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

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?

Prevent emergence of resistance: but of what resistance?

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

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

1-The case of target pathogens

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

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

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

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

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

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

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

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

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

When to start a treatment?

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

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

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

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)

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

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

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

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

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

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,

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

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

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

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

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

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?

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

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!

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

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

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….

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….

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

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% .

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

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 (~ 3 106 ufc/g 1 d). E. coli R followed to a slow decrease back to initial level after 12 days

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).

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

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.

Ecological consequences of the commensal flora exposure by antibiotic

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

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

Selectivity of antimicrobial drugs in veterinary medicine PD Narrow spectrum PK Selective distribution of the AB to its biophase

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

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

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

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,

Development of a selectivity index (SI)  

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)

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)

I there a selective window for the commensal flora

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

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 0 1 2 3 4 5 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: 17-25 (1997)

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

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

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

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

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

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

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

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

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

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

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

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%

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;30:243-254

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.

Traditional pharmacokinetic/ pharmacodynamic models Sensitive population Resistant population S R

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

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