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

Are we over-rating the risk of low-dose drug exposure on the

selection of resistant strains?

Pierre-Louis ToutainNational veterinary School

ToulouseFrance

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?

Target pathogens Zoonotics Commensal flora

Drug efficacy in animal:

A vet issue

Drug efficacy in

man

Resistance genereservoir

Global ecologicalproblem

Possible overuse of antibiotics

Natural eradication

Risk for permanent

colonisation

Individual issue Population issueAnimal issueAnimal issue



A vet issue

Drug efficacy in

man




Natural eradication

Risk for permanent

colonisation




A vet issue

Drug efficacy in

man




Natural eradication

Risk for permanent

colonisation


The priorities of a sustainable veterinary antimicrobial therapy is

related to public health issues, not to animal health issues


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

7

Selective pressure for antibiotic concentration lower than the MIC

CMI

Time

Concentration

Traditional hypothesis on emergence of AMR

Current view for the emergence and selection of resistance for the

target pathogen:The selective window

No antibiotics

Current view for the emergence and selection of resistance

With antibiotics

Mutation rate10-8

Mutation rate10-8

Mutation rate10-8

eradication

susceptible Mutants population résistant

Wild pop

Mutant pop

Current view for the emergence and selection of resistance: with antibiotic

10

Eradication of all bacteria

Low inoculum size No mutants

Large inoculum with ABfew mutants

Wild population Usual MIC

Mutant MIC=MPC

Large inoculum MIC<[AB]<MPC i.e.within the selective window

Large inoculumAB>MPC

Growth R

Growth S

Selective Window(SW)

S

R

Time in SW

Antibiotic concentration

Selection of R

Current view for the emergence and selection of resistance:

The selective window

MICWild population

MIC mutant=MPC

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

14

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

MIC50 MIC90 MPC50 MPC90 MPC/MIC

Ceftiofur 0.016 0.016 1 2 125Enrofloxacine 0.016 0.125 0.25 1 8Florfenicol 2 2 4 8 4Tilmicosine 2 8 16 >32 ≈8Tulathromycine 1 2 4 8 4

15

Consequences of a selective window associated to an inoculum effect for a

rational treatment for veterinary medicine

When to start a treatment?

Disease health

TherapyMetaphylaxis

(Control)Prophylaxis(prevention)

Growth promotion

The different uses of antibiotics in veterinary medicine

HighHighPathogen loadPathogen load

SmallSmall NoNoNANA

Antibiotic consumptionAntibiotic consumption

Only a risk factor

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

Inoculation of Pasteurella multocida

1500 CFU/lung

Model of pulmonary infection

Materials and methods

A strain of Pasteurella multocida isolated from the trachea of a pig with clinical symptoms of a bacterial lung infection

Inoculation of Pasteurella multocida1500 CFU/lung

• Model of pulmonary infection


Progression of infection

0 10 20 30 40 50

Time after infection (h)

Bacteria counts per lung (CFU/lung)

100

102

104

106

108

1010

18 control mice were used to assess the natural growth of Pasteurella multocida in the lungs.


Progression of infection

early (10h)Administration

Late (32h)Administration

Inoculation of Pasteurella multocida

1500 CFU/lung

0 10 20 30 40 50

Time (h) Bact

eria

cou

nts

per l

ung

(CFU

/lun

g)

100

102

104

106

108

1010no clinical

signs of infection

anorexia lethargy

dehydration


10 hours after the infection (n=14)

32 hours after the infection (n=14)

•A single administration of marbofloxacin

•Two doses tested for each group

1 mg/kg and 40 mg/kg

Inoculation of Pasteurella multocida1500 CFU/lung

0

20

40

60

80

100 %

1 mg/kg

Marbofloxacin doses

40 mg/kg

early late

Marbofloxacin administrations Po

urce

ntag

es o

f mic

e al

ive

control

1-Clinical outcome (survival) A low early dose better than a late high dose

2-Bacterial eradicationEarly low dose= late high dose

0

20

40

60

80

100 %

% o

f mic

e w

ith b

acte

rial

erad

icati

on

1 mg/kg

Marbofloxacin doses

40 mg/kg

Early Late

Marbofloxacin administrations

control

3-Selection of resistant target bacteriaAn 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

0

10

20

30

40

50 %

+38h

observation 16 hours after marbofloxacin administration= 48 hours after the infection = like early administration

1 mg/kg

Marbofloxacin doses

40 mg/kg

% o

f mic

e w

ith re

sist

ant

bact

eria

control

Early late

Marbofloxacin administrations

+38h1 mg/kg 40 mg/kg

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

28M V. Vasseur, A A. Ferran, M Z. Lacroix, PL Toutain and A Bousquet-Mélou,

Effect of amoxicillin (clinical cure )metaphylaxis vs. curative

29Dose mg/kg

Effect of amoxicillin (bacteriological cure)metaphylaxis vs. curative

30Dose mg/kg

Effect of cefquinome (clinical cure )metaphylaxis vs. curative

31Dose mg/kg

Effect of cefquinome (bacteriological cure)metaphylaxis vs. curative

32Dose 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?


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

1 mg Several Kg

Manurewaste

Food chain

Several tons

Soil, plant….

1µg

Test tube

Duration of exposure of bacteria exposed to antibiotics

Infected Lungs

Digestive tract

Few days

ManureSludgewaste

Food chain

Several weeks/months

Soil, plant….

24h

Test tube

Biophases & antibiorésistance

G.I.TProximal Distal

Résistance = lack of efficacy

Blood

Gut flora•Zoonotic (salmonella, campylobacter •commensal ( enterococcus)

1-F%

F%

Target biophaseBug of vet interest

AB: oral route

Résistance = public health concern

Food chain Environmental exposure

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 tractProximal Distal

Intestinal secretion Bile

Résistance = lack of efficacyRésistance =public health issue

BiophaseTarget pathogen

Blood

Food chain

Environment

Systemic Administration

QuinolonesMacrolidesTétracyclines


44

Fluoroquinolone impact on E. coli in pig intestinal flora(From P. sanders, Anses, Fougères)

• 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

IVIM 3 days

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).

0 1 2 3 4 5 6 7days

cop

ies/

g o

f fe

ces

oral route fed

oral route fasted

intramuscular route

control group1 E+5

1 E+6

1 E+7

1 E+8

1 E+9

1 E+10

1 E+4

• 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

Commensal floraZoonotic pathogensGene of resistance

one world, one health

Vet AB

Resistance is contagious!It will continue to spread even after infection has been cleared

Transmissible genetic elements allow antibiotic resistance genes to spread both to commensal bacteria and to strains that cause disease.

One world, one health

EnvironmentEnvironment

Food chain

AMR should be viewed as a global ecological problem with commensal flora as the turntable of the system

Greening our AB

Selectivity of antimicrobial drugs in veterinary medicine

- 52

Innovation: PK selectivity of antibiotics

environment

ProximalDistal

Blood


BiophaseRésistance = public health concern

Food chain

1-F=90%

F=10%

Animal health

Efflux

Quinolones, macrolides

IM

Kidney

Oral

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)

Selectivity of amoxicillin to eradicate a low a or a high inoculum size of P. multocida

SI=51 SI=5.54

Low: 105 CFU/mL High:107 CFU/mL

P. Multocida (105 or 107 CFU/ml)E coli (107 CFU/mL)

Selectivity of cefquinome to eradicate a low a or a high inoculum size of P. multocida

SI=2.9 SI=0.66

Low:105 CFU/mL High:107 CFU/mL

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

ClarithromycinClarithromycin AzithromycinAzithromycin

Selective Window

MAC

MIC

10.00

1.00

0.1

0.01

0.001Con

cent

ratio

n (

ug/m

l )

0 1 2 3 4 5Weeks

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

Guggenbichler JP, Kastner H Infect Med 14 Suppl C: 17-25 (1997)

Effect of Elimination Kineticson Bacterial Resistance

Selective window can be longer and delayed in the GIT

Plasma/Lung

GIT/commensal

A long half-life is desirable for convenience in vet medicine: two possible options

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!

We need to broaden the concept of selection of resistance when devising optimal dosing strategies – both for guidelines for future and existing antibiotics

Conclusions

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

• 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)

Conclusion

Early administrations were more favourable than late administrations

• 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 givenspecies to a given drug may be due to treatment of OTHER infections

Normal flora: Consequences

One world, one health

EnvironmentEnvironment

Food chain

Vet ABHazard

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

- 72

Innovation: PK selectivity of antibiotics

environment

G.I.TProximal Distal

Blood


BiophaseRésistance = public health concern

Food chain

90%0%

Animal health

Trapping or destruction of the antibiotic

Efflux

Quinolones, macrolides

IM

Kidney

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

Innovation pour une voie systémique

Tractus digestif

Proximal Distal

BiophasePathogène visé

sang

Chaîne alimentaire

EnvironmentAdministration

Elimination par efflux ou biliaire=0%

flore•Zoonotiques (salmonella, campylobacter )•commensaux ( enterococcus)

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.

S R

Sensitivepopulation

Resistantpopulation

Traditional pharmacokinetic/ pharmacodynamic models

S R

Sensitivepopulation

Resistantpopulation

Incorporating the immune response

S

I

Sensitivepopulation

Immuneresponse

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

Documents

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