Are we over-rating the risk of low-dose drug exposure on the selection of resistant strains?

1. Are we over-rating the risk of low-dose drug exposure on the selection of resistant strains? Pierre-Louis Toutain National veterinary School Toulouse France

2. The question: Are we over-rating the risk of low-dose drug exposure on the selection of resistant strains • But of what resistance are we speaking?

3. Prevent emergence of resistance: but of what resistance?

4. The priorities of a sustainable veterinary antimicrobial therapy is related to public health issues, not to animal health issues

5. The question: Are we over-rating the risk of low-dose drug exposure on the selection of resistant strains? • The public health issues being critical , we have to investigate both: • The case of target pathogens • The case of non-target pathogens • Zoonotic • Commensal flora • And also acknowledge possible conflict of interest

6. 1-The case of target pathogens

7. Traditional hypothesis on emergence of AMR Concentration CMI Selective pressure for antibiotic concentration lower than the MIC Time

8. Current view for the emergence and selection of resistance for the target pathogen:The selective window

9. Current view for the emergence and selection of resistance Mutation rate10-8 No antibiotics Mutant pop Mutation rate10-8 Wild pop With antibiotics Mutation rate10-8 eradication résistant Mutants population susceptible

10. Current view for the emergence and selection of resistance: with antibiotic Low inoculum size No mutants Wild population Usual MIC Mutant MIC=MPC Large inoculum MIC<[AB]<MPC i.e.within the selective window Large inoculum AB>MPC Large inoculum with AB few mutants Eradication of all bacteria

11. Current view for the emergence and selection of resistance: The selective window Antibiotic concentration Growth R Growth S R Selection of R S Selective Window (SW) MIC mutant=MPC MIC Wild population Time in SW

12. Linezolid Oxacillin Ciprofloxacin Daptomycin Gentamicin Vancomycin MICs estimated with different inoculmum densities, relative to that MIC at 2x105

13. MIC & MPC for the main veterinary quinolones for E. coli & S. aureus

15. Comparative MIC and MPC values for 285 M. haemolytica strains collected from cattle

16. Consequences of a selective window associated to an inoculum effect for a rational treatment for veterinary medicine

17. When to start a treatment?

18. The different uses of antibiotics in veterinary medicine Disease health Antibiotic consumption Metaphylaxis (Control) Prophylaxis (prevention) Growth promotion Therapy High Pathogen load Only a risk factor No Small NA

19. The inoculum effect and Very Early Treatment (VET) • Tested hypothesis • Efficacious dosage regimen is different when the pathogen load is large, low or null • Treatment should start as early as possible

21. Inoculation of Pasteurella multocida 1500 CFU/lung Materials and methods Model of pulmonary infection A strain of Pasteurella multocida isolated from the trachea of a pig with clinical symptoms of a bacterial lung infection

22. 1010 108 106 104 102 100 0 10 20 30 40 50 Materials and methods • Model of pulmonary infection Inoculation of Pasteurella multocida 1500 CFU/lung Bacteria counts per lung (CFU/lung) Progression of infection 18 control mice were used to assess the natural growth of Pasteurella multocida in the lungs. Time after infection (h)

23. 1010 108 106 104 102 100 0 10 20 30 40 50 Materials and methods anorexia lethargy dehydration no clinical signs of infection Progression of infection Bacteria counts per lung (CFU/lung) Inoculation of Pasteurella multocida 1500 CFU/lung Time (h) Late (32h) Administration early (10h) Administration

24. Materials and methods 10 hours after the infection (n=14) • A single administration of marbofloxacin • Two doses tested for each group • 1 mg/kg and 40 mg/kg 32 hours after the infection (n=14) Inoculation of Pasteurella multocida 1500 CFU/lung

25. 1-Clinical outcome (survival) A low early dose better than a late high dose Marbofloxacin administrations early late 100 % 80 60 Pourcentages of mice alive 40 20 0 1 mg/kg 40 mg/kg control Marbofloxacin doses

26. 2-Bacterial eradication Early low dose= late high dose Marbofloxacin administrations Late Early 100 % 80 60 % of mice with bacterial eradication 40 20 0 control 40 mg/kg 1 mg/kg Marbofloxacin doses

27. 3-Selection of resistant target bacteria An early 1 mg/kg marbofloxacin dose has no more impact on resistance than a high late treatment while this low dose is selecting resistance when administered later Marbofloxacin administrations 50 % late Early 40 observation 16 hours after marbofloxacin administration = 48 hours after the infection = like early administration % of mice with resistant bacteria 30 20 +38h 10 0 1 mg/kg 40 mg/kg control 40 mg/kg +38h 1 mg/kg Marbofloxacin doses

28. Metaphylaxis vs. curative • Pulmonary infectious model by inhalation (P multocida) • Amoxicillin & et cefquinome • Treatment during the prepatent (incubation) period (24h) vs. when symptoms are present M V. Vasseur, A A. Ferran, M Z. Lacroix, PL Toutain and A Bousquet-Mélou,

29. Effect of amoxicillin (clinical cure )metaphylaxis vs. curative Dose mg/kg

30. Effect of amoxicillin (bacteriological cure)metaphylaxis vs. curative Dose mg/kg

31. Effect of cefquinome (clinical cure )metaphylaxis vs. curative Dose mg/kg

32. Effect of cefquinome (bacteriological cure)metaphylaxis vs. curative Dose mg/kg

33. An early/low dose treatment is better for bacteriological cure than a late/high dose for three antibiotics: marbofloxacin, amoxicillin & cefquinome

34. Q:Are we over-rating the risk of low-dose drug exposure on the selection of resistant strains? • A: Apparently not for the target pathogen when an early treatment is initiated i.e when antibiotic only a low inoculum is exposed to an antibiotic • But what about other non-targeted bacteria?

35. The question: Are we over-rating the risk of low-dose drug exposure on the selection of resistant strains? • The public health issues being critical , we have to investigate both: • The case of target pathogens • The case of non-target pathogens • Zoonotics • Commensal flora • And acknowledge possible conflict of interest

36. Example of conflict of interest • the antimicrobial treatments should not only aim at curing the diseased animals but also at limiting the resistance on non target flora. Optimal dosing for treatment ≠ optimal to prevent resistance!

37. For AR, what are the critical veterinary ecosystems in terms of public health (commensals)

38. The critical animal ecosystems in terms of emergence and spreading of resistance • Open and large ecosystems • Digestive tract • Skin • Open but small ecosystem • Respiratory tract • Closed and small ecosystem • Mammary gland

39. Bacterial load exposed to antibiotics during a treatment Digestive tract Infected Lungs Test tube Manure waste 1µg Several tons 1 mg Several Kg Food chain Soil, plant….

40. Duration of exposure of bacteria exposed to antibiotics Digestive tract Manure Sludge waste Infected Lungs Test tube Few days Several weeks/months 24h Food chain Soil, plant….

41. AB: oral route 1-F% Environmental exposure Food chain Target biophase Bug of vet interest Résistance = public health concern Biophases & antibiorésistance G.I.T Proximal Distal • Gut flora • Zoonotic (salmonella, campylobacter • commensal ( enterococcus) F% Blood Résistance = lack of efficacy

42. Bioavailability of oral tetracyclins • Chlortetracycline: • Chickens:1% • Pigs Fasted or fed: 18 to 19% • Turkeys:6% • Doxycycline: • Chickens:41.3% . • Pigs :23% • Oxytetracycline: • Pigs:4.8% • Piglets, weaned, 10 weeks of age: by drench: 9%;in medicated feed for 3 days: 3.7% . • Turkeys: Fasted: 47.6% ;. Fed: 9.4% • Tetracycline: • Pigs fasted:23% .

43. Biophases & antibiorésistance Gastrointestinal tract Proximal Distal • Gut flora • Zoonotic (salmonella, campylobacter • commensal ( enterococcus) Intestinal secretion Bile Quinolones Macrolides Tétracyclines Food chain Systemic Administration Environment Blood Biophase Target pathogen Résistance =public health issue Résistance = lack of efficacy

44. Fluoroquinolone impact on E. coli in pig intestinal flora(From P. sanders, Anses, Fougères) IM 3 days IV • Before treatment : E. coli R (0.01 to 0.1%) • After IV. :Decrease of total E coli , slight increase of E. coli R (4 to 8 %) • Back to initial level • After repeated IM (3d) : Decrease below LoD E. coli (2 days), fast growth (~ 3 106 ufc/g 1 d). E. coli R followed to a slow decrease back to initial level after 12 days

45. Genotypic evaluation of ampicillin resistance:copy of blaTEM genes per gram of feces A significant effect of route of administration on blaTEM fecal elimination (p<0.001).

46. Performance-enhancing antibiotics (old antibiotics) • chlortetracycline, sulfamethazine, and penicillin (known as ASP250)] • phylogenetic, metagenomic, and quantitative PCR-based approaches to address the impact of antibiotics on the swine gut microbiota

47. It was shown that antibiotic resistance genes increased in abundance and diversity in the medicated swine microbiome despite a high background of resistance genes in nonmedicated swine. • Some enriched genes, demonstrated the potential for indirect selection of resistance to classes of antibiotics not fed.

48. Ecological consequences of the commensal flora exposure by antibiotic

49. one world, one health Transmissible genetic elements allow antibiotic resistance genes to spread both to commensal bacteria and to strains that cause disease. Vet AB Commensal flora Zoonotic pathogens Gene of resistance Resistance is contagious! It will continue to spread even after infection has been cleared

50. Greening our AB One world, one health Environment Food chain AMR should be viewed as a global ecological problem with commensal flora as the turntable of the system