European Food Safety Authority (EFSA), Pierre-Alexandre Beloeil, Beatriz Guerra and Anca-Violeta Stoicescu

TECHNICAL REPORT APPROVED: 25 January 2018 doi: 10.2903/sp.efsa.2018.EN-1369 Manual for reporting on antimicrobial resistance within the framework of Directive 2003/99/EC and Decision 2013/652/EU for information derived from the year 2017 European Food Safety Authority (EFSA), Pierre-Alexandre Beloeil, Beatriz Guerra and Anca-Violeta Stoicescu Abstract This manual provides guidance for reporting antimicrobial resistance under the framework of Directive 2003/99/EC and Commission Implementing Decision 2013/652/EU in food-producing animals and foodstuffs derived thereof. The objective is to harmonise and streamline the reporting made by the Member States to ensure that the antimicrobial resistance data collected are relevant and easy to analyse at the European Union level. Detailed guidelines are provided for the reporting of data and text forms. This guidance typically applies to Salmonella spp., Campylobacter coli and Campylobacter jejuni, and the animal populations and food categories to be reported on. Guidance is also provided on indicator commensal Escherichia coli, indicator Enterococcus and meticillin-resistant Staphylococcus aureus. The manual notably includes specific guidance for reporting mandatory data on Salmonella spp. and commensal indicator E. coli producers of ESBLs/AmpCs/carbapenemases obtained from the harmonised routine monitoring, and ESBL-/AmpC-/Carbapenemase-producing E. coli derived from specific monitoring, as well as voluntary data on specific monitoring of carbapenemase-producers. This manual is specifically aimed at guiding the reporting of information deriving from the year 2017. European Food Safety Authority, 2018 Key words: antimicrobial resistance, zoonotic bacteria, indicator bacteria, MRSA, reporting Requestor: European Food Safety Authority Question number: EFSA-Q-2017-00809 Correspondence: zoonoses_support@efsa.europa.eu www.efsa.europa.eu/publications EFSA Supporting publication 2018:EN-1369

Manual for reporting on 2017 antimicrobial resistance data Acknowledgements: EFSA wishes to thank the members of the Scientific Network for Zoonoses Monitoring Data who reviewed this report. Suggested citation: EFSA (European Food Safety Authority), Beloeil P-A, Guerra B and Stoicescu A V, 2018. Manual for reporting on antimicrobial resistance within the framework of Directive 2003/99/EC and Decision 2013/652/EU for information derived from the year 2017. EFSA supporting publication 2018:EN-1369. 27 pp. doi:10.2903/sp.efsa.2018.en-1369 ISSN: 2397-8325 European Food Safety Authority, 2018 Reproduction is authorised provided the source is acknowledged. www.efsa.europa.eu/publications 2 EFSA Supporting publication 2018:EN-1369

Summary This manual provides guidance on reporting antimicrobial resistance (AMR) under the framework of Directive 2003/99/EC and Commission Implementing Decision 2013/652/EU in food-producing animals and foodstuffs derived thereof. The objective is to harmonise and streamline the reporting made by the Member States (MSs) to ensure that the data collected are relevant and easy to analyse at the European Union (EU) level. Detailed guidelines are provided for the reporting of data and text forms in extensible Markup Language (XML) files, through the Data Collection Framework (DCF) of the EFSA. This manual typically applies to the bacterial agents, antimicrobial substances, animal populations and food categories to be reported. Instructions are given on the description of the sampling and monitoring schemes, as well as on the analyses of the results in the national reports. This manual specifically covers Salmonella, Campylobacter coli, C. jejuni, indicator commensal Escherichia coli, indicator Enterococcus and MRSA, as included in the current data collection. These instructions are applicable to reporting national data on AMR in bacterial agents and national overview information on the general evaluation of the AMR situation and the process of monitoring AMR in relative text forms, through the DCF. Specific guidance is also included for reporting on the prevalence, genetic diversity and AMR of meticillin-resistant Staphylococcus aureus (MRSA) from food-producing animals and food derived thereof. The manual incorporates specific guidance for the mandatory reporting of data on Salmonella spp. and commensal indicator E. coli producers of ESBLs/AmpC/carbapenemases obtained from the harmonised routine monitoring, and data on ESBL-/AmpC-/carbapenemase-producing E. coli derived from the specific monitoring as well as the voluntary reporting of data on the specific monitoring of carbapenemase-producers.this manual is aimed specifically at Member States (MSs) data providers to guide the reporting of information deriving from the year 2017. Technical information about AMR data reporting can be found in the Data dictionaries guidelines for reporting 2017 data on zoonoses, antimicrobial resistance and food-borne outbreaks. www.efsa.europa.eu/publications 3 EFSA Supporting publication 2018:EN-1369

Table of contents Abstract...1 Summary...3 Table of contents...4 1. Introduction...5 1.1. Background and Terms of Reference as provided by EFSA...5 1.2. Purpose of this manual...6 2. Monitoring and reporting antimicrobial resistance in bacteria from food-producing animals and food...6 3. Guidelines for reporting overview information in text forms...6 3.1. General evaluation of antimicrobial resistance situation...7 3.2. General description of antimicrobial resistance monitoring...7 3.2.1. Description of sampling design and strategy...8 3.2.2. Stratification procedure per animal population and food category...9 3.2.3. Randomisation procedure per animal population and food category...9 3.2.4. Analytical method used for detection and confirmation...9 3.2.5. Laboratory methodology used for detection of antimicrobial resistance...9 3.2.6. Results of investigation...9 3.2.7. Additional information...9 4. Guidelines for reporting antimicrobial resistance data...9 4.1. General recommendations...9 4.2. Reporting antimicrobial resistance data on Salmonella spp....10 4.2.1. Routine monitoring of antimicrobial resistance in Salmonella spp....10 4.2.2. Monitoring of presumptive ESBL-, AmpC- or carbapenemase-producing Salmonella derived from the routine monitoring...11 4.3. Reporting antimicrobial resistance data on Campylobacter spp...12 4.4. Reporting antimicrobial resistance data on indicator E. coli...12 4.4.1. Routine monitoring of antimicrobial resistance in E. coli...12 4.4.2. Monitoring of presumptive ESBL-, AmpC- or carbapenemase-producing E. coli derived from the routine monitoring...13 4.4.3. Monitoring of ESBL-, AmpC- or carbapenemase-producing E. coli derived from either routine monitoring or specific monitoring...13 4.5. Reporting data on antimicrobial resistance in indicator commensal Enterococcus spp...15 5. Guidelines for validating data before reporting...16 5.1. Validation against business rules...16 5.2. Scientific data validation...16 6. Guidelines for reporting meticillin-resistant Staphylococcus aureus data...17 6.1. General recommendations...17 6.2. Meticillin-resistant Staphylococcus aureus in animals and food...17 6.3. Reporting data on antimicrobial resistance in MRSA...19 References...20 Abbreviations...21 Appendix A General definitions...22 Appendix B Analytical methods for susceptibility testing...23 Appendix C Data reporting requirements...26 www.efsa.europa.eu/publications 4 EFSA Supporting publication 2018:EN-1369

1. Introduction 1.1. Background and Terms of Reference as provided by EFSA EFSA s mandates for the production of annual European Union (EU) Summary Reports (EUSRs): 1) on zoonoses and food-borne outbreaks, 2) on antimicrobial resistance (AMR) and on 3) Transmissible Spongiform Encephalopathies (TSE) are described in a unifying charter. 1 These mandates from the European Commission (EC) to EFSA also require the production of reporting manuals which need regular updating. The production of the EUSR on zoonoses and food-borne outbreaks as well as the EUSR on AMR is underpinned by the Directive 2003/99/EC 2 laying down the EU system for monitoring and reporting of information on zoonoses, which obligates the MSs to collect data on zoonoses, zoonotic agents, AMR and food-borne outbreaks. EFSA is assigned the tasks of examining the data collected and preparing the EUSRs in collaboration with the European Centre for Disease Prevention and Control (ECDC). Based on the data reported each year, EFSA and ECDC jointly produce an annual EUSR on zoonoses, zoonotic agents and food-borne outbreaks. Similarly, the two agencies will produce an annual EUSR on AMR. To support the MSs in their reporting, the existing reporting manuals for zoonoses, AMR and food-borne outbreaks need to be updated to take into account the latest recommendations and changes in the relevant EU legislation on reporting of AMR data and data on zoonoses and food-borne outbreaks. The production of the EUSR on TSE is underpinned by the Regulation (EC) No 999/2001 3 laying down laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. EFSA is assigned the tasks of data collection, data validation and preparing the EUSR on TSE. EFSA manages a Data Collection Framework (DCF), to which MS have the possibility of submitting data in Extensible Markup Language (XML). New XML reporting schemas are created before the start of the reporting period in April each year, and these schemas are described in reporting manuals. To successfully run and complete these recurrent three annual projects the BIOCONTAM and Evidence Management (DATA) units need to meet the following objectives: produce and publish: 1. The joint annual EUSR on Trends and Sources of Zoonoses, Zoonotic Agents and Foodborne Outbreaks, 2. The joint annual EUSR on AMR in zoonotic and indicator bacteria from humans, animals and food, and 3. The annual EUSR on TSE. produce and publish the National Reports on Zoonoses, Food-borne Outbreaks and AMR, containing raw data and statistics reported to EFSA by MSs (and non-mss); implement annual development improvements aimed at efficiency gains as regards IT, data collection (data models and related tools) and data analyses, so as to shorten the production cycle of these EUSRs. Support MSs in their activities of monitoring and reporting on zoonoses and AMR in animals and food, and TSE, and on food-borne outbreaks, as necessary. The Terms of Reference prescribes the delivery of six technical reports: Manual for reporting on zoonoses and zoonotic agents, within the framework of Directive 2003/99/EC, and on some other pathogenic microbiological agents for information derived from the year 2017 1 Available online: http://registerofquestions.efsa.europa.eu/roqfrontend/questionslistloader?mandate=m-2015-0231 2 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. OJ L 325, 12.12.2003, p. 31 40. 3 Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. OJ L 147, 31.5.2001, p. 1 40. www.efsa.europa.eu/publications 5 EFSA Supporting publication 2018:EN-1369

Manual for reporting on antimicrobial resistance within the framework of Directive 2003/99/EC and Decision 2013/652/EU for information derived from the year 2017 Data dictionaries guidelines for reporting 2017 data on zoonoses, antimicrobial resistance and food-borne outbreaks Data dictionaries guidelines for reporting 2017 prevalence sample based data in accordance with SSD2 data model User manual for mapping Member State zoonoses standard terminology to EFSA standard terminology for information derived from the year 2017 Data dictionaries guidelines for reporting surveillance data on Transmissible Spongiform Encephalopathies (TSE) in EU within framework of Regulation 999/2001/EC 1.2. Purpose of this manual The present manual provides primarily scientific guidance on reporting overview information and data on AMR pertaining to the 2017 data collection through the DCF (https://dcf.efsa.europa.eu/dcfwar/dc). Separate guidelines are also given on the technical details of the DCF reporting system elsewhere (EFSA, 2018). Quantitative isolate-based AMR data for the year 2017 should be submitted by the MSs to the EFSA Data Collection Framework (DCF) using XML files only by 31 May 2018. In addition, national overview information should be reported through narrative parts using the word template. 2. Monitoring and reporting antimicrobial resistance in bacteria from food-producing animals and food Directive 2003/99/EC on the monitoring of zoonoses and zoonotic agents set out generic requirements for the monitoring and reporting of AMR in isolates of zoonotic Salmonella spp. and Campylobacter spp., as well as in selected other bacteria as far as they present a threat to public health from food-producing animals and food in the EU MSs. Within the framework of AMR monitoring in foodproducing animals and food, the occurrence of AMR is typically defined as the proportion of bacterial isolates tested for a given antimicrobial and found to present reduced susceptibility, i.e. to display microbiological resistance. Epidemiological cut-off values (ECOFFs) are used as interpretative criteria of microbiological resistance. The AMR monitoring in food-producing animals and food was revised by Commission Implementing Decision 2013/652/EU implementing Directive 2003/99/EC which set out monitoring priorities from a public health perspective and described those combinations of bacterial species, antimicrobial substances, food-producing animal populations and food products which should be monitored from 2014 onwards, including the frequency with which monitoring should be performed. Since the implementation of this Commission Decision, the monitoring of AMR in zoonotic Salmonella spp. and Campylobacter jejuni, as well as in indicator E. coli from the major food-producing animal populations domestically-produced has become mandatory. The monitoring of AMR in zoonotic organisms focused on the animal populations to which the consumer is most likely exposed through food derived thereof, such as domestic fowl (mainly broilers), pigs and cattle. The Commission Implementing Decision 2013/652/EU ensures that all reporting countries submit data for a common core set of antimicrobials and bacteria. It consists of a concise set of substances selected according to their relevance to human therapeutic use (e.g. critically important antimicrobials (CIAs) with highest priority for human medicine) and/or of epidemiological relevance. Data for these combinations should therefore be comprehensive. 3. Guidelines for reporting overview information in text forms National overview information should be reported through narrative parts including concise but complete and comprehensible descriptions of the general evaluation of the AMR situation and the implementation of the AMR monitoring programme from which the reported data on AMR, www.efsa.europa.eu/publications 6 EFSA Supporting publication 2018:EN-1369

MRSA, ESBL, AmpC and carbapenemase derive. The reporting of overview information ensures that data/results are analysed, understood and interpreted correctly. The description should be detailed enough to give an accurate picture of the situation and the monitoring activities in place and to facilitate, where possible, the comparison of the results between different reporting years and countries. The harmonised text forms, whose titles are provided in the word template listed below, should be used to report corresponding information and draft the narrative parts of the national report. 3.1. General evaluation of antimicrobial resistance situation This text form is not mandatory to be filled in, but MSs are still recommended to provide a general evaluation of the national situation on AMR using the paragraphs presented in Table 1. Table 1: Recommended paragraphs to report general evaluation of the national situation on foodborne antimicrobial resistance No Paragraph 1 Situation and epidemiological evolution (trends and sources) regarding antimicrobial resistance (AMR) to critically important antimicrobials (a) (CIAs) over time until recent situation 2 Public health relevance of the findings on food-borne AMR in animals and foodstuffs 3 Recent actions taken to control AMR in food-producing animals and food 4 Any specific action decided in the Member State or suggestions to the European Union for actions to be taken against food-borne AMR threat 5 Additional information (a): The CIAs depends on the bacterial species considered and the harmonised set of substances tested within the framework of the harmonised monitoring: for Campylobacter spp., macrolides (erythromycin) and fluoroquinolones (ciprofloxacin); for Salmonella and E. coli, 3rd- and 4th-generation cephalosporins (cefotaxime) and fluoroquinolones (ciprofloxacin) and colistin (polymyxin); 3.2. General description of antimicrobial resistance monitoring Under this text form title, the general description of the national programme for AMR monitoring should be provided using the paragraphs presented in Table 2. The paragraphs Description of sampling designs, Stratification procedure per animal population and food category and Randomisation procedure per animal population and food category are mandatory to be reported, as required by Commission Implementing Decision 2013/652/EU. Table 2: Recommended/mandatory paragraphs to report a general description of antimicrobial resistance monitoring No Paragraph [To be filled in per combination of bacterial species/matrix ] 1 Description of sampling design and strategy (a) 2 Stratification procedure per animal population and food category 3 Randomisation procedure per animal population and food category 4 Analytical method used for detection and confirmation (b) 5 Laboratory methodology used for detection of antimicrobial resistance (c) 6 Results of investigation 7 Additional information (a): Method of sampling (description of sampling technique: stage of sampling, type of sample taken, sampler), Frequency of sampling, Procedure of selection of isolates for susceptibility testing, Method used for collecting data should be addressed. (b): Analytical method used for detection and confirmation: according to the legislation, the protocols developed by the EURL AR should be used and reported here. In the case of the voluntary specific monitoring on carbapenemase-producers, the selective media used (commercial plates, in house media) should be also reported here. In general, any variation with regard to the EURL-AR protocols should be stated here, as well as the number of isolates isolated per sample, in particular for Campylobacter spp. Analytical methods for susceptibility testing are present in Appendix B of this document. (c): Antimicrobials included and cut-off values used for interpreting resistance should be addressed. Antimicrobial substances, interpretative thresholds and concentration ranges to be used for testing are presented in Appendix B of this document. www.efsa.europa.eu/publications 7 EFSA Supporting publication 2018:EN-1369

3.2.1. Description of sampling design and strategy Description of sampling design and sampling strategy used in monitoring this part should describe, in general terms, the sampling design and the sampling strategy applied as well as the purpose of the sampling. The sampling design encompasses all aspects of how to group epidemiological units on the frame (e.g. stratification), determine the sample size, allocate the epidemiological unit/sample to the various classifications of frame units (e.g. proportional allocation of epidemiological units/samples per slaughterhouses classified by descending throughput), and finally, select the sample. For example, the definition of the targeted population and its elements should be reported (epidemiological units that make up the population under study from which information is sought) (EFSA, 2014). It is useful to state if the sampling covered the whole MS/target population or only parts of it. The target population whether a food-producing animal population or a food category should be identified. It should be explained, for example, whether the entire animal/foodstuff population was covered or only a subset of it and the reasons for choosing this subset for sampling. Similarly, the animal populations and categories of foodstuffs sampled should be identified. If the sampling was stratified, for example, by geographical regions or other criteria, such as throughputs of the slaughterhouses, this should be described. It is important to explain how the epidemiological units to be sampled were chosen, regardless of whether objective, selective, suspected, convenience or census sampling was applied or if several sampling methods were applied. The framework of the sampling is an important part of the strategy. It should be stated whether the sampling was part of a permanent or temporary monitoring programme, linked to monitoring or control programmes or if it was the result of a single survey. Method of sampling (description of sampling technique: stage of sampling, type of sample taken, sampler), frequency of sampling, procedure of selection of isolates for susceptibility testing, and method used for collecting data should also be addressed in general terms per combination of bacterial species/matrix in reporting. Method of sampling (description of sampling technique) the sampling technique, meaning the procedure(s) used to obtain the sample, should be described here. This should include information on the site of sampling (e.g. the part of a carcase, the part of the facilities for an environmental sample), the size of the sample taken (e.g. in g, cm² or ml), the use of swabs or other instruments in the sampling (where relevant), the number of (sub) samples/sample units taken, the pooling of samples if any (refer to the number of samples combined by pooling, if available), the possible storage of samples and the length of storage (where relevant). It is also essential to indicate the stage of sampling where the samples were taken, e.g. on farm, at the slaughterhouse or at a retail outlet. The stage of sampling can be any step in the animal rearing process or the food chain. For example, the sample may be taken at the animal rearing period, production period, before or after the chilling of a carcase in a slaughterhouse or before or after the expiration of the shelf-life of foodstuffs. Type of sample taken under this title, the sample taken from the epidemiological units sampled should be described. For example, in the case of food-producing animals, the specimen tested could be caecal sample. It should specify which kind of sampler was performing the sampling, e.g. samples taken by the competent authority as part of an official sampling, samples taken by owners of animals, food business operators, or by other representatives of private enterprises in the context of the hazard analysis critical control point (HACCP)/own checks. Frequency of sampling this part should be used to explain how often samples were intended to be taken and whether the sampling was evenly distributed over the year as required. Procedures for the selection of isolates for antimicrobial testing in the case that only part of the isolates identified at the national level were tested for antimicrobial resistance, for example countries where Salmonella prevalence is high, the procedure for the selection should be reported here. www.efsa.europa.eu/publications 8 EFSA Supporting publication 2018:EN-1369

Methods used for collecting data under this title the methods used for collecting data should be described. 3.2.2. Stratification procedure per animal population and food category The stratification procedures should be reported in detail under this title. This information is mandatory. 3.2.3. Randomisation procedure per animal population and food category The randomisation procedures should be reported in detail under this title. This information is mandatory. 3.2.4. Analytical method used for detection and confirmation Detection and confirmation of Salmonella, Campylobacter and E. coli. In the case of the voluntary specific monitoring on carbapenemase-producers, the selective media used (commercial plates, in house media) should be also reported here. In general, any variation with regard to the EURL-AR protocols should be stated here. 3.2.5. Laboratory methodology used for detection of antimicrobial resistance Antimicrobials included in monitoring and cut-off values used in testing can be reported. According to the legislation, the protocols developed by the EURL-AR should be used and reported here. 3.2.6. Results of investigation A short assessment of the reported results can be provided in the narrative part. This analysis may cover comparison of current results with those from previous years, in order to identify any trend. 3.2.7. Additional information Any other information relevant to the monitoring of the zoonoses in question can be provided. 4. Guidelines for reporting antimicrobial resistance data 4.1. General recommendations In accordance with Decision 2013/652/EU, MSs should report on AMR in (a) Salmonella spp., (b) C. jejuni, (c) indicator commensal E. coli, and (d) on extended-spectrum beta-lactamase (ESBL)-, AmpC- or carbapenemase-producing Salmonella spp. and E. coli, 4 derived from the routine monitoring and the specific monitoring. Based on the requirements from Commission Implementing Decision 2013/652/EU, it is mandatory that AMR data are reported for the animal populations/food categories listed in Table 3. More details on data reporting requirements for sample description and programme code for the mandatory and voluntary reporting of isolates from the EU harmonised sampling 2017 are presented in Table 19 and Table 20 (in Appendix C of this document). The monitoring of AMR in food-producing animals under Commission Implementing Decision 2013/652/EU covers the main food-producing animal species and where appropriate, includes different production sectors (for example, broilers and laying hens). Randomised, representative sampling is no longer stratified at the level of the different animal species (e.g. Gallus gallus, cattle, pigs) but rather is performed at the level of the major food-producing animal production categories, such as broilers, laying hens, fattening pigs, fattening turkeys and bovines under 1 year of age. The collection and reporting of data is performed at the isolate level, which enables in depth analyses to be conducted, in particular on the occurrence of multidrug resistance. The analysis of the results at individual isolate level also allows investigation of possible associations between the occurrence of 4 In 2017, the specific monitoring of ESBL-/AmpC-/carbapenemase-producing indicator commensal E. coli is mandatory. Specific monitoring focusing only on carbapenemase-producing isolates is voluntary in 2017. www.efsa.europa.eu/publications 9 EFSA Supporting publication 2018:EN-1369

isolates which are fully-susceptible to the panel of antimicrobials tested and the consumption of antimicrobials. Table 3: Mandatory categories to be used for the reporting of the origin of the isolates Bacteria Animal species/food categories Salmonella spp. Laying hens, broilers, fattening turkeys Carcases of broilers, fattening turkeys, fattening pigs and bovines under 1 year of age Campylobacter jejuni Broilers, fattening turkeys, fattening pigs (a) Indicator E. coli Broilers, fattening turkeys, fattening pigs, bovines under one year of age (b) Fresh broiler meat, pig meat and bovine meat (b) Indicator enterococci (c) Broilers, fattening turkeys, fattening pigs, bovines under 1 year of age Note: In the years 2014, 2016, 2018 and 2020: for laying hens, broilers and fresh meat thereof, and fattening turkeys. In the years 2015, 2017 and 2019: for pigs, bovines under 1 year of age, pig meat and bovine meat. (a): If a MS decides to test for AMR in C. coli on a voluntary basis. (b): For the purpose of monitoring of ESBL- or AmpC- or carbapenemase-producing E. coli. (c): If a MS decides to test for AMR in E. faecalis and E. faecium on a voluntary basis. The requirements for monitoring AMR by MSs are laid down in Commission Implementing Decision 2013/652/EC. In particular, as regards the information that must be collected by MSs, the categories are listed in Annex, Part B of the Decision and are presented in Table 4 below. Table 4: Information that must be collected and reported according to Decision 2013/652/EC Information to be reported Details General information Identifier or code of the isolate Bacterial species Serovar (for Salmonella spp.) Phage type of Salmonella Enteritidis and Salmonella Typhimurium (optional) Specific information with regard Food-producing animal population or food category to sampling Stage of sampling Type of sample Sampler The sampling strategy Date of sampling Date of isolation Specific information with regard Identifier or code of the isolate given by the laboratory performing to AMR testing antimicrobial susceptibility testing of the isolate Date of susceptibility testing Antimicrobial substance Specific information with regard Minimum inhibitory concentration (MIC) value (in mg/l) to dilution method results Synergy testing results Synergy testing with clavulanic acid for ceftazidime Synergy testing with clavulanic acid for cefotaxime 4.2. Reporting antimicrobial resistance data on Salmonella spp. 4.2.1. Routine monitoring of antimicrobial resistance in Salmonella spp. The monitoring of AMR in zoonotic Salmonella spp. from the major food-producing animal populations domestically-produced and meat derived thereof is mandatory (Table 5). More details on data reporting requirements for sample description and programme code for the mandatory and voluntary reporting of isolates from the EU harmonised sampling 2017 are presented in Table 19 and Table 20 (in Appendix C of this document). For Salmonella spp. from broilers, laying hens and fattening turkeys, isolates are included which originate from salmonella national control plans, as well as isolates from carcases of broilers and fattening turkeys, sampled as part of process hygiene criteria. For Salmonella spp. from fattening pigs and bovine animals under 1 year of age, isolates are included originating from carcases of these animals, sampled as part of process hygiene criteria. For 2017, it www.efsa.europa.eu/publications 10 EFSA Supporting publication 2018:EN-1369

is mandatory to report on fattening pigs and bovines under 1 year of age, as well as the meet thereof. The target number of organisms which should be examined is 170 from each type of domestic animal (this is reduced to 85 organisms from poultry and pigs, if production is less than 100,000 tonnes per annum). The Salmonella prevalence occurring in a given animal population/food category in a country may hamper the ability to collect 170 isolates per year. It is mandatory to report at the serovar level, but reporting the phagetypes of Salmonella Enteriditis and Salmonella Typhimurium is optional. Table 5: Relevant animal species/food categories to be reported for Salmonella spp. Animal species/food Categories Domestic fowl (Gallus gallus) Laying hens Broilers Turkeys Fattening turkeys Carcases Broilers Fattening turkeys Fattening pigs Bovines under one year of age (if the production of those bovines in the MS is more than 10,000 tonnes slaughtered per year) Note: In the years 2014, 2016, 2018 and 2020: laying hens, broilers and fattening turkeys; carcases of broilers and fattening turkeys. In the years 2015, 2017 and 2019: carcases of fattening pigs and bovines under 1 year of age. Mandatory antimicrobials to be reported (first panel): Ampicillin, Azithromycin, Cefotaxime, Ceftazidime, Chloramphenicol, Ciprofloxacin, Colistin, Gentamicin, Meropenem, Nalidixic acid, Sulfamethoxazole, Tetracycline, Tigecycline and Trimethoprim. 4.2.2. Monitoring of presumptive ESBL-, AmpC- or carbapenemase-producing Salmonella derived from the routine monitoring Extended susceptibility testing to the second panel All randomly selected isolates of Salmonella spp. recovered from non-selective media, that are resistant to cefotaxime or ceftazidime or meropenem (first panel), are further tested with a second panel of antimicrobial substances. This second panel of antimicrobials includes cefotaxime and ceftazidime with and without clavulanic acid (to investigate whether synergy is observed with clavulanate), as well as the antimicrobials cefoxitin, cefepime, temocillin, ertapenem, imipenem and meropenem. The second panel of antimicrobials is designed to enable phenotypic characterisation of presumptive ESBL-, AmpC- and carbapenemase-producers. It is of note that no resistance to meropenem but to other carbapenems would indicate the presence of other mechanisms different than carbapenemase-production. Mandatory antimicrobials to be reported (second panel): Cefepime, Cefoxitin, Ceftazidime, Ceftazidime + clavulanic acid, Cefotaxime, Cefotaxime + clavulanic acid, Ertapenem, Imipenem, Meropenem and Temocillin. In the case of discrepant results affecting the categorization of an isolate as resistant or susceptible to cefotaxime/ceftazidime/meropenem which is tested in both the first and the second panel, re-testing of the isolate concerned using both panels in parallel is recommended before reporting data. Similarly, whenever carbapenem resistance is registered, re-testing of the isolate concerned using both panels and bacterial species confirmation is recommended before reporting data. Inference of the presumptive ESBL-, AmpC- or carbapenemase-producing phenotypes Depending on the results obtained from the susceptibility testing performed with the second panel, the isolates can be subsequently classified by EFSA as presumptive ESBL-, AmpC-, ESBL/AmpC- or carbapenemase-producers (ESBL, AmpC or Carbapenemase-phenotypes) according to the criteria proposed by EUCAST (EUCAST, 2013). www.efsa.europa.eu/publications 11 EFSA Supporting publication 2018:EN-1369

If any additional molecular results on the resistance mechanisms conferring resistance to thirdgeneration cephalosporins and/or carbapenems are available, reporting of the corresponding data is recommended. 4.3. Reporting antimicrobial resistance data on Campylobacter spp. The monitoring and reporting of AMR in zoonotic Campylobacter jejuni from the major food-producing animal populations domestically-produced is mandatory. Campylobacter spp. isolates derive from active monitoring programmes, based on representative random sampling of carcasses of healthy animals sampled at the slaughterhouse to collect caecal samples (Table 6). More details on data reporting requirements for sample description and programme code for the mandatory and voluntary reporting of isolates from the EU harmonised sampling 2017 are presented in Table 19 and Table 20 (in Appendix C of this document). The target number of organisms of each bacterial species which should be examined is 170 from each type of domestic animal (this is reduced to 85 organisms from poultry and pigs, if production is less than 100,000 tonnes per annum). For 2017, it is recommended to report on resistance in C. coli from pigs and pork. It is also highly recommended to report the total number of epidemiological units tested to enable assessment of the prevalence of C. jejuni and C. coli in the animal populations investigated. Table 6: Relevant animal populations to be reported for Campylobacter coli and C. jejuni Campylobacter species Animal species Sample type/sample stage C. jejun isolates Broilers of Gallus gallus (mandatory) Fattening turkeys (a) C. col isolates (optional) Broilers of Gallus gallus Caecal samples gathered at slaughter Fattening pigs (a): where the production of turkey meat in the MS is more than 10,000 tonnes per year. Note: In the years 2014, 2016, 2018 and 2020: broilers and fattening turkeys. Mandatory antimicrobials to be reported: for C. jejuni and C. coli it is mandatory that results are reported for: Ciprofloxacin, Erythromycin, Gentamicin, Nalidixic acid, Tetracycline and on a voluntary basis Streptomycin. 4.4. Reporting antimicrobial resistance data on indicator E. coli 4.4.1. Routine monitoring of antimicrobial resistance in E. coli Since the implementation of Commission Decision 2013/652/EU, the monitoring and reporting of AMR in indicator commensal (non-pathogenic) E. coli from the major food-producing animal populations domestically-produced has become mandatory. Indicator E. coli isolates derive from active monitoring programmes, based on representative random sampling of carcasses of healthy animals sampled at the slaughterhouse to collect caecal samples (Table 7). More details on data reporting requirements for sample description and programme code for the mandatory and voluntary reporting of isolates from the EU harmonised sampling 2017 are presented in Table 19 and Table 20 (in Appendix C of this document). For 2017, it is mandatory to report on pigs and bovines under one year. The target number of organisms of each bacterial species which should be examined is 170 from each type of domestic animal (this is reduced to 85 organisms from poultry and pigs, if production is less than 100,000 tonnes per annum). Table 7: Relevant animal species/food categories to be reported for the monitoring of indicator commensal E. coli (non-pathogenic) Animal species Broilers of Gallus gallus Fattening turkeys (a) Fattening pigs Bovines under one year of age (a) Sample type/sample stage Caecal samples gathered at slaughter www.efsa.europa.eu/publications 12 EFSA Supporting publication 2018:EN-1369

(a): Where the production of turkey/bovine under one year age meat in the MS is more than 10,000 tonnes per year. Note: In the years 2014, 2016, 2018 and 2020, broilers and fattening turkeys. In the years 2015, 2017 and 2019, fattening pigs and bovines under one year of age. Mandatory antimicrobials to be reported (first panel): Ampicillin, Azithromycin, Cefotaxime, Ceftazidime, Chloramphenicol, Ciprofloxacin, Colistin, Gentamicin, Meropenem, Nalidixic acid, Sulfamethoxazole, Tetracycline, Tigecycline, and Trimethoprim. 4.4.2. Monitoring of presumptive ESBL-, AmpC- or carbapenemase-producing E. coli derived from the routine monitoring All randomly selected isolates of commensal E. coli recovered from non-selective media, that are resistant to cefotaxime or ceftazidime or meropenem (first panel), are further tested with a second panel of antimicrobial substances. This second panel of antimicrobials includes cefotaxime and ceftazidime with and without clavulanic acid (to investigate whether synergy is observed with clavulanate), as well as the antimicrobials cefoxitin, cefepime, temocillin, ertapenem, imipenem and meropenem. The second panel of antimicrobials is designed to enable phenotypic characterisation of presumptive ESBL-, AmpC- and carbapenemase-producers. It is of note that no resistance to meropenem but to other carbapenems would indicate the presence of other mechanisms different than carbapenemase-production. Mandatory antimicrobials to be reported (second panel): Cefepime, Cefoxitin, Ceftazidime, Ceftazidime + clavulanic acid, Cefotaxime, Cefotaxime + clavulanic acid, Ertapenem, Imipenem, Meropenem, Temocillin 4.4.3. Monitoring of ESBL-, AmpC- or carbapenemase-producing E. coli derived from either routine monitoring or specific monitoring Commission Implementing Decision 2013/652/EU stipulates that culture using selective media for cephalosporin-resistant E. coli should be performed. Caecal samples from broilers, fattening turkeys, fattening pigs and bovines under 1 year of age, as well as from broiler and turkey meat, pork and beef collected at retail should be examined for cefotaxime-resistant E. coli using selective media incorporating the 3rd-generation cephalosporin cefotaxime (Table 8). More details on data reporting requirements for sample description and programme code for the mandatory and voluntary reporting of isolates from the EU harmonised sampling 2017 are presented in Table 19 and Table 20 (in Appendix C of this document). This medium is selective for E. coli resistant to 3rd-generation cephalosporins and is expected to allow the growth of extended-spectrum beta-lactamase (ESBL), AmpC or carbapenemase enzyme producers which are resistant to cefotaxime at the microbiological cut-off (so called specific monitoring). Three hundred caecal samples should be examined from each type of livestock. All presumptive ESBL- or AmpC- or carbapenemase-producing E. coli isolates identified through the selective plating, as well as all those randomly selected isolates of indicator commensal E. coli recovered from non-selective media (routine monitoring) (see 4.4.2.), that are resistant to cefotaxime or ceftazidime or meropenem, are further tested with the second panel of antimicrobial substances. This second panel of antimicrobials includes cefotaxime and ceftazidime with and without clavulanic acid (to investigate whether synergy is observed with clavulanate), as well as the antimicrobials cefoxitin, cefepime, temocillin, ertapenem, imipenem and meropenem. The second panel of antimicrobials is designed to enable phenotypic characterisation of presumptive ESBL-, AmpC- and carbapenemase-producers according to the criteria proposed by EUCAST (EUCAST, 2013). www.efsa.europa.eu/publications 13 EFSA Supporting publication 2018:EN-1369

Table 8: Relevant animal species/food categories to be reported for the specific monitoring of ESBL-, AmpC- or carbapenemase-producing E. coli Animal species/food Sample type/sample stage Broilers of Gallus gallus Fattening turkeys (a) Fattening pigs Caecal samples gathered at slaughter Bovines under one year of age (a) Fresh 5 meat Broilers Fresh meat at retail Pigs Bovines (a): Where the production of turkey/bovine under one year age meat in the MS is more than 10,000 tonnes per year. Note: In the years 2014, 2016, 2018 and 2020: broilers and fattening turkeys and fresh meat of broilers. In the years 2015, 2017 and 2019: fattening pigs and bovines under one year of age and fresh pig fresh meat and bovine fresh meat. Specific monitoring of ESBL-/AmpC-/Carbapenemase-producing E. coli For the detection of ESBL- or AmpC-producing E. coli, the method should start with a pre-enrichment step, followed by inoculation on McConkey agar containing a third-generation cephalosporin in a selective concentration, in accordance with the most recent version of the detailed protocol for standardisation of the EU Reference Laboratory for Antimicrobial Resistance. 6 Using this protocol, also carbapenemase-producing isolates could be recovered. It is of note that, as the isolates are recovered from plates containing cefotaxime at 1 mg/l (ECOFF), resistance to at least this antimicrobial is expected, and the testing of the second panel for most of the isolates will be necessary. The microbial species E. coli should be identified using an appropriate method. This monitoring will be referred as the specific monitoring of ESBL-/AmpC-/Carbapenemase-producing E. coli. To facilitate the specific detection of ESBL-producing E. coli, the MS may decide, based on the epidemiological circumstances, to test, in parallel, an additional selective plate that inhibits the growth of AmpC-producing E. coli. When using this method, the results from this additional selective plate, which inhibits the growth of AmpC-producing E. coli, should be reported separately. Specific monitoring of carbapenemase-producing micro-organisms MSs may decide to detect for carbapenemase-producing micro-organisms by using pre-enrichment and subsequent selective plating on carbapenem-containing media, in accordance with the most recent version of the detailed protocol for standardisation of the EU Reference Laboratory for AMR. This monitoring will be referred as the specific monitoring of carbapenemase-producers and is voluntary. Within the framework of both specific monitoring, it is recommended to report the total number of epidemiological units tested to enable assessment of the prevalence of ESBL-/AmpC- producing E. coli and of carbapenemase-producing E. coli in the animal populations investigated. Monitoring of ESBL-/AmpC-/carbapenemase-producing E. coli derived from the routine monitoring One randomly selected E. coli isolate obtained from each positive caecal sample and meat sample should be tested for resistance to the first panel of antimicrobials and then submitted for extended susceptibility testing (second panel) if cefotaxime, ceftazidime or meropenem are resistant, based on the interpretative criteria (epidemiological cut-off values). Extended susceptibility testing to the second panel All presumptive ESBL-, AmpC- or carbapenemase-producing E. coli isolates, identified through selective plating (derived from specific monitoring), and E. coli that, after testing with the first panel of antimicrobials in the routine monitoring, are found to be resistant to cefotaxime, ceftazidime or meropenem, should be further tested with the second panel of antimicrobials. This panel includes 5 Within the framework of this sampling plan, fresh meat is understood as chilled meat (meaning that frozen meat is excluded), including meat that is wrapped, vacuum-wrapped or wrapped in a controlled atmosphere (EFSA, 2014). 6 Available online: www.crl-ar.eu www.efsa.europa.eu/publications 14 EFSA Supporting publication 2018:EN-1369

cefoxitin, cefepime and clavulanate in combination with cefotaxime and ceftazidime for the detection of ESBL and AmpC production. In addition, the second panel also contains imipenem, meropenem and ertapenem to phenotypically verify presumptive carbapenemase-producers. In the case of discrepant results affecting the categorization of a given isolate as resistant or susceptible to cefotaxime/ceftazidime/meropenem which are substances tested in both the first and the second panel, re-testing of the isolate concerned using both panels in parallel is recommended to resolve the discrepancy before reporting data. Similarly, whenever carbapenem resistance is registered, re-testing the isolate concerned by using both panels and confirming the bacterial species are both recommended before reporting data. Inference of the presumptive ESBL-, AmpC- or carbapenemase-producing phenotypes Depending on the results obtained from the susceptibility testing performed with the second panel, the isolates can be classified as presumptive ESBL-, AmpC-, ESBL/AmpC- or carbapenemase-producers (ESBL, AmpC or Carbapenemase-phenotypes) according to the criteria proposed by EUCAST (EUCAST, 2013). If any additional molecular results on the resistance mechanisms conferring resistance to thirdgeneration cephalosporins and/or carbapenems are available, reporting of the corresponding data is recommended. 4.5. Reporting data on antimicrobial resistance in indicator commensal Enterococcus spp. The Enterococcus species E. faecium and E. faecalis are suitable as indicators of antimicrobial resistance in Gram-positive bacteria, as both species are commonly isolated from animal faeces. These species of Enterococcus are also important in human medicine, especially vancomycin-resistant E. faecium strains that may be resistant to most or all antimicrobials effective for treatment of vancomycin-susceptible enterococci infections. Furthermore, the occurrence of E. faecium and E. faecalis in the intestinal tract of animals or on food, even if not directly significant for humans, may constitute a reservoir of resistance genes which may be transferred either to pathogenic bacteria or to other commensal bacteria. Determining the occurrence of resistance to antimicrobial agents in commensal enterococci also provides data useful for investigating the selective pressure exerted by the use of antimicrobials on the intestinal population of bacteria in food-producing animals. Harmonised monitoring of E. faecium and E. faecalis from food and animals (Table 9) is to be implemented and reported by the MSs on a voluntary basis in accordance with Commission Implementing Decision 2013/652/EU. More details on data reporting requirements for sample description and programme code for the mandatory and voluntary reporting of isolates from the EU harmonised sampling 2017 are presented in Table 19 and Table 20 (in Appendix C of this document). Table 9: Relevant animal species/food categories to be reported for the monitoring on a voluntary basis of Enterococcus spp.(non-pathogenic) Animal species Sample type/sample stage Broilers of Gallus gallus Fattening turkeys (a) caecal samples gathered at slaughter Fattening pigs Bovines under one year of age (a) (a): Where the production of turkey/bovine under one year age meat in the MS is more than 10,000 tonnes per year. Relevant agent species to be reported: Enterococcus faecium and E. faecalis should be reported separately. Mandatory antimicrobials to be reported*: Ampicillin, Ciprofloxacin, Chloramphenicol, Daptomycin, Erythromycin, Gentamicin, Linezolid, Quinopristin/dalfopristin, Teicoplanin, Tigecycline, Tetracycline, Vancomycin *If a MS decides to test E. faecalis and E. faecium, then the antimicrobials listed should be tested. www.efsa.europa.eu/publications 15 EFSA Supporting publication 2018:EN-1369

5. Guidelines for validating data before reporting Business rules and selection criteria for reference testing are circulated to the MSs so that they can be used by the MSs to validate AMR data before reporting to EFSA. The importance of reporting representative and fully validated AMR data to EFSA is highly emphasised. 5.1. Validation against business rules Before reporting data to EFSA, it is recommended that the MSs first check the data for usability against a series of business rules. For the list of business rules, please see EFSA, 2018. 5.2. Scientific data validation In addition, it is also highly recommended that the MSs perform a revision and validation of the data collected before submitting to EFSA by comparing between data reported for the same antimicrobials when tested by different panels. Special attention is given to carbapenems, colistin, azithromycin, tigecycline and to possible discrepancies between results for antimicrobials present in both panels (i.e. cefotaxime, ceftazidime, meropenem). If considered needed, MSs are asked to confirm the MIC results and the species identification of the reported isolates. To be considered as errors (revise and re-test AST and species confirmation): E. coli and/or Salmonella spp.: Resistance to third generation cephalosporins but susceptibility to ampicillin. MRSA: Discrepancy between the categorization of resistant vs. susceptible with two or more levels MIC difference between results from both panel 1 and panel 2 for cefotaxime, ceftazidime and meropenem (antimicrobials present in both panels). Susceptible to cefoxitin and penicillin. Odd phenotypes worth to be considered as a warning: E. coli and/or Salmonella spp.: MRSA: Resistance to meropenem (suspect of carbapenemase-producer, re-test AST and if confirmed, repeat species identification). Resistance to other carbapenems (if imipenem-r, re-test AST). Resistance to colistin with MIC>=8 mg/l (re-test AST) Resistance to tigecycline with MIC >=4 mg/l (re-test AST) Resistance to azithromycin is not very common. We would recommend to re-test randomly some isolates for confirmation (especially if MIC is close to the ECOFF, MIC=32 mg/l) Resistant to vancomycin (re-test AST and if confirmed, repeat species identification) Resistance to linezolid (re-test AST and if confirmed, repeat species identification). Please, take also into account: If the resistance to cefotaxime, ceftazidime and meropem is reported for panel 1 for an isolate collected within the routine monitoring, data for panel 2 shall be always reported. Isolates collected within the specific ESBL/AmpC/Carbapenemase monitoring (selective media with cefotaxime 1 mg/ml) are expected to be resistant to cefotaxime. If this resistance is confirmed by AST, data from both panel 1 and panel 2 shall be reported. www.efsa.europa.eu/publications 16 EFSA Supporting publication 2018:EN-1369