Combined therapy with ceftriaxone and doxycycline does not improve the outcome of meningococcal meningitis in mice compared to ceftriaxone monotherapy

Background: Meningococcal meningitis (MM) is a life-threatening disease associated with approximately 10% case fatality rates and neurological sequelae in 10-20% of the cases. Recently, we have shown that the matrix metalloproteinase (MMP) inhibitor BB-94 reduced brain injury in a mouse model of MM. The present study aimed to assess whether doxycycline (DOX), a tetracycline that showed a neuroprotective effect as adjuvant therapy in experimental pneumococcal meningitis (PM), would also exert a beneficial effect when given as adjunctive therapy to ceftriaxone (CRO) in experimental MM. Methods: BALB/c mice were infected by the intracisternal route with a group C Neisseria meningitidis strain. Eighteen h post infection (hpi), animals were randomised for treatment with CRO [100 mg/kg subcutaneously (s.c.)], CRO plus DOX (30 mg/kg s.c.) or saline (control s.c.). Antibiotic treatment was repeated 24 and 40 hpi. Mouse survival and clinical signs, bacterial counts in cerebella, brain damage, MMP-9 and cyto/chemokine levels were assessed 48 hpi. Results: Analysis of bacterial load in cerebella indicated that CRO and CRO + DOX were equally effective at controlling meningococcal replication. No differences in survival were observed between mice treated with CRO (94.4%) or CRO + DOX (95.5%), (p > 0.05). Treatment with CRO + DOX significantly diminished both the number of cerebral hemorrhages (p = 0.029) and the amount of MMP-9 in the brain (p = 0.046) compared to untreated controls, but not to CRO-treated animals (p > 0.05). Levels of inflammatory markers in the brain of mice that received CRO or CRO + DOX were not significantly different (p > 0.05). Overall, there were no significant differences in the parameters assessed between the groups treated with CRO alone or CRO + DOX. Conclusions: Treatment with CRO + DOX showed similar bactericidal activity to CRO in vivo, suggesting no antagonist effect of DOX on CRO. Combined therapy significantly improved mouse survival and disease severity compared to untreated animals, but addition of DOX to CRO did not offer significant benefits over CRO monotherapy. In contrast to experimental PM, DOX has no adjunctive activity in experimental MM

Bacterial counts in blood and spleen of BALB/c mice depleted of neutrophils or macrophages.

<p>Twelve groups of BALB/c mice (n = 3–9) were infected by the i.v. route with four different strains of <i>S. pneumoniae</i> (TIGR4, D39, DP1004 and G54) at the challenge dose of 2.5×10⁵ CFU/each strain. Bacterial counts over time of each pneumococcal strain in the blood (black lines) and in the spleen (grey lines) were reported for untreated mice (A1, A2, A3 and A4), clodronate liposomes treated mice (B1, B2, B3 and B4) and anti-GR1 mAb treated mice (C1, C2, C3 and C4). Samples were collected over 13 h, with the exception of mice treated with clodronate (8 h). The cut off is 20 CFU/ml. Data are reported as the mean ± SD of bacterial counts.</p

Co-infection of CD1 mice with three isogenic variants of <i>S. pneumoniae</i> TIGR4.

<p>A mixture of three isogenic <i>S. pneumoniae</i> TIGR4 variants (3×10⁵ CFU/each strain) was given to CD1 mice (n = 68) by the i.v. route. Bacterial counts were performed collecting blood at various time points. (A) Blood counts in the first 10 h after challenge. (B) Blood counts up to 72 h post-challenge, (including data reported in A). Each symbol indicates a single mouse. Blood cultures yielding all three variants are shown in downward white triangles, those yielding two variants as upward grey triangles, and samples yielding a monoclonal blood culture are shown as black squares. Samples from mice with negative blood cultures are shown as open circles. The ratio of infected over un-infected mice was 0.31 at 24 h, 0.37 at 48 h and 0.67 at 72 h. The cut off for detection is 100 CFU/ml. Data of two independent experiments are reported.</p

Pneumococcal strains.

<p>*see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004026#ppat-1004026-t003" target="_blank">Table 3</a> for detailed description.</p

Statistical analysis: number of blood cultures with one, two or three pneumococcal variants observed at 24 h in the experimental infection compared to those predicted from the statistical analysis.

<p>Statistical analysis: number of blood cultures with one, two or three pneumococcal variants observed at 24 h in the experimental infection compared to those predicted from the statistical analysis.</p

Pneumococcal sepsis and phagocytosis by splenic macrophages.

<p>Intravenous challenge of four outbred CD1 mice (A),four inbred BALB/c mice (B) and four inbred CBA/Ca mice (C) was performed by co-infection with D39 (red), G54 (blue) and TIGR4 (green). Bacterial counts in blood collected at different time points from each single mouse are reported (A–C). Cut off is 100 CFU/ml). Blood counts at 5 min (D) and 30 min (E) after i.v. infection of BALB/c mice (n = 6–12) with four pneumococcal strains: D39 (red bar), G54 (blue bar), TIGR4 (green bar) and a rough D39 derivative DP1004 (red open bar). Adhesion (F) and invasion (phagocytosis) (G) of pneumococcal strains in primary spleen macrophages from BALB/c mice. Independent experiments were run in triplicate and reported as mean ± SEM. Statistical analysis were performed with Kruskal-Wallis and Dunn's test. (H) Cytoflurimetric characterisation of surface markers of the primary spleen macrophages. A representative experiment is reported. Comparable data on splenic macrophages isolates from C57BL/6 mice are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004026#ppat.1004026.s004" target="_blank">Figure S4</a>.</p

Phenotypes of single-colony blood culture isolates.

<p>Growth profiles of wild type strains (coloured lines) and ATPase mutants (black lines) in standard laboratory media THY (A) and TSB (B). D39 is indicated in red while TIGR4, FP318 and FP321 in different shade of green. (C, D) Maximum OD₅₉₀ reached by wild type and ATPase mutants during growth with 80 mM K₂HPO₄ and pH 8.0 (D) and growth at pH 6.6 (E). Kinetics of opsono-phagocytosis assays in rotating blood with anti-type 4 serum (open symbols) and without antibodies (filled symbols) of parental strains (green) compared to a SP1507 <i>atpC</i> mutant (black). (F) Phagocytosis with primary cultures of spleen macrophages of TIGR4 and FP487 <i>atpC</i> mutant. (G) Bacterial counts in blood (squares) and spleen (triangles) at 72 h after i.v. infection of BALB/c mice (n = 6) with TIGR4 (green) and FP487 carrying a frame shifted SP1507 <i>atpC</i> gene (black). Data points below the cut off are negative. All data were analyzed by Student's <i>t</i>-test (<i>P</i><0.05).</p