Immunotherapy markedly increases the effectiveness of antimicrobial therapy for treatment of Burkholderia pseudomallei infection

Abstract

Burkholderia pseudomallei is a soil bacterium that is endemic in southeast Asia and northern Australia and that can cause both acutely lethal pneumonia and chronic systemic infections in humans. The effective treatment of infection with B. pseudomallei requires rapid diagnosis and prolonged treatment with high doses of antimicrobials, and even with appropriate antibiotic therapy, patient relapses are common. Thus, new approaches to the treatment of B. pseudomallei infections are needed. In the present study, we asked whether active immunotherapy with gamma interferon (IFN-gamma), a key cytokine regulating the intracellular replication of B. pseudomallei, could increase the effectiveness of conventional antimicrobial therapy for B. pseudomallei infection. Macrophage infection assays and in vivo pulmonary challenge models were used to assess the inhibitory effects of combined treatment with IFN-gamma and ceftazidime on B. pseudomallei infection. We found that treatment with even very low doses of IFN-gamma and ceftazidime elicited strong synergistic inhibition of B. pseudomallei growth within infected macrophages. In vivo, active immunotherapy markedly potentiated the effectiveness of low-dose ceftazidime therapy for the treatment of infected mice in a pulmonary challenge model of B. pseudomallei. Combined treatment was associated with a significant reduction in the bacterial burden and a significant lessening of bacterial dissemination. We concluded, therefore, that immunotherapy with either endogenous or exogenous IFN-gamma could significantly increase the effectiveness of conventional antimicrobial therapy for the treatment of acute B. pseudomallei infection.

FIG. 1.

CLDC supernatants synergize with ceftazidime to inhibit the intracellular replication of B. pseudomallei. AMJ.2 macrophages were infected in triplicate wells with B. pseudomallei strain 1026b for 1 h and were then treated with ceftazidime and/or CLDC spleen supernatants, alone or in combination, for 24 h, as described in Materials and Methods. The concentrations of intracellular bacteria were quantitated by serial dilution plating of macrophage lysates. The effects of the CLDC supernatants at the dilutions indicated, alone or in combination with 10 μg/ml of ceftazidime, on the intracellular replication of B. pseudomallei were assessed. The mean bacterial concentrations in each treatment group were plotted, and bars represent means ± standard deviations. Synergistic interactions were assessed statistically by two-way ANOVA (***, P < 0.001). The data presented here are representative of those from two independent experiments.

FIG. 2.

Synergistic inhibitory activity of CLDC-stimulated supernatants is mediated by IFN-γ. Macrophages were infected in triplicate wells in vitro with B. pseudomallei for 1 h and were then treated for 24 h with CLDC-stimulated spleen supernatants (1:5 dilution) alone or in combination with ceftazidime at 10 μg/ml. The CLDC supernatants were untreated or were treated with an IFN-γ-neutralizing antibody (Ab) or isotype control antibody for 30 min before they were added to the infected macrophages. The intracellular bacterial numbers were determined 24 h after infection, and the mean bacterial numbers (± standard deviations) were plotted. Assessments for synergistic interactions between ceftazidime and IFN-γ were performed statistically by two-way ANOVA (***, P < 0.001), as described previously (27). The data presented here are representative of those from two independent experiments.

FIG. 3.

rIFN-γ and ceftazidime reciprocally increase inhibition of intracellular replication of B. pseudomallei in vitro. Macrophages were infected in triplicate wells in vitro with B. pseudomallei for 1 h and were then treated with rIFN-γ and ceftazidime for 24 h, and the intracellular bacterial numbers were determined. (A) Decreasing concentrations of rIFN-γ were added to infected macrophages treated with 10 μg/ml of ceftazidime, and the effects on intracellular B. pseudomallei replication were assessed. For each treatment, the mean bacterial numbers were plotted, and the bars represent group means ± standard deviations. (B) Decreasing concentrations of ceftazidime (Ceft) were added to macrophages treated with a fixed concentration of rIFN-γ (100 U/ml), and the effects on intracellular B. pseudomallei replication were assessed. Synergistic interactions between ceftazidime and IFN-γ were assessed statistically by two-way ANOVA (***, P < 0.001), as described previously (27). The data presented here are representative of those from two independent experiments.

FIG. 4.

Time course of intracellular B. pseudomallei replication and killing following treatment with IFN-γ and ceftazidime. Macrophages were infected in triplicate wells in vitro with B. pseudomallei for 1 h and were then treated with rIFN-γ (100 U/ml) or ceftazidime (10 μg/ml), or with both agents in combination, for the indicated times, and the intracellular bacterial numbers were determined. The mean bacterial concentrations (± standard deviations) in the individual treatment groups were compared over time by repeated-measures ANOVA with the Bonferroni posttests (***, P < 0.001 for the group treated with ceftazidime plus IFN-γ combined versus all other groups). Similar results were obtained in one additional experiment.

FIG. 5.

Low-dose ceftazidime (Ceftaz.) plus CLDC immunotherapy effectively protects mice from acute and chronic infection with B. pseudomallei. (A) BALB/c mice (n = 10 mice per group) were challenged i.n. with 7.5 × 10³ CFU of B. pseudomallei, as described in Materials and Methods. Six hours later the mice were treated i.p. with 25 mg/kg ceftazidime or 20 μl CLDC, or with both agents in combination. The ceftazidime treatments were continued every 12 h for a total of six treatments over 3 days, while CLDCs were administered only once. Short-term (20-day) survival times were assessed. (B) Mice (n = 10 per group) that initially survived the 20-day short-term period were followed for an additional 40 days to assess the effects of treatment on chronic infection. At the end of the 60-day observation period, any surviving animals were euthanized and their organs (lung, spleen, liver) were cultured for B. pseudomallei, as described in Materials and Methods. Survival times were assessed as described in Materials and Methods. Statistical differences in survival times were determined by the use of Kaplan-Meier curves, followed by the log-rank test. The Bonferroni corrected threshold was applied for comparison of multiple survival curves, such that a P value of <0.02 was considered significant for these analyses (***, P < 0.001 for combination therapy versus CLDC treatment and for combination therapy versus ceftazidime treatment). The survival curves represent pooled data from two independent experiments.

FIG. 6.

Combined treatment with CLDCs and ceftazidime (Ceft.) significantly decreases the bacterial burden. BALB/c mice (n = 5 mice per group) were challenged i.n. with 8 × 10³ CFU of B. pseudomallei. Six hours later the mice were treated i.p. with 25 mg/kg ceftazidime or 20 μl CLDC, or with both agents in combination. The ceftazidime treatments were continued every 12 h for a total of four treatments, and the CLDCs were administered once. The mice were euthanized 48 h after the bacterial challenge; and the bacterial burdens in the lungs (a), spleen (b), and liver (c) were quantified. Organ bacterial burdens between combination treatments and single-agent treatments were compared statistically by one-way ANOVA and the Tukey multiple-means-comparison test (***, P < 0.001; **, P < 0.01). Significant reductions (P < 0.01) in bacterial counts in the lungs, livers, and spleens of single-drug-treated animals compared with those in the organs of untreated control animals were also observed but are not noted here.

FIG. 7.

Treatment with low-dose ceftazidime plus rIFN-γ protects mice from acute but not chronic B. pseudomallei infection. (A) BALB/c mice (n = 10 mice per group) were challenged with 7.5 × 10³ CFU of B. pseudomallei administered by the i.n. route. Six hours later the mice were treated i.p. with 25 mg/kg ceftazidime (Ceftaz.) or 3 × 10³ U rIFN-γ, or with both in combination. Ceftazidime treatments were continued every 12 h for a total of six treatments over 3 days. Treatment with rIFN-γ was administered twice, at 6 and 18 h postinfection. (B) Mice (n = 10 per group) that initially survived the 20-day short-term period were followed for an additional 40 days to assess the effects of the treatment on chronic infection. At the end of the 60-day observation period, any surviving animals were euthanized and their organs (lung, spleen, liver) were cultured for B. pseudomallei, as described in Materials and Methods. The survival times of the treated and the control mice were determined, and statistical differences in the survival times were determined by the use of Kaplan-Meier curves followed by the log-rank test. The Bonferroni corrected threshold was applied for comparison of multiple survival curves, such that a P value of <0.02 was considered significant for this analysis (***, P < 0.001 for combination therapy versus rIFN-γ treatment and for combination therapy versus ceftazidime treatment). The survival curves represent pooled data from two independent experiments.