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  • 1/10/2014EURL Campy1 DTU Food, Technical University of Denmark

    Monitoring of antimicrobial resistance in Campylobacter EURL AR activities in framework of the new EU regulation

    Lina Cavaco licav@food.dtu.dk

  • 1/10/2014EURL Campy2 DTU Food, Technical University of Denmark

    Outline EURL-AR presentation and tasks Tasks related to new monitoring Brief overview Campylobacter in the monitoring

    Sampling AST and panel of antimicrobials to be tested

    Ring trials for Campylobacter AST in EURL-AR network Results of most recent Campylobacter AST trial (2013) Challenges

  • 1/10/2014EURL Campy3 DTU Food, Technical University of Denmark

    The tasks of the EURL-AR Scientific advice and support to the Commission

    Co-ordination of National Reference Laboratories and provision of technical support

    Creation of the network of NRLs Workshops Dissemination of knowledge and information Collection of information on activities at the NRLs

    Ring trials, comparative testing and quality assurance

    Confirmatory testing

    Evaluation and development of analytic methods

  • 1/10/2014EURL Campy4 DTU Food, Technical University of Denmark

    Co-ordination of National Reference Laboratories and provision of technical support

    Creation of the network of NRLs The address list

    Workshops One annual workshop next April 2015

    Training courses aimed at the specific methods to apply in the monitoring (2013 was training on AST incl. Campylobacter , this year was focused on selective isolation of presumptive ESBL-, AmpC or carbapenemase producing E. coli

    Site visits Dissemination of knowledge and information

    Home page: www.eurl-ar.eu Newsletters E-learning site ( ongoing preparation)

    Collection of information on activities at the NRLs Questionnaires

    The Tasks of the EURL-AR

  • 1/10/2014EURL Campy5 DTU Food, Technical University of Denmark

    The tasks of the EURL-AR (The ring trial system - EQAS)

    AST of eight Salmonella, Campylobacter, E.coli, Staphylococcus andEnterococcus strains

    MRSA isolation identification and spa typing Genotyping (gene EQAS) New one on the way, awaiting approval of WP 2015 Supply labs with original ref. strains E. coli ATCC 25922, C jejuni ATCC

    33560, S. aureus ATCC 29213 and E. faecalis ATCC 29212

    Results and comments submitted through a web-based data entry program, using individual passwords

    Instant individual evaluation reports with suggestions for corrective actions

    Yearly over-all evaluation reports on the web

  • 1/10/2014EURL Campy6 DTU Food, Technical University of Denmark

    Work of EURL-AR and the monitoring according to EU 652/2013

    Participation in the EFSA Workgroups Scientific advice to the EU Commission and participation on

    the Preparation and discussion of the contents and methods used in the regulation

    Facilitating training Facilitating ring trials Development of methods and confirmatory testing

    Thus, the activities aim at implementing, from an analytical point of view, the provisions of monitoring of antimicrobial resistance set down the EU regulation 652/2013

  • 1/10/2014EURL Campy7 DTU Food, Technical University of Denmark

    Campylobacter monitoring acc EC 652/2013 Sampling

    (a) In the years 2014, 2016, 2018 and 2020 for laying hens, broilers and fresh meat thereof, and fattening turkeys.

    (b) In the years 2015, 2017 and 2019, for pigs, bovines under one year of age, pig meat and bovine meat.

    Sample nrs 170 isolates for AST for each combination of bacterial species and type of

    sample of animal population or food category listed in point 1(a), (b), (c), (e) and (f). in Member States with a production of less than 100 000 tonnes -85 isolates is minimum

    In Member States with low bacterial prevalence or low number of epidemiological units, all available isolates at the end of the monitoring period shall be included in the antimicrobial susceptibility testing.

    Disclaimer: for exact information and further info on sampling design and representative sampling - consult the regulation and the EFSA technicalspecifications for the sampling procedures!

  • 1/10/2014EURL Campy8 DTU Food, Technical University of Denmark

  • 1/10/2014EURL Campy9 DTU Food, Technical University of Denmark

    Campylobacter monitoring Testing

    ID Campylobacter C. jejuni /C. coli Recommended PCR method for speciation under:

    http://eurl-ar.eu/data/images/protocols/protocol%20for%20campylobacter_november_2013.pdf

    Acc: Denis, C. Soumet, K. Rivoal, G. Ermel, D. Blivet, G. Salvat and P. Colin. Development of a m-PCR assay for simultaneous identification of Campylobacter jejuniand C. coli. Letters in Applied Microbiology 1999, 29, 406410

    AST MIC testing Antimicrobial panel and ranges acc EFSA technical

    specifications not changed from previous reccomendations QC and storage of isolates (for 5 years)

    Data collection- isolate-based data - EFSA

  • 1/10/2014EURL Campy10 DTU Food, Technical University of Denmark

    Choice of antimicrobials and ranges Insert reasons from EFSA

  • 1/10/2014EURL Campy12 DTU Food, Technical University of Denmark

    Rationale for antimicrobials to be tested Macrolides (erythromycin, clarithromycin, azithromycin) are considered

    first choice drugs for therapy of human infections with Campylobacterspp. Acquired resistance is caused by single point mutations in the ribosomal DNA resulting in cross-resistance between the different macrolides. Macrolides are commonly used in food-animals and resistance to macrolides is reported, predominantly in C. coli. Erythromycin is commonly used in the test panels as representative of the macrolides.

    (Fluoro)quinolones (ciprofloxacin, nalidixic acid) Resistance to quinolones is due to mutation in the gyrase gene resulting in resistance.

    Both nalidixic acid and ciprofloxacin are used in existing monitoringprogrammes as indicators of acquired resistance to quinolones in Campylobacter spp.

  • 1/10/2014EURL Campy13 DTU Food, Technical University of Denmark

    Rationale for antimicrobials to be tested(cont) Tetracyclines are considered alternative choices for treatment of

    Campylobacteriosis. But acquired resistance does occur. Therefore inclusion of this class is relevant. Resistance in Campylobacter spp. is encoded by tet(O), a gene that expresses a ribosomal protection protein.

    Streptomycin Resistance to aminoglycosides in Campylobacter spp. is encoded by APH and AAD-genes.

    Although cross resistance between different aminoglycosides is incomplete, streptomycin resistance can be used as indicator for acquired genes.

  • 1/10/2014EURL Campy14 DTU Food, Technical University of Denmark

    Ring trials, comparative testing and quality assurance

    Ring trials, comparative testing and quality assurance Proficiency tests -include one annual ring trial on AST of

    Campylobacter

    Confirmatory testing Confirmation of results and characterization of monitoring

    isolates

    Evaluation and development of analytic methods Establishing reference strain collections for relevant research

    and requests Methods for detection of relevant resistances- very focused on

    the needs in monitoring.

    The tasks of the EURL-AR

  • 1/10/2014EURL Campy15 DTU Food, Technical University of Denmark

    Area: Antimicrobial ResistanceMicroorganisms:

    CampylobacterSalmonellaE. coliEnterococci Staphylococci(MRSA, Genotyping and ESBL/AmpC)

    Methods for phenotypic antimicrobial susceptibility testing (AST): Microbroth / Agar dilution (MIC-test)

    Focus of the EURL-AR EQASs

  • 1/10/2014EURL Campy16 DTU Food, Technical University of Denmark

    To have laboratories evaluate their performance of antimicrobial susceptibility testing (AST)

    To assess the quality of AST in European reference laboratories

    And improve the quality of monitoring data to EFSA

    To identify challenges within the methodsTo evaluate the effect of training courses

    Objectives of the EQAS

  • 1/10/2014EURL Campy17 DTU Food, Technical University of Denmark

    Accredited methods (relevant for the EQAS): MIC testing according to EN/ISO 17025 Provider of EQAS (serotyping and AR) according to ILAC G13

    (International Laboratory Accreditation Cooperation)

    International standards/guidelines ISO (International Organization for Standardization) CLSI (Clinical and Laboratory Standards Institute) EUCAST (The European Committee on Antimicrobial Susceptibility

    Testing www.eucast.org)

    Quality Assurance of the EQAS

  • 1/10/2014EURL Campy18 DTU Food, Technical University of Denmark

    18

    Campy EQAS AST- Participation 2013

    28 countries

    30 sets of Campylobacterresults

  • 1/10/2014EURL Campy19 DTU Food, Technical University of Denmark

    19

    Comparison to former EQASs

  • 1/10/2014EURL Campy20 DTU Food, Technical University of Denmark

    20

    Campylobacter results strains

    Test strain AST in total

    % correct

    C-8.1 (C. coli) 179 93.9C-8.2 (C. coli) 180 97.8C-8.3 (C. jejuni) 174 94.8C-8.4 (C. jejuni) 161 96.9C-8.5 (C. jejuni) 180 97.8C-8.6 (C. coli) 180 96.1C-8.7 (C. coli) 180 96.1C-8.8 (C. coli) 180 98.9

    Only 3 incorrect identifications out of 240 identifications reported > 98.8 % correct

  • 1/10/2014EURL Campy21 DTU Food, Technical University of Denmark

    21

    Campylobacter results antimicrobials

    Antimicrobial % of correct ASTCiprofloxacin 96.6Erythromycin 97.5Gentamicin 99.6Nalidixic acid 93.2Streptomycin 96.6Tetracycline 95.7

    Overall p

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