Scientific Reports on Harmonised Monitoring and Reporting of

Antimicrobial Resistance (AMR) in Food-producing Animals and Food:

Salmonella, Campylobacter, Indicator bacteria, MRSA

Pierre-Alexandre Beloeil Biological Monitoring Unit

7th EURL-AMR Workshop, Lyngby, Denmark, 4 April 2013

• AMR monitoring in Salmonella, Campylobacter + indicator E.coli, enterococci

• Focus on food-producing animal populations (vs animal species) and meat thereof:

o Prioritisation of certain combinations bacteria/animal populations (or food)

• Salmonella: laying hens, broilers, fattening turkeys, calves, fattening pigs

• Indicator E. coli: broilers, fattening pigs, calves

o Less common food-producing animal species (turkeys, ship and goat) threshold of production

• Relaxed monitoring (e.g. every 3rd year) for animal populations not primarily aimed at consumption or in which AMR is low laying hens, dairy cattle, breeding flock

Food animal populations and/or food products

Guiding principle: Potential exposure of consumers through food

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Harmonised sets of antimicrobial agents ‘First panel’

Salmonella Indicator E. coli • Ampicillin • Cefotaxime • Chloramphenicol • Ciprofloxacin • Gentamicin • Nalidixic acid • Streptomycin • Sulphonamides • Tetracycline • Trimethoprim* • Colistin • Ceftazidime • Meropenem • Florfenicol • Tigecycline •.Azithromycin

• Ampicillin • Cefotaxime • Chloramphenicol • Ciprofloxacin • Gentamicin • Nalidixic acid • Streptomycin • Sulphonamides • Tetracycline • Trimethoprim* • Colistin • Ceftazidime • Meropenem • Florfenicol • Tigecycline

• A two-step strategy including Salmonella and E. coli isolates resistant to ESC has been devised to characterise whether their phenotype is:

presumptive ESBL or AmpC or ESBL+AmpC or Carbapenemases

• For the purpose of harmonisation, the following criteria would apply:

Further characterisation of ESC resistant isolates

Stepwise strategy for testing isolates ‘microbiologically resistant’ to ESCs or meropenem:

• Detection of ESBL-/AmpC-producing E. coli in animals and food: o Use of selective (vs. non-selective) enrichment o Plating on selective agars: selective for ESBL E. coli vs. AmpC E. coli

• Further characterisation and classification of Salmonella and E. coli isolates showing resistance to third-generation cephalosporins and meropenem

• It is recommended as an optional measure to carry out a parallel quantification (enumeration) of the ESBL-/AmpC-producing E. coli in animal populations and food sector where a high prevalence has been observed.

o Enumeration allows the circumvention of any saturation effect issue in a high prevalence context and thus assessing temporal trends

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Monitoring of ESBL-/AmpC-producing E. coli

• Experts of the Working Groups: Marc Aerts, Bjorn Bengtsson, Sophie Granier,

Christopher Teale, Bernd-Alois Tenhagen, Benno Ter Kuile

• The EFSA Zoonoses Task Force and ECDC FWD-Net

• EURL on AMR

• EUCAST

• Peer reviewers

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Acknowledgments

Thank you for your attention!

Detection and confirmation of ESBL-, ampC and carbapenemase producing Enterobacteriaceae

EUCAST Subcommitee for Detection of Resistance

Mechanisms

Robert Skov and Christian Giske Statens Serum Institut / Karolinska University Hospital

and EUCAST

• EUCAST was asked by ECDC in 2012 to develop guidelines for detection of specific antimicrobial resistance mechanisms of clinical and/or epidemiological importance.

• The guidance will serve as a manual for EARS-Net and include: – Definition of the mechanisms. – Explanation of the clinical and/or public health need for detection

of the mechanisms. – An outline description of recommended methods of detection. – References to detailed descriptions of the methods.

Members of the SC • Christian G. Giske (Chair; Sweden, EUCAST and EARS-Net) • Luis Martinez-Martinez (Spain, EUCAST) • Rafael Canton (Spain, EUCAST) • Youri Glupczynski (Belgium) • Patrice Nordmann (France) • James Cohen-Stuart (Netherlands) • Marek Gniadkowski (Poland) • Vivi Miriagou (Greece) • Robert Skov (Denmark, EUCAST) • Stefania Stefani (Italy) • Mandy Wootton (UK) • Gunnar Skov Simonsen (Norway, EARS-Net) • Helena Zemlickova (Czech republic, EARS-Net)

Mechanisms and bacteria

• Extended-spectrum β-lactamase producing Enterobacteriaceae

• Acquired AmpC-producing Enterobacteriaceae • Acquired carbapenemases in Enterobacteriaceae • Methicillin-resistant S. aureus • Vancomycin low-level resistance in S. aureus

(VISA/heteroVISA) • Vancomycin-resistant enterococci • Penicillin non-susceptible S. pneumoniae

When should ESBL-testing be done?

Method Antibiotic Conduct ESBL-testing if

MIC

Broth or agar

dilution

Cefotaxime

or

Ceftazidime

MIC > 1 mg/L

Cefpodoxime MIC >1 mg/L

Disk diffusion

Cefotaxime (5 μg)

Ceftriaxone (30 μg)

Ceftazidime (10 μg)

Inhibition zone < 21 mm

Inhibition zone < 23 mm

Inhibition zone < 22 mm

Cefpdoxime (10 µg) Inhibition zone < 21 mm

cefoxitin ESBL SCREENING:

I/R to one or both of cefotaxime and ceftazidime, using EUCAST breakpoints

Group 1: E.coli, Klebsiella spp., P. mirabilis, Salmonella spp., Shigella spp.

Group 2: Enterobacteriaceae with inducible chromosomal AmpC: Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia stuartii, Serratia spp., Hafnia alvei.

Species dependent ESBL confirmation

Yes

No No ESBL

Method Antimicrobial agent (disk content) Confirmation is positive if

Broth microdilution

Cefotaxime +/- clavulanic acid (4 mg/L) MIC ratio ≥ 8

Ceftazidime +/- clavulanic acid (4 mg/L) MIC ratio ≥ 8

Cefepime +/- clavulanic acid (4 mg/L) MIC ratio ≥ 8

Etest ESBL

Cefotaxime +/- clavulanic acid MIC ratio ≥ 8 or deformed ellipse

present

Ceftazidime +/- clavulanic acid MIC ratio ≥ 8 or deformed ellipse

present

Combination disk

diffusion test (CDT)

Cefotaxime (30 µg) +/- clavulanic acid (10 µg) ≥ 5 mm increase in inhibition zone

Ceftazidime (30 µg) +/- clavulanic acid (10 µg) ≥ 5 mm increase in inhibition zone

Double disk synergy

test (DDST)

Cefotaxime, ceftazidime and cefepime disk +

amoxicillin-clavulanic acid disk

Expansion of indicator cephalosporin

inhibition zone towards amoxicillin-

clavulanic acid disk

Group 1: E.coli, Klebsiella spp., P. mirabilis, Salmonella spp., Shigella spp.

Method Antibiotic Confirmation is positive if

Etest ESBL Cefepime

+/- clavulanic acid

MIC ratio ≥ 8 or

deformed ellipse present

Combination disk

diffusion test

Cefepime (30 µg)

+/- clavulanic acid (10 µg) ≥ 5 mm increase in inhibition zone

Broth

microdilution

Cefepime

+/- clavulanic acid (4 mg/L) MIC ratio ≥ 8

Double disk

synergy test

(DDST)

Cefotaxime, ceftazidime and

cefepime disk + amoxicillin-

clavulanic acid disk

Expansion of indicator cephalosporin

inhibition zone towards amoxicillin-

clavulanic acid disk

Group 2: Enterobacteriaceae with inducible chromosomal AmpC: Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia stuartii, Serratia spp., Hafnia alvei.

cefoxitin ESBL SCREENING:

I/R to one or both of cefotaxime and ceftazidime, using EUCAST breakpoints

ESBL CONFIRMATION1 with ceftazidime and cefotaxime+/- clavulanic acid

Group 1: E.coli, Klebsiella spp., P. mirabilis, Salmonella spp., Shigella spp.

Group 2: Enterobacteriaceae with inducible chromosomal AmpC: Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia stuartii, Serratia spp., Hafnia alvei.

ESBL CONFIRMATION with cefepime +/- clavulanic acid

Species dependent ESBL confirmation

Negative: No ESBL Indeterminate2 Positive: ESBL Negative: no ESBL Positive: ESBL

Yes

No No ESBL

Indeterminate2

1If cefoxitin MIC > 8 mg/L, perform cefepime+/- clavulanic acid confirmation test 2Cannot be determined as either positive or negative (e.g. if the strip cannot be read due to growth beyond the MIC range of the strip.). 3Genotypic testing is required.

Control strains Strain Mechanism

K. pneumoniae ATCC 700603 SHV-18 ESBL

E. coli CCUG62975 CTX-M-1 group ESBL and acquired CMY AmpC

E. coli ATCC 25922 ESBL-negative

E. coli CCUG 58543 Acquired CMY-2 AmpC

E. coli CCUG62975 Acquired CMY AmpC and CTX-M-1 group ESBL

E. coli ATCC 25922 AmpC negative.

Algorithm for detection of plasmid-mediated AmpC

Cefotaxime R or ceftazidime R AND cefoxitin R1 Hos E. coli, K. pneumoniae, P. mirabilis, Salmonella spp,

Shigella spp

Cloxacillin synergy detected

Cloxacillin synergy

not detected

E. coli and Shigella spp: PCR is required to discriminate between plasmid-acquired and chromosomal AmpC

Other mechanisms (e.g. porin loss)

K.pneumoniae, P. mirabilis, Salmonella (lack chromo-

somal AmpC: plasmid- mediated AmpC detected

1 AmpC can also be present in isolates with positive ESBL-test (clavulanic acid synergy). Testing should therefore be done regardless of result in ESBL-testing.

When should screening be carried out?

Carbapenem MIC (mg/L) Disk diffusion (mm)

S/I breakpoint Screening cut off S/I breakpoint Screening cut off

Meropenem (10 µg)1 ≤2 >0.125 ≥22 <252

Imipenem (10 µg)3 ≤2 >1 ≥22 <23

Ertapenem (10 µg)4 ≤0.5 >0.125 ≥25 <25

1Best balance of sensitivity and specificity. 2In some cases OXA-48-producers have zone diameters up to 26 mm, so <27 mm may be used as a screening cut-off during outbreaks caused by OXA-48-producers, but with significant reduction in specificity. 3With imipenem the separation between the wild-type and carbapenemase-producers is relatively poor. Imipenem is therefore not recommended to use as a stand-alone screening test compound. 4High sensitivity, but low specificity, and therefore not routinely recommended.

Phenotypic confirmation methods

• For confirmation a range of inhibitor substances is used – DPA=dipicolinic acid (MBL, MBL + KPC) – EDTA=ethylenediaminetetraacetic acid (MBL) – APBA= aminophenyl boronic acid (KPC, ampC + porin loss) – PBA= phenyl boronic acid (KPC, ampC + porin loss) – CLX=cloxacillin (ampC + porin loss) – (Temocillin) – Oxa48 like

Algorithm for detection of carbapenemases

Meropenem <25 mm with disk- diffusion or MIC >0.12 mg/L

in all Enterobacteriaceae

Synergy with APBA/PBA only

Synergy with APBA/ PBA AND cloxacillin

Synergy with DPA only

KPC (or other class A carbapenemase)

AmpC (chromosomal and plasmid-acquired) AmpC plus porin loss

Metallo-beta- lactamase (MBL)

No synergy1

ESBL plus porin loss AND OXA-482

1 Combination of KPC and MBL can also produce no synergy. Normally these isolates will have very high resistance levels to carbapenems. They are easiest to detect with molecular methods. 2 High-grade temocillin resistance (>32 mg/L, temocillin (30 µg) zone diameter ≤11 mm) is a phenotypic indicator of OXA-48.

Phenotypic confirmation methods

β-lactamase

Synergy observed as increase in meropenem zone (mm)

with a 10µg disk

Temocillin MIC

> 32 mg/L

DPA/EDTA APBA/PBA DPA+APBA CLX

MBL ≥5 - - - NA1

KPC - ≥4 - - NA1

MBL+KPC2 Variable Variable ≥5 - NA1

OXA-48-like3 - - - - Yes

AmpC + porin loss - ≥4 - ≥5 NA1

ESBL + porin loss - - - - No

1 Temocillin is recommended only in cases where no synergy is detected, in order to differentiate between ESBL + porin loss and OXA-48-like enzymes. 2 There are no published reports with commercial disks or tablets containing double inhibitors (DPA plus APBA / PBA), and in-house tests have not been evaluated in multi-centre studies. This phenotype is rare outside of Greece and confers high-level resistance to carbapenems.

Control strains Strain Mechanism

E. cloacae CCUG 59627 AmpC + decreased porin expression

K. pneumoniae CCUG 58547 or K. pneumoniae NCTC 13439

Metallo-β-lactamase (VIM)

K. pneumoniae NCTC 13443 Metallo-β-lactamase (NDM-1)

E. coli NCTC 13476 Metallo-β-lactamase (IMP)

K. pneumoniae CCUG 56233 or K. pneumoniae NCTC 13438 K. pneumoniae carbapenemase (KPC)

K. pneumoniae NCTC 13442 OXA-48 carbapenemase

K. pneumoniae ATCC 25955 Negative control

ESBL detektion

AmpC detection

Carbapenemase detection

DPA MER

BOR CLX

DPA MER

BOR CLX

MBL KPC