Statens Serum Institut National Veterinary Institute, Technical University of Denmark National Food Institute, Technical University of Denmark

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1 DANMAP 3 DANMAP 3 DANMAP 3 - Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark Statens Serum Institut National Veterinary Institute, Technical University of Denmark National Food Institute, Technical University of Denmark

2 DANMAP 3 Editors: Birgitte Borck Høg (bibo@food.dtu.dk) Helle Korsgaard (hkor@food.dtu.dk) Yvonne Agersø (yvoa@food.dtu.dk) National Food Institute, Technical University of Denmark Mørkhøj Bygade 9, DK - 86 Søborg Line Skjøt-Rasmussen (lbs@ssi.dk) Anette M. Hammerum (ama@ssi.dk) Microbiology and Infection Control, Statens Serum Institut Artillerivej 5, DK - 3 Copenhagen Authors: National Food Institute Yvonne Agersø, Flemming Bager, Jeppe Boel, Birgitte Helwigh, Birgitte Borck Høg, Lars Bogø Jensen, Leonardo de Knegt, Helle Korsgaard, Lars Stehr Larsen, Anna Vedel Sørensen Statens Serum Institut Tine Dalby, Anette M. Hammerum, Steen Hoffmann, Katrin Gaardbo Kuhn, Anders Rhod Larsen, Maja Laursen, Eva Møller Nielsen, Stefan S. Olsen, Andreas Petersen, Line Skjøt-Rasmussen, Robert L. Skov, Hans-Christian Slotved, Mia Torpdahl DANMAP board: National Food Institute Yvonne Agersø, Flemming Bager National Veterinary Institute Svend Erik Jorsal Statens Serum Institut Anette M. Hammerum, Robert L. Skov Layout: National Food Institute Susanne Carlsson Photos: Colourbox and Mikkel Adsbøl Printing: Rosendahls-Schultz Grafisk A/S DANMAP 3 - September 4 ISSN 6-3 Text and tables may be cited and reprinted only with reference to this report: DANMAP 3. Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. ISSN 6-3 The report is available from This report is issued by DANMAP - The Danish Integrated Antimicrobial Resistance Monitoring and Rese Programme. It presents the results of monitoring the antimicrobial use and antimicrobial resistance in animals, food and humans in 3. The report is produced in collaboration between the National Food Technical University of Denmark and Statens Serum Institut. The DANMAP programme is funded jointly the Ministry of Health, the Ministry of Science, Innovation and Higher Education and the Ministry of Foo Agriculture and Fisheries.

3 DANMAP 3 DANMAP 3 - Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark

4 TABLE OF CONTENTS.. Introduction 6.. About DANMAP.. Acknowledgements.3. DANRES Summary.. 3. Sammendrag Summary 9 4 Background information 3. Populations 3. Marketed antimicrobial agents 4. 9 Antimicrobial consumption in animals Introduction 4 Totalantimicrobial consumption 5 Antimicrobial consumption by animal species 7 Antimicrobial consumption in humans Introduction Total consumption in both primary healthcare and hospitalcare 3 Textbox : Consumption of antimicrobial agents and incidence of multi-resistant bacteria 35 in Greenla Textbox : Maintained focus on the use of antimicrobial agents in the Faroe Islands37 Textbox 3: The Danish Council of Ethics statement on the use of antibiotics Primary healthcare 4 Textbox 4: Temporal and geographical variation in tetracycline prescription patterns in Denmark 5.4. Hospital care Resistance in zoonotic bacteria 6.. Salmonella 54 Textbox 5: Surveillance Salmonella of andcampylobacter in Denmark 6.. Campylobacter Resistance in indicator bacteria Enterococci 66 Escherichia coli coli Textbox 6: Occurrence of extended spectrum beta-lactamaseescherichia (ESBL)-producing E. coli in meat and slaughter pigs, but no sign of carbapenemase producing Resistance in human clinical bacteria Textbox Textbox Textbox 8.6. Textbox Escherichia coli 78 Klebsiella pneumoniae 8 7: Carbapenemase producing bacteria in Denmark 8 Pseudomonas aeruginosa 84 Streptococci 84 Enterococci 85 8:Increased occurrence of vancomycin resistant enterocooci in Danish hospitals 86 9:Neisseria gonorrhoeae 3 87 Staphylococcus aureus 89 : Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among humans 3 9 DANMAP 3 77

5 TABLE OF CONTENTS 9. Materials and Methods General information Data on antimicrobial consumption Collection of bacterial isolates Isolation and identification of bacteria Susceptibility testing Data handling Terminology.... List of abbreviations Glossary DANMAP 3 5

6 . INTRODUCTION. Introduction. About DANMAP The objectives of DANMAP are: to monitor the consumption of antimicrobial agents in Antimicrobial resistance is considered a major threat to food animals and humans; to human health. While antimicrobial agents are essential formonitor the occurrence of antimicrobial resistance in isolated from food animals, food of animal orig treating disease in humans and in animals, they are alsobacteria the (e.g. meat) and humans; main contributors to the selection and spread of antimicrobial into study associations between antimicrobial consumpti resistance. It is, therefore, essential to monitor trends and antimicrobial resistance; and antimicrobial resistance and consumption of antimicrobial toto identify routes of transmission and areas for further agents in order to identify the risk factors that contribute research the dissemination of resistance and the interaction between the studies. risk factors. The monitoring of antimicrobial resistance is based on thre categories of bacteria: Humans and animals constitute overlapping reservoirs of into human and animal pathogens that cause infections and resistance and an integrated approach that takes this account are thought to reflect resistance caused by the use of is therefore needed. The Danish Integrated Antimicrobial antimicrobial agents in the respective reservoirs; Resistance Monitoring and Research Programme, DANMAP, zoonotic bacteria that can develop resistance in the an has implemented the One Health approach, comprising the reservoir, which may subsequently compromise treatm entire chain from farm to fork to sickbed since 995. The effect organisation and collection of DANMAP data are presented in when causing infection in humans; coli) due to their indicator bacteria (enterococcie.and Figure.. ubiquitous nature in animals, food and humans, and th DANMAP was established at the initiative of the Danish ability to readily develop or transfer antimicrobial resist in response to selective pressure in both reservoirs. Ministry of Health and the Danish Ministry of Food, Agriculture and Fisheries. The programme participants are the All pathogens may be considered reservoirs of resistance National Food Institute and the National Veterinary Institute, determinants genes that may be disseminated indepen both at the Technical University of Denmark (DTU), as well of the bacterial hosts. as Statens Serum Institut (SSI). The DANMAP programme is funded jointly by the Ministry of Health, the Ministry of A web annex presenting minimum inhibitory concentration Science, Innovation and Higher Education, and the Ministry of (MIC) distributions, detailed tables of antimicrobial Food, Agriculture and Fisheries. consumption and other additional data are available for download at Current and previous DANMAP reports are also available at the website (PDF versions). Public health risks Bacteria become resistant either by spontaneous mutation or by transfer of resistance genes from other ba strains are favoured when use of antimicrobial agents provide a selective pressure. This occurs in humans a animals undergoing antimicrobial treatment. Resistant bacteria can spread between humans in the commu centres and at hospitals. Furthermore, resistant bacteria from animals can be transmitted to humans either contact with animals and their environment or through ingestion of contaminated food or other contaminat Antimicrobial treatment failure may occur if the ingested resistant bacteria are a direct cause of disease, or determinants are transferred to pathogenic bacteria causing the disease. Bacteria may be resistant to seve all antimicrobial agents available for treatment, leading to life-threatening illness. Currently, there is only a limited number of antimicrobial agents, with novel modes of actions, under develo the pharmaceutical industry. Therefore, it is vital for public health organisations to ensure the continued eff compounds considered critically important to human treatment by ensuring prudent use for both humans a Prudent use should include the restriction of critical antimicrobial agents for use in humans only, as well as of overuse, i.e. only humans and animals suffering from an infection responsive to antimicrobial treatment s exposed to antimicrobial agents. 6 DANMAP 3

7 INTRODUCTION.. Acknowledgements The DTU National Food Institute, would like to thank Statens the Serum Institut would like to thank the following: following: the Departments of Clinical Microbiology in the DANRES the meat inspection staff and the personnel at the group - Danish Study Group for Antimicrobial Resistanc participating slaughterhouses for collecting samples Surveillance from - for providing data on resistance in bacter animals at slaughter. Without their careful recording from of thehuman clinical samples; animals farm of origin, the results would be less useful; the staff of the Neisseria and Streptococcus Typing Unit the Laboratory of Swine Diseases, the Danish Agriculture at SSI; and Food Council, Kjellerup, and the DTU National the staff of the Foodborne Pathogens Unit at SSI; Veterinary Institute for making isolates of animal pathogens the staff of the Staphylococcus Laboratory at SSI; available to the programme; the staff of the Antimicrobial Resistance Referenc the staff of the Regional Veterinary and Food Control Laboratory and Surveillance Unit at SSI; Authorities for collecting food samples and isolating Erik Villadsen from the Department of Health bacteria; Documentation at SSI for providing data on hospital the Department of Medication Statistics and Research activities. Support at SSI (formerly the Danish Medicines Agency) for collecting and transmitting data on veterinary consumption of antimicrobial agents from the pharmacies; Finally, we would like to thank all reviewers of the DANMAP the Danish Veterinary and Food Administrationreport for for their careful proofreading and helpful feedback. collecting and transmitting data on veterinary consumption of antimicrobial agents from VetStat, including statistics on consumption measured in tonnage; and the Danish Agriculture and Food Council for cooperation regarding the estimation of live biomass of production animals. Figure.. Organisation of DANMAP DANMAP 3 7

8 . INTRODUCTION.3 DANRES The Danish Study Group for Antimicrobial Resistance Surveillance provides data from the Departments of Clinical Microbiology (DCM) in Denmark. DCM, Hvidovre Hospital: Jenny Dahl Knudsen Elly Kristensen Pia Littauer Kristian Schønning Henrik Westh DCM, Rigshospitalet: Maria Kristin Bjõrnsdottir Zhijun Song Michael Tvede DCM, Herlev Hospital: Magnus Arpi Hanne Wiese Hallberg Dennis Schrøder Hansen Ellen Larsen Tina Profft Larsen Lisbeth Nielsen DCM, Slagelse Hospital: Tine Besser Ram Dessau Ole Heltberg Barbara Holzknecht Bent Røder DCM, Odense University Hospital: Bente Gahrn-Hansen Anette Holm Thøger Gorm Jensen Ulrik Stenz Justesen DCM, Esbjerg Hospital: Esad Dzajic Jeanne Elin Storm Ute Wolff Sönksen DCM, Vejle Hospital: Jens Kjølseth Møller Claus Østergaard DCM Midt-Vest: Ingrid Astrup Berit Have Kallesøe Helga Schumacher Marianne Hedegaard Søndergaard DCM, Skejby Hospital: Svend Ellermann-Eriksen Lars Erik Lemming Marianne Bøgild Pedersen Marianne Kragh Thomsen Mikala Wang DCM, Aalborg Hospital: Lena Mortensen Henrik C. Schønheyder 8 DANMAP 3

9 SAMMENDRAG / SUMMARY DANMAP 3 9

10 . SUMMARY. Summary. Sammendrag Fjerkræ: Antibiotikaforbruget til fjerkræ var cirka.7 kg DANMAP (Danish Antimicrobial Resistance Monitoringaktivt and stof i 3, hvilket er 57 % højere end i. Den p årsag til denne stigning var en høj forekomst af luftvejssyg Research Program) har siden 995 beskrevet det årlige forbrug kalkunflokke i starten af 3, samt en øget forekomst af af antibiotika og forekomsten af antibiotikaresistens ihos dyr og diarre i slagtekyllingeflokke i 3. Forbruget af fluorkinolo mennesker i Danmark. Denne udgave beskriver udviklingen i i den danske fjerkræproduktion, inklusiv avl og opdræt, ha 3. været meget lavt siden 6, og der er ikke rapporteret br cefalosporiner til fjerkræ i mere end år. Antibiotikaforbrug til dyr Fisk: Siden er al anvendelse af receptordineret medicin til Det dyr totale antibiotikaforbrug til fisk i akvakultur var p 3.58 kg aktivt stof i 3, en generel stigning på 3 % i f registreret i det offentlige register VetStat. til. Antibiotikaforbruget til fisk er meget afhængig af vandtemperaturen, og de ekstraordinære høje temperatur I 3 blev der brugt 6,3 tons antibiotika (aktivt stof) til i juli og august måned i 3 medførte øget forekomst af dyr i Danmark. Fordelt på dyrearter står svin for ca. 78 % af bakterielle antibiotikaforbruget i 3, kvæg for %, akvakultur for 3 %, infektioner i forhold til de foregående år. Indust fortsat fjerkræ for %, pelsdyr for 4 % og kæledyr, heste oghar andre dyr fokus på brug af vaccine for at begrænse bruge antibiotika. for de resterende 3 %. Kæledyr og heste: Oplysningerne i VetStat vedrørende De overordnede ændringer i antibiotikaforbruget til dyr er primært styret af ændringer i forbrugsmønstret tilantibiotikaforbrug til kæledyr og heste er mindre detaljere end oplysningerne vedrørende produktionsdyr. Forbrug til svin. Svineproduktionen står for ca. 84 % af den danske kæledyr steg fra,449 kg aktivt stof i til,989 kg ak kødproduktion, men kun ca. 4 % af den totale levende i 3. Stigninger forekom for adskillige grupper af antibio biomasse. aminoglycosider, makrolider, penicilliner (beta-laktamase følsomme), sulfonamider, trimethoprim og tetracykliner. Svin: Det totale antibiotikaforbrug til svin i var på ca. Forbruget af kritisk vigtige antibiotika som cefalosporiner o 9 tons aktivt stof. I forhold til forbruget i, er der sket fluorkinoloner faldt i 3. Men, forbruget af kritisk vigtige en stigning på 5 % stigning når forbruget udregnes i DAPD antibiotika til behandling af alvorlige infektionssygdomme og der justeres for eksport. Antibiotikaforbruget til svin faldt hos mennesker er stadig højt i kæledyr sammenlignet med drastisk i perioden umiddelbart efter Gult kort-ordningen forbruget i produktionsdyr og giver stadig anledning til øge blev bekendtgjort den. juli. Ordningen, som har til bevågenhed. hensigt at reducere forbruget, er rettet specifikt mod de svinebesætninger, som har det højeste antibiotikaforbrug pr svin. Selvom stigninger blev observeret også i 3 er forbruget % lavere end i 9 og på niveau med forbruget i 8. Antibiotikaforbruget til dyr fortsatte med at stige i 3. Det totale forbrug til svin målt i kg aktivt stof steg med Stigningen i antibiotikaforbrug (DAPD) skyldtes især et 6 % i 3,svarende til en 5 % stigning i beregnet i øget forbrug af pleuromutiliner og tetracykliner, men også DAPD. Brugen af kritisk vigtige antibiotika i den danske stigninger i forbruget af sulfonamid/trimethroprim og svineproduktion er fortsat lav. Derimod er brugen af isæ penicilliner. Pleuromutiliner og tetracykliner tilsættes primært kritisk vigtige antibiotika til kæledyr høj sammenlignet til foder eller vand i forbindelse med mave-tarm lidelser. Visse med forbruget til produktionsdyr, men forbruget af disse typer antibiotika især fluorkinoloner og 3. og 4. generations stoffer til kæledyr var lavere i 3 end. cefalosporiner er kritisk vigtige i behandling af sygdom hos mennesker. I svin var forbruget af 3. og 4. generations cefalosporiner meget lavt (3 kg), hvilket er et resultat af, at svinebranchen i frivilligt indførte stop for brugen af disse stoffer. Brugen af fluorkinoloner var på det samme lave niveau, Antibiotikaforbrug til mennesker som det har været siden 3, hvor lovgivning om begrænsning Forbruget af receptordineret medicin på patientniveau er af fluorkinolonforbruget trådte i kraft. blevet overvåget siden begyndelsen af 99erne. Totalforbrug: I 3 steg det totale forbrug af antibiotika Kvæg: Det totale antibiotikaforbrug til kvæg har ligget relativt systemisk stabilt på ca. 4 tons aktivt stof om året siden 5, til men i 3 brug (primærsektoren og hospitalssektoren sammenlagt) til mennesker med, %. Forbruget i faldt forbruget til ca. tons. Målt i standard doser (DADD) primærsektoren udgjorde 9 % af det totale forbrug. Forbr steg forbruget til golddyrsbehandling med 7 %, hvorimod af bredspektrede antibiotika var 5 % højere i 3 samme antallet af DADDer til behandling af klinisk mastitis faldt med 9 % i forhold til. Forbruget af beta-laktamase følsomme med. Fra 4 til 3 er det totale forbrug af antibi penicilliner udgjorde som tidligere år størstedelen af iforbruget. Danmark steget med %. For bredspektrede antibiotika I relation til antibiotika kritisk vigtige i human behandling stigningen 7 %. har forbruget af fluorkinoloner siden 3 været stort set nul. Sammenlignet med faldt forbruget af 3. og 4. generations cefalosporiner til systemisk behandling med 4 %. DANMAP 3

11 SUMMARY. Salmonella Typhimurium er en af de mest almindelig Primærsektor: Det totale antibiotikaforbrug i primærsektoren forekommende serotyper i danske svin, dansk svinekød og steg med, % sammenlignet med. De mest udtaltestighumanesalmonella infektioner. Blandt S. Typhimurium fra ninger blev observeret for tetracykliner, kombinationssvin var 6-7 % af isolaterne resistente overfor ampicillin penicilliner, beta-laktamase resistente penicilliner og penistreptomycin, sulfonamid, og tetracyklin, og forekomsten cilliner med udvidet spektrum. Forbruget af makrolider faldt af resistens overfor disse fire antibiotika (ASSuT resistens) med % i forhold til og forsatte således det fald, der er steget over de sidste fem år. Dette kan primært tilskrive er blevet observeret siden. Forbruget af bredspektrede den stigende forekomst af monofasiske varianter, som ofte antibiotika steg 5,7 % fra til 3. er multiresistente, og i 3 var 5 % af de undersøgte S. Typhimurium isolater fra svin var monofasiske. Der blev I det seneste årti er forbruget af antibiotika i primærsektoren også S.Typhimurium blandt steget med 9 %. Denne stigning skyldes sandsynligvis enfundet høj forekomst af resistens fra dansk svinekød. Generelt var forekomsten af stigning i dosis (DDD) per behandlet patient samt enisolater øget dosis multiresistens blandt S.Typhimurium isolater fra danske svin per udskrevet medicinpakning. Årsagen til denne ændring (64 %) og svinekød (7 %), højeresalmonella end i generelt kendes ikke. (Salmonella spp.), hvor hhv. 37 % og 36 % var multiresistent Når resultaterne fra Salmonella overvågningsprogrammerne I Textbox 4 er forbruget af tetracykliner beskrevet nærmere. sammenholdes med disse forekomster, estimeres det at 9 Fra 5 til 3 er forbruget af tetracykliner (DDD pr.. af svinene indbygger-dage) steget med 54 %. Den største stigning sås for og,5 % af slagtekroppene havde multiresisten Salmonella de 4 årige (86 %) samt for de 5 9 årige (58 %). Dette. Som i de foregående år blev der ikke påvist resis overfori 3 cefalosporiner (ceftiofur og cefotaxim) eller kinolon betød, at 5.4 flere unge blev behandlet med tetracyklin (ciprofloxacin og nalidixansyre) Salmonella blandt isolater fra sammenlignet med 5 (justeret for populationsændringer). eller dansk svinekød. Størstedelen af udskrivningerne var sandsynligvis til svin behandling af akne. Det var oftere praktiserende læger end dermatologer, der udskrev tetracykliner. Ligesom for svin og svinekød, er der de seneste fem år ske en stigning i den relative forekomst af de monofasiske S. Hospitaler: På somatiske hospitaler steg det totale Typhimurium varianter blandt isolater fra patienter, som antibiotikaforbrug opgjort i DDD pr. sengedage (DBD) havde erhvervet infektionen i Danmark (både sporadiske med,4 % fra til 3. og udbrudstilfælde). I 3 blev der påvist multiresistens i 54 % af S. Typhimurium isolater fra sporadiske tilfælde Fra til 3 steg forbruget af kombinationspenicilliner, erhvervet i Danmark, og resistens niveauet var generelt på kombinationer af sulfonamid og trimethoprim, beta-laktamase niveau med. Resistensforekomsten var højere for fire resistente penicilliner, penicilliner med udvidet spektrum ud af og de 6 testede stoffer, når forekomsten i isolater fra carbapenemer. Der blev observeret et fald for andenpatienter generations med rejse-relaterede infektioner sammenlignes m cefalosporiner, fluorkinoloner, makrolider og tetracykliner. forekomsten i isolater fra sporadiske infektioner erhvervet Danmark. Det gælder også for fluorkinoloner, som bruges Fra 4 3 steg det totale antibiotikaforbrug med 66 %. behandling af patienter med alvorlige mave-tarm infektion Denne stigning skyldtes primært en kombination af stigning i af både forårsaget Salmonella og Campylobacter. Andelen af DDD og et fald i antallet af sengedage. I løbet af det multiresistente seneste årti isolater faldt i 3 blandt patienter med r er forbruget af bredspektret antibiotika på somatiskerelaterede hospitalerinfektioner, og i 3 var niveauet af multiresis steget med 4 %. sammenligneligt med niveauet i isolater fra sporadiske infektioner erhvervet i Danmark. Salmonella??? I 3 steg det totale forbrug af antibiotika til systemisk behandling af mennesker (primær- og hospitalssektoren S. Typhimurium, som ofte sammenlagt) med, % sammenholdt med. Forekomsten af monofasiske er multiresistent, er steget i svin, svinekød og blandt Forbruget i primærsektoren udgjorde 9 % af det totale forbrug, mens forbruget på hospitalerne udgjorde dehumane infektioner de seneste fem år. Blandt de huma resterende %. Over en -årig periode fra 4 tilinfektioner, var resistensforekomsten i 3 generelt højere, 3 steg det totale forbrug af antibiotika til mennesker i for 4 ud af 6 stoffer i testpanelet (herunder fluorkinoloner), blandt de rejserelaterede humane Danmark med %. tilfælde end blandt de hjemligt erhvervede infektioner. Selvom resistens var hyppigst forekommende blandt isolater fra patienter med rejserelaterede infektioner, faldt andelen af multiresistente isolater i 3 blandt Resistens i zoonotiske bakterier disse, og niveauet af multiresistens var sammenlignelig Zoonotiske bakterier som Salmonella og Campylobacter med niveauet i isolater fra sporadiske infektioner er sygdomsfremkaldende bakterier, som kan overføres fra erhvervet i Danmark. dyr til mennesker. Udvikles der resistens i disse bakterier i husdyrproduktionen, kan resistens overføres til mennesker via fødevarer, og kan i visse tilfælde medføre behandlingssvigt ved sygdom. DANMAP 3

12 . SUMMARY I 3 var resistensforekomsten Campylobacter i jejuni isolater fra kyllinger og kvæg på samme niveau som i, men en anelse højere end i. Selv om disse ændringer ikkeset vari et One Health perspektiv er der en direkte samm hæng mellem antibiotikaforbruget i kyllingeproduktionen statistisk signifikante, har det fulgt forbrugsmønstret for og forekomsten af antibiotikaresistente E. faecalis i dansk tetracyklin i fjerkræ. kyllingekød. Denne kobling findes ikke mellem danske svin iog dansk svinekød. I dansk svinekød er forekomsten Set i en europæisk sammenhæng er resistensforekomsten C. af resistens blandt E. faecalis generelt lavere end blandt jejunifra danske slagtekyllinger og kyllingekød C. coli samt fra isolater fra svin. Foruden dette, er resistens forekomsten svin blandt Europas laveste. svinekød faldende, en tendens som ikke observeres i svi Fluorkinolon resistens C. jejuni i var også højere blandt isolater fra importeret kyllingekød (53 %) end fra dansk kyllingekød ( %). IndikatorEscherichia coli fra slagtekyllinger var ofte Som i de foregående år var forekomsten af fluorkinolon resistens resistente overfor sulfonamid og ampicillin (begge 6 %), s i C. jejuniisolater fra patienter med rejse-relaterede infektioner typisk bruges til slagtekyllinger. Der blev påvist fluorkinolo (9 %) højere end i isolater fra patienter, hvor infektionen var (ciprofloxacin) resistens i 6 % af isolaterne, og resistens erhvervet i Danmark (4 %). overfor 3. generations cefalosporiner (ceftiofur) blev påvis i to isolater fra slagtekyllinger. Resistensforekomsten E. coli i fra slagtekyllinger og dansk kyllingekød var sammenlignel Fluorkinolon resistens C. jejuni i er fortsat højere blandtresistensforekomsten i isolater fra kvæg og dansk oksekød som foregåede år ganske lav. Resistens E. colifraisvin var den isolater fra importeret kyllingekød sammenlignet med dansk kyllingekød, og blandt C. jejunifra patienter med højeste blandt produktionsdyrene og forblev på samme hø niveau som i. rejse-relaterede infektioner i forhold til patienter, hvor infektionen var erhvervet i Danmark og denne forskel er mere udtalt i 3 end i. Blandt isolaterne fra kød havde isolaterne fra importeret kyllingekød generelt de højeste resistensforekomster ogs når det gjaldt de kritisk vigtige antibiotika. Sammenlignet med dansk kyllingekød havde E. coliisolater fra importeret kyllingekød højere resistens overfor 4 af de 6 testede Resistens i indikatorbakterier antibiotika. For svinekød havde E. coliisolater fra dansk kød Indikatorbakterier er inkluderet i DANMAP overvågningen for en lavere resistensforekomst overfor kinoloner (ciprofloxac at kunne give et indblik i den generelle forekomst af resistens i og nalidixin syre) end isolaterne fra importeret svinekød. raske husdyr og i kød. ESBL-producerende bakterier er et af de hurtigst vokse I E. faecalisfra danske slagtekyllinger var forekomsten af resistensproblemer verden over. Flere nyere studier finder tetracyklin resistens størst (38 %) efterfulgt af resistens overfor de samme ESBL gener, plasmider og kloner E. coliisolater af erythromycin og salinomycin. i både dyr og i mennesker med infektioner, hvilket tyder på et zoonotisk link. Forekomsten af Enterobacteriaceae E. faecalis Højeste forekomster af resistens blev fundet blandt bakterier, der er resistente overfor carbapenemer, er også isoleret fra svin sammenlignet med isolater fra slagtekyllinger, voksende trussel, idet carbapenemer er sidste mulighed fo hvilket følger forbrugsmønsteret i Danmark til disse dyrearter. antibiotikabehandling af mennesker med infektioner, der BlandtE. faecalis isoleret fra svin var forekomsten af tetracyklin skyldes multiresistente Gram-negative bakterier. højest (9 %). Tetracykliner er og har været de mest anvendte antimikrobielle stoffer til danske svin i en årrække og har E. coli, primært været brugt til behandlinge.af coli infektioner. Seks procent af slagtesvinene havde ESBL-producerende Forekomst af erythromycin resistense.blandt faecalis isoleret hvilket var lavere end i og signifikant lavere end i 9 før landbrugets frivillige stop for brug af cefalosporin fra svin var 45 % og faldt i forhold til forekomsten i. trådte i kraft. Den højeste prævalens of ESBL-producerend E. Forekomst af resistens var højere i importeret end i dansk coli i kødprøver blev fundet i importeret kyllingekød (5 %) produceret svinekød. hvilket er på samme niveau som i og. Forekom af ESBL-producerende E. colii prøver af dansk kyllingekød var Blandt isolater fra kyllingekød, blev den højeste forekomst signifikant lavere i 3 sammenlignet med. Desude af resistens overfor flere stofgrupper fundet i importeret var andelen af prøver positive for ESBL producerende E. kyllingekød. Ligeledes blev der fundet højere forekomst coliiafdansk kyllingekød signifikant lavere end forekomsten multiresistens blandt isolater fra importeret kyllingekød det importerede kyllingekød. Dette skyldes sandsynligvis e (39 % og 4 % for henholdsvis E. faecalis og E. faecium ) ved frivilligt stop af brug af 3. generations cefalosporiner i topp sammenligning med forekomster i dansk produceret af kød. I alt avlspyramiden i udlandet, hvilket resulterer i der overfø 7 % af de multiresistente E. faecalis fra importeret kyllingekød færre ESBL producerende E. colifra importerede forældredyr havde samme resistensprofil (erythromycin, kanamycin, til de danske slagtekyllinger. I lighed med sidste år er der s streptomycin og tetracyklin). ingen fund af carbapenemase producerende E. coli. Generelt er forekomsten af resistens overfor kritisk vigtige antibiotika lav, men et dansk E. faecalis isolat var resistent overfor fluorkinoloner (ciprofloxacin). DANMAP 3

13 SUMMARY. (ciprofloxacin) resistens var 7 %, og mecillinam resistens v %, hvilket også er på samme niveau som i. Sulfon Det frivillige stop i brugen af cefalosporiner i resistens faldt fra 6 % i til % i 3. svineproduktionen resulterer fortsat i en lav forekomst af ESBL producerende E. colii slagtesvin. Ligeledes ses Blandt Pseudomonas aeruginosa isolater fra blod var E. colii dansk et signifikant fald i ESBL producerende niveauet af resistens for alle testede antibiotika ikke signifi kyllingekød, hvilket sandsynligvis skyldes et frivilligt forskelligt fra niveauet i, men der sås en stigende ten stop i brug af cefalosporiner i toppen af avlspyramiden for aminoglykosid (gentamicin) resistens i perioden 7 i udlandet. 3. Forekomsten af carbapenemase producerende bakter i Danmark er beskrevet i Textbox 7. I 3 blev der fund Resistens i bakterier fra diagnostiske 8 carbapenemase producerende Enterobacteriaceae (CPE) indsendelser fra mennesker sammenlignet med 9 CPE i hele perioden 8. I Rapporteringen af antibiotikaresistens i bakterier fra 3 var der spredning af NDM- producerende Citrobacter diagnostiske indsendelser fra mennesker er baseret på frivillig freundii imellem fire patienter på en hospitalsafdeling i Regi indsendelse af data fra DANRES-gruppen, som dækker de Nordjylland. Ingen af patienterne havde været ude at rejse Klinisk Mikrobiologiske Afdelinger (KMA) i Danmark. for nylig, og kilden til den NDM- producerende C. freundii Undtagelser omfatter methicillin-resistente Staphylococcus C. med freundii var ukendt. To af de fire patienter havde også aureus(mrsa) og invasive Streptococcus pneumoniae, som NDM- producerende K. pneumoniae. Der blev detekteret er anmeldepligtige. Data vedr. disse bakterier kommer Acinetobacter baumannii syvfra OXA-3 producerende isolater i referencelaboratorierne på SSI. 3. BlandtEscherichia coli isolater fra blod var forekomsten Derafblev i to tilfælde set spredning af OXA-3 producerend 3. generations cefalosporin resistens 8 % i 3, hvilket er det mellem to patienter. I 3 blev der fundet tre A. baumannii samme niveau som i, men højere end i de andre nordiske producerende OXA-4-like A. baumannii. Her var der også lande i. Ciprofloxacin resistensen faldt til % sket i 3 smittespredning fra en patient til en anden. Derudove sammenlignet med 4 % i. Aminoglykosid resistensen blev der detekteret to NDM- producerende A. baumannii. (gentamicin) var 7 % i 3, hvilket er samme niveau som iblev. I 3 der fundet tre VIM producerende P. aeruginosa, Resistensdata for piperacillin/tazobactam blev rapporteret og en NDM og VIM producerende P. aeruginosa. For første for første gang i denne DANMAP rapport. Data omfatter deblev der detekteret en IMP producerende gang P. aeruginosa sidste fem år (9 3); i denne periode var forekomsten af Patienterne med de to sidstnævnte i Danmark. P. aeruginosa resistens på omkring 4 %. havde begge forinden været indlagt på udenlandske hospi BlandtE. coliisolater fra urin fra patienter på hospitalerne I 3 var forekomsten af resistens for penicillin og makro var forekomsten af 3. generations cefalosporin resistens 6%i Streptococcus pneumoniae (erythromycin) stadig lav blandt 3, hvilket er det samme niveau som i. Forekomsten af A, B, C og G streptokokker. og gruppe fluorkinolon (ciprofloxacin) resistens var % i 3, hvilket er samme niveau som i, men der har været en Forekomsten stigende Enterococcus faecium af ampicillin resistens i forekomst fra 3 % i 4. Aminoglykosid (gentamicin) resistens isolater fra blod var 93 % i 3. Vancomycin resistens v var 5 % i 3 og sulfonamid resistens var 33 %, begge var påog, % i E. faecium Enterococcus i faecalis isolater fra blod. I samme niveau som i. 3 modtog Reference laboratoriet for Antibiotikaresisten øget antal vancomycin resistente enterokokker (VRE) (Tex BlandtE. coliisolater fra urin fra patienter i almen8); praksis 58 isolater fra infektioner og 68 fra fækale screening er forekomsten af fluorkinolon (ciprofloxacin) resistens stegetalle VRE var Næsten vana E. faecium isolater. VRE isolaterne støt fra 3 % i 4 til % i 3. Forekomsten af 3.var generations primært fra hospitaler i Region Hovedstaden, men der cefalosporin resistente E. colivar 4 %, sulfonamid resistensvar varogså isolater fra hospitaler i Region Sjælland samt Reg 33 % og ampicillin resistens var 4 % i 3, hvilket Midtjylland. for alle tre Der blev tillige fundet VRE i de to andre region antibiotika var på samme niveau som i. men i meget lavere antal. Pulsfelt gelelektroforese typning at der var sket spredning af flere forskellige vana typer VRE BlandtKlebsiella pneumoniae isolater fra blod var forekomsten både inden for hospitalerne samt imellem hospitalerne. af resistens den samme i 3 som i. Forekomsten af 3. generations cefalosporin resistens var 9 %, aminoglykosid Forekomsten af fluorkinolon (ciprofloxacin) resistens i (gentamicin) resistens var 4 %, og fluorkinolon (ciprofloxacin) Neisseria gonorrhoeae steg støt fra 3 % i 3 til 75 % i 9 resistens var 9 %. efterfulgt af et fald til 56 % i 3. Penicillinase produktion blandt gonokok isolater svingede imellem 4 % i 3 og BlandtK. pneumoniae isolater fra urin fra patienteri på 3. Der blev ikke rapporteret hverken ceftriaxon resiste hospitalerne faldt forekomsten af resistens for mecillinam isolater eller tilfælde af ceftriaxon behandlingssvigt i denn ( %), sulfonamid ( %), gentamicin (4 %),. generations periode. I 3 var forekomsten af makrolid (azithromycin cefalosporiner (cefuroxim) (9 %) og 3. generations cefalosporiner resistens 45 %, cefixim resistens var 9 %, og der var ingen (7 %) fra til 3. Fluorkinolon (ciprofloxacin) resistens spectinomycin resistente isolater (Textbox 9). var på samme niveau i 3 som i. I 3 blev der indrapporteret.769 tilfælde Staphylococcus af BlandtK. pneumoniae isolater fra urin fra patienter iaureus almen bakteriæmier svarende til en incidens på 3,9 tilfæld praksis var forekomsten af 3. generations cefalosporin per resistens. indbyggere. Antallet af methicillin-resistente S. 6 %, hvilket er samme niveau som i. Fluorkinolon aureus(mrsa) fra bakteriæmier var 3 (,7 %), hvilket er på DANMAP 3 3

14 . SUMMARY Summary samme niveau som tidligere år og blandt de laveste. incidenser DANMAP (Danish Antimicrobial Resistance Monitoring and observeret i Europa. Den højeste forekomst af resistens ud Research over penicillin resistens var resistens for fusidinsyre (5 %), Program) has monitored antimicrobial resistance of antimicrobial agents in food animals and i erythromycin (7 %), clindamycin (6 %) og norfloxacinconsumption (5 %). humans in Denmark since 995. This report describes cha Niveauet af resistens for de testede antibiotika var det samme and trends in 3. som i. Antallet af nye MRSA tilfælde (både koloniserede og Antimicrobial inficerede consumption in animals personer) steg i 3 til.94 sammenlignet med,556. Datai on all medicines prescribed by veterinarians have be Stigningen blev primært set for MRSA tilhørende klonal registered at the farm and species level by the official VetS kompleks CC398 (LA-MRSA), hvor der blev observeret 643 programme since. tilfælde i 3 sammenlignet med 3 tilfælde i. CC398 udgjorde 3 % af alle nye MRSA tilfælde i 3. En stor af consumption of antimicrobial agents in 3 Thedel total stigningen skyldes formentlig inklusionen af kontakt amounted til svin to 6.3 tonnes of active compounds, an increa som en risikofaktor medførende screening ved indlæggelse på of 4% increase compared with 3. Pigs accounted for hospitalerne. Størstedelen (87 %) af de personer, som havde approximately 78%, cattle for approximately %, fur anim CC398 (både infektion og kolonisation), havde haft kontakt for 4%, aquaculture for 3%, and poultry for % of the tota med svin eller boede sammen med personer, der havde kontaktconsumption of antimicrobials measured in kg veterinary med svin. Der var dog ingen tegn på betydelig spredning activeafcompounds. The remaining 3% was used in pets, ho CC398 til byområder, hvilket indikerer, at fødevarer ikke and udgør others. en væsentlig smittekilde. The overall changes in veterinary consumption are genera Blandt alle MRSA tilfælde var forekomsten af infektioner lavere driven by changes in consumption in pig production. Pigs i 3 end i (45 % versus 57 %). Antallet af hospitalsaccount for approximately 84% of the meat production in erhvervede tilfælde var fortsat lavt i 3 og udgjorde kun but only about 4% of the total live biomass. Denmark, %. I 3 er der ikke foretaget undersøgelser for MRSA i dyr og fødevarer. Pigs: The total consumption of veterinary antimicrobial agents in Danish pig production was approximately 9 ton Measured in DAPD, we observed a 5% increase from to 3. This follows a decrease in, which was probably the result of the introduction of legislation to red I 3 blev der fundet 8 carbapenemase producerende overuse of antimicrobials in the pig production. Even thou Enterobacteriaceae (CPE) sammenlignet med 9 CPE i hele the antimicrobial consumption increased in 3, it remai perioden 8. I 3 var der spredning af NDM- producerende Citrobacter freundii imellem fire patienter approximately % lower than in 9 and was at the sam as in 8. på en hospitalsafdeling i Region Nordjylland. Ingen level af patienterne havde været ude at rejse for nylig, og kilden til In 3, the increase in consumption (measured in DAPD) NDM- producerende C. freundii var ukendt. was attributed mainly to an increase in pleuromutilins and tetracyclines and to a lesser extent, to the use of penicillin Et stigende antal vancomycin resistente enterokokker blev and sulfonamides/trimethroprim. Pleuromutilins and modtaget på SSI i 3; 58 isolater fra infektioner og 68 tetracyclinesare mainly used in feed or water medication f fra fækale screeninger. Næsten alle VRE isolater vana var E. faecium. VRE isolaterne var primært fra hospitalergastrointestinal disease. Some types of antimicrobials incl i Region Hovedstaden, men der var også isolater frafluoroquinolones and 3rd and 4th generation cephalospori are considered critically important for treatment of severe hospitaler i Region Sjælland samt Region Midtjylland. infections in humans. The use of 3rd and 4th generation cephalosporins in pigs remained very low (3 kg), as a resu Antallet af nye MRSA tilfælde (både koloniserede oga voluntary ban on cephalosporins introduced by the Dani inficerede personer) steg i 3 til.94 sammenlignet pig industry in. The use of fluoroquinolones remained med.556 i Stigningen blev primært set for MRSA the same low level as has been observed since legal restri tilhørende klonal kompleks CC398 (LA-MRSA), hvor were der enforced in 3. blev observeret 643 tilfælde i 3 sammenlignet med 3 tilfælde i. CC398 udgjorde 3 % af alle nye MRSA Cattle: Overall, the antimicrobial consumption in cattle ha tilfælde i 3. Antallet af hospitals-erhvervede MRSA tilfælde var fortsat lavt og udgjorde kun % i 3.remained stable at around 4 tonnes since 5, but in it declined to about tonnes. Measured in standard dose (DADD) the consumption for drying-off treatment increase by 7% whereas, the number of DADDs for treatment of clinical mastitis decreased by 9% compared to. The lactamase sensitive penicillins account for the majority of consumption. For critically important antimicrobials, the u of fluoroquinolones has been close to zero since 3. The use of 3rd and 4th generation cephalosporins for systemic treatment decreased by 4% compared with. 4 DANMAP 3

15 SUMMARY. spectrum. The consumption of macrolides was % lower Poultry: In 3, the overall consumption of antimicrobial compared with, continuing the decrease observ agents in poultry was approximately,7 kg active3 compound, since which represents a 57% increase compared with. The. The consumption of broad-spectrum agents increased main reason for this appears to be widespread problems with by 5.7% from to 3. respiratory disease in turkey flocks produced in early 3 and an increased occurrence of diarrhea in broiler flocks. The During the past decade, the consumption of antimicrobial reported use of fluoroquinolones in poultry has beenagents low since in the Danish primary sector has increased by 9% 6, and they were not used in the poultry production in 3. seems to be caused by the fact that the dosage prescribed Furthermore, use of cephalosporins has not been reported in each patient and in each package has increased significan Danish poultry production for more than a decade. over the years, however, the underlying medical reasons f this (if any) are unclear. Aquaculture: The overall antimicrobial consumption in aquaculture was 3,58 kg in 3, an overall increase of increased 3%. The consumption of tetracyclines has been furth. From The relatively large increase in consumption in 3described is explained in Textbox 4 5 to 3, the consumption mainly by extraordinary high temperatures in July and August (DDDs per, inhabitant-days) of tetracyclines for all ag 3, leading to higher water temperatures and an increase inby 54% with large increases observed for the increased the occurrence of bacteriological infections. There is,year however, olds (86%) and 5 9 year olds (58%). When adjuste in the aquaculture industry, still focus on vaccinationpopulation to reduce increases, approximately 5,4 more persons in the risk of diseases that may require antibiotic treatment. these age groups were treated with tetracyclines in 3 t General Practioners (GPs) rather than dermatologists in 5. prescribed Pets and horses: The information available on antimicrobialthe majority of the prescriptions, presumably for acne. consumption in pet animals and horses is less detailed as for production animals. The overall antimicrobial consumption for pets increased from,449 kg in to Hospitals: In 3, the consumption of antimicrobial agen,989 kg active compound in 3. The increase wasinseen for hospitals (expressed as DBDs, i.e. DDDs per somatic several antimicrobial classes; aminoglycosides, macrolides occupied bed-days) was.4% higher than in. and penicillin s (b-lactamase sensitive), sulfonamides and trimethoprim, as well as for tetracyclines. The consumption From to 3, a higher consumption was observed fo of antimicrobials critical for human treatment such as combination penicillins, combinations of sulfonamide and cephalosporins and flouroquinolones decreased in 3. trimethoprim, beta-lactamase resistant penicillins, penicill Nonetheless, consumption of broad-spectrum antimicrobials with extended spectrum and carbapenems, while decrease in pet animals and the use of antimicrobial agents critical for consumption was seen for nd generation cephalosporins, treatment of human infections remains high compared with fluoroquinolones, macrolides and tetracyclines. both production animals and is a matter of concern. The consumption in hospitals has steadily increased by 66 in the past decade, primarily caused by an increase in DDD The antimicrobial consumption in animals continued and a decrease in the number of hospital bed-days. This h to increase in 3. The total consumption (measured especially been seen in the consumption of broad-spectrum in kg active compound) in pigs increased by 6% in antimicrobial agents which increased by 4%. 3, corresponding to a 5% increase in DAPD. Use of critically important antimicrobials in the pig production remains low. However, the use of critically important antimicrobials in pets remains high compared with In humans, the overall consumption of antimicrobial other species, but decreased from to 3. agents for systemic use increased slightly (. %) from to 3. Antimicrobial consumption in the primary healthcare sector represented 9% of the total consumption and the hospital sector accounted for the Antimicrobial consumption in humans remaining %. From 4 to 3, the total consumpti In Denmark, the Department of Data Delivery and Medicinal of antimicrobial agents by humans in Denmark increased Product Statistics at Statens Serum Institut register theby %. consumption of antimicrobial agents to humans. Total consumption: In 3, the total consumption of Resistance in zoonotic bacteria antimicrobial agents for systemic use (primary healthcare and Salmonella Zoonotic bacteria such as andcampylobacter can hospital care) was.% higher than in. The consumption in develop resistance in the animal reservoir. The resistant ba primary healthcare accounts for 9% of the total antimicrobial maywas be transferred to humans via food and may subseque consumption. The proportion of broad-spectrum agents compromise 5% higher in 3 compared with. Since 4, the overall treatment effect when causing infection in consumption of antimicrobial agents in humans has humans. increased by %. For broad-spectrum, agents the increase has been 7%. Resistance in zoonotic bacteria Salmonella Zoonotic bacteria such as and Campylobacter Primary healthcare: In 3, the total consumption of develop resistance in the animal reservoir. The resista antimicrobial agents for systemic use in the primary can sector bacteria may be transferred to humans via food and may was.% higher than observed in. Particular increases subsequently were observed for tetracyclines, combination penicillins, beta- compromise treatment effect when they cau disease in humans. lactamase resistant penicillins and penicillins with extended DANMAP 3 5

16 . SUMMARY Salmonella Typhimurium is one of the most common In serovars a European context, Denmark reports the lowest levels in Danish pigs and pork as well as in human infections. antimicrobial resistance among C. jejunifrom broilers and AmongS. Typhimurium from pigs, 6 7% of the isolates broiler meat andc. forcoliisolates obtained from pigs. were resistant to ampicillin, streptomycin, sulfonamide, and tetracycline; and the occurrence of resistance to these Forfour several years, the level of fluoroquinolone resistance C. in antimicrobial agents have increased over the last five years. jejuni has been higher among isolates from imported broile This can mainly be attributed to an increasing prevalence meat of (53%), compared with isolates from Danish broiler m monophasic S.Typhimurium that has a strong tendency(%). to be multi-resistant. In 3, 5% of S. Typhimurium the isolates from pigs were of the monophasic variants. High levels of As observed in previous years, the C. jejuni isolates from t resistance S. intyphimurium were also found among isolates travel-associated cases continued to have a significantly h from Danish pork. In general, we found higher levels of multilevel of fluoroquinolone resistance (9%) compared with resistance among S.Typhimurium (including the monophasic domestic cases (4%). variants) isolates from Danish pigs (64%) and pork (7%) compared to other Salmonella spp. isolates from pigs and pork (37% and 36%, respectively). Based on the relative occurrence of resistance from DANMAP samples and Salmonella the The level of fluoroquinolone(ciprofloxacin) resistance prevalence from the national control programmes, it was in C. jejuniremains higher among isolates from estimated that 9% of Danish pigs and.5% of the pig carcasses imported broiler meat compared with isolates from were positive with multi-resistant Salmonella. As in previous Danish broiler meat, and among C. jejuni from travelyears, resistance to cephalosporins (ceftiofur or cefotaxim) or associated cases compared with domestic cases and quinolones (ciprofloxacin or nalidixic acid) was not detected this difference became more pronounced from amongsalmonella from Danish pigs or pork. to 3. As in isolates from pigs and pork, the occurrence of monophasic variants of S.Typhimurium in humans increased in prevalence among both domestic sporadic cases and outbreaks Resistance over the in indicator bacteria last five years. In 3, multi-resistant isolates were recovered Indicator bacteria, enterococci and Escherichia coli, are from 54% of the domestic sporadic cases, however, generally included in the DANMAP programme to provide informatio the levels of resistance was comparable to. Resistance to 4 about the general levels of resistance in healthy productio of the 6 tested compounds was higher among travel-associated animals and in meat. cases than domestic human cases, including resistance to fluoroquinolones, which are used for empiric treatment of Enterococcus faecalis from Danish broilers, resistance adults with severe bacterial gastroenteritis caused byinboth to tetracycline (38%) was the most dominant, followed by Salmonella and Campylobacter. Among the travel-associated resistance to erythromycin and salinomycin. cases, the occurrence of multi-resistance decreased in 3 to a level comparable to the occurrence among the sporadic Higher prevalence of resistance to antimicrobials was obse domestic cases. among E. faecalis isolated from pigs compared to poultry, whic reflects the usage pattern of antimicrobials in these anima species. Among E. faecalis from pigs, the highest occurrence of resistance was to tetracycline (9%). Tetracycline has b The occurrence of monophasic S. Typhimurium, which theand most widely used antimicrobial agent in the Danish pig are often multi-resistant, has increased in pigs, pork production among isolates from human infections the past five years. for more than a decade and has primary been for treatmente. ofcoliinfections. Erythromycin resistance in Among isolates from human infections, the resistance E. faecalis from pigs was 45% a decline since. Occurren levels were higher for four out of 6 compounds tested of resistance were higher in imported pork when compare (including fluoroquinolones) among the travel related Danish produced pork. human cases compared to the domestically acquired cases. Although resistance was most common among isolates from travel related human cases the amount Among isolates from broiler meat, the highest level of of multi-resistance isolates decreased in 3 to aresistance level to several compounds was observed in importe comparable to isolates from domestically sporadicbroiler meat, similar to previous years. Moreover, higher cases. prevalences of multi-resistance was observed in isolates fr imported broiler meat (39% and 34% E. faecalis for and E. faecium, respectively) compared to broiler meat produced in Denmark. Among the multi-resistant E. faecalis isolates from In 3,Campylobacter jejuni isolates from Danish broilers imported broiler meat, 7% had an identical resistance pro and cattle were similar to the observed levels in, and (erythromycin, kanamycin, streptomycin and tetracycline) slightly higher than in. The observed changes in In general, resistance to antimicrobial agents of critical resistance to tetracycline were not statistically significant; importance for human treatment was low, but fluoroquino nonetheless they correspond well to changes in consumption (ciprofloxacin) resistance was observed in E.one faecalis of tetracycline in the broiler production. isolate from a Danish pig. 6 DANMAP 3

17 SUMMARY. In a One Health perspective, there appears to be a direct The voluntary stop in usage of cephalosporins in the link between the antimicrobial consumption in the Danish pig production continuously results in a low broiler production and the level of resistance E. faecalis in E. coli occurrence of ESBL producing in pigs at slaughter. isolates from Danish broilers. A similar link between Also a significant decrease in the occurrence of ESBL Danish pork and pigs has not been observed. E. faecalis producing E. coliin Danish produced broiler meat was isolates from pork were generally more susceptible observed, which is most likely due to a voluntary stop when compared with isolates from pigs. Furthermore,in usage in cephalosporins in the top of the breeding resistance in Danish pork has been declining, a trendpyramid not abroad. seen in Danish pigs. Indicator Escherichia coli from broilers were most oftenresistance in human clinical bacteria resistant to sulfonamide and ampicillin (both 6%), Data on antimicrobial resistance in bacteria from diagnost which can be explained by the usage pattern. Resistance to submissions from human patients were gathered by volun fluoroquinolones was observed in 6% of the isolatesreporting and from the DANRES group, which covers the ceftiofur (3rd generation cephalosporin) resistance was Departments of Clinical Microbiology (DCM) in Denmark. observed in two E. coliisolates from broilers. ResistanceExceptions in were methicillin-resistant Staphylococcus aureus isolates from Danish broiler meat was similar to the (MRSA) findingsand invasivestreptococcus pneumoniae that are in broilers. Resistance in isolates from cattle and beef was notifiable. Data on these bacteria were obtained from the generally low. The highest occurrence among production reference laboratories at SSI. animals was observed in pigs, and resistance was at the same high level as in. In Escherichia coli blood isolates, resistance to 3rd generati cephalosporins was 8% in 3, the same level as reporte In isolates from meat, the highest occurrence of resistance,, but above the level reported in the other Nord including resistance to critically important antimicrobials, countries. The occurrence of ciprofloxacin resistance decre was found in imported broiler meat. Compared with from Danish 4% in to % in 3. Aminoglycoside (gentam broiler meat, we found higher levels of resistance forresistance 4 of 6 was 7% and at the same level as reported in tested antimicrobial agents from imported broiler meat. For Resistance data on piperacillin/tazobactam was reported f E. colifrom pork of domestic origin, resistance to quinolones first time in the present DANMAP report. Data were obtaine (ciprofloxacin and nalidixic acid) was significantly lower than for five years (9 3). For all five years, the resistanc in imported pork. at the same level (around 4%). Extended spectrum beta-lactamase (ESBL)-producing, 3rd In E. coli urine isolates from hospital patients generation bacteria is one of the fastest emerging resistance problems cephalosporin resistance was 6% - the same level as in worldwide. Lately, several studies have found the same ESBL The occurrence of ciprofloxacin resistance (%) was at th genes, plasmids and clones E. coli ofisolates originating from same level as in, but a steady increase has been see animals and isolates involved in human infections, suggesting ciprofloxacin resistance from 3% in 4. Aminoglycoside a zoonotic link. The occurrence of Enterobacteriaceae resistant resistance was 5% and sulfonamide resistanc (gentamicin) to carbapenems is a growing threat in human medicine 33%, which are at the same levels as in. because carbapenems are the last resort for treatment of infections caused by multidrug resistant Gram-negative In E. coli urine isolates from primary healthcare, ciprofl bacteria in humans. resistance has increased steadily from 3% in 4 to % The occurrence of 3rd generation cephalosporin resistance In 3, six percent of pigs at slaughter had ESBL-producing 4%, sulfonamide resistance was 33% and ampicillin resista E. coli, which was lower than in and and 9. Fromin 3, all at same levels as reported in. was 4% meat samples, the highest prevalence of ESBL E. producing coliwas found among imported broiler meat (5%), similar, the resistance levels In Klebsiella pneumoniae blood isolates level as in to. The occurrence of meat samples were the same as reported in. Resistance to 3rd gene positive for ESBL producing E. colifrom Danish broiler cephalosporins was 9%, aminoglycoside (gentamicin) resis meat was significantly lower (5%) than in (36%). The was 4%, and ciprofloxacin resistance was 9%. occurrence of ESBL producing E. coliin meat samples was significantly higher in imported broiler meat when compared, In K. pneumoniae urine isolates from hospital patients to Danish broiler meat. This is most likely due to a voluntary resistance to mecillinam (%), sulfonamide (%), genta stop in the usage of 3rd generation cephalosporins in the (4%), nd generation cephalosporins (cefuroxime) (9%) an top of the breeding pyramid abroad resulting in a reduced transmission of ESBL producing E. colifrom imported parent3rd generation cephalosporins (7%) decreased from 3. Ciprofloxacin resistance was at the same level as rep animals to the Danish broilers. As in no carbapenemase in. producing E. coliwere found. DANMAP 3 7

18 . SUMMARY In K. pneumoniae urine isolates from primary healthcare, In 3,,769 casesstaphylococcus of aureus bacteraemia were resistance to 3rd generation cephalosporins was 6%,reported, which is corresponding to 3.9 cases per, inhabit -resistant similar to the level reported in. Resistance to ciprofloxacin The number of methicillin S. aureus (MRSA) from was 7% and resistance to mecillinam was %, which were bacteraemia was 3 (.7%), a level similar to previous yea similar to the levels reported in. Sulfonamide resistance among the lowest incidences recorded in Europe. The high decreased from 6% in to % in 3. frequency of resistance in addition to penicillin was observ for fusidic acid (5%), erythromycin (7%), clindamycin (6% and In Pseudomonas aeruginosa blood isolates, resistance to allnorfloxacin (5%). Susceptibility to the tested antimicro was at the same level as in. the tested antimicrobial agents was not significantlyagents different from the levels reported in, but an increasing trend has been observed for gentamicin resistance during 7 3. The number of new cases of MRSA (both infected and colo persons) increased in 3 to,94 compared to,556 in The increase was primarily seen in livestock associated MR The occurrence of carbapenemase producing bacteria in belonging to clonal complex 398 (CC398), with 643 cases Denmark is described in Textbox 7. In 3, 8 carbapenemase 3 vs 3 in. In 3, CC398 constituted 3% of a producing Enterobacteriaceae (CPE) were detected compared new MRSA cases in Denmark. A significant part of the incre to 9 during 8. In 3, spread of NDM- producing Citrobacter freundii was detected between four patientswas at associated with inclusion of contact to pigs as a risk fa requiring screening for MRSA when being admitted to hosp a hospital ward in the North Denmark Region. None of the The majority (87%) of persons infected or colonized with four patients had been travelling recently and the origin of CC398 had close contact to pigs or were household memb the NDM- producing C. freundii was unknown. Besides the to persons who had pig contact. There were, however, no NDM- producing C. freundii, two of the four patients had of significant spread of CC398 to urban areas, which indica NDM- producing K. pneumoniae. During 3, seven OXAthat 3 producing A. baumannii isolates were detected. Spread of food does not constitute an important transmission ro Among all MRSA cases, the proportion of cases presenting OXA-3 producing A. baumannii was detected twice between patients. Furthermore, OXA-4-like producing A. baumannii with infection was lower in 3 compared to (45% v respectively). The number of hospital-acquired (HA) (n = 3) was detected, two of these isolates were part57%, of the same transmission chain. Two NDM- producing A. baumannii cases continued to be low and constituted only % of the t isolates were detected. In 3, three VIM producing P. number of MRSA cases in 3. In 3, no monitoring of MRSA in animals and meat was performed. aeruginosa isolates were detected. Furthermore, an NDM and VIM producing P. aeruginosa was detected. For the first time, an IMP producing P. aeruginosa isolate was detected in Denmark. Both patients had been hospitalized abroad prior to detection of these isolates. In 3, 8carbapenemase producing Enterobacteriaceae (CPE) were detected compared to 9 CPE during 8 Resistance to penicillin and erythromycin Streptococcus in C. freundiiwas pneumoniae and in group A, B, C and G streptococci. Spread of NDM- producing detected between four patients at a hospital ward in the. remained low in 3 North Denmark Region. None of the four patients had been travelling recently and the origin of the NDM- In 3, resistance to ampicillin was 93% Enterococcus in producing was unknown. C. freundii faecium isolates from blood. Vancomycin resistance was 3.4% in E. faecium and.%enterococcus in faecalis blood isolates. An increasing number of vancomycin resistant During 3, an increasing number of vancomycin resistant enterococci were received at SSI during 3. Twoenterococci were referred to the Antimicrobial Resistance hundred-four-eight of the VRE isolates were from Reference Laboratory at SSI (Textbox 8). Of the VRE isolates, clinical infections and 68 were faecal screening isolates 48 were from clinical infections and 68 were faecal screening. They were Nearly all VRE isolates vana were E. faecium isolates. Nearly all VRE isolates vana were E. faecium isolates. primarily detected at hospitals in the Capital Region, bu The VRE isolates were primarily from hospitals in the Capital also from the Zealand Region and the Central Denmark Region, but also from the Zealand Region and the Central Region. Denmark Region. VRE was detected in the two other regions of Denmark too, but to a much lower extent. Pulsed-field gel electrophoresis typing showed spread of vana several E. The number of new cases of MRSA (both infected faecium types both within hospitals and between hospitals. and colonized persons) increased in 3 to,94 compared to,556 in. The increase was primarily seen in livestock associated MRSA, belonging to clonal Ciprofloxacin resistance Neisseria in gonorrhoeae increased 398 (CC398), with 643 cases in 3 vs 3 in steadily from 3% in 3 to 75% in 9, followed bycomplex a. CC398 constituted 3% of all new MRSA cases decrease to 56% in 3. Penicillinase production among in Denmark in 3. The number of hospital-acquired gonococcus isolates fluctuated between 4% in 3 and MRSAofcases continued to be low and constituted only % in 3, and no ceftriaxone resistant isolates or cases. in 3 % of ceftriaxone treatment failure, were reported in this period. In the total number of MRSA cases 3, azithromycin resistance was 45%, cefixime resistance was 9%, and no spectinomycin resistant isolates were detected (Textbox 9). 8 DANMAP 3

19 BAckGROUND INFORMATION DANMAP 3 3 9

20 3. BACKGROUND INFORMATION 3. Background information Therethe was a 6% increase in the Danish broiler production The following section presents general information about from to 3 (Table 3.). Approximately 8% of the human population in Denmark in 3, and the production produced in 3 were exported for slaughter, a s of food animals in Denmark, over the past decade. Itbroilers also provides an overview of the antimicrobial agents for decrease systemic compared to, but a marked increase from of the production in 3. The annual production of turkey and intramammary therapeutic use in humans and animals in. has fluctuated considerably over the last decade. Since 3 more than 99% of the turkeys produced have been export slaughter, thus all turkey meat available in Denmark is list 3. Populations imported. The distribution of the Danish human population, which could antimicrobial agents potentially have received antimicrobial treatment in 3. 3,Marketed is displayed in Figure 3., together with the 5 healthcare regions Table 3. shows the antimicrobial agents that are registere and the Departments to treat bacterial infections in humans and animals. Some The production of food animals and the production ofthese meatantimicrobial agents are considered critically import for humans and milk is presented in Table 3.. While the number pigs was by WHO. An antimicrobial agent is considered critically important if it is the only compound, or one of lim approximately % lower than in, the number of fattening available pigs (5 5 kg) exported increased by 5%, and the export hastherapy, to treat serious human disease. Criticall important increased by more than five-fold since 4. As in the previousantimicrobial agents are also used in food anima and pets to treat veterinary diseases, and bacteria that are years, the amount of milk produced increased (%). resistant to these critically important agents may be trans to humans. Figure 3. The five healthcare regions and Departments of Clinical Microbiology (DCM) in Denmark DANMAP 3 North Denmark Region No. of inhabitants of inhabitants/km No. of inhabitants/gp 58,7 No Central Denmark Region DCM AALBORG No. of inhabitants No. of inhabitants/km No. of inhabitants/gp DCM MIDT-VEST (Viborg) Capital Region of Denmark No. of inhabitants No. of inhabitants/km No. of inhabitants/gp DCM AARHUS DCM MIDT-VEST (Herning) DCM HERLEV DCM HVIDOVRE DCM ESBJERG No. of inhabitants of inhabitants/km No. of inhabitants/gp 86,359 No. 63 Region of Southern Denmark DCM ODENSE Slagelse No. of inhabitants DCM REGION ZEALAND No. of inhabitants/km No. of inhabitants/gp DCM SØNDERBORG Nykøbing F. DANMAP 3,73, Region Zealand DCM VEJLE,7, ,, Source: Statistics Denmark ( and the Danish Medical Association ( GP=general practitioner

21 BACKGROUND INFORMATION 3. Bacteria that cause human disease may acquire resistance Growthgenes promoters, which are no longer used for animals in from bacteria of animal origin. Fluoroquinolones, 3rdDenmark, and 4th are shown in parentheses in Table 3.. Most of t generation cephalosporins, macrolides and glycopeptides are antimicrobial agents used for growth promotion in Denma among the antimicrobial agents considered criticallyhad important effects on Gram-positive bacteria. Since 995, the ind for humans [AGISAR, WHO 9]. enterococci from animals and meat (and in some years fro healthy humans) have been used as a measure of resistan growth promoters. Table 3.. Production of food animals and the production of meat and milk, Denmark Year Broilers Turkeys, (b) mill. kg heads, mill. kg heads Cattle (slaughtered), heads mill. kg Dairy cows, mill. kg heads milk DANMAP 3 (a) Farmed fish Fresh Marine water Pigs Export,, mill. kg mill. kgmill. kg heads (c) heads , % -33% % % -% % % 8% % 3 Increase (d) 6% 5% <% 5% Source: Statistics Denmark ( and The Danish AgriFish Agency. Production data for farmed fish was not availabl exported prior to slaughter are included in number of animals and amount of meat produced. Export data for poultry from (personal communication) and export of 5-5 kg live pigs from Danish Agriculture and Food a) The numbers for 3 are not final. The production of farmed fish includes fish transferred from one production facility t b) Assume a final slaughtered weight of.5 kg per broiler produced (Danish Agriculture and Food, 3) c) Export of 5-5 kg live pigs. These are included in total number of heads, but antimicrobial use after export until slaugh takes place outside of Denmark d) Increase from to 3 DANMAP 3

22 3. BACKGROUND INFORMATION Table 3.. Antimicrobial agents marketed for systemic and veterinary intramammary therapeutic use in animals and huma Denmark 3 DANMAP 3 (a) ATC / ATCvet codes Therapeutic group Antimicrobial agents within the therapeutic groups Animals Humans JAA / QJAA,QJ5AA Tetracyclines lymecycline, Chlortetracycline, doxycycline,doxycycline, oxytetracycline, tetracycline, oxytetracycline tigecycline JBA / QJBA Amphenicols Florfenicol Chloramphenicol Ampicillin, pivampicillin, amoxicillin, bacampcillin, pivmecillinam, piperacillin, mecillinam JCA / QJCA Penicillins with extended spectrumampicillin, amoxicillin JCE / QJCE Benzylpenicillin, Benzylpenicillin, phenoxymethylpenicillin, Beta-lactamase sensitive penicillins phenoxymethylpenicillin, procaine benzathine benzynpenicillin, penicillin, penethamate hydroiodide procaine penicillin JCF / QJ5CF Beta-lactamase resistant penicillins Cloxacillin, nafcillin Dicloxacillin, flucloxacillin, cloxacillin, methicillin Comb. of penicillins, incl. betaamoxicillin/clavulanic acid, Amoxicillin/clavulanate lactamase inhibitors piperacillin/tazobactam JDB / QJDB,QJ5DB First-generation cephalosporins Cefalexin, cefadroxil, cefapirincefalexin, cefalotin JDC Second-generation cephalosporins Cefuroxime, cefoxitime Cefotaxime, ceftazidime, JDD / QJDD,QJ5DD Third-generation cephalosporins Cefoperazone, ceftiofur, cefovecin ceftriaxone, cefpodoxime JDE / QJ5DE Fourth-generation cephalosporins Cefquinome Cefepime JDF Monobactams Aztreonam Meropenem, ertapenem, JDH Carbapenems imipenem/cilastatin, doripenem JEA Trimethoprim and derivatives Trimethoprim JEB / QJEQ Short-acting sulfonamides Sulfadimidine Sulfamethizole JCR / QJCR JEE / QJEW Comb.of sulfonamides and Sulfadiazine/trimethoprim, Sulfamethoxazole/trimethoprim trimethoprim, incl. derivativessulfadoxine/trimethoprim, sulfamethoxasol/trimethoprim JFA / QJFA Macrolides JFF / QJFF (b) JFG / QJXX Lincosamides Streptogramins JG / QJRA,QA7AA Aminoglycosides Spiramycin, tylosin, tilmicosin,erythromycine, roxithromycine, tylvalosintartrat, tulathromycin, clarithromycine, azithromycine, gamithromycin, tildiprocin spiramycine Clindamycin, lincomycin Clindamycin (Virginiamycin) Streptomycin, dihydrostreptomycin, Tobramycin, gentamicin, amikacin, gentamicin, neomycin, apramycin netilmicin JMA / QJMA Fluoroquinolones Enrofloxacin, marbofloxacin, difloxacin, ibafloxacin, pradofloxacin QJMB QJMQ(b) Other quinolones Quinoxalines Oxolinic acid (Carbadox, olaquindox) JXA,A7AA / (b,c) Not in ATCvet (b) JXB / QA7AA JXC (c) JXD,PAB JXE Glycopeptides (Avoparcin) JXX / QJFF Other antibacterials QJXQ QP5AG4 (b) Not in ATCvet (b) Not in ATCvet Pleuromutilins Tiamulin, valnemulin Antiprotozoals, sulfonamides Sulfaclozine Oligosaccharides (Avilamycin) Flavofosfolipols (Flavomycin) Polypeptides (incl. polymyxins) Colistin, bacitracin Steroid antibacterials Imidazole derivatives Nitrofurane derivatives Spectinomycin Ofloxacin, ciprofloxacin, norfloxacin, fleroxacin, grepafloxacin, trovafloxacin, moxifloxacin Nalidixic acid Vancomycin, teicoplanin Colistin Fusidic acid Metronidazole Nitrofurantoin Methenamine, linezolid, daptomycin a) ATCvet codes starts with a Q b) Animal growth promoters used before 999 are listed in parentheses c) Although intestinal antiinfectives (A7AA) and imidazole derivatives for protozoal diseases (PAB) are used to treat hu are not reported by DANMAP DANMAP 3

23 4 ANTIMIcROBIAL consumption IN ANIMALS DANMAP 3 3

24 4. ANTIMICROBIAL CONSUMPTION IN ANIMALS 4. Antimicrobial consumption in animals optimize antimicrobial use, and 3) enforcement of the so called cascade rule [Order (DK) 4/993], which limits thehas usebeen of (cheaper) extemporaneously produced medicin The use of antimicrobial agents in humans and animals - thisthe particularly affected the use of tetracyclines from monitored by the DANMAP programme since 995. Since Another important intervention was the restriction on the, there has been both political and public focus early 99s on use of fluoroquinolones in production animals through the use of antimicrobial agents in the Danish animal production. legislation This resulted in discontinued use of several antimicrobial agentsimplemented in and 3. Furthermore,, the pig industry imposed a voluntary ban on th, July used for growth promotion from , and more recently use of cephalosporins, due to concerns regarding extende a voluntary ban of use of cephalosporins in the pig production, resistance (ESBL). as well as regulatory legislation regarding therapeuticbeta-lactamase use [DANMAP ]. From to, consumption decreased following theand introduction of threshold values for antimicrobial Overall, the antimicrobial consumption for both humans animals has increased since the late 99s. While theconsumption consumptionadopted within the yellow card initiative. enforces for humans has gradually increased throughout the period, thelegal actions on pig farmers with high antimicrob agent use consumption in animals has fluctuated notably. The increase in per pig [DANMAP ]. Effects from other par of the legislation may be less obvious, but are important veterinary consumption can partly be explained by the increase keepofinthe mind, when interpreting the veterinary prescriptio in pork production, which constitutes approximately 84% patterns. meat production in Denmark (Table 3.). Figure 4. shows the total antimicrobial consumption in animals and humans since Official guidelines for the selection of antimicrobial agent 994 and 997, respectively. that veterinarians may choose from for pigs and cattle ha been available since 996. The guidelines provide specifi The prescription pattern has been clearly influenced by implemented legislation. For example, the decrease inrecommendations for the selection of the appropriate antimicrobial agents for treatment of all common indicati antimicrobial consumption after 994 was likely the result of in major ) limitation of veterinarians profit from sales of medicine ) production animal species. Initially, guidelines w developed by the National Veterinary Laboratory (presen implementation of preventive veterinary strategies with herd National Veterinary Institute, DTU). Since 5, the health contracts and regular monthly visit from the veterinarian guidelines have been updated by the Danish Veterinary a in order to promote preventive veterinary strategies and Food Administration (DVFA) in collaboration with National 4. Introduction Figure 4.. Prescribed antimicrobial agents for humans, and for animals compared with the number of pigs produced, Den DANMAP 3 35 Antimicrobial agents (tonnes) Pigs produced (mill. heads) Prescribed veterinary antimicrobials Antimicrobial growth promoters Pigs produced (mill. heads) 3 Prescribed human antibacterials Sources: Human therapeutics: The Danish Medicines Agency. Veterinary consumption: Until, data are based on repor pharmaceutical industry of total annual sales from the Federation of Danish pig producers and slaughterhouses ( Medicines Agency and Danish Plant Directorate (996 ). Data from 3 originate from VetStat 4 DANMAP 3

25 ANTIMICROBIAL CONSUMPTION IN ANIMALS4. Veterinary Institute DTU, National Food Institute DTU,DAPD the (DADD per, animals per day) - estimated treatment proportion Practicing Veterinarians Organization, university experts, Trends in veterinary consumption, both within and across the Danish Association of the Veterinary Pharmaceutical species, Industry and the Danish Agriculture and Food Council. The are presented in DAPD, allowing for comparison between sectors and adjusting for changes in live biomass latest update was in, when new dynamic evidence-based estimated treatment guidelines for pigs were launched [DANMAP, live biomass is expressed as the number of stan animals with an estimated average weight on a given day. In, the Danish Veterinary Association mayofalso be referred to as the standard-animals-at-risk, a published treatment guidelines to promote prudent use into account species differences in body-mass and li antimicrobials in dogs and cats, prepared by clinical takes specialists span. and expert scientists from the Faculty of Health and Medical Sciences at the University of Copenhagen and National Food. Institute, DTU DAPD or estimated treatment proportion is a statistical measure, providing a rough estimate of the proportion of animals treated daily with a particular antimicrobial agent 4.. Data sources For been example, DAPDs indicate that an estimated % of Data on antimicrobial use at the product the level have population (e.g. broiler population), on average, receiv collected in Denmark since 996, including historicalthe data back a certain treatment on a given day (Section 4.3 and Chapt to 99. In Denmark, all therapeutic medicine is by prescription Materials and Methods). Furthermore, presenting the only, and since, data on all medicine prescribed9,for use veterinary consumption in DAPD allows comparisons with in animals, including vaccines, have been collected (at end antimicrobial consumption in the human sector as express users) in a national database (VetStat). Data on consumption in defined daily dose per, inhabitants per day (DID), coccidiostatic agents (non-prescription) and antimicrobial Chapter, Terminology, for a description of DID. growth promoters (no longer used), are also collected by VetStat. At the European level, the ESVAC (European Surveillance o Veterinary Antimicrobial Consumption) project monitors Consumption data for 3 - for use in DANMAP - were usage in a number of countries. ESVAC monitor extracted from VetStat by the Danish Veterinary andveterinary Food based on the quantity by weight of antimicrobials, using Administration (DVFA) on June 4. The NationalisFood correction unit (PCU) as denominator to adjus Institute, DTU has carried out no further validation of population the. for changes in size of the production animal population received data within the respective countries over time. A description of methodology used by ESVAC is contained in the first repor 4.. Methods Trends in the sales of veterinary antimicrobial agents in n Metrics of antimicrobial consumption are numerous, each European countries 5-9 [ with its own advantages and limitations. Therefore, the chosen measures must depend on the purpose and the available In the context of DANMAP, we base our comparison on dos information. in order to keep in focus the newer, potent antimicrobials s as fluoroquinolones and cephalosporins that are critically The overall amount of antimicrobial agents is measured in kg important in the treatment of human infections. Furthermo active compound and is used in Section 4. for the purpose of an the biomass of the live population is used as denominator overall crude comparison of antimicrobial use in the veterinary allow for comparisons of selection pressure between anim and human sectors and to enable international comparisons populations. (Figure 4.). In the text below, unless otherwise stated, DAPD will be us In DANMAP, we introduced two new metrics to follow to describe patterns in veterinary antimicrobial consumpti trends in antimicrobial consumption to ensure the robustness of the analyses over time and to facilitate comparisons between Total antimicrobial consumption animal species, as well as comparisons between the 4. veterinary and human sectors. The new metrics are defined below, and for In 3, more information on methodology, please refer to Chapter 9 the total veterinary consumption of antimicrobial agents, including agents used for companion animals, and the web annex [ tonnes active compound (Table 4.), amounted to 6 representing a 4% increase compared with. The incre DADD (Defined animal daily dose) mainly DADD is the average maintenance dose per day for awas drug used attributed to a 6% increase in the amount used pigs. The two major species, cattle and pigs, comprise equ for its main indication in the appropriate animal species. proportions of live biomass. However, the vast proportion The DADD is not defined at product level but for each biomass consists of dairy cows, which have a very lo antimicrobial agent, administration route and animalcattle species; consumption of antimicrobial agents compared with growi and when appropriate, also age group. DADD has been specifically defined for use in DANMAP and does not animals. always In 3, the antimicrobial consumption in pigs, %, and cattle and poultry comprised 78%, ~ ~% of the total completely match the prescribed daily dose or the recommended veterinary dosage in the Summaries of Product Characteristics (SPC) or in consumption, respectively (Figure 4.).. the VetStat database Since, the DADD has replaced the ADD (as defined in VetStat), which had been used since DANMAP 3. For more details, see Chapter 9, Materials and Methods. The DADDs used in DANMAP 3 are presented in the web annex. DANMAP 3 5

26 4. ANTIMICROBIAL CONSUMPTION IN ANIMALS Figure 4.. Live biomass (mill kg) and antimicrobial consumption (kg) in main animal species, Denmark DANMAP 3 Cattle< yr., 7% Cattle, % Pets, % Cattle > yr., 4% Horses, pets and others 3% Horses, 5% Pigs, 78% Poultry, % Poultry, % Fur Animals, % Fur Animals, 4% Aquaculture, % Aquaculture, 3% Pigs, 43% Live biomass Kg active compound Note: The live biomass is estimated from census (pigs, cattle and pet animals) and production data (poultry, fur animals, a estimation procedures are described in Chapter 9. For poultry: the figures comprise only the biomass for the main producti and broiler production) Weaners Finishers Cattle, total Total Others Tetracyclines Pleuromutilins Macrolides Lincosamides Amphenicols QJAA 565 QJE Sulfonamides and (b) trimethoprim QJCA (a) Penicillin's, others Penicillin's, QJCE b-lactamase sensitive QJMB Other quinolones 3 88 QJXQ QJMA Fluoroquinolones Therapeutic group QJFA QJD Cephalosporins 63 -Sows and piglets ATCvet code QJFF QJG (c) Pigs, total QJB Aminoglycosides Table 4.. Antimicrobial agents sold (kg active compound) by animal species and age group, Denmark DANMAP 3 -Intramammaries Calves < mdr Cows and bulls Heifers and steers Poultry, total Poultry incl. broilers, layers and other poultry 8 Turkeys Other production animal 85 species Aquaculture 85 Fur animals Companion animals 5 Horses 6 Pets 44 9 Total (d) Note: Only the ATCvet group contributing mostly to the antimicrobial group is mentioned. Combination drugs are divided in a) Penicillins with extended spectrum, cloxacillin and amoxicillin/clavulanic acid b) Sulfaclozin (a prescription coccidiostat) is included in the sulfonamide/trimethoprim group c) Fluoroquinolones reported for use in pigs were used in a clinical trial approved by the Danish Medicine Agency [Source: d) Antimicrobials used for companion animals: DVFA has allocated kg active compound to the appropriate target species (h on knowlegde of which products are used for the particular species 6 DANMAP 3

27 ANTIMICROBIAL CONSUMPTION IN ANIMALS4. DAPD (DADD per animals per day) (a) Figure 4.3. Antimicrobial consumption in the pig production, Historically, the overall consumption measured as kg active and the distribution on age groups, Denmark compound - was 43% lower in 3 compared with while the total meat production increased by 3% during this period (Table 3. and Figure 4.). A major part of the decrease in consumption can be explained the by discontinued use of growth promoters ( ). However, from to 9, the antimicrobial consumption in production animals increased by 36% (Figure 4.). This 8 increase was driven mainly by an increase in consumption in pigs and should be seen in the context that the number of pigs produced increased by 9% (Table 3.). At the same time 6 the proportion of live pigs (approx. 3 kg) being exported has increased and thus resulted in a decrease in the overall biomass of the pig population Antimicrobial consumption by animal species 4.3. Antimicrobial consumption in pigs In 3, the total antimicrobial consumption in pigs was 9.6 Sows and piglets Weaners.5 tonnes tonnes active compound (Table 4.), an increase of 5 Finishers Total, adjusted (6%) compared with. The treatment proportions (DAPD) in the pig population overall and by age group are presented inadjusted total is adjusted for the increasing export o Note: The Figure 4.4 and in the web annex (Table A4.). at 3 kg (see text). Sows includes treatment in piglets pre-wea a) The DAPD is calculated as the number of standard doses for o animal divided by the estimated live biomass in the age group at The treatment proportion (DAPD) is much higher in the total population (in tonnes) weaning pigs, compared with finishers and sows (Figure 4.3). However, the biomass of the weaning pigs is very small (7.5-3 administered kg, 4 weeks), compared with the finishers (3-7 kg, weeks) orally, and particularly used for treatment of gastrointestinal disease in weaning pigs and finishers. The and the sows. overall treatment proportion (DAPD) of pleuromutilins increased by 8%, while the use of tetracyclines increased The DAPD of the total population should reflect the trends 4%. in selection pressure in the population. Due to the differences in treatment proportion between age groups, the DAPD of the total For thee.g. critically important antimicrobial agents, the use population is affected by changes in population structure, of flouroquinolones increased for the second year running, increased export of live pigs at 3 kg. The increase in the export however remains at a very low level and constitutes less th would in itself cause an increase in DAPD for the remaining. The per mille of the total consumption in pigs increase in 3 population, because the DAPD for finishers is relatively low. is largely explained by a clinical trial approved by the DVFA Thus, a true impression of the antimicrobial consumption use of cephalosporins in pigs was low (3 kg), however, pattern and selection pressure in the pig production The requires increased that changes in export and productivity are taken into account.from kg in (Figure 4.5)., the antimicrobial consumption in pigs increased by Overall Over the 5% to approximately 3 DAPD (Figure 4.3) when adjusted for last decade, the treatment proportion (DAPD) increased from 4 to 9. However, in and, changes in export. Overall the number of pigs produced in a decrease 3 was similar to ; however the number of pigs exportedin DAPD compared with 9 was observed, probably as a response to the DFVA s implementation of th increased by 5% (Table 3.). yellow card initiative a special provision for reduction o consumption in pig production (See DANMAP Within the different age groups, the DAPD increased antimicrobial in all age for further details). The reductions in antimicrobial us, and 5% in both finishers and.weaners groups; 9% in sow herds were associated with increasing use of vaccines and a sligh The increase in consumption was associated primarily with the temporary decrease in productivity in some herds, but dise use of pleuromutilins and tetracyclines, and to a lesser extent, outbreaks did not increase [Danish Veterinary Bulletin no. to the use of penicillins and sulfonamides/trimethroprim ]. (Figure 4.4). Tetracyclines and pleuromutilins have been thewith the increase in antimicrobial consumption in p in 3, most commonly used antimicrobial agents in the Danish pig the consumption is still % lower than in 9, a similar to the 8 level (Figure 4.3). production for a decade (Figure 4.4). They are almost entirely DANMAP 3 7

28 4. ANTIMICROBIAL CONSUMPTION IN ANIMALS (a) (b) Figure 4.4. Antimicrobial consumption in the total pig production, and in finishers, weaners, sows and piglets, Denmark DANMAP 3.. Finishers DAPD (DADD per animals per day) DAPD (DADD per animals per day). Total pigs produced Weaners Sows and piglets 45. DAPD (DADD per animals per day) DAPD (DADD per animals per day) Macrolides Tetracyclines Pleuromutilins Lincosamides Penicillins, b-lact. sen. (c) Penicillins, other Cephalosporins Aminoglycosides Sulfonam./trimeth. 3 Penicillin/streptomycin Note: Amphenicols, colistin, fluoroquinolones, intramammaries, gynecologicals and topical drugs are not included in the fig veterinary practice are not included (<% of the consumption in pigs) a) The DAPD is calculated as the number of standard doses for one kg animal divided by the estimated live biomass in the population (in tonnes) b) Total pigs produced includes pigs exported at 3 kg, which has increased in numbers from.7 million in 4 to 9. mil Consumption in these pigs is included only from birth to 3 kg body weight. See discussion in the DANMAP. c) Beta-lactamase sensitive penicillins 8 DANMAP 3

29 ANTIMICROBIAL CONSUMPTION IN ANIMALS Antimicrobial consumption in poultry4.3.3 Antimicrobial consumption in cattle DANMAP we expressed reservations about data on In Denmark, poultry production comprises mainly theinbroiler antimicrobial consumption in cattle. However, analyses of production (Gallus gallus), followed by egg layers (Gallus gallus) and turkey production. In addition, there is a this veryissue smallin collaboration with representatives from the ca industry and the veterinary authorities has shown that the production of ducks, geese, and game birds. quality of reporting of use in cattle has increased markedly in recent years and is now as high as that for other major In Denmark, the antimicrobial consumption in the broiler production production is generally low compared with other species and, animal species. a few disease outbreaks in some farms can seriously affect the The data shows that use of antimicrobials in cattle has dec national consumption, causing considerable fluctuations in to a total of about tonnes active compound in 3. Du annual consumption data. this period, the veal and beef production has remained at same In 3, the total antimicrobial consumption in poultry (alllevel, although with minor fluctuation from year to y and, poultry) was 7 kg active compound, an increase of 57%the milk production has increased slightly. compared with. In poultry (other than turkeys), the In general, the majority of the parenteral use in cattle is fo increase was mainly in the use of simple penicillins (from (Table 4.), and is mainly prescribed for mastitis. The kg in to kg in 3), but also in the use of cows tetracyclines (36 kg in and kg in 3). In turkeys there systemic was also use an of fluoroquinolones in cattle was close to zer 3, and has been at a low level since 3. In 3, increase in the use of simple penicillins, but the mostinsignificant increase was seen in the use of tetracyclines, which systemic increaseduse of cephalosporins in cattle declined to 4 kg from to 47 be kg in (Figure 4.5). Use of 3rd and 4th genera from 7 kg to 49 kg in 3. The main reason appears cephalosporins in intramammaries has declined steadily fr widespread problems with respiratory disease in turkey flocks a peak at 7 kg in 7 to 7 kg in 3 (Table 4.). This is produced early in 3. Furthermore, an increased occurrence a result of diarrhea in broiler flocks in 3 may be the reason for theof increased focus by the Danish Cattle Association and veterinary practitioners, on the public health aspects increased consumption of penicillins. antimicrobial resistance caused by high consumption of th For broilers, amoxicillin has been the most commonlygroup usedof compounds. antimicrobial agent for at least a decade. Fluoroquinolones were the second most commonly used antimicrobial Data agenton intramammary use show a slight reduction in ove level of intramammary treatment from 5 to 3 (Tabl until 7 (when new medicines were approved for poultry),. 4.). However, drying-off treatment has increased steadily but has not been used in broilers in -3 during the same period, while therapeutic treatment has decreased (Table 4.3). A milk quality campaign conducte the Danish Cattle Association (Agriculture and Food Counc Table 4.. Use of antimicrobial agents for intramammary application in cattle, Denmark Doses per antimicrobial class 9 DANMAP 3 3 DADDs ('s) (a) Penicillins Aminoglycoside-benzylpenicillin (b) combinations Cephalosporins, st generation Cephalosporins, 3rd and 4th generation (c) Others Total Total DADD per cow per year Note: For intramammary treatment, DADD is defined as the dose to treat two teats for 4 hours a) Includes benzylpenicillin, cloxacillin, and cloxacillin-ampicillin combinations (QJ5CE, QJ5CF, QJ5RC) b) Mainly dihydrostreptomycin-benzyl benicillin combinations; includes also combinations of penicillin/aminoglycoside with nafcillin (QJ5RC) c) Lincosamides, neomycin-lincomycin combinations and trimethoprim-sulfonamide combinations Table 4.3. Number of treatments with antimicrobial agents for intramammary application in cattle, Denmark DANMAP (a) Total doses per indication Drying off treatment (4 teats) 9 3 DADDs ('s) 73 Therapeutic treatment ( teats) Note: For intramammary therapeutic treatment, DADD is defined as the dose to treat two teats for 4 hours. For drying o is defined as the dose to treat 4 teats. One product used for both indications is included as half drying off and half therape DANMAP 3 9

30 4. ANTIMICROBIAL CONSUMPTION IN ANIMALS Figure 4.5. consumption of 3rd and 4th generation cephalosporins in pigs and cattle, Denmark 4 DANMAP 3 Kg active compound Systemic treatment of pigs Systemic treatment of cattle 3 The information available on antimicrobial consumption in companion animals is not as detailed as for production animals. In 3 the overall antimicrobial consumption in pets amounted to,989 kg active compound (,449 kg in ). The increase was seen for several antimicrobial clas aminoglycosides, macrolides and penicillin s (b-lactamase sensitive), sulfonamides and trimethoprim, as well as for tetracyclines (Table 4.). The use of fluoroquinolones for use in pets was kg in This means that the total use of fluoroquinolones constitut nearly 4% the total veterinary use (kg) of fluoroquinolone Similarly, they account for a significant proportion of the use of cephalosporins. In a One Health perspective, this is a cause for concern, because the close contact with their owners poses a risk for transmission of extended spectrum beta lactamase resistance to humans. Presently, however, there is no information available concerning the prevalence antimicrobial resistance in pet animals. Intramammary treatment of cattle A large proportion of antimicrobials used for pet animals ar prescribed for treatment chronic or recurrent disease, main dermatitis. Particularly the consumption of critically import since, most likely has contributed to this trend.antimicrobial The goals agents in pet animals could pose an importan of the campaign are to reduce treatment of clinical mastitis by risk to owners of diseased dogs that are frequently treated 5%, mainly through a reduction of treatment of subclinical mastitis, but also by increased monitoring of cell counts to Acknowledgement determine the need for treatment. The methodology for calculating veterinary consumption in DAPD, as first applied in DANMAP, was developed and Furthermore, Order (DK) 785/ provides legal regulations describedofby Vibeke Frøkjær Jensen. use of antimicrobial agents for mastitis in cattle (recommending using simple penicillins) and the industry has also emphasized Leonardo de Knegt, Birgitte Borck Høg and Flemmin that farmers should use narrow spectrum penicillins to treat mastitis caused by Gram-positive bacteria, unless sensitivity testing reveals resistance towards these antimicrobials Antimicrobial consumption in aquaculture, fur animals and companion animals The antimicrobial consumption in aquaculture increased by 3% to,58 3 kg in 3 compared with (Table 4.). Measured in kg active compound sulfonamide/trimethoprim comprised 7%, quinolones 5% and amphenicols 5%.The antimicrobial consumption in both and was very low compared to previous years, probably due to cold summers in, and the relatively large increase in consumption in 3 is explained mainly by extraordinary high temperatures in July and August 3, leading to higher water temperatures and an increase in the occurrence of bacteriological infections [personal communication: N.H. Henriksen, Danish Aquaculture]. There is, however, in the aquaculture industry, increased focus on vaccination to reduce the risk of diseases that may require antibiotic treatment. In 3, the production of mink increased slightly to 7.5 million mink from 7 million in (Source: Kopenhagen Fur). Antimicrobial consumption in fur animals decreased by approximately % to 4.9 tonnes, kg active compound compared with (Table 4.). This reverses the continuous increase in consumption for fur animals seen over the past five years (from 8-9 consumption increased by 6%). Use of fluoroquinolones and cephalosporins in fur animal production is close to zero. 3 DANMAP 3

31 5 ANTIMIcROBIAL consumption IN HUMANS DANMAP 3 3

32 5. ANTIMICROBIAL CONSUMPTION IN HUMANS 5. Antimicrobial consumptions in humans 5. Introduction Table 5.. classification of antimicrobial agents for systemic use in humans into narrow-spectrum and broad-spectrum agents, Denmark DANMAP 3 humans In Denmark, the use of antimicrobial agents for (a) has been monitored since 995 and presently all data ongroup ATC Therapeutic group consumption in both the primary healthcare and hospital care is registered by the Department of Data Delivery and MedicinalNarrow-spectrum Product Statistics at Statens Serum Institut. JCE Beta-lactamase sensitive penicillins JCF Beta-lactamase resistant penicillins In this section, the term antimicrobial agents covers only First-generation cephalosporins (included in data JDB antibacterial agents for systemic use in humans, agents used from primary healthcare as a broad-spectrum agent the group JD) for treatment of infections caused by virus or fungi are not included. Currently available antimicrobial agents forjdf systemic Monobactams treatment in humans (and in animals) are listed in Table JEA3.. Trimethoprim and derivatives Antimicrobial agents have been classified as either narrowjeb Short-acting sulfonamides spectrum or broad-spectrum agents according to thejfa spectrum Macrolides of the activity (Table 5.). JFF JXA 5. Total consumption (primary healthcare JXC and hospital care) JXD JXE The overall consumption of antimicrobial agents in Denmark JXX Lincosamides Glycopeptides Steroid antibacterials (fusidic acid) Imidazol derivatives Nitrofuran derivatives (nitrofurantoin) Other antibacterials is calculated as a combined measure of the consumption in the Broad-spectrum primary healthcare and hospital care. This measure is presented in DID and is intended for comparison between sectors and for JAA Tetracyclines illustration of the consumption in hospital care without taking JCA Penicillins with extended spectrum account of hospital activity (discharges) (Figure 5.). Since the Combinations of penicillins, incl. beta-lactamase JCR first publication of DANMAP, the consumption in primary inhibitors healthcare has accounted for 9% of the total antimicrobial Cephalosporins and related substances (primary JD consumption in Denmark. healthcare only) JDC Second-generation cephalosporins Third-generation cephalosporins Carbapenems Combinations of sulfonamides and trimethoprim, incl. derivatives JGB Aminoglycosides JMA Fluoroquinolones The proportion of broad-spectrum agents was 5% higher JXB Polymyxins than in (Figure 5.). This continues the trend from the a)by From the 3 edition of the Anatomical Therapeutic Chemica past decade where broad-spectrum agents increased 7%, (ATC) classification system comprising 3% of the total consumption in 4 and 46% in In 3, the total consumption of antimicrobial agents for JDD systemic use (primary healthcare and hospital care) was.% JDH higher than the previous year (Figure 5.). Since 4, the overall consumption of antimicrobial agents has increased JEE by %. 3. The distribution of DIDs between primary healthcare and hospital care differed between antimicrobial agents (Figure 5.3). For most of the antimicrobial agents, the consumption was higher in primary healthcare, with exception of cephalosporins and other β-lactams, aminoglycosides and imidazole derivatives. In 3,,5 5 kg of antimicrobial agents for systemic use were used in humans in Denmark. This is the highest level observed,5of since 995, representing an increase kg6(4%) since 4 (Table A5. in web annex). 3 DANMAP 3

33 ANTIMICROBIAL CONSUMPTION IN HUMANS 5. Figure 5.. Total consumption of antimicrobial agents (J) in humans in primary healthcare vs hospital care, Denmark DDD/ inhabitant-days DANMAP Primary health care Hospital care Figure 5.. Total consumption of antimicrobial agents (J) in humans by narrow-spectrum and broad-spectrum agents, D DANMAP DDD/ inhabitant-days Narrow-spectrum Broad-spectrum Note: Narrow-spectrum includes: beta-lactamase sensitive penicillins, beta-lactamase resistant penicillins, trimethoprim, macrolides, lincosamides, glycopeptides, fusidic acid, imidazol derivatives, nitrofuran derivatives, and other antibiotics Broad-spectrum includes: tetracyclines, penicillins with extended spectrum, combinations of penicillins incl. beta-lactam cephalosporins and related substances, combinations of sulfonamides and trimethoprim, aminoglycosides, fluoroquinolone DANMAP 3 33

34 5. ANTIMICROBIAL CONSUMPTION IN HUMANS Figure 5.3. Distribution of DIDs between primary healthcare and hospital care, Denmark DANMAP 3 JCR Penicillins, incl. beta-lactamase inhibitors JD Cephalosporins and other β-lactam antibiotics JE Sulfonamides and trimethoprim JG Aminoglycosides JMA Fluoroquinolones JXA Glycopeptides JXB Polymyxins JXC Steroid antibacterials (fusidic acid) JXD Imidazol derivatives Primary health care JXE Nitrofuran derivatives (nitrofurantoin) Hospitals JXX Other antibacterials DDD/ inhabitant-days JAA Tetracyclines JCA Penicillins with extended spectrum JCE Beta-lactamase sensitive penicillins JCF Beta-lactamase resistant penicillins Primary health care Hospitals JF Macrolides, lincosamides and streptogramins 3 4 DDD/ inhabitant-days 34 DANMAP 3 5 6

35 Textbox ANTIMICROBIAL CONSUMPTION IN HUMANS 5. consumption of antimicrobial agents and incidence of multi-resistant bacteria in Green Background: Greenland has a population of inhabitants (January 3) and Nuuk is the capital with inhabitants. Greenland has its own Ministry of Health and the country is divided into five health regions: Qeq former health districts Sisimiut and Maniitsoq), Disko (the former health districts Aasiaat, Kangaatsiaq, Qeqe Qasigiannguit), Avannaa (the former health districts Ilulissat, Uummannaq, Upernavik and Qaanaaq), Serme former health districts Nuuk, Paamiut/Ivittuut, Tasiilaq and Illoqqortoormiut), and Kujataa (the former health Qaqortoq, Nanortalik and Narsaq). The largest hospital, Dronning Ingrids Hospital, is situated in Nuuk (85 beds). There are several smaller hos healthcare centers in the five health regions. Around 5-6. persons are admitted to hospital once or se year. The primary healthcare is organized different from that in Denmark; there are no general practitioners practice, and the hospital clinics are used for patients from the primary healthcare. In Nuuk, a large healthc combined function as medical clinic, emergency room and primary healthcare with doctors and nurses. The have nursing stations (supervised by doctors via phone or telemedicine and doctors visiting three to four tim Medication on prescription is free of charge. Patients with specific/serious diseases are transferred to Denma for further treatment (e.g. hemodialysis, cancer treatment, brain surgery etc.). to 3, Resistant bacteria: From 4 patients have been diagnosed with MRSA, 33 patients with ESBL-produ Enterobacteriaceae Clostridium difficile, and 9 out of 85 patients with infection had the 7 type. Most of these resistant bacteria were imported from Denmark or abroad, but in some cases, especially in patients with an ESBL-pro Enterobacteriaceae, treatment with broad-spectrum antimicrobial agents in Greenland has probably selected for C. difficile bacteria. Since there has been an increasing problem with infections (mainly type 7) in the hospitals C. and transmission within the country has occurred. A project focusing on identification of risk factors for acqu difficileinfections, mapping C. of difficile types and prevention strategies is planned for the near future. Consumption of antimicrobial agents: All antimicrobial agents in Greenland are purchased and dissemin the National Pharmacy. The total purchase of selected antimicrobial agents from 7 to 3 are shown in 3, an increase of narrow-spectrum (8%) and broad-spectrum penicillins (%) has been seen From 7 to 3, an increase in piperacillin-tazobactam (37%) and a decrease in broad-spectrum antimicrobial as tetracyclines (6%), macrolides (7%), and fluoroquinolones (6%) have been seen. Meropenem has increa whereas cephalosporins (mainly ceftriaxone) have been at the same level from to 3. Conclusion: Frequent change in workforce and hospitalization abroad are challenges for maintaining a rest policy and of these reasons continued focus on the use of broad-spectrum antimicrobial agents - both in hos primary healthcare - and the incidence of multi-resistant bacteria in Greenland is very important in the futu Anne Kjerulf, Jette Holt, Anne Birgitte Jensen, Turid B. Skifte, Peter Poulsen, Ing For further information: Anne Kjerulf (alf@ssi.dk) DANMAP 3 35

36 5. ANTIMICROBIAL CONSUMPTION IN HUMANS Figure. consumption of selected antimicrobial agents in humans in Greenland (DDD/, inhabitants/day) 7 3: (a) consumption of narrow- and broad-spectrum penicillins, macrolides and tetracyclines; and (b) consumptio of cephalosporins, meropenem, fluoroquinolones, piperacillin/tazobactam and gentamicin. (Note: Narrow-spectrum penicillins include benzylpenicillin, phenoxymethylpenicillin and dicloxacillin, and broad-spectrum penicillins includ ampicillin, pivampicillin, amoxicillin and amoxicillin with enzyme inhibitor). (a) DANMAP DDD/ inhabitant days Penicillins (narrow-spectrum) Penicillins (broad-spectrum) Macrolides Tetracyclines DANMAP 3 (b) DDD/ inhabitant days Cephalosporins 36 DANMAP 3 Meropenem Fluoroquinolones Piperacillin/tazobactam Gentamicin

37 Textbox ANTIMICROBIAL CONSUMPTION IN HUMANS 5. Maintained focus on the use of antimicrobial agents in the Faroe Islands Background: The Faroe Islands (FI) consists of 8 islands, inhabited by approx. 48, inhabitants, 9, in the capital Tórshavn. The main hospital (Landssjúkrahúsið, LS, with 7 beds) is located in Tórshavn, and two smaller hospitals in Klaksvik (36 beds) and Súðuroy (6 beds). The healthcare system is comparable to healthcare system with general practitioners responsible for primary care and secondary care provided by has a local as well as a centralized function. In the case of specified diseases, patients are referred to hospi or other foreign hospitals. The healthcare staff constitutes a stable workforce but consultants, mainly from perform specialized treatment where the number of patients is too small to support full-time specialist emp Data and data sources: Data for antimicrobial consumption for FI and for LS were supplied by the Chief P Office, beginning from 7. Bed-days and data on MRSA and ESBL-producing bacteria were obtained from Antimicrobial consumption in primary healthcare: Total antimicrobial consumption outside the hospit 4.3 DDD/ inhabitants/day; this is unchanged since, but represents a 6.6% decrease compare The use of tetracyclines has increased by 3.8% compared to 7 whereas the consumption of macrolide with 3.% from 7 to 3. The reason for this shift remains to be elucidated. Regarding antimicrobial a used for urinary tract infections, marked changes have been seen mainly reflecting identification of a poten fatal carnitine transporter gene defect in /3.6 inhabitants of the t al.fi 6. [Joensen Ugeskr Laeg 68: ] which led to an almost total stop in the use of pivampicillin and pivmecillinam in. In 3, pivampicill still not used, whereas pivmecillinam use outside hospitals increased to 3.3% of the 7 level compared in (Figure ). Data from hospitals show the same trend. This increase in consumption, although still at level, is probably due to increased screening for the gene defect. The decreased use of pivmecillinam has l increase in the use of sulfamethizole (43.%), trimethoprim (4.%), and ciprofloxacin (46.7%) compared t Antimicrobial consumption in hospital care: The consumption of antimicrobial agents in 3 was 39. (DDD/ bed-days). This is a decline from (4.45 DBD) and the consumption is now back at the sam in 7 (Table ). However, whereas the total consumption of antimicrobial agents at LS is at the 7 leve three antimicrobials, which are well known for their effect on selection of virulent binary-toxin C. difficile -positive cefuroxime, ciprofloxacin, and meropenem - has increased to 37.4%, 44.5%, and 75.% of the 7 lev The consumption of cefuroxime, ciprofloxacin, and meropenem and piperacillin with enzyme inhibitor, in a between LS and Danish hospitals for 7 to 3 are shown in Figure. It is thus evident that LS has been curve of increase as Danish hospitals in general, only with some 7-8 years delay. This is also illustrated by the consumption of the three above antimicrobials together increased from 8.99 DBD in 7 to 4.54 in 39.8 to 58.8% of the use in Danish hospitals. However, it seems that the use in LS is now at least stabilizing Resistant microorganisms. Since the first case of MRSA in 4, a total of 36 cases of MRSA have been ( with infection and 4 carriers). From 6 3, 5 ESBL-producing Escherichia coli and four ESBL-producing Klebsiella pneumoniae have been detected, furthermore one patient had both an ESBL-producing E. coliand an ESBLproducing K. pneumoniae. Conclusion: Increased focus on the use of broad-spectrum antimicrobial agents and resistant bacteria is m the current strategy of the Faroe Islands. Elsebeth Tvenstrup Jensen, Anne Kjerulf, William Smith, Niels Joensen, Hjørdis Reinert and For further information: Elsebeth Tvenstrup Jensen (etj@ssi.dk) DANMAP 3 37

38 5. ANTIMICROBIAL CONSUMPTION IN HUMANS Figure. consumption of antimicrobial agents used prophylaxis and treatment of urinary tract infections in primary healthcare (7-3) DID 3.5 DANMAP Ciprofloxacin Methenamin. Nitrofurantion Pivmecillinam.5 Sulfamethizol Trimethoprim Table. Total consumption of antimicrobials and consumption of cefuroxime, ciprofloxacin, meropenem, and piperacillin w. enzyme inhibitor at LS 7-3, shown as DBD (DDD/ bed-days) and as %DANMAP of 73 level Cefuroxime Ciprofloxacin Meropenem Piperacillin + inhibitor Total (.) (9.) (.8) (3.) (37.5) (4.9) (37.4) (.) (396.4) (339.3) (7.) (53.6) (5.8) (44.5) (.) (83.7) (.) (8.4) (35.) (4.9) (75.) (.) (37.) (8.4) (99.5) (4.) (8.) (59.) (.) (3.4) (3.5) (4.) (8.3) (5.7) (.3) Figure. consumption of cefuroxime, ciprofloxacin, meropenem, and piperacillin with enzyme inhibitor at the LS hospital and in Danish hospitals (7-3) DANMAP 3 35 DDD/ bed-days 3 5 Piperacillin + enzyme inhibitor Meropenem 5 Ciprofloxacin Cefuroxime FaroeIslands (LS) 38 DANMAP Denmark (all hospitals)

39 Textbox 3 ANTIMICROBIAL CONSUMPTION IN HUMANS 5. The Danish council of Ethics statement on the use of antibiotics Background: In January 4, The Danish Council of Ethics published a set of recommendations on the et antibiotics in a Danish context. The recommendations set out to prescribe action as a response to two specific ethical dilemmas. These are below. Despite many years of efforts to the contrary, antibiotics use and antimicrobial resistance (AMR) problems in a steady increase in Denmark and elsewhere. In the view of the Danish Council of Ethics, the presence o dilemmas associated with combating AMR is likely to be an important factor behind the challenges encoun Ethical dilemmas Dilemma : Rationing antibiotics The need to prescribe human antibiotics reflects the general living condition of a population, including hyg conditions in homes, in public spaces and at workplaces, day-care facilities and hospitals. The consumption in farming is largely a result of the production methods chosen. Owing to the great mobility of people, animals and foodstuffs, the effect of a cautious approach to using an the local resistance situation is uncertain. However, the extent of resistance is inextricably bound up with the consumption of antibiotics. Even well- j promotes resistance development. Within a fairly short span of years, increasing resistance to antibiotics w patients access to potent and effective antibiotics. It is crucial, therefore, to exercise reticence regarding the use of antibiotics, in spite of the fact that greater involve a greater risk for patients, animals and livestock. Examples of obviously unjustifiable use - that is to antibiotics are prescribed even though the treatment is known to be ineffective, and where reticence is the to have no impact - are probably rare. How should the doctor, veterinarian and farmer balance consideration for the patient, animal or herd with r future patients? Dilemma : Preventing infection Being a carrier of antibiotic-resistant bacteria entails a risk of infection. Infection can entail medical risks an strains and stresses. Those not infected should be protected against infection. That may call for the use of isolation, restraining Those not infected should also have the option of deciding which infection risks they want to expose thems advocates openness around infection sources, e.g. by informing the public about infected animal herds or h to report knowledge of infected individuals. For carriers of resistant bacteria, however, such initiatives can be stigmatizing, offensive and involve interf individual s freedom. Furthermore, experience has shown that carrier status, as a consequence of stigma, h concealed to a greater extent, thus causing the risk of infection to rise. Recommendations In guidelines the authorities should acknowledge the ethical dilemmas in which doctors, veterinarians, f and ordinary citizens are placed as a result of endeavours to ration antibiotics and prevent the spread o resistant bacteria The authorities efforts to combat antibiotic resistance should be intensified in the health sector with a v reducing the problem both nationally and internationally. Particular heed should be paid to specialist gu for the use of antibiotics and their implementation DANMAP 3 39

40 5. ANTIMICROBIAL CONSUMPTION IN HUMANS The authorities should minimize the stigmatization, isolation and discrimination that may attach to being antibiotic-resistant bacteria. First and foremost, the risk of infection should be limited by improving gene Use of antibiotics to be on the safe side or to reduce discomfort should be avoided in interaction betwe and patients The authorities should redouble their efforts to combat antibiotic consumption in farming. This can be do example, by promoting and demanding stricter criteria for healthier forms of production and by limiting t herd medication Nationally and internationally, the authorities should work to promote consumers scope for choosing pro made with limited consumption of antibiotics Statement, working papers, an interview and more (in Danish): On behalf of the Danish Council of Ethics, professor Gorm Gr chairman of the antibiotics working group 4 DANMAP 3

41 ANTIMICROBIAL CONSUMPTION IN HUMANS Primary healthcare 5.3. Measures at treated patient level In 3, each treated patient received.3 DDDs (Table 5 an increase of 3.4% compared to. The number of pat 5.3. Total consumption in primary healthcare treated was, however,.3% less (Table A5. and A5.3 in w In 3, the consumption of antimicrobial agents in primary annex), which means that in 3, more DDDs were presc healthcare was.% higher than that observed in (Table for each package. During the past decade, the number of 5.) and thus reversed a large part of the positive decline seen in per package has increased by %, reaching the highest l. During the past decade, the consumption of antimicrobial in.6 3.DID Similarly, the number of DDDs per patient has als agents in Danish primary healthcare has increased by increased significantly since 4 while the number of pac (9%). per patient has remained relatively constant (Table 5.3)..39was The proportion of broad-spectrum agents in 3 Considering the leading antimicrobial groups, each treated DID (5.7%) higher than in (Figure 5.4). Since 4, the patient received.8.6 DDDs in.4.7 packages w proportion of broad-spectrum agents has increased by 7%. the exception of tetracyclines (Table 5.3). For tetracyclines both the number of DDDs per patient and DDDs per packa As observed in previous years, beta-lactamase sensitive are significantly higher than any other antimicrobial group penicillins represented the largest therapeutic groupprimarily of due to the fact that tetracyclines are commonly u antimicrobial agents consumed (8%) and penicillins forin acne treatment; there higher dosages are given for lon general accounted for 64% of the total consumption periods. in 3 (Figure 5.5). For the other therapeutic groups, several changes in consumption were observed between and 3 (Table When examining three different indicators at the treated 5.). Especially noteworthy are an % increase in tetracyclines patient level (Figure 5.7), a marked increase is observed fo (see Textbox 4) and a 6% increase in combination penicillins. DIDs while the number of packages and treated patients h The consumption of macrolides was % lower in 3. In4 relatively constant since other words, the compared to, upholding the decrease observedremained since dosage prescribed for each patient and in each package h. (Figure 5.6) increased significantly during the past years. The reasons these changes are as yet. unclear Table 5.. consumption of antimicrobial agents for systemic use in primary healthcare (DDD/ inhabitant-days), Denma DANMAP 3 Year ATC Therapeutic group (a) group JAA Tetracyclines JCA Penicillins with extended spectrum JCE Beta-lactamase sensitive penicillins JCF Beta-lactamase resistant penicillins Combinations of penicillins, including betajcr lactamase inhibitors JD Cephalosporins and other β-lactam. antibiotics JEA Trimethoprim and derivatives JEB Short-acting sulfonamides Combinations of sulfonamides and JEE trimethoprim, including derivatives JFA Macrolides JFF Lincosamides JGB Aminoglycosides JMA Fluoroquinolones JXA Glycopeptides JXB Polymyxins JXC Steroid antibacterials (fusidic acid) JXD Imidazole derivatives JXE Nitrofuran derivatives (nitrofurantoin) JXX Other antibacterials (methenamine.3 >99%) J Antibacterial agents for systemic use (total) a) From the 3 edition of the Anatomical Therapeutic Chemical (ATC) classification system DANMAP 3 4

42 5. ANTIMICROBIAL CONSUMPTION IN HUMANS Table 5.3. Number of DDDs and packages per treated patient among leading groups of antimicrobial agents in primary he Denmark DANMAP 3 ATC (a) group Therapeutic group Year Indicator DDDs / patient Packages / patient DDDs / package JCA DDDs / patient Penicillins with extended Packages / patient spectrum DDDs / package DDDs / patient Beta-lactamase sensitive JCE Packages / patient penicillins DDDs / package DDDs / patient Beta-lactamase resistant JCF Packages / patient penicillins DDDs / package DDDs / patient Combinations of JCR penicillins, incl. beta- Packages / patient lactamase inhibitors DDDs / package DDDs / patient JFA Macrolides Packages / patient DDDs / package DDDs / patient JMA Fluoroquinolones Packages / patient DDDs / package DDDs / patient Antibacterial agents for Packages / patient J systemic use (total) DDDs / package a) From the 3 edition of the Anatomical Therapeutic Chemical (ATC) classification system JAA Tetracyclines Figure 5.4. consumption of antimicrobial agents (J) in primary healthcare by narrow-spectrum and broad-spectrum agen Denmark DANMAP DDD/ inhabitant-days Narrow-spectrum Broad-spectrum Note: Narrow-spectrum includes: beta-lactamase sensitive penicillins, beta-lactamase resistant penicillins, trimethoprim, macrolides, lincosamides, glycopeptides, fusidic acid, imidazol derivatives, nitrofuran derivatives, and other antibiotics Broad-spectrum includes: tetracyclines, penicillins with extended spectrum, combinations of penicillins incl. beta-lactam cephalosporins and related substances, combinations of sulfonamides and trimethoprim, aminoglycosides, fluoroquinolone 4 DANMAP 3

43 ANTIMICROBIAL CONSUMPTION IN HUMANS 5. Figure 5.5. Distribution of the total consumption of antimicrobial agents in primary healthcare, Denmark DANMAP 3 Tetracyclines (JAA), % Beta-lactamase resistant penicillins (JCF), 8% Macrolides, lincosamides and streptogramins (JF), % Comb. of penicillins, incl. beta-lactamase inhib. (JCR), 7% Sulfonamides and trimethoprim (JE), 5% Penicillins with extended spectrum (JCA), % Fluoroquinolones (JMA), 3% Other antibacterials (JD,G,X), 5% Beta-lactamase sensitive penicillins (JCE), 8% Note: Bold highlights indicate broad-spectrum antimicrobial agents Figure 5.6. consumption of leading antimicrobial groups for systemic use in primary healthcare, Denmark 6 DANMAP 3 Beta-lactam. sens. penicillins (JCE) DDD/ inhabitant-days 5 Penicillins with extend. spectrum (JCA) 4 Macrolides (JFA) Tetracyclines (JAA) 3 Beta-lactam. resis. penicillins (JCF) Fluoroquinolones (JMA) Combinations of penicillins, including beta-lactamase inhibitors (JCR) Figure 5.7. Indicators of antimicrobial consumption (J) in primary healthcare, Denmark DANMAP 3 3% Index: 4 = % DDD/ inh./day % Packages/ inh./year % Treated pat./ inh./year % 9% DANMAP 3 43

44 5. ANTIMICROBIAL CONSUMPTION IN HUMANS Tetracyclines Combination penicillins (primarily amoxicillin/clavulanic acid) are currently advocated for broad treatment of From to 3, the overall consumption of tetracyclines infections, including patients with exacerbation increased by.4%, primarily due to increase in userespiratory of chronic obstructive pulmonary disease (COPD). The highes doxycycline which is now the most used substance (Table 5.). consumption of these drugs is observed in people aged 65 Since 4, the consumption of tetracyclines has increased older, suggesting that the increase may be related to an in by 68%, primarily driven by significant increases in both rate of diagnosis and treatment of respiratory infections an doxycycline and lymecycline and a more conservative increase COPD 4in this age group. Further, the increase may also par in tetracycline consumption (Figure 5.8). See also Textbox about tetracycline prescription patterns in Denmark.be explained by an increasing tendency of GPs to prescrib sweet-tasting amoxicillin mixture for respiratory infections children rather than benzylpenicillin (beta-lactamase sens Penicillins penicillins). The overall consumption of penicillins in 3 was 3% higher than that observed in (Table 5.), continuing the generally increasing trend observed during the past decade with an Macrolides From 3, the consumption of macrolides decrease increased consumption of % from 4 3. by % (Table 5.), and this decrease was apparent for all ).5. substances (Figure The high consumption of macrolides For individual penicillin groups, the consumption of narrow in and may be explained by two epidemic wave spectrum penicillins (i.e. phenoxymethylpenicillin) continued pneumoniae infection (DANMAP & ) to decrease whereas increases in consumption were Mycoplasma observed and the return to a generally lower consumption in a for combination penicillins (6%), beta-lactamase resistant onwards is most likely related to the absence of significant penicillins (7.4%) and penicillins with extended spectrum disease outbreaks (DANMAP ). (.4%). The consumption of these three groups in particular has also increased significantly over the past decade replacing the use of narrow spectrum penicillins (Table 5.). From 4-3, the consumption of roxithromycin, clarithromycin and azithromycin increased (Figure 5.). T consumption of erythromycin decreased substantially, mo At the substance-level in 3, phenoxymethylpenicillin likely in response to changes in national guidelines which continues to be the most commonly consumed penicillin 4 substituted the first-choice macrolide in primary care despite decreasing usage. The consumption of flucloxacillin, erythromycin to roxithromycin and subsequently also amoxicillin and enzyme inhibitor, and pivmecillinam from increased clarithromycin in 7. (Figure 5.9). The consumption of ampicillin decreased by 8%. Figure 5.8. consumption of tetracyclines in primary healthcare, Denmark DANMAP 3 DDD/ inhabitant-days..9 Doxycycline (JAA).6 Lymecycline (JAA4).3 Tetracycline (JAA7) DANMAP

45 ANTIMICROBIAL CONSUMPTION IN HUMANS 5. Figure 5.9. consumption of leading penicillins in primary healthcare, Denmark DANMAP Pivampicillin (JCA) DDD/ inhabitant-days Amoxicillin (JCA4).5 5. Pivmecillinam (JCA8) 4.5. Phenoxymethylpenicillin (JCE).5 Dicloxacillin (JCF). Amoxicillin and enzyme inhibitor (JCR).5 Flucloxacillin (JCF5) Figure 5.. consumption of macrolides in primary healthcare, Denmark DANMAP 3 DDD/ inhabitant-days. Erythromycin (JFA).9 Roxithromycin (JFA6).6 Clarithromycin (JFA9).3 Azithromycin (JFA) DANMAP 3 45

46 5. Textbox 4 ANTIMICROBIAL CONSUMPTION IN HUMANS Temporal and geographical variation in tetracycline prescription patterns in Denmark Background: In Denmark, tetracyclines are used as treatment for acne vulgaris in young adults. In DANMAP shown that a large increase in the consumption of antimicrobial agents in primary healthcare was driven by with particular increases for tetracyclines. In this age group, tetracyclines are prescribed almost as often as p the level of consumption is steadily increasing [DANMAP ]. In the present study, Danish tetracycline prescriptions were investigated more closely with particular focus o through time (5-3) and at the geographical level for different age groups. Methods: Data on consumption of tetracyclines (DID, number of persons treated and number of prescription to 3, the type (GP versus dermatologist) and geographical location of the prescriber were obtained from D and Medicinal Product Statistics at Statens Serum Institut. Data were analysed by descriptive statistics and tw using the STATA software. (Statacorp., Lakeway, TX, USA). Results: From 5 to 3, the consumption (DID) of tetracyclines for all ages increased by 54% with large observed for 4 year olds (86%) and 5 9 year olds (58%). When adjusted for population increases, app more persons in these age groups were treated with tetracyclines in 3 than in 5. A large difference at the municipality level (kommuner) in tetracycline consumption was seen for 9 yea in six Danish municipalities had a consumption which was 7 to % higher than the national average (Fig lowest consuming municipalities were 7 to 4% below average. Discussion: The current results show that that the observed increases in tetracycline consumption are main Danish adolescents. In the two age groups -4 and 5-9 years, an extra 5.4 persons were treated with 5 to 3. We further show that the majority of prescriptions, presumably for acne [DANMAP ], are p GPs rather than dermatologists, highlighting the ease of access to GPs in comparison to dermatologists wher needed. The geographical pattern of tetracycline consumption is highly varied with some municipalities showing a co consumption in the adolescent age groups and other municipalities having a below-average consumption thr Resistance to broad-spectrum antimicrobial agents is an important problem in Denmark. There is currently a to firstly investigate the extent of tetracycline resistance in bacteria from young adults and secondly to estab treatment guidelines for adolescents presenting to a GP with acne problems. Katrin G. Kuhn For further information: Katrin G. Kuhn (kuh@ssi.dk) Figure. The consumption of (DDD/ inhabitants) of tetracyclines in 3-9 year olds in Danish municipalities DANMAP 3 46 DANMAP 3

47 ANTIMICROBIAL CONSUMPTION IN HUMANS Hospital care The observed increases in combination penicillins is most likely attributable to a change in empiric treatment from cephalosporins to piperacillin-tazobactam for severe 5.4. Introduction Hospital care includes all hospitals (i.e. rehabilitationinfections centres, and an increasing usage of amoxycillin-clavulan acid for treatment of acute exacerbation of chronic obstru hospices, private-, psychiatric-, specialized-, and somatic pulmonary diseases (COPD). hospitals). Somatic hospitals account for approximately 97% of the antimicrobial consumption within hospital care. As specialized hospitals (psychiatric hospitals, hospices and Other measures of somatic hospital rehabilitation centres) contribute a large proportion of bed-days consumption and admissions but only a small proportion of antimicrobial DDD per admissions (DAD) consumption, the antimicrobial consumption for hospital care of the observed changes in the number of hospita Because in Denmark is related only to bed-days and admissions in bed-days over time, the consumption of antimicrobial age somatic hospitals. in Danish hospitals may also be measured in relation to admissions (i.e. DDD per admissions, DAD). The consumption of antimicrobial agents in hospital care is presented as DDD per occupied bed-dayswhen (DBD)expressed as DAD, the total consumption of antimic and as DDD per admissions (DAD) to account foragents hospital in somatic hospitals was.6% higher in 3 comp activity. Further, data are also presented as DID purely enable to to (Table 5.5). During the past decade, DAD increase comparison with primary healthcare. by 9.3%; an increase primarily driven by a higher numbe DDDs but counterbalanced by an increase in the number o During the past decade, the hospitalization pattern in hospital Denmark admissions. has changed notably: more people are admitted to somatic hospitals but the average length of stay has been shortened With respect to individual antimicrobial groups, increases (Figure 5., Table A5.4 in web annex) and outpatient were observed for: combination penicillins, combinations treatment has increased significantly. Selection pressure for the of sulfonamide and trimethoprim, beta-lactamase resistan emergence of antimicrobial resistance increases withpenicillins increasingand carbapenems. As observed in, the hospital activity (admissions) and thus the selection consumption pressure of nd generation cephalosporins and has increased considerably from 4 to 3. fluoroquinolones also decreased in 3 by 3% and.% respectively (Table 5.5). The same trends were seen when 5.4. Somatic hospitals DDD per occupied consumption was expressed as DBD (Table 5.4). bed-days (DBD) DDD per, inhabitants per day (DID) In 3, the consumption of antimicrobial agents in somatic The of antimicrobial agents in somatic hospit hospitals was.4% higher than in (Table 5.4). In theconsumption past higher in 3 compared to. In the past de decade the consumption has increased by 66%. Thiswas was.5% caused the DIDs by a combination of an increase in DDDs and a decrease in consumed in hospitals have increased by 3% (Ta A5.5 in web the number of hospital bed-days. This was particularly seen for annex). During the same time, broad-spectrum %, 7 comprising 68% of the total broad spectrum agents which increased by 4%. agents have increasedby consumption in 3 compared to 5% in 4 (Figure 5. Extended spectrum penicillins represented the largest group Katrin Gaardbo Kuhn and Maja Laurse of antimicrobial agents consumed and penicillins in general accounted for 5% of the total consumption (Figure 5.). Cephalosporins (4%) and fluoroquinolones (%) were also among the most commonly consumed antimicrobial agents in hospital care. From to 3, a higher consumption was observed for combinations of sulfonamide and trimethoprim (6%), combination penicillins (4%), beta-lactamase resistant penicillins (8.4%), carbapenems (4.%) and penicillins with extended spectrum (.%). A lower consumption was observed for nd generation cephalosporins (3%), macrolides (4.8%), tetracyclines (3.8%) and fluoroquinolones (.5%) (Table 5.4). The changes in leading groups of antimicrobial agents used in somatic hospitals during 4 3 are shown in Figure 5.3. Large increases were observed for combinations of penicillins (5%), carbapenems (37%) and fluoroquinolones (%). The consumption of beta-lactamase sensitive penicillins, however, decreased 6% during the past decade. DANMAP 3 47

48 5. ANTIMICROBIAL CONSUMPTION IN HUMANS Figure 5.. Number of bed-days and admissions in somatic hospitals, Denmark DANMAP No. admissions (mill.) No. bed-days (mill.) Bed-days 3 Admissions Figure 5.. Distribution of the total consumption of antimicrobial agents in somatic hospitals, Denmark DANMAP 3 Beta-lactamase sensitive penicillins (JCE), % Beta-lactamase resistant penicillins (JCF), % Fluoroquinolones (JMA), % Comb. of penicillins, incl. beta-lactamase inhib. (JCR), 4% Carbapenems (JDH), 4% Penicillins with extended spectrum (JCA), 6% Macrolides, lincosamides and streptogramins (JF), 4% Sulfonamides and trimethoprim (JE), 5% Cephalosporins (JDB,DC,DD), 4% Other antibacterials (JA,DF,X), 9% Note: Bold highlights indicate broad-spectrum antimicrobial agents 48 DANMAP 3 Aminoglycosides (JG), %

49 ANTIMICROBIAL CONSUMPTION IN HUMANS 5. Figure 5.3. Total somatic hospital consumption (DBD) by leading groups of antimicrobial agents (J), Denmark DANMAP 3 8 Combinations of penicillins. incl. beta-lactamase inhibitors (JCR) Cephalosporins (JDB, DC, DD) DDD/ occupied bed-days 7 6 Carbapenems (JDH) 5 Fluoroquinolones (JMA) 4 Aminoglycosides (JGB) 3 Macrolides (JFA) Beta-lactamase sensitive penicillins (JCE) Penicillins with extended spectrum (JCA) Figure 5.4. consumption of antimicrobial agents (J) in hospital care by narrow-spectrum and broad-spectrum agents, D DDD/ inhabitant-days..38 DANMAP Narrow-spectrum Broad-spectrum Note: Narrow-spectrum antibiotics includes: beta-lactamase sensitive penicillins, first-generation cephalosporins, beta-la penicillins, monobactams, trimethoprim, sulfonamides, macrolides, lincosamides, glycopeptides, fusidic acid, imidazol deri derivatives, and other antibiotics Broad-spectrum includes: tetracyclines, penicillins with extended spectrum, combinations of penicillins incl. beta-lactam second-generation cephalosporins, third-generation cephalosporins, carbapenems, combinations of sulfonamides and trim aminoglycosides, fluoroquinolones, and polymyxins DANMAP 3 49

50 5. ANTIMICROBIAL CONSUMPTION IN HUMANS Table 5.4. consumption of antimicrobial agents for systemic use in somatic hospitals (DDD/ occupied bed-days), Denma DANMAP 3 (a) ATC group Therapeutic group Year Tetracyclines Penicillins with extended.5 spectrum Beta-lactamase sensitive. penicillins Beta-lactamase resistant6.78 penicillins Combinations of penicillins. incl. betajcr lactamase inhibitors JDB First-generation cephalosporins JDC Second-generation cephalosporins JDD Third-generation cephalosporins JDF Monobactams JDH Carbapenems JEA Trimethoprim and derivatives JEB Short-acting sulfonamides Combinations of sulfonamides and JEE trimethoprim. incl. derivatives JFA Macrolides JFF Lincosamides JGB Aminoglycosides JMA Fluoroquinolones JXA Glycopeptides JXB Polymyxins JXC Steroid antibacterials (fusidic. acid) JXD Imidazole derivatives JXE Nitrofuran derivatives (nitrofurantoin) JXX5 Methenamine JXX8 Linezolid JXX9 Daptomycin Antibacterial agents for systemic use J (total) a) From the 3 edition of the Anatomical Therapeutic Chemical (ATC) classification system JAA JCA JCE JCF 5 DANMAP 3

51 ANTIMICROBIAL CONSUMPTION IN HUMANS 5. Table 5.5. consumption of antimicrobial agents for systemic use in somatic hospitals (DDD/ admitted patients), Denma DANMAP 3 (a) ATC group Therapeutic group JAA JCA JCE JCF JCR JDB JDC JDD JDF JDH JEA JEB JEE JFA JFF JGB JMA JXA JXB JXC JXD JXE JXX5 JXX8 JXX9 J Year (b) Tetracyclines Penicillins with extended5. spectrum Beta-lactamase sensitive54.53 penicillins Beta-lactamase resistant3.77 penicillins Comb. of penicillins. incl. beta lactamase inhibitors First-generation cephalosporins Second-generation cephalosporins Third-generation cephalosporins Monobactams Carbapenems Trimethoprim and derivatives Short-acting sulfonamides Comb. of sulfonamides and trimethoprim. incl. derivatives Macrolides Lincosamides Aminoglycosides Fluoroquinolones Glycopeptides Polymyxins Steroid antibacterials (fusidic.acid) Imidazole derivatives Nitrofuran derivatives (nitrofurantoin) Methenamine Linezolid Daptomycin Antibacterial agents for systemic use (total) a) From the 3 edition of the Anatomical Therapeutic Chemical (ATC) classification system b) The number of admissions was affectedly low in 8 due to a major hospital strike DANMAP 3 5

52 5. ANTIMICROBIAL CONSUMPTION IN HUMANS 5 DANMAP 3

53 6 RESISTANcE IN ZOONOTIc BAcTERIA DANMAP 3 53

54 6. RESISTANCE IN ZOONOTIC BACTERIA 6. Resistance in zoonotic bacteria In Denmark and the rest of European Union, S. Enteritidis Zoonoses are infectious diseases that can be transmitted S. Typhimurium are the serovars most frequently found between animals and humans, either through direct and contact to be associated with human illness. Human cases caused with animals or indirectly by contaminated food. Zoonotic bacteria, suchsalmonella as andcampylobacter, can develop by S. Enteritidis are most commonly associated with the consumption S. where resistance towards antimicrobial agents as a result of treatment of contaminated eggs or poultry meat, Typhimurium cases are mostly associated with the consum of both animals and humans, which subsequently may lead of contaminated pork, beef or poultry meat. to limited treatment possibilities or even treatment failure of human infectious diseases. Especially successful multisalmonella resistant clones have spread extensively during Forthe Salmonella, DANMAP 3 includes isolates from broiler, last years, resulting in a very complex relationship between layer and cattle farms infected during 3 as well as isola antimicrobial use and levels of resistance. from Danish and imported broiler meat, turkey meat, beef and pork collected as part of national surveillance and con programmes. From pigs, Salmonella isolates recovered from A more detailed description of the trends and sources of sow herds as well as multiplier and breeding herds as part zoonoses in Denmark and of national surveillance and control thesalmonella control programme are included. In addition, programmes can be found in the Annual Report on Zoonoses caecum samples from healthy pigs were collected at the in Denmark 3 [ slaughterhouses and cultured Salmonella. for Isolates from all reported human cases are included. Only one isolate pe 6. Salmonella farm, meat sample or human case was included, and data presented in the report where a sufficient number of isolat Salmonella is the second most important zoonotic bacterial were obtained (>5) from a given source. For details on pathogen in Denmark and can have a severe impactmethodology on both see Chapter 9, Materials and Methods. animal production and human health. In DANMAP 3, we primarily present resistance S.among Typhimurium. During the last ten years, the numbers of po flocks and meat infected or contaminated with S. Enteritid has decreased, and therefore resistance S.Enteritidis in will not be presented in this report. The occurrence of resistance a spp. from pigs and Danish pork is presented (Tab Table 6.. Resistance (%) among Salmonella Spp. from pigssalmonella 6.). and pork, Denmark DANMAP 3 Pigs Pork Antimicrobial agent % Danish Tetracycline Chloramphenicol Florfenicol Ampicillin Cefotaxime Ceftiofur Trimethoprim Sulfonamide Streptomycin Gentamicin Neomycin Apramycin Ciprofloxacin Nalidixic acid Colistin Spectinomycin Fully sensitive Multi-resistant In DANMAP, S. Typhimurium includes the monophasic % variants with antigenic formulas S.4, [5],:i:, as recommended by the European Food Safety Authority [EFSA journal 8(): 86]. In the text, generic S. Typhimurium indicates results only covering isolates of the non-monophasic varia Since, routine analyses S. Typhimurium of phage types are no longer performed in Denmark. MIC distributions for S. Typhimurium from pigs, pork and humans, as well assalmonella for spp. from pigs and Danish pork in 3 are presented in the web annex (Tables A6. A6.5). An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the test panel, and multiresistant if resistant to three or more of the ten antimicrob classes (see Table.3). 6.. Salmonella spp. in pigs and domestic produced pork In 3, S.Typhimurium (including the monophasic variants) ands. Derby were the most common serotypes isolated fro Danish pigs (44% and 43%, respectively) and pork (46% a Number of isolates %, respectively). In general, the serotype distribution in Danish pork has been similar to the distribution among Note: An isolate is considered fully sensitive if susceptible the to all antimicrobial agents included in the test panel and multi-resistant isolates iffrom pigs. resistant to three or more of the ten antimicrobial classes (see Table 9.3) Note that the isolates from pigs in Table 6. and 6. includ isolates from pen faecal samplings from the national contr programme as well as isolates from the sampling of health 54 DANMAP 3

55 RESISTANCE IN ZOONOTIC BACTERIA 6. S. resistant, at slaughter. Thus the serotype distributions differ slightly fromthe prevalence of pig herds with multi-resistant Typhimurium amounts to approximately 7% compared to 3 the ones presented for the slaughter pig herds. in Textbox 5 in. Salmonella Among all the isolates from Danish pigs (n = 5), In Danish we observed high levels (range: 33% - 47%) of resistance to pork (n = 68), 53% of the isolates were of the monophasic variants and high levels (4%) of ASSuT resist ampicillin, streptomycin, sulfonamide, and tetracycline (Table observed. AsS.inTyphimurium isolates from pigs, the 6.). Co-resistance of these four antimicrobial agentswere is often occurrence of monophasic variants as well as multi-resista called the ASSuT resistance-profile, also when resistant to remained at the same level as in, but has generally additional antimicrobial agents. Resistance to tetracycline increased from to 3, whereas resistance to increased the other since 9, accompanied with a reduced occurr of isolates fully sensitive to all the antimicrobial agents inc tested antimicrobial agents was similar to levels reported in in the test panel (Figure 6.3). (Figure 6.). Salmonella The occurrence of resistance in spp. from Danish For the isolates from pigs and Danish pork, resistance to th pork resembles the occurrences in the isolates from other pigs tested antimicrobial agents was similar to levels rep. in, except for an increase in resistance to trimethopr ((Figure 6.) amongs. Typhimurium in pigs (from 6% to 3%). Levels of S. Typhimurium isolates were comparable among The majority of the S. Derby isolates were fully sensitive,resistance and from pigs and Danish pork (Table 6.). the overall occurrences of resistance Salmonella among spp. in pigs and Danish pork were lower than observed among the S. Typhimurium isolates from Danish pigs and S. Typhimurium isolates only. Even though S. Derby is very None of the pork were resistant to cephalosporins (ceftiofur, cefotaxim S.Derby cases (N=) common among pigs, only few human. quinolones (ciprofloxacin, nalidixic acid) or colistin (Table were reported in Denmark in 3 [ 6.). Only in, a few DANMAP isolates from pigs were Overall, it was estimated that in 3 multi-resistant Salmonella resistant to ciprofloxacin, resulting in a decreasing trend o was present in 9% of the tested pigs Salmonella (4% positive the last five years (Figure 6.). Among the ten EU Member S. Typhimurium in from pork in Salmonella pigs) and on.5% of the tested pig carcasses (.3% States reporting resistance, most Member States reported no resistance to third positive carcasses, Textbox 5 and Table 6.). generation cephalosporins (cefotaxime), whereas quinolon None of the Salmonella spp. isolates from pigs or pork resistance was more common ranging up to 9% of the iso [EUor Summary report on AMR ]. were resistant to cephalosporins (ceftiofur, cefotaxime) quinolones (ciprofloxacin, nalidixic acid). S.Typhimurium It is also important to note that the occurrence of isolates fully sensitive to all included antimicrobial agents 6.. S. Typhimurium in,pigs domestic been decreasing since 9, this is the case for isolates bo produced and imported pork from pigs and Danish pork (Figure 6.3). Among the S. generic S. Typhimurium isolated from pigs (n = 48) had verytyphimurium high isolates from pigs, 4% of the isolates were f levels of resistance to ampicillin (6%), streptomycinsensitive (66%), isolates, a level comparable to previous years, w sulfonamide (66%), and tetracycline (7%, Table 6.), andof the monophasic isolates from pigs were fully sensi none the occurrence of resistance for all four antimicrobialinagents 3. increased over the last five years (Figure 6.). The occurrence of ASSuT resistance as well as multi-resistance in general was few S. Typhimurium isolates from imported pork Relatively comparable with the levels in ; however ASSuT (n resistance = ) are available (Table 6.), however higher levels of amongs.typhimurium in pigs has been increasing since 9, to tetracycline, chloramphenicol, flourfenicol an resistance partly due to an increase in the prevalence of the monophasic spectinomycin was observed compared to isolates from Da variants ofs. Typhimurium often carrying the ASSuT pork. resistance-profile (Figure 6.3) Salmonella in humans S. 4,[5],:i:- is The increased occurrence of monophasic In 3, Salmonella continued to be the second most frequent not an isolated Danish phenomenon, but related to new cause of bacterial intestinal infections in Denmark. A total pandemic strains Salmonella of in Europe [Hopkins et al.,36 human laboratory-confirmed cases of salmonellosis, Eurosurveillance 3;]. As the changes in occurrences reported (.3 cases per, inhabitants) [Annual rep of antimicrobial resistance among Salmonella are highly Zoonoses in Denmark 3]. The most common serotype -resistant influenced by the spread of such successful multi S. Enteritidis and a total of 346 confirmed S. Enteritidis cases clones, the relationship between antimicrobial use and levels of were reported (6. cases per, inhabitants), of whic resistance becomes very complex. 79% were associated with travel outside Denmark especia Turkey. This year, onlys. Typhimurium isolates from humans S.Typhimurium variants constituted In 3, the monophasic were systematically susceptibility tested. 5% of the total numbers.oftyphimurium isolates from pigs, representing 7% of the multi-resistant isolates. This is As in previous years, SSI collected information on travel comparable to the occurrence in. history through phone interviews. Patients were asked abo the date of disease onset and whether they had travelled The prevalences.of Typhimurium infected slaughter pig within herds a seven-day period prior to the onset of the disease was.6% in 3 [Annual report on Zoonoses in Denmark Furthermore, patients who had been abroad were asked ab 3], and as 64% ofs.the Typhimurium isolates were multitheir destinations. Cases were categorised as domesticall DANMAP 3 55

56 6. RESISTANCE IN ZOONOTIC BACTERIA were reported several domestic foodborne outbreak acquired if the patients had not travelled within thethere last week withs.typhimurium, and compared with the proportio prior to the onset of the disease. Patients were categorised as of outbreak related cases (n = 69) increased from 3% to of unknown origin if no telephone interview was conducted 3, only 3% of isolates from the outbreak related cas and travel information had not been reported to the In general wasfor of68% the multi-resistant monophasic variants compared w practitioners. In 3, travel information was obtained 9% of all reported Salmonella cases [Annual report on Zoonoses inin. Denmark 3]. Among the S. Typhimurium isolates from domestic as well asreported travel-associated cases, we observed very high levels o All human cases associated with a detected outbreak resistance to ampicillin, streptomycin, sulfonamide, and in the Annual Report on Zoonoses in Denmark in 3 were considered outbreak-related and all other domestictetracycline. cases were High levels of ASSuT resistance occurred amo isolates from sporadic domestic cases (43%, n = 6) as w considered sporadic domestic in this report.. n = 5) and travel-associated cases (47%, 6..4 Salmonella Typhimurium in humans Occurrences of the resistance to the other antimicrobial ag S. Typhimurium was for the first time since 8 onlywere the similar to most levels reported in for the domes sporadic second most common serotype among the human cases (337cases and similar for all antimicrobial agents for t. travel-associated cases cases), and isolates with valid results from susceptibility testing of all antimicrobial agents included in the test panel (n = 39) There were no significant changes in levels of resistance fr were included. to 3 for either domestic sporadic or travel-associa cases. The increasing trend in resistance to ampicillin, Among the reported human S.Typhimurium isolates included streptomycin, sulfonamide and tetracycline, observed prev in DANMAP, 6% of the cases were categorised as travelyears discontinued, and for both domestic sporadic cases a associated whereas 33% and % most likely had acquired as among the travel associated cases (Figure 6.). Am their infection in Denmark as sporadic incidences or well as part thethe domestic sporadic cases, fluoroquinolone (ciprofloxacin of detected outbreaks, respectively (Table 6.). Among resistance has decreased over the last five years. In gener cases where the origin of infection was unknown (9%), the levels of resistance for the domestic cases were similar the occurrence of resistance falls within the ranges of both to those of Danish pork, except for the low occurrence of. domestic cases and travel-associated cases fluoroquinolone resistance, a resistance not observed amo the isolates from Danish pork. (a) Table 6.. Resistance (%) among Salmonella Typhimurium from pigs, Danish pork and human(b),cases Denmark Pigs Pork Antimicrobial agent % Danish % Imported % Tetracycline Chloramphenicol Florfenicol Ampicillin Cefotaxime Ceftiofur Trimethoprim Sulfonamide Streptomycin Gentamicin Neomycin Apramycin Ciprofloxacin Nalidixic acid Colistin Spectinomycin Fully sensitive Multi-resistant Domestic sporadic % Number of isolates DANMAP 3 Human Domestic Unknown Travel abroad outbreak origin % % % Note: An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the test panel and multi-r three or more of the ten antimicrobial classes (see Table 9.3) a) Include isolates verified as monophasic S. variants Typhimurium of with antigenic formulas S. 4,[5],:i:b) An isolate was categorised as domestic sporadic if the patient did not travel outside Denmark one week prior to the on was not reported as being part of an outbreak 56 DANMAP 3

57 RESISTANCE IN ZOONOTIC BACTERIA 6. % resistant isolates Since 9, the proportion of multi-resistant isolatesfigure from 6.. Resistance (%) in Salmonella Spp. in pigs and Danish pork, Denmark sporadic domestic cases has increased (no significant change DANMAP 3 from to 3), probably as a consequence of an increasing Pork Pigs occurrence of multi-resistant monophasic variants S. of 5 Danish Typhimurium (Figure 6.3). In 3, the monophasic variants represented 44% of all domestic sporadic isolates and 7%45of all multi-resistant isolates. Also in line with this, the proportion 4 of fully sensitive S.Typhimurium isolates of domestic sporadic origin (8% in 3) has decreased over the last five years35(no significant change from to 3). 3 Among thes. Typhimurium from travel-associated cases, 5 resistance to chloramphenicol, ciprofloxacin, florfenicol and nalidixic acid was more frequent than among isolates of domestic sporadic origin, whereas the level of resistance to5the other agents included in the panel as well as the proportion of. multi-resistant isolates were comparable 5 In most of the Member States reporting data on monophasic variants S. of Typhimurium from human to the European Food Safety Authority, the number of reported cases was higher in compared with. Overall, the monophasic variants represented 7.% of all human Salmonella cases in EU in. [EU Summary Report on Zoonoses, EFSA, 3] 3 3 Tetracycline Chloramphenicol Ampicillin Sulfonamide Ciprofloxacin Note: The number of isolates varies between years (pigs: n = 34 5, Danish pork: n = 8-48). Before, not all serotypes w susceptibility tested (a) (b) Figure 6.. Resistance (%) in Salmonella Typhimurium pigs,inpork and human cases, Denmark DANMAP 3 Pork Danish Pigs 8 Humans Domestic sporadic Humans Travel abroad 7 % resistant isolates Tetracycline 3 Ampicillin 9 Streptomycin 3 9 Sulfonamide 3 Ciprofloxacin 9 3 Trimethoprim Note: The number of isolates varies between years (pigs: n = , Danish pork: n = 6-7, domestic sporadic human travel related human cases: n = 5-95) b) An isolate is categorised as domestic sporadic if the patient did not travel outside Denmark one week prior to the onse not reported as being part of an outbreak DANMAP 3 57

58 6. RESISTANCE IN ZOONOTIC BACTERIA S. Typhimurium infections may reflect a Regarding resistance to antimicrobial agents critical the for travel-associated higher prescription of fluoroquinolones in production anim treatment of human infections, resistance to 3rd generation the countries of destination. In Denmark, fluoroquinolon cephalosporins (ceftiofur) was low and only found in in isolates are rarely from one travel-associated case, one sporadic domestic case used in animal husbandry (except for pet anima since the implementation of legal restrictions in -3 and two cases of unknown origin. [DANMAP ]. As also observed in previous years, a marked difference in fluoroquinolone (ciprofloxacin) resistance was found Although between the MIC of the observed fluoroquinolone resistan isolates was below the clinical breakpoint, it should be not domestically acquired infections (3%) and travel-associated that ciprofloxacin or other fluoroquinolones are often used infections (%). The higher level of ciprofloxacin resistance in empiric treatment of adults with severe bacterial gastroen Helle Korsgaard, Lars Stehr Larsen and Mia Torp (a b) and monophasic variants (c) in Salmonella Typhimurium in pigs, pork and human Figure 6.3. Occurrence (%) of multi-resistance (d) cases, Denmark DANMAP 3 Pork Danish Pigs Human Domestic sporadic Human Travel abroad % of isolates Fully sensitive 9 3 Multi-resistant 9 ASSuT Monophasic Note: The number of isolates varies between years (pigs: n = , Danish pork: n = 6 7, domestic sporadic human and travel related human cases: n= 5-95) a) An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the test panel and multi-resi three or more of the ten antimicrobial classes (see Table.3). Data on resistance to colistin and trimethoprim are not ava the proportions of multi-resistant or fully sensitive are not calculated b) ASSuT isolates are resistant to ampicillin, streptomycin, sulfonamide and tetracycline, but can include resistant to oth such as chloramphenicol S.Typhimurium c) Recording of the monophasic variants of with antigenic formulas S. 4,[5],:i:- in the database was not fully impl 7 and 8, thus data are not presented d) An isolate is categorised as domestic sporadic if the patient did not travel outside Denmark one week prior to the ons not reported as being part of an outbreak 58 DANMAP 3

59 Textbox 5 RESISTANCE IN ZOONOTIC BACTERIA 6. Surveillance of Salmonella and Campylobacter in Denmark Background: In Denmark, all flocks of laying hens, broilers and turkeys, including breeder flocks, are mon for Salmonella according to the EU requirements and the Danish legislation. Salmonella surveillance A and control programme is also running in the Danish pig production, as S. Dublin well asmonitoring a programme in cattle. Since January, a mandatory surveillance Campylobacter of in broiler flocks at the farm has also been in place. Salmonella andcampylobacter in fresh meat are surveyed at the slaughterhouses and at the retail level, and fina intensified controlsalmonella of andcampylobacter in fresh meat, based on a case-by-case risk assessment, has bee in place for Danish and imported meat ready for retail since 7. Human salmonellosis and campylobacte are notifiable illnesses in Denmark, and all cases are reported to SSI and recorded in a national database. M information regarding trends and sources of zoonotic infections in humans and animals in 3 is available Annual Report on Zoonoses in Denmark at Salmonella : In layer and broiler flocks,salmonella the prevalence has been low for more than a decade and only.% and.%, respectively, of flocks were positive in 3. From 8, all Danish flocks that test positive farm are heat treated at slaughter, and as it was in and, none of the slaughter batches tested a weresalmonella positive in 3. The prevalencesalmonella of in cattle is low, and in 3 only.3% of the cattle carcasses tested at slaughter w positive. Furthermore, 95.5% of the non-milk producing herds and 9.% of the milk-producing herds were as probably S. Dublin free. Among pigs sampled prior to slaughter, 4.%Salmonella were found positive, while.3% of the pig carcasses tested at slaughter were positive. As in previous years, the most common seroty and pork in 3 S. was Derby (6% and 39%, respectively) followed S. Typhimurium by (3% and 44%, respectively) when including the monophasic variants with antigenic S. 4,[5],:iformulas (Figure ). Only,36 cases of human salmonellosis were reported in 3 with an incidence on.3 cases per, inhabitants. This is the lowest number for several decades and a result Salmonella of thecontrol intensive programmes in animal production. In total, six domestic Salmonella outbreaks were reported, and five of these were S. caused by Typhimurium of which two of the outbreaks were caused by Danish pork. As in previous years, the Salmonella source account estimated that approximately half of the human cases of salmonellosis were acquired during international travel. Similar to previous years, more S. Enteritidis than 75% of the cases were acquired abroad whereas the majority S. Typhimurium of the cases was acquired in Denmark. The model could not attribute around a quarter of the humane cases to the included food sources, and as previous ye pork was estimated to be the most important domestic food source, followed by different types of importe (Figure ). Campylobacter : The proportioncampylobacter of positive broiler flocks has decreased since, however in 3 the decrease leveled out and 3.% of all broiler flocks Campylobacter tested positive. At retail, Campylobacter was detected in 7.8% of the chilled broiler meat of Danish origin, compared with 9.7% in (Figure 3). The common Campylobacter species in broilersc.are jejuni (9.7% in 3). Broilers were also tested at slaughter, where 8.% of the conventional and 9.3% of the organic-free-range birds Campylobacter were positive. is also found in cattle and pigs, where C. jejuniis dominant in the cattlec.and coliis dominant in pigs. Campylobacter is the most frequently reported foodborne pathogen in Denmark; however, the number of hum campylobacteriosis cases in 3 (67. cases per, inhabitants) at a a level comparable to. S approximately one-third of the cases have been related to international travel. Consumption and handling meat is assumed to be the most important source of human campylobacteriosis (estimated more than 5% domestic sporadic cases), however other sources such as contaminated water, vegetables and direct conta animals cattle exist. Helle Korsgaard, Birgitte Helwigh and Anna Vedel Sørens For further information: Birgitte Helwigh (bhel@food.dtu.dk) DANMAP 3 59

60 6. RESISTANCE IN ZOONOTIC BACTERIA (a) Figure. Occurrence (%) of Salmonella serovars in pigs and at farm in Danish pork, Denmark 5% % positve samples % positive slaughter pig herds DANMAP 3.5% 3% % 5% %.%.5% 5% % (n=834) (n=833).% 3 (n=5) (n=7,45) (n=4,385) (n=,485) (n=,5) (n=8,655) (n=8,5) Pigs Typhimurium Danish pork 4,[5],:i:- Derby Infantis Livingstone Other and non typeable a) Faecal samples from healthy pigs collected at the slaughterhouses Salmonella and cultured as partfor of the DANMAP programme (a),36 Figure. Estimated sources of cases of human salmonellosis, Denmark 3 Sporadic cases, source unknown (6.8-3.%) DANMAP 3 Domestic pork (.9-9.7%) Domestic outbreak-related cases, source unknown (7.%) Domestic pork, outbreak-associated (5.6%) Domestic beef (.3-5.%) Travel-related outbreak cases, source unknown (9.9%) Domestic table eggs (.6-.7%) Domestic ducks (.-.8%) Imported pork (.3-4.4%) Imported beef (.-.9%) Imported broilers (.-.3%) Imported turkey (-.6%) Travel ( %) Source: Danish Zoonosis Centre, National Food Institute a) Sporadic and outbreak-related cases with unknown source include all sources not in the model (b), Denmark Figure 3. Occurrence of Campylobacter in broiler(a)flocks and fresh chilled broiler meat DANMAP 3 % 5% % 5% % (n=3,3) (n=3,379) (n=3,376) 3 (n=3,58) (n=5) 3 (n=884) At farm Chilled Broiler flocks Danish broiler meat a) Boot swabs collected in the stable 7- days before slaughter (detection limit: < cfu/g) b) Non-heat treated chilled broiler meat at retail (detection limit: < cfu/g). The prevalence was calculated as a mean prevalences 6 DANMAP 3

61 RESISTANCE IN ZOONOTIC BACTERIA Campylobacter The consumption of antimicrobial agents in broilers is gene low, but tetracycline has been one of the most commonly Campylobacter Thermotolerant are the most commonly antimicrobial agents in Danish broilers over the last five ye Theinconsumption of tetracycline increased considerably fro reported causes of gastrointestinal bacterial infections 8 to, decreased from to, but increased humans in Denmark as well as in the European Union [ECDC, 3. For C. jejuniisolates from broilers, the resistance and 4]. The species most commonly associated with in human consumption patterns for tetracycline appear to follow eac infections C. is jejuni, but other species may also cause infections. other (Figure 6.4). We did not observe a similar agreement In Denmark, 85%-95% of the human campylobacteriosis cases between the consumption and resistance patterns for. are caused C. byjejuni fluoroquinolones (ciprofloxacin) C. jejuni in broiler isolates. Even though fluoroquinolones have not been used in the b Campylobacter are widespread in nature and the most important production since 9, the resistance level has increased t reservoirs are the alimentary tract of wild and domesticated moderate level (6%) during the last five years (Figure 6.4 birds and mammals. Among sporadic human cases, broilers have been identified as the primary source of infection, though In C. jejuni isolates from Danish broiler meat (n = 7), we other sources also exist, e.g. water from untreated water sources observed levels of resistance to ciprofloxacin (%) and and other infected animals. tetracycline (%) that were slightly lower than in. T resistance to ciprofloxacin and tetracycline has fluctuated For Campylobacter, DANMAP 3 includes randomly the last five years, however the ciprofloxacin resistance lev collected isolates from broilers and cattle at slaughter and observed in was the highest since 7. from fresh broiler meat sold at wholesale and retail outlets. Isolates from human cases originates from three out of five The meat levels of antimicrobial resistance were comparable be geographical regions in Denmark. One isolate per farm, jejuniisolated from Danish broilers and Danish broiler me sample or human case is included, and data are onlyc.presented (Figure if a sufficient number of isolates were obtained (>5) from a6.4), and macrolide (erythromycin) resistance has remained at a very low level for a decade. given source. For details see Chapter 9, Materials and Methods. 6.. C. jejuni from imported meat C. jejunifrom broilers and cattle, broiler MIC distributions for meat and humans, as well C. as coli forfrom pigs and broilerin C. jejuniisolates from imported broiler meat (n = 3), meat in 3 are presented in the web annex (Tablesthe A6.6-A6.9). levels of resistance to tetracycline (8%) and quinolon An isolate is considered fully sensitive if susceptible (nalidixic to all acid and ciprofloxacin, 53% and 57%, respective antimicrobial agents included in the test panel, and multiremained high (Table. 6.3) From 9 to 3 there has been resistant if resistant to three or more of the six included an overall decrease in the proportion of fully C.sensitive jejuni antimicrobial classes (see Table.3). isolates among isolates from imported broiler meat (from 3 to 3%). 6.. C. jejuni in broilers and domestic produced broiler meat Also, over the past five years, the level of resistance to tet and ciprofloxacin has generally been higher in isolates from In 3, we observed moderate levels of resistance to imported broiler meat compared with Danish broiler meat. fluoroquinolones (ciprofloxacin) and tetracycline (6% and %, respectively) among C. jejuniisolates from broilers (n This = corresponds with the data reported by EFSA, where Denmark has reported the lowest proportions of resistance 54, Table 6.3) compared with (5% for both compounds). amongc. jejuniisolates from broiler meat [EFSA, 3]. Table 6.3. Resistance (%) in Campylobacter jejuni from animals, meat of Danish and imported origin (a) and, Denmark human cases DANMAP 3 Cattle Broilers Broiler meat Antimicrobial agent Danish % Danish % Danish % Import % Tetracycline Chloramphenicol Erythromycin Streptomycin Gentamicin Ciprofloxacin Nalidixic acid Fully sensitive Multi-resistant Number of isolates Humans Domestically Travel acquired abroad % % Note: An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the test panel and multi-r three or more of the six antimicrobial classes (see Table.3) a) An isolate is categorised as domestic sporadic if the patient did not travel outside Denmark one week prior to the onse DANMAP 3 6

62 6. RESISTANCE IN ZOONOTIC BACTERIA Figure 6.4. Resistance (%) in Campylobacter jejuni from broilers, broiler meat and(a)human, Denmark cases DANMAP Broilers Broiler meat Danish % Broiler meat Imported Human Domestic Human Travel abroad 9% % resistant isolates 8% 7% 6% 5% 4% 3% % % % Tetracycline 9 3 Erythromycin Ciprofloxacin Note: The number of isolates varies between years (broilers: n = 4 75, Danish broiler meat: n = 6 7, imported broiler domestic sporadic human cases: n = 4 4 and travel-associated human cases: n = 4 78) a) An isolate was categorised as domestic if the patient did not travel outside Denmark one week prior to the onset of th 6..3 C. jejuni in cattle 6..4 C. jejuni in humans In 3,Campylobacter continued to be the most frequent In C. jejuniisolates from cattle (n= 86), we observed moderate cause of bacterial intestinal infections in Denmark. A total levels (%) of resistance to fluoroquinolones (ciprofloxacin) 3,766 human laboratory confirmedcampylobacteriosis cases of and low levels (3%) of resistance to tetracycline (Table 6.3). were reported (67. per, inhabitants) [Annual repo Resistance to fluoroquinolones (ciprofloxacin) among C. on Zoonoses in Denmark 3]. jejunifrom cattle has remained at a moderate level (6%-%) A subset (n = 66) ofc.the jejuni isolates submitted to SSI were since 6 (Figure 6.5). As described in previous DANMAP selected for susceptibility testing continuously over the ye reports, we observed an increase in the level of fluoroquinolone isolates were randomly selected from Campylobacter all of the resistance in 5 despite low consumption of fluoroquinolones isolated from stool samples in the three geographical regio in cattle. In, only one of the fluoroquinolone resistant isolates was also resistant to tetracycline, indicatingincluded that co- in the surveillance. Among the tested isolates, 37 from travel-associated cases and 63% were consider selection by tetracycline (one of the major drugs forwere treatment be domestically acquired. As in previous years, SSI collecte of calves) was not the explanation for the observed levels of information on travel history through phone interviews. fluoroquinolone resistance. It has been discussed [DANMAP Patients were asked about the date of disease onset and w 7] that clonal spread, particularly between farms, could be they had travelled abroad within a seven-day period prior an explanation for the observed resistance to fluoroquinolones. onset of disease. Furthermore, patients who had been abro Initially, fluoroquinolone-resistant C. jejuni isolates came from wereisolates asked about their destinations. Cases were categoris cattle farms in Southern Jutland, but as in recent years, domestically acquired if the patients had not travelled w were obtained from farms distributed throughout Jutland and the week prior to the onset of disease. other parts of Denmark. From 4 to, we observed a general increase in the resistance to tetracycline. However, this trend was discontinued in, and in 3 the level of tetracycline resistance is comparable to 6-9 (3%, Figure 6.4). 6 DANMAP 3

63 RESISTANCE IN ZOONOTIC BACTERIA 6. Figure 6.5. Resistance (%) in Campylobacter jejuni from cattle, Among the domestically acquired infections, 67% were ful Denmark sensitive to all the antimicrobial agents tested, while the 35 3 % resistant isolates 5 5 DANMAP 3 percentage of fully sensitive isolates was much lower (8% among isolates from travel associated cases (Table 6.3). T level of multi-resistance was low (%-4%) among both gro but among the travel-associated cases with C.resistant jejuni, all (/) were resistant to fluoroquinolones (ciprofloxacin compared with 7% (/4) among isolates from the. domestically acquired infections The occurrence of resistance to ciprofloxacin and tetracyc continued to be significantly higher in travel-associated C. jejuniisolates (9% and 54%, respectively) compared to isolates from domestically acquired infections (4% and respectively, Table 6.3 and Figure 6.4). Ciprofloxacin or other fluoroquinolones are often used for empiric treatment of adults with severe bacterial gastroen and the level of resistance to these antimicrobial agents is therefore of major importance. In Denmark, fluoroquinolon are rarely used in animal husbandry (except for pet anima since the implementation of legal restrictions in - Tetracycline Erythromycin [DANMAP ]. Travelling to, or eating meat from countri where fluoroquinolone restrictions are not implemented ca Ciprofloxacin be associated with a higher risk of acquiring infection with Note: The number of isolates varies between years (n = 4 98). ciprofloxacin-resistant C. jejuni 5 Birgitte Borck Høg, Lars Stehr Larsen, Mia Torp DANMAP 3 63

64 6. 64 RESISTANCE IN ZOONOTIC BACTERIA DANMAP 3

65 7 RESISTANcE IN INDIcATOR BAcTERIA DANMAP 3 65

66 7. RESISTANCE IN INDICATOR BACTERIA 7. Resistance in indicator bacteria E. among The MIC distributions and occurrence of resistance Indicator bacteria (Enterococcus faecalis, Enterococcus ande. faecalis are presented in the web annex (Tables faeciumand Escherichia coli ) have been included in the faecium A7. - A7.3). DANMAP programme since 995. Enterococci are included to monitor resistance in Gram-positive bacteria E. coliand as representative of Gram-negative bacteria. These bacteria 7..were E. faecalis from broilers and domestic selected as indicators for occurrence of antimicrobialproduced resistance broiler meat for several reasons: they are ubiquitous and present as major In E. faecalis isolates from broilers (n = 4), 38% of isolates commensals in both the animal and human reservoirs, they were tetracycline resistant followed by erythromycin (% can acquire antimicrobial resistance as response to selective and salinomycin (5%). Antimicrobial resistance to salinomy pressures and finally they have the potential for transferring increased from % in to 5% in 3, a level compara resistance to pathogenic bacteria and between reservoirs and. Only four isolates (4%) were multithe occurrence in (4%). can cause infection in humans resistant while 5% were susceptible to all tested antimicr (Table 7.), a level comparable to. All four multi-resis 7. Enterococci isolates were resistant to tetracycline and erythromycin. For Enterococci, DANMAP 3 includes randomly collected E. faecalis The levels of resistance in isolates from broiler meat Enterococcus isolates from healthy pigs and broilers at slaughter E. to faecalis were comparable isolates from broilers (Figure 7.) (E. faecalis only) and from domestic fresh broiler meat,except pork for streptomycin where significantly higher prevale and beef sold at wholesale and retail outlets E. faecalis (both (%) was found in broiler meat. Only three isolates (5%) w and E. faecium ). In addition, enterococci from imported multi-resistant and all of these were resistant to tetracyclin broiler meat, beef and pork were included. We included only Prevalence of multi-resistance has been lowered from 3% one isolate per farm or meat sample, and data are presented in 9 toin5% in 3, while resistance to erythromycin ha the report where a sufficient number of isolates wereincreased obtained from 7% in to 3% in 3. (>5) from a given source. For details on methodology, see Chapter 9, Materials and Methods. 7.. E. faecalis from pigs and domestic produced pork An isolate is considered fully sensitive if susceptible to all Very high occurrence of resistance to tetracycline (9%) an antimicrobial agents included in the test panel, and multioccurrence of resistance to erythromycin (45%), streptomy resistant if resistant to three or more of the ten included (34%) and kanamycin (8%) was found ine. faecalis antimicrobial classes (see Table 9.3). isolates from pigs (n = 9). Resistance to fluoroquinolone Figure 7.. Resistance (%) in Enterococcus faecalis from animals and meat of Danish and imported origin, Denmark DANMAP 3 Broiler meat Danish Broilers Broiler meat Imported Pigs Pork Danish Pork Imported % resistant isolates Tetracycline 93 Streptomycin 93 Erythromycin 93 Kanamycin 93 Salinomycin Note: The number of isolates varies between years (broilers: n =, Danish broiler meat: n = 34 75, imported broile = 69 4, Pigs: n = 9 57, Danish pork: n = 84 5 and imported pork: n = 45 4) 66 DANMAP 3

67 RESISTANCE IN INDICATOR BACTERIA 7. Table 7.. Resistance (%) among Enterococcus faecalis from animals and meat of Danish and imported origin, Denmark DANMAP 3 Antimicrobial agent Tetracycline Tigecycline Chloramphenicol Ampicillin Penicillin Erythromycin Streptomycin Gentamicin Kanamycin Ciprofloxacin Vancomycin Teicoplanin Linezolid Salinomycin Fully sensitive Multi-resistant Number of isolates Broilers Danish % Broiler meat Danish Imported % % Danish % Beef Imported % Pigs Danish % Pork Danish Imported % % Note: An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the test panel and multi-re three or more of the ten antimicrobial classes (see Table.3) Table 7.. Resistance (%) among Enterococcus faecium from meat of Danish and imported origin, Denmark DANMAP 3 Antimicrobial agent Tetracycline Tigecycline Chloramphenicol Ampicillin Penicillin Erythromycin Quinupristin/dalfopristin Streptomycin Gentamicin Kanamycin Ciprofloxacin Vancomycin Teicoplanin Linezolid Salinomycin Fully sensitive Broiler meat Danish Imported % % Pork Danish % 5 95 Imported % Multi-resistant 5 34 Number of isolates Note: An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the test panel and multi-r three or more of the ten antimicrobial classes (see Table.3) DANMAP 3 67

68 7. RESISTANCE IN INDICATOR BACTERIA (ciprofloxacin) was observed in one isolate (%) fromwere pigs more frequently resistant (Table 7.). Only resistance (Table 7.). The levels of resistance were comparable with. (5%), streptomycin (6%) and chloramphenico tetracycline Tetracyclines and macrolides are the most commonly(%) usedwas detected in imported beef. Only one isolate (%) antimicrobial agents in the Danish pig production, whereas the isolates from imported beef were multi-resistan the tested. very use of fluoroquinolones cephalosporins in pigs was low compared to 3% among isolates from Danish beef. Overa 75% of the tested isolates from imported beef were fully to all tested antimicrobials compared to 7% in All the chloramphenicol (n = 9), kanamycin (n = 3)susceptible and produced beef. gentamicin (n = 7) resistant isolates from pigs weredanish resistant to both tetracycline and erythromycin, while all erythromycin resistant isolates (n = 49) were resistant to tetracycline E. faecium from domestic produced Overall, 34% of the isolates were multi-resistant (Table 7.). broiler meat and pork Nine percent of the tested isolates were fully sensitive to all the High and increased occurrence of salinomycin resistance antimicrobial agents tested. (64%) was observed E. in faecium isolates from Danish broiler meat (n = 66, Table 7.) followed by resistance to tetracyc Since the occurrence of macrolide (erythromycin) (9%) and erythromycin (8%). Only three isolates (5%) from resistance among E. faecalis isolated from pigs has increased the Danish broilers were multi-resistant, and most of the from 44% to 56%, but from to 3 a decline toresistant 45% was isolates (n = 4) were only resistant to salinomyc observed (Figure 7.). Prevalence of multi-resistant E. faecalis (78%). Occurrences of the resistance and multi-resistance (34%) from pigs was comparable to, but lower than were in similar to levels reported in. However since (4%). resistance to erythromycin and tetracycline has decreased whereas resistance to salinomycin has increased (from 38% Most of the E. faecalis isolates from Danish pork (n = 5)9 wereto 64% in 3, Figure 7.). fully sensitive to all of the antimicrobial agents tested (88%, Table 7.). The highest prevalence of resistance was In seen for isolates (n = ) recovered from Danish pork on E. faecium tetracycline (%) followed by erythromycin (5%), and onethe isolate was resistant (to salinomycin only, Table 7.). T aminoglycosides streptomycin (4%) and kanamycin (4%). occurrences of resistance were lower than reported in Resistance to flouroquinolones (ciprofloxacin) was observed in. no significant differences were detected one isolate (%) from Danish pork. Resistance to tetracycline has been declining from % in 9 to % in 3, trend Asain, none of the isolates from domestic broiler mea that is not observed in the isolates from pigs where tetracycline and pork were resistant to fluoroquinolone (ciprofloxacin) resistance has been 8%-9% over the last ten years. or glycopeptides (vancomycin) in 3. Two isolates from Danish broiler meat and none from pork were resistant to 7..3 E. faecalis from domestic produced beef virginiamycin (quinupristin/dalfopristin, Table 7.). In E. faecalis from beef (n = 4), 9% of the isolates were resistant to tetracycline, % resistant to erythromycin ande. faecium from imported meat 7..5 streptomycin and 8% resistant to chloramphenicol and As in, resistance to tetracycline, ampicillin, penicillin, kanamycin (Table 7.). Levels of antimicrobial resistances were erythromycin, quinupristin/dalfopristin, streptomycin and comparable with. kanamycin, as well as multi-resistance was significantly m frequent in E. faecium from imported broiler meat (n = 64) compared with isolates from Danish broiler meat.(n = 66) 7..4 E. faecalis from imported meat Compared with Danish broilere. meat, faecalis from imported Resistance to salinomycin was higher in Danish broiler me with imported (Table 7.). Overall, 34% of the broiler meat (n = 93) had higher levels of resistance compared to E. faecium tetracycline, erythromycin, kanamycin and streptomycin and isolates from imported broiler meat were multiwere more often multi-resistant (Figure 7.). Overall, resistant, 39% of and among the resistant isolates (n = 4), 3% o isolates were resistant to both erythromycin and. tetracycli thee. faecalis from imported broiler meat were multi-resistant In isolates imported pork, 77% of E.the faecium isolates (n = 3) were (all tetracycline resistance) compared with 5% among sensitive to the tested antimicrobial agents (Table 7. from Danish broiler meat. Among the multi-resistant fully isolates andthe only resistance to tetracycline (9%) and erythromyci from imported broiler meat 7% (6 of 36 isolates) had same (3%) were resistance profile (erythromycin, kanamycin, streptomycin and detected. Occurrences of resistance were simila between tetracycline) coming from multiple countries in Europe. For Danish and imported pork, except for a higher lev of resistance to tetracycline in imported pork. salinomycin, occurrence of resistance was higher in Danish. (Table 7.) broiler meat compared with imported broiler meat 7..6 One Health perspective Compared with Danish pork, E. faecalis isolates from imported pork (n = 4) were more frequently resistant to tetracycline, Occurrence of antimicrobial resistance in enterococci whereas resistance levels to all other antimicrobial agents isolated were from production animals and food are used as comparable. Fifty-nine percent E. of faecalis the isolates from indicators for prevalence and transmission of antimicrobia imported meat were fully sensitive to all of the antimicrobial resistance through the food chain to the consumer. Chang agents tested compared to 88% in isolates from Danish in occurrences pork. of antimicrobial resistance in enterococci fr All resistant isolates from imported pork were at leastproduction resistant animals are thought to be directly related to th. to tetracycline. Only % were multi-resistant compared of antimicrobial with agents. 5% in isolates from Danish pork (Table 7.) The level of resistant E. faecalis is generally higher in pigs than The levels of resistance E. in faecalis isolates in Danish (n =in broilers, indicating that pig production is a potential gre 4) and imported beef (n = 5) were comparable, except reservoir for for resistance genes. Tetracycline is predominant erythromycin and kanamycin where Danish producedused beefagainst E. coliinfections in pigs and since resistance 68 DANMAP 3

69 RESISTANCE IN INDICATOR BACTERIA 7. in previous years, the occurrence of antimicrobial resist amonge. coliisolates from pigs is still low (35%, Table As 7.3) faecalis from Danish pork is much lower than from tetracycline can still be used. However, a side effect in of E. this usage faecalis from broiler faecalis has given resistance levels ofe.9% in and most likelydanish pigs. This is not observede.among meat where in other bacterial species isolated from pigs (6% resistance in equal levels of resistance are observed excep streptomycin. These results may indicate that enterococc E. faecium from pigs in ). A very high and increasing level of resistance to tetracycline (8%-9%) has occurredpopulations over the in the live animal and on pork constitute differ sub populations. Pork cuts for sampling are collected from last years. wholesale and retail outlets. Possibly, enterococci on the product may reflect the processing environment, rather, Apart from tetracycline significantly higher resistance to than direct contamination of the meat during slaughter an chloramphenicol, erythromycin, streptomycin, gentamicin E. faecalis and kanamycin was found among isolated from dressing. In contrast, cutting of broilers is done in slaughte pigs when compared to broilers; reflecting the higherplants, usagewhich may explain why the enterococcal population from of antimicrobials in pigs. All these antimicrobial agents arelive broilers and from broiler meat do not appear too used for human treatment (chloramphenicol for eye dissimilar. infections only). Higher occurrence of salinomycin resistance was found In isolates from imported broiler meat, especially the preva in isolates from broilers when compared to pig isolates (5% of fluoroquinolone resistance is noticeable and could be of vs %). Salinomycin is not used to treat human infections, so importance for human treatment but also the presence of salinomycin resistance in itself does not pose a public health (erythromycin, kanamycin, streptomycin problem. However, continuously growing prevalenceresistant and co-e. faecalis tetracycline) among poultry meat isolates from multip resistance with other antimicrobial agents can be of and importance, and in 3, 4 out of 6 salinomycin-resistant isolatescountries were alsoraise concern. Imported broiler meat contains resistant Enterococcus isolates more often than Danish broiler resistant to other antimicrobial agents, especially tetracycline. meat, especially tetracycline, erythromycin, streptomycin E. faecium in ). No vancomycin resistant enterococci were detected kanamycin in Danish (also ampicillin and penicillin produced meat in 3 and only very few vancomycin resistant The tendency that imported meat contains higher prevalen DANMAP isolates have been reported from pigs during the resistance is also seen for pork (tetracycline), but not fo last decade. An increased occurrence of vancomycinof resistant beef. There is a significantly higher occurrence of resistanc E faecium infections has been observed in Danish hospitals erythromycin and kanamycin in Danish beef compared wit (Textbox 8), however, it does not seem likely that, these imported beef. infections are related to Danish meat or pigs. The clones causing the hospital infections are all resistant to ampicillin, in contrast to the vancomycin resistant E. faecium previous Lars Bogø Jensen, Lars Stehr Larsen og Helle Kor isolates from pigs. Figure 7.. Resistance (%) in Enterococcus faecium from Danish and imported broiler meat, Denmark DANMAP 3 Broiler meat Imported Broiler meat Danish % resistant isolates Tetracycline Streptomycin Erythromycin Kanamycin Salinomycin Ampicilin 3 Note: The number of isolates varies between years (Danish broiler meat: n = 66 45, imported broiler meat: n = 64 7) DANMAP 3 69

70 7. RESISTANCE IN INDICATOR BACTERIA 7. Indicator Escherichia coli Ampicillin resistance conveys cross-resistance to amoxicill which has been the most frequently used antimicrobial For indicatore. coli,danmap 3 includes randomly agent in the broiler production for at least a decade where collected isolates from healthy pigs, broilers and cattle at sulfonamides have only been used in the last -3 years. Hi slaughter and from fresh broiler meat, beef and porkuse soldofatsulfonamide and co-selection by ampicillin may par wholesale and retail outlets. We included only one isolate per, however in explain the occurrence of sulfonamide resistance farm or meat sample, and present only data where a3 sufficient sulfonamide use in poultry increased markedly. number of isolates were obtained (>5). For details on methodology see Chapter 9, Materials and Methods. We found 3% of E. thecolibroiler isolates resistant to nalidixic acid and 6% to ciprofloxacin. During the period 3-7 fluoroquinolone consumption in poultry was significantly The MIC distributions and occurrence of resistance E. among coliare presented in the web annex (Tables A7.4 and higher A7.5). than for the other production animal species in because antimicrobial agents approved for poul Data for each of the figures are also presented in thedenmark, web werebut limited to amoxicillin and fluoroquinolones. However, annex. Carbapenems are not included in the test panel, since 8, fluoroquinolone usage in the broilers has been E. coliisolated after selective enrichment with 3rd generation low. One isolate was resistant to colistin but with,a MIC < cephalosporins are examined for carbapenemase and ESBL which is seen occasionally. resistance genes (see Textbox 6). Among An isolate is considered fully sensitive if susceptible to all E. coliisolates (n = 6) from domestic broiler meat, found the highest levels of resistance for ampicillin (4%) antimicrobial agents included in the test panel, and multisulfonamide (8%). Resistance was comparable to the leve resistant if resistant to three or more of the ten included..3) observed in and the the occurrence of ciprofloxacin a antimicrobial classes (see Table nalidixic acid resistance remained at a low level (5%). 7.. Indicator E. coli from broilers and domestic E. coli The levels of resistance infrom Danish broiler meat were broiler meat to what was found among isolates from Danish bro E. coliisolates from broilers (n = 5) were most oftensimilar resistant (Table 7.3). to sulfonamide and ampicillin (both 6%, Table 7.3). Resistance was comparable to the levels observed in, except for an Among E. coliisolates from Danish broilers and broiler meat, increase in trimethoprim resistance. Over the last five years is noteworthy resistance to ampicillin and sulfonamide has increased whereas that the level of multi-resistance has more t doubled within the past five years; reaching 6% - 8% in resistance to fluoroquinolones (ciprofloxacin) has declined while the level of fully sensitive strain has remained relativ (Figure 7.3). In 3, resistance to tetracycline increased, stable. apparently reflecting the increased use of this antibiotic in The increase in multi-resistance is mainly driven by poultry production. Table 7.3. Resistance (%) among Escherichia coli from animals and meat of Danish and imported origin, Denmark DANMAP 3 Antimicrobial agent Tetracycline Chloramphenicol Florfenicol Ampicillin Cefotaxime Ceftiofur Trimethoprim Sulfonamide Streptomycin Gentamicin Neomycin Apramycin Ciprofloxacin Nalidixic acid Colistin Spectinomycin Fully sensitive Multi-resistant Number of isolates Broilers Danish % Broiler meat Danish Imported % % Cattle Danish % Beef Danish Imported % % Pigs Danish % Pork Danish Imported % % Note: An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the test panel and multi-re three or more of the ten antimicrobial classes (see Table.3) 7 DANMAP 3

71 RESISTANCE IN INDICATOR BACTERIA 7. Figure 7.3. Resistance (%) in Escherichia coli from animals and meat of Danish and imported origin, Denmark DANMAP 3 % resistant isolates 8 Broilers Broiler meat Danish Pigs Broiler meat Imported Pork Danish Pork Imported Tetracycline 93 Ampicillin 93 Streptomycin 93 Sulfonamide 93 Ciprofloxacin Note: The number of isolates varies between years (broilers: n = 5-5, Danish broiler meat: n = 6 97, imported bro pigs: n = 46 6, Danish pork: n = 68 6 and imported pork: n = 3 65) increase in occurrence of the ASSuTr phenotype (Figure )Indicator E. coli from pigs and pork and the increased use of tetracycline in poultry may Resistance be a driverto ampicillin (9%), streptomycin (4%), in this trend. sulfonamide (37%) and tetracycline (35%) was common in E. coliisolates from pigs (n = 46, Table 7.3). Resistance wa Resistance to cephalosporins (ceftiofur and cefotaxime) was comparable to the levels observed in, and the trend observed in %-% ofe.the coliisolates from Danish broiler over the period 9-3 shows an increasing occurrence meat. Based on a more sensitive selective enrichment method,to ampicillin (Figure 7.3). resistance a high level of cephalo sporinase producing E. coli(esc) was also observed in broiler meat (see Textbox 6). Overall, we found 35% of the isolates from pigs were multi resistant (Table 7.3. The level of multi-resistance E. colifrom in Danish pigs has been comparable during 8-3. This 7.. Indicator E. coli from cattle and domestic coincides with an increasing use of antibiotics, among them beef tetracyclines, in Danish pig production in recent years. In cattle, we found similar levels of resistance E. coliisolates in (n = 3) as in. The highest levels of resistance were found E. coliisolates (n = 46) from Danish pork the leve Among the for tetracycline (%), sulfonamide (8%) and streptomycin of resistance to streptomycin (44%), tetracycline (34%), an (8%, Table 7.3). The level of multi-resistance has remained at sulfonamide the same level, fluctuating between % and 8% during the past (34%), ampicillin (7%) as well as multi-resist was similar to (Figure 7.3 and Table 7.3). five years. As in cattle, the levels of resistance E. coliin isolates (n = 4)The level of fluoroquinolone (ciprofloxacin) resistance rema very from Danish beef were very low, and at the same level aslow in at one percent. This reflects the low consumption fluoroquinolones in pigs since 3 (web annex, Tab. A4.), when the use in production animals was restricted by legislation. Also, occurrence of cephalosporin (ceftiofur None of thee. coliisolates from cattle or Danish beef were and resistance remained very low; we observe resistant to cephalosporins (ceftiofur and cefotaxime) or cefotaxime) to only one such resistant isolate from Danish pork. The use o quinolones (ciprofloxacin and nalidixic acid). The use of cephalosporins in pigs has been close to zero since July fluoroquinolones in cattle has been negligible since 3, and when the use of 3rd and 4th generation cephalosporins has beena voluntary ban was adopted by the industry. gradually decreased since 8 (Figure 4.5). Surveillance based on selective methods has shown a decr in the number of cephalosporin resistant (ESC) in samples from pigs at slaughter in 3 compared with 9 (see Textbox 6). DANMAP 3 7

72 7. RESISTANCE IN INDICATOR BACTERIA (a b c) Figure 7.4. Occurence (%) of multi-resistant and fully sensitive Escherichia coli from animals and meat of Danish and imported origin, Denmark DANMAP 3 Broilers Broiler meat Danish Broiler meat Imported Pigs Pork Danish Pork Imported % of isolates Fully sensitive 93 Multi-resistant ASSuT ASuTr Note: The number of isolates varies between years (broilers: n = 5 5, Danish broiler meat: n = 6 97, imported br n = 36, pigs: n = 46-6, Danish pork: n = 68 6, imported pork: n = 3 65) a) An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the test panel and multi-resis three or more of the ten antimicrobial classes (see Table.3) b) ASSuT isolates are resistant to ampicillin, streptomycin, sulfonamide and tetracycline, but can include resistant to othe such as chloramphenicol c) ASuTr isolates are resistant to ampicillin, sulfonamide and trimethoprim, but can include resistant to other antimicrobia chloramphenicol 7..4 Indicator E. coli from imported meat In imported beef, 86% of E. the coliisolates (n = 35) were fully The level of multi-resistance (67%) in imported broiler meat and three isolates (9%) multi-resistant (Table 7.3) sensitive was considerably higher compared to Danish broiler level meatof fully sensitive and multi-resistant isolates in import (6%, Table 7.3), and also compared to other Danishbeef meat has not changed during the past 5 years, and is simila. types included in the DANMAP programme to what we find in Danish beef. One isolate was resistant to quinolones (ciprofloxacin and nalidixic acid) and one to 3rd generation cephalosporins (cefotaxime). The highest occurrences of resistance E. coliisolates in (n = 36) from imported broiler meat were found for ampicillin (64%), sulfonamide (57%), tetracycline (53%) and streptomycin E. coliisolates The highest occurrence of resistance in (n = 5) (48%). One isolate was resistant to colistin with a MIC <6. from imported pork was for tetracycline and streptomycin For almost all antimicrobial agents in the test panel (except for (44%), ampicillin and sulfonamide (36%), and trimethoprim gentamycin, apramycin and colistin) the level of resistance was 7.3). Overall, 38% of the isolates were multi(34%), Table significantly higher for imported broiler meat than inresistant, broiler a decline from 47% in (Figure 7.4). Resistan meat of domestic origin (Figure 7.3, Table 7.3). The exceptions to fluoroquinolones (ciprofloxacin) was found in five isolate were gentamycin, apramycin og colistin, where onlywhereas one isolate cephalosporin (ceftiofur cefotaxime) resistance w among the isolates from imported broiler meat was resistant not observed. Compared with Danish pork, resistance to and none from the Danish broiler Meat. quinolones (ciprofloxacin and nalidixic acid) was significan higher in imported pork (Figure 7.4). 7 DANMAP 3

73 RESISTANCE IN INDICATOR BACTERIA One Health Perspective Multi-resistance is of importance, because high levels of We usee. colias an indicator organism for antimicrobial resistance decrease the number of good first choice antibi resistance because it is a commensal in both mammals and for treatment of infections in humans and becau available birds and commonly present on raw meat. Using phenotypic increases the risk of selection of antimicrobial resistance. A resistance as a marker, our data indicate E. coliinthat slaughter an example, if resistance to fluoroquinolone (chromosoma animals and in the derived meat constitute overlapping bacterial or ESBL (plasmid) develops, the risk of co-selection throug populations (Table 7.3). Therefore, meat has the potential use ofto old antimicrobial agents (such as tetracycline, act as a vehicle transferring antimicrobial resistance sulfonamide from food and penicillin) in the same bacterium increase animals to humans. Furthermore, somee.ofcoli these strains with the occurrence of multi-resistance. Resistance to thes are virulent with a zoonotic potential for disease in humans antimicrobial agents is common E. coliin from Danish pigs and [DANMAP, Textbox 6]. pork, as well as imported broiler meat, where we also obse high levels (>3%) of multi-resistance (Figure 7.3 and 7.4) Transfer of genes coding for resistance to antimicrobial agents that are critically important in human medicine such With co-resistance to critically important antimicrobial age as third generation cephalosporins is especially worrisome, the risk of maintenance and spread of the critical importan as is transmission through food ofe. coli resistant to antimicrobial resistance through use of another class of fluoroquinolones. Resistance to fluoroquinolones is generally, in particular when antimicrobial agents increases markedly low in Danish animals, but higher in imported meats,resistance in determinants are located on mobile genetic ele particular broiler meat (Figure 7.4). This also applies High to levels of resistance and multi-resistance contribute to cephalosporin resistance. Resistance to cephalosporins is reduced number of antimicrobial agents available for use i presently increasing internationally in the animal reservoir, human medicine, thus limiting the solutions for treating hu causing great concern both nationally and internationally.. illness, se section 8. regardinge.human coliinfections Cephalosporin resistance commonly resides on mobile genetic elements, e.g. plasmids, and therefore may be transferred Flemming Bager, Lars Stehr Larsen and Helle Kors colistrains. between bacteria, in addition to clonal E. spread of DANMAP 3 73

74 7. RESISTANCE IN INDICATOR BACTERIA Textbox 6 Occurrence of Extended spectrum beta-lactamase (ESBL)-producing Escherichia coli in meat and slaughter pigs, but no sign of carbapenemase producing E. coli Background: Extended spectrum beta-lactamase (ESBL)-producing bacteria is one of the fastest emerging problems worldwide in both humans and production animals. Lately, several studies have found the same E plasmids and clones E. coli of isolates originating from animals and involved in human infections, suggesting a zo link. The occurrence of Enterobacteriaceae resistant to carbapenems is a growing threat in human medicine as c are the last line antimicrobial agents for treatment of infections caused by multidrug resistant Gram-negati this has been pointed out by both EFSA and ECDC. The presence of carbapenemase producing bacteria in f producing animals is not known, but recently carbapenemase E. coli producing has been detected in livestock pigs in Germany, and carbapenemase producing Salmonella enterica subsp. enterica has been detected in both livestock pigs and poultry in Germany [Fischer et al.. J. Antimicrob Chemother. 67:793-5]. The presence and possible spre of carbapenemase producing bacteria in production animals is thus considered extremely important for the of potential zoonotic risks. Carbapenems are not used in the Danish animal production. Both ESBL and carbapenem resistance can be use of cephalosporins. In July, the use of cephalosporins in the Danish pig production was discontinue still used for systemic and intramammary treatment in cattle. Cephalosporins have not been used in the Da production for at least a decade, but was used outside of Denmark in the production of grandparent animal Danish broilers before. The aim of this study was to investigate the occurrence of ESBL/carbapenemase-producing E. coliin pigs at slaugther and in meat at retail (see Chapter. for definition of ESBL). Materials and methods: During January through December 3, faecal samples were taken from pigs a = 38). One animal represented one herd and no herds were sampled more than once in the same month. period, broiler meat (Danish: n = 46, imported: n = 7), beef (Danish: n = 7, imported: n = ) and po = 38, imported: n = 5) samples were collected randomly in retail stores and outlets in E. allcoli regions of Den was isolated from g of faecal sample or 5 g of meat after selective enrichment in McConkey media with ce µg/ml). The genetic background for ESBL-resistance was revealed by use of whole genome sequencing (WG were assembled de novo prior to prediction of genes. The web-server ResFinder ( used to identify acquired ESBL and carbapenemase genes in the WGS data. For isolates where no genes we the sequences were investigated for up-regulation of ampc chromosomal by use of CLCbio Genomic Workbench v6. Results: For pigs at slaughter, 5.8% (/38) contained ESBL-producing E. coli, which was significantly lower than in 9 (%) and in (%). Among the ESBLE.producing coliisolates from pigs the most commonly detected gene was, as in previous years, CTX-M- (59%) followed by CMY- (8%), ampc upregulation (4%) and CTX-M-4 (Figure ). From meat samples, the highest prevalence of ceftriaxone E. coliwas resistant found among imported broiler meat (5%, 89/7) and were at the same level as in to (between 5 % to 6%). The prevalence of ceftriaxone resistant E. coliin Danish broiler meat was significantly lower than in and a decreasing trend since, where the occurrence of ceftriaxone E. resistant coli(44%) was the highest observed. The occurrence of ceftriaxone resistant E. coliwas significantly higher in imported broiler meat when compared with Danish broiler Among ESBL producing E. coliisolates the most commonly detected gene in Danish broiler meat was, as found previous years, CMY- (83%), whereas CTX-M- (37%) and CMY-(4%) were almost equally present in impo broiler meat. Eight isolates of the ESBL-producing isolates from broiler meat contained two ESBL genes. In t meat types the prevalence was generally low (-.4%, Figure ). None of the isolates from meat and slaugh contained any known carbapenemase genes. Discussion: The usage of cephalosporins was still low (approximately 3 kg in total, Table 4.) compared wi other antimicrobial agents, and the occurrence of cephalosporinase E. coli producing in slaughter pigs was significantly lower than than before the voluntary ban of cephalosporin usage in the Danish pig production was effectua The occurrence of cephalosporinase producing E. coliin Danish produced broiler meat was significantly lower in than in, which could be explained by the voluntary discontinuation of the use of 3rd generation cephalosporins in the top of the breeding pyramid (in the country producing the grandparents for the Danis broilers) leading to a reduction in imported parentflocks, but other explanations may also exist. 74 DANMAP 3

75 RESISTANCE IN INDICATOR BACTERIA 7. Since none of the tested ESBL-producing E. coliisolates contained known carbapenemase genes, meat or food-produ animals in Denmark are most likely not a source to carbapenemase producing bacteria causing human infec Denmark; however monitoring of carbapenemase producing bacteria in animals and meat is still important may change over time. E.coli Conclusion: The voluntary ban of cephalosporins in the pig production seems to retain theinesbl pigsproducing E. of coli at slaughter at a low level. For the first time a significant reduction in the occurrence ESBL in Danish producing E. coli broiler meat was observed, which could be due to less introduction of ESBL producing from imported parent animals, but this was not investigated in this study and also other factors may influence the occurence. ESBL genes found in ESBL-producing E. colifrom human infections are still present in pork and pigs, and broiler me from both Danish and imported origin. A possible zoonotic link will be studied further in the coming years. Currently, and based on the data presented here, carbapenemase E. colifrom producing meat or food-producing animals are considered a low,if any, risk to human health. Yvonne Agersø, Rolf Sommer Kaas, Henrik Hasma For further information: Yvonne Agersø (yvoa@food.dtu.dk) Figure. Occurrence (%) of ESBL Escherichia coli and genes in pig from samples collected at farm and slaughterhou level, Denmark 4 DANMAP 3 Unknown % positive samples AmpC upregulation TEM-35 TEM- 8 SHV- CMY- 6 CTX-M-55 CTX-M-5 4 CTX-M-4 CTX-M- CTX-M- (n=99) (n=78) Farm level 9 (n=786) (n=47) (n=777) (n=787) 3 (n=38) At slaugther Note:E. coliwas isolated after selective enrichment in McConkey media with ceftriaxone ( μg/ml). The genetic backg ESBL resistance was revealed by use of PCR, micro array and DNA sequencing DANMAP 3 75

76 7. RESISTANCE IN INDICATOR BACTERIA (a), in Figure. Occurrence (%) of ESBL Escherichia coli and genes Denmark meat 7 DANMAP 3 % positive samples Danish Import Pork Danish Import Beef Danish Import Broiler meat Unknown AmpC upregulation TEM-5 TEM- SHV- SHV-a CMY- and (CTX-M-, TEM-,-3-5, -6 or SHV-) CMY- CTX-M-4 CTX-M- CTX-M- and (TEM-, -3 or SHV-) or TEM-3 and SHV- CTX-M- Note:E. coliwas isolated after selective enrichment in McConkey media with ceftriaxone ( μg/ml). The genetic backg for ESBL resistance was revealed by use of PCR, microarray and DNA sequencing a) Each year, approximately, samples are collected, evenly distributed between the six categories of meat 76 DANMAP 3

77 8 RESISTANcE IN HUMAN clinical BAcTERIA DANMAP 3 77

78 8. RESISTANCE IN HUMAN CLINICAL BACTERIA 8. Resistance in human clinical bacteria 8. Escherichia coli Blood isolates from hospital patients DANMAP received data on the antimicrobial susceptibility o approximately 4, E. coliisolates from blood (Table 8. and Escherichia coli is part of the normal intestinal flora of both Figure 8.). humans and animals but also cause infections. In E.humans, coliis the most frequent cause of bacteraemia and communityand hospital-acquired urinary tract infections. E. For coli, In 3, resistance to nd generation cephalosporins outof Departments DANMAP 3 includes data from (cefuroxime) was 9% and resistance to 3rd generation of Clinical Microbiology (DCM), representing 95% of the cephalosporins 8%. Both levels are similar to the levels rep Danish population. Nine of the DCM working within primary. The level of 3rd generation cephalosporin resistanc healthcare isolates contributed data on antimicrobialinresistance Denmark was above the level reported to EARS-Net by t in urine isolatese.ofcolifrom primary healthcare (Table 8.). other Nordic countries and corresponded to the occurrence reported by other European countries in [EARS-Net ]. Table 8.. Resistance (%) in Escherichia coli isolates from humans, Denmark DANMAP 3 Substance Blood isolates, hospitals Urine isolates, hospitals Urine isolates, primary healthcare % % % Ampicillin Mecillinam 8 6 Piperacillin/tazobactam 4 Sulfonamide Gentamicin 7 5 Ciprofloxacin # Cefuroxime 9 6 a) 3rd generation cephalosporins Meropenem < < < Max. number of isolates tested #) A number sign indicates a significant decrease from to 3 a) Tested 3rd generation cephalosporins were ceftazidime, ceftriaxone, cefpodoxime and cefotaxime Figure 8.. Resistance (%) in Escherichia coli blood isolates from humans, Denmark 5 DANMAP 3 6 % resistant isolates % resistant isolates Ampicillin (n=3964) Piperacillin/tazobactam (n=3963) Ciprofloxacin (n=396) 3rd gen. cephalosporin (n=393) Mecillinam (n=3475) Gentamicin (n=3886) Cefuroxime (n=365) Meropenem (n=83) Note: The number (n) in parentheses represents the number of isolates tested for susceptibility in 3 78 DANMAP 3

79 RESISTANCE IN HUMAN CLINICAL BACTERIA 8. One carbapenem (meropenem) resistant E. coliblood isolate In 3, carbapenem (meropenem) resistance was observ fivee. coliurine isolates from hospitalised.patients was reported in. occurrence of ciprofloxacin resistance was at the same The occurrence of ciprofloxacin resistance decreasedthe from 4% as in, in to % in 3. This level was the same as reported by but a steady increase has been seen in ciproflo many other countries in Europe [EARS-Net ]. resistance from 3% in 4 to % in 3 (Figure 8.). Aminoglycoside (gentamicin) resistance was 7%, which is at Aminoglycoside (gentamicin) resistance was 5% and the same level as reported in. This level was the same as resistance was 33%, which are at the same le sulfonamide reported by many other countries in Europe [EARS-Net as ]. in. Urine isolates from primary healthcare Mecillinam resistance was % and ampicillin resistance received data on the antimicrobial susceptibility was 46% in 3, which are at the same levels as in DANMAP. of approximately 44, E. coliisolates from urinary tract infection Resistance data on piperacillin/tazobactam was reported for in patients from primary healthcare (Table 8. an Figure 8.3). the first time in the present DANMAP report. The DCMs were asked to report resistance data for five years (9-3) (Table 8. and Figure 8.). In all five years, the resistance was the Theatoccurrence of resistance to 3rd generation cephalospo same level around 4%. was 4% and this was the same level as reported in. In the -year period from 4 to 3, resistance E. in Since 3, the level of ciprofloxacin resistance has increa coliblood isolates has increased steadily: resistance to nd from 3% in 4 to % in 3 (Figure 8.3), whic steadily generation cephalosporins from 3% to 9%; ciprofloxacin parallels the increasing trend observed in the consumption resistance from 5% to %; aminoglycoside (gentamicin) fluoroquinolones during the last decade (Table 5.). resistance from % to 7%. These increases parallel the increased antimicrobial consumption, which has been seen in the Thesame occurrence of ampicillin resistance was 4% and period (Table 5.4). sulfonamide resistance was 33%, both levels were similar levels reported in. Urine isolates from hospital patients DANMAP received data on the antimicrobial susceptibility of In 3, carbapenem (meropenem) resistance was observ approximately 4, E. coliisolates from hospital patients with fivee. coliurine isolates from primary healthcare. a urinary tract infection (Table 8. and Figure 8.). Line Skjøt-Rasmussen, Stefan S. Ols and Anette M. Hammerum The occurrence of resistance to nd generation cephalosporin (cefuroxime) (6%) and 3rd generation cephalosporin (6%) were at the same level as in. Figure 8.. Resistance (%) in Escherichia coli urine isolates from humans in hospitals, Denmark DANMAP % resistant isolates % resistant isolates Ampicillin (n=33894) Sulfonamide (n=8979) Gentamicin (n=3865) 3rd gen. cephalosporin (n=3955) Mecillinam (n=3976) Ciprofloxacin (n=39365) Cefuroxime (n=3583) Note: The number (n) in parentheses represents the number of isolates tested for susceptibility in 3 DANMAP 3 79

80 8. RESISTANCE IN HUMAN CLINICAL BACTERIA Figure 8.3. Resistance (%) in Escherichia coli urine isolates from humans in primary healthcare, Denmark DANMAP 3 4 % resistant isolates % resistant isolates Ampicillin (n=438) Mecillinam (n=4368) Ciprofloxacin (n=38878) 3rd gen. cephalosporin (n=4354) Sulfonamide (n=3474) Note: The number (n) in parentheses represents the number of isolates tested for susceptibility in 3 8. Klebsiella pneumoniae In general, the level of antimicrobial resistance in 3 wa similar to (Figure 8.4). Klebsiella pneumoniae is part of the normal intestinal flora in humans but also cause infections such as urinary tract-, Resistance to nd generation cephalosporins (cefuroxime) respiratory tract-, wound- and bloodstream infections. Many % andofresistance to 3rd generation cephalosporins was these infections are hospital acquired and can be lifein threatening, 3, 3rd generation cephalosporin resistance was abo especially if the strains are resistant to antimicrobial K. agents. level reported to EARS-Net by the other Nordic countries a pneumoniae is intrinsically resistant to aminopenicillins corresponded (e.g. to the occurrence reported by other Europea ampicillin). Therefore, infections caused K. pneumoniae by countries in [EARS-Net ]. are treated with broad spectrum antimicrobial agents such as ciprofloxacin, gentamicin, cephalosporins and carbapenems. In 3, two carbapenem (meropenem) K. pneumoniae resistant For K. pneumoniae, DANMAP 3 includes data from. blood isolates were detected out of DCM, representing 95% of the Danish population. Nine of the DCM working with primary healthcare isolates resistance (9%), and resistance to aminoglyc contributed data on antimicrobial resistance in urine Ciprofloxacin isolates of (gentamicin) (4%) were similar to the level reported in K. pneumoniae from primary healthcare (Table 8.). The level of resistance to ciprofloxacin was above the leve Blood isolates from hospital patients reported DANMAP received data on the antimicrobial susceptibility of from the other Nordic countries and the same approximately 9 K. pneumoniae isolates from blood (Tableas reported to EARS-Net by other European countries in [EARS-Net ]. Whereas, the level of resistance to 8. and Figure 8.4). aminoglycosides was the same as in the other Nordic coun reporting to EARS-Net [EARS-Net ]. Table 8.. Resistance (%) in Klebsiella pneumoniae isolates from humans, Denmark Substance Mecillinam Piperacillin/tazobactam Sulfonamide Gentamicin Ciprofloxacin Cefuroxime a) 3rd generation cephalosporins Meropenem Max. number of isolates tested #) A number sign indicates a significant decrease from to 3 a) Tested 3rd generation cephalosporins were ceftazidime, ceftriaxone, cefpodoxime and cefotaxime 8 DANMAP 3 DANMAP 3 Blood isolates, hospitals Urine isolates, hospitals Urine isolates, primary healthcare % % % 8 # 6 # # 4 4# # 9 7# 6 < < <

81 RESISTANCE IN HUMAN CLINICAL BACTERIA 8. Figure 8.4. Resistance (%) in Klebsiella pneumoniae blood Urine isolates from hospital patients isolates from humans, Denmark DANMAP received data on the antimicrobial susceptibility DANMAP 3 of approximately 5,9 K. pneumoniae isolates from hospital patients with a urinary tract infection (Table 8. and Figure 8.5). % resistant isolates 5 Resistance to mecillinam (%), sulfonamide (%), gentamicin (4%), nd generation cephalosporins (cefuroxime) (9%) and to 3rd generation cephalosporins (7%) decreased from to 3. Ciprofloxacin resistance was at the same level as reported in. In 3, carbapenem (meropenem) resistance was observed5 in eightk. pneumoniae urine isolates from hospitalised patients. Urine isolates from primary healthcare DANMAP received data on the antimicrobial susceptibility of approximately 3,6 K. pneumoniae isolates from urinary tract infection in patients from primary healthcare (Table 8. and Figure 8.6) Mecillinam (n=778) Piperacillin/tazobactam (n=874) Ciprofloxacin (n=876) Gentamicin (n=865) Cefuroxime (n=796) 3rd gen. cephalosporin (n=87) Meropenem (n=645) In 3, resistance to 3rd generation cephalosporins was 6%, which is similar to the level reported in. In 3, three carbapenem (meropenem) resistant K. Note: The number (n) in parentheses represents the number of i pneumoniae urine isolates from patients in primary healthcare tested for susceptibility in 3 were found. Resistance to ciprofloxacin was 7% and resistance to mecillinam was %, which were similar to the levels reported in. Sulfonamide resistance decreased from 6% in to %. in 3 Line Skjøt-Rasmussen, Stefan S. Olsen and Anette M. Hammerum Figure 8.5. Resistance (%) in Klebsiella pneumoniae urine isolates from humans in hospitals, Denmark DANMAP Mecillinam (n=59) 3 DANMAP 3 4 % resistant isolates % resistant isolates Figure 8.6. Resistance (%) in Klebsiella pneumoniae urine isolates from humans in primary healthcare, Denmark 9 3 Mecillinam (n=3649) Sulfonamide (n=858) Ciprofloxacin (n=585) Gentamicin (n=4553) Sulfonamide (n=334) Ciprofloxacin (n=378) Cefuroxime (n=4659) 3rd gen. cephalosporin (n=586) 3rd gen. cephalosporin (n=366) Note: Note: The number (n) in parentheses represents the number of The number (n) in parentheses represents the number of isolates tested for susceptibility in 3 isolates tested for susceptibility in 3 DANMAP 3 8

82 8. RESISTANCE IN HUMAN CLINICAL BACTERIA Textbox 7 carbapenemase producing bacteria in Denmark Background: Carbapenems comprise one of the only classes of antimicrobial agents that can be used for of infections with multi-resistant bacteria Klebsiella such as pneumoniae, Escherichia coli, Pseudomonas aeruginosa andacinetobacter baumannii. Treatment options for infections with carbapenem resistant bacteria are often non or suboptimal. Resistance can be caused by the presence of various carbapenemases of which the most fre occurring are K. pneumoniae carbapenemase (KPC), Oxacillinase (OXA), Verona integron-encoded metallo-βlactamase (VIM), and New Delhi metallo-β-lactamase (NDM). Furthermore, Imipenemase (IMP) can be detec P. aeruginosa isolates. In recent years, Danish Departments of Clinical Microbiology (DCM) have, on a voluntary basis, submitted c resistant isolates for verification and genotyping at the Antimicrobial Resistance Reference Laboratory at S Institut. The present textbox describes carbapenemase Enterobacteriaceae producing (CPE), and carbapenemase producing P. aeruginosa anda. baumannii. During 3, 35 carbapenemase producing bacteria were detected. More than one isolate from the same p included, if the isolates belonged to different bacterial species and/or if the isolates harboured different car In many of the cases, the carbapenemase producing isolates were related to travelling, but spread between Denmark was also reported for Enterobacteriaceae both anda. baumanii in 3. Enterobacteriaceae: In 3, 8 CPE were detected compared to 9 CPE during 8 (Figure ). Fifteen o isolates harboured NDM genes K. pneumoniae ( (n = 4), Citrobacter freundii (n = 4), E. coli(n = ), Providencia stuartii (n = ) and Proteus mirabilis (n = )), OXA-48 was detected in 3 isolates K. pneumonia ( (n = ), E. coli(n = )), and KPC (K. pneumoniae (n = )) and VIM Klebsiella ( oxytoca (n = )) in one isolate each. During 3, the first spread of CPE in Denmark was confirmed by comparing epidemiological information molecular data. The first spread of CPE was between two patients, who had been admitted to the same wa in the North Denmark Region in. None of these patients had a prior history of travel noted in their hos records. In 3, it was shown by molecular typing that the patients shared the same E. coli.vim-4 This was producing the first detected spread of CPE in Denmark. The origin of VIM-4 E. coli producing was unknown. In 3, NDM- producing C. freundii isolates were detected from four patients at the same hospital ward in the N Denmark Region. Spread of NDM- C. freundii was shown by molecular typing and comparison of epidemiological data. None of the four patients had been travelling recently and the origin of the NDM- C. freundii producing was unknown. Besides the NDM- producing C. freundii, two of the four patients had NDM- producing K. pneumoniae. A. baumannii: During 3, seven OXA-3 producing A. baumannii isolates were detected. Spread of OXA-3 producing A. baumannii was detected two times from one patient to another patient. Furthermore, three OXA-4 like producing A. baumannii were detected, two of these being part of the same transmission chain. Furthermore NDM- producing A. baumannii isolates were detected. P. aeruginosa: In 3, three VIM producing P. aeruginosa isolates were detected. Furthermore, an NDM and VIM producingp. aeruginosa was detected. For the first time, an IMP producing P. aeruginosa isolate was detected in Denmark. Both patients had been hospitalized abroad prior to detection of these isolates. Conclusion: The occurrence of carbapenemase-producing bacteria in Denmark is increasing. Most isolates multi-resistant, which makes infections caused by these bacteria extremely difficult to treat with antimicrob The increasing trend of carbapenemase producing bacteria is therefore worrying. Especially the spread of C concern, since Enterobacteriaceae can be carried in the intestine for a long time without any symptoms of infection which makes infection control difficult. Anette M. Hammerum, Frank Hansen and Lotte Jakobse For further information: Anette M. Hammerum (ama@ssi.dk) 8 DANMAP 3

83 RESISTANCE IN HUMAN CLINICAL BACTERIA 8. Figure. Numbers of carbapenemase-producing Enterobacteriaceae (cpe) DANMAP OXA-48 KPC NDM 8 VIM Note: More than one isolate was included from the same patient, if the isolates belonged to different bacterial species harboured different carbapenemases. DANMAP 3 83

84 8. RESISTANCE IN HUMAN CLINICAL BACTERIA 8.3 Pseudomonas aeruginosa Figure 8.7. Resistance (%) in Pseudomonas aeruginosa blood isolates from humans, Denmark DANMAP 3 of 8 % resistant isolates Pseudomonas aeruginosa is an opportunistic pathogen immunocompromised individuals. P. aeruginosa typically infects the pulmonary tract, urinary tract, burns, wounds, 7and also causes other bloodstream infections. It is the most frequent coloniser of medical devices (e.g. catheters). P. aeruginosa 6 infection is a serious problem in patients hospitalised with cancer, cystic fibrosis and burns. The case fatality rate in these 5 patients is high. P. aeruginosa is intrinsically resistant to the majority of antimicrobial agents. The antimicrobial classes4 which can be used for treatment include some fluoroquinolones (e.g. ciprofloxacin and levofloxacin), aminoglycosides (e.g.3 gentamicin, tobramycin and amikacin), some beta-lactams (piperacillin-tazobactam, ceftazidime, and carbapenems) and colistin. P. aeruginosa blood isolates obtained from hospitalised patients ForP. aeruginosa, DANMAP 3 includes data from out of Departments of Clinical Microbiology (DCM), representing Ciprofloxacin (n=48) 95% of the Danish population. DANMAP received data on the Gentamicin (n=48) antimicrobial susceptibility of approximately P. aeruginosa 44 Ceftazidime (n=35) isolates from blood. Resistance to all the tested antimicrobial Meropenem (n=4) agents was not significantly different from the level in, Piperacillin / Tazobactam (n=44) but an increasing trend has been observed for gentamicin during 7 3 (Figure 8.7). The occurrence of resistance to Note: The number (n) in parentheses represents the number of i fluoroquinolones, carbapenems, ceftazidime and piperacillin/ tested for susceptibility in 3 tazobactam was at the same level or lower than most of the other countries reporting to EARS-Net in [EARS-Net Streptococcus pneumoniae ]. S. pneumoniae is a leading cause of bacterial pneumonia, otit media, bacteraemia and meningitis. In 3, susceptibility S. pneumoniae Meropenem resistance was observed for 3% (n =P.)testing of the was performed on 84 non-duplicate isolates from invasive infections (blood and spinal fluid) aeruginosa isolates in 3. A Danish study P. aeruginosa of (Figure 8.8). carbapenem non-susceptible isolates from showed that carbapenemases were present in a minority of the isolates (7%) [Textbox, DANMAP ]. As in previous years, A total of 4 (5.%) S. pneumoniae invasive isolates from blood presumable carbapenemase producing P. aeruginosa isolates and cerebrospinal fluid were non-susceptible (resistant an were sent on a voluntary basis from the DCM to SSIintermediary for resistant) to erythromycin in 3 compared national surveillance on carbapenemase producing bacteria, 5.4% in (Figure 8.8). The 4 isolates belonged to including not only isolates from bloodstream infections but serotypes and the most commonly found erythromycin no also from other origins. During 3, three VIM producing P. susceptible serotypes were type 5A (n = 9), 9A (n = 7) aeruginosa isolates and an NDM and VIM producing isolate 4F (n = 4). were identified. For the first time an IMP producing isolate was detected in Denmark [Textbox 7]. Regarding penicillin, 5 (6.3%) invasive isolates from blood and cerebrospinal fluid were non-susceptible (resistant an Anette M. Hammerum, Stefan S.intermediary Olsen resistant) in 3 compared to 5.% in and Line Skjøt-Rasmussen (Figure 8.8). The 5 isolates belonged to different serot 8.4 Streptococci and the most commonly found penicillin non-susceptible serotypes were type 5A (n = ), 3B (n = ) 9A (n = Streptococci are part of the normal commensal flora 4F of (n = 4). None of the isolates were resistant to penicilli according to EUCAST. the mouth, skin, intestine, and upper respiratory tract of humans, however they also cause infections such as otitis Four serotypes were particularly non-susceptible to either media, tonsillitis, bacterial pneumonia, bacteraemia/sepsis, penicillin or erythromycin or both, namely 5A ( of 3 endocarditis and meningitis. received isolates), 3B ( of 6 received isolates), 9A (8 received isolates) and 4F (4 of received isolates). In this report, data on resistance in invasive streptococcal isolates (only from blood or cerebrospinal fluid) were obtained The levels of erythromycin (macrolide) and penicillin nonfrom the Neisseria and Streptococcus Reference laboratory susceptibility in Denmark were similar to the levels reporte covering all DCM in Denmark. Infections with streptococci are to EARS-Net by the neighboring countries Norway primarily treated with penicillins and macrolides. All in invasive Sweden, Germany and the United Kingdom. Many other non-duplicate Streptococcus pneumoniae and group A, B, C and European countries reported considerably higher levels of G streptococci were susceptibility tested against erythromycin resistance in [EARS-Net ]. and penicillin. 84 DANMAP 3

85 RESISTANCE IN HUMAN CLINICAL BACTERIA 8. Figure 8.8. Nonsusceptibility (%) in Streptococcus pneumoniae 8.5 blood and spinal fluid isolates from humans, Denmark 7 DANMAP 3 % resistant isolates Enterococci are part of the normal intestinal flora of both humans and animals but also cause infections. Important clinical infections caused by Enterococcus species include urinary tract infections, bacteraemia and bacterial endoca E. faecalis ande. faecium can cause life-threatening infections in humans, especially in the hospital environment. The natur high level of antimicrobial resistance found E. faecalis in and E. faecium makes infections difficult to treat. Antimicrobial therapy for serious enterococcal infections requires the use of synergistic combinations of a cell-wall-active agent such penicillin (ampicillin) and an aminoglycoside (gentamicin) a glycopeptide (vancomycin). ForE. faecalis ande. faecium, data from of the DCM were obtained, representing 95% of the Danish population Enterococci Enterococcus faecium andenterococcus faecalis blood isolates obtained from hospitalised patients DANMAP received data on the antimicrobial susceptibility o 657E. faecium isolates and 579 E. faecalis isolates from blood. Penicillin (MIC >=.5 ug/ml) As in previous years, most of the E. faecium isolates from bloodstream infections were ampicillin resistant. In 3, 9 of thee. faecium isolates were resistant to ampicillin. Treatmen with fluoroquinolones, cephalosporins or carbapenems has been described as a risk factor for development E. faecium of an Group A streptococci An increasing consumption of these antimicrobia In 3, susceptibility testing was performed on 67infection. nonduplicate group A streptococci (GAS, Streptococcus pyogenes ) agents has been observed in hospitals in Denmark during last decade. The antimicrobial selection pressure in a hosp isolates from invasive infections (blood and spinal fluid). Erythromycin resistance was detected in five isolatesenvironment (3.%) as might be a reason for the high level of ampic resistant E. faecium as a cause of bloodstream infections. compared to one isolate in (.6% of 67). No resistance to penicillin was detected. Only one of the DCMs (Aalborg) tested all enterococcal blo Group B, C and G streptococci isolates for high-level gentamicin resistance (HLGR). Amon In 3, susceptibility testing was performed on 9the non, 7% were HLGR, whereas 68% of tested E. faecalis isolates duplicate group B streptococcistreptococcus (GBS, agalactiae ) the tested E. faecium isolates were HLGR. The level ofe.hlgr isolates from invasive infections (blood and spinal fluid). faecalis and HLGRE. faecium was similar to or higher than the Erythromycin resistance was detected in isolates level (7.detected %) in many European countries reporting to EA as compared to 7 in (.7%.ofNo 34) resistance tonet in [EARS-Net ].. penicillin was detected Erythromycin (MIC > ug/ml) In 3, vancomycin resistance was detected in 3.4% of In 3, susceptibility testing was performed on 66 nonthee. faecium isolates (n = ) and.% (n = ) ofe.the duplicate group C streptococcistreptococcus (GCS, equisimilis faecalis isolates from bloodstream infections. E. The faecium ands. zooepidemicus ) isolates from invasive infections (blood bloodstream isolates were part of outbreaks with vancomy and spinal fluid). Erythromycin resistance was detected in vana resistant ( ) E. faecium, which is described in Textbox 8.. No of The 3 isolates (4.5%) as compared to 4 in (5.5% 73) level of vancomycin resistant E. faecium was above the resistance to penicillin was found. level reported to EARS-Net by the other Nordic count [EARS-Net ]. In 3, susceptibility testing was performed on 54 nonduplicate group G streptococci isolates from invasive infections Anette M. Hammerum, Stefan S. Olse (blood and spinal fluid). Erythromycin resistance was detected and Line Skjøt-Rasmussen in 6 isolates (.4%) as compared to 7 in (.6% of 6). No resistance to penicillin was found. Tine Dalby, Hans-Christian Slotved and Steen Hoffmann DANMAP 3 85

86 8. RESISTANCE IN HUMAN CLINICAL BACTERIA Textbox 8 Increased occurrence of vancomycin resistant enterococci in Danish hospitals Background: Enterococcus faecalis andenterococcus faecium are commensal bacteria in the intestinee. offaecalis humans. ande. faecium can also cause urinary tract infections (UTI) and fatal infections like sepsis and endocarditis, es among older patients. Enterococci are intrinsically resistant to a number of first-line antimicrobial agents in cephalosporins. Therefore therapy of enterococcal infections may be difficult. Severe enterococcal infection treated with vancomycin, but recently an increase in the occurrence of vancomycin resistant enterococci (V observed in Denmark and internationally. Many of the VRE are also resistant to ampicillin and gentamicin lim treatment possibilities. Newer antibiotics such as linezolid and daptomycin can be used for treatment of VR antimicrobial agents have many side effects. Surveillance of VRE: Since 5, Danish Departments of Clinical Microbiology (DCM) have, on a voluntar submitted vancomycin resistant enterococci for species identification, vana, genotyping vanb, vanc ) and ( surveillance to the Antimicrobial Resistance Reference Laboratory at Statens Serum Institut (Figure ). In and, an increase in the number vana E. of faecium was detected at hospital wards in the Central Denmark Region and screening of faecal samples was initiated [DANMAP and DANMAP ]. In, 54 VRE isolates from clinical infections (UTI, wounds and bloodstream infections) were received at one isolate per patient was included) (Figure ). In 3, the number increased further to 48 clinical isolat 68 faecal screening isolates were received. Most of the patients with VRE were 6 years of age or older. Ne isolates from and 3 vana weree. faecium isolates (Figure ). They were primarily detected at hospitals in the Capital Region, but also from The Zealand Region and the Central Denmark Region. VRE was detected in th regions of Denmark too, but to a much lower extent. Pulsed-field gel electrophoresis typing showed spread vana E. faecium types both inside hospitals and between hospitals. Conclusion: The increasing number and spread of VRE in Denmark is worrying, since only limited number antimicrobial agents are left for treatment of infections. VRE can be carried in the intestine for a long period symptoms of infection and likewise persist in the hospital environment, which makes infection control diffic infection control guidelines are needed to contain the current increase and spread of VRE. The guidelines sh proper cleaning, good hand hygiene, screening for VRE and isolation of patients. Lotte Jakobsen, Brian Kristensen, Robert Skov and Anette M. Ham For further information: Lotte Jakobsen (lja@ssi.dk) Figure. Numbers of vancomycin resistant Enterococcus faecium and Enterococcus faecalis isolates and van genes, Denmark DANMAP 3 3 No. of isolates 5 E. faecalis vanb E. faecalis vana 5 E. faecium vanb E. faecium vana DANMAP

87 Textbox 9 RESISTANCE IN HUMAN CLINICAL BACTERIA 8. Neisseria gonorrhoeae 3 Neisseria gonorrhoeae Background: is the causative agent of the sexually transmitted infection gonorrhoea, which N. in gonorrhoeae usually located in the urethra in males and cervix females.(gonococci) may sometimes be demonstrated in specimens from the pharynx or rectum in either gender, although infections in these sites are generally a Complications of gonorrhoea include e.g. salpingitis, epididymitis, orchitis, and prostatitis. Further, conjunc may occur in newborns after transmission from an infected mother during labour and rarely in adults follow inoculation. Methods: Through decades, all Departments of Clinical Microbiology in Denmark have submitted their isola gonococci to the Neisseria and Streptococcus Reference (NSR) laboratory at Statens Serum Institut for natio of antimicrobial resistance. Most of the received isolates are from urethra or cervix, while specimens from re pharynx are only rarely obtained by clinicians. Occasionally, the NSR laboratory receives strains isolated fro anatomical sites, such as conjunctivae, joint fluid, blood, Bartholin s abscess, etc. At the NSR laboratory ceftriaxone and ciprofloxacin MICs were determined using the Etest on chocolate ag at 35 C in 5% CO. The breakpoints used were those defined by the EUCAST, as modified by ECDC. Both fully a intermediary resistant isolates were categorized as resistant. Penicillinase production was tested using the technique. As part of NSRs participation in ECDCs surveillance of sexually transmitted infections since 9, approxima gonococcus isolates are investigated twice per year for susceptibility to an expanded panel of antimicrobial addition to ceftriaxone and ciprofloxacin, this panel includes azithromycin, cefixime, spectinomycin, and ge Results and discussion: The ciprofloxacin resistance rate increased steadily from 3% in 3 reaching a in 9, followed by a decrease to 56% in 3 (Figure ). The percentage of strains producing penicillinase between 4% in 3 and % in 3. Ceftriaxone resistant gonococci (MIC >.5 mg/l) as well as ceftriaxone treatment failure in patients with have been reported from several countries during recent years. No cases from Denmark have ever been rep 3 through 9 the proportion of isolates with MIC.8 mg/l gradually increased from 4% to near ), but during recent years this shift has nearly reversed. Thus, there is no evidence of emerging ceftriaxone Denmark. In 3, azithromycin resistance including intermediary resistance (MIC >.5 mg/l) in gonococci was 45% to the rate in 9 (Table ). It is worth noting that several recent guidelines for the treatment of gonorrhoe the combination of high dose ceftriaxone and azithromycin ( g or g). Resistance against cefixime (MIC >.5 mg/l) was 9% in 3, i.e. unchanged compared to. Cefixim oral cephalosporin which has never been used in Denmark. During 9 3 none of the isolates were res spectinomycin (MIC > 64 mg/l); however, this parenteral drug is not marketed in Denmark and is not readil in most countries. Gentamicin has been used successfully for the treatment of gonorrhoea in several countries in Africa but no clinical trials evaluating its efficacy have been published. Gentamicin breakpoints for gonococci have not be In through 3, 98% of the examined strains from Denmark had gentamicin MIC 4 mg/l. Conclusions: The centralised national surveillance of antimicrobial resistance in gonococci should be conti Steen Hoffmann For further information: Steen Hoffmann (hof@ssi.dk) DANMAP 3 87

88 8. RESISTANCE IN HUMAN CLINICAL BACTERIA Figure. ciprofloxacin resistance and penicillinase production in gonococci, Denmark, % resistant isolates Number of isolates DANMAP Ciprofloxacin resistant 8 9 Penicillinase producing 3 Number of isolates Figure. Distribution of ceftriaxone MIc values in gonococci, Denmark, 3 3 DANMAP 3 % 9% 8%.5 7%.64 6%.3 5%.6 4%.8 3%.4. % % % Table. Resistance rates (%) against azithromycin, cefixime and spectinomycin in gonococci, Denmark, 9 3 DANMAP 3 Antimicrobial agent 9 3 Azithromycin Cefixime Spectinomycin Number of isolates 88 DANMAP

89 RESISTANCE IN HUMAN CLINICAL BACTERIA Staphylococcus aureus The CC398 cases have in the past years constituted an in part of the CA cases and due to these increasing numbers to CC398 are analysed as a separate group as b Staphylococcus aureus is part of the normal flora from skinbelonging and epidemiology and exposition are different. mucosa in approximately 5% of humans. Some people only S. carry aureus carrys. aureus intermittently whereas others for Surveillance of bacteraemia S. aureus longer time. However, in addition also cause infections S. aureus In 3, bacteraemia cases, corresponding ranging from superficial skin infections i.e. impetigo and boils,769 to 3.9 per, inhabitants, were reported from the to invasive infections such as post-operative wound infections, Departments of Clinical Microbiology (DCM) in Denmark. infections related to intravenous catheters and prosthetic This is somewhat higher than in the previous five years (c. devices, bacteraemia, osteomyelitis, endocarditis and arthritis.,5 annual cases) and may likely reflect underreporting previous years rather than a true increase. Thirty (.7%) o In Denmark, a voluntary surveillance programmes.of all the cases were caused by MRSA. This is at the same level aureusbacteraemia cases was established in 957. Methicillin, and very low compared to most other countrie previous years resistants. aureus (MRSA) has been both laboratory and reporting to clinical notifiable since November 6. At SSI, all isolates are EARS-Net [EARS-Net ]. spatyping and epidemiological information registered. typed by resistance S. aureus in bacteraemia isolates from Based on this information each case is classified withantimicrobial respect 8 3 is presented in Table 8.3. The highest frequenc to possible place of acquisition: hospital (HA), community resistance to other than penicillin was observed for fusidic (CA), healthcare-associated with a community onset (HACO) and import (IMP). MRSA belonging to clonal complex (5%), 398 erythromycin (7%), clindamycin (6%) and norfloxac (5%). Susceptibility to all tested antimicrobial agents was (CC398) has in recent years attracted special attention as this same level as in, but both fusidic acid and norfloxaci type has been closely connected to livestock animals, especially resistance pigs, and increasingly affects people in direct contact with pigs.have increased steadily since 8 (Table 8.3). Resistance to at least, or 3 other antimicrobial agents addition to penicillin was demonstrated in %, 8% and 4% the cases, respectively. Table 8.3. Resistance (%) in isolates from Staphylococcus aureus bacteraemia cases, Denmark Antimicrobial agent Methicillin Penicillin Erythromycin Clindamycin Tetracycline Fusidic acid Rifampicin Norfloxacin Kanamycin Linezolid Mupirocin Trimethoprim-sulfamethoxazole Number of isolates. Note: nt = not tested DANMAP 3 8 % < < nt 9 % < < nt % < 3 < nt % < 4 < < < % < 4 < 3 % < Figure 8.9. Number of MRSA cases, with a three years moving average, Denmark 5 DANMAP 3 5 Number of cases Number of cases 3 years moving average DANMAP 3 89

90 8. RESISTANCE IN HUMAN CLINICAL BACTERIA Surveillance of methicillin-resistant S. aureus The total number of cases and the number of cases presen In 3,,94 new MRSA cases were detected (37.4 per, with infection according to epidemiological classification a inhabitants). This is the highest number of cases observed shownin in Table 8.4. Most of the cases (83%) were acquired over 5 years (Figure 8.9). A case was a person foundinpositive Denmark. The epidemiological classification of MRSA for the first time with a specific MRSA strain regardless whether7 3 is shown in Figure 8.. Despite the infections the patient was infected or colonised only. increasing total number of cases, both the number of hosp acquired infections and the number of healthcare-associat In 3, the number of MRSA increased by 3% compared with community onset (HACO) infections were at a stable to. The number of new cases was more than three verytimes low level. The number of CA infections continued the as high as in 7. In 3, 4 persons were found with their trend in 3 and was by far the largest group increasing second case of MRSA (i.e. MRSA of a new subtype). At the(figure 8.). The proportion of bloodstream infection 44) time of diagnosis, 45% (n = 94) of cases had infection withwhich MRSAiswas.7% in 3 (see surveillance S. aureus of lower than in (57%). CC398 cases increased substantially bacteraemia). in 3 and constituted 3% of new MRSA cases in 3 (Table 8.4). The increase was for a large part due to inclusion of contact, requiring to pigs as a risk factor in December screening for MRSA when admitted to hospitals. This also explains the lower fraction of infections seen for CC398 compared to other. CC groups. See also Textbox Table 8.4. Epidemiological classification of new MRSA cases, Denmark Epidemiologic classification Imported (IMP) Hospital-acquired (HA) Healthcare associated, community onset (HACO) DANMAP 3 3 No. (%) of No. (%) of No. of cases No. of cases cases with cases with (% of total) (% of total) infections infections 34 () 8 (67) 366 (7) (6) 67 (4) 4 (63) 5 () 7 (5) Exposure with healthcare risk 78 () with known exposure without known exposure 6 8 Healthcare worker 9 () Community-acquired (CA) without healthcare risk 76 (47) with known exposure 378 without known exposure 348 CC398 3 (5) Note: Numbers shown in bold are totals Unresolved cases in 3 (notifications not received): 58 (8) 4 (4) 88 (75) 5 (7) () 834 (4) (3) 69 (8) 3 (86) 9 (4) 7 (44) 96 (8) 7 (43) 75 (8) 39 (77) 57 (4) Figure 8.. Number of MRSA infections according to epidemiological classification, Denmark DANMAP No. of infections DANMAP Imported (IMP) Hospital-acquired (HA) Health-care associated, community onset (HACO) CC398 Community-acquired (CA) 3

91 RESISTANCE IN HUMAN CLINICAL BACTERIA 8. Molecular typing of the MRSA strains Resistance among MRSA isolates spa In total,spatyping revealed 7 different strain types of The which resistance patterns varied considerably between types spatype t34 isolates 94 types were associated with clinical infections. The (Table number 8.6). In 3, % of CC398 of isolates belonging to the dominating spatypes isolated in were resistant to tetracycline and 94% of t34 were resista 3 is shown in Table 8.5. They constituted 6% of to theclindamycin. total In contrast, the majority of t9, a primaril spatype, and t34 were susceptible to number of MRSA isolates. spa Ten types constituted 56% ofcommunity-acquired the 94 clinical infections with MRSA. Most prevalent spatypes all tested antimicrobial agents except for beta-lactams. It i causing clinical infections at time of presentation were noteworthy t34 (n that 47% (34 of 643) of CC398 isolates had th = 6), t (n = 9), t8 (n = 7), t9 (n = 67), unusual t7 (n = phenotype 43), of being susceptible to erythromycin w t4 (n = 8), t44 (n = 8), t34 (n = 6), t437 (n = resistant 5) andtot3 clindamycin. Whole genome sequencing of 5 (n = ). The PVL encoding lukf-pv gene was demonstrated CC398 in isolates showed that this was due to presence lnu(b) of th 36% of the infections and in 4% of the asymptomatic gene carriers a gene previously reported in MRSA CC398 strains and most often in relation to isolates spawith types t8 (n =Spain, Portugal and USA. Whether lnu(b)is responsible for 97), t9 (n = 9), t (n = 59) and t44 (n = 45). this phenotype in general needs to be investigated. Resist to at least, or 3 other antimicrobial agents in addition t β-lactam antibiotics (cefoxitin/penicillin) was demonstrated 73%, 6% and 43% of the cases, respectively. Andreas Petersen, Robert L. Skov an Anders Rhod Larsen Table 8.5. The ten most prevalent spa types demonstrated in MRSA cases, Denmark DANMAP 3 spatype (a) CC group No. of cases No. causing infections (%) t34 CC (4) t CC5 6 9 (56) t8 CC8 3 7 (64) t9 CC (73) t7 CC (49) t34 CC (34) t CC (6) t3 CC 49 (43) t3 CC 48 6 (33) t44 CC (6) a) CC = Clonal complex Table 8.6. Resistance (%) in the six most prevalent spa types demonstrated in MRSA cases compared with all MRSA cases, 3 DANMAP 3 spatype Clonal complex Erythromycin Clindamycin Tetracycline Fusidic acid Rifampicin Norfloxacin Kanamycin Linezolid Mupirocin Trimethoprim-sulfamethoxazole Number of isolates t34 CC398 % < 57 t CC5 % < 6 t8 CC8 % t9 CC3 % 5 9 t7 CC % t34 CC8 % All cases % < < 3 94 DANMAP 3 9

92 8. Textbox RESISTANCE IN HUMAN CLINICAL BACTERIA Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among humans in 3 Background: Since 3, livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged worldwide. LA-MRSA primarily belong to clonal complex 398 (CC398) and is especially associated with pigs. MRSA CC398 has especially been found in persons with contact to pigs. A new type of MRSA carrying a different resistance determinant mecchas been found in humans, cattle and sheep in Denmark and internationally but do not appear to have the same close relationship to animals as CC398 [G et al.. Lancet Infect Dis. : ]. Livestock associated MRSA in humans: The number of persons positive for MRSA CC398 steeply increa in 3 (4 in 9, in, 64 in and 3 in ). A significant part of the increase was asso inclusion of contact to pigs as a risk factor requiring screening for MRSA when admitted to hospitals in Dece [ Thespa most typefrequent related to CC398 was type t34 (n = 57). The majority of CC398 cases (n = 56, 87%) were in persons with documented clo pigs or being a household member to a person with direct contact. This resembles the proportion from prev which means that the actual number of CC398 cases without documented contact to livestock increased co from the previous years. There were however, no signs of significant spread of CC398 to urban areas. One hundred and fifty-seven CC398 cases (4%) presented with infections. In 3, four bacteraemia cases CC398. Two of the patients died within 3 days. In both cases, the patients had severe comorbidity. The pat have any direct contact to pigs. MRSA isolates carrying themec new A homologue mec C, were demonstrated in 4 cases (%) in 3 (9 in 9, i, 37 in, and 4 in ). Twenty-eight of the cases (68%) had infections, including one bacteraem of diagnosis. Only one possible livestock contact was registered mec C cases. for the 4 In 3, no animal surveillance of LA-MRSA was performed, but sampling at farm level is ongoing and expe finalized in 4. Conclusions: In 3, a steep increase in CC398 cases was detected and CC398 became the most commo among human MRSA cases. An increasing number of CC398 cases had no documented contact to pigs. The MRSA isolates carryingmec the C gene seems to have stabilized. Andreas Petersen, Anders Rhod Larsen and Robert L. For further information: Robert L. Skov (rsk@ssi.dk) DANMAP 3 9 DANMAP 3

93 9 MATERIALS AND METHOD DANMAP 3 93

94 9. MATERIALS AND METHODS 9. Materials and methods 9. General information The pharmacy or company either sells the medicines to veterinarians for own use in practice or for re-sale to farme For the DANMAP 3 report, population sizes and sells the medicines directly to the animal holder on presen of a prescription. By law, the profit that veterinarians may geographical data were obtained from Statistics Denmark [ and data on general practitioners fromon thethe sale of medicine is very small (5%), thereby limiting economic incentive to sell medicine. Danish Medical Association [ In 3, the animal owners and veterinarians purchased th The epidemiological unit for pigs and cattle was defined agents almost equally between the pharmac as the individual farm, meaning that only one isolateantimicrobial per and the veterinary drug trading companies, while only % bacterial species per farm was included in the report. The purchased same epidemiological unit was applied for broilers, except for from the feed mills. Sales from feed mills additi comprised zinc chloride for the pig production on veterinar Salmonella where the individual flock of broilers was defined prescription, and non-prescription sales of coccidiostatic as the epidemiological unit. For food, the epidemiological unit agents for domestic fowl (Gallus gallus). was defined as the individual meat sample. For humans, the epidemiological unit was defined as the individual patient and the first isolate per patient per year was included. Data on all sales of veterinary prescription medicine from pharmacies, private companies, feed mills and veterinaria aretrends sent electronically to VetStat. Veterinarians are require Unless stated specifically, all differences and temporal law to report to VetStat all use and prescriptions for produ noted in the text are statistically significant (p<.5) using animals (monthly submissions). For most veterinarians, th either Chi-square tests or linear logistic regression (see Section registration of data is linked to the writing of invoices. How.6.4). errors in the veterinarians invoice system sometimes caus errors in amounts reported, and these data are not validat 9. Data on antimicrobial consumption entry to Vetstat. The electronic registration of the sales at pharmacies is linked to the billing process and stock accou Antimicrobial agents used for humans and animals inthe Denmark pharmacy, which ensures a very high data quality rega are presented in Table 3.. amounts and identity of drugs. 9.. Data on antimicrobial consumption in The VetStat database contains detailed information about source and consumption for each prescription item: date o animals sale, identity of prescribing veterinarian, source ID (identit Since, consumption data presented in this report have of the pharmacy, feed mill, or veterinarian practice reporti been obtained from the national monitoring programme package identity code and amount, animal species, age-gr VetStat, which is a database hosted by the Danish Veterinary disease and Food Administration. Prior to, data were based on category and code for farm-identity (CHR - Danish Central Husbandry Register). The package code is a uniqu national sales figures from the pharmaceutical industry. identifier, relating to all information on the medicinal produ such as active ingredient, content as number of unit doses Data registration number In Denmark, all therapeutic drugs are prescription-only and of tablets), package size, and code of the antimicr agent in the Veterinary Anatomical Therapeutic Chemical VetStat collects data on all medicines prescribed by veterinarians (ATCvet) for use in animals, except in a few instances when medicines classification system. are prescribed on special license (i.e. medicines not approved Knowledge of the target animal species enables the presen for marketing in Denmark). In addition, data on consumption ofand consumption data in defined animal daily doses - a na of coccidiostatics as feed additives (non-prescription) veterinary equivalent to the international defined daily dos antimicrobial growth promoters (not in use since ) are (DDDs) system applied in the human field [ collected by VetStat. See further description of the ADD system in the DANMAP 9 at report Until 7, antimicrobial agents could only be purchased the [ pharmacy or in medicated feed from the feed mills. From April Methods 7, the monopoly was suspended and private companies DANMAP, we want to compare consumption of (two in ) were permitted - on certain conditionsin (identical antimicrobials between different animal populations and conditions as for pharmacies) - to sell prescribed veterinary between medical products for animals. In addition, price setting was veterinary and human sectors. In order to do this need liberalised, which allowed for discounts corresponding to to take into account the quantity of antimicrobials us their potency, their formulation, the route of administratio lower administration costs related to sale of large quantities to and sometimes the age of the animals in which they ar the veterinarians. used. We also need to know the size of the populations to the antimicrobials are administered. 94 DANMAP 3

95 MATERIALS AND METHODS 9. The animal daily doses (ADDs) used in previous DANMAP Denominator reports are an integrated part of the VetStat database Trends and have in veterinary consumption, both within and across been described elsewhere [DANMAP 9; etjensen al., Prev species, are presented in DADD per, animals per Vet Med. vol: 64, -5, 4]. In principle, the ADD dayshould DAPD. The number of animals in a population (in be identical for all products within medicinal groups and epidemiological terms: the population at risk) is represente defined by the active compound, route of administration by their andlive biomass. The biomass of a species is calculate the formulation. However, in VetStat, the ADDs are defined taking into by account average live body-weight and the aver the dosage level that was part of the product registration, life-span andof the species. over time doses for products within the same medicinal group sometimes have changed. The greatest variation occurs when the ADD for some products have been defined solely based on the approved dosage. For example, the approved dosage of a DAPD - DADD per, animals per day product registered in may differ from a similar product registered in. The number of DADDs administered to a specific animal species during a year (in thousands) divided by the In DANMAP, we therefore introduced two new metrics to follow trends in antimicrobial consumption to ensurenumber of standard animals at risk per day. The number robustness of the analyses over time. The new metrics,standard DADD animals at risk per day takes into account spec differences in average body-mass and lifespan. When and DAPD, are defined below in the highlighted boxes. relevant, the numbers of DADDs and standard animals at risk are estimated for specific age groups, or simply a number of doses (DADDs) used to treat one kg of anima divided with the total estimated biomass (in tonnes). DADD - Defined animal daily dose DAPD is a statistical measure, providing a rough estimat DADD is the average maintenance dose per day for aofdrug the proportion (in thousands) of animals treated used for its main indication in the appropriate animaldaily with an average maintenance dose of a particular species. The DADD is not defined at product level butantimicrobial for agent. For example, DAPDs indicate tha each antimicrobial agent, administration route and animal an estimated % of the population, on average, receives species and when appropriate, also age group. DADDa has certain treatment on a given day. The DAPD is also been specifically defined for use in DANMAP and does referred to as the treatment proportion. not always completely match the prescribed daily dose or the recommended dosage in the Summaries of Product In principle, the metric DAPD is parallel to the metric Characteristics (SPC). used in pharmaco-epidemiology for the human sector, Defined daily dose per, inhabitants per day (DID), see Section The DADDs used in DANMAP 3 are presented in the web annex. Due to a relative high number of pigs exported around 3 (3% of pigs produced in 3, Table 3.), an adjusted me The basic principles for the DADD are similar to the principles consumption per pig was calculated. The adjustment is previously described for the ADD. The designation ofofthe on DADD is based on the VetStat ADDs, but re-defined for the assumption that pigs exported at 3 kg, on average received the same amount of antimicrobial agents before each group of antimicrobial agents, i.e. for each combination export as other pigs from farrowing to 3 kg. of active compound, administration route, formulation, considering the following principles: Antimicrobial use per pig produced (adjusted) = [DADDs +DADDw + (+Q)*DADDf ] / (biomass-days-total+ Nw*. Minor inconsistencies, e.g. due to rounding of numbers, 58(kg*days)), where DADDs = amount of antimicrobial have been corrected; agents used in sows; DADDw = amount of antimicrobial. Approved dosage for the most widely used antimicrobial agents used in weaners; DADDf = amount of antimicrobial products were given priority above dosage for products agents used in finishers; Q is the proportion of weaning pig that are rarely used; exported 3. Approved dosage for older products within the group are around 3 kg. Nw = number of pigs exported at 3 kg bodyweight, and Nw*58 is the number of biomass da maintained as the common DADD even if a new product the exported pigs would have contributed to the live biom is approved with a higher dosage; not exported. 4. In determining the dosage for a group with large if variation between approved dosages of the products, the dosages 9.. Estimation of live biomass of animals in accordance with the dose given in The Veterinary Formulary [British Veterinary Association, 5, The 6th estimation of live biomass and thus the number of sta edition] are applied; animals at risk per day depends on the available data sour 5. Dosages may vary within active compound and for each species. administration route, if different dosages have been approved for different age group/indication or formulation. Broiler and layer production (Gallus gallus). The live biomass is estimated based on number of broilers When principle and 3 are conflicting, principle 4 is produced applied. (Table 3.), and an average live weight at slaugh DANMAP 3 95

96 9. MATERIALS AND METHODS relatively.97 kg [Statistics Denmark, 3] after an estimated averagestable during the last ten years [Danish Dog regi ]. life span of 3 days. The mean live biomass per broiler is The average live weight for cats and dogs were est to 4 kgthe and kg, respectively (based on pedigree registra assumed to be half of the weight at slaughter. In addition, data). biomass of the parental animals (rearing and breeding) for the broiler production was estimated for based on number of hens per year (,9 mill) annual rotations, length of the empty Aquaculture. The estimation is based on data from the D periods, percentage of cocks, and average weight ofagrifish the cocks Agency (NaturErhvervstyrelsen) on produced and hens in rearing and in breeding [DANHATCH, 3; amounts in each subtype of production, and information o S. Kabell, Danish Agriculture and Food Council, personal the typical lifespan and entrance and exit body weights, a communication]. For the other years, the biomass ofwere rearing calculated in cooperation with Danish Aquaculture [N and breeders was assumed to be proportionate to the broiler Danish Aquaculture]. Data from were not Henriksen, production as in. available at the time of publication. In the layer production chain, the biomass of the parent flocks is estimated separately for each production type, based on Data on antimicrobial consumption in 9..3 number of eggs produced, eggs per hen,and average production humans length [Danish Poultry Producers, Statistics Denmark]. We onon consumption of antibacterial agents in humans we have assumed an average weight of.3 kg per hen, Data based obtained from Statens Serum Institut (SSI), National Regist slaughter weight [Statistics Denmark, 3]. Medicinal Products Statistics. SSI has the legal responsibili monitoring the consumption of all human medicinal produ Turkey production. The live biomass is estimated based on This is performed by monthly reporting of consumption fro the number of turkeys produced (Table 3.) and an average all pharmacies in Denmark, including hospital pharmacies, live weight at slaughter of kg for male turkeys and kg to SSI. Data from the primary healthcare sector have been for hens after an estimated average life span of weeks and collected since 994, whereas data on consumption in hos 5.5 weeks, respectively [Danish Agro; S. Astrup, personal are available from 997. communication]. The estimated mean live biomass per turkey is assumed to be half of the weight at slaughter. Certain categories of hospitals were excluded when the consumption was measured by occupied bed-days and Pig production. The estimation was based on number of admissions. Data from private hospitals and clinics, psychi pigs produced, including exports at different ages [Statistics hospitals, specialised non-acute care clinics, rehabilitation Denmark; Danish Agriculture and Food Council], productivity centres and hospices were excluded from DANMAP data for each year [Danish Agriculture and Food Council] (representing approximately 3% of the antimicrobial and census data for breeding animals [Statistics Denmark]. consumption at hospitals and of the number of bed-days). The average weight and lifespan for the growing animals In Denmark, all antimicrobial agents for human use are (piglets, weaners and finishers) were estimated from the prescription-only medicines and are sold by pharmacies in productivity number. The estimation methods were developed defined packages. Each package is uniquely identified by a in cooperation with Danish Agriculture and Food Council. code, which can be related to package size, content as num There are no statistics on average weight of breeding animals of unit doses (e.g. number of tablets), content as number o available, so an estimated average weight had to be assumed. Defined Daily Doses (DDDs), code of the antimicrobial age However, the size of the breeding animals has probably in the Anatomical Therapeutic Chemical (ATC) classificatio increased over the last decade, but this could not be accounted system, and producer. In addition, the following informatio for. is collected for each transaction: social security number (C number) of the patient, code identifying the prescribing Cattle production. The live biomass of the cattle population physician,isdate and place (pharmacy, hospital pharmacy, estimated from census data [Statistics Denmark, 4] and the of the transaction, and information regarding institution) average live weight of the different age groups. The reimbursement Danish cattle of cost, if applicable. The data are transfer population is mainly dairy, particularly Holstein Friesian, but to SSI in an electronic format. monthly also other breeds such as Jersey and a small population of beef cattle. Most of the cattle slaughtered are dairy cows and bull From DANMAP and onwards, the consumption calves of dairy origin. The average live weight was estimated of certain infusion substances, such as cephalosporins, for different age and gender categories. carbapenems and trimethoprim, has been directly reporte the hospital pharmacies to SSI. In previous DANMAP report Fur animals. The live biomass of mink is estimated the from consumption of these substances was corrected by dir production data [Statistics Denmark, 3; Kopenhagen data collection from all Danish hospital pharmacies; howev Fur, 3] and the average weight at pelting was.45 kg DANMAP all data were delivered only by SSI. since [Kopenhagen Fur, 3]. The progeny live for approximately 7 months. The biomass for the breeding animals (female) was The present report includes data on the consumption of estimated based on census data and an assumed average live antibacterial agents for systemic use, or group J, of the weight of kg. 3 update of the ATC classification, in primary healthcar and in hospitals. As recommended by the World Health Pet animals. Only dogs and cats are taken into account, as the (WHO), consumption of antibacterial agents i Organization other population sizes are negligible in Denmark, and relatively primary healthcare is expressed as DIDs, i.e. the number o rare in veterinary practice. The population is based DDDson per, inhabitants per day (DDD/, inhabitan census data [Statistics Denmark, ] estimating 65, cats days). Consumption in primary healthcare is also reported and 55, dogs. The number of dogs in Denmark has been a number of packages per, inhabitants. Consumption 96 DANMAP 3

97 MATERIALS AND METHODS 9. of antibacterial agents in hospitals is expressed as DIDs, for and broilers is presented in Table 9.. The isolation C. rates o comparison with primary healthcare, and DBDs, the jejuni number from pigs and C. colifrom cattle and broilers were low of DDDs per occupied beds per day (DDD/ occupied and therefore MIC-analyses were not performed. bed-days). Salmonella. National Food Institute DTU is the national Since antimicrobial consumption expressed as DDD/ reference laboratorysalmonella for in animals and food and occupied bed-days does not necessarily reflect changes in all isolates for typing. Only one isolate per serotyp receives hospital activity and production, consumption in hospitals is was selected for the DANMAP report, except for per farm also presented as DAD (the number of DDDs per isolates admitted from broilers, where one isolate per flock was inclu patients). Isolates of S. Typhimurium include the monophasic variants with antigenic formulas S. 4,5,:i:- and S. 4,:i:-. The number of occupied bed-days is calculated as the date of discharge minus the date of admission (minimum one day), The Salmonella isolates from pigs originated both from and the number of admissions is calculated as one admission the random sampling of healthy animals at slaughter for whenever a patient is admitted to one specific ward DANMAP (one patient and from the nationalsalmonella surveillance can be registered as admitted multiple times if transferred programme where the results of a serological surveillance between wards during one hospital stay). Data on the number the slaughterhouses and in all breeding herds appoint risk of occupied bed-days (or patient-days) and number of herds to be further examined by microbiological analysis o admissions in each hospital were obtained from the National pen-faecal samples. Patient Registry at the National Board of Health [ NoSalmonella data from layers, broilers and cattle are presen 9.3. collection of bacterial isolates in DANMAP 3 due to the low findings of serotype S. Enteritidis ands. Typhimurium.Salmonella isolates from diagnostic submissions were not included in DANMAP Animals Samples from animals are collected from healthy production animals randomly selected at slaughter. From pigs, isolates Further of details on the sampling procedures and the finding Escherichia coli, Enterococcus faecium, Enterococcus faecalis, the Danish Salmonella surveillance programs are presented in and thermophilic Campylobacter spp. were collected. FromTextbox 5, and in the Annual Report on Zoonoses in Denm Campylobacter cattle, isolatese.ofcoliand thermophilic spp. 3 [ E. coli were collected, and from broilers isolates, thermophilic of Campylobacter spp.,e. faecalis ande. faecium were collected.isolates from diagnostic submissions were specifically In addition, isolates of E. colio49, were collected fromcollected for the DANMAP programme at the Laboratory of diagnostic submissions. Swine Diseases, the Danish Agriculture and Food Council, KjellerupE. ( colio49 from diarrheic pigs). Only one isolate Campylobacter spp., indicator E. coliand enterococci. Samples per farm was included. from healthy pigs, cattle and broilers were collected for the DANMAP programme at slaughter by meat inspection9.3. staff or Meat abattoir personnel and sent for examination at DTU National Campylobacter, indicator E. coliand enterococci. The meat Food Institute. For broilers, cloacal swabs were collected weekly isolates originated from meat samples collected at wholes from May through October and the sampling programme and retail outlets in all regions of Denmark. All samples we represented 86% of all broiler farms in Denmark. A Danish collected by the Danish Veterinary and Food Administratio broiler farm is typically comprised of more than one unit each (DVFA) Food Control Offices and were collected during the generating several flocks per year, but even though routine a farm was inspection by the authorities or on specific request sampled more than once through the sampling period, only from the for the DANMAP programme. The sampling includ one isolate per farm of each bacterial species was included. both Danish and imported meat. Only one isolate per bact species per meat sample was selected for DANMAP. For pigs and cattle, the slaughter plants included in the DANMAP programme accounted for 99% and 95% ofsalmonella the. The Salmonella isolates from Danish pork total number of animals slaughtered in Denmark during 3, from the national Salmonella originated surveillance respectively. The number of pigs and cattle samples programme from each (swab samples from pork and beef carcasses t slaughter plant was proportional to the annual number of slaughterhouse after cooling). at the Salmonella isolates from animals slaughtered at the plant. Samples were collected oncepoultry meat and other imported fresh meats de imported a month from January through November as caecumfrom samples a case-by-case risk assessment control programme from pigs and rectum samples from cattle. Only one (DFVA), isolate per are not presented due to the low number of isolate farm of each bacterial species was included.. Textbox (<5). Further details on findings are presented in Only one isolate per positive swab sample or batch of mea Accordingly, the bacterial isolates from the Danish production included for DANMAP. IsolatesS. oftyphimurium include animals may be regarded as representing a stratifiedthe random S. monophasic variants, antigenic S. formula 4,5,:i:- and sample of the respective populations, and the observed 4,:i:-. prevalence of resistant isolates provides an estimate of the true occurrence in the population Humans S.Typhimurium, S.Enteritidis and C. jejuni. Antimicrobial An overview of the number of samples analysed, isolates susceptibility was performed on human faecal isolates sub obtained and MIC determinations performed for pigs, cattle DANMAP 3 97

98 9. MATERIALS AND METHODS to Statens Serum Institut (SSI). Campylobacter isolates were E. faecium were identified by motility- and arginine dihydrola submitted from Departments of Clinical Microbiologytests (DCM) and the ability to ferment mannitol, sorbitol, arabino covering three geographical regions: Northern Jutland, and Funen raffinose. All isolates E.of faecium and E. faecalis were and Roskilde/Køge. Information on travel history wasstored obtained at -8 C. for the patients. Salmonella isolates were submitted from all DCM in Denmark. Exact figures of the proportion tested andmeat 9.4. the sampling strategy for the different species can be found inwas isolated by the regional laboratories at the Salmonella Sections 6. and 6.. DVFA according to the open reference methods issued by t NMKL (NMKL No. 87, 7 or NMKL No. 7, 999), the. All blood isolates were referred to Staphylococcus aureus ISO 6579: or alternative methods validated against th the Staphylococcus reference laboratory at SSI on a reference voluntary method according to ISO 64:. Sero-typin basis. In November 6, methicillin-resistant S. aureus was performed at the National Food Institute, DTU. (MRSA) became a notifiable disease in Denmark and it became mandatory to submit all MRSA isolates to the reference Campylobacter was isolated according to the guidelines laboratory. for microbiological examination of food (NMKL No. 9, 7). Identification was performed by microscopy and by Invasive Streptococcus pneumoniae, Streptococcus pyogenes oxidase activity, catalase activity and the ability to hydroly (group A streptococci), group B, C and G streptococci. indoxyl acetate and hippurate. Isolation and identification Invasive pneumococcal disease is a notifiable disease in performed by the laboratories at the DVFA in Ringsted was Denmark, and therefore all invasive isolates nationwide are sent Denmark. All isolates C. of jejuni, C. coliandc. lariwere sent to to SSI for identification or confirmation as well as susceptibility the National Food Institute, DTU for MIC-testing and storag testing and typing. Invasive group A, B, C and G streptococcal at -8 C. isolates are referred to SSI on a voluntary basis. Traditionally, only isolates from blood and spinal fluid are includedindicator in the E. coliwas isolated by the regional laboratories at DANMAP report. the DVFA by adding 5 g of the sample to 45 ml of MacConk or laurylsulphate-broth, which was incubated o/n at 44 C, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, subsequently streaked onto violet red bile agar and incuba invasive E. faecium and invasive E. faecalis. Data were for 4 h at 44 C. Presumptive E. coliwas identified using TBX provided on all isolates recorded from either blood E. samples ( agar (β-glucoronidase activity) incubated o/ne.atcoli 44 C. coli, K. pneumoniae, P. aeruginosa, E. faecium ande. faecalis ) isolates were sent to DTU National Food Institute for MICor urine samples E. (coliand K. pneumoniae ) submitted for testing and storage at -8 C. susceptibility testing to the DCM at the following hospitals: Rigshospitalet, Hvidovre, Herlev/Hillerød, Region Zealand, Indicator enterococci were isolated by the regional labo Odense, Esbjerg, Vejle, Herning/Viborg, Aarhus and Aalborg. at the DVFA by adding 5 g of the sample to 45 ml azide de No samples were collected from healthy humans. broth, incubated o/n at 44 C and subsequently streake ont Slanetz-Bartley agar. After incubation at 44 C for 48 h, colo 9.4 Isolation and identification of bacteriatypically for E. faecium ande. faecalis were identified by a real9.4. Animals time PCR assay, and sent to DTU National Food Institute fo MIC-testing and storage at -8 C. Campylobacter. The samples from broilers was inoculated Humans directly onto mccd agar (Oxoid, Denmark) and incubated Salmonella isolates were serotyped by slide agglutination in microaerophilic atmosphere for -3 days at 4.5 C. For samples from cattle, selective enrichment in Prestonaccording broth at to the Kauffman-White Scheme. a ratio of : incubated in microaerophilic atmosphere for 4 h at 4.5 C was performed followed by inoculation ofcampylobacter µl. Species identification was performed using a of the enrichment broth to mccd agar. Campylobacter -like species-specific PCR assay [Klena et al.4. J Clin Microbiol. colonies were verified by microscopy and species identification 4: ]. of C. jejuniandc. coliwas performed by a real-time PCR assay [Mayret al., J Food Prot. 73():4-5]. All isolates C. Staphylococcus of aureus. Sequencing ofs.the aureus specific spa jejuniandc. coliwere stored at -8 C. gene was performed for species confirmation and Spa- typing. negative isolates were confirmed S. aureus as by MALDI-TOF. Indicator E. coli. The material was inoculated directly onto Thespa-typing [Harmsen et al.3. J Clin Microbiol. 4: 544 Drigalski agar (SSI Diagnostica, Denmark) and incubated 5448]o/n and additional typing by multi locus sequence typin at 37 C. Yellow colonies were inoculated onto BBL CHROM (MLST) [Enright et al.. J Clin Microbiol. 38: 8 5] agar Orientation Medium (Becton Dickinson, Germany) and were annotated using eburst v.3 software (www. mlst.net red colonies were collected E. coli as after o/n incubation atbased on the spaand MLST typing, each isolate was assigned 37 C. All isolates were stored at -8 C. to a clonal complex (CC). For MRSA isolates, presence of th mecaor meccmethicillin resistance genes was confirmed by PCR [Larsenet al.8. Clin Microbiol Infect. 4: 6 64; Indicator enterococci. An adequate amount of material Stegger et al.. Clin Microbiol Infect. 8: 395 4]. For all suspended in ml of sodium chloride (.9%) was inoculated isolates, presence lukf-pv of gene (PVL) was demonstrated by on Slanetz Bartley agar and incubated two days at 4 C. Three colonies resembling typical E. faecalis and E. faecium PCR [Larsenet al.8. Clin Microbiol Infect. 4: 6 64; et al.. Clin Microbiol Infect.8: 395 4]. morphology were sub-cultivated on blood E. faecalis agar.and Stegger 98 DANMAP 3

99 MATERIALS AND METHODS 9. Invasive Streptococcus pneumoniae. Serotype identificationdiscs of (Oxoid, Roskilde, Denmark), respectively, on Müllerinvasive S. pneumoniae was performed by latex agglutination Hinton agar (Müller-Hinton plate, 5% blood, mg beta(immulex Pneumotest Kit, SSI Diagnostica, Hillerød, NAD, SSI Diagnostica, Hillerød, Denmark). Penicillin and Denmark) and further with factor specific antisera byerythromycin the MICs were determined using STP6F plate, Neufeld quellung test (SSI Diagnostica, Hillerød, Denmark). Sensititre (Trek Diagnostic Systems Ltd., East Grinstead, UK as recommended by the manufacturer. All breakpoints use were as defined by the EUCAST. Both fully and intermediar Invasive Streptococcus pyogenes (group A streptococci), resistant isolates were defined as resistant. group B, C and G streptococci. Identification of groups were performed by latex agglutination (Streptococcal Grouping Invasive Streptococcus pyogenes (group A), group B, C Reagent, Oxoid, Roskilde, Denmark). and G streptococci from humans. Screening for penicil erythromycin- and clindamycin-resistant streptococci was 9.5 Susceptibility testing performed with unit penicillin G, 5 discs μg erythromycin discs and µg clindamycin discs (Oxoid, Roskilde, Denmark Antimicrobial susceptibility testing of Salmonella, respectively, on Müller-Hinton agar (Müller-Hinton plate, 5% Campylobacter, indicator E. coli, Enterococcus, Staphylococcus blood, mg beta-nad, SSI Diagnostica, Hillerød, Denmar aureusand the veterinary pathogens was performed as Isolates were simultaneously tested for inducible clindamy microbroth dilution MIC with Sensititre (Trek Diagnostic resistance. Non-sensitive streptococci were tested further Systems Ltd., East Grinstead, UK). Inoculation and incubation the respective E-tests (Biomérieux), either benzylpenicillin procedures were in accordance with the CLSI guidelines erythromycin or clindamycin on Müller-Hinton agar. The [Clinical and Laboratory Standards Institute, USA] and the breakpoints used were as defined by the EUCAST. Both full European standard ISO 776-:6. and intermediary resistant isolates were defined as resista The quality control strains Staphylococcus aureus ATCC 93,, K. pneumoniae, invasive P. aeruginosa, invasive E. Escherichia coli ATCC 59, Pseudomonas aeruginosa ATCC E. coli ande. faecalis from humans. The DCM performed 7853, Enterococcus faecalis ATCC 9 and Campylobacterfaecium either disk (Oxoid, Basingstoke, UK) or tablet (Neo-Sensita jejuniatcc 3356 were used. A/S Rosco) diffusion susceptibility testing on a number of media. As per September, all DCM except Rigshospit Isolates from animals and meat were tested at DTU National used breakpoints defined by EUCAST. Food Institute, and the Salmonella, Campylobacter and Staphylococcus aureus isolates of human origin were tested at Data on antimicrobial resistance from private hospitals and SSI. MIC-testing at DTU National Food Institute is accredited clinics and from psychiatric hospitals were excluded. All by DANAK (the national body for accreditation). submitting laboratories participate in national and internat quality assurance collaborations such as the United Kingdo One isolate per bacterial species per farm, per meat National sample External Quality Assessment Schemes (NEQAS). or per patient was tested for antimicrobial susceptibility. For Salmonella isolates from poultry, one isolate per serotype per Data handling flock was tested. For isolates in excess numbers (e.g.9.6 isolates from healthy animals), a random selection was appointed to 9.6. Animal MIC. Table. presents the interpretation of MIC-values The usedresults for from the analysis of all animal samples positi any combination of bacteria and antimicrobial agent.assince well as negative findings - and of the bacteria isolated a 7, data were interpreted by EUCAST epidemiological cut-off the susceptibility testing were stored in an Oracle Databas values (ECOFFs) with a few exceptions described in Table.. Edition at DTU National Food Institute. The 9i Enterprise The corresponding clinical breakpoints validated by EUCAST susceptibility data were stored as continuous values as we are presented both in Table. and in the MIC-distribution categorised as susceptible or resistant as defined by the re tables to visualize the impact of the use of ECOFFs contra ECOFF. Each isolate was identified by the bacterial species clinical breakpoints. In general, if ECOFFs were re-validated subtype as applicable and by the date of sampling and spe and changed by EUCAST during the past year, all data presented of animal. Information on the farm of origin was also record from previous years were interpreted using the changed All handling and evaluation of results were carried out usin ECOFFs. Data from susceptibility testing Staphylococcus of SAS Software, SAS Enterprise Guide 4.3. aureuswere interpreted using EUCAST clinical breakpoints. All MIC-distributions are presented in the web annex9.6. at www. Meat danmap.org. Each of the tables provides informationresults on the from the analysis of food samples were reported vi number of isolates, the applied interpretation of MIC-values the database administrated by the DVFA, except for the da and the estimated level of resistance and confidenceon intervals. Salmonella, which were reported to and extracted from Multi-resistance was defined as resistance to three or more the laboratory database at the National Food Institute, DTU of the antimicrobial classes listed in Table.3. Isolates were bacterial isolate, information was available on foo For each considered fully sensitive if susceptible to all the antimicrobial type, bacterial species, date and place of sampling, date o agents included in the test. examination, country of slaughter, and an identification number, which makes it possible to obtain further informat Invasive Streptococcus pneumoniae from humans. Screening about the isolate from the relevant authorities. Furthermor for penicillin- and erythromycin-resistant S. pneumoniae was information about the country of origin was recorded when performed with μg oxacillin discs and 5 μg erythromycin possible. DANMAP 3 99

100 9. MATERIALS AND METHODS Human E. coli P. aeruginosa, K. pneumoniae, invasive, invasive. Data on E. faecium and invasive E. faecalis. Ten out of eleven DCM Salmonella andcampylobacter Salmonella and in Denmark provided data on resistance levels E. coli,in K. Campylobacter infections are stored in the Danish Registry pneumoniae, invasivee. faecium and of Enteric Pathogens (SQL database) maintained by SSI. This, invasive P. aeruginosa E. faecalis isolates. Data were extracted from the register includes only one isolate per patient within ainvasive window following of six months and includes data on susceptibility testing of laboratory information systems: gastrointestinal pathogens. ADBakt (Autonik AB, Skoldinge, Sweden) for the DCM a Staphylococcus aureus. For MRSA, data on the characteristics Hvidovre, Herlev and Aalborg Hospitals. of the isolates and the clinical/epidemiological information were MADS (DCM Skejby Hospital, Aarhus, Denmark) for obtained from the Danish MRSA register at SSI (mandatory the DCM at Rigshospitalet and Slagelse/Region Zealand reportable). Patients were registered, regardless of whether it Esbjerg, Vejle, Herning/Viborg, and Aarhus Odense, was colonisation or infection, the first time they were diagnosed (Skejby) Hospitals. with MRSA or when a new subtype was demonstrated. Based on the reported information, MRSA cases were classified Resistance data on the first isolate per patient per year as colonization/active screening (i.e. surveillance samples were included. Generally, resistance data were excluded if to detect nasal, throat, gut or skin colonization), imported susceptibility to a certain antimicrobial agent was tested o infection (i.e. acquired outside Denmark), infection acquired only a selected number of isolates. in a Danish hospital, defined as diagnosed >48 hours after hospitalisation with no sign of infection at admittance (HA- Statistical tests MRSA) or infection diagnosed outside hospitals (community Significance tests of differences between proportions of onset). resistant isolates were calculated using SAS Software, SA Enterprise Guide 4.3 or StatCalc in EpiInfo v. 6. Differenc MRSA cases with community onset were further classified in pair-wise comparisons were tested using Chi-square, or according to risk factors during the previous months as Exact Test when the number of samples is low (< Fisher s either healthcare associated with community onset (HACO) When appropriate, significance of temporal trends is tested or community-acquired (CA). Healthcare associated using risk linear logistic regression using Proc LOGISTC procedu factors included prior hospitalizations or stay in long-term in SAScare (Likelihood ratio test). facilities within months prior to MRSA isolation and being a healthcare worker. Community risk factors included known In the text, commented differences imply statistically signi MRSA-positive household members or other close contacts. where p<.5. Due to the increasing numbers of cases belonging todifferences CC398 were treated separately as both epidemiology and exposition When comparing proportions between years, the EUCAST are different from other CA cases. epidemiological cut-off values for 3 were also used for Streptococcus pneumoniae, Streptococcus pyogenes (group interpretation of previous years MICs. A streptococci), group B, C and G streptococci. Data on susceptibility testing of isolates were stored as MICs in a Microsoft Access database placed on a SQL server at SSI. Analysis including selection of isolates from blood and spinal fluid samples and removal of duplicate isolates was performed in Microsoft Access. DANMAP 3 Jeppe Boel and Line Skjøt-Rasmusse

101 MATERIALS AND METHODS 9. Table 9.. Number of DANMAP samples, number of isolates and MIc-tests from healthy production animals at slaughter, De DANMAP 3 E. coli Pigs(a) E. faecalis No. of samples analysed ( per farm) No. of isolates obtained No. of isolates MIC-tested/reported 46 9 C. jejuni Cattle No. of samples analysed ( per farm) 66 No. of isolates obtained 5 No. of isolates MIC-tested/reported 3 Broilers No. of samples analysed (no. of flocks) No. of farms represented 5 34 No. of isolates MIC-tested/reported 5 4 Note: Data in this table should not be used for reporting of prevalences of the bacterial species a) From, the DANMAP samples from pigs were also part of the surveillance Salmonella programme for Table 9.. Interpretation criteriae for MIc-testing by EUcAST epidemiological cut-off values (blue fields) and the correspon EUcAST clinical breakpoints (white fields) DANMAP 3 Antimicrobial agent Salmonella E. coli E. faecium E. faecalis C. jejuni C. coli Clinical Clinical Clinical Clinical Clinical Clinical ECOFF ECOFF ECOFF ECOFF ECOFF ECOFF breakpoint breakpoint breakpoint breakpoint breakpoint breakpoint μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml Ampicillin >8* >8* >8* >8* >4* >8* >4* >8* Apramycin >6 >6 Cefotaxime >.5* >* >.5* >* Cefoxitin Ceftiofur >* >* Chloramphenicol >6* >8* >6* >8* >3* >3* >6* >6* (b) Ciprofloxacin >.6 * >* >.6* >* >6 >8(b) >.5* >.5* >.5* >.5* (c) Colistin >*/ >8 >* >* >* Erythromycin >4* >4* >4* >4* >8* >8* Florfenicol >6* >6* Gentamicin >* >4* >* >4* >3* >3* >* >* Kanamycin >,4 >,4 Linezolid >4* >4* >4* >4* Nalidixic acid >6* >6* >6* >6* Neomycin >4* >8* Penicillin >6* >6* Quinupristin/ (a) >4 >4* dalfopristin Salinomycin >4 >4 Spectinomycin >64 >64* Streptomycin >6* >6* >8* >5* >4* >4* Sulfonamide >56 >64* Teicoplanin >* >* >* >* Tetracycline >8* >8* >4* >4* >* >* >* >* Tiamulin Tigecycline >.5* >.5* >.5* >.5* Trimethoprim >* >4* >* >4* Vancomycin >4* >4* >4* >4* * EUCAST epidemiological cut-off values (ECOFFs) and EUCAST clinical breakpoints. Changes in ECOFF values since DANM highlighted by orange. a) The EUCAST ECOFF (>) was not applied for quinopristin/dalfopristin (tradename synercid) according to investigations p 6. b) The EUCAST ECOFF (>4) was not applied for ciprofloxacin. The aim was to look for high level ciprofloxacin-resistance as al, (Int J Antimicob Agents;35:9-5). DANMAP 6. c) The EUCAST ECOFF (>) for colistin was applied S. Typhimurium for and other serotypes, except S. Enteritidis for and S. Dublin where ECOFF >8 was applied as recommended by Agersøe et al, (DANMAP, Textbox 6). DANMAP 3

102 9. MATERIALS AND METHODS Table Definitions of antimicrobial classes for calculation of multi-resistance (MR) in zoonotic and indicator bacteria, D DANMAP 3 Antimicrobial classes Tetracyclines Phenicoles Salmonella and (a) E. coli Tetracycline Chloramphenicol and/or florfenicol Penicillins Ampicillin Cephalosporins Ceftiofur and/or cefotaxime Sulfonamides Sulphonamides Trimethoprim Trimethoprim Aminoglycosides I Aminoglycosides II Quinolones Polymycins Macrolides Glycopeptids Ionophores Oxazolidinones Gentamicin Campylobacter Enterococcus Tetracycline Tetracycline Chloramphenicol Chloramphenicol (b) (a) Ampicillin and/or penicillin Gentamicin Gentamicin and/or kanamycin and/or streptomycin Streptomycin Streptomycin Ciprofloxacin and/or nalidixic Ciprofloxacin and/or nalidixic Ciprofloxacin and/or nalidixic acid acid acid Colistin Erythromycin Erythromycin Vancomycin and/or teicoplanin Salinomycin Linezolid Glycylcyclines Tigercycline Note: An isolate is considered fully sensitive if susceptible to all antimicrobial agents included in the panel for the selected a) An isolates is considered multi-resistant if resistant to three or more of the ten antimicrobial classes b) An isolates is considered multi-resistant if resistant to three or more of the six antimicrobial classes DANMAP 3

103 TERMINOLOGY DANMAP 3 3