Dr. Vicent Yusà. yusa_vic@gva.es / Vicent.Yusa@uv.es

CONTENTS

Introduction

European Framework

Organization of the Monitoring System in Spain

Sampling PlanSampling strategyRepresentativeness of samplingSampling procedures

Analytical Methods

Results (2014)

Conclusions

USE OFANTIMICROBIALS

IN FARMS

Presence of Antimicrobials

Residues in food at levels > MRL

AMRChemical hazard

BiologicalHazard

USE OF ANTIMICROBIALS: A FOOD SAFETY ISSUE

Very few non-compliant samples Low riskHigh budget

Raising threat for public health.

Very low budget

~ 25, 000 isolates (2014)

~123, 000 samples (2014)

Control of

Veterinary Residues in

Food

HORMONS

(88.600 samples)

β-AGONISTS

(41,300 samples)

ANTIBACTERIALS

(122,959 samples)

Prohibited substances

(80,570 samples)

Others

(100.392)

Monitoring of veterinary medicinal product residues in food (2014)

6600

18221593

2038

629367 342

208 179 89

0

1000

2000

3000

4000

5000

6000

7000

Pigs Bovines Poultry Sheep andgoats

Milk Rabbit meat Eggs Honey Aquaculture Horse

Number of total samples for antibacterial residues in food (Spain 2014). Total: 13,947

MONITORING OF ANTIBACTERIAL RESIDUES (B1) (Directive 96/23/EC)

EUROPE: 122,959 samples analysed for antibacterials; only 0.18 % non-compliant

Nº isolates for AMR: ~ 1000 (2014)

0,15

0,27

0,19

0,93

0,16

0,27

0

0,96

0

0

0 0,2 0,4 0,6 0,8 1 1,2

Pigs

Bovines

Poultry

Sheep and goats

Milk

Rabbit meat

Eggs

Honey

Aquaculture

Horse

Europe Spain

Non-compliant samples (%) for antibacterials (B1). 2014

MONITORING OF ANTIBACTERIAL RESIDUES (Directive 96/23/EC)

ANIMAL Substance Number samples

Non-compliant

BOVINES Amoxycillin (P) 628 2 (0.3 %)

Benzylpenicillin(Penicillin G)

6 1 (16.7%)

Doxycycline (T) 556 1 (0.2 %)

Enrofloxacin (FQ) 596 1 (0.2%)

Oxytetracycline (T) 642 2 (0.3%)

PIGS Amoxicillin (P) 1910 1 (0.1%)

Doxycycline (T) 1947 7 (0.4 %)

Lincomycin (L) 1314 1 (0.1%)

Oxytetracycline (T) 1948 1 (0.1%)

POULTRY Doxycycline (T) 290 2 (0.7 %)

Enrefloxacin (FQ) 250 1 (0.4 %)

MILK Spiramycin (M) 319 1 (0.3%)

Antibacterials (B1) detected in non-compliant samples . Spain, 2014

THERE IS A LOW EXPOSURE TO ANTIMICROBIALS RESIDUES FROM THE DIET LOW RISK FOR THE GENERAL POPULATION

CONTROL OF VETERINARY RESIDUES IN FOOD

(Directive 96/23/EC)

AMR in food(Decision 2013/652/EU)

ANTIMICROBIALS IN FOOD SAFETY

FARM ABATTOIR RETAIL

Modules of the food (meat) chain

ANTIBIOTICS7982 tonnes

/year (EU)

Antibiotic Resistant Bacteria in animals and food

Foodborne pathogens: Salmonella

Campylobacter

Indicator bacteria:

E.Coli; Enterococci

How muchFOOD CHAIN

contributesto

resistance transfer

from animals to

human?

Reservoir of resistance genes with potential for transferring resistance to pathogenic bacteria.

Permit a more accurate study of the trends and the effects of patterns of antimicrobials use (all animal carry them)

Infection agents transferred from animals to human

One Health

ANTIBIOTICS in humans: 3399 tonnes per year (EU)

RESISTANT BACTERIA IN HUMANS (≥25 % o more)25.00 people die each year in Europe

The link is well documented

European Surveillance of Veterinary Antimicrobial Consumption (ESVAC). October 2016population correction unit (PCU)

SALES

AMR monitoring should cover both zoonotic agents (Salmonella and Campylobacter, and indicator organisms of the commensal flora

.

Commensal bacteria that contaminate food may be also considered a potential AMR hazard, as they can harbour transferable resistance genes

LEGAL AND TECHNICAL REFERENCES

Legal Reference Title

Reg. 882/2004 Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules

Dec. 2013/652/EU 2013/652/EU: Commission Implementing Decision of 12 November 2013 on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria

Dir. 2003/99/EC Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents, amending Council Decision 90/424/EEC and repealing Council Directive 92/117/EEC

Reg. 2073/2005 Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs

EU framework

EFSA Guidance Documents

EFSA. 2012 - Technical specifications on the harmonised monitoring and reporting of antimicrobial resistance in Salmonella, Campylobacter and indicator Escherichia coli and Enterococcus spp. bacteria transmitted through food.In EFSA Journal. http://www.efsa.europa.eu/en/efsajournal/pub/274.htm

EFSA. 2012 - Technical specifications for the analysis and reporting of data on antimicrobial resistance (AMR) in the European Union Summary Report.In EFSA Journal. http://www.efsa.europa.eu/en/efsajournal/pub/2587.htm

EFSA. 2014 - Technical specifications on randomised sampling for harmonised monitoring of antimicrobial resistance in zoonotic and commensal bacteria.In EFSA Journal. http://www.efsa.europa.eu/en/efsajournal/pub/3686.htm

EFSA. 2015 - Data dictionaries-guidelines for reporting data on zoonoses, antimicrobial resistance and food-borne outbreaks using the EFSA data models for the Data Collection Framework (DCF) to be used in 2015, for 2014 data.In EFSA. http://www.efsa.europa.eu/en/supporting/doc/776e.pdf

EFSA. 2015 - Manual for reporting on antimicrobial resistance within the framework of Directive2003/99/EC and Decision 2013/652/EU for information deriving from the year 2014.In EFSA. http://www.efsa.europa.eu/en/supporting/pub/771e.htm

PREVALENCE OF FODDBORNE AMR

DETERMINATION OF TRENDS AND SOURCES OF

AMR

DATA FOR CONDUCTING RISK ANALYSIS

(humans and animals)

INFORMATION FOR EVALUATING AM

PRESCRIPTIONS AND PRACTICES

MONITORING OF AMR IN

FOOD

EU framework

HARMONIZED MONITORING

of AMR

COMBINATIONS

OF

BACTERIAS

ANIMALS/FOODS

SUSCEPTIBILITY TESTING METHOD

(phenotypic methods; panel

of antimicrobials)

SAMPLING PLANS

REPORTING OF THE DATA

Decision 2013/652/EU

EU framework

Randomised sampling strategy

(reduction of sampling bias)

Representativeness

(data from a sample reflect

the population under study)

Stratified

sampling approach

(allocation of the sample numbers per strata: slaughterhouses, NUTS-3,

laboratories)

Sampling time

Even distribution of collected samples (quarters)

Sampling strategy(Prospective sampling/Retrospective

sampling)

QUALITY OF THE SAMPLING

Ministry of

Agriculture, Food

and Environment

(MAGRAMA)

Ministry of Health

(MSSSI)

SGSE (PIFs)

AGRICULTURAL DEPARTMENT OF

AACC

HEALTH DEPARTMENT OF

AACC

AUTONOMOUS

COMMUNITIES

(AACC)

Official control of primary production and food quality

Official control of food safety after primary production

Sallmonella andESBL-E.Coli

sampling at slaughterhouses

and retail

Monitoring of AMR in primary production including caecalsamples at slaughterhouses

ORGANIZATION OF THE MONITORING SYSTEM

CENTRAL AUTHORITIES

COMPULSORY SAMPLING PLAN 2014-2020(domestic production)

Year Animal populations/Type of meat

Where to collect Salmonella(number of ISOLATES)

ESBL-or AmpC- producing E.Coli(number of samples)

2014, 2016, 2018, 2020

Broilers Slaughterhouse Carcases (170)

Retail Fresh meat (300)

Fattening turkeys

Slaughterhouse Carcases (170)

2015,2017, 2019

Fattening pigs Slaughterhouse Carcases (170)

Retail Meat (300)

Calves under 1 year

Slaughterhouse Carcases (170)

Retail Meat (300)

SALMONELLA isolates from carcasses

at slaughterhouses(≥60% of the specific

processed population)

ESBL-AmpC producing E. COLI

from meat at retail

Retrospectivesampling strategy

Isolates from official samples

collected to verify FBO compliance

withProcess Hygiene

Criteria(R 2073/2005)

AntimicrobialSusceptibility Testing (AST)

Sampledby FBO

(R. 2073/2005)

FBO: food business operators

Collected by Competent Authorities

If not sufficient

Prospective sampling plan

Fresh meat (chilled meat) including wrapped,

vacuum-wrapped/controlled

atmosphere

SAMPLING STRATEGY

Food category Micro-organisms

Sampling plan (1)

Limits Analytical referencemethod

Stage where thecriterion applies

2.1.3 Carcasses of cattle

Salmonella n=50 (5)c=2 (6)

Absence in the area tested per carcase

EN/ISO 6579 Carcases after dressingBut before chilling

2.1.4. Carcases of pig

Salmonella n=50 (5)c=5 (6) Absence in the

area tested per carcase

EN/ISO 6579 Carcases after dressingbut before chilling

2.1.5 Poultry carcases of broilers and turkeys

Salmonella n=50 (5)c=7 (6) Absence in 25

g of a pooled sample of neck skin

EN/ISO 6579 Carcases after chilling

(1) n = number of units comprising the sample; c = number of sample units giving values between m and M.(5) The 50 samples are derived from 10 consecutive sampling sessions in accordance with the sampling rules and frequencies laid down in this Regulation ; (6) The number of samples where the presence of salmonella is detected. The c value is subject to review in order to take into account the progress made in reducing the salmonella prevalence. Member States or regions having low.

SAMPLING at SLAUGHTERHOUSES (R. 2073/2005)

Isolation of Salmonella

EN/ISO 6579

SALMONELLA

Send to

AntimicrobialSusceptibility Testing (AST)(ISO 20776)

Public Health Laboratories of the AACC ISO 17025

National Reference Laboratory (CNA)(ISO 17.025)

SEROTYPES (Typhimurium, Enteritidis,…)

Broilers, turkeys.

Pig and cattle

SAMPLING IN ABATTOIR(ISO 17604)

ISOLATION (EN/ISO 6579)

GEOGRAPHICAL DISTRIBUTION OF SAMPLES OF PIG AND CATTLE CARCASES (300 + 300)

13896

100

116

32

24

24

24

22

24

The 600 samples from slaughterhouses distributed between 13 provinces (NUTS-3) that represent more than 60 % of total animal production(should cover at least 60% of the animal production)

National Reference Laboratory (CNA)(ISO 17.025)

Samples collected at retail byAACC inspectors

Send to NRL

< 24 hTransport : +2 ºC/ +8ºCBefore Date of ExpireEach sample from different lot

ESBL- E. Coli

SAMPLING at retail

Isolation (McConkey + cefotaxime) and specific identification

ESBL: extended spectrum β-lactamase

Antimicrobial Susceptibility Testing (AST)

GEOGRAPHICAL DISTRIBUTION OF ESBL-E.Coli FROM RETAIL (300 samples)

69

48

50

58

16

12

12

12

1212

The 300 samples from retail distributed between 13 provinces (NUTS-3) that represent more than 60 % of total population(should cover at least 80% of the population)

SAMPLING PROCEDURE FOR POULTRY CARCASES

EXAMPLES OF SAMPLING SESSIONS

CARCASES AFTER CHILLING

< 3.5 ºC

Carcasses shall be sampled at random during each sampling session and after chilling

1 sample: 25 g of a pooled sample of neck skin

Each sample contains neck skin from 3 carcases from the same flock (Epidemiological unit)

A minimum of 15 carcases shall be sampled at random during each sampling session

A piece of approx. 10 g from each carcase

Each sampling session includes 5 samples (n=5) from different flocks

Transport the samples in an insulated box with frozen freezer blocks

Transport: 1-8 ºC

Put the container with the sample in an plastic bag and close it with the official seal

NON-DESTRUCTIVE SAMPLING METHOD (ISO 17604): Abrasive sponge sampling

SAMPLING KIT

Peptone water (25 mL)

Before sampling

Moist the sponge in 10 mL of peptone water

After sampling

Pour the rest of the peptone water

SAMPLING PROCEDUE FOR PIGS

spongeSquare template

SAMPLING SITES(ISO 17604)

NON-DESTRUCTIVE SAMPLING METHOD (ISO 17604): Abrasive sponge sampling

Five carcases shall be sampled at random during each sampling session.Sampling sites (4) should be selected taking into account the slaughter technology used in each plant and the recommendations of ISO 17604

Template: 100 cm2

Press the template hard on to the surface.

Rub the sponge over the whole area 10 times in each direction (horizontally and vertically)

More than one site may be sampled using the same swab

NON-DESTRUCTIVE SAMPLING METHOD (ISO 17604): Abrasive sponge sampling

Five carcases shall be sampled at random during each sampling session.Sampling sites (4) should be selected taking into account the slaughter technology used in each plant and the recommendations of ISO 17604

ANALAYTICAL METHODS IN ROUTINE MONITORINGCLSI method (ISO 20776-1:2006)

Antimicrobial susceptibility testing (AST)

Antimicrobial susceptibility testing is performed with phenotypic methods.

The basis of phenotypic methods is the Minimum Inhibitory Concentration (MIC, mg/l).

DILUTION procedures are the reference methods for AST

DILUTION METHOD shall be performed according with ISO 20776-1:2006

Panel 1 of antimicrobial substances to be included in AMR monitoring

Epidemiological cut-off (mg/L)

ANTIMICROBIAL Salmonella E.Coli

Ampicillin >8 >8

Cefotaxamine >0,5 >0,25

Ceftazidime >2 >0.5

Meropenem >0.125 >0.125

Nalidixic acid >16 >16

Ciprofloxacin >0.064 >0.064

Tetracicline >8 >8

Colistin >2 >2

Gentamicin >2 >2

Trimethoprim >2 >2

Sulfamethoxazole NA >64

Chloramphenicol >16 >16

Azithromycin NA NA

Tigecycline >1 >1

Stepwise strategy for testing for specific monitoring of ESBL-producing Salmonella and E.Coli

14 antimicrobials

8antimicrobials

REPORTING

Data Collection Framework(EFSA)

Submission of data in the form of raw isolate-data in the electronic collection form provided by EFSA.The submitted reports include the compulsory information for each individual isolate

RESULTS (2014)

132 isolates from broilers 101 res ≥1 31 sus to all

Salmonella spp.- S. Kentucky-S. Enteritidis-S. Anatum

resistance rate (%)

RESULTS (2015)

128 isolates from pigs 104 res ≥1 24 sus to all

Salmonella spp.- S. Typhimurium-S. Derby-S. Rissen

Resistance rate (%)

RESULTS (2015)

E.Coli

ESBL: 27 (all res ≥1 )ESBL: 36 (all res ≥1)

301 isolates from pig meat299 isolates from bovine meat

resistance rate (%)resistance rate (%)

TAKE HOME MESSAGES

The sales of antibiotics in Spain for food producing animals are the highest in Europe (29) and need to be reduced

To ensure representativeness, the sampling program should be distributed evenly over the year

The samples taken at retail should include the necessary information to exclude isolates from the same repeated epidemiological unit (batch of product)

Increase the number of official labs accredited for AST

Increase the participation of food business operators with isolates from their own control

Include more zoonotic and commensal bacterial in the food monitoring program

Reduce ANTIMICROBIAL residues control, and INCREASE AMR monitoring