Animal models for the study of. staphylococci. Niels Frimodt Møller Professor, MD DMSc Dept. of Clinical Microbiology Hvidovre Hospital Denmark
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1 Animal models for the study of antibiotic PKPD against staphylococci Niels Frimodt Møller Professor, MD DMSc Dept. of Clinical Microbiology Hvidovre Hospital Denmark
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
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
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5 AAC, 56:
6 AAC, 56:
7 Andes & Craig AAC 7 ECCMID 9 Abs 67
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9 The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus Inoculation: Intraperitoneal injection of S.aureus 9 Sandberg et al., Antimicrob Agents Chemother (9) 53:
10 The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus Inoculation: Intraperitoneal injection of S.aureus peritonitis ( hr) 9 Sandberg et al., Antimicrob Agents Chemother (9) 53:
11 The mouse peritonitis model Electron microscopy of peritoneal fluid post infection with S. aureus Extracellular S. aureus 9 Intracellular S. aureus Sandberg et al., Antimicrob Agents Chemother (9) 53:
12 The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus + Antibiotic treatment Intraperitoneal injection of S.aureus Subcutaneous injection of antibiotic 9 Sandberg et al., Antimicrob Agents Chemother (9) 53:
13 The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus + Sampling: Euthanasia Intraperitoneal injection of HBSS ( ml) and mix 9 Sandberg et al., Antimicrob Agents Chemother (9) 53:
14 The mouse peritonitis model Intra- and extracellular activity of antibiotics against S. aureus + Sampling: Euthanasia Intraperitoneal injection of HBSS ( ml) and mix Collection of peritoneal fluid through incision 9 Sandberg et al., Antimicrob Agents Chemother (9) 53:
15 Separation of intra- and extracellular bacteria A) Sampling of peritoneal fluid B) : dilution with HBSS C) Total colony count Division of sample into two equal fractions D) Division of sample into two equal fractions F) Centrifugation E) Admixture of lysostaphin G) Supernatant: Extracellular colony count H) Incubation 5 min K) Intracellular colony count J) Re-suspension in HO I) Centrifugation and re-suspension in HBSS. Four repetitions 9 Sandberg et al., Antimicrob Agents Chemother (9) 53:
16 Separation of intra- and extracellular bacteria A) Sampling of peritoneal fluid D) Division of sample into two equal fractions F) Centrifugation B) : dilution with HBSS C) Total colony count E) Admixture of lysostaphin Division of sample into two equal fractions Fraction A: Extracellular S. aureus estimated from supernatant after centrifugation G) Supernatant: Extracellular colony count H) Incubation 5 min K) Intracellular colony count J) Re-suspension in HO I) Centrifugation and re-suspension in HBSS. Four repetitions 9 Sandberg et al., Antimicrob Agents Chemother (9) 53:
17 Separation of intra- and extracellular bacteria A) Sampling of peritoneal fluid D) Division of sample into two equal fractions F) Centrifugation B) : dilution with HBSS C) Total colony count E) Admixture of lysostaphin Division of sample into two equal fractions Fraction A: Extracellular S. aureus estimated from supernatant after centrifugation G) Supernatant: Extracellular colony count K) Intracellular colony count J) Re-suspension in HO H) Incubation 5 min I) Centrifugation and re-suspension in HBSS. Four repetitions Fraction B: Intracellular S. aureus estimated after incubation with lysostaphin, lysostaphin wash-out, and lysis with H O 9 Sandberg et al., Antimicrob Agents Chemother (9) 53:
18 Dose-response studies DICLOXACILLIN vs. S. aureus 4 IN VIVO 4 IN VITRO extracellular intracellular LOG (CFU) mg/kg (log ) mg/l (log ) log(cfu) = changes in colony counts compared to the original inoculum (treatment outcome) 9 Sandberg et al., Antimicrob Agents Chemother () 54:39-4
19 Dose-response studies DICLOXACILLIN vs. S. aureus 4 IN VIVO 4 IN VITRO extracellular intracellular LOG (CFU) mg/kg (log ) mg/l (log ) Extracellular activity: dissimilar results were obtained in vitro and in vivo 9 Sandberg et al., Antimicrob Agents Chemother () 54:39-4
20 Dose-response studies DICLOXACILLIN vs. S. aureus 4 IN VIVO 4 IN VITRO extracellular intracellular LOG (CFU) mg/kg (log ) mg/l (log ) Intracellular activity: similar results were obtained in vitro and in vivo 9 Sandberg et al., Antimicrob Agents Chemother () 54:39-4
21 PK/PD studies: Dicloxacillin vs S. aureus LOG (CFU) -4hr EXTRACELLULAR R.4 INTRACELLULAR R.5 3 fauc/mic 4 hr 3 fauc/mic 4 hr No correlation between treatment outcome and the AUC/MIC index 9 Sandberg et al., Antimicrob Agents Chemother () 54:39-4
22 PK/PD studies: Dicloxacillin vs S. aureus EXTRACELLULAR INTRACELLULAR LOG (CFU) 4hr 3 3 fc max /MIC fc max /MIC No correlation between treatment outcome and the Cmax /MIC index 9 Sandberg et al., Antimicrob Agents Chemother () 54:39-4
23 PK/PD studies: Dicloxacillin vs S. aureus LOG (CFU) 4hr EXTRACELLULAR R.8 INTRACELLULAR R.89 3 ft>mic% 3 ft>mic% Correlation between treatment outcome and the T>MIC index 9 Sandberg et al., Antimicrob Agents Chemother () 54:39-4
24 PK/PD studies: Dicloxacillin vs S. aureus LOG (CFU) 4hr EXTRACELLULAR R.8 INTRACELLULAR R.89 3 ft>mic% 3 ft>mic% T>MIC is the predicting PK/PD index both intra- and extracellularly 9 Sandberg et al., Antimicrob Agents Chemother () 54:39-4
25 PK/PD studies: Dicloxacillin vs S. aureus LOG (CFU) 4hr EXTRACELLULAR R.8 INTRACELLULAR R.89 log reduction 3 ft>mic% 3 ft>mic% A reduction of logs was obtained intracellularly with optimal dosing 9 Sandberg et al., Antimicrob Agents Chemother () 54:39-4
26 Dose-response studies LINEZOLID vs. S. aureus 4 3 IN VIVO IN VITRO 4 3 intracellular extracellular log CFU 3 mg/kg (log ) mg/l (log ) 9 Sandberg et al., J. Antimicrob. Chemother () 65:96-973
27 Dose-response studies LINEZOLID vs. S. aureus 4 3 IN VIVO 4 3 IN VITRO intracellular extracellular log CFU 3 mg/kg (log ) mg/l (log ) No decreased intracellular activity in vitro 9 Sandberg et al., J. Antimicrob. Chemother () 65:96-973
28 Dose-response studies LINEZOLID vs. S. aureus 4 3 IN VIVO 4 3 IN VITRO intracellular extracellular log CFU 3 mg/kg (log ) mg/l (log ) No reduction of the original intracellular inoculum in vivo 9 Sandberg et al., J. Antimicrob. Chemother () 65:96-973
29 PK/PD studies: Linezolid vs S. aureus EXTRACELLULAR INTRACELLULAR log cfu 4hr - log CFU 4hr - fc max /MIC fc max /MIC No correlation between treatment outcome and the Cmax /MIC index 9 Sandberg et al., J. Antimicrob. Chemother () 65:96-973
30 PK/PD studies: Linezolid vs S. aureus EXTRACELLULAR EXTRACELLULAR R =.55 R =.5 log cfu 4hr log cfu 4hr fauc 4h /MIC ft>mic% Both AUC and T>MIC correlated equally to the extracellular outcome 9 Sandberg et al., J. Antimicrob. Chemother () 65:96-973
31 PK/PD studies: Linezolid vs S. aureus INTRACELLULAR INTRACELLULAR R =.9 R =.3 log cfu 4hr log cfu 4hr ft>mic% fauc 4h /MIC Poor correlation between PK/PD indices and the intracellular outcome 9 Sandberg et al., J. Antimicrob. Chemother () 65:96-973
32 PK/PD studies: Linezolid vs S. aureus Conventional dose: 6 mg twice daily AUC/MIC = 8 EXTRACELLULAR INTRACELLULAR R =.55 R =.3 log cfu 4hr log cfu 4hr fauc 4h /MIC fauc 4h /MIC Acceptable extracellular effect but questionable intracellular effect with conventional dose 9 Sandberg et al., J. Antimicrob. Chemother () 65:96-973
33 Intracellular peritonitis mouse model: Fusidic acid 3 Total count Extracellular count 3 Intracellular count 3 log(cfu/ml) logauc/mic log(cfu/ml) logauc/mic log(cfu/ml) logauc/mic Sandberg et al. unpublished
34 Intracellular peritonitis mouse model: Fusidic acid log(cfu/ml) 3 - Total count log(cfu/ml) Extracellular count 3 - log(cfu/ml) Intracellular count T>MIC T>MIC T>MIC Sandberg et al. Unpublished
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)
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