Geoffrey W Coombs, Denise A Daley, Yung Thin Lee, Stanley Pang, Julie C Pearson, J Owen Robinson, Paul DR Johnson, Despina Kotsanas, Jan M Bell, John D Turnidge for the Australian Group on Antimicrobial Resistance
From 1 January to 31 December 2014, 27 institutions around Australia participated in the Australian Enterococcal Sepsis Outcome Programme (AESOP). The aim of AESOP 2014 was to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial resistant, and to characterise the molecular epidemiology of the Enterococcus faecium isolates. Of the 952 unique episodes of bacteraemia investigated, 94.4% were caused by either E. faecalis (54.9%) or E. faecium (39.9%). Ampicillin resistance was detected in 0.6% of E. faecalis and in 89.4% of E. faecium. Vancomycin non-susceptibility was reported in 0.2% and 46.1% of E. faecalis and E. faecium respectively. Overall 51.1% of E. faecium harboured vanA or vanB genes. For the vanA/B positive E. faecium isolates, 81.5% harboured vanB genes and 18.5% vanA genes. The percentage of E. faecium bacteraemia isolates resistant to vancomycin in Australia is significantly higher than that seen in most European countries. E. faecium consisted of 113 pulsed-field gel electrophoresis pulsotypes of which 68.9% of isolates were classified into 14 major pulsotypes containing 5 or more isolates. Multilocus sequence typing grouped the 14 major pulsotypes into clonal cluster 17, a major hospital-adapted polyclonal E. faecium cluster. The geographical distribution of the 4 predominant sequence types (ST203, ST796, ST555 and ST17) varied with only ST203 identified across most regions of Australia. Overall 74.7% of isolates belonging to the four predominant STs harboured vanA or vanB genes. In conclusion, the AESOP 2014 has shown enterococcal bacteraemias in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin resistant vanA or vanB E. faecium, which have limited treatment options. Commun Dis Intell 2016;40(2):E236–E243.
Keywords: antimicrobial resistance surveillance, Enterococcus faecium, Enterococcus faecalis, vancomycin resistant enterococci, bacteraemia
Globally enterococci are thought to account for approximately 10% of all bacteraemias, and in North America and Europe are the 4th and 5th leading causes of sepsis respectively.1,2 Although in the 1970s healthcare-associated enterococcal infections were primarily due to Enterococcus faecalis, there has been a steadily increasing prevalence of E. faecium nosocomial infections.3–5 Worldwide, the increase in nosocomial E. faecium infections has primarily been due to the expansion of polyclonal hospital-adapted clonal complex (CC) 17 strains. While innately resistant to many classes of antibiotics, E. faecium has demonstrated a remarkable capacity to evolve new antimicrobial resistances. In 2009, the Infectious Diseases Society of America highlighted E. faecium as one of the key problem bacteria or ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens requiring new therapies.6
The Australian Group on Antimicrobial Resistance (AGAR) is a network of laboratories located across Australia that commenced surveillance of antimicrobial resistance in Enterococcus species in 1995.7 In 2011, AGAR commenced the Australian Enterococcal Sepsis Outcome Programme (AESOP).8 The objective of AESOP 2014 was to determine the proportion of E. faecalis and E. faecium bacteraemia isolates demonstrating antimicrobial resistance with particular emphasis on:
- assessing susceptibility to ampicillin;
- assessing susceptibility to glycopeptides; and
- molecular epidemiology of E. faecium.
Twenty-seven laboratories from all 8 Australian states and territories participated in 2014.
From 1 January to 31 December 2014, the 27 laboratories collected all enterococcal species isolated from blood cultures. Enterococci with the same species and antimicrobial susceptibility profiles isolated from a patient’s blood culture within 14 days of the first positive culture were excluded. A new enterococcal sepsis episode in the same patient was recorded if it was confirmed by a further culture of blood taken more than 14 days after the initial positive culture. Data were collected on age, sex, date of admission and discharge (if admitted), and mortality at 30 days from date of blood culture collection. To avoid interpretive bias, no attempt was made to assign attributable mortality. Each episode of bacteraemia was designated as ‘hospital onset’ if the first positive blood culture(s) in an episode was collected more than 48 hours after admission.
Enterococcal isolates were identified to the species level by the participating laboratories using one of the following methods: API 20S (bioMérieux), API ID32Strep (bioMérieux), Vitek2® (bioMérieux), Phoenix™ (BD), matrix-assisted laser desorption ionization (MALDI) Biotyper (Bruker Daltonics), Vitek-MS (bioMérieux), polymerase chain reaction (PCR), or conventional biochemical tests. Antimicrobial susceptibility testing was performed by using the Vitek2® (bioMérieux, France) or the Phoenix™ (BD, USA) automated microbiology systems according to the manufacturer’s instructions. Minimum inhibitory concentration (MIC) data and isolates were referred to the Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research. Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were utilised for interpretation.9,10 Isolates with either a resistant or an intermediate category were classified as non-susceptible. Linezolid and daptomycin non-susceptible isolates and selected vancomycin susceptible isolates were retested by Etest® (bioMérieux, France) using the Mueller-Hinton agar recommended by the manufacturer. E. faecalis ATCC® 29212 was used as the control strain. Molecular testing including vanA/B PCR, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) was performed as previously described.11–13
A chi-square test for comparison of 2 proportions was performed and 95% confidence intervals (95% CI) were determined using MedCalc for Windows, version 12.7 (Medcalc Software, Ostend Belgium).
Approval to conduct the prospective data collection was given by the research ethics committee associated with each participating laboratory.
From 1 January to 31 December 2014, 952 unique episodes of enterococcal bacteraemia were identified. Although 10 Enterococcus species were identified, 54.9% (523 isolates) were E. faecalis and 39.9% (380 isolates) were E. faecium. Forty-nine enterococci were identified either as E. casseliflavus (19 isolates), E. gallinarum (13), E. avium (9), E. hirae (2) E. raffinosus (3), E. durans (1), E. cecorum (1), and E. mundtii (1).
A significant difference was seen in patient sex (P < 0.0001) with 613 (64.4%) being male (95% CI, 61.4–67.5). The average age of patients was 63 years ranging from 0 to 100 years with a median age of 67 years. Of the 952 episodes, 474 (49.8%) were hospital onset (95% CI, 46.5–52.9). However, a significant difference was seen between E. faecium and E. faecalis, with 71.8% (95% CI, 67.0–76.3) of E. faecium episodes being hospital onset compared with 36.5% (95% CI, 32.4–40.8) for E. faecalis (P < 0.0001). All-cause mortality at 30 days was 18.5% (95% CI, 15.9–21.3). There was a significant difference in mortality between E. faecalis and E. faecium episodes (13.2% [95% CI, 10.3–16.6] vs 27.6% [95% CI, 22.9–32.7] respectively, P < 0.0001) and between vancomycin susceptible and vancomycin non-susceptible E. faecium episodes (22.8% [95% CI, 16.9–29.6] vs 32.9% [95% CI, 25.7–40.7] respectively, P = 0.05).
Enterococcus faecalis phenotypic susceptibility
Apart from erythromycin, tetracycline, ciprofloxacin and high-level gentamicin, acquired resistance was rare among E. faecalis (Table 1). Ampicillin resistance was detected in 3 isolates and only 1 isolate was vancomycin non-susceptible. Thirty-six (6.9%) E. faecalis, were initially reported as linezolid non-susceptible (CLSI breakpoint > 2 mg/L). However by Etest®, 22 of the 35 isolates available for MIC testing by Etest® had a linezolid MIC of ≤ 2 mg/L and were therefore considered linezolid susceptible. Thirteen isolates with an MIC of 4 mg/L, although non-susceptible by CLSI guidelines, were considered susceptible by EUCAST guidelines. Eight (1.6%) isolates were initially reported non-susceptible to daptomycin (CLSI and EUCAST breakpoint > 4 mg/L). However by Etest®, 7 of the 8 isolates had an MIC of < 4 mg/L and were therefore considered susceptible. One isolate had an MIC of 8 mg/L, which was considered non-susceptible. All isolates were susceptible to teicoplanin.
* Clinical and Laboratory Standards Institute (CLSI) non-susceptible breakpoint.
† European Committee on Antimicrobial Susceptibility Testing (EUCAST) non-susceptible breakpoint.
‡ CLSI and EUCAST non-susceptible breakpoint.
|High level gentamicin||519||>128*||198||38.2|
Enterococcus faecium phenotypic susceptibility
The majority of E. faecium were non-susceptible to multiple antimicrobials (Table 2). Most isolates were non-susceptible to ampicillin, erythromycin, tetracycline, ciprofloxacin, nitrofurantoin and high-level gentamicin. Overall, 175 (46.1%) were phenotypically vancomycin non-susceptible (MIC > 4 mg/L). Thirty-one (8.2%) and 33 (8.8%) isolates were teicoplanin non-susceptible by CLSI and EUCAST guidelines respectively. Nine (2.4%) isolates were initially reported as linezolid non-susceptible (CLSI breakpoint > 2 mg/L). However by Etest®, 7 of the 9 isolates had a linezolid MIC of ≤ 2 mg/L. One isolate had an MIC of 4 mg/L, which was considered susceptible by EUCAST guidelines but non-susceptible by CLSI guidelines. One isolate had an MIC of 8 mg/L, which was considered non-susceptible by CLSI and EUCAST guidelines.
* Clinical and Laboratory Standards Institute (CLSI) non-susceptible breakpoint.
† European Committee on Antimicrobial Susceptibility Testing (EUCAST) non-susceptible breakpoint.
‡ CLSI and EUCAST non-susceptible breakpoint.
|High level gentamicin||377||>128*||233||61.5|
Genotypic vancomycin susceptibility
VanA/vanB PCR was performed on 512 of the 523 E. faecalis isolates. Overall, 7 (1.4%) of the 512 isolates harboured a vanA or vanB gene. The vancomycin non-susceptible E. faecalis isolate (Vitek® vancomycin MIC ≥ 32 mg/L) harboured a vanB gene. One phenotypically vancomycin/teicoplanin susceptible isolate (Vitek® vancomycin MIC = 1 mg/L, teicoplanin MIC = ≤ 0.5 mg/L) harboured vanA. A further 5 phenotypically vancomycin susceptible E. faecalis isolates (Vitek® vancomycin MIC = 1) harboured vanB genes.
VanA/B PCR was performed on 370 of the 380 E. faecium isolates, including 171 of the 175 vancomycin non-susceptible isolates and 199 of the 205 vancomycin susceptible isolates. Overall, 189 (51.1%) of the 370 isolates harboured a vanA or vanB gene.
Thirty-one of the vancomycin non-susceptible E. faecium isolates harboured vanA (Vitek® vancomycin MIC = 8 mg/L [1 isolate] and > 16 mg/L [30 isolates]). A further 140 E. faecium vancomycin non-susceptible isolates harboured vanB (Vitek® vancomycin MIC = 8 mg/L [2 isolates] and > 16 mg/L [135 isolates]).
VanA or vanB genes were detected in 18 vancomycin susceptible E. faecium isolates. Four isolates harboured vanA (Vitek® vancomycin MIC ≤ 0.5 mg/L [2 isolates], MIC = 1 mg/L [1 isolate] and MIC = 2 mg/L [1 isolate], teicoplanin ≤ 1 mg/L [4 isolates]). Fourteen isolates harboured vanB (Vitek® vancomycin MIC ≤ 0.5 mg/L [7 isolates], MIC = 1 mg/L [6 isolates] and MIC = 2 mg/L [1 isolate]).
Of the 154 vanB E. faecium isolates, 3 were teicoplanin resistant (MIC > 32 mg/L).
Enterococcus faecium molecular epidemiology
Of the 380 episodes, 369 E. faecium isolates were available for typing. By PFGE, 367 isolates were classified into 113 pulsotypes, including 14 major pulsotypes with 5 or more isolates (Table 3). Two isolates were not typable by PFGE. Of the 99 pulsotypes with less than 5 isolates, 90 had only 1 isolate. Overall 253 (68.9%) of the 367 isolates were grouped into the 14 major pulsotypes from which 9 multilocus sequence types (STs) were identified. Using eBURST, the 9 STs were grouped into CC 17.
ND Not done.
Geographical distribution of the 9 STs varied (Table 3). For the 4 most prominent STs, ST203 (69 isolates) was identified across most of Australia, ST796 (65 isolates) primarily in Victoria, ST555 (45 isolates) primarily in South Australia and Western Australia and ST17 (34 isolates) primarily in New South Wales. For the remaining 5 STs, ST117 (16 isolates) was found in New South Wales, Queensland and the Australian Capital Territory, ST 761 (7 isolates) in New South Wales and Queensland, ST192 (6 isolates) in Victoria and Tasmania, ST80 (7 isolates) in New South Wales and Western Australia and ST341 (5 isolates) in New South Wales and the Australian Capital Territory.
VanA was detected in two major pulsotypes (29 isolates, Efm18 and Efm85), and vanB in 8 major pulsotypes (137 isolates, Efm1, Efm2, Efm3, Efm18, Efm74, Efm75, Efm76, Efm77) (Table 4). Efm18 (ST17) harboured vanA and vanB genes. Twelve minor pulsotypes (14 isolates) also harboured vanB genes. In addition, vanA genes were detected in 6 minor pulsotypes (6 isolates).
Enterococci are intrinsically resistant to a broad range of antimicrobials including the cephalosporins and sulphonamides. Due to their ability to acquire additional resistance through the transfer of plasmids and transposons and to disseminate easily in the hospital environment, enterococci have become difficult to treat and provide major infection control challenges.
As the AGAR programs are similar to those conducted in Europe14 comparison of Australia antimicrobial resistance data with other countries is possible.
In the 2013 European Centre for Disease Prevention and Control and Prevention (ECDC) Enterococci surveillance program the European Union/European Economic Area (EU/EEA) population-weighted mean percentage of E. faecium resistant to vancomycin was 8.9% (95% CI, 7–12), ranging from 0.0% (95% CI, 0–9) in Estonia, Lithuania, Malta and Sweden to 42.7% (95% CI, 38–48) in Ireland. Cyprus (23.3%), United Kingdom (23.3%), Portugal (22.0%) and Greece (21.2%) were the only other EU/EEA countries to report above 20%.15
In AESOP 2014 approximately 40% of enterococcal bacteraemia were due to E. faecium, of which 46.1% (95% CI, 41.0–51.2) were phenotypically vancomycin non-susceptible by Vitek2® or Phoenix™. However, 51.1% of E. faecium isolates tested (189/370) harboured vanA/vanB genes, of which 81.5% were vanB. Overall, 9.5% (35/370) of E. faecium isolates harboured a vanA gene, which is a significant increase from the 2.6% (8/310) of isolates reported in AESOP 2013 (P = 0.0005).16 The majority of E. faecium isolates were also non-susceptible to multiple antimicrobials, including ampicillin, erythromycin, tetracycline, ciprofloxacin and high level gentamicin. In AESOP 201117 and 2013,16 37.0% and 48.6% of E. faecium harboured vanA/vanB respectively confirming the incidence of vancomycin resistant E. faecium bacteraemia in Australia is increasing.
Fourteen (9.1%) of the 154 vanB E. faecium isolates had a vancomycin MIC at or below the CLSI and the EUCAST susceptible breakpoint (≤ 4 mg/L) and would not have been identified using routine phenotypic antimicrobial susceptibility methods. Furthermore, 6 vanA/B E. faecalis were also phenotypically vancomycin susceptible (MIC 1 mg/L).
By PFGE, E. faecium was shown to be very polyclonal, consistent with the known plasticity of the enterococcal genome. The 14 major E. faecium pulsotypes formed part of CC17, a global hospital-derived lineage that has successfully adapted to hospital environments. CC17 is characteristically ampicillin and quinolone resistant and subsequent acquisition of vanA– or vanB-containing transposons by horizontal transfer in CC17 clones has resulted in vancomycin resistant enterococci with pandemic potential. In AESOP 2014, 4 E. faecium STs predominated: ST203 (of which 77% of isolates harboured vanB genes); ST796 (100% harboured vanB); ST555 (37% harboured vanB); and ST17 (71% harboured vanA and 3% harboured vanB). Two minor PFGE pulsotypes identified in AESOP 2013 have become major pulsotypes in AESOP 2014: Efm80–ST117 (16 isolates) found in New South Wales (11 isolates), the Australian Capital Territory (4 isolates) and Queensland (1 isolate) and Efm85–ST80 (6 isolates), in New South Wales (5 isolates) and Western Australia (1 isolate). The majority of Efm85–ST80 isolates harboured vanA genes.
The AESOP 2014 study has shown that although predominately caused by E. faecalis, enterococcal bacteraemia in Australia is frequently caused by ampicillin-resistant high-level gentamicin-resistant vanB E. faecium. Furthermore, the percentage of E. faecium bacteraemia isolates resistant to vancomycin in Australia is significantly higher than that seen in almost all European countries. Although the vanB operon continues to be the predominant genotype, the number of vanA E. faecium identified in AESOP 2014 has significantly increased when compared with AESOP 2013. In addition to being a significant cause of healthcare-associated sepsis, the emergence of multiple multi-resistant hospital-adapted E. faecium strains has become a major infection control issue in Australian hospitals. Further studies on the enterococcal genome will contribute to our understanding of the rapid and ongoing evolution of enterococci in the hospital environment and assist in preventing their nosocomial transmission.
This study was primarily funded by a grant from the Australian Government Department of Health.
We gratefully acknowledge Yung Ching Lee from the Department of Microbiology, PathWest Laboratory Medicine – WA, Fiona Stanley Hospital.
Members of the AGAR in 2014 were:
Australian Capital Territory
Peter Collignon and Susan Bradbury, The Canberra Hospital
New South Wales
Thomas Gottlieb and Graham Robertson, Concord Hospital
James Branley and Donna Barbaro, Nepean Hospital
George Kotsiou and Peter Huntington, Royal North Shore Hospital
Sebastian van Hal and Bradley Watson, Royal Prince Alfred Hospital
David Mitchell and Lee Thomas, Westmead Hospital
Rod Givney and Ian Winney, John Hunter Hospital
Rob Baird and Jann Hennessy, Royal Darwin Hospital
Enzo Binotto and Bronwyn Thomsett, Pathology Queensland Cairns Base Hospital
Graeme Nimmo and Narelle George, Pathology Queensland Central Laboratory
Petra Derrington and Sharon Dal-Cin, Pathology Queensland Gold Coast Hospital
Robert Horvath, Pathology Queensland Prince Charles Hospital
Naomi Runnegar and Joel Douglas, Pathology Queensland Princess Alexandra Hospital
Jenny Robson and Georgia Peachey, Sullivan Nicolaides Pathology
Kelly Papanaoum and Nicholas Wells, SA Pathology (Flinders Medical Centre)
Morgyn Warner and Kija Smith, SA Pathology (Royal Adelaide Hospital)
John Turnidge and Jan Bell, SA Pathology (Women’s and Children’s Hospital)
Louise Cooley and Rob Peterson, Royal Hobart Hospital
Denis Spelman and Amanda Dennison, the Alfred Hospital
Benjamin Howden and Peter Ward, Austin Hospital
Tony Korman and Despina Kotsanas, Monash Medical Centre
Andrew Daley and Gena Gonis, Royal Women’s Hospital
Mary Jo Waters and Linda Joyce, St Vincent’s Hospital
David McGechie and Rebecca Wake, PathWest Laboratory Medicine – WA Fremantle Hospital
Barbara Henderson and Ronan Murray, PathWest Laboratory Medicine – WA Queen Elizabeth II Hospital
Owen Robinson, Denise Daley and Geoffrey Coombs, PathWest Laboratory Medicine – WA Royal Perth Hospital
Sudha Pottumarthy-Boddu and Fay Kappler, St John of God Pathology
Prof Geoffrey W Coombs1,2
Ms Denise A Daley3
Ms Yung Thin Lee1
Dr Stanley Pang1,2
Ms Julie C Pearson2
J Owen Robinson1,2,
Prof Paul DR Johnson4
Ms Despina Kotsanas5
Ms Jan M Bell6
Prof John D Turnidge6,7
for the Australian Group on Antimicrobial Resistance
1. Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research, School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia
2. Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine-WA, Fiona Stanley Hospital, Murdoch, Western Australia
3. Australian Group on Antimicrobial Resistance, Fiona Stanley Hospital, Murdoch, Western Australia
4. Microbiology and Infectious Diseases Departments, Austin Health, Heidelberg, Victoria
5. Infectious Diseases, Monash Health, Monash Medical Centre, Clayton, Victoria
6. SA Pathology, Department of Microbiology and Infectious Diseases, Women’s and Children’s Hospital, North Adelaide, South Australia
7. Departments of Pathology, Paediatrics and Molecular and Biomedical Sciences, University of Adelaide, Adelaide, South Australia
Corresponding author: A/Prof Geoffrey Coombs, Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research, School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia. Telephone: +61 8 6152 2397. Email: email@example.com
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