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

Animal models for the study of antibiotic PKPD against staphylococci Niels Frimodt ‐ Møller Professor, MD DMSc Dept. of Clinical Microbiology Hvidovre Hospital Denmark

Animal models for antibiotic acitivity against S. aureus General screening Specialised infection models: models: • Peritonitis/sepsis • Endocarditis – – mouse rabbit, rat • Thigh (myositis) • Osteomyelitis – – mouse pig, rabbit, rat • Wax moth larva (Galleria • Skin infection mellonella) – mouse • Pneumonia – rat, mouse

Animal models for antibiotic PKPD acitivity against S. aureus General screening Specialised infection models: models: • Peritonitis/sepsis • Endocarditis – – mouse rabbit, rat • Thigh (myositis) • Osteomyelitis – – mouse pig, rabbit, rat • Wax moth larva (Galleria • Skin infection mellonella) – mouse • Pneumonia – rat, mouse

AAC 2012, 56: 1568 ‐ 73

AAC 2012, 56: 1568 ‐ 73

ECCMID 2009 Abs 2267 Andes & Craig AAC 2007

The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus Inoculation: • Intraperitoneal injection of S.aureus Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883 11 ‐ 09 ‐ 2012

The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus Inoculation: • Intraperitoneal injection of S.aureus peritonitis (2 hr) Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883 11 ‐ 09 ‐ 2012

The mouse peritonitis model Electron microscopy of peritoneal fluid post infection with S. aureus Extracellular S. aureus Intracellular S. aureus Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883 11 ‐ 09 ‐ 2012

The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus + Antibiotic treatment • Intraperitoneal injection of S.aureus • Subcutaneous injection of antibiotic Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883 11 ‐ 09 ‐ 2012

The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus + Sampling: • Euthanasia • Intraperitoneal injection of HBSS (2 ml) and mix 11 ‐ 09 ‐ 2012 Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus + Sampling: • Euthanasia • Intraperitoneal injection of HBSS (2 ml) and mix • Collection of peritoneal fluid through incision Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883 11 ‐ 09 ‐ 2012

Separation of intra- and extracellular bacteria Division of sample into two A) Sampling of peritoneal fluid B) 1:1 dilution with HBSS equal fractions C) Total colony count D) Division of sample into two equal fractions E) Admixture of lysostaphin F) Centrifugation H) Incubation 15 min G) Supernatant: Extracellular colony count I) Centrifugation and re-suspension in HBSS. K) Intracellular Four repetitions colony count J) Re-suspension in H 2 O Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883 11 ‐ 09 ‐ 2012

Separation of intra- and extracellular bacteria Division of sample into two A) Sampling of peritoneal fluid B) 1:1 dilution with HBSS equal fractions C) Total colony count D) Division of sample into Fraction A: two equal fractions Extracellular S. aureus estimated from supernatant E) Admixture of lysostaphin after centrifugation F) Centrifugation H) Incubation 15 min G) Supernatant: Extracellular colony count I) Centrifugation and re-suspension in HBSS. K) Intracellular Four repetitions colony count J) Re-suspension in H 2 O Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883 11 ‐ 09 ‐ 2012

Separation of intra- and extracellular bacteria Division of sample into two A) Sampling of peritoneal fluid B) 1:1 dilution with HBSS equal fractions C) Total colony count D) Division of sample into Fraction A: two equal fractions Extracellular S. aureus estimated from supernatant E) Admixture of lysostaphin after centrifugation F) Centrifugation H) Incubation 15 min Fraction B: G) Supernatant: Extracellular Intracellular S. aureus colony count estimated after incubation with lysostaphin, lysostaphin I) Centrifugation and wash-out, and lysis with H 2 O re-suspension in HBSS. K) Intracellular Four repetitions colony count J) Re-suspension in H 2 O 11 ‐ 09 ‐ 2012 Sandberg et al., Antimicrob Agents Chemother (2009) 53:1874-1883

Dose-response studies DICLOXACILLIN vs. S. aureus IN VIVO IN VITRO 4 4 extracellular intracellular 2 2  LOG (CFU) 0 0 ‐ 2 ‐ 2 ‐ 4 ‐ 4 ‐ 4 ‐ 2 0 2 4 ‐ 4 ‐ 2 0 2 4 mg/kg (log 10 ) mg/L (log 10 ) ∆ log(CFU) = changes in colony counts compared to the original inoculum (treatment outcome) Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400 11 ‐ 09 ‐ 2012

Dose-response studies DICLOXACILLIN vs. S. aureus IN VIVO IN VITRO 4 4 extracellular intracellular 2 2  LOG (CFU) 0 0 ‐ 2 ‐ 2 ‐ 4 ‐ 4 ‐ 4 ‐ 2 0 2 4 ‐ 4 ‐ 2 0 2 4 mg/kg (log 10 ) mg/L (log 10 ) Extracellular activity: dissimilar results were obtained in vitro and in vivo Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400 11 ‐ 09 ‐ 2012

Dose-response studies DICLOXACILLIN vs. S. aureus IN VIVO IN VITRO 4 4 extracellular intracellular 2 2  LOG (CFU) 0 0 ‐ 2 ‐ 2 ‐ 4 ‐ 4 ‐ 4 ‐ 2 0 2 4 ‐ 4 ‐ 2 0 2 4 mg/kg (log 10 ) mg/L (log 10 ) Intracellular activity: similar results were obtained in vitro and in vivo Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400 11 ‐ 09 ‐ 2012

PK/PD studies: Dicloxacillin vs S. aureus EXTRACELLULAR INTRACELLULAR 2 2 R 2 0.52 R 2 0.40  LOG (CFU) 0-24hr 1 1 0 0 ‐ 1 ‐ 1 ‐ 2 ‐ 2 ‐ 3 ‐ 3 1 10 100 1000 1 10 100 1000 f AUC/MIC 24 hr f AUC/MIC 24 hr No correlation between treatment outcome and the AUC/MIC index 11 ‐ 09 ‐ 2012 Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400

PK/PD studies: Dicloxacillin vs S. aureus INTRACELLULAR EXTRACELLULAR 2 2 1 1  LOG (CFU) 0 ‐ 24hr 0 0 ‐ 1 ‐ 1 ‐ 2 ‐ 2 ‐ 3 ‐ 3 1 10 100 1000 1 10 100 1000 f C max /MIC f C max /MIC No correlation between treatment outcome and the C max /MIC index Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400 11 ‐ 09 ‐ 2012

PK/PD studies: Dicloxacillin vs S. aureus EXTRACELLULAR INTRACELLULAR 2 2 R 2 0.81 R 2 0.89 1 1  LOG (CFU) 0 ‐ 24hr 0 0 ‐ 1 ‐ 1 ‐ 2 ‐ 2 ‐ 3 ‐ 3 1 10 100 1 10 100 f T>MIC% f T>MIC% Correlation between treatment outcome and the T>MIC index Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400 11 ‐ 09 ‐ 2012

PK/PD studies: Dicloxacillin vs S. aureus EXTRACELLULAR INTRACELLULAR 2 2 R 2 0.81 R 2 0.89 1 1  LOG (CFU) 0 ‐ 24hr 0 0 ‐ 1 ‐ 1 ‐ 2 ‐ 2 ‐ 3 ‐ 3 1 10 100 1 10 100 f T>MIC% f T>MIC% T>MIC is the predicting PK/PD index both intra- and extracellularly Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400 11 ‐ 09 ‐ 2012

PK/PD studies: Dicloxacillin vs S. aureus EXTRACELLULAR INTRACELLULAR 2 2 R 2 0.81 R 2 0.89 1 1  LOG (CFU) 0 ‐ 24hr 0 0 2 log reduction ‐ 1 ‐ 1 ‐ 2 ‐ 2 ‐ 3 ‐ 3 1 10 100 1 10 100 f T>MIC% f T>MIC% A reduction of 2 logs was obtained intracellularly with optimal dosing Sandberg et al., Antimicrob Agents Chemother (2010) 54:2391-2400 11 ‐ 09 ‐ 2012

Dose-response studies LINEZOLID LINEZOLID vs. S. aureus IN VIVO IN VITRO 4 4 intracellular 3 3 extracellular 2 2  log 10 CFU 1 1 0 0 ‐ 1 ‐ 1 ‐ 2 ‐ 2 ‐ 4 ‐ 3 ‐ 2 ‐ 1 0 1 2 3 4 ‐ 3 ‐ 2 ‐ 1 0 1 2 mg/kg (log 10 ) mg/L (log 10 ) Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973 11 ‐ 09 ‐ 2012

Dose-response studies LINEZOLID LINEZOLID vs. S. aureus IN VIVO IN VITRO 4 4 intracellular 3 3 extracellular 2 2  log 10 CFU 1 1 0 0 ‐ 1 ‐ 1 ‐ 2 ‐ 2 ‐ 4 ‐ 3 ‐ 2 ‐ 1 0 1 2 3 4 ‐ 3 ‐ 2 ‐ 1 0 1 2 mg/kg (log 10 ) mg/L (log 10 ) No decreased intracellular activity in vitro Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973 11 ‐ 09 ‐ 2012

Dose-response studies LINEZOLID vs. S. aureus LINEZOLID IN VIVO IN VITRO 4 4 intracellular 3 3 extracellular 2 2  log 10 CFU 1 1 0 0 ‐ 1 ‐ 1 ‐ 2 ‐ 2 ‐ 4 ‐ 3 ‐ 2 ‐ 1 0 1 2 3 4 ‐ 3 ‐ 2 ‐ 1 0 1 2 mg/kg (log 10 ) mg/L (log 10 ) No reduction of the original intracellular inoculum in vivo Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973 11 ‐ 09 ‐ 2012

PK/PD studies: Linezolid vs S. aureus EXTRACELLULAR INTRACELLULAR 2 2  log 10 CFU 0 ‐ 24hr  log 10 cfu 0 ‐ 24hr 1 1 0 0 -1 ‐ 1 -2 ‐ 2 1 10 1 10 f C max /MIC f C max /MIC No correlation between treatment outcome and the C max /MIC index Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973 11 ‐ 09 ‐ 2012

PK/PD studies: Linezolid vs S. aureus EXTRACELLULAR EXTRACELLULAR R 2 = 0.51 R 2 = 0.55 2 2  log 10 cfu 0 ‐ 24hr  log 10 cfu 0 ‐ 24hr 1 1 0 0 ‐ 1 ‐ 1 ‐ 2 ‐ 2 1 10 100 1 10 100 f T>MIC% f AUC 24h /MIC Both AUC and T>MIC correlated equally to the extracellular outcome Sandberg et al., J. Antimicrob. Chemother (2010) 65:962-973 11 ‐ 09 ‐ 2012