1. STATENS SERUM INSTITUT Spread of Resistant E.coli and Resistance Genes from Animals to Humans Anette M. Hammerum National Center for Antimicrobials and Infection Control Statens Serum Institut, Copenhagen, Denmark

2. STATENS SERUM INSTITUT Agenda Danmap- brief history Olaquindox Resistance in Indicator E. coli from Animals and Humans Normat-study Comparison of Indicator E. coli from Pigs, Pork and Humans Detection of sul genes in pig, pork and humans Flouroquinolones in animals and humans TRAINAU

3. STATENS SERUM INSTITUT DANMAP – brief history The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) was established by the Danish Ministry of Food, Agriculture and Fisheries and the Danish Ministry of Health in 1995 Since 1996, DANMAP reported annually on the occurrence of antimicrobial resistance in pathogenic and indicator bacteria from animals, foods and humans in Denmark Statens Serum Institut

4. STATENS SERUM INSTITUT DANMAP Danish Integrated Antimicrobial Monitoring and Research Programme DANMAP pdf-files: www.danmap.org

5. STATENS SERUM INSTITUT http://www.strama.se/news.asp http://www.danmap.org http://www.vetinst.no/Arkiv/Zoonosesenteret/ NORM_NORM-VET_2003.pdf http://www.swab.nl

6. STATENS SERUM INSTITUT Consumers are increasingly concerned about food safety Animal to human transfer of bacteria (or resistance determinants) carrying resistance to antimicrobials considered critically important in human medicine

7. STATENS SERUM INSTITUT Transfer of antimicrobial resistance between animals and humans Statens Serum Institut National Center for Antimicrobials and Infection Control Environment Direct contact

8. STATENS SERUM INSTITUT Public Health Concerns Salmonella & E. coli Fluroquinolones 3rd Gen. Cephalosporins Aminoglycosides Sulphonamides Campylobacter Fluroquinolones Macrolides Most G- Concerns relate to the THERAPEUTIC use of antibiotics in food animals Enterococci, Streptococci, Staphylococci Vancomycin Streptogramins Most G+ concerns relate to the GROWTH PROMOTER use of antibiotics

9. STATENS SERUM INSTITUT Antimicrobial Growth Promoters (AGP) Antimicrobial substances used as a supplement in animal feed in sub-therapeutic concentrations Avoparcin (G+) Spiramycin (G+) Bacitracin (G+) Avilamycin (G+) Virginiamycin (G+) Flavomycin (G+) Tylosin (G+) Carbadox (G-) Olaquindox (G-)

10. STATENS SERUM INSTITUT Olaquindox Resistance in Indicator E. coli from Animals and Humans It has recently been shown that olaquindox resistance is encoded by oqxA and oqxB (efflux pump system) This system has been found in E. coli isolates from pigs. Hansen et al. 2005 AAC

11. STATENS SERUM INSTITUT Olaquindox resistence in E. coli isolates from humans Olaquindox resistance can been found in persons with close contact to animals and in person with no contact to pigs

12. STATENS SERUM INSTITUT Trends in consumption of antimicrobials in food animals in Denmark AGP for Finisher pigs and broilers terminated AGP for Weaner pigs terminated Profit on therapeutics reduced

13. STATENS SERUM INSTITUT Effect of the AGP Stop on Antimicrobial Consumption and Occurrence of Resistance in Food Animals in Denmark Total antimicrobial consumption in animals reduced by >50% from 1994 to 2002 Slow increase in consumption of antimicrobials for therapy in pigs - No increase in broilers Substantial quantitative reduction of antimicrobial resistance in bacteria from animals and food

14. STATENS SERUM INSTITUT Veterinary practice Private Laboratories Regional Food Control Laboratories Regional hospital laboratories General practice Danish Institute for Food and Veterinary research Danish Medicines Agency Samples Isolates Diagnosticsubmission Isolates Samples Samples Danish Zoonosis Centre Slaughter plants Samples Humans Data Data Foods Food animals Data Data Isolates Data Data Data DANMAP - data flow VetStat Data Statens Serum Institut Data Data

15. STATENS SERUM INSTITUT Frederiksberg Municipality Ringkoebing Frederiksborg Funen Storstroem Bornholm Viborg North Jutland Aarhus Ribe South Jutland Roskilde Copenhagen Municipality West Zealand Vejle Copenhagen County -16 counties in DK -15/16 clinical microbiology laboratories delivered data in 2004

16. STATENS SERUM INSTITUT Data Included in the DANMAP Integrated surveillance

17. STATENS SERUM INSTITUT Indicator bacteria Easily obtained from various animal species as well as from humans Good ability to acquire resistance to various antimicrobial substances Easily obtained from healthy individuals Gram+ : E. faecium / E. faecalis Gram - : E. coli

18. STATENS SERUM INSTITUT Indicator Bacteria in DANMAP E. coli (and Enterococcus faecalis og Enterococcus faecium) from meat and healthy animals has been sampled since 1996, whereas a continuously sampling of faces from non- hospitalized persons was initiated in 2002

19. STATENS SERUM INSTITUT Normal Flora Study at SSI Methods: Volunteers were found through the Danish Civil Registry system, which is a continuously updated register of all residents in DK. The scientific ethics committee for Copenhagen and Frederiksberg municipalities approved the protocol Age and gender profile of the invited volunteers were in compliance with the total Danish population

20. STATENS SERUM INSTITUT Selection of persons via CPR (DZC) 1st Letter: Invitation + Declaration of consent Declaration of consent to DZC 2nd Letter: Material for faeces sample + questionnaire (with code) Faeces sample sent to SSIE-mail to DZC with code Telephone interview / webDatabase (DZC) NOR-MAT, 2002- ongoing  One random E. coli, E. faecium and E. faecalis isolate / sample  MIC determination (Sensititre) for growth promoters and antibiotics used for therapy  A selective method to detect vancomycin res. enterococci  Presence of enteric pathogens Identification and testing

21. STATENS SERUM INSTITUT Respons rate YearInviteredAnswarsRespons rate 2002 (from Marts) 76011114.6 % 2003105513012.3 % 200498811912.0 %

22. STATENS SERUM INSTITUT From March 2002 to December 31, 2003, 1815 individuals were invited to participate in the study 248 (13.7%) responded to the letter In total, 93 E. faecium isolates, 119 E. faecalis isolates, and 195 E. coli isolates were obtained NOR-MAT: Results, 2002-2003

23. STATENS SERUM INSTITUT NOR-MAT: Results, 2002-2003 A single E. faecalis isolate was gentamicin-resistant and one vancomycin resistant E. faecium isolate was detected (selective method) 10% E. coli isolates were multi-resistant The carrier-rate of Salmonella and Campylobacter in the normal population is lower than 1%, however several pathogenic E. coli was identified

24. STATENS SERUM INSTITUT Comparison of Indicator E. coli from Pigs, Pork and Humans

25. STATENS SERUM INSTITUT Trends in Ampicillin Resistance among Indicator E. coli

26. STATENS SERUM INSTITUT Trends in Sulphomanide Resistance among Indicator E. coli

27. STATENS SERUM INSTITUT Detection of sul1, sul2 and sul3 in sulphonamide resistant Escherichia coli isolates obtained from healthy humans, pork and pigs in Denmark (Hammerum et al. accepted for publication)

28. STATENS SERUM INSTITUT Background In 2003, antibiotic treatment of pigs accounted for almost 80% of the total amount of antibiotics administered to food animals in Denmark. The combination of sulphonamide and trimethroprim was the second most used antimicrobial therapy for sows and piglets in 2003 (approx. 4 tonnes)

29. STATENS SERUM INSTITUT Sulphonamide or trimethroprim are used for treatment of the majority uncomplicated urinary tract infections of humans in Denmark (approx. 3 tonnes) Potential transfer of sulphonamide resistant (sulR) Escherichia coli from animals, directly or via handling of raw meat, to humans is therefore undesirable. SulR is often encoded by sul1 and sul2 in Enterobacteriaceae. Recently a third gene, sul3, was found to encode for sulR.

30. STATENS SERUM INSTITUT Materials: 998 E. coli isolates, obtained from pig faeces collected at slaughter, Danish pork collected at retail outlets and from faeces from healthy persons in Denmark All sulphonamide resistant E. coli isolates were investigated for the presence of sul1, sul2, and sul3 genes by PCR

31. STATENS SERUM INSTITUT Prevalence of sul1, sul2 and sul3 in 236 Sulphonamide Resistant (sulR) E. coli isolates OriginNo. of isolates tested No. of isolates PCR-positive for : Number of strains with more than one sul R gene sul1sul2sul3 Humans3540%80%020% Pork3923%74%5% Pigs16253%50%11%14%

32. STATENS SERUM INSTITUT Discussion and Conclusion: The proportion of sulR E. coli isolates obtained from faeces of pigs increased significantly from 23% to 31% from 2002 to 2003 (DANMAP 2003). This coincided with an increased consumption of sulphonamide and trimethroprim by pigs. Urinary tract infections are generally thought of as infections caused by bacteria from the host’s own faecal flora (Murray et al. 2004). A recent study has demonstrated a close resemblance between certain food borne and human isolates of extra intestinal pathogenic E. coli, which suggests a possible transmission of E. coli causing urinary tract infections through food products (Johnson et al. 2005).

33. STATENS SERUM INSTITUT Discussion and Conclusion: The presence of sulR E. coli in faeces from pigs, in pork and in the gut flora of healthy humans is therefore of concern The origin of the sulphonamide resistance genes found in isolates from healthy human E. coli isolates is speculative, but spread via the food chain seems very likely

34. STATENS SERUM INSTITUT Fluoroquinolone consumption in DK – Primary health care, hospital and veterinary use Year Veterinary use (kg) Primary health care (kg) Hospital (kg)Total 2001 185 220 115520 2002 97 238 143478 2003 53 348 154555 Source: VetStat and The Danish Medicines Agency, 2004 Statens Serum Institut

35. STATENS SERUM INSTITUT Use of fluoroquinolones in animals in Denmark 2001 and 2002 Source: VETSTAT 2001 & 2002

36. STATENS SERUM INSTITUT Companion animals Investigation of previously neglected reservoirs: Companion animals (dogs, cats, etc.): VETSTAT data indicates large consumption of fluoroquinolones and cephalosporins in companion animals In 2003 danes had in total 550,000 dogs, 650,00 cats 24 milllion slaughter pigs, 130 million broiler chickens, 1.2 million cattle and dairy cows were produced during 2003 AntimicrobialsConsumption in companion animals (kg) Consumption in food animals and companion animals (kg)* Cephalosporins254461 Fluoroquinolones2453 ( Heuer, Jensen and Hammerum 2005) EID, vol.11, no 2.)

37. STATENS SERUM INSTITUT Consumption of ciprofloxacin and other fluoroquinolones in human therapy Source: The Danish Medicines Agency, 2004 Statens Serum Institut

38. STATENS SERUM INSTITUT Ciprofloxacin consumption in primary health care - Price versus consumption Source: Muscat M, et al. EPI-NEWS 2004, week 41. Available at: URL: http://www.ssi.dk/graphics/en/news/epinews/2004/pdf/2004_41.pdf

39. STATENS SERUM INSTITUT DANMAP 2003: Ciprofloxacin resistance in Escherichia coli urine isolates from primary health care and hospital Primary health care Hospital Source: DANMAP report 2003 Data: The average number of ciprofloxacin resistant E. coli isolates from 7 clinical microbiology laberatories (with 95 % c.i.) Statens Serum Institut

40. STATENS SERUM INSTITUT Source: Kahlmeter et al. 2003. JAC vol. 52, p. 1005-1010 and ESAC Fluoroquinolone use and nalidixan-R Escherichia coli, Europe (year 2000) Statens Serum Institut

41. STATENS SERUM INSTITUT WWW.TRAINAU.dk

42. STATENS SERUM INSTITUT What is TRAINAU? TRAINAU is a multidisciplinary early-stage research training (EST) site on identification, characterisation and assessment of public health risks associated with non-human antimicrobial usage, i.e. antimicrobial use in animals The project will bring together young researchers with different backgrounds (veterinary and medical microbiology, epidemiology, statistics, pharmacology, and analytical chemistry) and provide them with the scientific and technological competences necessary to obtain substantial progress in this field

43. STATENS SERUM INSTITUT

44. Seven PhD Fellowships in Denmark Open for Applications Location: Students will primarily be enrolled at the Royal Veterinary and Agricultural University, but the research activities will also be carried out at one or more of the TRAINAU institutions: The Royal Veterinary and Agricultural University, Statens Serum Institut, The Danish University of Pharmaceutical Sciences & Danish Institute for Food and Veterinary Research all situated in Copenhagen, Denmark.

45. STATENS SERUM INSTITUT List of proposed PhD projects: P1-Transmission of antimicrobial resistant bacteria and resistance genes through the food chain P2-Transmission of antimicrobial resistant bacteria and resistance genes by contact with animals and by environmental exposure P3- In vivo and in vitro models for investigatigating the impact of different veterinary treatment strategies on development of antimicrobial resistance P4-Associations between non-human antimicrobial resistance usage and occurence of resistant bacteria in animals and food

46. STATENS SERUM INSTITUT P5-Animal and human models for studying the consequences of antimicrobial resistance on human health P6-Mathematical models for assessing the impact of non- human antimicrobial resistance P7-the effect of antimicrobial drug metabolites on selection of resistant bacteria

47. STATENS SERUM INSTITUT Is it relevant to consider animals in relation to antimicrobial resistance in humans?… Yes! Antimicrobial resistance in bacterial populations comes from exposure to selective pressure Substantial amounts of antimicrobials are used in animals worldwide (Therapy, Prophylaxis and Growth Promotion) creating large resevoirs of antimicrobial resistance Antimicrobial resistance in bacterial populations comes from exposure to selective pressure Substantial amounts of antimicrobials are used in animals worldwide (Therapy, Prophylaxis and Growth Promotion) creating large resevoirs of antimicrobial resistance

48. STATENS SERUM INSTITUT Thanks to: Karin Sixhøj Pedersen, Frank Hansen, Stine Frese-Madsen, Aviaja Maya Sicka, Rikke Kubert and Inge Hansen for technical assistance Lone Jannok Porsbo, Dorthe Sandvang, Ole E. Heuer, Line Bagger-Skjøt, Christian Brandt, Lotte Jakobsen, Sigrid R. Andersen, Anne Mette Seyfarth,Eleni Galanis, Katharina E. P. Olsen, Jens S. Andersen, Vibeke F. Jensen, Jakob Neimann, Lars Bogø Jensen, Lars Hestbjerg Hansen, Dominique L. Monnet, Henrik C. Wegener and Niels Frimodt-Møller for help with data handling and scientific input

49. STATENS SERUM INSTITUT http://www.danmap.org