K. Loens1,2, BB. Xavier 2, J. Coppens2, V. Matheeussen1,2, S. Malhotra-Kumar2, H. Goossens1,2

1 Laboratory of Microbiology, National Reference Centre for Enterococci, University Hospital Antwerp, Edegem,

Belgium

2 Laboratory of Medical Microbiology, Vaccine & Infectious Disease Institute (VAXINFECTIO), University of

Antwerp, Wilrijk, Belgium

Clonal spread of vancomycin-resistant Enterococcus faecium ST80, ST117 and ST203 across Belgian hospitals

Transparency Declaration

• The authors have no conflict of interest to declare

• Enterococcus spp. is of the leading pathogens in hospitalized patients,

accounting for nosocomial infections:

• Including endocarditis, bloodstream infections, and urinary tract

infections,

• associated with peritonitis and intra abdominal abscesses.

Enterococci: Introduction

PhenotypeCharacteristic VanA VanB VanC VanD VanE/G

Vancomycin MIC (µg/ml) 64->1000 4->1000 2-32 16-64 16

Teicoplanin MIC (µg/ml) 16-512 0.5 > 32 0.5-1 2-4 0.5

gene vanA vanB vanC1-C2 vanD vanE

frequent E. faecium E. faecium E. gallinarum E. faecium E. faecalis

enterococcal species E. faecalis E. faecalis E. casseliflavus/

E. flavescens

determinant Acquired Acquired Intrinsic Acquired Acquired

species

characteristic

transferable Yes Yes No No No

The VRE problem in Europe: Enterococcus faecium

source: ECDC Surveillance Atlas, Antimicrobial Resistance

2012 2017

VRE E. faecium outbreaks are increasing across Europe

• Portugal: increase among haemodialysis patients Correia et al. 2014 Enferm Infecc Microbiol Clin.

• Ireland: rise from 33% in 2007 to 45% in 2012, mainly ST17, ST18, ST78 and ST203 Ryan et al. 2015. J.

Antimicrob. Chemother.

• Denmark: increase since 2012 Pinholt et al. 2015. J. Antimicrob. Chemother.

• Germany: increase over of the years; most prevalent: ST117, ST192, ST78, ST80 Werner et al. 2016,

ECCMID. P0658

• The Netherlands: increase since 2011, mainly ST117, ST203, ST18 Willems et al. , 2014, ECCMID O114

• Norway and Poland: mainly ST117 Werner et al. 2016, ECCMID. P0658

• Spain: spread of ST117 across several regions De Benito et al. 2013, ECCMID R2632; Pedrosa-Tedim et al. 2016, ECCMID,

P0657

• Spain: increase in VRE 0.33% in 1996 to 1.33% in 2014, ST18 replaced by ST80, ST117 and ST203

Tedim et al. 2017 J. Antimicrob. Chemother.

• Denmark: increase ST203, ST80 Hammerum et al. 2017 J. Antimicrob. Chemother.

• Hungary: increase ST17, ST18, ST80, ST117, ST203 Melegh et al 2018 Microb. Drug. Resist.

Materials and methods

• Species identification by MaldiTof (Bruker)

• Confirmation of vanA/vanB by monoPCR (Depardieu et al. 2004) and

duplex real-time PCR (in-house)

• Strains

• 368 vancomycine resistant E. faecium , (2002-2019), blood isolates and outbreak strains

• WGS:

• MasterPure complete DNA and RNA purification kit (Epicentre)

• Nextera XT (2x250bp paired end sequencing) and MiSeq (Illumina)

• Bacpipe pipeline (Xavier et al., under revision),

• Comparative genome analysis (Mauve) and CLC genomics Workbench v9,5,3 (clcbio,

Denmark)

• Core genome phylogenetic analysis by ParSNP

• Gene by gene analysis by Chewbbacca (https://github.com/B-UMMI/chewBBACA)

• Microreact (http://microreact.org)

Enterococci received at the NRC

0

5

10

15

20

25

30

35

40

2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

Nr of outbreaks

0

100

200

300

400

500

600

700

2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

Nr of strains received

Geographical spread of ST80, ST117 and ST203 across Belgium

Efm ST80 vanA (n=94, 2011-2019) Efm ST117 vanB (n=91, 2010-2018)

Efm ST117 vanA (n=51, 2012-2018) Efm ST203 vanA (n=49, 2011-2018)

Hospital 1Hospital 2Hospital 3Hospital 4Hospital 5Hospital 6Hospital 7Hospital 8Hospital 9Hospital 10Hospital 11Hospital 12

Hospital 14Hospital 15Hospital 16

Hospital 13

Postal code:

same area as

H1 and H2

Belgian ST80 vanA positive E. faecium strains

West-Flanders Liège province

Pairwise comparison E. faecium ST80 vanA Liège region

Belgian ST117 vanB positive E. faecium strains

Hospital 1Hospital 2Hospital 3Hospital 4Hospital 5Hospital 6Hospital 7Hospital 8Hospital 9Hospital 10Hospital 11Hospital 12Hospital 13Hospital 14Hospital 15Hospital 16Hospital 17Hospital 18Hospital 19Hospital 20Hospital 21Hospital 22Hospital 23Hospital 24Hospital 25Hospital 26Hospital 27Hospital 28

Brussels Capital Region West-Flanders

Hospital 5Hospital 1

Northeast of Belgium

*vanAB

Belgian ST117 vanA positive E. faecium strains

Hospital 1Hospital 2Hospital 3Hospital 4Hospital 5Hospital 6Hospital 7Hospital 8Hospital 9Hospital 10Hospital 11Hospital 12Hospital 13Hospital 14Hospital 15Hospital 16Hospital 17

*vanAB

Belgian ST203 vanA positive E. faecium strains

Hospital 1Hospital 2Hospital 3Hospital 4Hospital 5Hospital 6Hospital 7Hospital 8Hospital 9Hospital 10

Unit UNK

ICU

ONCOL

DIALNEFINTMED

SURG

Conclusion

• Belgian E. faecium ST80, ST117 and ST203 isolates can be divided into

distinct (sub)clusters.

• Clonal spread of E. faecium ST80 vanA over at least 5 hospitals within the

Liège province.

• Clonal spread of E. faecium ST117 vanB over 3 hospitals in West-Flanders,

6 hospitals in Brussels Capital region, and 7 hospitals in the North-east of

Belgium.

• Typing of E. faecium is critical to monitor further spread of dominant clones.

• If patients are transferred from one hospital to another, information on VRE-

carriage status should be provided.

Acknowledgement

• The National Reference Centre is partially supported by the Belgian Ministry

of Social affairs through a fund within the Health Insurance System.