Animal models for the study of antibiotic PKPD against staphylococci - - PowerPoint PPT Presentation

▶
animal models for the study of antibiotic pkpd against
SMART_READER_LITE
LIVE PREVIEW

Animal models for the study of antibiotic PKPD against staphylococci - - PowerPoint PPT Presentation

Mar 05, 2023 273 likes •632 views

Animal models for the study of antibiotic PKPD against staphylococci Niels Frimodt Mller Professor, MD DMSc Dept. of Clinical Microbiology Hvidovre Hospital Denmark Animal models for antibiotic acitivity against S. aureus General screening

slide-1
SLIDE 1

Animal models for the study of antibiotic PKPD against staphylococci

Niels Frimodt‐Møller

Professor, MD DMSc

  • Dept. of Clinical Microbiology

Hvidovre Hospital Denmark

slide-2
SLIDE 2

Animal models for antibiotic acitivity against S. aureus

General screening models:

  • Peritonitis/sepsis

– mouse

  • Thigh (myositis)

– mouse

  • Wax moth larva (Galleria

mellonella) Specialised infection models:

  • Endocarditis

– rabbit, rat

  • Osteomyelitis

– pig, rabbit, rat

  • Skin infection

– mouse

  • Pneumonia

– rat, mouse

slide-3
SLIDE 3

Animal models for antibiotic PKPD acitivity against S. aureus

General screening models:

  • Peritonitis/sepsis

– mouse

  • Thigh (myositis)

– mouse

  • Wax moth larva (Galleria

mellonella) Specialised infection models:

  • Endocarditis

– rabbit, rat

  • Osteomyelitis

– pig, rabbit, rat

  • Skin infection

– mouse

  • Pneumonia

– rat, mouse

slide-4
SLIDE 4
slide-5
SLIDE 5

AAC 2012, 56: 1568‐73

slide-6
SLIDE 6

AAC 2012, 56: 1568‐73

slide-7
SLIDE 7

ECCMID 2009 Abs 2267

Andes & Craig AAC 2007

slide-8
SLIDE 8
slide-9
SLIDE 9

11‐09‐2012

Inoculation:

  • Intraperitoneal injection of S.aureus

The mouse peritonitis model

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

Intra- and extracellular activity of antibiotics against S. aureus

slide-10
SLIDE 10

11‐09‐2012

Inoculation:

  • Intraperitoneal injection of S.aureus peritonitis (2 hr)

The mouse peritonitis model

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

Intra- and extracellular activity of antibiotics against S. aureus

slide-11
SLIDE 11

11‐09‐2012

Electron microscopy of peritoneal fluid post infection with S. aureus

Extracellular S. aureus Intracellular S. aureus

The mouse peritonitis model

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

slide-12
SLIDE 12

11‐09‐2012

Antibiotic treatment

  • Intraperitoneal injection of S.aureus
  • Subcutaneous injection of antibiotic

+

The mouse peritonitis model

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

Intra- and extracellular activity of antibiotics against S. aureus

slide-13
SLIDE 13

11‐09‐2012

+

Sampling:

  • Euthanasia
  • Intraperitoneal injection of HBSS (2 ml) and mix

The mouse peritonitis model

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

Intra- and extracellular activity of antibiotics against S. aureus

slide-14
SLIDE 14

11‐09‐2012

+

Sampling:

  • Euthanasia
  • Intraperitoneal injection of HBSS (2 ml) and mix
  • Collection of peritoneal fluid through incision

The mouse peritonitis model

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

Intra- and extracellular activity of antibiotics against S. aureus

slide-15
SLIDE 15

11‐09‐2012

Separation of intra- and extracellular bacteria

Division of sample into two equal fractions

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

B) 1:1 dilution with HBSS G) Supernatant: Extracellular colony count C) Total colony count I) Centrifugation and re-suspension in HBSS. Four repetitions J) Re-suspension in H2O A) Sampling of peritoneal fluid D) Division of sample into two equal fractions E) Admixture of lysostaphin F) Centrifugation H) Incubation 15 min K) Intracellular colony count

slide-16
SLIDE 16

11‐09‐2012

B) 1:1 dilution with HBSS G) Supernatant: Extracellular colony count C) Total colony count I) Centrifugation and re-suspension in HBSS. Four repetitions J) Re-suspension in H2O A) Sampling of peritoneal fluid D) Division of sample into two equal fractions E) Admixture of lysostaphin F) Centrifugation H) Incubation 15 min K) Intracellular colony count

Fraction A: Extracellular S. aureus estimated from supernatant after centrifugation

Separation of intra- and extracellular bacteria

Division of sample into two equal fractions

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

slide-17
SLIDE 17

11‐09‐2012

B) 1:1 dilution with HBSS G) Supernatant: Extracellular colony count C) Total colony count I) Centrifugation and re-suspension in HBSS. Four repetitions J) Re-suspension in H2O A) Sampling of peritoneal fluid D) Division of sample into two equal fractions E) Admixture of lysostaphin F) Centrifugation H) Incubation 15 min K) Intracellular colony count

Fraction A: Extracellular S. aureus estimated from supernatant after centrifugation Fraction B: Intracellular S. aureus estimated after incubation with lysostaphin, lysostaphin wash-out, and lysis with H2 O Division of sample into two equal fractions

Separation of intra- and extracellular bacteria

Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

slide-18
SLIDE 18

11‐09‐2012

‐4 ‐2 2 4 ‐4 ‐2 2 4

extracellular intracellular

IN VITRO mg/L (log10) ‐4 ‐2 2 4 ‐4 ‐2 2 4 IN VIVO mg/kg (log10) LOG (CFU)

∆log(CFU) = changes in colony counts compared to the original inoculum (treatment outcome)

DICLOXACILLIN vs. S. aureus

Dose-response studies

Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

slide-19
SLIDE 19

11‐09‐2012

‐4 ‐2 2 4 ‐4 ‐2 2 4

extracellular intracellular

IN VITRO mg/L (log10) ‐4 ‐2 2 4 ‐4 ‐2 2 4 IN VIVO mg/kg (log10) LOG (CFU)

Extracellular activity: dissimilar results were obtained in vitro and in vivo DICLOXACILLIN vs. S. aureus

Dose-response studies

Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

slide-20
SLIDE 20

11‐09‐2012

‐4 ‐2 2 4 ‐4 ‐2 2 4

extracellular intracellular

IN VITRO mg/L (log10) ‐4 ‐2 2 4 ‐4 ‐2 2 4 IN VIVO mg/kg (log10) LOG (CFU)

Intracellular activity: similar results were obtained in vitro and in vivo DICLOXACILLIN vs. S. aureus

Dose-response studies

Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

slide-21
SLIDE 21

11‐09‐2012

EXTRACELLULAR INTRACELLULAR

‐3 ‐2 ‐1 1 2

R2 0.52

1 10 100 1000

fAUC/MIC24 hr

‐3 ‐2 ‐1 1 2

R2 0.40

1 10 100 1000

fAUC/MIC24 hr LOG (CFU)0-24hr

No correlation between treatment outcome and the AUC/MIC index

PK/PD studies: Dicloxacillin vs S. aureus

Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

slide-22
SLIDE 22

11‐09‐2012

‐3 ‐2 ‐1 1 2 1 10 100 1000

fCmax/MIC LOG (CFU)0‐24hr

‐3 ‐2 ‐1 1 2 1 10 100 1000

fCmax/MIC

EXTRACELLULAR INTRACELLULAR

No correlation between treatment outcome and the Cmax /MIC index

PK/PD studies: Dicloxacillin vs S. aureus

Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

slide-23
SLIDE 23

11‐09‐2012

Correlation between treatment outcome and the T>MIC index

PK/PD studies: Dicloxacillin vs S. aureus

‐3 ‐2 ‐1 1 2

R2 0.81

1 10 100

fT>MIC% LOG (CFU)0‐24hr

‐3 ‐2 ‐1 1 2

R2 0.89 fT>MIC%

1 10 100

EXTRACELLULAR INTRACELLULAR

Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

slide-24
SLIDE 24

11‐09‐2012

T>MIC is the predicting PK/PD index both intra- and extracellularly

PK/PD studies: Dicloxacillin vs S. aureus

‐3 ‐2 ‐1 1 2

R2 0.81

1 10 100

fT>MIC% LOG (CFU)0‐24hr

‐3 ‐2 ‐1 1 2

R2 0.89 fT>MIC%

1 10 100

EXTRACELLULAR INTRACELLULAR

Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

slide-25
SLIDE 25

11‐09‐2012

A reduction of 2 logs was obtained intracellularly with optimal dosing 2 log reduction

PK/PD studies: Dicloxacillin vs S. aureus

‐3 ‐2 ‐1 1 2

R2 0.81

1 10 100

fT>MIC% LOG (CFU)0‐24hr

‐3 ‐2 ‐1 1 2

R2 0.89 fT>MIC%

1 10 100

EXTRACELLULAR INTRACELLULAR

Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

slide-26
SLIDE 26

11‐09‐2012

Dose-response studies

‐4 ‐3 ‐2 ‐1 1 2 3 4 ‐2 ‐1 1 2 3 4

extracellular intracellular

IN VITRO mg/L (log10)

LINEZOLID

‐3 ‐2 ‐1 1 2 ‐2 ‐1 1 2 3 4

IN VIVO mg/kg (log10)  log10 CFU

LINEZOLID vs. S. aureus

Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973

slide-27
SLIDE 27

11‐09‐2012

No decreased intracellular activity in vitro

Dose-response studies

‐4 ‐3 ‐2 ‐1 1 2 3 4 ‐2 ‐1 1 2 3 4

extracellular intracellular

IN VITRO mg/L (log10)

LINEZOLID

‐3 ‐2 ‐1 1 2 ‐2 ‐1 1 2 3 4

IN VIVO mg/kg (log10)  log10 CFU

LINEZOLID vs. S. aureus

Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973

slide-28
SLIDE 28

11‐09‐2012 ‐4 ‐3 ‐2 ‐1 1 2 3 4 ‐2 ‐1 1 2 3 4

extracellular intracellular

IN VITRO mg/L (log10)

LINEZOLID

‐3 ‐2 ‐1 1 2 ‐2 ‐1 1 2 3 4

IN VIVO mg/kg (log10)  log10 CFU

No reduction of the original intracellular inoculum in vivo

Dose-response studies

LINEZOLID vs. S. aureus

Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973

slide-29
SLIDE 29

11‐09‐2012

EXTRACELLULAR INTRACELLULAR

  • 2
  • 1

1 2 1 10

fCmax/MIC  log10 cfu0‐24hr

‐2 ‐1 1 2 1 10

fCmax/MIC  log10 CFU0‐24hr No correlation between treatment outcome and the Cmax /MIC index

PK/PD studies: Linezolid vs S. aureus

Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973

slide-30
SLIDE 30

11‐09‐2012

EXTRACELLULAR

‐2 ‐1 1 2

R2 = 0.55

1 10 100

fAUC24h/MIC  log10 cfu0‐24hr

Both AUC and T>MIC correlated equally to the extracellular outcome

‐2 ‐1 1 2

R2 = 0.51

1 10 100

fT>MIC%  log10 cfu0‐24hr EXTRACELLULAR

PK/PD studies: Linezolid vs S. aureus

Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973

slide-31
SLIDE 31

11‐09‐2012

INTRACELLULAR

‐2 ‐1 1 2

R2 = 0.23

1 10 100

fAUC24h/MIC  log10 cfu0‐24hr Poor correlation between PK/PD indices and the intracellular outcome

‐2 ‐1 1 2

R2 = 0.29

1 10 100

fT>MIC%  log10 cfu0‐24hr INTRACELLULAR

PK/PD studies: Linezolid vs S. aureus

Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973

slide-32
SLIDE 32

11‐09‐2012

EXTRACELLULAR INTRACELLULAR

‐2 ‐1 1 2

R2 = 0.55

1 10 100

fAUC24h/MIC  log10 cfu0‐24hr

‐2 ‐1 1 2

R2 = 0.23

1 10 100

fAUC24h/MIC  log10 cfu0‐24hr

Conventional dose: 600 mg twice daily AUC/MIC = 80 Acceptable extracellular effect but questionable intracellular effect with conventional dose

PK/PD studies: Linezolid vs S. aureus

Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973

slide-33
SLIDE 33

Intracellular peritonitis mouse model: Fusidic acid

Total count

1.5 2.0 2.5 3.0 3.5

  • 2
  • 1

1 2 3

logAUC/MIC

log(CFU/ml) Extracellular count

1.5 2.0 2.5 3.0 3.5

  • 2
  • 1

1 2 3

logAUC/MIC

log(CFU/ml) Intracellular count

1.5 2.0 2.5 3.0 3.5

  • 2
  • 1

1 2 3

logAUC/MIC

log(CFU/ml)

Sandberg et al. unpublished

slide-34
SLIDE 34

Intracellular peritonitis mouse model: Fusidic acid

Total count

50 100 150

  • 2
  • 1

1 2 3

T>MIC

log(CFU/ml) Extracellular count

50 100 150

  • 2
  • 1

1 2 3

T>MIC

log(CFU/ml) Intracellular count

50 100 150

  • 2
  • 1

1 2 3

T>MIC

log(CFU/ml)

Sandberg et al. Unpublished

slide-35
SLIDE 35

Conclusion

  • S. aureus infection primarily intracellularly
  • Still, antibiotic effect better (lower doses) in

normal (non‐neutropenic) mice

  • PKPD studies of antibiotics against S. aureus

should include both intra‐ and extracellular compartments

  • Surprisingly, dicloxacillin best effect in vivo

(vs. linezolid, fusidic acid, macrolides)