1. Pierre-Louis Toutain National veterinary School Toulouse France
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. But of what resistance are we speaking?
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
No antibiotics
Mutation rate10-8
Mutant pop
Mutation rate10-8
Wild pop
With antibiotics
Mutation rate10-8
eradication
Mutants population
résistant
susceptible

10. Eradication of all bacteria
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. MICs estimated with different inoculmum densities, relative to that MIC at 2x105
Ciprofloxacin
Gentamicin
Linezolid
Daptomycin
Oxacillin
Vancomycin

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

15. Comparative MIC and MPC values for 285 M
Comparative MIC and MPC values for 285 M. haemolytica strains collected from cattle
MIC50
MIC90
MPC50
MPC90
MPC/MIC
Ceftiofur
0.016 1 2
125
Enrofloxacine
0.125
0.25 8
Florfenicol 4
Tilmicosine 16
>32 ≈8
Tulathromycine

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

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

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

24. Materials and methods A single administration of marbofloxacin
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. Pourcentages of mice alive
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
control
1 mg/kg
40 mg/kg
Marbofloxacin doses

26. 2-Bacterial eradication Early low dose= late high dose
Marbofloxacin administrations
Early
Late
100 % 80
% of mice with bacterial eradication 60 40 20
control
1 mg/kg
40 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 %
Early
late 40
% of mice with resistant bacteria
observation 16 hours after marbofloxacin administration
= 48 hours after the infection = like early administration 30 20
+38h 10
1 mg/kg
40 mg/kg
control
1 mg/kg
40 mg/kg
+38h
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
Infected
Lungs
Digestive
tract
Test
tube
Manure
waste
1µg
1 mg
Several Kg
Several tons
Food chain
Soil, plant….

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

41. Biophases & antibiorésistance
G.I.T
Proximal
Distal
AB: oral route
1-F%
Gut flora
Zoonotic (salmonella, campylobacter
commensal ( enterococcus)
Résistance = public health concern
Food chain
Environmental exposure F%
Blood
Target biophase
Bug of vet interest
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
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 (~ 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
This graph shows the average amount of fecal blaTEM genes for each mode of treatment.
Treatment had a significant effect on the excretion of blaTEM genes, and oral administration to fed pigs led to a higher excretion of blaTEM genes than the intramuscular administration.
treatment=“tritment”
led=“laide”
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. One world, one health Greening our AB Commensal flora Environment
Genes of resistance
(zoonotic pathogens)
Environment
Food chain
AMR should be viewed as a global ecological problem with commensal flora as the turntable of the system

51. Selectivity of antimicrobial drugs in veterinary medicinePD
Narrow spectrum PK
Selective distribution of the AB to its biophase

52. Innovation: PK selectivity of antibiotics
Proximal
Distal
1-F=90%
Oral
Gut flora
Zoonotic (salmonella, campylobacter
commensal ( enterococcus)
Efflux
F=10%
Food chain
Quinolones, macrolides
environment IM
Blood
Kidney
Biophase
Résistance = public health concern
Animal health

53. Currently no veterinary antibiotic is selective of target pathogens and our hypothesis was that a low dose would be more selective than a high, regular, dose

54. In vitro assessment of the selectivity of antibiotics on the target pathogen vs. commensal flora: eradication of a low vs. high inoculum size of P multocida

55. Selectivity of amoxicillin & cefquinome
Using killing curves selectivity was tested using E.coli, as a commensal bacterium in condition for which the two tested antibiotics were able to eradicate a low or a large inoculum of P.multocida,

56. Development of a selectivity index (SI)

57. P. Multocida (105 or 107 CFU/ml)
Selectivity of amoxicillin to eradicate a low a or a high inoculum size of P. multocida
Low: 105 CFU/mL
High:107 CFU/mL
SI=51
SI=5.54
P. Multocida (105 or 107 CFU/ml)
E coli (107 CFU/mL)

58. P. Multocida (105 or 107 CFU/ml)
Selectivity of cefquinome to eradicate a low a or a high inoculum size of P. multocida
Low:105 CFU/mL
High:107 CFU/mL
SI=2.9
SI=0.66
P. Multocida (105 or 107 CFU/ml)
E coli (107 CFU/mL)

59. I there a selective window for the commensal flora

60. All macrolides are not equals
The normal flora is disturbed more or less according to the pharmacokinetic profiles of the respective macrolides.
85% of patients treated with azithromycin were colonized by macrolide-resistant organisms 6 weeks after therapy, compared to 17% treated with clarithromycin

61. Effect of Elimination Kinetics on Bacterial Resistance
10.00
1.00
0.1
0.01
0.001
Clarithromycin
Azithromycin
Selective Window
Concentration ( ug/ml )
MIC
MAC
Weeks
Longer half-life antibiotics may create a greater window
of opportunity for the development of resistance
Guggenbichler JP, Kastner H Infect Med 14 Suppl C: (1997)

62. Selective window can be longer and delayed in the GIT
GIT/commensal
Plasma/Lung

63. A formulation property
A long half-life is desirable for convenience in vet medicine: two possible options
Long HL
A substance property
Low clearance
High MW
lipophilic
Likely lower degradability
Excretion by the GIT
Large volume of distribution
Large diffusion
A formulation property
Slow absorption
(flip-flop)
High clearance
hydrophilic
Likely higher degradability
Excretion by the kidney
Macrolides/FQ
Beta-lactams/sulfonamides

64. Longer half-life antibiotics may create a greater window of opportunity for the development of resistance

65. One size does NOT fit all!
Conclusions
One size does NOT fit all!
We need to broaden the concept of selection of resistance when devising optimal dosing strategies – both for guidelines for future and existing antibiotics

66. When to finish a treatment?
ASAP
Should be determined in clinics
Should be when clinical cure is actually achieved
Should not be a hidden prophylactic treatment for a possible next infectious episode

67. Conclusion
For a same dose of marbofloxacin, early treatments (10 hours after the infection) were associated to
more frequent clinical cure
more frequent bacteriological cure
less frequent selection of resistant bacteria
than late treatments (32 hours after the infection)
Early administrations were more favourable than late administrations

68. Normal flora: Consequences
Treatment exerts selection on innocent bacteria
Most of the harm done by use of a drug may be on species OTHER than the target of treatment
Most of the exposure of a given
species to a given drug may be
due to treatment of OTHER
infections

69. One world, one health Commensal flora Vet AB Hazard Environment
Genes of resistance
zoonotic pathogens
Environment
Food chain
AMR should be viewed as a global ecological problem with commensal flora as the turntable of the system

70. New Eco-Evo drugs and strategies should be considered in vet medicine

72. Innovation: PK selectivity of antibiotics
Trapping or destruction of the antibiotic
G.I.T
Proximal
Distal
Efflux
90% 0%
Gut flora
Zoonotic (salmonella, campylobacter
commensal ( enterococcus)
Food chain
Quinolones, macrolides
environment IM
Blood
Kidney
Biophase
Résistance = public health concern
Animal health

73. My view of an ideal antibiotic for vet medicine
High plasma clearance
Rapidly metabolized (in vivo, environment) to inactive metabolite(s)
High renal clearance
Elimination by non-GIT route (not bile or enterocyte efflux)
volume of distribution not too high
Pathogens are extracellular; half-life rather short; not too short to compensate a relatively high clearance
High bioavailability by oral route
To avoid to expose distal GIT to active AB
Low binding to plasma protein
Only free antibiotic is active; to reduce the possible nominal dosage regimen and environmental load
High binding to cellulosis
To inactivate AB in large GIT
High potency
To be able to select a low dose
High PK selectivity (biophase)
To distribute only to target biophase

74. Innovation pour une voie systémique
Tractus digestif
Proximal
Distal
flore
Zoonotiques (salmonella, campylobacter )
commensaux ( enterococcus)
Elimination par efflux ou biliaire=0%
Chaîne alimentaire
Administration
Environment
sang
Biophase
Pathogène visé
Elimination rénale=100%

75. Renal clearance of different quinolones
Drugs
% of total clearance
Ofloxacin 70
Levofloxacin 65
Ciprofloxacin 50
Sparfloxacin 13
Grepafloxacin 10
Trovafloxacin
5-10
Hooper DC CID 2000;30:

76. Conclusions
Appropriate use of antibiotics should not only include knowledge of the pathogen and its susceptibility, but also the spectrum and pharmacokinetic properties of the respective antimicrobial drug.

77. Traditional pharmacokinetic/ pharmacodynamic models
Sensitive
population
Resistant
population S R

78. Incorporating the immune response
Sensitive
population
Sensitive
population
Resistant
population S S R I
Immune
response

79. Possible pathogen dynamics
Unregulated bacterial dynamics: Commensal bacteria
that uses body as a habitat
Regulated bacterial dynamics: Bacteria and the immune
response settles an equilibrium