1. Austrian Agency for Health and Food Safetywww.ages.at Antimicrobial resistance in poultry (laying hens, broilers, turkeys) and pigs Heimo Lassnig, AGES, Institute for Veterinary disease control Graz, Austria Workshop on Monitoring and reporting system of Zoonoses and Control of Salmonella Belgrade, 15.-16.3.2012

2. www.ages.at EU Directives 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 2

3. www.ages.at Chapter III Antimicrobial resistance Article 7, Monitoring of antimicrobial resistance 1. Member States shall ensure, in accordance with the requirements set out in Annex II, that monitoring provides comparable data on the occurrence of antimicrobial resistance in zoonotic agents and, in so far as they present a threat to public health, other agents. 2. Such monitoring shall supplement the monitoring of human isolates conducted in accordance with Decision No 2119/98/EC. 3. Detailed rules for the implementation of this Article shall be laid down in accordance with the procedure referred to in Article 12(2). 3

4. www.ages.at Chapter V Exchange of information Article 9 Assessment of trends and sources of zoonoses, zoonotic agents and antimicrobial resistance 1. Member States shall assess trends and sources of zoonoses, zoonotic agents and antimicrobial resistance in their territory. Each Member State shall transmit to the Commission every year by the end of May a report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance, covering the data collected pursuant to Articles 4, 7 and 8 during the previous year. Reports, and any summaries of them, shall be made publicly available. 4

5. www.ages.at ANNEX II Requirements for monitoring of antimicrobial resistance pursuant to Article 7 General requirements Member States must ensure that the monitoring system for antimicrobial resistance provided for in Article 7 provides at least the following information: 1. animal species included in monitoring; 2. bacterial species and/or strains included in monitoring; 3. sampling strategy used in monitoring; 4. antimicrobials included in monitoring; 5. laboratory methodology used for the detection of resistance; 6. laboratory methodology used for the identification of microbial isolates; 7. methods used for the collection of the data. 5

6. www.ages.at ANNEX II Requirements for monitoring of antimicrobial resistance pursuant to Article 7 Specific requirements Member States must ensure that the monitoring system provides relevant information at least with regard to a representative number of isolates of Salmonella spp., Campylobacter jejuni and Campylobacter coli from cattle, pigs and poultry and food of animal origin derived from those species. 6

7. www.ages.at 2007/407/EC COMMISSION DECISION monitoring of antimicrobial resistance on a harmonised monitoring of antimicrobial resistance in Salmonella in poultry and pigs FAO/OIE/WHO workshop on scientific assessment on non- human usage of antimicrobials and antimicrobial resistance in 2003 Clear evidence of adverse human health consequences due to resistant organisms resulting from non-human usage of antimicrobials: increased frequency of infections, increased frequency of treatment failures increased severity of infections, 7

8. www.ages.at 2007/407/EC COMMISSION DECISION monitoring of antimicrobial resistance as documented for instance by fluoroquinolone- resistant human Salmonella infections. Evidence shows that the amount and pattern of non-human usage of antimicrobials affect the occurrence of resistant bacteria in animals and food and thereby human exposure to these resistant bacteria However, it should be noted that most of the resistance problems in human medicine are caused by human usage and over usage of antimicrobial agents for therapy and prophylaxis 8

9. www.ages.at 2007/407/EC COMMISSION DECISION monitoring of antimicrobial resistance European Food Safety Authority (EFSA) relatively high proportion of Campylobacter and Salmonella isolates from animals and food were resistant to antimicrobials commonly used in treatment of human diseases. Food-borne infections caused by these resistant bacteria pose a particular risk to humans due to possible treatment failure. 9

10. www.ages.at 2007/407/EC COMMISSION DECISION monitoring of antimicrobial resistance Scientific Panel on Biological Hazards Scientific Panel an Animal Health and Welfare of the EFSA Opinion on ‘Review of the Community Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Antimicrobial Resistance in the European Union in 2004’ Opinion indicates the importance to provide detailed information on the Salmonella serovar for each isolate and to harmonise the breakpoints applied for resistance assessment and reporting. 10

11. www.ages.at 2007/407/EC COMMISSION DECISION monitoring of antimicrobial resistance The Task Force on Zoonoses Data Collection of the EFSA adopted a ‘Report including a proposal for a harmonised monitoring scheme of antimicrobial resistance in Salmonella in fowl (Gallus gallus), turkeys and pigs and Campylobacter jejuni and C. coli in broilers’ The report makes recommendations on a harmonised monitoring scheme and harmonised methodology for susceptibility testing. 11

12. www.ages.at 2007/407/EC COMMISSION DECISION monitoring of antimicrobial resistance Article 2 Collection and analyses of isolates The collection of isolates of Salmonella spp. referred to in Article 1 and the analysis thereof shall be performed by the competent authority or under its supervision in accordance with the technical specifications set out in the Annex. 12

13. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX TECHNICAL SPECIFICATIONS REFERRED TO IN ARTICLE 2 1. Origin of isolates Salmonella isolates collected through control and monitoring programmes, set up in accordance with Article 5 of Regulation (EC) No 2160/2003 of the European Parliament and of the Council (1) and/or Commission Decisions 2006/662/EC (2), 2006/668/EC (3), shall be collected for antimicrobial resistance monitoring in accordance with Table 1. 13

14. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX Table 1: Years certain agents selected for antim resist testing 14 Year All Salmonella serovars Laying hensBroilersTurkeysSlaughter pigs 2007xx 2008x 2009xx 2010xxx 2011xxxx 2012xxxx

15. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX Table 1: Years certain agents selected for antim resist testing Not more than one isolate per Salmonella serovar from the same epidemiological unit per year shall be included in the monitoring. The epidemiological unit for laying hens, broilers, and turkeys is the flock. For pigs, the epidemiological unit is the holding. 15

16. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX Number of isolates to be tested 16 Target population Salmonella sppCampylob spp Animal speciesStudy population Fowl (Gallus gallus) Flocks laying hens170 Broiler flocks170 TurkeysFlocks170 PigsSlaughter pig herds170

17. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX Number of isolates to be tested In those Member States where, in any given year, a lower number of isolates than the target sample size is available from the monitoring or control programmes, all these isolates shall be included in the antimicrobial resistance monitoring. In those Member States where a higher number of isolates is available all isolates, or a representative random selection equal or larger than the target sample size, shall be included. 17

18. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX Antimicrobial susceptibility testing Member States shall test at least the antimicrobials that are specified in Table 2, using the cut-off values given and an appropriate concentration range to determine the susceptibility of Salmonella. Dilution methods shall be performed according to the methods described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI), accepted as international reference method (ISO standard 20776-1:2006). It is recommended that the selected isolates of S. Enteritidis and S. Typhimurium are phage typed. 18

19. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX Antimicrobial susceptibility testing, Table 2 19 AntimicrobialCut-off-value SalmonellaCefotaxime0,5 Nalidixic acid16 Ciprofloxacin0,06 Ampicillin4 Tetracycline8 Chloramphenicol16 Gentamicin2 Streptomycin32 Trimethoprim2 Sulphonamides256

20. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX Collection of the data and reporting The results of the antimicrobial resistance monitoring shall be assessed and reported, in accordance with Article 9 of Directive 2003/99/EC, in the yearly report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance. Without prejudice to the provisions of Annex IV of Directive 2003/99/EC the following information shall be reported for Salmonella in laying hens, broilers, turkeys and pigs: 20

21. www.ages.at 2007/407/EC COMMISSION DECISION ANNEX Collection of the data and reporting — Origin of isolates i.e. baseline study, control programme, passive surveillance — number of isolates susceptibility tested — number of isolates found to be resistant per antimicrobial — number of fully-susceptible isolates and number of isolates resistant to 1, 2, 3, 4 and > 4 antimicrobials listed in Table 2. 21

22. www.ages.at Salmonella monitoring All salmonella serotypes with public health significance Laying hens: — rearing flocks — day-old chicks — pullets two weeks before moving to laying phase or laying unit — laying flocks — every 15 weeks during the laying phase 22

23. www.ages.at Salmonella monitoring All salmonella serotypes with public health significance Broilers — birds leaving for slaughter Turkeys — birds leaving for slaughter 23

24. www.ages.at Salmonella monitoring All salmonella serotypes with public health significance Herds of pigs: — breeding pigs — animals leaving for slaughter or carcases at the slaughterhouse — slaughter pigs — animals leaving for slaughter or carcases at the slaughterhouse No final decision made 24

25. www.ages.at Diagnostic method Validated method Annex D of ISO 6579/2007 Pre enrichment puff Peptonwater 37°C 24 h Enrichment MSRV 42°C 2 x 24 h Culture XLD, SM2, Rambach,… 37°C Confirmation Agglutination Antisera biochemical 25

26. www.ages.at Diagnostic method Serotyping: isolates selected for antimicrobial susceptibility testing should be identified at serovar level Kauffmann-White Schema -HPA Colindale, London Phage Typing it is recommended that all the isolates of S. Enteritidis and S. Typhimurium selected for antimicrobial susceptibility testing are phage typed Colindale phage typing scheme Antibiotische Resistenztestung -CLSI Standards -EUCAST Standards (2011) 26

27. www.ages.at Diagnostic method Strains isolated in these programmes are to be stored for a minimum of 2 years. The number of culture passages, the storage, and the transportation methods should ensure integrity of the strains. Further details concerning detection and isolation of microbial isolates and quality assurance are ddressed separately by Community Reference Laboratories. 27

28. www.ages.at Diagnostic method Methodology for susceptibility testing Because this monitoring scheme aims at optimum sensitivity for detection of acquired resistance, epidemiological cut-off values are proposed as interpretive criteria and not clinical breakpoints. These criteria have been developed by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) for the interpretation of MICs only. Disk diffusion is not advocated for European monitoring because different methodologies are used with different criteria and epidemiological cut-off values have not been defined. 28

29. www.ages.at Diagnostic method Methodology for susceptibility testing Moreover, one set of interpretive criteria would not be sufficient for harmonisation of the results, because differences in methods used for disk diffusion exist. Therefore, to improve the comparability of the data provided by Member States, only quantitative data on MIC will be accepted. 29

30. www.ages.at Diagnostic method Methodology for susceptibility testing To control the quality and comparability of MIC- results, the laboratories performing susceptibility testing should successfully participate in proficiency testing conducted regularly by the Community Reference Laboratory for Antimicrobial Resistance. It is recommended that all susceptibility testing is carried out at the national reference laboratory. 30

31. www.ages.at Diagnostic method Methodology for susceptibility testing For non-fastidious micro-organisms, such as Salmonella spp, the EUCAST and CLSI methods for determining MICs have been accepted as an international reference method through CEN and ISO. Dilution methods should be performed according to these methods as described in [ISO 20776-1:2006] ISO 20776-1:2006 describes one reference method, broth microdilution, for determination of MICs. The MIC reflects the activity of the drug under the described test conditions, and can be interpreted for clinical management purposes by taking into account other factors, such as drug pharmacology or bacterial resistance mechanisms. This allows categorization of bacteria as susceptible (S), intermediate (I), or resistant (R). 31

32. www.ages.at Diagnostic method Methodology for susceptibility testing For non-fastidious micro-organisms, such as Salmonella spp, the EUCAST and CLSI methods for determining MICs have been accepted as an international reference method through CEN and ISO. Dilution methods should be performed according to these methods as described in [ISO 20776-1:2006] ISO 20776-1:2006 describes one reference method, broth microdilution, for determination of MICs. The MIC reflects the activity of the drug under the described test conditions, and can be interpreted for clinical management purposes by taking into account other factors, such as drug pharmacology or bacterial resistance mechanisms. This allows categorization of bacteria as susceptible (S), intermediate (I), or resistant (R). 32

33. www.ages.at Diagnostic method Methodology for susceptibility testing Antimicrobials and cut-off values The antimicrobial prioritisation is based on the relevance to public health and/or on epidemiological relevance. Member States are asked to test and report as a minimum the priority antimicrobials that are specified in Table 2 above. This should not prevent Member States to include additional antimicrobials in their monitoring. 33

34. www.ages.at Diagnostic method Antimicrobials and cut-off values The minimum concentration range to be tested and epidemiological cut-off values to be used to classify bacteria as resistant (R) are specified in Table 3. It is suggested that for a certain antimicrobial agent the concentration range should include the susceptible wild- type population and at least four concentrations above the cut-off values. Because of the format of a microtitre plate the range would consist of a minimum of eight two-fold dilutions. On occasions wider ranges are advised in order to detect high level resistance (e.g. ciprofloxacin). 34

35. www.ages.at Diagnostic method Antimicrobials and cut-off values The objective of the relatively wide range is that by including the wild-type susceptible population within this range, isolates with acquired resistance can be detected with optimum sensitivity. Because it is the specific purpose of this monitoring programme to detect acquired resistance mechanisms in bacterial isolates and not to give clinical advice for therapy, the cut off values for the wild-type distributions are used and not the clinical breakpoints. 35

36. www.ages.at Diagnostic method Antimicrobials and cut-off values Table 3. 36 Adviced opt CR 0,06-8 2-256 0,008-8 0,5-64 2-256 0,25-32 2-256 0,25-32 8-1024 mg/L

37. www.ages.at Diagnostic method Antimicrobials and cut-off values Interpretation based on epidemiological cut-off values by EUCAST (www.eucast.org) - seewww.eucast.org Annex 3. Rationale for inclusion of antimicrobials in antimicrobial resistance monitoring and cut-off values to be used. In those cases where epidemiological cut-off values have not yet been defined by EUCAST, CLSI MIC breakpoints or MIC breakpoints advised in the ARBAO-II network are used. 37

38. www.ages.at Data and reports Member States are asked to report the number of isolates resistant to each antimicrobial separately. In addition, they are asked to report, only for antimicrobials in table 3, the number of isolates that are fully sensitive, and the number of isolates resistant to 1, 2, 3, 4 and > 4 antimicrobials. Additionally tested antimicrobials should not be included to ensure comparability of the number of multi-drug resistant isolates from different Member States. 38

39. www.ages.at Data and reports Only resistance to different antimicrobials for which the resistance mechanisms are not related should be reported: For Salmonella this includes resistance to ampicillin, cefotaxime, nalidixic acid (or ciprofloxacin), streptomycin, gentamicin, tetracycline, chloramphenicol, trimethoprim and sulphamethoxazole. For Salmonella it is requested to report separately the number of isolates with the pentaresistance (ACSSuT) phenotype. This includes resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline. It is recommended that also the serovar and phage type of these isolates is reported. 39

40. www.ages.at Data and reports The data should be collected and evaluated on national level and presented within the obligations of Article 9 of Directive 2003/99/EC. Member States have to submit their report each year by 31st May. EFSA has established a web-based reporting system and database to streamline and harmonize the reporting under Directive 2003/99/EC. This system is used also for the purpose of reporting on antimicrobial resistance. 40

41. www.ages.at Data and reports In line with Annexes II and IV to Directive 2003/99/EC the national report should summarise the following topics: 1) Overall description in text form 2) Results of antimicrobial susceptibility testing in table form for every study population, broken down by Campylobacter species or Salmonella serovars: -a) Qualitative tables to report the result for each antimicrobial tested: -b) Quantitative tables to report MIC distributions for the following study populations in certain bacterial species, if available: 41

42. www.ages.at Data and reports 1) Overall description in text form Overview of the number of isolates available and the selection strategy. Isolates normally should come from national control programmes. Description of the source where samples were taken from (i.e. faeces, dust, carcasses, lymph nodes). The definitions and interpretation criteria (if they deviate from the recommended susceptibility testing methods) and a description of quality assurance systems in place. Control measures for antimicrobial resistance in legislation, recent actions to control antimicrobial resistance, suggestions to Community for actions to be taken. 42

43. www.ages.at Data and reports 1) Overall description in text form Evaluation of the results, which may include any of the following: trends, emerging resistance, discussion of encountered difficulties and inherent biases. Relevance of findings, comparison of the situation along the food chain, that is in isolates of feed, animal, food and human origin. Additional information, e.g. changes in drug licensing or in the amount of antimicrobials used. 43

44. www.ages.at Data and reports 2) Results of antimicrobial susceptibility testing in table form for every study population, broken down by Campylobacter species or Salmonella serovars: -a) Qualitative tables to report the result for each antimicrobial tested: Number of isolates tested. Number of resistant isolates. Number of fully-susceptible and number of isolates resistant to 1, 2, 3, 4 or >4 antimicrobials. 44

45. www.ages.at Data and reports 2) Results of antimicrobial susceptibility testing in table form for every study population, broken down by Campylobacter species or Salmonella serovars: -b) Quantitative tables to report MIC distributions for the following study populations in certain bacterial species, if available: 45 S.E.S.T.S.DerbyOther S. Laying hensXXX BroilersXXX TurkeysXXX PIGSXXX

46. www.ages.at Data and reports These MIC-distribution tables have been selected after taking into account a number of different factors; they are considered a minimum for satisfactory reporting and interpretation of the data collected by Member States. Resistance to some antimicrobials can be associated with particular Salmonella serovars or phage types. Consideraton of the number of isolates of a given serovar available -a minimum number of isolates is required to provide an informative table -fluctuations in the prevalence of resistance are to be expected when only low numbers of isolates are available. 46

47. www.ages.at Data and reports These MIC-distribution tables have been selected after taking into account a number of different factors; they are considered a minimum for satisfactory reporting and interpretation of the data collected by Member States. Resistance to some antimicrobials can be associated with particular Salmonella serovars or phage types. Consideraton of the number of isolates of a given serovar available -a minimum number of isolates is required to provide an informative table -fluctuations in the prevalence of resistance are to be expected when only low numbers of isolates are available. 47

48. www.ages.at Data and reports S. Typhimurium and S. Enteritidis are included in the analysis because of their public health significance and because in many countries these serovars are likely to be relatively frequently detected in the monitoring scheme. Clonal spread of resistant Salmonella serovars or phage types of S. Typhimurium is an important phenomenon in some countries. 48

49. www.ages.at Data and reports S. Derby is specifically included for pigs because it occurs at a reasonable prevalence in this species in many countries and it may also provide some additional information on the relative antimicrobial selective pressure being exerted on the study population. The remaining Salmonella serovars are grouped in a separate table, in which emergent resistance in these serovars to antimicrobials such as cefotaxime or ciprofloxacin will be readily evident. 49

50. www.ages.at Data and reports The tables described should therefore allow comparison across the study population and between countries, whilst at the same time minimising the amount of information Member States are requested to provide. The levels of resistance in bacterial species can fluctuate and serovars may wax and wane in terms of both prevalence and relative public health significance; for these reasons it is likely that the reporting requirements may change in future. Annex 4 provides example tables as well as some considerations for national data collection which is the competence of each Member State. 50

51. www.ages.at Example 51 MIC (mg/l) Antimicrobial% resistant<= 0.0310.06250.1250.250.512481632641282565121024 Amoxillin/Clavulanic acid0.0 89.7 10.3 Ampicillin10.3 75.913.8 10.3 Apramycin0.0 100 Ceftiofur0.0 96.63.4 Cephalotin0.0 69.017.213.8 Chloramphenicol0.0 24.175.9 Ciprofloxacin0.0100 Colistin0.0 100 Florfenicol0.0 96.63.4 Gentamicin0.0 100 Nalidixic acid0.0 100 Neomycin0.0 100 Spectinomycin3.4 86.210.3 3.4 Streptomycin3.4 48.344.83.4 Sulfamethoxazol6.9 89.73.4 6.9 Tetracycline0.0 100 Trimethoprim0.0 100 TABLE 8: Results* of antimicrobial susceptibility testing of Salmonella spp.

52. www.ages.at Die Entwicklung der letzten Jahrzehnte Humane Erstisolate 1983-2011 52

53. www.ages.at Danke für Ihre Aufmerksamkeit 53