Antimicrobial susceptibility patterns of enterococci in intensive care units in Sweden evaluated by different MIC breakpoint systems

Anita Hällgrena,*, Hossein Abednazarib, Christer Ekdahlb, Håkan Hanbergerb, Maud Nilssona, Annika Samuelssona, Erik Svenssona, Lennart E. Nilssona and the Swedish ICU Study Group{dagger}

a Divisions of Clinical Microbiology and b Infectious Diseases, Department of Health and Environment, Faculty of Health Sciences, SE-581 85 Linköping, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three hundred and twenty-two (322) clinical isolates were collected from patients admitted to intensive care units (ICUs) at eight Swedish hospitals between December 1996 and December 1998. Of the isolates, 244 (76%) were Enterococcus faecalis, 74 (23%) were Enterococcus faecium and four (1%) were other Enterococcus spp. MICs of ampicillin, imipenem, meropenem, piperacillin/tazobactam, ciprofloxacin, trovafloxacin, clinafloxacin, gentamicin, streptomycin, vancomycin, teicoplanin, quinupristin/dalfopristin, linezolid and evernimicin were determined by Etest. Susceptible and resistant isolates were defined according to the species-related MIC breakpoints of the British Society for Antimicrobial Chemotherapy (BSAC), the National Committee for Clinical Laboratory Standards (NCCLS) and the Swedish Reference Group for Antibiotics (SRGA). Tentative breakpoints were applied for new/experimental antibiotics. Multidrug resistance among enterococci in ICUs is not uncommon in Sweden, particularly among E. faecium, and includes ampicillin resistance and concomitant resistance to fluoroquinolones. Almost 20% of E. faecalis isolates showed high-level resistance to gentamicin and concomitant resistance to fluoroquinolones. Vancomycin-resistant enterococci were only found sporadically. Among the new antimicrobial agents, linezolid and evernimicin showed the best activity against all enterococcal isolates. There was good concordance between the BSAC, NCCLS and SRGA breakpoints in detecting resistance. When applying the SRGA breakpoints for susceptibility, isolates were more frequently interpreted as intermediate. This might indicate earlier detection of emerging resistance using the SRGA breakpoint when the native population is considered susceptible, but with the risk that isolates belonging to the native susceptible population will be incorrectly interpreted as intermediate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nosocomial infections caused by Gram-positive bacteria, including enterococci, are an increasing problem. Owing to invasive procedures, high antibiotic consumption and a patient population with serious underlying diseases, nosocomial infections are more frequent in intensive care units (ICUs)1 compared with general hospital wards2 and outbreaks often originate in ICUs.3

Enterococci have been regarded as pathogens that seldom cause serious infections.4 However, in the last decade they have emerged as an important cause of nosocomial infections, with an increasing frequency of multidrug resistance, including high-level resistance to gentamicin and resistance to ampicillin.58 Glycopeptide resistance among enterococci has become increasingly common in many European countries and in North America.911 However, in Swedish hospitals only sporadic findings of glycopeptide-resistant enterococci have been reported.12,13

In order to achieve early detection of emerging resistance, species-related breakpoints have been introduced in recent years with the aim of setting the breakpoint for susceptibility as close as possible to the upper border of the MIC distribution of the native population of a specific species.1417

The aim of this study was to investigate the MIC distribution of 14 different antibiotics for isolates of enterococci collected from patients admitted to ICUs and to evaluate the outcome by applying the breakpoint systems of the British Society for Antimicrobial Chemotherapy (BSAC),16 the National Committee for Clinical Laboratory Standards (NCCLS)17 and the Swedish Reference Group for Antibiotics (SRGA).15 The purpose was also to determine the MIC distribution of some new antibiotics, i.e. trovafloxacin, clinafloxacin, quinupristin/dalfopristin, linezolid and evernimicin.1823


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design and setting

A Swedish multicentre susceptibility study was performed on enterococcal isolates collected between December 1996 and December 1998 from patients admitted to ICUs at eight Swedish hospitals. Four hospitals were tertiary care (university) hospitals and four were secondary care (county) hospitals.

Bacterial cultures

Specimens collected on the basis of clinical indication, were cultured at each hospital (Table IGo). When enterococci had been identified, they were sent to the Division of Clinical Microbiology at the Faculty of Health Science in Linköping for further analysis. All initial isolates were included. Repeat isolates of the same species from the same patient were only included if there was a difference in MICs of >=2 dilution steps in at least one antibiotic compared with the initial isolate.


View this table:
[in this window]
[in a new window]
 
Table I. Source of isolates
 
Identification of the isolates

Identification of enterococci to the species level was based on a series of conventional biochemical tests.24,25 Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 35667 were used as control strains. When biochemical tests were inconclusive and/or the MIC of vancomycin was >4 mg/L, detection of vancomycin resistance genes (vanA, vanB, vanC1 or vanC2/C3) with multiplex PCR was used as described by Monstein and co-workers.26 The specimens were grown on paper disc method (PDM) agar plates (AB Biodisk, Solna, Sweden) with 5% defibrinated horse blood and with a vancomycin disc (AB Biodisk).15 Bacterial colonies close to the vancomycin disc were chosen for DNA extraction to make sure that they had not lost their resistance genes. The PCR amplifications were performed in a programmable thermal controller (GeneAmp PCR system 9600; Perkin Elmer, Beaconsfield, UK). The PCR products were analysed by 1.2% (w/v) agarose gel electrophoresis (E-Gel; Invitrogen, Groningen, The Netherlands) and stained with ethidium bromide. The gel was photographed in UV light and compared visually with positive controls for vanA (E. faecium HJ 827), vanB (E. faecalis HJ 227), vanC1 (Enterococcus gallinarium CCUG 18658) and vanC2/C3 (Enterococcus casseliflavus CCUG 18657/ATCC 25788).

Susceptibility testing

MICs of ampicillin, imipenem, meropenem, piperacillin/ tazobactam, ciprofloxacin, trovafloxacin, clinafloxacin, gentamicin, streptomycin, vancomycin, teicoplanin, quinupristin/dalfopristin, linezolid and evernimicin were determined by Etest (AB Biodisk), using a suspension equivalent to a 0.5 McFarland standard and PDM agar. Plates were examined after 24 h incubation at 37°C for all antibiotics except gentamicin and streptomycin, which were examined after 48 h.28 All isolates were tested for ß-lactamase production with the chromogenic nitrocefin disc test (AB Biodisk).

Breakpoints

Resistant and susceptible strains were defined according to the species-related MIC breakpoints of the BSAC,16 the NCCLS17 and the SRGA15 (Table IIGo). For antibiotics where BSAC, NCCLS or SRGA breakpoints were not available, breakpoints suggested in earlier studies or pharmaceutical company breakpoint recommendations were applied (Table IIIGo).21,23,29


View this table:
[in this window]
[in a new window]
 
Table II. Species-related MIC breakpoints (mg/L) according to the BSAC, the NCCLS and the SRGA
 

View this table:
[in this window]
[in a new window]
 
Table III. Frequency of resistant and susceptible isolates using tentative MIC breakpoints
 
Statistics

With respect to resistance to ampicillin or high-level resistance to gentamicin or streptomycin, isolates with or without concomitant resistance to fluoroquinolones were analysed using the {chi}2 test. Results were considered significant when P < 0.01.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the 322 enterococcal isolates, 244 were E. faecalis, 74 were E. faecium and four were other Enterococcus spp. Of these, three carried the vanC1 gene, and one the vanC2 gene. The sources of the isolates were mainly urine, respiratory tract and gastrointestinal tract (Table IGo).

Percentages of susceptible and resistant isolates according to BSAC, NCCLS and SRGA, or tentative breakpoints, are shown in Tables III and IVGoGo, respectively. The MIC distributions of the antibiotics are shown in Table VGo.


View this table:
[in this window]
[in a new window]
 
Table IV. Frequency (%) of susceptible and resistant isolates according to the BSAC, NCCLS and SRGA
 

View this table:
[in this window]
[in a new window]
 
Table V. MIC distribution of E. faecalis (n = 244) and E. faecium (n = 74)
 
Susceptibility to ß-lactam antibiotics

No ß-lactamase production was detected. Fifty-five isolates of E. faecium (74%) were resistant to ampicillin, but all isolates of E. faecalis were susceptible (Tables IV and VGoGo). When applying the SRGA and the BSAC breakpoints for susceptibility to the MIC distribution of imipenem, 37% and 97%, respectively, of the E. faecalis isolates were interpreted as susceptible (Tables IV and VGoGo). Similar results were seen with meropenem, where 9% were interpreted as susceptible using the SRGA breakpoints and 69% with the BSAC breakpoints (Tables IV and VGoGo).

Susceptibility to quinolones

None of the enterococci were susceptible to ciprofloxacin using the SRGA breakpoint. Applying the NCCLS breakpoints, 12% of E. faecium isolates and 23% of E. faecalis isolates were susceptible (Tables IV and VGoGo). Compared with ciprofloxacin, a higher number of isolates, 77% of E. faecalis and 20% of E. faecium, were susceptible to clinafloxacin (Tables III–VGoGoGo). Nevertheless, a large number of isolates with resistance to the new quinolones, clinafloxacin and trovafloxacin, were found among E. faecalis (23%, both antibiotics) and E. faecium (62% and 68%, respectively). All but two isolates resistant to the new quinolones were also resistant to ciprofloxacin (Tables III–VGoGoGo).

Of the 55 ampicillin-resistant isolates, 44 (80%) were resistant to all fluoroquinolones tested. In comparison, only one (5%) of the 19 ampicillin-susceptible E. faecium isolates was resistant to all of the fluoroquinolones ({chi}2 = 33.10, P < 0.0001).

High-level resistance to aminoglycosides

Of the E. faecium isolates, 54% showed high-level streptomycin resistance (HLSR), but no high-level gentamicin resistance (HLGR) was found (Tables III–VGoGoGo). Twenty per cent of the E. faecalis isolates showed HLGR, and 22% showed HLSR. All but one (98%) of the 48 E. faecalis isolates with HLGR were also resistant to all fluoroquinolones, compared with the E. faecalis isolates with no HLGR, where only eight of 196 isolates (4%) were resistant to all quinolones ({chi}2 = 194.4, P < 0.0001). No significant difference in quinolone resistance was found between E. faecalis with HLSR and without HLSR ({chi}2 = 0.91, P = 0.36). Among E. faecium, 31 of 40 isolates (77%) with HLSR were resistant to all quinolones tested, compared with the E. faecium isolates with no HLSR, where 14 of 34 (41%) were quinolone resistant ({chi}2 = 10.17, P = 0.001).

Susceptibility to glycopeptides

Among the E. faecium isolates, two with vancomycin MICs of 16–32 mg/L were detected. Both of these were of the vanB genotype. Of the four enterococcal isolates that were non-E. faecium–non-E. faecalis, three carried the vanC1 genotype (MICs = 8) and one the vanC2/3 genotype (MIC = 8). None of the enterococcal isolates were resistant to teicoplanin according to the BSAC, NCCLS and SRGA breakpoints for resistance (Tables IV and VGoGo).

Susceptibility to the new antibiotics

All strains of E. faecalis were resistant to quinupristin/ dalfopristin, except three isolates (1%) that were susceptible using the BSAC breakpoint and intermediate using the NCCLS breakpoints. For E. faecium, 78% and 98% were susceptible according to the NCCLS and the BSAC breakpoints, respectively, and 1.4% were resistant to quinupristin/dalfopristin irrespective of which breakpoint was used (Tables IV and VGoGo).

All enterococcal isolates were susceptible to linezolid and evernimicin using the tentative MIC breakpoint of 4 mg/L (Tables III and VGoGo).21,29


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic resistance of isolates from ICUs has been shown to be at a lower level in Sweden compared with other European countries.30 This study demonstrates that multidrug resistance is common among E. faecium isolates, and at levels similar to other European countries, although vancomycin resistance is still sporadic in Sweden.10,31,32 Of the 322 isolates collected, 74 were E. faecium (23%), more than elsewhere in Europe,31,32 although these studies were not based on the ICU patient population.

Four isolates were vanC-carrying species other than E. faecalis or E. faecium. The significance of vanC-carrying enterococcal species has recently been reviewed.33 Although serious infections may occur, many treatment options most often remain available, in contrast to enterococci with acquired resistance to vancomycin. On the basis of these conclusions we chose to exclude the four vanC- carrying isolates.

MICs in this study were performed by Etest according to the manufacturer's instructions and the SRGA's recommendations.15 The recommended growth medium is PDM or IsoSensitest agar (Oxoid, Basingstoke, UK) by the SRGA15 and Iso-Sensitest agar by the BSAC.16 Etest MICs correlate well with the reference method (agar dilution) using these media.15 Etest has been approved by the Food and Drug Administration using Mueller–Hinton agar, and correlates well with reference methods recommended by the NCCLS.28,34,35

Ampicillin and fluoroquinolones

Enterococci in general, and E. faecium in particular, are intrinsically relatively resistant to penicillin and ampicillin compared with other streptococci, with MICs of up to 8 mg/L for E. faecalis or higher for E. faecium.3638 In the late 1980s an increasing number of enterococcal isolates, mainly E. faecium, were reported to have even higher MICs (>100 mg/L),4,8 due to overproduction of a lowaffinity penicillin-binding protein (PBP) and a further decrease in the affinity of one of these PBPs.8,39 In our study, 55 isolates (74%) of E. faecium were resistant to ampicillin according to all breakpoints used, and 40 isolates (54%) showed MICs >= 100 mg/L (Table VGo). This is in agreement with studies performed mainly on hospitalized patients in the USA and Europe in the 1990s, although ICU wards were not specifically assessed.5,6 Forty-four (80%) of these 55 isolates with resistance to ampicillin in our study were concomitantly resistant to all fluoroquinolones tested. This is in agreement with the Swedish colonization study by Torell et al.,13 who showed that 91% of ampicillin-resistant enterococci were concomitantly resistant to ciprofloxacin. Trovafloxacin and clinafloxacin have both recently been restricted or withdrawn from clinical use because of toxicity. Although these new quinolones showed better activity (Table VGo) compared with ciprofloxacin, resistance was not uncommon and was, with two exceptions, only present in isolates resistant to ciprofloxacin (Tables III and IVGoGo), suggesting cross resistance.

High-level aminoglycoside resistance

In a recent study in 27 European countries, HLGR was as frequent in E. faecium (23%) and E. faecalis (20%).32 In contrast to these results, HLGR was not detected in any strain of E. faecium in our study. Twenty per cent of E. faecalis demonstrated HLGR, and all but one isolate with HLGR was resistant to all fluoroquinolones, in accordance with earlier studies.40,41 In the European study by Schouten et al.,32 resistance to ciprofloxacin and HLGR was found in enterococcal isolates from all 27 participant countries. Although clonal spread has been discussed, no explanation for this concomitant resistance has been confirmed.32

Vancomycin and teicoplanin

Only two isolates (both E. faecium) were resistant to vancomycin when applying the SRGA breakpoints, in accordance with sporadic reports of vancomycin-resistant enterococci (VRE) in Swedish hospitals12,13 and in contrast to findings from many other countries.6,10,11 This might be the result of the restricted use of vancomycin or because outbreaks have been limited by infection control interventions.15 It may also be influenced by the fact that use of antibiotics as growth promoters in animal food has been banned in Sweden since 1986.42

The vancomycin-resistant E. faecium strains carried the vanB genotype, and although isolated from the same patient, had different antibiograms with MICs of ampicillin of 8 and 512 mg/L, respectively. When applying the NCCLS breakpoint, one of the isolates (VRE no. 2) was interpreted as intermediate. To distinguish enterococci with acquired resistance (VanA or VanB) from intrinsically resistant isolates (VanC), the NCCLS recommends biochemical tests (motility and pigmentation tests) when the vancomycin MIC is 8–16 mg/L in order to identify isolates of E. gallinarium and E. casseliflavus.17 In our study the series of biochemical tests performed23,24 identified this isolate (VRE no. 2) as E. faecium, indicating an acquired resistance to vancomycin, and vanB was confirmed with multiplex PCR. However, biochemical tests are sometimes inconclusive in identifying enterococci to the species level,26,33 and identification of vancomycin resistance genes is of value in the identification of enterococci with MICs of vancomycin of 8–32 mg/L.33

Quinupristin/dalfopristin

E. faecalis is intrinsically resistant to quinupristin/dalfopristin;43 however, it has been used successfully to treat infections due to E. faecium.44,45 Recent studies, however, have described the emergence of resistance during therapy.46 In our study all E. faecalis were resistant using the NCCLS breakpoint, and only 1% were susceptible when applying the BSAC breakpoint. Of the E. faecium isolates, 78% or 98% were susceptible using the NCCLS or BSAC breakpoint, respectively, and one isolate was resistant (1.4%). These results are in accordance with most of the recent international and European studies where susceptibilities between 79 and >95% for E. faecium isolates have been reported,18,47,48 although susceptibility as low as 45% was reported in one study.49

Linezolid

All enterococcal isolates were susceptible at the tentative MIC breakpoint of 4 mg/L (Table IIGo). The range of linezolid MICs was narrow (1–4 mg/L). Similar narrow ranges have been noted by others, with Johnson et al.29 reporting most as 4 mg/L.

Evernimicin

All isolates in our study were susceptible to evernimicin using the tentative MIC breakpoint of 4 mg/L21 (Tables III and VGoGo), which is in agreement with earlier studies.20 Evernimicin has recently been withdrawn from further development because of toxicity.

Conclusions

Multidrug-resistant enterococci, particularly E. faecium, in ICUs in Sweden are not uncommon. Almost 20% of the E. faecalis isolates showed high-level resistance to gentamicin and concomitant resistance to all fluoroquinolones tested. VRE is still sporadic in Swedish ICUs. Among the new antimicrobial agents, evernimicin and linezolid showed the best activity for both E. faecalis and E. faecium. There was good concordance between the BSAC, NCCLS and SRGA breakpoints in determining resistant isolates, although SRGA breakpoints for susceptibility indicated more intermediate strains. This was especially evident for the carbapenems and the quinolones. This might indicate that emerging resistance is detected earlier when applying the SRGA species-related breakpoints when the native population is considered susceptible,15 but there is a risk with the SRGA breakpoints that isolates belonging to the susceptible native population will be incorrectly interpreted as intermediate.


    Acknowledgments
 
The authors thank Mats Fredrikson and Anders Magnusson, Department of Health and Environment, Division of Occupational and Environmental Medicine, Faculty of Health Sciences, Linköping, for kind assistance with the statistical analyses. The study was supported by Park-Davis, Division of Warner Lambert, Schering Plough, Pharmacia Upjohn and Rhône-Polenc Rorer and by a Medical School Grant from Merck.

Swedish ICU Study Group members are as follows. Coordinators: Lennart E. Nilsson, Håkan Hanberger. Participants: Berndt Claesson (Skövde), Anders Kärnell (Huddinge), Erik Kjellberg (Falun), Peter Larsson (Göteborg), Margareta Rylander (Stockholm), Lars Sörén (Jönköping).


    Notes
 
* Corresponding author. Tel: +46-1322-2000; Fax: +46-1322-4596; E-mail: aniha{at}ihm.liu.se Back

{dagger} Members of the Swedish ICU Study Group are listed in the Acknowledgements. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Erlandsson, C. M., Hanberger, H., Eliasson, I., Hoffman, M., Isaksson, B., Lindgren, S. et al. (1999). Surveillance of antibiotic resistance in ICUs in southeastern Sweden. ICU Study Group of the South East of Sweden. Acta Anaesthesiologica Scandinavica 43, 815–20.[ISI][Medline]

2 . Vincent, J. L., Bihari, D. J., Suter, P. M., Bruining, H. A., White, J., Nicolas-Chanoin, M. H. et al. (1995). The prevalence of nosocomial infection in intensive care units in Europe. Results of the European Prevalence of Infection in Intensive Care (EPIC) Study. Journal of the American Medical Association 274, 639–44.[Abstract]

3 . Asensio, A., Oliver, A., Gonzalez-Diego, P., Baquero, F., Perez-Diaz, J. C., Ros, P. et al. (2000). Outbreak of a multiresistant Klebsiella pneumoniae strain in an intensive care unit: antibiotic use as risk factor for colonisation and infection. Clinical Infectious Diseases 30, 55–60.[ISI][Medline]

4 . Murray, B. E. (1990). The life and times of the Enterococcus. Clinical Microbiology Reviews 3, 46–65.[ISI][Medline]

5 . Gray, J. W., Stewart, D. & Pedler, S. J. (1991). Species identification and antibiotic susceptibility testing of enterococci isolated from hospitalized patients. Antimicrobial Agents and Chemotherapy 35, 1943–5.[ISI][Medline]

6 . Huycke, M. M., Sahm, D. F. & Gilmore, M. S. (1998). Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. Emerging Infectious Diseases 4, 239–49.[ISI][Medline]

7 . Silverman, J., Thal, L. A., Perri, M. B., Bostic, G. & Zervos, M. J. (1998). Epidemiologic evaluation of antimicrobial resistance in community-acquired enterococci. Journal of Clinical Microbiology 36, 830–2.[Abstract/Free Full Text]

8 . Grayson, M. L., Eliopoulos, G. M., Wennersten, C. B., Ruoff, K. L., De Girolami, P. C., Ferraro, M. J. et al. (1991). Increasing resistance to beta-lactam antibiotics among clinical isolates of Enterococcus faecium: a 22-year review at one institution. Antimicrobial Agents and Chemotherapy 35, 2180–4.[ISI][Medline]

9 . Lavery, A., Rossney, A. S., Morrison, D., Power, A. & Keane, C. T. (1997). Incidence and detection of multi-drug-resistant enterococci in Dublin hospitals. Journal of Medical Microbiology 46, 150–6.[Abstract]

10 . Kjerulf, A., Pallesen, L. & Westh, H. (1996). Vancomycin-resistant enterococci at a large university hospital in Denmark. Acta Pathologica, Microbiologica et Immunologica Scandinavica 104, 475–9.

11 . Goossens, H. (1998). Spread of vancomycin-resistant enterococci: differences between the United States and Europe. Infection Control and Hospital Epidemiology 19, 546–51.[ISI][Medline]

12 . Torell, E., Fredlund, H., Tornquist, E., Myhre, E. B., Sjoberg, L. & Sundsfjord, A. (1997). Intrahospital spread of vancomycin-resistant Enterococcus faecium in Sweden. Scandinavian Journal of Infectious Diseases 29, 259–63.[ISI][Medline]

13 . Torell, E., Cars, O., Olsson-Liljequist, B., Hoffman, B. M., Lindback, J. & Burman, L. G. (1999). Near absence of vancomycin-resistant enterococci but high carriage rates of quinolone-resistant ampicillin-resistant enterococci among hospitalized patients and nonhospitalized individuals in Sweden. Journal of Clinical Microbiology 37, 3509–13.[Abstract/Free Full Text]

14 . Hanberger, H., Nilsson, L. E., Claesson, B., Kärnell, A., Larsson, P., Rylander, M. et al. (1999). New species-related MIC breakpoints for early detection of development of resistance among Gram-negative bacteria in Swedish intensive care units. Journal of Antimicrobial Chemotherapy 44, 611–9.[Abstract/Free Full Text]

15 . Kahlmeter, G. (1999). The Swedish Reference Group for Antibiotics (SRGA) and its subcommittee on methodology (SRGA-M). [On-line, Version 3, SRGA homepage] www.srga.org (2 April 2001, date last accessed).

16 . The British Society for Antimicrobial Chemotherapy (BSAC). (2000). Standardized Disc Testing Method. [On-line, version: February 2000] www.bsac.org.uk (25 April 2001, date last accessed).

17 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Tests for Bacteria that Grow Aerobically: Approved Standard M7–A5. NCCLS, Villanova, PA.

18 . Dowzicky, M., Nadler, H. L., Feger, C., Talbot, G., Bompart, F. & Pease, M. (1998). Evaluation of in vitro activity of quinupristin/ dalfopristin and comparator antimicrobial agents against worldwide clinical trial and other laboratory isolates. American Journal of Medicine 104, 34S–42S.[Medline]

19 . Patel, R., Rouse, M. S., Piper, K. E. & Steckelberg, J. M. (1999). In vitro activity of linezolid against vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae. Diagnosic Microbiology and Infectious Disease 34, 119–22.

20 . Urban, C., Mariano, N., Mosinka-Snipas, K., Wadee, C., Chahrour, T. & Rahal J. J. (1996). Comparative in-vitro activity of SCH 27899, a novel everninomicin, and vancomycin. Journal of Antimicrobial Chemotherapy 37, 361–4.[Abstract]

21 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (1999). In vitro activities of SCH27899 alone and in combination with 17 other antimicrobial agents. Antimicrobial Agents and Chemotherapy 43, 2996–7.[Abstract/Free Full Text]

22 . Sefton, A. M., Maskell, J. P., Rafay, A. M., Whiley, A. & Williams, J. D. (1997). The in-vitro activity of trovafloxacin, a new fluoroquinolone, against Gram-positive bacteria. Journal of Antimicrobial Chemotherapy 39, Suppl. B, 57–62.[Abstract/Free Full Text]

23 . Itokazu, G. S., Nathan, C., Hariharan, R., Kostman, J. R., Kabins, S. A. & Weinstein, R. A. (1996). The comparative in vitro activity of clinafloxacin and other antimicrobials against vancomycin-susceptible and vancomycin-resistant enterococci. Chemotherapy 42, 235–9.[ISI][Medline]

24 . Facklam, R. R., Sahim, D. F. & Martins-Teixteira, L. (1999). Enterococcus. In Manual of Clinical Microbiology, 7th edn, (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 297–305. ASM Press, Washington, DC.

25 . Devriese, L. A., Pot, B., Kersters, K., Lauwers, S. & Haesebrouch, F. (1996). Acidification of methyl-ß-d-glucopyranoside: a useful test to differentiate Enterococcus casseliflavus and Enterococcus gallinarum from Enterococcus faecium species group and from Enterococcus faecalis. Journal of Clinical Microbiology 34, 2607–9.[Abstract]

26 . Monstein, H. J., Johansson, Y. & Jonasson, J. (2000). Detection of vancomycin resistance genes combined with typing of Enterococci by means of multiplex PCR amplification and multiple primer DNA sequencing. Acta Pathologica, Microbiologica et Immunologica Scandinavica 108, 67–73.

27 . Monstein, H. J., Quednau, M., Samuelsson, A., Ahrne, S., Isaksson, B. & Jonasson, J. (1998). Division of the genus Enterococcus into species groups using PCR-based molecular typing methods. Microbiology 144, 1171–9.[Abstract]

28 . Schulz, J. E. & Sahm, D. F. (1993). Reliability of the Etest for detection of ampicillin, vancomycin and high-level aminoglycoside resistance in Enterococcus spp. Journal of Clinical Microbiology 31, 3336–9.[Abstract]

29 . Johnson, A. P., Warner, M. & Livermore, D. M. (2000). Activity of linezolid against multi-resistant Gram-positive bacteria from diverse hospitals in the United Kingdom. Journal of Antimicrobial Chemotherapy 45, 225–30.[Abstract/Free Full Text]

30 . Hanberger, H., Diekema, D., Fluit, A., Jones, R., Struelens, M., Spencer, R. et al. (2001). Surveillance of antibiotic resistance in European ICUs. Journal of Hospital Infection, in press.

31 . Vandamme, P., Vercauteren, E., Lammens, C., Pensart, N., Ieven, M., Pot, B. et al. (1996). Survey of enterococcal susceptibility patterns in Belgium. Journal of Clinical Microbiology 34, 2572–6.[Abstract]

32 . Schouten, M. A., Voss, A. & Hoogkamp-Korstanje, J. A. (1999). Antimicrobial susceptibility patterns of enterococci causing infections in Europe. The European VRE Study Group. Antimicrobial Agents and Chemotherapy 43, 2542–6.[Abstract/Free Full Text]

33 . Nelson, R. R. (1999). Intrinsically vancomycin-resistant gram-positive organisms: clinical relevance and implications for infection control. Journal of Hospital Infection 42, 275–82.[ISI][Medline]

34 . Endtz, H. P., Van Den Braak, N., Van Belkum, A., Goessens, W. H., Kreft, D., Stroebel, A. B. et al. (1998). Comparison of eight methods to detect vancomycin resistance in enterococci. Journal of Clinical Microbiology 36, 592–4.[Abstract/Free Full Text]

35 . Huang, M. B., Baker, C. N., Banerjee, S. & Tenover, F. C. (1992). Accuracy of the E test for determining antimicrobial susceptibilities of staphylococci, enterococci, Campylobacter jejuni and gram-negative bacteria resistant to antimicrobial agents. Journal of Clinical Microbiology 30, 3243–8.[Abstract]

36 . Fontana, R., Ligozzi, M., Pittaluga, F. & Satta, G. (1996). Intrinsic penicillin resistance in enterococci. Microbial Drug Resistance 2, 209–13.[ISI][Medline]

37 . Atkinson, B. A., Abu-Al-Jaibat, A. & LeBlanc, D. J. (1997). Antibiotic resistance among enterococci isolated from clinical specimens between 1953 and 1954. Antimicrobial Agents and Chemotherapy 41, 1598–600.[Abstract]

38 . Murray, B. E. (1998). Diversity among multidrug-resistant enterococci. Emerging Infectious Diseases 4, 37–47.[ISI][Medline]

39 . Fontana, R., Aldegheri, M., Ligozzi, M., Lopez, H., Sucari, A. & Satta, G. (1994). Overproduction of a low-affinity penicillin-binding protein and high-level ampicillin resistance in Enterococcus faecium. Antimicrobial Agents and Chemotherapy 38, 1980–3.[Abstract]

40 . Caballero-Granado, F. J., Cisneros, J. M., Luque, R., Torres-Tortosa, M., Gamboa, F., Díez, F. et al. (1998). Comparative study of bacteremias caused by Enterococcus spp. with and without high-level resistance to gentamicin. The Grupo Andaluz para el estudio de las Enfermedades Infecciosas. Journal of Clinical Microbiology 36, 520–5.[Abstract/Free Full Text]

41 . Schaberg, D. R., Dillon, W. I., Terpenius, M. S., Robinson, K. A., Bradley, S. F. & Kauffman, C. A. (1992). Increasing resistance of enterococci to ciprofloxacin. Antimicrobial Agents and Chemotherapy 36, 2533–5.[Abstract]

42 . Björnerot, L., Franklin, A. & Tysén, E. (1996). Usage of antibacterial and antiparasitic drugs in animals in Sweden between 1988 and 1993. Veterinary Record 139, 282–6.[ISI][Medline]

43 . Low, D. E. (1995). Quinupristin/dalfopristin: spectrum of activity, pharmacokinetics, and initial clinical experience. Microbial Drug Resistance 1, 223–34.[ISI][Medline]

44 . Linden, P. K., Pasculle, A. W., McDevitt, D. & Kramer, D. J. (1997). Effect of quinupristin/dalfopristin on the outcome of vancomycin-resistant Enterococcus faecium bacteraemia: comparison with a control cohort. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 145–51.[Abstract/Free Full Text]

45 . Dever, L. L., Smith, S. M., Dejesus, D., Masurekar, M., Patel, D., Kaminski, Z. C. et al. (1996). Treatment of vancomycin-resistant Enterococcus faecium infections with an investigational streptogramin antibiotic (quinupristin/dalfopristin): a report of fifteen cases. Microbial Drug Resistance 2, 407–13.[ISI][Medline]

46 . Chow, J. W., Donahedian, S. M. & Zervos, M. J. (1997). Emergence of increased resistance to quinupristin/dalfopristin during therapy for Enterococcus faecium bacteremia. Clinical Infectious Diseases 24, 90–1.[ISI][Medline]

47 . Elsner, H.-A., Sobottka, I., Feucht, H.-H., Claussen, M., Kaulfers, P.-M., Laufs, R. et al. (2000). In vitro susceptibilities of enterococcal blood culture isolates from the Hamburg area to ten antibiotics. Chemotherapy 46, 104–10.[ISI][Medline]

48 . Jones, R. N., Ballow, C. H., Biedenbach, D. J., Deinhart, J. A. & Schentag, J. J. (1998). Antimicrobial activity of Quinupristin– Dalfopristin (RP 59500, Synercid®) tested against over 28000 recent clinical isolates from 200 medical centers in the United States and Canada. Diagnostic Microbiology and Infectious Disease 30, 437–51.

49 . Jones, R. N., Hare, R. S., Sabatelli, F. J. and the Ziracin Susceptibility Testing Group. (2001). In vitro Gram-positive antimicrobial activity of evernimicin (SCH 27899), a novel oligosaccharide, compared with other antimicrobials: a multicentre study. Journal of Antimicrobial Chemotherapy 47, 15–25.[Abstract/Free Full Text]

Received 30 November 2000; returned 6 February 2001; revised 22 March 2001; accepted 5 April 2001