Presentation at the 20th NMFP meeting held on 4-5 June 2019 at ECDC in Stockholm, Sweden.

1. Molecular epidemiology of
carbapenemase-producing
Klebsiella pneumoniae in Hungary
Ákos Tóth, Laura Jánvári, Erika Ungvári, Ivelina
Damjanova
National Public Heath Center, Hungary
ECDC National Focal Points for Microbiology – 20th Meeting
ECDC, Solna, 4-5 June 2019

2. Resistance to 3rd gen. cephalosporins and carbapenems in
K.pneumoniae isolated from blood, Hungary, 2003-2017
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Proportionofresistanceisolates
Year (number of isolates)
carbapenem non-S 3rd gen ceph. R
Source: National Bacteriological Surveillance, NPHC, Hungary

3. Resistance to 3rd gen. cephalosporins and carbapenems in
K.pneumoniae isolated from blood, Hungary, 2003-2017
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Proportionofresistanceisolates
Year (number of isolates)
carbapenem non-S 3rd gen ceph. R
Source: National Bacteriological Surveillance, NPHC, Hungary
1st outbreaks by SHV-type
ESBL-producing Kpn

4. Resistance to 3rd gen. cephalosporins and carbapenems in
K.pneumoniae isolated from blood, Hungary, 2003-2017
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Proportionofresistanceisolates
Year (number of isolates)
carbapenem non-S 3rd gen ceph. R
Source: National Bacteriological Surveillance, NPHC, Hungary
1st CTX-M-15 ESBL-roducing
Kpn isolates
1st outbreaks by SHV-type
ESBL-producing Kpn

5. Resistance to 3rd gen. cephalosporins and carbapenems in
K.pneumoniae isolated from blood, Hungary, 2003-2017
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Proportionofresistanceisolates
Year (number of isolates)
carbapenem non-S 3rd gen ceph. R
Source: National Bacteriological Surveillance, NPHC, Hungary
CTX-M-15 prevalent ESBL in
Kpn
1st outbreaks by SHV-type
ESBL-producing Kpn
1st CTX-M-15 ESBL-roducing
Kpn isolates

6. Occurence and spread of MDR K. pneumoniae high-risk
clones in Hungary, 2005 (n=196, 13 counties, 35 HCFs)
CTX-M-15-producing ST15 (HEC- Hungarian
Epidemic Clone)
CTX-M-15-producing ST147
CTX-M-15-producing ST11
Damjanova I et al. J Antimicrob Chemother. 2008;62:978-985.

7. K. pneumoniae high-risk clones
Wyres, KL & Kathryn E. Holt KE. Trends in Microbiology. 2016; 24: 944-956.

8. Distribution of prevalent MDR K. pneumoniae
epidemic clones in Hungary, 2005-2016
(n=2498)
27%
22%
11%
3%
37%
ST15
ST11
ST525
ST147
ST258
others

9. Resistance to 3rd gen. cephalosporins and carbapenems in
K.pneumoniae isolated from blood, Hungary, 2003-2017
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Proportionofresistanceisolates
Year (number of isolates)
carbapenem non-S 3rd gen ceph. R
Source: National Bacteriological Surveillance, NPHC, Hungary
Only CTX-M-15 in ST15

10. ESBL (CTX-M-15)
Distribution of multiresistant K. pneumoniae ST15 epidemic clone in
Hungary, 2005-2009 (n=152, 14 counties, 35 HCFs)
35% of all typed
MDR K. pneumoniae were HEC

11. Resistance to 3rd gen. cephalosporins and carbapenems in
K.pneumoniae isolated from blood, Hungary, 2003-2017
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Proportionofresistanceisolates
Year (number of isolates)
carbapenem non-S 3rd gen ceph. R
Source: National Bacteriological Surveillance, NPHC, Hungary
Only CTX-M-15 in ST15 VIM-4 +/- CTX-M-15 in ST15

12. Distribution of multiresistant K. pneumoniae ST15 epidemic clone in
Hungary, 2010-2016 (n=683, 16 counties, 99 HCFs - 20% GPs)
VIM (VIM-4) +/-CTX-M-15
ESBL (CTX-M-15)
OXA-48-like+CTX-M-15
NDM
DHA
*Melegh Sz et al. Clin Microbiol Infect. 2013
* 25% of all typed
MDR K. pneumoniae were HEC (+KP046)

13. Hypothesis – Occurence and possible molecular
evolution of ST15 K. pneumoniae clones in Hungary
year
ESBL+
(n=652)
HEC
(n=10)
PFGE-type
ESBL+
KP046
(n=6)
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 201620152014
VIM+ (+ESBL)
OXA-162 (ESBL+)
OXA-48
(ESBL+)
VIM-4+ P. aeruginosa
(Aeromonas sp.)*
* Libisch B et al. Antimicrob Agents Chemother. 2004, 48:3576-8.; Libisch B et al. J Clin Microbiol. 2008,46:1878-80.
In238b

14. Molecular epidemiology of K. pneumoniae ST15
by WGS, Hungary, 2003-2016
• Strain collection (n=27):
– ESBL +/- producing K. pneumoniae ST15, Hungary, 2003-
2016:
• 1 OXA-162 (+CTX-M-15) (KP046)
• 3 OXA-48 (+CTX-M-15) (HEC)
• 13 CTX-M-15 (HEC) – 7 (2003-2009), 6 (2010-2016)
• 10 VIM-4 (+CTX-M-15) (HEC)
• Whole genome sequencing: Miseq (Illumina)
• Analysis: cgMLST (SeqSphere+), resistome, plasmids
(BIGSdb, Resfinder, Plasmidfinder, pMLST)
• ST15 in European context: Comparison with 103 K.
pneumoniae ST15 genomes from EuSCAPE project
(GenBank)

15. Acquired antimicrobial resistance genes & plasmid RTs
Antibiotics
b-lactams Aminoglycosides
Macrolides
Phenicols
Sulfonamids
Trimethoprim
Tetracycline
Main plasmid
replicon types
In238bclassIintegron
ARGpatterns
No.ofstrains
blaCTX-M-15
blaVIM-4
blaOXA-48
blaOXA-162
blaTEM-1
blaOXA-1
aac(3)-IIa
aac(6’)-Ib
aac(6’)-Ib-cr
aph(3’)-Ia
aph(3’’)-Ib
aph(6’)-Id
msr(E)
mph(E)
catA1
catB4
sul1
sul2
dfrA14
dfrA30
tet(A)
IncF
IncL/M
IncR
K1:A-B-
K2:A-:B-
K9:(A13):B-
F2:A-:B-
I 1
II 2
IIa 1
IIb 1
IIc 1
IId 1
III 5
IIIa 1
IV 5
IVa 3
IVb 1
IVc 1
V 2
Va 1
VI 1

16. Minimum spanning tree based on cgMLST of K. pneumoniae
ST15 (n=27), Hungary, 2003-2016
cluster: < 15 allele difference (2,358 genes of the K. pneumoniae cgMLST scheme)
OXA-162+CTX-M-15
CTX-M-15
VIM-4+CTX-M-15
CTX-M-15
CTX-M-15
OXA-48+CTX-M-15
year
Cluster I
Cluster II
ARG patterns

17. Conclusion
ESBL+
(n=652)
HEC
(n=10)
PFGE-type
ESBL+
KP046
(n=6)
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 201620152014
VIM+ (+ESBL)
OXA-162 (ESBL+)
OXA-48
(ESBL+)
?
year
VIM-4+ P. aeruginosa
(Aeromonas sp.)*
In238b

18. ST15 in European context - (Hungary: n=27, European countries:
n=103)
CTX-M-15
VIM-4
+
CTX-M-15
CTX-M-15
OXA-162
Cluster II
Cluster I

19. Thank you for your attention!
Acknowledgments
• All participating Hungarian
microbiological laboratories
•Szilvia Melegh (University of Pécs,
Pécs, Hungary)
•Sophia David (Wellcome Sanger
Institute, Cambridge, UK)

20. ST15 in European context - (Hungary: n=27, European countries:
n=103)
Cluster II
Cluster I
OXA-162