Abstract
BackgroundSpecific antibodies are likely to be present before S. pneumoniae infection. We explored cefditoren (CDN) total and free values of serum concentrations exceeding the MIC (t>MIC) related to efficacy in a mice sepsis model, and the effect of specific gammaglobulins on in-vitro phagocytosis and in-vivo efficacy.Methodology/Principal FindingsWe used three pneumococcal isolates (serotype, MIC of CDN): Strain 1 (6B, 1 µg/ml), Strain 2 (19F, 2 µg/ml) and Strain 3 (23F, 4 µg/ml). Hyperimmune serum (HS) was obtained from mice immunized with heat-inactivated strains. In-vitro, phagocytosis by HS diluted 1/10 in presence/absence of sub-inhibitory concentrations was measured by flow cytometry including fluorescent bacteria and a neutrophil cell line. In-vivo dose-ranging experiments with HS (dilutions 1/2–1/16) and CDN (6.25 mg/kg–100 mg/kg tid for 48 h) were performed to determine the minimal protective dilution/dose (highest survival) and the non-protective highest dilution/dose (highest mortality: HS-np dilution and CDN-np dose) over 7 days. Efficacy of CDN-np in animals pre-immunized with HS-np (combined strategy) was explored and blood bacterial clearance determined. The CDN measured protein binding was 86.9%. In-vitro, CDN significantly increased phagocytosis (vs. HS 1/10). In non pre-immunized animals, t>MIC values for CDN of ≈35% (total) and ≈19% (free) were associated with 100% survival. Significant differences in survival were found between HS-np alone (≤20%) or CDN-np alone (≤20%) vs. the combined strategy (90%, 60% and 60% for Stains 1, 2 and 3), with t>MIC (total/free) of 22.8%/14.3%, 26.8%/16.0%, and 22.4%/12.7% for Strains 1, 2 and 3, respectively. Prior to the second dose (8 h), median bacterial counts were significantly lower in animals surviving vs. dead at day 7.Conclusions/SignificanceIn mice (CDN protein binding similar to humans) total t>MIC values of ≈35% (≈19% free) were efficacious, with a decrease in the required values in pre-immunized animals. This reinforces that immunoprotection to overcome resistance may provide lifesaving strategies.
Highlights
Differences between in vitro activity and in vivo efficacy of antimicrobials may result from the participation of the immune system in bacterial eradication and/or the limitation of antibiotic activity by the binding of antibiotics to serum proteins
The incubation of S. pneumoniae with subinhibitory concentrations of cefditoren in the presence of specific antibodies significantly increased the phagocytosis against the three strains compared to the phagocytosis mediated by the 1/10 dilution of hyperimmune serum or the subinhibitory antibiotic concentrations independently, both using HL-60 (p,0.03) (Fig. 1A–C) and mice neutrophils (p#0.001) (Fig. 2A–C)
This was not the case of ceftriaxone and Strain 2 where the phagocytosis mediated by HL-60 cells was not statistically different (p = 0.2) from the one mediated by the 1/10 dilution of hyperimmune serum itself (Fig. 1D), in experiments with mice neutrophils the phagocytosis with 0.256 MIC was significant (p = 0.012) but not with 0.56 MIC (p = 0.1) (Fig. 2D)
Summary
Differences between in vitro activity and in vivo efficacy of antimicrobials may result from the participation of the immune system in bacterial eradication and/or the limitation of antibiotic activity by the binding of antibiotics to serum proteins (protein binding). Since colonisation is to some extend a B-cell immunising event [1,2] and preventive measures as pneumococcal vaccination are increasingly being used, antibodies to capsular polysaccharides (a surrogate marker of immunity) are likely to appear before infection. In this situation, the appearance of pneumococcal sepsis indicates defective protection against pneumococcal invasion. We explored cefditoren (CDN) total and free values of serum concentrations exceeding the MIC (t.MIC) related to efficacy in a mice sepsis model, and the effect of specific gammaglobulins on in-vitro phagocytosis and in-vivo efficacy
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