AMR Monitoringaccording to Dec 20013/652/EC:Experiences from NRL-AR Italy

Antonio Battisti, DVMEURL-AR Meeting

Kgs Lyngby, 24-26 September 2014

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IZSLT Rome, NRL-AR Italy aims1

• Veterinary Public Health Institution under the Ministryof Health

• As a government institution, involved in monitoring of zoonotic pathogens, indicator bacteria, animalpathogens in:

• Primary production (priority)• Food of animal origin• Companion animals (pets, horses, etc.)

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IZSLT Rome, NRL Italy aims2

Some activities on AMR monitoring:• Transferable resistance-Beta-lactamases: ESBLs, and AmpC like: e. g. CMY-2; -Plasmid mediated Quinolone R in Salmonella, E. coli,

Klebsiella etc.• R determinants spill over through clonal spread(e. g. Campylobacter: Macrolide, FQs and

Amynoglycoside R, some Salmonella serotypes, MRSA)

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Emerging issues in AR in Enterobacteriaceae (e. g. Salmonella, E. coli, Klebsiella)

Transferable resistance

• Beta-lactamases: ESBLs, AmpC like, Carbapenemases

• Plasmid mediated Quinolone R (PMQR)

• Carbapenemases…

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ESBL/AmpC: phenotype-based screening

How it was….MIC screening : EUMVS(2) panel, EUCAST ecoffs

• Salmonella (Ceftazidime=2 mg/L; Cefotaxime =0.5 mg/L)

• E. coli (Ceftazidime=0.5 mg/L; Cefotaxime =0.25mg/L)

• [ESBLs phenotype confirmation by DD synergytest, CLSI standards]

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Molecular confirmation of ESBL/AmpC resistance

• PCR for CTX-M groups (ALL known CTX-Ms), SHV and TEM groups

• Then “sub-group-specific” PCR, with sequencing• AMPC-like investigated when phenotype suspicious

(i.e. difference inhib Ø CTX and Ø inhib CTX-CLA <5mm; Cefoxitin R);

• Multiplex PCR (MOX, LAT, CMY, ACC, DHA, BIL, MIR,

FOX…) or specific PCRs (e. g. CMY-2 )

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Further molecular typing

• Inc Group determination (PCR-based Replicon Typing approach);

• Plasmid analysis and transferability assays:-Plasmid DNA preparations used to transformcompetent cells.-Identification of the plasmid(s) carrying the ESBLsor AmpC genes, by selection of the transformantson selective agar plates.

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How it is now…

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2014: Study Design in broilers & turkeys at slaughter

• Six Regions involved (>90% productions in Italy)• Stratification of total number of samples (one sample per flock) by:

-Region (production)-Slaughterhouse (capacity)

Broiler capi % per Regione N campioni Regione N medio campioni mese N medio campioni meseVeneto 198658064 45 379 32 42Emilia-Romagna 85048112 19 162 14 18Lombardia 58806334 13 112 9 12Marche 44540473 10 85 7 9Abruzzo 37667713 8 72 6 8Piemonte 20785544 5 40 3 4Totale Top Regioni 445506240 100 850 71 94

Totale Italia 479053581

% Top regioni su totale Italia92,99716309

Tacchini capi % per Regione N campioni Regione N medio Campioni mese N medio Campioni meseVeneto 18493659 62 528 44 59Emilia-Romagna 8073168 27 230 19 26Lombardia 3206473 11 92 8 10Toscana 2059213 7 59 5 7Totale Top Regioni 29773300 100 850 76 101

Totale Italia 31968859

% Top regioni su totale Italia93,13219468

-850 samples from broiler flocks

(300 for ESBL/AmpC/CPEs)

-850 samples from turkey flocks

(300 for ESBL/AmpC/CPEs)

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Sampling & shipping activity

• N.10 caeca per broiler flock sampled;• N. 3 caeca per turkey flock sampled;• Shipped and delivered by courier service with 24

hours from sampling• Metadata from the National Register of Holdings;

other data entered by the Vet Services in an online form.(SINVSA web system, Ministry of Health, Vet Department, collaboration with IZSAM, Teramo)

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Printout of the sampling form

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CTX-MAC*, LAC+ve colonies 22h incubation

* CTX-MAC: Mac Conkey Agar (EP) + 1mg/L cefotaxime

Cultures caecal samples BPW on selective chromogenic agar biplate forCarbapenemases (SCA)

Subcultures from SCA on MAC (LAC+ve & LAC-ve)

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LAC+ve on MAC (see previous picture) not so obviuosly “RED” con chromogenic biplate

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Animal Species % pos C. jejuni % pos C. coli

Broiler chicken 45.4 35.0 Turkey 25.2 69.3 Overall 36.8 49.6

Some experiences on Campylobacter at the NRL-AR Italy…

Campylobacter jejuni & C. coli isolation rates

Statistically significant differences (same lab procedures, same personnel etc. …)

Chi-square p<0.001 for each combination animal species/agent

Practical information for future sampling size (exp. prevalences…)

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Thank you for attention!Special thanks to all personnel at the Diagnostic Dept.,

IZSLT, NRL-AR Italy

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