amr monitoring according to dec 20013/652/ec: experiences ... · turkey 25.2 69.3 overall 36.8 49.6...
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AMR Monitoringaccording to Dec 20013/652/EC:Experiences from NRL-AR Italy
Antonio Battisti, DVMEURL-AR Meeting
Kgs Lyngby, 24-26 September 2014
A. Battisti 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
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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
A. Battisti 2014