“update carbapenemases with animal origin„ … · hydrolisis: carbanp, bluecarba, maldi-tof ....

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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


What do we know…?


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

Presenter

Presentation Notes

Short introduction Carbapenems … … are used as last choice antimicrobials for infections with mdr Considered Hydrolize Retain susceptibility Confer resistance against


Carbapenem resistance EARS-Net Database, 2012

E. coli Klebsiella pneumoniae

Pseudomonas aeruginosa

R

R+I

http://www.ecdc.europa.eu


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

Presenter

Presentation Notes

Short introduction Carbapenems … … are used as last choice antimicrobials for infections with mdr Considered Hydrolize Retain susceptibility Confer resistance against


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

Presenter

Presentation Notes

In all four defined groups Most prevalent ones in .... Oxa-group – oxa variants with carbapenemase activity MBLs with some more important types Varaints/ types frequent When we have a look And one of these genes found its way into some german livestock farms....

http://www.lahey.org/Studies


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


What did we do and what have we found…?


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)

Presenter

Presentation Notes

There is a national ESBL surveilance project called RESET….. Longitudinal and cross sectional studies We collect samples using Cefotaxim selectiv media to Screen for ESBL + isolates We find typical … That we expected to find 3 salm, 2 e.c. from 3 different farms that harboured an acc-1 pAMpC showd in addition an Atypical resistace phenotype That means….


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

Presenter

Presentation Notes

These isolated showed / exhibits in contrast to – controll and to all other 200 iolates analized in stis stude A slightly decreased zone dioameter for the tested carbapenems But when we look / regard / check the With the guidelines They are just grouped / characterized as S or I Nevertheless we did


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

Presenter

Presentation Notes

…. Salm three diferent farms highly related patterns S. Infantis PFGE!!!! ST32 typical for Salm. Serovar Infantis … E. coli isolates at three different time points, all having a similar pattern like this selection Highly similar Higly related distance Relation farms origin? This is a story with a still a lot questions to answer. Unfortunately this is not the only story we have to tell…..


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

Presenter

Presentation Notes

We let them grow in Was (not) able to grow Whereas Even Indeed Have a second lokk Look again Really resistant Fully resistant


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

Presenter

Presentation Notes

Vim-1 was responsible for the phenotype Encoded Gene cassetes Locaed on Linked by Carried by Flanked by Harboured a Additional / in additopn to that Furthermore Moreover Whereas ST32 typical for Salm. Serovar infanits Infantis Belong Recovered Highly similar Higly related distance Level of expression seemed to be very low…


What have we learnt from the human side…


Kindly provided by Hajo Grundman (Glasner et al. Eurosurv. 2013).

Carbapenemase producers: human


Taken from Glasner et al. Eurosurv. 2013

Carbapenemase-encoding genes in humans:

mailto:[email protected]


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


From the human side:


What has happened in the animal/food side since then…?


VIM-1 isolates in these farms (S1, S2, S3, G1)

S1: Salmonella S2: Salmonella, E.coli

S1: Salmonella

G1: Salmonella

S2: E. coli


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.


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


First description of Carbapenemase-producers in foods: imported squibs

Pseudomonas VIM-1

Canada 2014

Rubin et al. 2014


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


Problem


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


?

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


From the animal/food side


Conclusions


• 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


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

Presenter

Presentation Notes

What we all should keep in mind regarding the history of antimicrobial and their res: After every design / discovery of e new drug it follows the spread of its resistance genes. First in human isolates and always a few yeras after in livestoock bacteria. And just some month ago we were happy because we at least did not find carbapenemases in livest But today I have to depict already two publications that demonstrate their occurence in livestock bacteria nowadays. And now we have also in animal farms …… And these are some really worrying news.


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


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

Presenter

Presentation Notes

More reports on carb prod in animals: Reports from livestock carrying ndm ….. Food producing animals in china….. A lot of these cases (head lices and pets) may just reflect this problem in humans! But Livestock must not be underestimated! So what do we learn from this? ….

“update carbapenemases with animal origin„ … · hydrolisis: carbanp, bluecarba, maldi-tof ....

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