(Question Nº EFSA-Q ) Adopted by The Task Force on 20 February 2007

Transcription

1 Report of the Task Force on Zoonoses Data Collection including a proposal for a harmonized monitoring scheme of antimicrobial resistance in Salmonella in fowl (Gallus gallus), turkeys, and pigs and Campylobacter jejuni and C. coli in broilers 1 (Question Nº EFSA-Q ) Adopted by The Task Force on 20 February For citation purposes: Report of the Task Force of Zoonoses Data Collection including a proposal for a harmonized pigs and Campylobacter jejuni and C. coli in broilers, the EFSA Journal (2007), 96,

2 Summary Presently many Member States monitor antimicrobial resistance in zoonotic agents, including Salmonella and Campylobacter. According Directive 2003/99/EC, Member States shall ensure that the monitoring provides comparable data on the occurrence of antimicrobial resistance. The European Commission has asked the European Food Safety Authority (EFSA) to prepare detailed specifications for harmonized schemes on antimicrobial resistance. The objective of these specifications is to lay down provisions for a monitoring and reporting scheme in Salmonella in fowl (Gallus gallus), turkeys, and pigs and Campylobacter jejuni and C. coli in broilers. This should help Member States to provide antimicrobial resistance data that is comparable and enable risk managers to consider provisions for harmonised monitoring. The current specifications are considered as a first step towards a gradual implementation of comprehensive antimicrobial resistance monitoring at the Community level. These specifications propose to test a common set of antimicrobials using common epidemiological cut-off values and a specified concentration range to determine the susceptibility of Salmonella and Campylobacter. Using isolates collected through programmes to estimate the prevalence of zoonotic agents, the target number of Salmonella isolates to be included in the antimicrobial resistance monitoring per Member State per year is 170 for each study population (i.e. laying hens, broilers, turkeys and slaughter pigs). The target number of Campylobacter isolates to be included in the antimicrobial resistance monitoring per Member State per year is 170 for each study population (i.e. broilers). The results of the antimicrobial resistance monitoring are assessed and reported, in accordance with Directive 2003/99/EC, in the yearly national report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance. 2

3 Table of contents Summary...2 A. Background and terms of reference...5 B. Specifications for a harmonized monitoring scheme of antimicrobial resistance in Salmonella in fowl (Gallus gallus), turkey, pigs and Campylobacter jejuni and C. coli in broilers...7 C. Underlying rationale for the specifications Scope Design of monitoring scheme Study population Sampling plan Sample size Methods Detection and identification and storage of microbial isolates Methodology for susceptibility testing Methodology for susceptibility testing Salmonella Methodology for susceptibility testing thermophilic zoonotic Campylobacter Antimicrobials to be monitored and cut-off values to be used Data and Reporting...20 D. Literature...22 E. Task Force on Zoonoses Data Collection members...23 F. Acknowledgments...23 Annex 1. Sample size calculations for monitoring antimicrobial resistance...24 Annex 2. Phage Typing of Salmonella Isolates...27 Annex 3. Rationale for inclusion of antimicrobials in antimicrobial resistance monitoring and cut-off values to be used...28 Annex 4. Example tables to report antimicrobial test results and some considerations for national data collection

4 Acronyms and definitions ARBAO CEN CRL CLSI EFSA EMEA EUCAST EU FAO ISO MIC NCCLS OIE WHO Antibiotic Resistance in Bacteria of Animal Origin European Committee for Standardization Community Reference Laboratory Clinical and Laboratory Standards Institute formerly known as NCCLS European Food Safety Authority European Medicines Agency European Committee on Antimicrobial Susceptibility Testing European Union Food and Agriculture Organization International Organisation for Standardization Minimum Inhibitory Concentration The National Committee for Clinical Laboratory Standards, now CLSI World Organization for Animal Health World Health Organization Epidemiological terminology Flock Holding National control programmes Specifications The epidemiological terminology used in this document can be found in the glossary of terms of the EFSA guidance document on the conduct of surveys (EFSA Journal 2006, 93). All poultry of the same health status kept on the same premises or in the same enclosure and constituting a single epidemiological unit; in the case of housed poultry, this includes all birds sharing the same airspace (Regulation 2160/2003/EC). Any establishment, construction or, in the case of an open-air farm, any place in which animals are held, kept or handled [Directive 92/102/EC, Art. (2) b]. Programmes referred to in Article 5 of Regulation 2160/2003/EC, for each zoonosis and zoonotic agent which shall be established by the Member States to achieve the Community targets provided for in Article 4 of the same Regulation. Specifications for a harmonized monitoring scheme of antimicrobial resistance that the European Commission asked EFSA to develop. 4

5 A. Background and terms of reference A FAO/OIE/WHO workshop on scientific assessment on non-human usage of antimicrobials and antimicrobial resistance in 2003 concluded that there is 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 (in some cases death) and increased severity of infections, 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 (Joint FAO/OIE/WHO Expert Workshop, 2003). 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 (European Parliament, October 2006). According to Article 7 of Directive 2003/99/EC 2 of the European Parliament and of the Council on the monitoring of zoonoses and zoonotic agents, Member States must have a monitoring system that provides comparable data on the occurrence of antimicrobial resistance in zoonotic agents originating from animals, food and feed and, insofar as they present a threat to public health, other agents. Such monitoring is to supplement the monitoring of human isolates conducted in accordance with Decision 2119/98/EC. In order to obtain such comparable data, it is appropriate to make recommendations on minimum requirements that need to be met. EFSA convened a working group under the Task Force on Zoonoses Data Collection that, taking into account the CRL-E work as well as recommendations on surveillance of antimicrobial resistance that were issued in the framework of a Concerted Action (ARBAO, 2002), drafted this report providing the rationale of the choices made in drafting the specifications. Source of isolates Presently many Member States do have a system in place to monitor zoonotic agents, including Salmonella and Campylobacter although these systems are not harmonised. Regulation 2160/2003/EC 3 lays down the requirement to establish national control programmes for Salmonella and specifies that Community targets for the reduction of prevalence are to be established for Salmonella in flocks of poultry and turkeys, and in herds of pigs. Within these control programmes, food business operators must have samples taken and analysed and official controls (including sampling schemes) are required. This entails that the total population or a representative subset of it has to be regularly tested. Another possible source of isolates, although limited in time, may be coordinated monitoring programmes that are set up according to Article 5 of Directive 2003/99/EC to estimate the prevalence of zoonotic agents in each Member State. At present, the Commission is discussing with the Member States technical specifications for an EU monitoring scheme for 2 OJ L325, , p OJ L325, , p.1. 5

6 Campylobacter in broilers. In addition, a proposal for a harmonised monitoring scheme for Salmonella and Campylobacter in broiler meat was developed by EFSA (EFSA Journal 2006, 92). Because Salmonella and Campylobacter are presently isolated from monitoring activities in many Member States and will be isolated in the framework of national control programmes it is opportune to use these isolates for the purpose of antimicrobial resistance monitoring, as the underlying schemes through which they are collected are harmonised across Member States. Terms of Reference The European Commission has asked (EFSA-Q ) the European Food Safety Authority (EFSA) to prepare certain detailed specifications for harmonized schemes on antimicrobial resistance (hereinafter referred to as specifications ) indicating that the work done by the former Reference Laboratory for the Epidemiology of Zoonoses (CRL-E, 2004) could help development of such specifications. The Task Force on Zoonoses Data Collection and its working group were asked to prepare a proposal for a harmonized monitoring scheme of antimicrobial resistance in Salmonella and Campylobacter in certain animal species. The proposal should provide guidance to Member States, using isolates collected in the framework of national control programmes that meet the requirements laid down in Regulation 2160/2003/EC, to provide antimicrobial resistance data that is comparable. This should enable risk managers to consider detailed rules on the implementation of a harmonised monitoring of antimicrobial resistance. 6

7 B. Specifications for a harmonized monitoring scheme of antimicrobial resistance in Salmonella in fowl (Gallus gallus), turkey, pigs and Campylobacter jejuni and C. coli in broilers Objective and general provisions According to Article 7 of Directive 2003/99/EC, Member States shall ensure that the 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. The objective of these specifications is to lay down provisions for a monitoring and reporting scheme in Salmonella in fowl (Gallus gallus), turkeys, and pigs and Campylobacter jejuni and C. coli in broilers to obtain comparable data from Member States to be able to assess trends and sources of antimicrobial resistance in the Community. Origin of isolates to be tested Salmonella and Campylobacter isolates collected through control and monitoring programmes, set up in accordance with Article 5 of Regulation 2160/2003/EC and/or Article 5 of Directive 2003/99/EC, will be collected for possible inclusion in the antimicrobial resistance monitoring. In case there are no such programmes in place, isolates from other national control or monitoring programmes for Salmonella or Campylobacter in the animal species, or isolates from other sources (i.e. routine testing, passive laboratory surveillance, etc.) might be used for a transitional period. In case the control or monitoring programmes cover both the breeding and production animals, only isolates coming from the production animal population are used. Not more than one isolate per Salmonella serovar or Campylobacter species 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. Laying hens, broilers, turkeys and slaughter pigs are considered as separate study populations. 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, are to be included. 7

8 Number of isolates to be tested The number of Salmonella isolates to be included in the antimicrobial resistance monitoring per Member State per year is 170 for each study population (i.e. laying hens, broilers, turkeys and slaughter pigs). The number of Campylobacter isolates to be included in the antimicrobial resistance monitoring per Member State per year is 170 for each study population (i.e. broilers). Antimicrobial susceptibility testing Member States shall test the antimicrobials that are specified in Table A, using the cut-off values given and an appropriate concentration range to determine the susceptibility of Salmonella and Campylobacter. For Salmonella spp, 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 :2006) shall be used. For Campylobacter spp. dilution methods shall be performed according to the methods described in CLSI guidelines M31-A3 - Third Edition, Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals and M100 S16, Performance Standards for Antimicrobial Susceptibility testing; Sixteenth International Supplement. The Community Reference Laboratory for Antimicrobial Resistance is laid down in Commission Regulation 776/2006/EC, and the task of the National Reference Laboratories in Article 33 of Regulation 882/2004/EC. Collection of the data and reporting The results of the antimicrobial resistance monitoring are 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 and Campylobacter in broiler flocks: number of isolates susceptibility tested; number of isolates found to be resistant per antimicrobial; and number of fully-susceptible isolates and number of isolates resistant to 1, 2, 3, 4 and >4 antimicrobials listed in Table A. 8

9 Table A. Antimicrobials to be included in the antimicrobial resistance monitoring for each zoonotic agent and the cut-off value for each antimicrobial to be used to determine susceptibility. Antimicrobial Cut-off value (mg/l) R> Salmonella Campylobacter jejuni Campylobacter coli Cefotaxime 0.5 Nalidixic acid 16 Ciprofloxacin 0.06 Ampicillin 4 Tetracycline 8 Chloramphenicol 16 Gentamicin 2 Streptomycin 32 Trimethoprim 2 Sulphonamides 256 Erythromycin 4 Ciprofloxacin 1 Tetracycline 2 Streptomycin 2 Gentamicin 1 Erythromycin 16 Ciprofloxacin 1 Tetracycline 2 Streptomycin 4 Gentamicin 2 9

10 C. Underlying rationale for the specifications 1 Scope The scope of the specifications is to propose minimum requirements for the harmonised monitoring of antimicrobial resistance in Salmonella and Campylobacter in the Community, using isolates collected through programmes to estimate the prevalence of zoonotic agents. This should provide guidance to Member States to meet the requirement to provide data that is comparable and enable risk managers to consider detailed provisions on the implementation of a harmonised monitoring of antimicrobial resistance. Annex IIA of Directive 2003/99/EC provides general requirements for monitoring antimicrobial resistance including a list with information that minimally needs to be provided. The working group considered these aspects as well as other aspects relevant to achieve the stated objectives. Objectives Article 9 of Directive 2003/99/EC states that Member States shall assess and report on trends and sources of antimicrobial resistance. Such a monitoring scheme ideally should allow the determination of the proportion of resistance and assessment of temporal trends as well as the identification of the emergence of resistance or emergence of specific patterns (e.g. DT104) of resistance. The first objective of the monitoring scheme is to determine the proportion of resistance to different antimicrobials among Salmonella spp., Campylobacter jejuni, and C. coli so as to be able to assess temporal trends and emergence of resistance for each of them. The proportion of resistance is defined as the proportion of bacteria isolates that are tested for a given antimicrobial and found to be resistant. Resistance against an antimicrobial is considered to be present in a bacteria isolate if the Minimum Inhibitory Concentration (MIC) exceeds the epidemiological cut-off value. As for the second objective, i.e. the identification of sources of resistance and their contribution to human infections, the resistance monitoring scheme in combination with the schemes to monitor the prevalence of the zoonotic agents and the serovars distributions should enable to assess, at Member State level, the relative contribution to antimicrobial resistance of each study population that is considered. Limitations A monitoring scheme can also contribute to provision of information regarding the causes of emergence and/or spread of antimicrobial resistance and how to control resistance at the farm or production level. However, to assess these, information would be needed for example on usage of antimicrobials, and on animals or products harbouring germs with resistance genes. These aspects, although considered important, do not fall within the present scope and are not covered in these specifications. Other limitations that have been considered are: 10

11 Annex IIB of Directive 2003/99/EC lays down that the Member States monitoring system must provide information 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 from those species. In addition, the existing and forthcoming monitoring and control programmes pertain to Salmonella and Campylobacter. This is why other micro-organisms that also may form a threat to public health are not covered in the current specifications. Regulation 2160/2003/EC refers specifically to programmes for the reduction of prevalence of Salmonella in poultry and pigs. This is why the scope of the current specifications does not explicitly cover cattle nor does it cover food of animal origin. These may be addressed subsequently. However, many aspects of the laboratory methodology (susceptibility test methods, choice of the antimicrobials and interpretive criteria) as well as sample size are applicable regardless of the source of the isolate of a particular bacterial species. At present there is no Community guidance on control programmes for Campylobacter, but draft technical specifications for an EU monitoring scheme for Campylobacter in broilers are being discussed by the Commission and the Member States. For this reason specifications for Campylobacter are made for broilers only. However, the specifications made for broilers can also be used to monitor Campylobacter in turkeys. Meat and meat production animals (e.g. pigs) may be exported and slaughtered outside the country of origin and may contribute to the observed level of resistance in the host country. It is considered that specific studies would be required to understand their relative contribution to the local resistance problem. This falls outside the scope of this monitoring scheme. A number of Member States have few, if any, breeding flocks/herds and import adult breeders (e.g. turkeys). In such cases imported breeders or contaminated hatching eggs that are infected with Salmonella or Campylobacter can influence the percentage of the resistant isolates found subsequently in commercial flocks/herds. Although it may be important to test isolates for control purposes (in line with Regulation 1168/2006/EC), breeders are not included in this monitoring scheme because it will be very difficult to ensure representative sampling. Taking account of the above, specifications are drafted for a harmonized monitoring scheme of antimicrobial resistance in Salmonella in fowl (Gallus gallus), turkeys, and pigs and Campylobacter jejuni and C. coli in broilers. The current specifications are considered as a first step towards a gradual implementation of comprehensive antimicrobial resistance monitoring at the Community level. 11

12 2 Design of monitoring scheme 2.1 Study population Active monitoring following the same principles in all Member States will guarantee the availability of isolates to generate comparable data on antimicrobial resistance in zoonotic agents. Therefore, the most cost-effective and unbiased way to monitor antimicrobial resistance in the EU is considered to be through national control programmes. Although it will take time before the national control programmes are implemented, already the design of the antimicrobial resistance monitoring scheme is largely determined by these programmes. Annexes I and II of Regulation 2160/2003/EC specify a number of common elements and minimum sampling rules for national control programmes for Salmonella. As discussed above, the current specifications are limited to the study populations specified in Table 1 for Salmonella and to broiler flocks for Campylobacter. Whereas it may be of interest to monitor antimicrobial resistance in various poultry production phases, greatest benefit may result from focussing on those populations which the consumer will most likely be exposed to. This means isolates from broilers, turkeys, and pigs should preferably be collected close to or at slaughter, whereas isolates from laying hens should be periodically collected throughout the egg production cycle. For this reason the current specifications are limited to the production phases specified for the different study populations in Table 1. Table 1. Common elements in design of national control programmes for Salmonella (adapted from Annex IIB of Regulation 2160/2003/EC) Target population Location of sampling Animal species Fowl (Gallus gallus) Study population flocks of laying hens broiler flocks Stage of food chain primary production primary production Turkeys all flocks primary production Pigs slaughter pig herds primary production Production phase every 15 weeks during the laying phase animals leaving for slaughter animals leaving for slaughter animals leaving for slaughter or carcasses at slaughterhouse 2.2 Sampling plan In the programmes to determine the prevalence of Salmonella, details on collection of type of material and where the sampling is to take place are already fixed. To provide an unbiased estimate of the proportion of resistance the sampling frame should cover all epidemiological 12

13 units (flocks or holdings) of the national production. This is most readily achieved if isolates originate from national control programmes. In the absence of, or in addition to, such programmes, isolates from other monitoring systems can be used, provided that they were sampled in accordance with the methodology described in this document. In case there are no national control programmes in place yet for Salmonella or Campylobacter in certain animal species, isolates from other sources (i.e. routine testing, passive laboratory surveillance, etc.) might be used for a transitional period. It is the intention to draft additional monitoring schemes to cover Salmonella and Campylobacter in these animal species. The epidemiological unit for laying hens, broilers, and turkey is the flock, because most holdings practise all-in-all-out production. For pigs, the epidemiological unit is the holding, because many farms do not practice strict all-in-all-out production. It is assumed that Salmonella isolates of the same serovar or Campylobacter from the same species from the same epidemiological unit show a similar pattern of resistance. To ensure representativeness, susceptibility testing should be done for no more than one isolate per Salmonella serovar or Campylobacter species from the same epidemiological unit per year. If two different Salmonella serovars or Campylobacter species are included from the same epidemiological unit some clustering will occur. However, between serovars or species the pattern of resistance may vary considerably, therefore the effect of clustering will be minimal. 2.3 Sample size The sample size (i.e. number of isolates to be susceptibility tested) should allow within a predetermined accuracy to calculate the proportion of resistance to a particular antimicrobial in the Member States and to detect changes in this proportion over time. As explained in Annex 1, the target sample size may vary depending on whether the sample size is calculated for the purpose of determining the proportion of resistance or for the purpose of determining a trend. In addition, the sample size differs greatly with the magnitude in the change one wishes to be able to detect or the accuracy of the estimate. Which magnitude of change is relevant depends on the initial resistance situation. If resistance is already widespread, only a relatively large decrease or increase in proportion of resistance is considered relevant. For the emergence of resistance, on the other hand, an increase by a few percent should already be detectable. It was concluded from a public health perspective that an adequate target number of isolates to be susceptibility tested per study population per Member State per year is 170. As detailed in Annex 1, this sample size allows to: detect a change of 15% in the situation of widespread resistance (50% proportion of resistance) and to detect an increase of 5% in the situation of few pre-existing resistant isolates (0.1% proportion of resistance); and provide an accuracy of +/- 8% for the purpose of determining a proportion of resistance in the worst case scenario of 50% resistant isolates. 13

14 In case a linear trend exists within a country, smaller changes in proportion can be detected over time. In case of three years continuous monitoring: starting from an initial proportion of resistance of 50%: a 5% decrease in proportion of resistance per year can be detected; and starting from an initial proportion of resistance of 0.1%: an increase by 2% per year can be detected. As detailed in Table 2, Member States are asked to include 170 Salmonella isolates from each study population (i.e. laying hens, broiler flocks, turkeys, and pigs) as well as 170 Campylobacter isolates from each study population (i.e. broiler flocks). Table 2: Desirable number of isolates to include in susceptibility testing Target population Salmonella spp. Animal species Study population Fowl (Gallus gallus) flocks laying hens 170 Campylobacter spp. (i.e. jejuni and coli) broiler flocks Turkeys flocks 170 Pigs slaughter pig herds 170 Nevertheless, the number of isolates available from national control programmes may vary considerably between Member States, study populations and bacteria species, therefore: If, in low prevalence countries, the selection of one isolate per epidemiological unit per Salmonella serovar or Campylobacter species results in 170 or fewer available isolates, all these isolates should be submitted to susceptibility testing. If more than 170 isolates of a bacteria species are available from a study population, a minimum of 170 isolates should be randomly select for susceptibility testing. With this approach, Salmonella serovars as well as the Campylobacter species that are most frequently isolated will have the highest likelihood to be selected for susceptibility testing, and random selection will ensure representativeness also in case there would be a change in the pattern of serovars and/or species. The laboratory may also choose to test all available isolates, as long as only one isolate per epidemiological unit and serovar or species is included in the sample. In Member States with a low prevalence of Salmonella and Campylobacter or a small livestock production, the number of available isolates may be substantially smaller than the target sample size. This will result in a decreased precision of the estimate of the proportion of resistance and it will be more difficult to detect trends over time within the respective Member States. However, it is considered that these Member States do not contribute substantially to the problem of antimicrobial resistant Salmonella and Campylobacter at European level. Therefore, this should not impair the overall estimate of risk of antimicrobial resistance for European consumers. 14

15 If resources are limited, a sampling interval of two years can be applied for each study population. In one year, antimicrobial resistance testing could be performed in Salmonella from laying hens and slaughter pigs. In the following year, Salmonella and Campylobacter isolates from broiler and turkey production could be analysed. 15

16 3 Methods 3.1 Detection and identification and storage of microbial isolates For isolation and confirmation of bacteria validated methods should be followed, as defined in the national control programmes. 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 addressed separately by Community Reference Laboratories. For Salmonella all the isolates selected for antimicrobial susceptibility testing should be identified at serovar level. For S. Enteritidis and S. Typhimurium it is recommended that all the isolates selected for antimicrobial susceptibility testing are phage typed to allow proper interpretation of the observed proportion of resistance. For more detail, see Annex 2. For Campylobacter all the isolates selected for antimicrobial susceptibility testing should be identified at species level. Monitoring is restricted to C. jejuni and C. coli which are the most important species causing infections in humans. Other Campylobacter species should therefore not be included in the antimicrobial resistance monitoring. 3.2 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. Moreover, one set of interpretive criteria would not be sufficient for harmonisation of the results, because differences in methods used for disk diffusion exist. For Campylobacter spp. the use of disk diffusion is not advocated by CLSI because of the poor reproducibility of the results. Therefore, to improve the comparability of the data provided by Member States, only quantitative data on MIC will be accepted. 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. In case other designated laboratories participate in the monitoring they should comply with the quality requirements as detailed in Article 12 of Regulation 2160/2003/EC. Tasks and responsibilities of reference laboratories are laid down under Title III of Regulation 882/2004/EC. 16

17 3.2.1 Methodology for susceptibility testing Salmonella 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 :2006] Methodology for susceptibility testing thermophilic zoonotic Campylobacter For Campylobacter spp. dilution methods should be performed according to methods as described by CLSI (CLSI, 2006). At present, the CLSI documents are the only international standards giving guidance on broth micro-dilution testing and quality assurance for Campylobacter. 17

18 4 Antimicrobials to be monitored and cut-off values to be used 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 3. This should not prevent Member States to include additional antimicrobials in their monitoring. A detailed rationale for including antimicrobials in the monitoring is available in Annex 3. 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). 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. Interpretation should be based according to the current epidemiological cut-off values presented by EUCAST ( as described in Annex 3. The epidemiological cut off value is the highest MIC value of the wild-type population (WT) appropriate to detect biological resistance, expressed as WT X mg/l. 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. When enough MIC data will be available from reporting Member States, epidemiological cut-off values will be established to replace the presently used clinical breakpoints. Qualitative results should be reported as susceptible (S) or resistant (R), expressed as S X mg/l or R > X mg/l. 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. Only resistance to different antimicrobials for which the resistance mechanisms are not related should be reported: For Campylobacter this includes resistance to tetracycline, erythromycin, ciprofloxacin, gentamicin, and streptomycin. For Salmonella this includes resistance to ampicillin, cefotaxime, nalidixic acid (or ciprofloxacin), streptomycin, gentamicin, tetracycline, chloramphenicol, trimethoprim and sulphamethoxazole. 18

19 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. Table 3. Antimicrobials to be included in the antimicrobial resistance monitoring for each zoonotic agent, the epidemiological cut-off value for each antimicrobial to be used to determine susceptibility, and the concentration range to be tested for each antimicrobial. Salmonella Campylobacter jejuni Campylobacter coli Antimicrobial Cut-off value (mg/l) Advised optimum Concentration range to be tested (mg/l) R> Cefotaxime Nalidixic acid Ciprofloxacin Ampicillin Tetracycline Chloramphenicol Gentamicin Streptomycin 32** Trimethoprim* Sulphonamides 256*** Erythromycin Ciprofloxacin Tetracycline Streptomycin Gentamicin Erythromycin Ciprofloxacin Tetracycline Streptomycin Gentamicin * Trimethoprim is often used in combination with sulphonamides because of synergy in clinical treatment. However, for susceptibility testing it is important to test and report both substances separately. ** Breakpoint advised by ARBAO-II. *** CLSI breakpoint. 19

20 5 Data and Reporting 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 31 st 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. 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: 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. 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. 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. b) Quantitative tables to report MIC distributions for the following study populations in certain bacterial species, if available: S. Enteritidis S. Typhimurium S. Derby Other Salmonella serovars grouped together C. jejuni laying hens X X X broilers X X X X X turkeys X X X pigs X X X C. coli 20

21 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. For Salmonella an important consideration is the number of isolates of a given serovar available for analysis, because a minimum number of isolates is required to provide an informative table and because large fluctuations in the prevalence of resistance are to be expected when only low numbers of isolates are available. 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. 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. For C. jejuni and C. coli there are marked differences in the prevalence of resistance shown by each of these bacterial species to different antimicrobials and it is therefore inappropriate to consider reporting both species together. 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. Further guidance for reporting officers is available in the Manual for Reporting on Zoonoses, Zoonotic agents, antimicrobial resistance and food-borne outbreaks in the framework of Directive 2003/99/EC. 21

22 D. Literature ARBAO, Antibiotic Resistance in Bacteria of Animal Origin. Surveillance of antimicrobial resistance. Final Report. Concerted action PL CLSI, Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Sixteenth Informational Supplement (M100-S16); and Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Tentative guideline M31-A3 Third Edition. CRL-E, Community Reference Laboratory for the Epidemiology of Zoonoses. Draft ( ) Monitoring programme on the occurrence and trend of antimicrobial resistance. DANMAP, The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme. Consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. ISSN EFSA Journal 2006, 92. Report of Task Force on Zoonoses Data Collection on proposed technical specifications for a coordinated monitoring programme for Salmonella and Campylobacter in broiler meat in the EU, The EFSA Journal (2006), 92, eat.html. EFSA Journal 2006, 93. Report of Task Force on Zoonoses Data Collection on Guidance Document on Good Practices for Design of Field Surveys, The EFSA Journal (2006), 93, European Parliament, October DG for Internal Politics of the Union. Report on antibiotic resistance. EP IP/A/STOA/ST/ ISO :2006. International Organisation for Standardization. Clinical laboratory testing and in vitro diagnostic test systems -- Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices -- Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. ISO, Geneva, Switzerland. Joint FAO/OIE/WHO Expert Workshop on Non-Human Antimicrobial Usage and Antimicrobial Resistance: Scientific assessment. Geneva, December 1 5, Accessed 14 December

23 E. Task Force on Zoonoses Data Collection members José Ignacio Arraz Recio, Harry Bailie, Marta Bedriova, Pierre-Alexandre Beloeil, Sofia Boqvist, Birgitte Borck, Karen Camilleri, Georgi Chobanov, Adriana Costache, Kris De Smet, Katica Florjanc, Matthias Hartung, Birgitte Helwigh, Merete Hofshagen, Vaidotas Kiudulas, Terhi Laaksonen, Peter Much, Lisa O Connor, Rob A.A. Van Oosterom, Jacek Osek, José Luis Paramio Lucas, Manca Pavsič, Christodoulos Pipis, Antonia Ricci, Valentina Rizzi, Petr Šatrán, Joseph Schon, Jelena Sõgel, Petra Szabados, Patricia Tavared Santos, Kilian Unger, Luc Vanholme, Dimitris Vourvidis and Nicole Werner-Keišs. F. Acknowledgments The Task Force on Zoonoses Data Collection wishes to thank the working group for drafting the report: Antonio Battisti, Bjorn Bengtsson, Segundo Piriz Duran, Hanne-Dorthe Emborg, Dik Mevius, Getraud Regula, Pascal Sanders, Christopher Teale, and Dariusz Wasyl. Stef Bronzwaer, Hubert Deluyker, and Pia Mäkelä. Observers: Kris De Smet, Jordi Torren Edo, and Peet Tüll. 23

24 Annex 1. Sample size calculations for monitoring antimicrobial resistance Depending on the objective of the monitoring programme, several statistical methods exist to calculate the required sample size (number of bacteria isolates to be tested). For these calculations, the sample size depends on: The prevalence of resistance in the bacteria species and study population to be monitored. The magnitude of the change in prevalence of resistance which should be detected by the monitoring programme (effect size). The desired precision for estimating a prevalence of resistance, i.e. the maximum acceptable error of the prevalence estimate. The statistical confidence level, i.e. the degree of certainty that a statistical prediction on the change in prevalence of resistance is accurate, or the chance to obtain a prevalence estimate within the desired precision. The required statistical power, i.e. the chance to obtain a statistically significant result if a change in prevalence exists. The following examples explore the required sample size for different objectives and requirements on the effect size or precision to be measured. All sample size calculations were performed for the Member State level, i.e. the sample size refers to the number of isolates which should be tested per Member State, study population and bacteria species. At the European level, the monitoring programme would be much more precise than stated in the tables. Thus, the monitoring programme would also provide valuable information on subpopulations with a small sample size per Member State, such as rare Salmonella serovars or rare combinations of resistance against several antimicrobials. The following assumptions were used for the calculations: (1) infinite population size for the number of bacteria isolates in each study population and Member State; (2) a confidence level of 95% and a power of 80%; (3) perfect sensitivity and specificity of the diagnostic test (susceptibility testing). Objective 1. Detection of difference between proportion of resistance (from year to year) The magnitude of change in the proportion of resistance which can be detected with a given sample size depends on the initial proportion of resistance. The table below lists the required sample size to detect changes in the proportion of resistance for the situation of widespread resistance (50% proportion of resistance) and rare resistance (0.1% proportion of resistance): Decrease from 50% proportion of resistance to: 35% 30% 25% Sample size Increase from 0.1% proportion of resistance to: 2% 3% 5% 10% Sample size

25 If the same number of isolates is tested each year, a change in the resistance situation within one Member State from one year to the following year can be detected with the sample size listed in the table (e.g. a change from 50% to 35% proportion of resistance with a sample size of 168 isolates). Objective 2. Detection of trend in proportion of resistance over several years If the monitoring programme is only required to detect changes in the proportion of resistance after two or more years, the sample size required to detect an ongoing trend decreases. A 5% decrease in proportion of resistance per year can be detected after two years with a monitoring programme designed to detect a 10% decrease, or after three years with a programme designed to detect a 15% decrease. The table lists the required sample size to detect changes in proportion of resistance for the situation of widespread resistance (50% proportion of resistance) and rare resistance (0.1% proportion of resistance): Sample size over 2 years 3 years 4 years 5 years Initial proportion of resistance 50% Decrease by 5% per year Initial proportion of resistance 50% Decrease by 10% per year Initial proportion of resistance 50% Decrease by 15% per year Initial proportion of resistance 0.1% Increase by 1% per year Initial proportion of resistance 0.1% Increase by 2% per year Initial proportion of resistance 0.1% Increase by 3% per year Initial proportion of resistance 0.1% Increase by 5% per year In the calculations, it was assumed that the change in proportion of resistance over time within one member state is linear, and that it is sufficient to demonstrate statistical significance of the change in proportion of resistance after 2, 3, 4 or 5 years, respectively. This could be achieved by applying an interval of two or more years for testing antimicrobial resistance in a given study population. Nevertheless, testing samples every year is recommended, because trends in proportion of resistance might be detected earlier, even if the statistical power is not sufficient to reliably detect a trend after that time. For example, with a sample size of 168 there is a 12% chance (power) that a decrease of 5% per year from an initial prevalence of 50% will already be detected as statistically significant after one year. In the second year of monitoring the power to detect this decrease as significant is 41%, and in the third year 80%. The same sample size results in a power of 21% for the detection of a 2% increase from an initial prevalence of 0.1% after one year, and in a power of 57% to detect the same trend after two years. 25