JAC Advance Access published online on September 1, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn360
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Original research |
Emergence of ampicillin-resistant Enterococcus faecium in Danish hospitals
1 Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark 2 Department of Clinical Microbiology, Aalborg Hospital, Aarhus University Hospital, Hobrovej 18-22, DK-9000 Aalborg, Denmark 3 Department of Clinical Microbiology, Herlev Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark 4 Department of Clinical Microbiology, Hillerød Hospital, Helsevej 2, DK-3400 Hillerød, Denmark 5 Department of Clinical Microbiology, Odense University Hospital, Sdr Boulevard 29, DK-5000 Odense C, Denmark
* Corresponding author. Tel: +45-32-68-36-68; Fax: +45-32-68-32-31; E-mail: cal{at}ssi.dk
Received 6 May 2008; returned 2 July 2008; revised 4 August 2008; accepted 5 August 2008
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Abstract |
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Background: Ampicillin-resistant Enterococcus faecium isolates are reported in increasing numbers in many European hospitals. The clonal complex 17 (CC17) characterized by ampicillin resistance has been associated with nosocomial E. faecium outbreaks and infections in five continents. The aim was to investigate how prevalent ampicillin resistance is in clinical E. faecium isolates from Denmark and to investigate their clonal affiliation, especially to CC17.
Methods: Microbiology data from 2002 through 2006 on E. faecium and Enterococcus faecalis blood isolates was received from Departments of Clinical Microbiology in 11 Danish counties. From January 2004 through December 2004, we collected 275 clinical enterococci from four of these departments. Multilocus sequence typing (MLST) and PFGE were performed on the 84 ampicillin-resistant E. faecium isolates from this collection.
Results: A 68% increase in the number of infections caused by enterococci was observed from 2002 through 2006. The increase was mainly caused by E. faecium isolates, which tripled, whereas the number of E. faecalis isolates increased by only 23% during the same period. There was also a significant increase in the number of ampicillin-resistant E. faecium isolates. MLST showed that 98% of the tested ampicillin-resistant E. faecium isolates belonged to CC17. PFGE showed eight different clusters and we found indications of clonal spread within the hospitals.
Conclusions: Ampicillin-resistant E. faecium isolates have increased in frequency in Denmark during 2002–2006. Most of the ampicillin-resistant E. faecium isolates belong to complex CC17.
Key Words: multilocus sequence typing , MLST , clonal complex 17 , PFGE
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Introduction |
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Enterococci are becoming an important cause of nosocomial infections, including bacteraemia, endocarditis and surgical wound infections. Optimal antimicrobial therapy for serious enterococcal infections requires the use of synergistic combinations of a cell wall-active agent such as penicillin or a glycopeptide and an aminoglycoside. Enterococci with ampicillin resistance and high-level gentamicin resistance (HLGR) are therefore the cause of considerable therapeutic problems; acquired vancomycin resistance has further aggravated these problems.
The clonal complex 17 (CC17) characterized by ampicillin resistance has been associated with nosocomial Enterococcus faecium outbreaks in five continents.1 An increase in the number of infections caused by ampicillin-resistant E. faecium belonging to CC17 has also been observed in many countries and this has markedly changed the Enterococcus faecalis/E. faecium ratio for enterococcus infections.1
The aim of the present study was to investigate how prevalent ampicillin resistance is in clinical E. faecium isolates from Denmark. We also performed multilocus sequence typing (MLST) to determine whether these isolates are a part of the CC17 and PFGE to investigate to what extent the isolates were clonally related.
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Materials and methods |
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Microbiology data
Microbiology data on E. faecium and E. faecalis blood culture isolates were received from Departments of Clinical Microbiology in 11 of the 16 Danish counties, representing 73% of the Danish population. The data were received at Statens Serum Institut as part of The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) from 2002 through 2006.
From January 2004 through December 2004, 275 human enterococcal isolates were consecutively collected from 4 of 15 Danish Departments of Clinical Microbiology (Aalborg, Odense, Herlev and Hillerød) and sent to Statens Serum Institut. The isolates were obtained from normally sterile body sites. The isolates were determined to be of clinical significance by the local departments, and only one isolate per patient was included in the study.
Antimicrobial susceptibility testing
The MICs of ampicillin, gentamicin, linezolid, quinupristin/dalfopristin and vancomycin for the isolates were determined (Trek Diagnostic Systems, UK). The CLSI breakpoints were used.2 The ATCC strain E. faecalis 29212 was used as a quality control strain.
MLST was performed on the 84 ampicillin-resistant E. faecium isolates. A standard set of E. faecium MLST primers was used for amplification and sequencing (http://efaecium.mlst.net).
Whole-cell DNA was prepared according to Turabelidze et al.3 The PFGE patterns were compared using BioNumerics 5.1 (Applied Maths, Kortrijk, Belgium). Similarity analysis was performed with the Dice coefficient, and clustering was performed by UPGMA. Strains having a similarity of more than 84% were considered to be possibly related as described previously.4,5
The HLGR E. faecium isolates was studied by PCR for genes encoding aminoglycoside-modifying enzymes. The vancomycin-resistant isolate was tested for the presence of vancomycin resistance genes.
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Results |
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The data from the 11 Danish counties showed a large increase in the number of enterococcal blood culture isolates from 2002 to 2006 (Figure 1a). In 2002, the total number of enterococcal infections was 542, whereas in 2006 the number had increased by 68% to 911. The increase in infections was mainly caused by a large increase in the number of bacteraemias with E. faecium, whereas the numbers of E. faecalis infections did not increase as much. The total number of E. faecalis isolates increased by 23% from 405 in 2002 to 497 in 2006, whereas the number of E. faecium isolates increased by 202% in the same period from 137 in 2002 to 414 in 2006 (Figure 1a). The E. faecalis/E. faecium ratio was 3:1 in 2002, whereas in 2006 the ratio was 1.2:1. Data also showed a significant increase in the number of ampicillin-resistant E. faecium isolates. In 2002, 65% of the tested E. faecium isolates were ampicillin-resistant, whereas 87% of the tested isolates were ampicillin-resistant in 2006 (P < 0.001) (Figure 1b).
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During the 12 month study period, 275 clinical isolates were received for further characterization. The isolates were mainly obtained from blood (80%) and the collection consisted of 162 E. faecalis and 113 E. faecium isolates. Antimicrobial susceptibility testing showed that 84 (74%) of the 113 E. faecium isolates were resistant to ampicillin. Furthermore, 58 of the 84 ampicillin-resistant isolates were also resistant to high-levels of gentamicin. All 58 HLGR E. faecium isolates were PCR-positive for the aac(6’)-Ie-aph(2’’)-Ia gene; none of the other genes encoding aminoglycoside-modifying enzymes was detected. One of the ampicillin-resistant isolates was also resistant to vancomycin. This isolate was PCR-positive for the vanB gene. None of the ampicillin-resistant isolates was resistant to linezolid or quinupristin/dalfopristin.
MLST revealed 10 different sequence types (STs) for the 84 tested ampicillin-resistant E. faecium isolates (Figure 2). The STs were ST18 (n = 22), ST17 (n = 22), ST306 (n = 22), ST262 (n = 11), ST203 (n = 2), ST275 (n = 1), ST178 (n = 1), ST64 (n = 1), ST379 (n = 1) and ST380 (n = 1). ST379 and ST380 are new STs and have not yet been assigned to a clonal complex; however, ST379 is a double-locus variant of ST22 and therefore probably belongs to CC22, and ST380 is a single-locus variant of ST18 and therefore probably belongs to CC17. The most common STs were ST18, ST17 and ST306, which accounted for almost 80% of the isolates. Among the 84 tested isolates, 82 (98%) belonged to CC17. The only vancomycin-resistant isolate was ST203, which is also a part of CC17.
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Eight major PFGE clusters were identified with a similarity of >84% (data not shown). Many of the isolates in each cluster were from the same hospital. Comparison of the MLST typing and the PFGE typing showed that seven of the eight clusters in the PFGE dendrogram were dominated by one specific ST. However, single-locus variants and double-locus variants were also found within the same PFGE cluster, and few isolates even had a difference of more than two alleles in the same PFGE cluster. The last cluster contained eight isolates with four different STs.
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Discussion |
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The data from the 11 Danish counties showed a large increase in the number of clinically important enterococcal isolates. We do not believe that this was due to a general trend of an increase in the number of nosocomial infections, since the number of E. coli blood culture isolates actually decreased slightly in the same 11 counties during the same period (data not shown). The increase was mainly due to E. faecium, which tripled during the study period, whereas the increase in E. faecalis isolates in the same period was modest (23%). This change in the E. faecalis/E. faecium ratio has been reported in other countries.1 The increasing frequency of E. faecium as a cause of infection is of great concern as bacteraemia caused by E. faecium has been associated with higher mortality than bacteraemia caused by E. faecalis.6 A reason for the increasing frequency of E. faecium as a cause of bloodstream and other infections could be its ability to acquire many different resistance genes; the antimicrobial pressure in a hospital environment therefore allows for its selection. The consumption of broad-spectrum antimicrobial agents results in the elimination of the normal flora, leading to colonization of strains endemic in the environment of hospitals; this could be an ampicillin-resistant E. faecium belonging to CC17. This is followed by overgrowth of the strain and, ultimately, clinical infection when host defences are overwhelmed. Treatment with fluoroquinolones, cephalosporins or carbapenems has been described as a risk factor for the development of an E. faecium infection.6,7 An increasing consumption of these antimicrobial agents has also been observed in hospitals in Denmark during the past 5 years.8 This might be an explanation for the changing E. faecalis/E. faecium ratio.
Of the MLST-typed ampicillin-resistant E. faecium 98% belonged to CC17. Only two isolates did not belong to CC17: one isolate was part of another clonal complex characterized as being from non-hospitalized humans, and the other isolate was a new ST probably belonging to CC22 (Figure 2). This indicates that CC17 is prevalent among human clinical E. faecium isolates from Denmark. This is in accordance with studies from other European countries.9,10
The majority of ampicillin-resistant E. faecium isolates were also resistant to high-levels of gentamicin. This is not common in the other Nordic countries where HLGR is more predominant in E. faecalis.11 Only one of the 275 tested enterococcal isolates obtained in 2004 was vancomycin-resistant; it was a vanB- positive E. faecium isolate obtained in December 2004, which was followed by five more vanB-positive E. faecium isolates with highly similar PFGE profiles, thus constituting an outbreak in the same hospital (data not shown). Only a few isolates containing either the vanA or vanB gene have been detected at the different hospitals in this period. The low prevalence of VRE in the Danish hospitals might be explained by the low consumption of glycopeptides. However, in the recent years, an increased consumption of linezolid and glycopeptides has been recorded in Danish hospitals.8
In conclusion, infections caused by ampicillin-resistant E. faecium are an increasing problem in Denmark. This may necessitate a change in the treatment of enterococcal infections from ampicillin to vancomycin, which in turn will increase the risk of spread of VRE in Danish hospitals.
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Funding |
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This study was supported by the Danish Ministry of Family and Consumer Affairs and the Danish Ministry of the Interior and Health as part of the Danish Integrated Antimicrobial Resistance and Research Programme (DANMAP) and the European Union Sixth Framework Programme ‘Approaches to Control Multiresistant Enterococci (ACE): Studies on molecular ecology, horizontal gene transfer, fitness and prevention’ under contract LSHE-CT-2007-037410.
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Transparency declarations |
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None to declare.
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Footnotes |
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Present address. Novozymes, Kroghøjsvej 36, DK-2880 Bagsværd, Denmark. 
Participants are listed in the Acknowledgements section.
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Acknowledgements |
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Karin Sixhøj Pedersen is thanked for technical assistance. Janetta Top is thanked for providing us with Figure 3 from Top et al.9 and giving us permission to modify and present it in this paper as Figure 2.
The following members of the DANRES Study Group are thanked for providing data: A. Friis-Møller, Department of Clinical Microbiology, Hvidovre Hospital; O. Heltberg, Department of Clinical Microbiology, Næstved Hospital; K. T. Jensen, Department of Clinical Microbiology, Esbjerg Hospital; J. K. Møller, Department of Clinical Microbiology, Aarhus University Hospital; B. Røder, Department of Clinical Microbiology, Slagelse Hospital; H. Schumacher, Department of Clinical Microbiology, Herning Hospital; M. K. Thomsen, Department of Clinical Microbiology, Viborg Hospital; M. Tvede, Department of Clinical Microbiology, Rigshospitalet.
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References |
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1 . Leavis HL, Bonten MJ, Willems RJ. Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance. Curr Opin Microbiol (2006) 9:454–60.[CrossRef][Web of Science][Medline]
2 . Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15 (2005) Wayne, PA, USA: CLSI.
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