Tissue Cage Fluid Research Articles

Moxidectin is a candidate for use as an in vivo internal standard in pharmacokinetic studies, as demonstrated with use in simultaneous tissue cage and ultrafiltration fluid collection.

In vivo ultrafiltration has been used in veterinary pharmacokinetics since the early 2000's as an improvement on the tissue cage model which enables sampling of fluids from extra-circulatory compartments. Variability in analyte recovery from ultrafiltration samples, due to membrane fouling or tissue inflammation, has been a concern for this technique. Internal standards may be used to scale or verify the unknown result, such as is common in analytical extractions and in vivo microdialysis. Eight merino sheep were implanted with subcutaneous tissue cages and 2 weeks prior to the initiation of the study the sheep were injected with 0.2 mg/kg moxidectin subcutaneously. On the day of the study ultrafiltration probes were inserted subcutaneously. At time zero 4 mg/kg of carprofen was injected intravenously. Plasma, tissue cage, and ultrafiltration samples were taken 30 min before and 0.5, 1, 2, 3, 4, 5, 7, 24, 36, 48, 72 h after dosing. Carprofen and moxidectin concentrations were measured by LC-MS/MS. Pharmacokinetic parameters were estimated using Monolix for both the carprofen concentrations and the moxidectin corrected carprofen concentrations. The ultrafiltration probes failed to consistently produce enough sample volume to analyse. Moxidectin concentrations in the plasma and tissue cage fluid were stable throughout the 72 h sampling window. Moxidectin proved to be suitable as an in vivo internal standard for pharmacokinetic research using, tissue cages, plasma sampling and ultrafiltration probes, but the application of ultrafiltration techniques requires refinement.

Pharmacokinetic/pharmacodynamic integration of tilmicosin against Pasteurella multocida in a piglet tissue cage model.

Tilmicosin is a semi-synthetic macrolide for veterinary use with strong antibacterial effect on respiratory bacteria. In this study, the pharmacokinetic/pharmacodynamic (PK/PD) integration of tilmicosin against Pasteurella multocida (P. multocida) was evaluated by establishing a piglet tissue cage infection model. Concentration of tilmicosin and bacterial numbers of P. multocida in the tissue-cage fluid were monitered. After the population of P. multocida was equal to or greater than 107 CFU/mL in a tissue cage, piglets received an oral administration of tilmicosin at a dose of 30, 40, 50, and 60 mg/kg b.w., once daily for 3 days, respectively. Bacteria were counted every 24 h after drug administration and at 48 and 72 h after the last administration. A sigmoidal Emax model was used to fit the relationship between PK/PD parameters and the antibacterial effect. AUC24h/MIC was the best PK/PD index that correlated with effectiveness of tilmicosin against P. multocida. The magnitude of AUC24h/MIC required for continuous 1/3-log, 1/2-log, and 3/4-log reductions were 19.65 h, 23.86 h, and 35.77 h, respectively, during each 24 h treatment period. In this study, when the dosage was >50 mg/kg, the AUC24h/MIC was still >35.77 h in the period of 24-48 h after the last administration due to the slow elimination, that is, tilmicosin exhibited a potent antibacterial effect against P. multocida after three successive daily administrations. The data provide meaningful guidance to optimize regimens of tilmicosin to treat respiratory tract infections caused by P. multocida.

Kill Rate and Evaluation of Ex Vivo PK/PD Integration of Cefquinome Against Actinobacillus pleuropneumoniae.

We wished to study the detailed and precise antibacterial activity of cefquinome against Actinobacillus pleuropneumoniae (APP) in vitro and ex vivo. We analyzed the relationships between kill rate and cefquinome concentration in broth and between pharmacokinetic/pharmacodynamic (PK/PD) parameters and antibacterial effect in serum and tissue cage fluid (TCF) of piglets. Cefquinome exhibited time-dependent antibacterial activity against APP according to the kill rate. The maximum kill rate was 0.48 log10 CFU/mL/h at the 0-9-h period in broth. In the ex vivo PK/PD study, the maximum concentration (Cmax), time to reach the maximum concentration (Tmax), terminal half-life (T1/2β), and area under the concentration time curve (AUCinfinity) were 5.65 μg/ml, 0.58 h, 2.24 h, and 18.48 μg·h/ml in serum and 1.13 μg/ml, 2.60 h, 12.22 h, and 20.83 μg·h/ml in TCF, respectively. The values of area under the curve during 24 h/minimum inhibitory concentration (AUC24h/MIC) for bacteriostatic, bactericidal, and bacterial eradication effects were 18.94, 246.8, and 1013.23 h in serum and 4.20, 65.81, and 391.35 h in TCF, respectively. Our findings will provide a valuable basis for optimization of dosage regimens when applying cefquinome to treat APP infection.

In vitro and in vivo synergistic effects of tigecycline combined with aminoglycosides on carbapenem-resistant Klebsiella pneumoniae

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) infections represent severe threats to public health worldwide. The aim of this study was to assess potential synergistic interaction between tigecycline and aminoglycosides via in vitro and in vivo studies. Antibiotic resistance profiles and molecular characteristics of 168 CR-KP clinical isolates were investigated by susceptibility testing, PCR and MLST. Chequerboard tests and time-kill assays were performed for 20 CR-KP isolates to evaluate in vitro synergistic effects of tigecycline combined with aminoglycosides. A tissue-cage infection model of rats was established to evaluate in vivo synergistic effects. Different doses of tigecycline and aminoglycosides alone or in combination were administered for 7 days via tail vein injection. Antibiotic efficacy was evaluated in tissue-cage fluid and emergence of resistance was screened. The chequerboard tests showed that this combination displayed synergistic or partial synergistic activity against CR-KP. The time-kill assays further demonstrated that strong synergistic effects of such a combination existed against isolates that were susceptible to both drugs but for resistant isolates no synergy was observed if clinical pharmacokinetics were taken into consideration. The in vivo study showed that the therapeutic effectiveness of combination therapies was better than that of monotherapy for susceptible isolates, suggesting in vivo synergistic effects. Furthermore, combinations of tigecycline with an aminoglycoside showed significant activity in reducing the occurrence of tigecycline-resistant mutants. Compared with single drugs, tigecycline combined with aminoglycosides could exert synergistic effects and reduce the emergence of tigecycline resistance. Such a combination might be an effective alternative when treating CR-KP infections in clinical practice.

Pharmacokinetics and ex vivo pharmacodynamics of Minocycline against Salmonella abortus equi in donkey plasma and tissue cage fluid

Tissue Cage (TC) model was used to evaluate the pharmacokinetics and ex vivo pharmacodynamics of Minocycline (MINO) after intramuscular (IM) administration to donkeys at 4 mg/kg body-weight. The Cmax of MINO with 1.79 and 2.63 μg mL−1 was obtained at 2.96 and 1.41 h in TCF (tissue cage fluid) and plasma respectively. The absorption half-lives (t1/2ka) of MINO were calculated to be 0.71 h in TCF and 0.32 h in plasma, whereas the elimination half-lives (t1/2ke) were 10.46 h in TCF and 5.95 h in plasma. The distribution volume (Vd/F) of MINO was estimated to be 1.84 L kg−1 in TCF and 1.28 L kg−1 in plasma. The total clearance (CLb/F) of MINO was computed as 0.12 and 0.15 L/ (h·kg) in TCF and plasma respectively. The area under the concentration-time curve (AUC) of MINO was 32.77 μg mL−1h in TCF and 25.27 μg mL−1h in plasma, respectively.The ex vivo time-kill curves were established for plasma and TCF samples using Salmonella abortus equi. The MIC and MBC of MINO against salmonella were 0.08 and 0.16 μg mL−1 for plasma, 0.04 and 0.08 μg mL−1 for TCF. The plasma Cmax/MIC and AUC/MIC values after IM administration were 32.88 ± 9.87 and 315.88 ± 42.65 h, respectively. The TCF Cmax/MIC and AUC/MIC values after IM administration were 44.75 ± 9.32 and 819.25 ± 65.23 h, respectively. The values of T > MIC were approximately >36 h in plasma and > 65 h in TCF. These findings from this study suggest that MINO may be therapeutically effective in diseases of donkeys caused by salmonella when used at a dose of 4 mg/kg IM administration.

Pharmacokinetic/pharmacodynamic integration of cefquinome against Pasteurella Multocida in a piglet tissue cage model

To explore the in vivo antimicrobial activity of cefquinome against Pasteurella multocida in piglets, a piglet tissue cage infection model was used in this study. After the population of P.multocida reached 107 CFU/mL in a tissue cage, piglets received an intramuscular administration of cefquinome at 0.2, 0.4, 0.8, 1, 2, and 4mg/kg once daily for 3days. To assess the tissue cage pharmacokinetics (PKTCF) of cefquinome, tissue cage fluid was collected for cefquinome analysis at 1, 3, 6, 9, 12, and 24hr after each of the 3 daily drug administrations. Bacteria were counted every 24hr after drug administration and at 48 and 72hr after the last administration. Evaluation of the relationship between pharmacokinetic/pharmacodynamic (PK/PD) parameters and the antibacterial effect showed that the surrogate of %T>minimum inhibitory concentration (MIC) (R2 =0.981) was the best PK/PD index that correlated with effectiveness of cefquinome against P. multocida. The respective values of %T>MIC required for continuous 1/3-log, 1/2-log, and 1-log reductions were 14.23, 34.45, and 73.44%, respectively, during each 24-hr treatment period. In conclusion, cefquinome exhibited a potent antibacterial effect against P.multocida. When %T>MIC reached 73.44%, cefquinome exhibited a bactericidal effect against P.multocida after three successive daily administrations.

Acrylic microparticles increase daptomycin intracellular and in vivo anti-biofilm activity against Staphylococcus aureus

Daptomycin (DAP) is a cyclic lipopeptide antibiotic with potential clinical application in orthopedic infections caused by staphylococci. However, it failed to eradicate Staphylococcus aureus in vitro, in intracellular infection studies, as well as in vivo in an experimental model of implant-associated biofilm infections. In this study, the antimicrobial effect of DAP encapsulated in poly(methyl methacrylate)-Eudragit (PMMA-EUD) microparticles (DAP-MPs) on intracellular S. aureus was evaluated in human osteoblast cells using fluorescence in situ hybridization (FISH) analysis. Encapsulated DAP was able to reduce the amount of intracellular S. aureus by 73% compared to blank microparticles (MPs). Then, the advantage of treating with DAP-MPs versus free DAP was evaluated in a murine model of implant-associated biofilm infection. Free DAP showed a >3 log10 decrease in planktonic and adherent bacteria but failed to eradicate adherent methicillin-resistant S. aureus (MRSA), whereas DAP-MPs showed a clearance of planktonic MRSA, significantly reduced adherent MRSA by more than 3 log10 and cured the infection in 60%. This was linked to the prolonged higher DAP concentration within the tissue cage fluid compared to free DAP. To our knowledge, this study provides the first evidence for the high intracellular and in vivo anti-biofilm efficacy of DAP-MPs to target staphylococcal infections.

Drawbacks of the use of cotrimoxazole in foreign-body infections

IntroductionThe anti-staphylococcal efficacy of cotrimoxazole in the setting of difficult-to-treat infections seems to be compromised by large amounts of pus and devitalized tissue, and, therefore, high levels of thymidine. Our objective was to evaluate the activity of cotrimoxazole against a staphylococcal foreign-body infection experimental model, which also yields significant quantities of thymidine. Material and methodsWe used a rat tissue-cage model of infection (with high inherent thymidine levels) caused by a strain of methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 29213). MIC values were determined (microdilution method) and compared in the presence or absence of tissue-cage fluid samples. ResultsThe inefficacy of cotrimoxazole was found to be similar to that of the control group. The MIC of cotrimoxazole was 4•8 fold higher in the presence of rat tissue-cage fluid. ConclusionsThe inefficacy of cotrimoxazole in our foreign-body infection model by MSSA, and the probable negative impact of the presence of thymidine on its efficacy, challenge the use of this drug in acute phases of foreign-body infections. It should be reserved as an alternative treatment when the infection is more controlled.

Pharmacokinetic/pharmacodynamic assessment of cefquinome against Actinobacillus Pleuropneumoniae in a piglet tissue cage infection model

To evaluate the relationship between the pharmacokinetic/pharmacodynamic (PK/PD) parameters and the antibacterial effect of cefquinome against Actinobacillus pleuropneumoniae, a tissue cage infection model was established in piglets. In this model, an initial count of A. pleuropneumoniae of approximately 106 CFU/mL was exposed to different concentrations of cefquinome after multiple administration at dosages of 0.2, 0.4, 0.8, 1, 2, 4 mg/kg body weight once a day for 3 days. Concentration of cefquinome and bacterial numbers of A. pleuropneumoniae in the tissue-cage fluid (TCF) were monitered. An inhibitory form of sigmoid maximum effect (Emax) model was used to estimate the relationship between the antibacterial effect and PK/PD indices of cefquinome against A. pleuropneumoniae. The minimum inhibitory concentration of cefquinome against A. pleuropneumoniae was 0.016 μg/mL in TCF. The total maximum antibacterial effect was a 3.96 log10 (CFU/mL) reduction. In addition, the cumulative percentage of time over a 24 h period that the drug concentration exceeds the MIC (%T > MIC) was the pharmacokinetic-pharmacodynamic (PK-PD) index that best correlated with the antibacterial efficacy (R2 = 0.967). The estimated %T > MIC values were 11.59, 27.49, and 59.81% for a 1/3-log10 (CFU/mL) reduction, a 2/3-log10 (CFU/mL) reduction, and a 1-log10 (CFU/mL) reduction, respectively, during the 24h administration period of cefquinome. In conclusion, cefquinome exhibits excellent antibacterial activity and time-dependent characteristics against A. pleuropneumoniae in vivo. Furthermore, these data provide meaningful guidance to optimize regimens of cefquinome to treat respiratory tract infections caused by A. pleuropneumoniae.

Drawbacks of the use of cotrimoxazole in foreign-body infections

IntroductionThe anti-staphylococcal efficacy of cotrimoxazole in the setting of difficult-to-treat infections seems to be compromised by large amounts of pus and devitalized tissue, and, therefore, high levels of thymidine. Our objective was to evaluate the activity of cotrimoxazole against a staphylococcal foreign-body infection experimental model, which also yields significant quantities of thymidine. Material and methodsWe used a rat tissue-cage model of infection (with high inherent thymidine levels) caused by a strain of methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 29213). MIC values were determined (microdilution method) and compared in the presence or absence of tissue-cage fluid samples. ResultsThe inefficacy of cotrimoxazole was found to be similar to that of the control group. The MIC of cotrimoxazole was 4–8 fold higher in the presence of rat tissue-cage fluid. ConclusionsThe inefficacy of cotrimoxazole in our foreign-body infection model by MSSA, and the probable negative impact of the presence of thymidine on its efficacy, challenge the use of this drug in acute phases of foreign-body infections. It should be reserved as an alternative treatment when the infection is more controlled.

Pharmacokinetic-pharmacodynamic integration and modelling of oxytetracycline for the calf pathogens Mannheimia haemolytica and Pasteurella multocida.

A calf tissue cage model was used to study the pharmacokinetics (PK) and pharmacodynamics (PD) of oxytetracycline in serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids. After intramuscular administration, the PK was characterized by a long mean residence time of 28.3hr. Based on minimum inhibitory concentrations (MICs) for six isolates each of Mannheimia haemolytica and Pasteurella multocida, measured in serum, integration of in vivo PK and in vitro PD data established area under serum concentration-time curve (AUC0-∞ )/MIC ratios of 30.0 and 24.3hr for M.haemolytica and P.multocida, respectively. Corresponding AUC0-∞ /MIC ratios based on MICs in broth were 656 and 745hr, respectively. PK-PD modelling of in vitro bacterial time-kill curves for oxytetracycline in serum established mean AUC0-24hr /MIC ratios for 3log10 decrease in bacterial count of 27.5hr (M.haemolytica) and 60.9hr (P.multocida). Monte Carlo simulations predicted target attainment rate (TAR) dosages. Based on the potency of oxytetracycline in serum, the predicted 50% TAR single doses required to achieve a bacteriostatic action covering 48-hr periods were 197mg/kg (M.haemolytica) and 314mg/kg (P.multocida), respectively, against susceptible populations. Dosages based on the potency of oxytetracycline in broth were 25- and 27-fold lower (7.8 and 11.5mg/kg) for M.haemolytica and P.multocida, respectively.

Pharmacokinetic/pharmacodynamic analysis by Monte Carlo simulation of cefquinome in llamas, following intravenous, intramuscular and subcutaneous administration in serum and tissue cage fluid

The objective of this study was to determine the pharmacokinetic behavior of cefquinome (CFQ) in serum and tissue cage fluid (TCF) in llamas, after intravenous (IV), intramuscular (IM) and subcutaneous (SC) administration of 2mg/kg. Two tissue cages (TC) per animal were implanted in the subcutaneous tissue, on the pectoralis major muscles area. Serum and TCF concentrations of CFQ were determined by microbiological assay using Klebsiella pneumoniae ATCC 10031 as the test microorganism. Minimum inhibitory concentrations (MIC) of CFQ against Escherichia coli and Staphylococcus aureus strains isolated from llamas were determined by broth microdilution method. S. aureus ATCC 29213 and E. coli ATCC 25922 strains were used as controls. Finally, a pharmacokinetic/pharmacodynamic (PK/PD) analysis by Monte Carlo simulation, based on pharmacokinetic data obtained from IV, IM and SC administration of CFQ in llamas and MIC values, was carried out to evaluate the antimicrobial efficacy of the dose regimen. CFQ showed a limited distribution, with values of Vdss=0.10±0.01 and Vz=0.18±0.04L/kg. Total body clearance (0.05±0.01L*kg/h) was lower than reported in other species. Absolute bioavailability of IM and SC routes was complete (124±45% and 94±35%, respectively). Moreover, CFQ presented a AUCTCF/AUCserum ratio <1 after IV administration, but≈2 after IM and SC routes and showed a higher permanence in TCF compared with serum. These findings could determine higher T>MIC values, resulting in a better antimicrobial efficacy of CFQ in this biological compartment. MIC90 values for E. coli and S. aureus were 0.06μg/mL and 0.50μg/mL, respectively, with a MIC90/MIC50=2. Based on the PK/PD analysis, a dose regimen of CFQ of 2mg/kg q12h by IV, IM and SC routes, could be effective for the treatment of infections caused by pathogens with MIC values ≤0.50μg/mL. Therefore, CFQ appears to be a good choice against E. coli and S. aureus in llamas.

Relationship between Cefquinome PK/PD Parameters and Emergence of Resistance of Staphylococcus aureus in Rabbit Tissue-Cage Infection Model.

In order to explore the relationship between different antibiotic dosing regimens and selective enrichment of resistant strains, tissue-cage infection model was established in rabbits to study relationship between cefquinome pharmacokinetic/pharmacodynamic parameters and the change of susceptibility of Staphylococcus aureus (S. aureus). In this model, above 108 CFU/mL of S. aureus culture were exposed to cefquinome concentrations below the MIC99 (the minimal concentration that inhibits colony formation by 99% in vitro, 0.3 μg/mL), between the MIC99 and the MPC (the mutant prevent concentration in vitro, 1.6 μg/mL), and above the MPC after intramuscular injection with cefquinome at doses of 4, 8, 16, and 32 mg/kg of body weight (bw) once daily for 5 days or 4, 8, 16, and 24 mg/kg of bw twice daily for 2.5 days. Samples of tissue-cage fluid were collected from the tissue-cage at 2, 4, 6, 8, 10, 12, 24 h after each dosing (one dosing daily) or at 2, 4, 6, 8, 10, and 12 h (two dosing daily). Cefquinome concentration, susceptibility of S. aureus to cefquinome, and bacterial numbers at the infected site were monitored. The MICs of S. aureus and the fraction of resistant bacteria both increased when cefquinome concentrations fluctuated between the MIC99 and MPC. Resistant bacteria were selected in vivo when %T > MPC was < 58% of administration interval or %T > MIC99 was ≥70% of administration interval. These findings demonstrate that low-level, cefquinome-resistant S. aureus were selected predominantly when drug concentrations fell inside a concentration window in in vivo model, which was evidenced by pulsed-field gel electrophoresis. The selection of resistant bacteria arose from both susceptible bacteria being killed and resistant bacteria re-growth. Keeping drug concentrations above the MPC for ≥58% of administration interval provides a strategy to achieve effective antibacterial activity and minimize the emergence of resistance to cefquinome.

Response of a clinical Escherichia coli strain to repeated cefquinome exposure in a piglet tissue-cage model.

BackgroundIn order to provide some basis for effective dosage regimens that optimize efficacy with respect to bacteriological and clinical cures, the in vivo activity of cefquinome against a clinical Escherichia coli (E.coli) strain (the minimum inhibitory concentration value for this strain equals to the MIC90 value of 0.25 μg/ml for 210 E.coli strains isolated from pigs) was investigated by using a piglet tissue-cage infection model. The aim was to elucidate the pharmacokinetic/pharmacodynamics (PK/PD) index associated with cefquinome efficacy, and then to identify the magnitude of the PK/PD parameter required for different degree of efficacy in clinical treatment.ResultsTissue-cage infection model was established in piglets, and then the animals received intramuscular injection of cefquinome twice a day for 3 days to create a range of different drug exposures. The tissue-cage fluid was collected at 1, 3, 6, 9 and 12 h after every drug administration for drug concentration determination and bacteria counting. Different cefquinome regimens produced different percentages of time during that drug concentrations exceeded the MIC (%T > MIC), ranging from 0 % to 100 %. Cefquinome administration at 0.2, 0.4, 0.6, 0.8, 1, 2 and 4 mg/kg reduced the bacterial count (log10 CFU/mL) in tissue-cage fluid by −1.00 ± 0.32, −1.83 ± 0.08, −2.33 ± 0.04, −2.96 ± 0.16, −2.99 ± 0.16, −2.93 ± 0.11, −3.43 ± 0.18, respectively. The correlation coefficient of the PK/PD index with antibacterial effect of the drug was 0.90 for %T > MIC, 0.62 for AUC0–12/MIC, and 0.61 for Cmax/MIC, suggesting the most important PK/PD parameter was %T > MIC. A inhibitory form of sigmoid maximum effect (Emax) model was used to estimate %T > MIC, and the respective values required for continuous 1/6-log drop, 1/3-log drop and 1/2-log drop of the clinical E.coli count during each 12 h treatment period were 3.97 %, 17.08 % and 52.68 %.ConclusionsThe data derived from this study showed that cefquinome exhibited time-dependent killing profile. And from the results of the present study, it can be assumed that when %T > MIC reached 52.68 %, cefquinome could be expected to be effective against a clinical E.coli strain for which the MIC value is below 0.128 μg/ml (3-log drop of bacteria count can be achieved after six successive administrations for 3 days).

Daptomycin combinations as alternative therapies in experimental foreign-body infection caused by meticillin-susceptible Staphylococcus aureus

Whilst levofloxacin (LVX) in combination with rifampicin (RIF) is considered the optimal treatment for prosthetic joint infection (PJI) caused by meticillin-susceptible Staphylococcus aureus (MSSA), no therapeutic alternatives have been accurately evaluated. Based on the high effectiveness of the combination of daptomycin (DAP) plus RIF against meticillin-resistant S. aureus (MRSA) in this setting, in this study the efficacy of DAP+RIF and DAP+LVX combinations was tested as alternative therapies for foreign-body infections (FBIs) caused by MSSA. A tissue-cage infection model was performed using an MSSA strain. Male Wistar rats were treated for 7 days with LVX, DAP, RIF or the combinations LVX+RIF, DAP+RIF and DAP+LVX. Antibiotic efficacy was evaluated by bacterial counts from tissue cage fluid (TCF) and the cure rate was determined from adhered bacteria. Resistance was screened. Monotherapies were less effective than combinations (P<0.05), and resistance to DAP and RIF emerged. DAP+RIF (decrease in bacterial counts in TCF, −4.9logCFU/mL; cure rate, 92%) was the most effective therapy (P<0.05). There were no differences between LVX+RIF (−3.4logCFU/mL; 11%) and DAP+LVX (−3.3logCFU/mL; 47%). No resistant strains appeared with combined therapies. In conclusion, the combinations DAP+RIF and DAP+LVX showed good efficacy and prevented resistance. DAP+RIF provided higher efficacy than LVX+RIF. These DAP combinations were efficacious alternatives therapies for MSSA FBI. Further studies should confirm whether DAP+RIF may be useful as a first-line therapy in the setting of PJI caused by MSSA.

Pharmacokinetic/pharmacodynamic integration and modelling of amoxicillin for the calf pathogens Mannheimia haemolytica and Pasteurella multocida

The antimicrobial properties of amoxicillin were determined for the bovine respiratory tract pathogens, Mannheima haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves were established. Pharmacokinetic (PK)/pharmacodynamic (PD) modelling of the time-kill data, based on the sigmoidal Emax equation, generated parameters for three levels of efficacy, namely bacteriostatic, bactericidal (3log10 reduction) and 4log10 reduction in bacterial counts. For these levels, mean AUC(0-24 h) /MIC serum values for M. haemolytica were 29.1, 57.3 and 71.5 h, respectively, and corresponding values for P. multocida were 28.1, 44.9 and 59.5 h. Amoxicillin PK was determined in calf serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids, after intramuscular administration of a depot formulation at a dosage of 15 mg/kg. Mean residence times were 16.5 (serum), 29.6 (exudate) and 29.0 h (transudate). Based on serum MICs, integration of in vivo PK and in vitro PD data established maximum concentration (Cmax )/MIC ratios of 13.9:1 and 25.2:1, area under concentration-time curve (AUC0-∞ )/MIC ratios of 179 and 325 h and T>MIC of 40.3 and 57.6 h for P. multocida and M. haemolytica, respectively. Monte Carlo simulations for a 90% target attainment rate predicted single dose to achieve bacteriostatic and bactericidal actions over 48 h of 17.7 and 28.3 mg/kg (M. haemolytica) and 17.7 and 34.9 mg/kg (P. multocida).

Testing the mutant selection window hypothesis in vitro and in vivo with Staphylococcus aureus exposed to fosfomycin.

The purpose of this study was to test the mutant selection window (MSW) hypothesis in vitro and in vivo with Staphylococcus aureus exposed to fosfomycin. With the in vitro time-kill studies, S. aureus ATCC 29213 [with a minimal concentration that inhibits colony formation by 99% (MIC99) of 2.2 μg/mL and a mutant prevention concentration (MPC) of 57.6 μg/mL] lost fosfomycin susceptibility at antibiotic concentrations (2×, 4×, and 8× MIC) that are between the lower and upper boundaries of the MSW. In the tissue-cage model, S. aureus was exposed to fosfomycin pharmacokinetics at concentrations below the MIC99, between the MIC99 and the MPC, and above the MPC, respectively. Changes in susceptibility and counts of total and resistant viable bacteria were monitored in tissue-cage fluid obtained daily. However, the selection of resistant mutants was not observed during antibacterial treatment and 48 h after the termination of fosfomycin treatment, regardless of the fosfomycin dosage. Besides, we found no differences between the in vitro-isolated mutant and its sensitive parental strain, which indicates the absence of fitness cost of fosfomycin resistance in S. aureus ATCC 29213. These findings demonstrate that agar plate determinations do not fit the MSW for fosfomycin treatment of rabbits infected with S. aureus ATCC 29213; therefore, the existence of the window must be demonstrated not only in vitro but also in vivo. Further research is needed on the exact mechanism of resistance.

Foreign Body Infection Models to Study Host-Pathogen Response and Antimicrobial Tolerance of Bacterial Biofilm.

The number of implanted medical devices is steadily increasing and has become an effective intervention improving life quality, but still carries the risk of infection. These infections are mainly caused by biofilm-forming staphylococci that are difficult to treat due to the decreased susceptibility to both antibiotics and host defense mechanisms. To understand the particular pathogenesis and treatment tolerance of implant-associated infection (IAI) animal models that closely resemble human disease are needed. Applications of the tissue cage and catheter abscess foreign body infection models in the mouse will be discussed herein. Both models allow the investigation of biofilm and virulence of various bacterial species and a comprehensive insight into the host response at the same time. They have also been proven to serve as very suitable tools to study the anti-adhesive and anti-infective efficacy of different biomaterial coatings. The tissue cage model can additionally be used to determine pharmacokinetics, efficacy and cytotoxicity of antimicrobial compounds as the tissue cage fluid can be aspirated repeatedly without the need to sacrifice the animal. Moreover, with the advance in innovative imaging systems in rodents, these models may offer new diagnostic measures of infection. In summary, animal foreign body infection models are important tools in the development of new antimicrobials against IAI and can help to elucidate the complex interactions between bacteria, the host immune system, and prosthetic materials.

Secreted Gaussia princeps Luciferase as a Reporter of Escherichia coli Replication in a Mouse Tissue Cage Model of Infection

Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.

Pharmacokinetics and ex vivo pharmacodynamics of cefquinome in porcine serum and tissue cage fluids

A tissue cage (TC) model was used to evaluate the pharmacokinetics and ex vivo pharmacodynamics of cefquinome after intravenous (IV) and intramuscular (IM) administration to piglets at 2mg/kg bodyweight. The mean values of area under the concentration–time curve (AUC) were 21.28 (IV) and 21.37 (IM)μgh/mL for serum, and 17.40 (IV) and 16.57 (IM)μgh/mL for TC fluid (TCF), respectively. Values of maximum concentration (Cmax) were 6.15μg/mL (serum) and 1.15μg/mL (TCF) after IM administration. The elimination half-lives (t1/2β) in TCF (10.63h IV and 11.81h IM) were significantly higher than those in serum (2.33h IV and 2.30h IM) (P<0.05). The values of AUCTCF/AUCserum (%) after IV and IM administration were 82.4% and 80.7%, respectively.The ex vivo time-kill curves were established for serum and TCF samples using Escherichia coli ATCC 25922. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration values of cefquinome against E. coli were 0.030 and 0.060μg/mL in Mueller–Hinton broth, and 0.032 and 0.064μg/mL in both serum and TCF, respectively. The ex vivo growth inhibition data of TCF after IM administration were fitted to the sigmoid Emax model; AUC24h/MIC was 35.01h for bactericidal activity and 44.28h for virtual eradication, respectively. The findings from this study suggest that cefquinome may be therapeutically effective in diseases of pigs caused by E. coli when used at a dose rate of 1.33mg/kg administered every 24h for organisms with MIC90⩽0.50μg/mL.