Performance of The EUCAST Disk Diffusion Method, CLSI Agar Screen. and VITEK 2 Automated Antimicrobial Susceptibility Testing System in

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1 JCM Accepts, published online ahead of print on 5 March 2014 J. Clin. Microbiol. doi: /jcm Copyright 2014, American Society for Microbiology. All Rights Reserved Performance of The EUCAST Disk Diffusion Method, CLSI Agar Screen and VITEK 2 Automated Antimicrobial Susceptibility Testing System in Detection of Clinical Isolates of Enterococci with Low and Medium Level VanB-type Vancomycin Resistance: a Multicentre Study Kristin Hegstad 1,2#, Christian G. Giske 3, Bjørg Haldorsen 1, Erika Matuschek 4, Kristian Schønning 5, Truls M. Leegaard 6, Gunnar Kahlmeter 4, and Arnfinn Sundsfjord 1,2# on behalf of the NordicAST VRE detection study group Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North-Norway, Tromsø, Norway 2 Research group for Host-Microbe Interactions, Faculty of Health Sciences, University of Tromsø The Arctic University of Norway, Tromsø, Norway 3 Department of Clinical Microbiology, MTC - Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden 4 EUCAST Laboratory for Antimicrobial Susceptibility Testing (AST), Växjö, Sweden 5 Department of Clinical Microbiology, Hvidovre Hospital, Denmark 6 Department of Microbiology, Akershus University Hospital, Oslo, Norway # Corresponding authors: Kristin.Hegstad@uit.no and Arnfinn.Sundsfjord@uit.no Running title: Comparing methods for VanB-type VRE detection 1

2 ABSTRACT Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci was evaluated in a Scandinavian multicentre study (n=28). A pheno- and genotypically well-characterized diverse collection of Enterococcus faecalis (n=12) and Enterococcus faecium (n=18) with and without non-susceptibility to vancomycin was examined blindly in Danish (n=5), Norwegian (n=13) and Swedish (n=10) laboratories using the EUCAST disk diffusion method (n=28), and the CLSI agar screen (n=18) or the VITEK 2 system (n=5; biomérieux). The EUCAST disk diffusion method (VME 7.0%, sensitivity 0.93, ME 2.4% and specificity 0.98) and CLSI agar screen (VME 6.6%, sensitivity 0.93, ME 5.6% and specificity 0.94) performed significantly better (p=0.02) than VITEK 2 (VME 13%, sensitivity 0.87, ME 0% and specificity 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel to interpret fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid (p<0.0001) and Merck MH agar (p=0.027) for disk diffusion performed significantly better compared to laboratories using the BBL MH II medium. Laboratories using Difco BHI for CLSI agar screen performed significantly better (p=0.017) than those using Oxoid BHI. In conclusion, both the EUCAST disk diffusion and CLSI agar screen methods perform acceptable (sensitivity 0.93 and specificity ) in detection of low-level vanb VRE. Importantly, the use of CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel are trained in interpreting zone edges. 2

3 INTRODUCTION Enterococci are now recognized as an important cause of hospital-acquired infections worldwide (1, 2). Notably, recent European surveys have documented a pronounced yearly increase in bloodstream infection caused by multidrug resistant (MDR) E. faecium represented by High Risk Clones (3). The relative increase in infections caused by MDR enterococci is related to several characteristics. This includes their intrinsic ability to withstand exposure to our broadspectrum antibiotics and environmental extremes, as well as the capacity to acquire new genetic determinants promoting gastrointestinal colonisation and survival in the hospital environment (4, 5). Moreover, their remarkable ability to acquire new antimicrobial resistance determinants poses substantial therapeutic problems and physicians are forced to use last resort therapeutic options (4, 6). Hence, it is important that the clinical laboratories have the ability to deliver rapid, accurate antimicrobial susceptibility data for enterococci to support appropriate therapy and infection control measures. Currently the vancomycin resistance cluster (van) alphabet in enterococci consist of eight acquired gene clusters (vana, vanb, vand, vane, vang, vanl (7), vanm (8) and vann (9)). The vana genotype is the most prevalent VRE-genotype worldwide, but infections with vanbtype VRE (mainly E. faecium) have shown a dramatic increase in several European countries and are predominant in Australia (10-16). The vanb ligase gene has been divided into three subtypes, vanb1-3, based on phylogenetic diversity (17-19). The vanb-type VRE is inducible and expresses various levels of resistance to vancomycin (Minimum Inhibitory concentration (MIC) mg/l) and susceptibility to teicoplanin (MIC 2 mg/l) in vitro (7, 20). The wide range of vancomycin MIC in VanBtype enterococci is well known and has also been observed within the same clone during outbreaks (12, 21) (Sivertsen et al., submitted for publication). The European Committee on 3

4 Antimicrobial Susceptibility Testing (EUCAST) MIC clinical breakpoints for Enterococcus spp. are susceptible (S) 4 mg/l and resistant (R) > 4 mg/l for vancomycin and S 2 mg/l and R > 2 mg/l for teicoplanin (22). The inducible moderate to low vancomycin MIC phenotypes of vanb-type VRE challenges current phenotypic detection methods. It is important to detect these VRE isolates as glycopeptide treatment of infections caused by such isolates may lead to treatment failure due the increased MIC itself or selection of a constitutively expressed vanb cluster also showing resistance to teicoplanin (23-26). The purpose of this study was to examine the ability of different antimicrobial susceptibility testing methods to detect low and medium level vancomycin resistant enterococci (VRE). It was organized as a multicentre study where Danish, Norwegian and Swedish laboratories were invited to blindly examine a pheno- and genotypic wellcharacterized diverse collection of E. faecalis (n=12) and E. faecium (n=18) with and without non-susceptibility to vancomycin. The collection was examined by the EUCAST disk diffusion method in all laboratories and by one alternative method that could include the vancomycin-bhi-agar screen method referred to as Clinical and Laboratory Standards Institute (CLSI) agar screen, a commercial chromogenic agar screen, or an automated antimicrobial susceptibility test system MATERIAL AND METHODS Bacterial strains used in this study All isolates were previously genotyped by multiplex ID-PCR (27), vana/b/e/g PCRs (17, 28, 29), SmaI PFGE (30) and/or MLST to ensure species identification, van-genotype and genetic diversity. The isolates cover the whole range of vancomycin MIC values. The vancomycin MIC of the isolates was confirmed by both gradient test (Etest, biomérieux, Marcy l'etoile, 4

5 France) on MH agar as described by the manufacturer and broth microdilution according to International Standards Organisation recommendations (31). The isolate panel consisted of well-characterized vancomycin susceptible (n=3; three copies of E. faecalis ATCC 29212) and resistant (n=27) strains of E. faecalis and E. faecium (Table 1). The resistant isolates expressed various vancomycin MIC levels (Fig. 1). Twelve of the isolates were E. faecalis (vanb1 n=3; vanb2 n=1; vane n=1; vang n=1; ATCC n=3 vancomycin susceptible; ATCC vanb n=3) and 18 E. faecium (vanb1 n=1; vanb2 n=17) of diverse geographical origins (Norway (n=12), USA (n=11), Sweden (n=6) and Australia (n=1)). Seven of the isolates represented Nordic outbreak strains from Bergen (MIC 32 mg/l) (1996) (32, 33), Örebro (MIC 32 and 256 mg/l) ( ) (15), Stockholm (MIC 8 mg/l) (2007) and Västerås (MIC 16 mg/l) (2008) (Sivertsen et al., submitted for publication). The remaining 21 resistant strains expressed low-level (4-8 mg/l; n=5), medium-level (16-32 mg/l; n=13) or high-level vancomycin resistance (MIC 64 mg/l; n=2). The panel consisted of 23 PFGE types and two subtypes within type XII. MLST analysis of 13 isolates displayed 5 ST types within the HiRiCs E. faecium CC17 (ST16, n=1; ST17, n=5; ST192, n=1; ST262, n=1; ST313, n=1) as well as E. faecalis ST type 14 (n=1) and 30 (ATCC n=3) (Table 1). Study design. The study was organized through the NordicAST (Nordic Committee on Antimicrobial Susceptibility Testing) network ( All Danish (n=13), Swedish (n=23) and Norwegian (n=24) diagnostic laboratories in clinical microbiology were invited to participate in the study examining the bacterial panel as listed (Table 1). All laboratories were asked to perform the reference agar disk diffusion method as recommended by EUCAST (34) and at least one alternative method; the CLSI agar screening method (35) using brain heart infusion (BHI) agar supplemented with vancomycin 6 mg/l, 5

6 and/or a commercial automated test available in the laboratory (VITEK 2). The laboratories were also welcomed to test the performance of commercial VRE selective agars. Each participating laboratory used their own supply of test reagents, discs and agar. They were instructed to avoid any testing with a confirmation method (van-genotyping or gradient tests), as the objective was for the strains to be examined in a blinded, non-biased approach. The timeframe for the labs to complete the susceptibility test was maximum 3 weeks. A result data form was filled in for each participating laboratory for each strain and included; zone diameter and zone edge quality (fuzzy or sharp zone edge) for the agar disc diffusion, growth/no-growth for the agar screening, results for the commercial automated antimicrobial susceptibility test systems as reported, interpretation (S or R) according to EUCAST clinical breakpoints (22), and a comment on whether the laboratory under normal circumstances would refer the sample to confirmatory testing in a reference laboratory. Phenotypic methods used for antimicrobial susceptibility testing. The disk diffusion test was performed with 5-μg vancomycin 6 mm disks and Mueller-Hinton (MH) agar plates according to EUCAST methodology (34) and clinical breakpoints (22). Each laboratory participating in the study used its own supply of MH agar, which was either Oxoid (Oxoid Limited/Thermo Fisher Scientific, Cambridge, UK) (n=16), BBL MH II (Becton Dickinson Diagnostics, Baltimore, MD, USA) (n=10), or Merck (Merck KGaA, Darmstadt, Germany) (n=2). The CLSI agar screen method was performed with BHI agar with 6 mg/l vancomycin and read after 24 hours as described by Swenson et al. (35). The BHI agar used was either Difco (Becton Dickinson Diagnostics) (n=8), Oxoid (n=5), BBL (Becton Dickinson Diagnostics) (n=3), Scharlau (Scharlab SL, Barcelona, Spain) (n=1) or Acumedia (Neogen corporation, Lansing, Mi) (n=1). 6

7 Growth on commercial chromogenic culture media read after 48 h was tested using chromid VRE (biomérieux, Marcy l Etoile, France) (n=7) or CHROMagar VRE (CHROMagar, Paris, France) (n=5). Automatic susceptibility testing by the VITEK 2 system was performed using either the AST-P592 (n=2), AST-P586 (n=2) or AST-594 (n=1) susceptibility cards (biomérieux). Evaluation of results. The test results were categorized as either correct (S reported as S and R reported as R), very major error (VME; R reported as S) or major error (ME; S reported as R), as EUCAST has not defined any intermediate category for clinical vancomycin breakpoints (thus excluding the possibility of minor errors occurring). According to the EUCAST disk diffusion method the isolates were categorised as resistant when the zone diameter was less than 12 mm. Also, according to the method, resistance should be suspected when the vancomycin zone edge is fuzzy (examples in Fig. 2c and d) or colonies growing within the inhibition zone (example in Fig. 2b). Thus, zone edge quality was also taken into account. The isolates were reported as vancomycin susceptible only when the zone edges were sharp and 12 mm. Statistical methods and interpretation. Sensitivities (conditional probabilities that resistant isolates are correctly categorised), specificities (conditional probabilities that susceptible isolates are correctly categorised) and confidence intervals (CI) were calculated using Clinical research calculator 1 at vassarstats.net/index.html. Fisher's exact test with twotailed p-value using the online calculator ( was used to compute statistically significant difference (p<0.05) between pairs of methods or media RESULTS 7

8 A total of 34 Scandinavian laboratories agreed to participate and delivered data to the study. The results from six laboratories were excluded because they did not deliver data on the EUCAST disk diffusion method. Thus, results from 28 laboratories were included; 13 Norwegian, 10 Swedish, and five Danish. The laboratories delivered datasets on the following methods; EUCAST disk diffusion (n=28), CLSI agar screen (n=18), agar screen using commercial chromogenic VRE media (n=12), and VITEK 2 (n=5; biomérieux). Routine phenotypic testing for vancomycin resistance in enterococci is done by CLSI agar screen in Norwegian laboratories while the EUCAST disk diffusion is the preferred method in Sweden. Three of the Danish laboratories were also familiar with the EUCAST disk diffusion method in detection of VRE. EUCAST disk diffusion and CLSI agar screen performed better than VITEK 2. VME, ME, sensitivity and specificity were calculated for each method or agar type used (Table 2 show calculations for n 5). None of the tested methods scored perfect. The sensitivity ( ) and specificity (0.94-1) were high for the EUCAST disk diffusion method, the CLSI agar screen and the VITEK 2 system (Table 2). Overall the EUCAST disk diffusion and CLSI agar screen methods performed better than the VITEK 2 system (Table 2 and S1). Comparing each method with one another showed that both the disk diffusion method and CLSI agar screen performed significantly better than VITEK 2 in identifying the isolates as resistant or susceptible (Table S1). The distribution of VME related to reference MIC values of the isolates (data not shown) revealed that the troublesome isolates for the disk diffusion and CLSI agar screen methods mainly expressed low MIC (4-8 mg/l), whereas the VITEK 2 system had more problems concerning detection of moderate resistant levels (MIC mg/l). Mean, median, lowest and highest numbers of errors per laboratory for each method are reported in Table 3. The mean and median values are similar and low for disk diffusion and CLSI agar screen. 8

9 Experience and media influence the results by EUCAST disk diffusion. The observed numbers of VME and ME as well as sensitivity and specificity for disk diffusion differed between laboratory country origins. Swedish laboratories, experienced in using disk diffusion for testing vancomycin resistance, in general performed better at detecting the resistant isolates by the EUCAST disk diffusion method (Table 4). Dividing the results by type of MH agar used for disk diffusion indicated that BBL MH II was not performing as well as Oxoid MH (Table 2) and Merck MH. Statistical analyses showed that laboratories using Oxoid MH (p<0.0001) and Merck MH (p=0.027) performed significantly better compared to laboratories using BBL MH II. The disk diffusion inhibition zones were on average read to be 0.8 mm larger using the BBL MH II agar compared to Oxoid (Fig. 3). Resistant isolates more often had a zone size 12 mm on BBL MH II than on other agars (Fig. 3). Nine of 10 laboratories using BBL MH II were inexperienced with the agar disk diffusion method in detection of VRE. Thus, the number of experienced users that reported results for BBL MH II was too small to get a clear picture whether these errors were due to inexperience or agar characteristics (Tables S2 and S3). In line with this, the four inexperienced laboratories using Oxoid MH agar were not significantly better at identifying the isolates than the nine inexperienced laboratories using BBL MH II agar. Three laboratories performed disk diffusion using Neo-Sensitabs 9 mm disks (Rosco Diagnostica A/S, Taastrup, Denmark). The VME (14%) and specificity (1) for Neo-Sensitabs were higher than for 6 mm disks (VME 6.5% and specificity 0.98), while the ME (0%) and sensitivity (0.86) were lower than for 6 mm disks (ME 2.2% and sensitivity 0.93). Agar disk diffusion using 6 mm disks performed significantly better (p=0.042) than Neo-Sensitabs in identifying the isolates as resistant or susceptible. However, the VME and sensitivity for Neo- Sensitabs were slightly different compared to inexperienced personnel using 6 mm disks 9

10 (VME 11% and sensitivity 0.89), and agar disk diffusion with inexperience personnel using 6 mm disks did not perform significantly better than Neo-Sensitabs in identifying the isolates as resistant or susceptible (data not shown). Media influence the results by CLSI agar screen The observed number of VME and ME, sensitivity and specificity for CLSI agar screen revealed that the Norwegian laboratories, which are experienced in using agar screen for testing vancomycin resistance, did not appear better at detecting the resistant isolates by this method compared to the only participating Swedish laboratory reporting for this method (Table 4). The laboratories using Oxoid (Table 2) and BD BBL BHI (data not shown) for agar screen performed less well in revealing the susceptibility category of the isolates compared to Difco BHI. Laboratories using Difco BHI performed significantly better (P=0.017) than those using Oxoid BHI, while just insignificantly (P=0.052) better than those using BD BBL BHI (data not shown). Comparing only the experienced laboratories, Difco BHI performed significantly better at identifying the isolates correctly than Oxoid BHI in agar screen (Table S3). The numbers of laboratories using Scharlau or Acumedia BHI for agar screen was too small to give any significant difference compared to both Oxoid and Difco BHI. Agar screening using commercial chromogenic VRE media. Agar screening using commercial chromogenic VRE media (n=12) showed higher ME (11%) and sensitivity (0.98) and lower specificity (0.89) corresponding to those obtained by disk diffusion, CLSI agar screening and VITEK 2 (Table 2). The high ME and lower specificity of commercial chromogenic VRE agars indicate that their potential use in screening of clinical strains for vancomycin resistance would results in a high number of false positive results. 10

11 Comparison of the two different chromogenic VRE media used showed a slightly better performance of chromid VRE compared to VRE CHROMagar (Table 2) but these results were not statistically significant. Different performance of AST cards used in the VITEK 2 system. The VITEK 2 system results using different type of antimicrobial susceptibility test cards show that card AST-P592 (VME 7.4%, sensitivity 0.93) was better at identifying the isolates correctly than card AST-P586 (VME 20%, sensitivity 0.80) and AST-P594 (VME 11%, sensitivity 0.89). However, these results were not statistically significant DISCUSSION We have examined the ability of EUCAST disk diffusion, the CLSI agar screen and VITEK 2 automated antimicrobial susceptibility system to detect VanB-type VRE in a blinded panel of well-characterized E. faecalis and E. faecium strains with or without low to medium level resistance to vancomycin. The study was performed in a multicentre design involving clinical laboratories in Denmark, Norway and Sweden. All laboratories tested the EUCAST agar disk diffusion method and in addition one alternative method that included the CLSI agar screen, commercial chromogenic VRE agar screens, and/or automated systems. In projects like these it is possible that project samples receive more attention than routine samples and that this introduces a bias in the evaluation of performance. On the other hand, the results will disclose the best achievement level and knowing this is valuable. Although none of the methods were perfect, the CLSI agar screen and EUCAST agar disk diffusion methods showed comparably high and acceptable sensitivity and specificity levels. Our results show that the ability to detect VRE by agar disk diffusion is influenced by the experience of the personnel in reading inhibition zones while experience in interpretation of 11

12 CLSI agar screen results is not that critical. For the agar screen method the choice of BHI agar and quality controlling the vancomycin concentration, seemed to be more important for the correct identification of the VRE. The notion that experience is more important when using disk diffusion was supported by the numbers calculated for EUCAST disk diffusion and CLSI agar screen for experienced versus inexperienced laboratories (Table 4 and S2). Laboratories experienced in the disk diffusion method performed significantly better (p<0.0001) at identifying the isolates as resistant or susceptible by disk diffusion than inexperienced personnel (data not shown). Moreover, the mean and median error values are similar and low for EUCAST disk diffusion and CLSI agar screen while the highest numbers of VME for EUCAST disk diffusion were all reported from laboratories that have no previous experience with disk diffusion to detect low-level vancomycin resistance. Moreover, comparing the different media for agar disk diffusion showed that the resistant isolates more often had a zone size 12 mm on BBL MH II media (Fig. 3) which will require an experienced reader to check the zone edge quality in order to correctly categorize the isolate as resistant. In this study chromogenic VRE media gave the lowest number of VME (Table 2) but the highest number of ME. In a recent study by Klare et al. the VMEs of these chromogenic VRE screening media were higher than those observed in our study (36). The higher VMEs observed by Klare and co-workers could be due to their selection of strains where 43/129 (33%) of vanb type E. faecium strains showed an MIC 4 mg/l thus pushing the detection limits more than our collection where only one of 27 VRE isolates showed an MIC of 4 mg/l (Table 1). It is important to have rapid routine AST methods which are easily interpreted and communicated to the clinician. We have shown that the results can be influenced by the level of experience of the laboratory personnel and/or media used. In this study the participating laboratories were informed about the van-status in the examined strain collection after 12

13 reporting their results. Subsequently, many participants examined the samples again and reported that they had read the troublesome samples wrongly the first time. This was specifically the case for the disk diffusion method. Detection of low-level vancomycin resistant isolates with disk diffusion relies not only on evaluation of the zone size but also on evaluation of the zone edges which requires experience. Hence, it is important to note that EUCAST stress that the zone edges should be examined with transmitted light (plate held up to light) and resistance suspected when the vancomycin zone edge is fuzzy (Fig. 2C and D) or colonies grow within the inhibition zone (Fig. 2B) and that glycopeptide susceptibility tests on enterococci should be incubated for 24 h to ensure visibility of resistant colonies. These advices are specific for evaluating VRE and different from how disk diffusion is read with most other antimicrobials and species. The susceptible isolates sharp zones (Fig. 2A) are very characteristic when comparing them to the fuzzy zones. Thus, positive and negative controls for comparisons eases the evaluation of low-level resistant VRE. Moreover, the CLSI agar screen method also requires experienced personnel that can prepare and store the agar plates correctly so that the vancomycin concentration in the plates is accurate. It is well known that VanB-type VRE can be difficult to detect due to the inducible mechanism of resistance and the variable levels of vancomycin MIC expressed (12, 37). The strain collection was designed to be genetically and epidemiologically diverse and include troublesome isolates with low vancomycin MIC in addition to ATCC reference strains (Table 1). The vanb E. faecalis ATCC and E. faecalis ATCC strains are recommended as a positive and negative quality controls, respectively, both for the EUCAST disk diffusion (34) and CLSI agar screen (38). Twenty-one of the 28 laboratories in this study reported to use vanb E. faecalis ATCC while 5 laboratories used various CCUG VanB-positive enterococcal strains as a positive control. A previous evaluation of the vanb E. faecalis ATCC strain showed a vancomycin MIC of mg/l after 24 h incubation. However, after 13

14 h a vancomycin MIC of 128 mg/l was observed (38). A valid positive control is supposed to challenge the detection limits of the method. In this study the vanb E. faecalis ATCC was included in three copies in the blinded test collection. According to the disk diffusion zone sizes recorded in the different laboratories this reference strain does not seem to challenge the test conditions. All laboratories were able to place this isolate well within the resistant category with zone diameters ranging from 6-9 mm. A more relevant reference strain with a lower inducible vancomycin MIC should be considered for quality control purposes. In conclusion, our results demonstrate acceptable performance by both the EUCAST disk diffusion and the CLSI agar screen method. Both reliably detect low-level vanb VRE. The high ME and lower specificity of commercial chromogenic VRE agars indicate that their potential use in screening of clinical strains for vancomycin resistance would results in a high number of false positive results. However, confirmation of the ME and specificity calls for another study with a higher number of vancomycin susceptible strains included within the strain collection. Importantly, the use of agar disk diffusion method requires trained personnel in the interpretation of zone edges and use of the CLSI agar screen methods requires careful selection of control strains for monitoring the vancomycin concentration in the plates ACKNOWLEDGEMENT We thank Bettina Aasnæs and Tracy Munthali Lunde for excellent technical assistance. The study was performed in collaboration with the diagnostic laboratories in clinical microbiology in Norway, Sweden and Denmark forming the NordicAST VRE detection Study Group. Representatives of this study group included Kirsten Paulsen (Aalborg Hospital), Lars Erik Lemming (Aarhus University Hospital), Siri Haug Hänsgen (Akershus University Hospital), Marianne Bäckman (Aleris Medilab), Jenny Åhman (EUCAST Laboratory for AST), Thomas 14

15 Ahlqvist (Central Hospital Karlstad), Elisabeth Sirnes (Central Hospital Førde), Ann Cathrine Petersson (Dept Clinical Mircobiology, Laboratory Medicine Lund), Håkan Janson (Dept Clinical Mircobiology, Laboratory Medicine Malmö), Ingegerd Sjögren (Hallands Hospital Halmstad), Kristin Stenhaug Kilhus (Haukeland University Hospital), Magnus Arpi (Herlev Hospital), Inger Brock (Hillerød Hospital), Pia Littauer (Hvidovre Hospital), Sara Gustavsson (Kalmar County Hospital), Hong Fang (Karolinska University Hospital Huddinge), Kirsti Jalakas Pörnull (Karolinska University Hospital Solna), Lennart E. Nilsson (Linköping University Hospital), Margreet Boer (Molde Hospital), Hege Elisabeth Larsen (Nordland Hospital Bodø), Anette Holm (Odense University Hospital), Pia Langseth (Rikshospitalet University Hospital), Ia Adlerberth (Sahlgrenska University Hospital), Ole Heltberg (Slagelse Hospital), Anette M. Hammerum (Statens Serum Institute), Anita Løvås Brekken (Stavanger University Hospital), Kjersti Wik Larssen (St. Olavs Hospital), Dagfinn Skaare (Vestfold Hospital), Anita Kanestrøm (Østfold Hospital), Ståle Tofteland (Sørlandet Hospital), Thea Bergheim (Ullevål University Hospital), Gunnar Skov Simonsen (University Hospital of North-Norway), Angela Lagerqvist Vidh (Uppsala University Hospital), and Claus Østergaard (Velje Hospital). 15

16 REFERENCES 1. Werner, G., T. M. Coque, C. M. Franz, E. Grohmann, K. Hegstad, L. Jensen, W. van Schaik, and K. Weaver Antibiotic resistant enterococci - Tales of a drug resistance gene trafficker. Int. J. Med. Microbiol. 303: Gilmore, M. S., F. Lebreton, and W. van Schaik Genomic transition of enterococci from gut commensals to leading causes of multidrug-resistant hospital infection in the antibiotic era. Curr. Opin. Microbiol. 16: de Kraker, M. E., V. Jarlier, J. C. Monen, O. E. Heuer, N. van de Sande, and H. Grundmann The changing epidemiology of bacteraemias in Europe: trends from the European Antimicrobial Resistance Surveillance System. Clin. Microbiol. Infect. 19: Gilmore, M. S. and J. J. Ferretti Microbiology. The thin line between gut commensal and pathogen. Science 299: Zhang, X., J. Top, B. M. de, D. Bierschenk, M. Rogers, M. Leendertse, M. J. Bonten, T. van der Poll, R. J. Willems, and W. van Schaik Identification of a genetic determinant in clinical Enterococcus faecium strains that contributes to intestinal colonization during antibiotic treatment. J. Infect. Dis. 207: Arias, C. A., G. A. Contreras, and B. E. Murray Management of multi-drug resistant enterococcal infections. Clin. Microbiol. Infect. 16: Hegstad, K., T. Mikalsen, T. M. Coque, L. B. Jensen, G. Werner, and A. Sundsfjord Mobile genetic elements and their contribution to the emergence of antimicrobial resistant Enterococcus faecalis and E. faecium. Clin. Microbiol. Infect. 16:

17 Xu, X., D. Lin, G. Yan, X. Ye, S. Wu, Y. Guo, D. Zhu, F. Hu, Y. Zhang, F. Wang, G. A. Jacoby, and M. Wang vanm, a new glycopeptide resistance gene cluster found in Enterococcus faecium. Antimicrob. Agents Chemother. 54: Lebreton, F., F. Depardieu, N. Bourdon, M. Fines-Guyon, P. Berger, S. Camiade, R. Leclercq, P. Courvalin, and V. Cattoir D-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium. Antimicrob. Agents Chemother. 55: Werner, G., T. M. Coque, A. M. Hammerum, R. Hope, W. Hryniewicz, A. Johnson, I. Klare, K. G. Kristinsson, R. Leclercq, C. H. Lester, M. Lillie, C. Novais, B. Olsson-Liljequist, L. V. Peixe, E. Sadowy, G. S. Simonsen, J. Top, J. Vuopio- Varkila, R. J. Willems, W. Witte, and N. Woodford Emergence and spread of vancomycin resistance among enterococci in Europe. Euro. Surveill. 13: Granlund, M., C. Carlsson, H. Edebro, K. Emanuelsson, and R. Lundholm Nosocomial outbreak of vanb2 vancomycin-resistant Enterococcus faecium in Sweden. J. Hosp. Infect. 62: Werner, G., I. Klare, C. Fleige, U. Geringer, W. Witte, H. M. Just, and R. Ziegler Vancomycin-resistant vanb-type Enterococcus faecium isolates expressing varying levels of vancomycin resistance and being highly prevalent among neonatal patients in a single ICU. Antimicrob. Resist. Infect. Control 1: Bourdon, N., M. Fines-Guyon, J. M. Thiolet, S. Maugat, B. Coignard, R. Leclercq, and V. Cattoir Changing trends in vancomycin-resistant enterococci in French hospitals, J. Antimicrob. Chemother. 66: Söderblom, T., O. Aspevall, M. Erntell, G. Hedin, D. Heimer, I. Hökeberg, K. Kidd-Ljunggren, Å. Melhus, B. Olsson-Liljequist, I. Sjögren, J. Smedjegård, J. 17

18 Struwe, S. Sylvan, K. Tegmark-Wisell, and M. Thore Alarming spread of vancomycin resistant enterococci in Sweden since Euro. Surveill 15:pii= Bjørkeng, E. K., G. Rasmussen, A. Sundsfjord, L. Sjöberg, K. Hegstad, and B. Söderquist Clustering of polyclonal VanB-type vancomycin-resistant Enterococcus faecium in a low-endemic area was associated with CC17-genogroup strains harbouring transferable vanb2-tn5382 and prum-like repa containing plasmids with axe-txe plasmid addiction systems. APMIS 119: Johnson, P. D., S. A. Ballard, E. A. Grabsch, T. P. Stinear, T. Seemann, H. L. Young, M. L. Grayson, and B. P. Howden A sustained hospital outbreak of vancomycin-resistant Enterococcus faecium bacteremia due to emergence of vanb E. faecium sequence type 203. J. Infect. Dis. 202: Dahl, K. H., G. S. Simonsen, Ø. Olsvik, and A. Sundsfjord Heterogeneity in the vanb gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci. Antimicrob. Agents Chemother. 43: Gold, H. S., S. Unal, E. Cercenado, C. Thauvin Eliopoulos, G. M. Eliopoulos, C. B. Wennersten, and R. C. Moellering, Jr A gene conferring resistance to vancomycin but not teicoplanin in isolates of Enterococcus faecalis and Enterococcus faecium demonstrates homology with vanb, vana, and vanc genes of enterococci. Antimicrob. Agents Chemother. 37: Patel, R., J. R. Uhl, P. Kohner, M. K. Hopkins, J. M. Steckelberg, B. Kline, and F. R. 3. Cockerill DNA sequence variation within vana, vanb, vanc-1, and vanc- 2/3 genes of clinical Enterococcus isolates. Antimicrob. Agents Chemother. 42: Courvalin, P Vancomycin resistance in Gram-positive cocci. Clin. Infect. Dis. 42 (suppl 1):S25-S34. 18

19 Boyce, J. M., S. M. Opal, J. W. Chow, M. J. Zervos, G. Potter-Bynoe, C. B. Sherman, R. L. Romulo, S. Fortna, and A. A. Medeiros Outbreak of multidrug-resistant Enterococcus faecium with transferable vanb class vancomycin resistance. J. Clin. Microbiol. 32: The European Committee on Antimicrobial Susceptibility Testing Breakpoint tables for interpretation of MICs and zone diameters. Version 3.1: (last access ). 23. Hayden, M. K., G. M. Trenholme, J. E. Schultz, and D. F. Sahm In vivo development of teicoplanin resistance in a VanB Enterococcus faecium isolate. J. Infect. Dis. 167: Kawalec, M., M. Gniadkowski, J. Kedzierska, A. Skotnicki, J. Fiett, and W. Hryniewicz Selection of a teicoplanin-resistant Enterococcus faecium mutant during an outbreak caused by vancomycin-resistant enterococci with the vanb phenotype. J. Clin. Microbiol. 39: San Millan, A., F. Depardieu, S. Godreuil, and P. Courvalin VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin. Antimicrob. Agents Chemother. 53: Holmes, N. E., S. A. Ballard, M. M. Lam, P. D. Johnson, M. L. Grayson, T. P. Stinear, and B. P. Howden Genomic analysis of teicoplanin resistance emerging during treatment of vanb vancomycin-resistant Enterococcus faecium infections in solid organ transplant recipients including donor-derived cases. J. Antimicrob. Chemother. 68:

20 Dutka-Malen, S., S. Evers, and P. Courvalin Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33: Fines, M., B. Perichon, P. Reynolds, D. F. Sahm, and P. Courvalin VanE, a new type of acquired glycopeptide resistance in Enterococcus faecalis BM4405. Antimicrob. Agents Chemother. 43: Depardieu, F., B. Perichon, and P. Courvalin Detection of the van alphabet and identification of enterococci and staphylococci at the species level by multiplex PCR. J. Clin. Microbiol. 42: Dahl, K. H., E. W. Lundblad, T. P. Røkenes, Ø. Olsvik, and A. Sundsfjord Genetic linkage of the vanb2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences. Microbiol. 146: International Standards Organisation Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. ISO Dahl, K. H., T. P. Røkenes, E. W. Lundblad, and A. Sundsfjord Nonconjugative transposition of the vanb-containing Tn5382-like element in Enterococcus faecium. Antimicrob. Agents Chemother. 47: Harthug, S., A. Digranes, O. Hope, B. E. Kristiansen, A. G. Allum, and N. Langeland Vancomycin resistance emerging in a clonal outbreak caused by ampicillin-resistant Enterococcus faecium. Clin. Microbiol. Infect. 6: The European Committee on Antimicrobial Susceptibility Testing EUCAST Disk Diffusion Test Manual. Version 3.0: 20

21 (last access ). 35. Swenson, J. M., N. C. Clark, M. J. Ferraro, D. F. Sahm, G. Doern, M. A. Pfaller, L. B. Reller, M. P. Weinstein, R. J. Zabransky, and F. C. Tenover Development of a standardized screening method for detection of vancomycin-resistant enterococci. J. Clin. Microbiol 32: Klare, I., C. Fleige, U. Geringer, W. Witte, and G. Werner Performance of three chromogenic VRE screening agars, two Etest((R)) vancomycin protocols, and different microdilution methods in detecting vanb genotype Enterococcus faecium with varying vancomycin MICs. Diagn. Microbiol. Infect. Dis. 74: Grabsch, E. A., K. Chua, S. Xie, J. Byrne, S. A. Ballard, P. B. Ward, and M. L. Grayson Improved detection of vanb2-containing Enterococcus faecium with vancomycin susceptibility by Etest using oxgall supplementation. J. Clin. Microbiol 46: Swenson, J. M., N. C. Clark, D. F. Sahm, M. J. Ferraro, G. Doern, J. Hindler, J. H. Jorgensen, M. A. Pfaller, L. B. Reller, M. P. Weinstein, and Molecular characterization and multilaboratory evaluation of Enterococcus faecalis ATCC for quality control of screening tests for vancomycin and high-level aminoglycoside resistance in enterococci. J. Clin. Microbiol 33: Kim, E. B., L. M. Kopit, L. J. Harris, and M. L. Marco Draft genome sequence of the quality control strain Enterococcus faecalis ATCC J. Bacteriol. 194: Depardieu, F., M. G. Bonora, P. E. Reynolds, and P. Courvalin The vang glycopeptide resistance operon from Enterococcus faecalis revisited. Mol. Microbiol. 50:

22 Carias, L. L., S. D. Rudin, C. J. Donskey, and L. B. Rice Genetic linkage and cotransfer of a novel, vanb-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate. J. Bacteriol. 180: Rosvoll, T. C., T. Pedersen, H. Sletvold, P. J. Johnsen, J. E. Sollid, G. S. Simonsen, L. B. Jensen, K. M. Nielsen, and A. Sundsfjord PCR-based plasmid typing in Enterococcus faecium strains reveals widely distributed pre25-, prum-, pip501- and phtbeta-related replicons associated with glycopeptide resistance and stabilizing toxinantitoxin systems. FEMS Immunol. Med. Microbiol. 58: Nallapareddy, S. R., H. Wenxiang, G. M. Weinstock, and B. E. Murray Molecular characterization of a widespread, pathogenic, and antibiotic resistancereceptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island. J. Bacteriol. 187:

23 TABLE 1 Isolate ID, species, van genotype, gradient test and broth microdilution (BMD) MIC in mg/l for vancomycin as well as PFGE and MLST types of the blinded material used in this study Isolate ID Species Origin van Etest BMD MIC PFGE types b MLST types References no. subtype MIC VAN a VAN 1 K09-03 / V1-38 E. faecium Norway B I This study 2 K25-21 E. faecium Norway B II This study 3 K26-39 E. faecium Norway B III This study 4 K30-42 E. faecium Norway B2 8 4 IV This study 5 K33-53 E. faecium Norway B V This study 6 K46-59 E. faecium Norway B2 8 8 VI This study 7 ATCC E. faecalis Neg 4 2 VII Efs ST30 This study (39) 8 ATCC E. faecalis B VIII This study 9 K53-60 E. faecalis Norway B IX This study 10 K57-77 E. faecium Norway B X This study 11 K61-59 E. faecium Norway B2 8 8 XI This study 12 K55-34 (Ekkr0683) E. faecium Stockholm, Sweden B XIIa 192 (DLV 17) Sivertsen et al., submitted 13 K55-41 (Ekkr0881) E. faecium Västerås, Sweden B XIIb 17 Sivertsen et al., submitted 14 A2-46 (BM4518) E. faecalis Australia G XIII This study (40) 15 K55-41 (Ekkr0881) E. faecium Västerås, Sweden B XIIb 17 Sivertsen et al., submitted 16 ATCC E. faecalis Neg 4 2 VII Efs ST30 This study (39) 17 ATCC E. faecalis B VIII This study 18 A2-48 (BM4405) E. faecalis US E XIV This study (28) 19 TUH 12-1 (C68) E. faecium US B XV 16 (SLV 17) (41, 42) 20 K55-41 (Ekkr0881) E. faecium Västerås, Sweden B XIIb 17 Sivertsen et al., submitted 21 K45-3 (03T468) E. faecium Örebro, Sweden B XVI 262 (SLV 18) (15) 22 K45-12 (02T895) E. faecium Örebro, Sweden B XVII 17 (15) 23 TUH 1-3 (V583) E. faecalis US B1 32 XVIII Efs ST14 (17, 43) 24 TUH 1-79 E. faecium Norway B XIX (17) 25 TUH 2-18 E. faecium Bergen, Norway B2 32 XX 17 (17, 42) 26 TUH 4-65 E. faecium US B XXI 313 (SLV ST18) (17, 42) 27 TUH 7-13 E. faecalis US B XXII (17) 28 ATCC E. faecalis Neg 4 2 VII Efs ST30 This study (39) 29 ATCC E. faecalis B VIII This study KRE E. faecalis Norway B2 4 8 XXIII This study 23

24 a VAN vancomycin b Previous PFGE type designations have been changed to consecutive roman numbering in this study for pedagogic reasons 24

25 TABLE 2 Number of very major (VME) and major errors (ME), sensitivity and specificity calculated for each detection method as well as for each type of agar used for n 5 Method (n=no. of labs) No. VME (%) Sensitivity (95% CI) No. ME (%) Specificity (95% CI) EUCAST disk diffusion (n=28) Oxoid MH (n=16) BBL MH II (n=10) 53/756 (7.0%) 14/432 (3.2%) 37/270 (14%) 0.93 (0.91, 0.95) 0.97 (0.94, 0.98) 0.86 (0.81, 0.90) 2/84 (2.4%) 0/48 2/30 (6.7%) 0.98 (0.91, 1) 1 (0.91, 1) 0.93 (0.76, 0.99) CLSI agar screen (n=18) BHI Difco (n=8) BHI Oxoid (n=5) 32/486 (6.6%) 9/216 (4.2%) 15/135 (11%) 0.93 (0.91, 0.95) 0.96 (0.92, 0.98) 0.89 (0.82, 0.93) 3/54 (5.6%) 0/24 0/ (0.84, 0.99) 1 (0.83, 1) 1 (0.75, 1) Chromogenic agar screen (n=12) chromid VRE (n=7) VRE CHROMagar (n=5) 7/324 (2.2%) 3/189 (1.6%) 4/135 (3.0%) 0.98 (0.95, 0.99) 0.98 (0.95, 1) 0.97 (0.92, 0.99) 4/36 (11%) 1/21 (4.8%) 3/15 (20%) 0.89 (0.73, 0.96) 0.95 (0.74, 1) 0.8 (0.51, 0.95) VITEK 2 (n=5) 18/135 (13%) 0.87 (0.79, 92) 0/15 (0%) 1 (0.75, 1) VME; classified as S when containing van genotype, ME; classified as R when containing no van genotype. According to EUCAST rules for disk diffusion with vancomycin the sample was considered R even though the zone size suggested S if the zone edge was fuzzy or colonies grew within the zone. 25

26 TABLE 3 Mean, median, lowest and highest numbers of errors per laboratory for each method Method (n=no. of labs) No. VME/lab No. ME/lab Number Mean Median Lowest Highest Mean Median Lowest Highest EUCAST disk diffusion (n=28) CLSI agar screen (n=18) VITEK2 (n=5) 3.2 2, VME; classified as S when containing van genotype, ME; classified as R when containing no van genotype. The median number is the middle number in the numerically sorted list of numbers

27 TABLE 4 Number of very major (VME) and major errors (ME), sensitivity and specificity calculated for EUCAST disk diffusion and CLSI agar screen divided into laboratories country origin or the laboratories experience in using the detection methods 534 Method Disk diffusion Country/ experience (no. of labs) Sweden (10) Norway (13) Denmark (5) Experienced (13) Inexperienced (15) Agar screen Sweden (1) Norway (13) Denmark (4) Experienced (13) Inexperienced (5) VME in % (No. VME/ total number R) 1.9 (5/270) 10 (36/351) 8.9 (12/135) 2.6 (9/351) 11 (44/405) 3.7 (1/27) 6.8 (24/351) 6.5 (7/108) 6.8 (24/351) 5.9 (8/135) Sensitivity (95% CI) 0.98 (0.95, 0.99) 0.90 (0.86, 0.93) 0.91 (0.85, 0.95) 0.97 (0.95, 0.99) 0.89 (0.86, 0.92) 0.9 (0.79, 1) 0.93 (0.90, 0.95) 0.94 (0.87, 0.97) 0.93 (0.90, 0.95) 0.94 (0.88, 0.97) ME in % (No. ME/ total number S) 0 (0/30) 2.6 (1/39) 6.7 (1/15) 0 (0/39) 4.4 (2/45) 0 (0/3) 0 (0/39) 25 (3/12) 0 (0/39) 20 (3/15) Specificity (95% CI) 1 (0.86, 1) 0.97 (0.85, 1) 0.93 (0.66, 1) 1 (0.89, 1) 0.96 (0.84, 0.99) 1 (0.31, 1) 1 (0.89, 1) 0.75 (0.43, 0.93) 1 (0.89, 1) 0.8 (0.51, 0.95) 535 VME; classified as S when containing van genotype, ME; classified as R when containing no van genotype

28 FIG 1 MIC distribution of the collection of blinded isolates (n=30) FIG 2 Examples of disk diffusion inhibition zones for Enterococcus spp. with 5 μg vancomycin disk. A) Sharp zone edge and zone diameter 12 mm should be reported as susceptible. B-D) Fuzzy zone edge or colonies within zone which should be reported as resistant even if the zone diameter is 12 mm FIG 3 Average zone mm including standard deviation calculated for each isolate from the laboratories using either Oxoid MH (white bars) or BBL MH II agar (black bars) recorded results

29 14 12 Number of isolates MIC Etest MIC broth microdilu on MIC (mg/l)

30 A B C D

31 Zone diameter (mm) Isolate number