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BU
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FÜR
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“Update carbapenemases with animal origin„
Beatriz Guerra et al.
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 2
Outline
• What do we know?
• What did we do and what have we found?
• What has happened since then?
• What have we learnt?
• Problems
• Conclusions
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 3
What do we know…?
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 4
Carbapenem
• ß-lactams: Ertapenem, Imipenem, Meropenem, Doripenem
• Clinically Important Antimicrobials, CIA (WHO)
• Antimicrobials of last choice
• Treatment only for severe infections with multidrug resistant (nosocomial) pathogens
• Resistance mechanisms:
- porin loss + ESBL/AmpC
- Carbapenemases
• Acepted term: Carbapenem non-susceptible isolates
Ertapenem
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 5
Carbapenem resistance EARS-Net Database, 2012
E. coli Klebsiella pneumoniae
Pseudomonas aeruginosa
R
R+I
http://www.ecdc.europa.eu
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 6
Carbapenemases • Confer resistance to almost all β-lactams
• Produced by several gram-negative bacteria: E. coli, Klebsiella, Acinetobacter, Pseudomonas...
• Can be acquired but also intrinsic in some bacterial species
• Cause serious problems in hospitals
• Found mostly in humans (hospital, comunity)
• Seem to be frequent in the environment
• KPC, OXA-48, Metallo-β-Lactamases: NDM, VIM
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 7
http://www.lahey.org/Studies (June 2013)
CTX-M, 160
CMY, 121
IMP, 48 VIM, 42
KPC, 22
SHV, 189 TEM, 219 OXA, 426:
Classification, some numbers…
Group 1 (C)
AmpC enzymes
Group 2 (A) Groups 2d, 2de, 2df (D) Group 3 (B)
Bush & Jacoby (Ambler)
NDM, 12
OXA-48 OXA-23 OXA-40
…
„MβLs“
ACC, 5
Diversity of beta-lactamasas
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 8
Molecular detection:
Commercial Kits
PCR-amplification/sequencing Fischer et al. JAC, 2012, 2013. Dallene et al. JAC, 2010 Poirel et al. Diagn Microbiol Methods 2010 Nordman et al. CMI 2012 (Review)
(d) Multiplex PCR assay for the blaVIM/blaIMP /blaKPC genes,
amplification from control strains. Dallene et al. JAC 2012.
EUCAST and CLSI guidelines
Phenotypical tests Inhibition: EDTA, dipicolinic acid, pheyl boronic acid Hydrolisis: CarbaNP, BlueCarba, MALDI-TOF
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 9
What did we do and what have we found…?
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 10
0
50
100
150
200
250
300
cattle
swine
poultry
5 isolates (3 farms)
AmpC CTX-M TEM SHV
cattle
swine poultry
longitudinal and cross sectional studies in animal farms (472 E. coli, 3 Salmonella)
Results: RESET project (National ESBL surveillance project)
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 11
Phenotypic β-lactam susceptibility test
5 isolates from 3 farms (1x poultry, 2x swine): 3 Salmonella, 2 E. coli
E. coli R178 (swine)
DD cut-offS I R S R (WT ≥ Y mm)
Imipenem ≥ 23 20-22 ≤ 19 ≥ 22 mm < 16 mm ≥ 24 mmErtapenem ≥ 23 20-22 ≤ 19 ≥ 25 mm < 22 mm ≥ 29 mmMeropenem ≥ 23 20-22 ≤ 19 ≥ 22 mm < 16 mm ≥ 26 mm
CLSI (2011) EUCAST (2011) breakpoints breakpoints
Salmonella R3 (poultry)
IMP: 20 mm ETP: 24 mm
E. coli ATCC 25922
IMP 30 mm ETP 32 mm IMP 22 mm ETP 24 mm
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 12
Br 1 2 3 4 5 6 7 8 Br
Br - Braenderup H9812 1 - R3 2 - R25 3 - R27 4 - V-36 5 - R29 6 - R178 7 - V-39 8 - V-34 Br - Braenderup H9812
XBaI-PFGE
S2 G1 S1
• One poultry and three swine farms
• farms independent from each other
• Salmonella Infantis ST32, non motile
•E. coli ST88, phylogroup A, O118:NM
Molecular characterization
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 13
Carbapenem non-susceptible (CNS) vs. Carbapenem resistant
MHK cut-offS I R S R (WT ≤ X mg/l)
Imipenem ≤ 1 mg/l 2 ≥ 4 mg/l ≤ 2 mg/l > 8 mg/l ≤ 0.5 mg/lErtapenem ≤ 0.25 mg/l 0.5 ≥ 1 mg/l ≤ 0.5 mg/l > 1 mg/l ≤ 0.064 mg/lMeropenem ≤ 1 mg/l 2 ≥ 4 mg/l ≤ 2 mg/l > 8 mg/l ≤ 0,125 mg/l
CLSI (2011) EUCAST (2011)breakpointsbreakpoints
E. coli β1 ACC-1 MHK test
E. coli ATCC 25922
ETP [µg/ml]
0,5 1 2 4 8 16 0,5 1 2 4 8 16
IMP [µg/ml]
IMP/ ETP [µg/ml]
0,5 1 0,5 1
E. coli “R178” VIM-1+ACC-1
IMP [µg/ml]
0,5 1 2 4 8 16
Salmonella “R3” VIM-1+ACC-1
IMP [µg/ml]
0,5 1 2 4 8 16
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 14
Molecular characterization
Inc-HI2 plasmids E. coli: 220 kb, β-lactam-STR-SUL Salmonella: 300 kb, β-lactam-STR-SUL-CHL-TMP
blaACC-1
blaVIM-1 aacA4 aadA1
sul1
strA, strB
pRHR178 E. coli
blaACC-1
blaVIM-1 aacA4 aadA1 sul1
strA, strB
TMP-R
catA1
pRHR3 Salmonella
blaVIM-1
class 1 Integron In110 located in an In2-Tn402 element linked to Transposon Tn21
blaVIM-1 aacA4 aadA1 3´CS
intI1
5´CS
tnpM Tn21
Pc hybrid 2
IRi
IS1353
IS1326
orfA orfB
istB istA
tniA ∆tniB
Tn402
orf5 tnpR qacE∆1
sul1
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 15
What have we learnt from the human side…
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 16
Kindly provided by Hajo Grundman (Glasner et al. Eurosurv. 2013).
Carbapenemase producers: human
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 17
Taken from Glasner et al. Eurosurv. 2013
Carbapenemase-encoding genes in humans:
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 18
Quelle: Dr. med. Martin Kaase Nationales Referenzzentrum für gramnegative Krankenhauserreger Abteilung für Medizinische Mikrobiologie Ruhr-Universität Bochum
September 2009 – February 2012
Carbapenemases in Germany: human
2013
NDM-5, OXA-181KPC-2, VIM-1
OXA-23IMP-8
NDM-1, OXA-181IMI-1
TMB-1VIM-31
OXA-58NDM-5GIM-1
IMI-2NDM-7
OXA-162VIM-2
IMP-14NDM-1, OXA-48
OXA-244KPC-2, OXA-48
GES-5OXA-232
NDM-1, OXA-232VIM-4
OXA-181KPC-3
NDM-1KPC-2VIM-1
OXA-48
0 50 100 150 200 250 300
111111112222223333344
81010
5177
104113
283
OXA-48OXA-162OXA-181OXA-204OXA-244
VIM-1VIM-2VIM-4
VIM-19VIM-26
IMP-8IMP-4
IMP-13IMP-14NDM-1
NDM-1, OXA-48NDM-3GIM-1
KPC-2KPC-2, VIM-1
KPC-2, VIM-26KPC-3
GESIMI-2
0 50 100 150 200 250 300 350 400 450 500434
14411
2135108
22132
52116
2468
1122
31
Anzahl der StämmeSeptember 2009 – Januar 2013
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 19
From the human side:
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 20
What has happened in the animal/food side since then…?
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 21
VIM-1 isolates in these farms (S1, S2, S3, G1)
S1: Salmonella S2: Salmonella, E.coli
S1: Salmonella
G1: Salmonella
S2: E. coli
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 22
Acinetobacter OXA-23
France 2011
Belgium 2012
Other Carbapenemase-producers in animals
Senegal 2012
Fotos taken from different web pages via google
Acinetobacter NDM-1 China 2012 2013
Zhang et al. JAC 2013. Wang et al. PloS one 2012.
Poirel et al. EID 2012. Smet et al. JAC 2012.
Kempf et al. PloS one 2012.
Portugal 2009
Pomba et al. AAC 2014.
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 23
Other Carbapenemase-producers in animals
Fotos taken from different web pages via google
E. coli NDM-1
USA 2013
E. coli and Klebsiella OXA-48
Germany2013
Shaheen. AAC 2013. Stolle et al. JAC 2013.
Salmonella NDM-1 blaOXA-48 (Pets feed, DNA)
Seiffert et al. AAC 2014. Switzerland 2014
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 24
First description of Carbapenemase-producers in foods: imported squibs
Pseudomonas VIM-1
Canada 2014
Rubin et al. 2014
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 25
Genetic evolution in the farm: pRH-R27 vs. pRH-R178
Antimicrobial-R genes: blaVIM-1, blaACC-1, aacA4, aadA1, sul1, and strA/B; catA only in pRH-R27 Disinfectant-R genes: qacEΔ1 Heavy metals-R: ter-, mer- and sil-operons and pco genes; ars-operon only in pRH-27.
Kindly provided by L. Felgenhauer (University of Giessen,, Germany Felgenhauer et al. JAC submitted
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 26
Problem
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 27
How to detect them?
MHK cut-offS I R S R (WT ≤ X mg/l)
Imipenem ≤ 1 mg/l 2 ≥ 4 mg/l ≤ 2 mg/l > 8 mg/l ≤ 0.5 mg/lErtapenem ≤ 0.25 mg/l 0.5 ≥ 1 mg/l ≤ 0.5 mg/l > 1 mg/l ≤ 0.064 mg/lMeropenem ≤ 1 mg/l 2 ≥ 4 mg/l ≤ 2 mg/l > 8 mg/l ≤ 0,125 mg/l
CLSI (2011) EUCAST (2011)breakpointsbreakpoints
DD cut-offS I R S R (WT ≥ Y mm)
Imipenem ≥ 23 20-22 ≤ 19 ≥ 22 mm < 16 mm ≥ 24 mmErtapenem ≥ 23 20-22 ≤ 19 ≥ 25 mm < 22 mm ≥ 29 mmMeropenem ≥ 23 20-22 ≤ 19 ≥ 22 mm < 16 mm ≥ 26 mm
CLSI (2011) EUCAST (2011) breakpoints breakpoints
S. Corvallis12-1738 24 20 24 mm
S. Corvallis12-1738 0.25 mg/l 0.5 mg/l 0.125 mg/l
Etest
Oxoid discs, 10 µg
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 28
?
How to detect them?
For monitoring purposes: pre-enrichment, selective or not? Which antimicrobial? Which concentration? differences in specificity (low for ETP), sensitivity and stability (low for IMP). Probably meropenem the most appropiated Do comercial plates allow the growth of isolates with decreased susceptibility? Only some of them Is enough to test the ESBL/AmpC producers?: No detection of OXA-48 and other OXA-like producers
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 29
From the animal/food side
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 30
Conclusions
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 31
• Carbapenemases are nowadays also present in foods, food-producing animals, pets and wild animals
• We do not know to what extend...
• The question is, how to find them?
• Monitoring?
Conclusions
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 32
1950 1960 1970 1980 1990 2000 1940 2012
chromosom. AmpC, Metallo β-Lactamases Gram+ Penicillinases
Penicillin
S. aureus Penicillinases
Cephalosporin
Broad spectrum penicillins: Ampicillin
plasmidic broad spectrum β-lactamases
Cephamycine: Cefoxitin
Carbapenems β-Laktamase inhibitors
Aztreonam
ESBLs, Plasmid AmpC
livestock
Carbapenemases human
Carbapenemases livestock
Conclusion
?
Oxymino- (3rd gen. )
Cephalosporins: Cefotaxim
ESBLs, Plasmid AmpC
human
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 33
et al: J. Fischer, I. Rodríguez, S. Schmoger, S. Jahn, B. Baumann, R. Helmuth
Thank you !
Bundesinstitut für Risikobewertung Max-Dohrn Strasse 8-10, D-10589 Berlin, Germany. Tel. 0 30 - 184 12 – 2082; Fax 0 30 - 18412 - 4741 [email protected]; www.bfr.bund.de
Beatriz Guerra
RESET-Verbund (www.reset-verbund.de)
NRL-Salm (BfR) NRL-AR (BfR) Abt. 4 Dr. Roesler, Dr. Friese et al. (FU) Dr. Pfeifer (RKI) Dr. Kaase (NRZ-Nosokomiale Infektionen) Dr. H. Grundman (U. Groningen) Farmers Veterinary and Public Health Laboratories
Beatriz Guerra, EURL-Workshop, 24.09.2014 slide 34
Carbapenemase-producers in animals and their environment
Smet et al. JAC 2012. Belgium 2012
Pomba et al. AAC 2014, Portugal 2009
Acinetobacter OXA-23 Horse, Cat
E. coli and Klebsiella OXA-48 Dogs
Stolle et al. JAC 2013. Germany 2013
E. coli NDM-1 Dogs and cats
Shaheen. AAC 2013.
USA 2013
Poirel et al. EID 2012. France 2011
Acinetobacter OXA-23 Cattle
Kempf et al. PloS one 2012. Senegal 2012
Acinetobacter OXA-23 Human lice
Wang et al. PloS one 2012. Zhang et al. JAC 2013.
China 2012, 2013
Acinetobacter NDM-1
Broiler and swine
Pseudomonas VIM-2 Imported squibs
Rubin et al. EID 2014 Canada 2014
blaOXA-48 (Pets feed, DNA)
Seiffert et al. AAC 2014. Switzerland 2014