sign in sign up
<<
Home , Mezlocillin

Interpretation of ESBL and carbapenemase phenotypes

Yvonne Pfeifer FG13 Nosocomial Pathogens and Resistance Robert Koch Institute, Wernigerode, Germany

EURL-AR Training Course Copenhagen, 24th-26th of September 2014 1

Robert Koch Institute Wernigerode

Department 11: Bacterial Enteric Pathogens and Legionella National Reference Center Salmonella and other Enteric Pathogens

Department 13: Nosocomial Pathogens and Antibiotic Resistance National Reference Center Staphylococci (MRSA) and Enterococci

Working Group Enterobacteriaceae und Nonfermenter

Research Group A. baumannii

Research Group Salmonella

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 2 Working Group „Enterobacteriaceae und Nonfermenter“

Topic Molecular epidemiology of multidrug-resistant pathogens: Emergence and spread of resistance to , and fluoroquinolones in gram-negative in Germany

Tasks Susceptibility testing and identification of resistance genes Analysis of new (emerging) resistance genes / resistance mechanisms Characterisation of mobile genetic elements (plasmids, integrons, IS) Molecular typing of bacteria (PFGE-analyses, MLST etc.)

Third-party funded research:

RESET (ESBL- and Fluoroquinolone Resistance in Enterobacteriacaeae): 2011-2016

Molecular comparison of resistant Enterobacteriaceae from human/animal/environment

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 3 Phenotypic detection of β-lactamases

Which? Where? ESBL AmpC Enterobacteriaceae, A. baumannii, P. aeruginosa Carbapenemases

Why? Legislation: mandatory reporting Definition of „multidrug-resistance“

In-house surveillance

Outbreak investigations

Epidemiology national/international

Clinical relevance and therapeutic options

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 4 ESBL  Extended-Spectrum β-Lactamases

1960 Usage ; cephalosporins 1st/2nd generation , mezlocillin,  1965: Detection of β-lactamases (penicillinases) TEM-1 und SHV-1 in E. coli and K. pneumoniae

1980 Usage “new” cephalosporins 3rd generation , ,  1983: Detection of cephalosporinases Extended-Spectrum Beta-Lactamases TEM-ESBL n > 100 variants SHV-ESBL n > 60 variants „classic“ ESBL in CTX-M-ESBL n > 130 variants Enterobacteriaceae PER-ESBL GES-ESBL rare in Enterobacteriaceae A. baumannii, VEB-ESBL P. aeruginosa OXA-ESBL Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 5 β-lactamases class C = AmpC

Chromosomal-encoded Plasmid-encoded AmpC β-lactamases AmpC β-lactamases

● E. cloacae CMY FOX ACC LAT ● Citrobacter freundii ACT MOX ● Hafnia alvei ● ● Morganella morganii Horizontal gene transfer of formerly chromosomal ampC ● Aeromonas hydrophila ● Acquired by E. coli, K. pneumoniae, ● E. coli

Modifications in regulatory mechanisms ampC overexpression due to a promotor of an result in ampC overexpression insertion sequence upstream of the gene

→ Resistance to penicillins and → Resistance to penicillins und cephalosporins cephalosporins

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 6 Diagnostics: ESBL and AmpC β-lactamases

Phenotype: Resistance to penicillins (ampicillin) 1st, 3rd, 4th generation cephalosporins (cefotaxime, cefpodoxime, ceftazidime, ) Resistance to () Susceptible to () and carbapenems (, , ) ESBL inhibitors: , ,

Phenotype: Resistance to penicillins, 1st, 3rd gen. cephalosporins and cephamycins (cefoxitin, ) AmpC Susceptible to carbapenems Stable to ESBL inhibitors (clavulanic acid) AmpC inhibitor:

Screening: Chromogenic agar for selection of „ESBL-producing“ bacteria: with cefotaxime (1mg/L) or cefpodoxime (4mg/L) +/- cloxacillin Brilliance ESBL Agar (Oxoid); ChromID ESBL (Biomerieux), Chromagar ESBL (MAST)

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 7 Diagnostics: ESBL and AmpC β-lactamases

AST by automated systems:

„ESBL-positive“  cefotaxime- and/or ceftazidime-resistant and inhibition by an ESBL inhibitor (clavulanic acid)

„AmpC-positive“  „high-level-cephalosporinase“ oder „cephamycinase“ cefotaxime- and/or ceftazidime- and/or cefoxitin-resistant und keine No inhibition by an ESBL inhibitor (clavulanic acid)

ESBL/AmpC confirmation tests

Etest ESBL  cefotaxime/ceftazidime/cefepime + clavulanic acid Etest AmpC  cefotetan/cefoxitin + cloxacillin Disk-tests ESBL/AMPC  Combined-Disk-Tests (CDT)

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 8 CDT: ESBL+AmpC confirmation

Examples: D68C ESBL/AmpC ID (Mast); ESBL + AmpC Screen Kit 98008 (Rosco)

A cefpodoxime C cefpodoxime + AmpC inhibitor B cefpodoxime + ESBL inhibitor D cefpodoxime + ESBL- and AmpC inhibitor

ESBL-negative ESBL-positive AmpC-positive ESBL-positive AmpC-negative AmpC-positive

Limitations: Low -lactamase production  false-negative results Production of other -lactamases than ESBL/AmpC:  false positive/no results (e.g. K1(OXY) β-lactamase in Klebsiella oxytoca) Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 9 Limitations CDT: ESBL + AmpC confirmation

Phenotype: Hyperproducers of K1 β-lactamase can imitate the ESBL-phenotype K1 (OXY) But: consistently resistant to , –tazobactam and aztreonam, intermediate/resistant to cefotaxime and cefepime, K. oxytoca always fully susceptible to ceftazidime. Potz et al. JAC 2004

ESBL or AmpC or…? resistant subpopulation? “multidrug-resistant”?

K. pneumoniae CTX-M-15 + OXA-48 K. pneumoniae KPC-2

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 10 Etest: ESBL + AmpC confirmation

CT cefotaxime CTL cefotaxime + clavulanic acid TZ ceftazidime TZL ceftazidime + clavulanic acid

K. pneumoniae ESBL-type CTX-M-15 E. coli ESBL-type CTX-M-1

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 11 Rapid ESBL confirmation

Nordmann P, Dortet L, Poirel L. Rapid detection of extended-spectrum-β- lactamase-producing Enterobacteriaceae. J Clin Microbiol. 2012, 50:3016-22. This biochemical test (<1h)was based on the in vitro detection of a (cefotaxime) hydrolysis that is inhibited by tazobactam addition. The ESBL activity was evidenced by a color change (red to yellow) of a pH indicator (red phenol) due to carboxyl-acid formation resulting from cefotaxime hydrolysis that was reversed by addition of tazobactam (positive test).

ESBL-producer

Non-ESBL- producer

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 12 Mechanisms of resistance

FIG. sitemaker.umich.edu/.../files/resistance.gif Efflux-pumps Inner membrane

Periplasmic space

Outer membrane

Aktive transport of outwards; common in aeruginosa Loss of porins Porins = outer menbrane proteins (OMPs) Mutations in different porin genes lead to loss of porins  loss of permeability of the Porin loss + ESBL/AmpC production  carbapenem resistance (ETP, MPM) Common in aerogenes, K. pneumoniae

Carbapenemase production Beta-lactamase Hydrolysis of carbapenems by specific beta-lactamases = carbapenemases Outer membrane Periplasmic space Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 13 Carbapenemases

KPC  „Klebsiella pneumoniae Carbapenemase“ mainly in K. pneumoniae Common in „endemic areas“ (e.g. Greece, Israel, Italy) OXA-48  in Enterobacteriaceae; common in Turkey, North Africa, India VIM  „Verona Integron-borne Metallo-beta-lactamase“ in Enterobacteriaceae and P. aeruginosa common in Mediterranean countries (Italy, Greece) NDM  „New-Delhi Metallo-Beta-Lactamase“ in Enterobacteriaceae and A. baumannii from India, North Africa, Balkan states IMP  rare in E. cloacae, K. pneumoniae; more common in P. aeruginosa metallo-beta- GIM  „German Imipenemase“ single cases in E. cloacae, lactamases S. marcescens, P. aeruginosa, A. pittii (MBL) AIM  „Adelaide Imipenemase“ single cases in P. aeruginosa FIM  „Florence Imipenemase“ single cases in P. aeruginosa DIM  „Dutch Imipenemase“ single cases in Pseudomonas spp. SIM  „Seoul Imipenemase“ single cases in A. baumannii SPM  „São Paulo metallo-β-lactamase“ single cases in P. aeruginosa OXA-58/23/24  common in A. baumannii

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 14 Spread of carbapenemases in Europe

Canton R. et al. CMI, 2012

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 15 Carbapenemases in Germany

Cases of carbapenemase-producing gram-negative bacteria 2009-2013 Data from the National Reference Center of gram-negative nosocomial pathogens in Bochum (Dr. Martin Kaase) Enterobacteriaceae tested in 2013: n = 1831 Carbapenemase producers in 2013: n = 699 (K. pneumoniae n = 481; E. coli n = 73)

Carbapenemases

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 16 Phenotype: Carbapenemase producers

Antibiotics [mg/mL] NDM-1 VIM-1 KPC-2 OXA-48 + ESBL OXA-48 E. coli K. oxytoca K. pneumoniae K. pneumoniae E. coli Ampicillin R R R R R PIP/TAZ R R R R R Cefotaxime R R R R S Ceftazidime R R R R S Cefoxitin R R R R R Gentamicin R R I R R Amikacin R S R R R Oxytetracycline R R R R R Chloramphenicol R R R R R Ciprofloxacin R R R R R SXT R R R R R Meropenem R R R R I Imipenem R R R R I Ertapenem R R R R R MBL-Etest (IP/IPI) positiv positiv negativ negativ negativ Tigecycline I S I S S S S S S S Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 17 Diagnostics: Carbapenemases

Phenotype: Resistance to penicillins, 1st-4th gen. cephalosporins and Carbapenemase carbapenems producer Metallo-beta-lactamases (MBL)  susceptible to aztreonam OXA-48 carbapenemases  susceptible to 3rd gen. cephalosporins Stable to ESBL inhibitors (clavulanic acid) Carbapenemase inhibitors: EDTA (MBL); boric acid (KPC)

Screening: Chromogenic agar for selection of „Carbapenemase-producing“ bacteria: with Meropenem oder Ertapenem (Girlich et al., 2013, Diagn. Microbiol. Infect. Dis.) Brilliance CRE Agar (Oxoid) ChromID Carba (Biomerieux) Chromagar KPC (MAST) SUPERCARBA medium (Nordmann P.,et al., JCM, 2012)

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 18 Diagnostics: Carbapenemases

AST by automated systems:

„Carbapenemase producer (KPC or MBL) or loss of permeability + ESBL/high- level-cephalosporinase“  resistant/intermediate-resistant to imipenem/meropemen/ ertapenem  Manual confirmation tests is needed!

Carbapenemase confirmation tests:

Carbapenemase production

 mod. Hodge Test,  imipenem/meropenem-hydrolysis (Carba NP, MALDI)

Etest MBL  imipenem + imipenem/EDTA

Disk-Tests  CDT with carbapenemase inhibitors

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 19 Diagnostics: carbapenemase production

Mod. Hodge-Test Modified Hodge Test Disks: Imipenem Meropenem Ertapenem

K. pneumoniae K. pneumoniae carbapenem-susceptible carbapenemase producer E. coli – reference strain carbapenem-susceptible K. pneumoniae carbapenem-resistant but no carbapenemase production - Confirmation of any carbapenamase production (OXA-48, KPC, MBL)

- reliable for outbreak investigations

- low specifity - false positive results (e.g. AmpC producer) are common Girlich, D., Poirel, L., Nordmann, P., 2012. Value of the modified Hodge test for detection of emerging carbapenemases in Enterobacteriacae. J. Clin. Microbiol. 50, 477-479. Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 20 Rapid detection: Carbapenemase production

Carba NP Test Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis. 2012 ; 18:1503-7.

To rapidly identify carbapenemase producers in Enterobacteriaceae, we developed the Carba NP test. The test uses isolated bacterial colonies and is based on in vitro hydrolysis of a carbapenem, imipenem. It was 100% sensitive and specific compared with molecular-based techniques. This rapid (<2 hours), inexpensive technique may be implemented in any laboratory.

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 21 Rapid detection: Carbapenemase production

Blue Carba Test J. Pires, Â. Novais, and L. Peixe Blue-Carba, an Easy Biochemical Test for Detection of Diverse Carbapenemase Producers Directly from Bacterial Cultures J Clin Microbiol. Dec 2013; 51(12): 4281–4283

Advantages: direct use of colonies (instead of bacterial extracts that need the extraction buffer (B-PER II); reduced cost per reaction due to use of Tienam (ca. 10× cheaper than an imipenem monohydrate formula)

Imipenem/meropenem hydrolysis + confirmation with UV-Spec. or MALDI-TOF Bernabeu et al. DMID 2012; Hrabák et al. JCM 2012; Burckhardt et al. JCM 2011

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 22 CDT: Carbapenemase confirmation

„KPC/MBL/AmpC ID-Test“ (Rosco Diagn.) or „Carba-D70C“ Test (MAST Diagn.)

MRP Meropenem MR+BO Meropenem + KPC inhibitor MR+DP Meropenem + MBL inhibitor MR+CL Meropenem + AmpC inhibitor

KPC-positive MBL-positive (e.g. VIM, NDM)

Boric acid derivate = KPC inhibitor EDTA derivate = MBL inhibitor

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 23 Limitations CDT: Carbapenemase confirmation

MRP Meropenem MR+BO Meropenem + KPC inhibitor MR+DP Meropenem + MBL inhibitor MR+CL Meropenem + AmpC inhibitor

Carbapenemase or no carbapenemase?

P. aeruginosa carbapenem resistant K. pneumoniae carbapenem resistant Mod. Hodge-Test positive

P. aeruginosa without carbapenemase K. pneumoniae CTX-M-15 + OXA-48

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 24 Etest: Metallo-beta-lactamases (MBL)

K. pneumoniae VIM-1 E. coli NDM-1

Imipenem

Deformation of ellipse/phantom zone

Imipenem + EDTA (MBL inhibitor)

Metallo-β-lactamase-positive

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 25 Limitations: Etest metallo-beta-lactamases (MBL)

K. pneumoniae MIC imipenem 4mg/L S. marcescens MIC imipenem 1mg/L MIC meropenem 2mg/L MIC meropenem 1mg/L MIC ertapenem >32mg/L MIC ertapenem 4mg/L Mod. Hodge-Test positive Mod. Hodge-Test +/-?

Phantom zone

Metallo-β-lactamase-negative Metallo-β-lactamase-suspicious

K. neumoniae OXA-48 S. marcescens GIM-1 (MBL)

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 26 Limitations: Etest MBL and P. aeruginosa

A) B) C) Resistant subpopulation

Phantom zone

P. aeruginosa P. aeruginosa P. aeruginosa MBL-suspicious MBL-suspicious MBL-positive

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 27 Limitations: Etest MBL and Nonfermenter

IP Imipenem IPI Imipenem+EDTA General growth inhibition due to EDTA independently from MBL-production

The imipenem/EDTA inhibition zone is a „narrow ellipse“

 common in carbapenem-resistant P. aeruginosa or A. baumannii narrow ellipse producing OXA carbapenemases

 false-positive MBL-test

P. aeruginosa without carbapenemase

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 28 Carbapenemases and A. baumannii

> 95% of all carbapenem-resistant A. baumannii produce a carbapenemase

OXA-23 OXA-72 Common and wide spread in MDR-A. baumannii OXA-58 OXA-51+ISAba1 NDM-1 IMP Metallo-beta-lactamases (MBL) rare but increasing in A. baumannii VIM

Carbapenem resistance: intermediate/resistant to imipenem and meropenem

Mod. Hodge Test Mainly positive (or false positive due to AmpC-production) MBL-Etest Often false positive (growth inhibition by EDTA) Disk-Test No results or false positive results (growth inhibition by EDTA)

? Is phenotypic confirmation really necessary for carbapenem-resistant A. baumannii ?

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 29 Carbapenem resistance and porin loss

Cause: Porins = OMPs = outer menbrane proteins

Mutations in porin genes  porin loss (loss of permeability)

Porin loss + ESBL/AmpC- production  carbapenem resistance

Phenotype: AMP PIP/TAZ CTX CAZ FOX IPM MPM ETP R R R R R 0,25 - > 32 0,5 - > 32 R

Common resistance mechanism in Enterobacter aerogenes (>95%)

Common in K. pneumoniae (after therapy with carbapenems)

Ertapenem-resistant

PIP/TAZ-resistant

MIC imipenem <= MIC meropenem

AMP, ampicillin; PIP/TAZ, piperacillin/tazobactam; CTX, cefotaxime; CAZ, ceftazidime; ETP, ertapenem; MPM, meropenem; IPM, imipenem, FOX, cefoxitin

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 30 KPC = Klebsiella pneumoniae carbapenemase

Phenotype: AMP PIP/TAZ CTX IPM MPM ETP KPC R R R 0,5 - > 32 0,25 - > 32 R

Mainly in K. pneumoniae due to worldwide spread of clonal lineages (K. pneumoniae ST258/ST512 with KPC-2 orKPC-3)

Transfer of KPC-plasmids/transposons in other Enterobacteriaceae

Ertapenem-resistant

Mainly resistant to imipenem and meropenem (isolates of clonal lineages)

MIC imipenem >= MIC meropenem

PIP/TAZ-resistant

Phenotypical confirmation:

 mod. Hodge Test (Carba-NP-Test, MALDI-TOF)

 Disk-Tests with carbapenem + boric acid (KPC inhibitor)

AMP, ampicillin; PIP/TAZ, piperacillin/tazobactam; CTX, cefotaxime; CAZ, ceftazidime; ETP, ertapenem; MPM, meropenem; IPM, imipenem

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 31 OXA-48 Carbapenemase

Phenotype: AMP PIP/TAZ CTX CAZ IPM MPM ETP OXA-48 R R S/I S 0,5 - > 32 0,25 - > 32 R

Phenotype: AMP PIP/TAZ CTX CAZ IPM MPM ETP OXA-48 + ESBL R R R I/R 0,5 - > 32 0,25 - > 32 R

Common carbapenemase in K. pneumoniae, E. coli and other Enterobacteriaceae

Ertapenem-resistant

MIC imipenem >= MIC meropenem

PIP/TAZ-resistant

Susceptible to ceftazidime/cefotaxime (if there is no ESBL present!)

Resistance to !

Phenotypical confirmation  mod. Hodge Test (Carba-NP-Test, MALDI-TOF)

AMP, ampicillin; PIP/TAZ, piperacillin/tazobactam; CTX, cefotaxime; CAZ, ceftazidime; ETP, ertapenem; MPM, meropenem; IPM, imipenem

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 32 Example: Screening KPC + OXA-48

chromID® CARBA SMART

Study: Screening (rectal swabs) of patients with KPC-producer colonisation

KPC OXA-48

KPC/MBL OXA-48 selective selective

Red = KPC-2 E. coli Blue = KPC-2 K. pneumoniae Grey = KPC-2 C. freundii (outbreak strain)

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 33 Metallo-beta-lactamases

Phenotype: AMP PIP/TAZ CTX ATM IPM MPM ETP VIM/NDM R R R S 0,5 - > 32 0,25 - > 32 R

Common in Enterobacteriaceae (esp. E. cloacae, K. pneumoniae, K. oxytoca) and P. aeruginosa (VIM-type)

Ertapenem-resistant

MIC imipenem >= MIC meropenem

PIP/TAZ-resistant

Susceptible to aztreonam (if there is no ESBL present!)

Phenotypical confirmation

mod. Hodge Test (Carba NP test, Maldi-TOF etc.)

 MBL-Etest

 MBL-Disk tests with carbapenems + EDTA (MBL inhibitor)

AMP, ampicillin; PIP/TAZ, piperacillin/tazobactam; CTX, cefotaxime; CAZ, ceftazidime; ETP, ertapenem; MPM, meropenem; IPM, imipenem

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 34 Summary

• ESBL confirmation: Automated systems, Etest, disk-test are reliable tools • AmpC confirmation: Etest, disk-test, (automated systems) are reliable tools • No ESBL/AmpC confirmation is needed for A. baumannii/P. aeruginosa • Continuous problems: K. oxytoca (ESBL or K1 hyperproducer) low β-lactamase production (sensitivity) production of many β-lactamases

• Carbapenemase confirmation: Etest, disk-test (automated systems) are reliable tools • Continuous problems: mod. Hodge Test (low specifity) low carbapenemase production (sensitivity) no inhibitors for OXA carbapenemases

false-positive MBL-results A. baumannii/P. aeruginosa

Inconsistent results?  Confirmation of phenotype by molecular methods

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 35 Thank you

Robert Koch Institute Wernigerode:

Working group „Enterobacteriaceae and Nonfermenter“ Sibylle Müller-Bertling

National Reference Center Staphylococci (MRSA) / Enterococci PD Dr. Guido Werner

Robert Koch Institute Berlin Prof. Dr. Martin Mielke Dr. Tim Eckmanns

[email protected]

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 36