M. Durlik, L. Pa˛czek, A. Chmura, and W. Rowin´ski ABSTRACT The appearance of vancomycin-resistant enterococci (VRE) has caused serious therapeutic problems. In Poland, the frequency of VRE isolation is lower than in the United States or some other European countries. The aim of our work was to analyze the occurrence and characterization of VRE isolated from patients of 2 transplant medicine wards. These wards contained liver or kidney transplant patients. This study examined 5 years, including 235 to 313 enterococcal isolates per year. In 2001–2002, none of the isolated enterococci was confirmed as VRE, which appeared in 2003 (11 strains) and continued on a similar level (from 4% to 6%) in the next 2 years. Among all isolated enterococci, Enterococcus faecalis predominated. In 2003 and 2004, the numbers of E. faecium and E. faecalis among isolated VRE strains were similar, but in 2005, we observed significant predominance of E. faecium. Among VRE strains examined by polymerase chain reaction for the presence of vanA, vanB, vanD, vanE, and vanG ligases, only vanA was found in all cases. The examined strains represented several patterns of resistance to other antibiotics.
E
NTEROCOCCI ARE PART of the normal fecal flora of humans and animals. Despite their low pathogenic potential, they have become the third most common cause of nosocomial infections during the last 2 decades.1 The most common infections affect the urinary system, skin, and wounds as part of mixed floras as well as the blood. Infections caused by enterococci are often observed in patients who suffer from various types of immunodeficiencies, those using mechanically compromising devices, or those seriously ill.2 The increasing importance of this group may be due to an intrinsic resistance of enterococci to many antibiotics, eg, all cephalosporins, and their ability to acquire extrachromosomal elements encoding virulent traits or antibiotic resistance.3,4 Until recently, treatment of serious enterococcal infections relied on combination therapy with penicillin G or aminopencillin with an aminoglycoside. Unfortunately, the level of aminoglycoside resistance (HLAR) has increased to almost 100% among hospital-acquired enterococci, causing a lack of effectiveness of the combined therapy. Many strains, especially Enterococcus faecium, are also resistant to high doses of penicillins. Infections caused by these organisms are treated with glycopeptide antibiotics: vancomycin and teicoplanin. The appearance of glycopeptide-resistant enterococci (GRE; or more frequently, vancomycin-resistant
enterococci [VRE]) has caused considerable problems, forcing doctors to prescribe new, expensive, and not always safe drugs.5,6 The frequency and assortment of VRE among isolates from hospitalized and nonhospitalized individuals have often been investigated in most countries of the world.7 The aim of our work was to determine the frequency, predominant species, and determinants of resistance among VRE isolates from patients of 2 transplantology units. MATERIALS AND METHODS Bacterial Strains Bacteria were isolated from various specimens from patients of the 2 internal wards of the Transplantation Institute. Initial identificationand susceptibility testing were performed in the Routine From the Departments of Medical Microbiology (G.M., A.M., D.K., M.Ł.), Pharmaceutical Microbiology (S.T.), Transplant Medicine and Nephrology (M.D.), General and Transplantation Surgery (A.C., W.R.), and Immunology, Transplantology and Internal Diseases (L.P.), Warsaw Medical University, Warsaw, Poland; and Department of Antibiotics and Microbiology, National Medicines Institute, Warsaw, Poland (W.G., S.T.). Address reprint requests to Grazyna Młynarczyk, Department of Medical Microbiology, Warsaw Medical University, ul. Chalubinskiego 5, 02-004 Warsaw, Poland. E-mail: grazyna.mlynarczyk@ am.edu.pl
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.08.072
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2886
Transplantation Proceedings, 39, 2886 –2889 (2007)
Year 2001
2002
2003
2004
2005
Ward
All enterococci E. faecalis E. faecium Other species VRE E. faecalis VR E. faecium VR All VRE (%)
A
B
C
T
A
B
C
T
A
128 83 36 9
62 33 23 6
61 45 12 4
251 161 71 19
119 70 38 11
74 26 37 11
50 39 7 4
243 135 82 26
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
99 59 34 6
B
77 32 34 11
2 2 4 (4.0)
3 3 6 (7.8)
C
T
60 48 10 2
236 139 78 19
1 0 1 (1.7)
6 5 11 (4.7)
A
B
116 78 33 5
92 49 40 3
2 2 4 (3.4)
1 3 4 (4.3)
C
79 62 17 0 1 0 1 (1.3)
T
A
287 189 90 8
123 59 62 2
4 5 9 (3.1)
2 7 9 (7.3)
B
108 58 45 5
C
82 62 18 2
0 6 6 (5.6)
1 1 2 (2.4)
T
313 179 125 9 3 14 17 (5.4)
GLYCOPEPTIDE-RESISTANT ENTEROCOCCI
Table 1. The Number of Enterococci Isolated From Patients of Transplant Medicine Internal Wards in 2001–2005
Abbreviations: A, ward A; B, ward B; C, nephrologic clinic; T, total.
Table 2. Types of Specimens From Which Enterococci Were Recovered 2003
2004
2005
Type of Specimen
Enterococcus spp
VRE
% VRE
Enterococcus spp
VRE
% VRE
Enterococcus spp
VRE
% VRE
Urine Blood Wound/pus Drains Bile Sputum Feces Other
183 2 11 6 22 1 2 9
10 0 0 0 0 0 1 0
4.9 0 0 0 0 0 50.0 0
201 6 16 4 22 0 3 35
7 0 0 0 0 0 1 1*
3.4 0 0 0 0 0 33.3 2.9
260 5 15 3 8 6 2 14
12 2 0 0 0 0 1 2**
4.6 40.0 0 0 0 0 50.0 14.3
*Catheter. **Tongue, pleural fluid.
2887
2888
MŁYNARCZYK, GRZYBOWSKA, MŁYNARCZYK ET AL
Table 3. Characteristics of VRE Strains Isolated From Patients of Internal Wards of Transplantation Institute in 2003–2005 Identification
Type of van gene
The presence of vanB, vanD, vanE and vanG genes
MIC vancomycin (mg/L)
MIC teicoplanin (mg/L)
The pattern of the resistance*
E. faecium E. faecalis
vanA vanA
absent absent
128–⬎256 128–⬎256
64–⬎256 32–⬎256
5 different patterns 1 pattern
*Resistances to penicillin, amoxicillin, tetracycline, nitrofurantoine, ciprofloxacin, rifampicin and high level resistance to streptomycin and gentamycin were considered.
Diagnostic Laboratory of the Department of Microbiology. Initial identification of the strains was performed using API 20 Strep system (BioMerieux, France). For susceptibility testing ATB Enteroc 5 (BioMerieux) was used. Identification of VRE strains was confirmed by PCR reaction for ddl ligase genes of E. faecium and E. faecalis8 and correctness of glycopeptide resistance was confirmed by determining MIC of vancomycin and teicoplanin.
Determination of MIC All strains initially determined as VRE isolates were confirmed by determining MIC for vancomycin and teicoplanin. The E-tests with vancomycin and teicoplanin (AB Biodisk, Solna, Sweden) were performed. PCR. Total DNA was purified from the isolates using the Genomic DNA Prep Plus kit (A&A Biotechnology, Gdansk, Poland). The vanA, vanB, vanD, vanE and vanG genes were detected by specific PCR.8,9
RESULTS
The frequency of isolation of enterococci from patients of transplant and internal medicine wards in the subsequent five years was compared. Two hospital units (ward A and ward B) and the Nephrologic Clinic for outpatients were taken into consideration. The total numbers of enterococci isolated in the investigated years were similar and amounted from 236 to 313 per year. Among enterococci isolated up to 2003, no strains resistant to glycopeptides were detected (Table 1). In 2003, as many as 11 VRE were isolated from patients of the investigated wards. They constituted 4.7% of all enterococci. In 2003, about 50% of VRE were E. faecalis and 50% were E. faecium. In 2004 and 2005 VRE constituted 3.1% and 5.4% of all enterococci respectively. In 2005, E. faecium clearly predominated among VRE isolates (84%) but not among all enterococci. VRE were isolated from urine, however isolated strains were obtained from other specimens (Table 2). Most of VRE were examined for possessing genes of resistance to glycopeptides. All of examined strains carried genes for vanA type of ligase. Other known genes of the glycopeptide resistance were not found (Table 3). Other characteristics of the VRE strains s were presented in the Table 3. DISCUSSION
Despite enterococci being considered relatively nonpathogenic, considerable morbidity and mortality due to VRE infections has been demonstrated among immunocompromised patients, such as those treated in ICUs, those receiving dialysis, or those who underwent bone marrow and solid-organ transplantation.10,11
The survey of the results of bacteriological tests from our laboratory revealed that in our hospital, VRE had spread predominantly among those populations since 2003. Among all enterococci isolated from patients of transplant unit, E. faecalis definitely predominated. It is consistent with widely established data. Among 14 species of enterococci obtained from infected humans, E. faecalis usually accounts for 80 –90%, E. faecium 10 –15% and other species less than 5%.12,13 Different data were obtained from Brasil, where E. gallinarum accounted for 28.9% of colonized patients after renal transplantation.10 Among VRE isolated in investigated wards of our hospital during the first 2 years under study, the numbers of E. faecium and E. faecalis were similar, but after 2005 E. faecium predominated. The vanA determinant was present in all examined strains consistent with the MIC of vancomycin and teicoplanin. The VRE isolates were highly resistant to other antibiotics. The number of VRE isolates was similar in both investigated internal wards. While circulation of several epidemic and nonepidemic strains among patients is probable,14 spreading of the vanA determinant via conjugation between strains is also possible. To solve the problem we need to perform molecular epidemiological examination of isolated VRE strain.15 REFERENCES 1. Kuhn I, Iversen A, Finn M, et al: Occurrence and relatedness of vancomycin-resistant enterococci in animals, humans, and the environment in different European regions. Appl Environ Microbiol 71:5383, 2005 2. Alangaden GJ, Thyagarajan R, Gruber SA, et al: Infectious complications after kidney transplantation: current epidemiology and associated risk factors. Clin Transpl 20:401, 2006 3. Lee WG, Jernigan JA, Rasheed JK, et al: Possible horizontal transfer of the vanB2 gene among genetically diverse strains of vancomycin-resistant Enterococcus faecium in a Korean hospital. J Clin Microbiol 39:1165, 2001 4. Kawalec M, Pietras Z, Danilowicz E, et al: Clonal structure of Enterococcus faecalis isolated from Polish hospitals: characterization of epidemic clones. J Clin Microbiol 45:147, 2007 5. Maraha B, Bonten H, Van Hooff H, et al: Infectious complications and antibiotic use in renal transplant recipients during a 1-year follow-up. Clin Microbiol Infect 7:619, 2001 6. Weinstock DM, Conlon M, Iovino C, et al: Colonization, bloodstream infection, and mortality caused by vancomycinresistant enterococcus early after allogeneic hematopoietic stem cell transplant. Biol Blood Marrow Transplant 13:615, 2007 7. Torell E, Cars O, Olsson-Liljequist B, et al: Near absence of vancomycin-resistant enterococci but high carriage rates of quinoloneresistant ampicillin-resistant enterococci among hospitalized patients and nonhospitalized individuals in Sweden. J Clin Microbiol 37:3509, 1999
GLYCOPEPTIDE-RESISTANT ENTEROCOCCI 8. Dutka-Malen S, Evers S, Courvalin P: Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 33:24, 1995 9. Depardieu F, Perichon B, Courvalin P: Detection of the van alphabet and identification of enterococci and staphylococci at the species level by multiplex PCR. J Clin Microbiol 2:5857, 2004 10. Freitas MC, Pacheco-Silva A, Barbosa D, et al: Prevalence of vancomycin-resistant Enterococcus fecal colonization among kidney transplant patients. BMC Infect Dis 6:133, 2006 11. Zhou JD, Guo JJ, Zhang Q, et al: Drug resistance of infectious pathogens after liver transplantation. Hepatobiliary Pancreat Dis Int 5:190, 2006
2889 12. Ruoff KL, de la Maza L, Murtagh MJ, et al: Species identities of enterococci isolated from clinical specimens. J Clin Microbiol 28:435, 1990 13. Iwen PC, Kelly DM, Linder J, et al: Revised approach for identification and detection of ampicillin and vancomycin resistance in Enterococcus species by using MicroScan panels. J Clin Microbiol 34:1779, 1996 14. Leavis HL, Willems RJ, Top J, et al: Epidemic and nonepidemic multidrug-resistant Enterococcus faecium. Emerg Infect Dis 9:1108, 2003 15. Zirakzadeh A, Patel R: Vancomycin-resistant enterococci: colonization, infection, detection, and treatment. Mayo Clin Proc 81:529, 2006
E
NTEROCOCCI ARE PART of the normal fecal flora of humans and animals. Despite their low pathogenic potential, they have become the third most common cause of nosocomial infections during the last 2 decades.1 The most common infections affect the urinary system, skin, and wounds as part of mixed floras as well as the blood. Infections caused by enterococci are often observed in patients who suffer from various types of immunodeficiencies, those using mechanically compromising devices, or those seriously ill.2 The increasing importance of this group may be due to an intrinsic resistance of enterococci to many antibiotics, eg, all cephalosporins, and their ability to acquire extrachromosomal elements encoding virulent traits or antibiotic resistance.3,4 Until recently, treatment of serious enterococcal infections relied on combination therapy with penicillin G or aminopencillin with an aminoglycoside. Unfortunately, the level of aminoglycoside resistance (HLAR) has increased to almost 100% among hospital-acquired enterococci, causing a lack of effectiveness of the combined therapy. Many strains, especially Enterococcus faecium, are also resistant to high doses of penicillins. Infections caused by these organisms are treated with glycopeptide antibiotics: vancomycin and teicoplanin. The appearance of glycopeptide-resistant enterococci (GRE; or more frequently, vancomycin-resistant
enterococci [VRE]) has caused considerable problems, forcing doctors to prescribe new, expensive, and not always safe drugs.5,6 The frequency and assortment of VRE among isolates from hospitalized and nonhospitalized individuals have often been investigated in most countries of the world.7 The aim of our work was to determine the frequency, predominant species, and determinants of resistance among VRE isolates from patients of 2 transplantology units. MATERIALS AND METHODS Bacterial Strains Bacteria were isolated from various specimens from patients of the 2 internal wards of the Transplantation Institute. Initial identificationand susceptibility testing were performed in the Routine From the Departments of Medical Microbiology (G.M., A.M., D.K., M.Ł.), Pharmaceutical Microbiology (S.T.), Transplant Medicine and Nephrology (M.D.), General and Transplantation Surgery (A.C., W.R.), and Immunology, Transplantology and Internal Diseases (L.P.), Warsaw Medical University, Warsaw, Poland; and Department of Antibiotics and Microbiology, National Medicines Institute, Warsaw, Poland (W.G., S.T.). Address reprint requests to Grazyna Młynarczyk, Department of Medical Microbiology, Warsaw Medical University, ul. Chalubinskiego 5, 02-004 Warsaw, Poland. E-mail: grazyna.mlynarczyk@ am.edu.pl
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.08.072
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2886
Transplantation Proceedings, 39, 2886 –2889 (2007)
Year 2001
2002
2003
2004
2005
Ward
All enterococci E. faecalis E. faecium Other species VRE E. faecalis VR E. faecium VR All VRE (%)
A
B
C
T
A
B
C
T
A
128 83 36 9
62 33 23 6
61 45 12 4
251 161 71 19
119 70 38 11
74 26 37 11
50 39 7 4
243 135 82 26
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
99 59 34 6
B
77 32 34 11
2 2 4 (4.0)
3 3 6 (7.8)
C
T
60 48 10 2
236 139 78 19
1 0 1 (1.7)
6 5 11 (4.7)
A
B
116 78 33 5
92 49 40 3
2 2 4 (3.4)
1 3 4 (4.3)
C
79 62 17 0 1 0 1 (1.3)
T
A
287 189 90 8
123 59 62 2
4 5 9 (3.1)
2 7 9 (7.3)
B
108 58 45 5
C
82 62 18 2
0 6 6 (5.6)
1 1 2 (2.4)
T
313 179 125 9 3 14 17 (5.4)
GLYCOPEPTIDE-RESISTANT ENTEROCOCCI
Table 1. The Number of Enterococci Isolated From Patients of Transplant Medicine Internal Wards in 2001–2005
Abbreviations: A, ward A; B, ward B; C, nephrologic clinic; T, total.
Table 2. Types of Specimens From Which Enterococci Were Recovered 2003
2004
2005
Type of Specimen
Enterococcus spp
VRE
% VRE
Enterococcus spp
VRE
% VRE
Enterococcus spp
VRE
% VRE
Urine Blood Wound/pus Drains Bile Sputum Feces Other
183 2 11 6 22 1 2 9
10 0 0 0 0 0 1 0
4.9 0 0 0 0 0 50.0 0
201 6 16 4 22 0 3 35
7 0 0 0 0 0 1 1*
3.4 0 0 0 0 0 33.3 2.9
260 5 15 3 8 6 2 14
12 2 0 0 0 0 1 2**
4.6 40.0 0 0 0 0 50.0 14.3
*Catheter. **Tongue, pleural fluid.
2887
2888
MŁYNARCZYK, GRZYBOWSKA, MŁYNARCZYK ET AL
Table 3. Characteristics of VRE Strains Isolated From Patients of Internal Wards of Transplantation Institute in 2003–2005 Identification
Type of van gene
The presence of vanB, vanD, vanE and vanG genes
MIC vancomycin (mg/L)
MIC teicoplanin (mg/L)
The pattern of the resistance*
E. faecium E. faecalis
vanA vanA
absent absent
128–⬎256 128–⬎256
64–⬎256 32–⬎256
5 different patterns 1 pattern
*Resistances to penicillin, amoxicillin, tetracycline, nitrofurantoine, ciprofloxacin, rifampicin and high level resistance to streptomycin and gentamycin were considered.
Diagnostic Laboratory of the Department of Microbiology. Initial identification of the strains was performed using API 20 Strep system (BioMerieux, France). For susceptibility testing ATB Enteroc 5 (BioMerieux) was used. Identification of VRE strains was confirmed by PCR reaction for ddl ligase genes of E. faecium and E. faecalis8 and correctness of glycopeptide resistance was confirmed by determining MIC of vancomycin and teicoplanin.
Determination of MIC All strains initially determined as VRE isolates were confirmed by determining MIC for vancomycin and teicoplanin. The E-tests with vancomycin and teicoplanin (AB Biodisk, Solna, Sweden) were performed. PCR. Total DNA was purified from the isolates using the Genomic DNA Prep Plus kit (A&A Biotechnology, Gdansk, Poland). The vanA, vanB, vanD, vanE and vanG genes were detected by specific PCR.8,9
RESULTS
The frequency of isolation of enterococci from patients of transplant and internal medicine wards in the subsequent five years was compared. Two hospital units (ward A and ward B) and the Nephrologic Clinic for outpatients were taken into consideration. The total numbers of enterococci isolated in the investigated years were similar and amounted from 236 to 313 per year. Among enterococci isolated up to 2003, no strains resistant to glycopeptides were detected (Table 1). In 2003, as many as 11 VRE were isolated from patients of the investigated wards. They constituted 4.7% of all enterococci. In 2003, about 50% of VRE were E. faecalis and 50% were E. faecium. In 2004 and 2005 VRE constituted 3.1% and 5.4% of all enterococci respectively. In 2005, E. faecium clearly predominated among VRE isolates (84%) but not among all enterococci. VRE were isolated from urine, however isolated strains were obtained from other specimens (Table 2). Most of VRE were examined for possessing genes of resistance to glycopeptides. All of examined strains carried genes for vanA type of ligase. Other known genes of the glycopeptide resistance were not found (Table 3). Other characteristics of the VRE strains s were presented in the Table 3. DISCUSSION
Despite enterococci being considered relatively nonpathogenic, considerable morbidity and mortality due to VRE infections has been demonstrated among immunocompromised patients, such as those treated in ICUs, those receiving dialysis, or those who underwent bone marrow and solid-organ transplantation.10,11
The survey of the results of bacteriological tests from our laboratory revealed that in our hospital, VRE had spread predominantly among those populations since 2003. Among all enterococci isolated from patients of transplant unit, E. faecalis definitely predominated. It is consistent with widely established data. Among 14 species of enterococci obtained from infected humans, E. faecalis usually accounts for 80 –90%, E. faecium 10 –15% and other species less than 5%.12,13 Different data were obtained from Brasil, where E. gallinarum accounted for 28.9% of colonized patients after renal transplantation.10 Among VRE isolated in investigated wards of our hospital during the first 2 years under study, the numbers of E. faecium and E. faecalis were similar, but after 2005 E. faecium predominated. The vanA determinant was present in all examined strains consistent with the MIC of vancomycin and teicoplanin. The VRE isolates were highly resistant to other antibiotics. The number of VRE isolates was similar in both investigated internal wards. While circulation of several epidemic and nonepidemic strains among patients is probable,14 spreading of the vanA determinant via conjugation between strains is also possible. To solve the problem we need to perform molecular epidemiological examination of isolated VRE strain.15 REFERENCES 1. Kuhn I, Iversen A, Finn M, et al: Occurrence and relatedness of vancomycin-resistant enterococci in animals, humans, and the environment in different European regions. Appl Environ Microbiol 71:5383, 2005 2. Alangaden GJ, Thyagarajan R, Gruber SA, et al: Infectious complications after kidney transplantation: current epidemiology and associated risk factors. Clin Transpl 20:401, 2006 3. Lee WG, Jernigan JA, Rasheed JK, et al: Possible horizontal transfer of the vanB2 gene among genetically diverse strains of vancomycin-resistant Enterococcus faecium in a Korean hospital. J Clin Microbiol 39:1165, 2001 4. Kawalec M, Pietras Z, Danilowicz E, et al: Clonal structure of Enterococcus faecalis isolated from Polish hospitals: characterization of epidemic clones. J Clin Microbiol 45:147, 2007 5. Maraha B, Bonten H, Van Hooff H, et al: Infectious complications and antibiotic use in renal transplant recipients during a 1-year follow-up. Clin Microbiol Infect 7:619, 2001 6. Weinstock DM, Conlon M, Iovino C, et al: Colonization, bloodstream infection, and mortality caused by vancomycinresistant enterococcus early after allogeneic hematopoietic stem cell transplant. Biol Blood Marrow Transplant 13:615, 2007 7. Torell E, Cars O, Olsson-Liljequist B, et al: Near absence of vancomycin-resistant enterococci but high carriage rates of quinoloneresistant ampicillin-resistant enterococci among hospitalized patients and nonhospitalized individuals in Sweden. J Clin Microbiol 37:3509, 1999
GLYCOPEPTIDE-RESISTANT ENTEROCOCCI 8. Dutka-Malen S, Evers S, Courvalin P: Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 33:24, 1995 9. Depardieu F, Perichon B, Courvalin P: Detection of the van alphabet and identification of enterococci and staphylococci at the species level by multiplex PCR. J Clin Microbiol 2:5857, 2004 10. Freitas MC, Pacheco-Silva A, Barbosa D, et al: Prevalence of vancomycin-resistant Enterococcus fecal colonization among kidney transplant patients. BMC Infect Dis 6:133, 2006 11. Zhou JD, Guo JJ, Zhang Q, et al: Drug resistance of infectious pathogens after liver transplantation. Hepatobiliary Pancreat Dis Int 5:190, 2006
2889 12. Ruoff KL, de la Maza L, Murtagh MJ, et al: Species identities of enterococci isolated from clinical specimens. J Clin Microbiol 28:435, 1990 13. Iwen PC, Kelly DM, Linder J, et al: Revised approach for identification and detection of ampicillin and vancomycin resistance in Enterococcus species by using MicroScan panels. J Clin Microbiol 34:1779, 1996 14. Leavis HL, Willems RJ, Top J, et al: Epidemic and nonepidemic multidrug-resistant Enterococcus faecium. Emerg Infect Dis 9:1108, 2003 15. Zirakzadeh A, Patel R: Vancomycin-resistant enterococci: colonization, infection, detection, and treatment. Mayo Clin Proc 81:529, 2006