Combating Multidrug-Resistant Pathogens with Host-Directed Nonantibiotic Therapeutics

Abstract

Earlier, we reported that three Food and Drug Administration-approved drugs, trifluoperazine (TFP; an antipsychotic), amoxapine (AXPN; an antidepressant), and doxapram (DXP; a breathing stimulant), identified from an in vitro murine macrophage cytotoxicity screen, provided mice with 40 to 60% protection against pneumonic plague when administered at the time of infection for 1 to 3 days. In the present study, the therapeutic potential of these drugs against pneumonic plague in mice was further evaluated when they were administered at up to 48 h postinfection. While the efficacy of TFP was somewhat diminished as treatment was delayed to 24 h, the protection of mice with AXPN and DXP increased as treatment was progressively delayed to 24 h. At 48 h postinfection, these drugs provided the animals with significant protection (up to 100%) against challenge with the agent of pneumonic or bubonic plague when they were administered in combination with levofloxacin. Likewise, when they were used in combination with vancomycin, all three drugs provided mice with 80 to 100% protection from fatal oral Clostridium difficile infection when they were administered at 24 h postinfection. Furthermore, AXPN provided 40 to 60% protection against respiratory infection with Klebsiella pneumoniae when it was administered at the time of infection or at 24 h postinfection. Using the same in vitro cytotoxicity assay, we identified an additional 76/780 nonantibiotic drugs effective against K. pneumoniae For Acinetobacter baumannii, 121 nonantibiotic drugs were identified to inhibit bacterium-induced cytotoxicity in murine macrophages. Of these 121 drugs, 13 inhibited the macrophage cytotoxicity induced by two additional multiple-antibiotic-resistant strains. Six of these drugs decreased the intracellular survival of all three A. baumannii strains in macrophages. These results provided further evidence of the broad applicability and utilization of drug repurposing screening to identify new therapeutics to combat multidrug-resistant pathogens of public health concern.

Keywords: Acinetobacter baumannii; Clostridium difficile; Klebsiella pneumoniae; Yersinia pestis; bubonic plague; in vitro assays; mouse models; new therapeutics; pneumonic plague.

FIG 1

Survival analysis of Y. pestis CO92-infected mice treated with TFP, AXPN, or DXP at delayed time points after infection in a model of pneumonic plague. Mice were challenged by the i.n. route with 8 LD₅₀ (1 LD₅₀ = 500 CFU) of WT strain CO92 and administered TFP (1.5 mg/kg; n = 7 mice) (A), AXPN (3 mg/kg; n = 7 mice) (B), or DXP (20 mg/kg; n = 7 mice) (C) by the i.p. route at the indicated time points p.i. A group of untreated, infected animals (which were treated with PBS; n = 5 mice) served as a control. The animals were dosed with DXP and AXPN for 3 days (at 24-h intervals) or received only 1 dose of TFP at the indicated times. Mice were monitored for signs of morbidity and mortality for 21 days. The data were analyzed for significance by the use of Kaplan-Meier survival estimates. The P values were determined on the basis of a comparison of the results for each of the drug treatment groups to the results for the untreated and infected control group of mice (which were treated with PBS).

FIG 2

Survival analysis of Y. pestis CO92-infected mice treated with TFP, AXPN, or DXP alone or in combination with levofloxacin (Levo) at 48 h p.i. in a model of pneumonic plague. Mice were challenged by the i.n. route with 9 LD₅₀ of WT strain CO92 (n = 5 to 7 mice per group) and administered TFP (A), AXPN (B), or DXP (C) alone or in combination with levofloxacin (5 mg/kg or 0.25 mg/kg) by the i.p. route at the indicated time points p.i. The animals were dosed for up to 3 days (at 24-h intervals) beginning at 48 h p.i. (as described in the Fig. 1 legend). Untreated and infected mice (which were treated with PBS) served as the control group. Mice were monitored for signs of morbidity and mortality for 14 days. The data were analyzed for significance by the use of Kaplan-Meier survival estimates. The P values were determined on the basis of a comparison of the results for each of the drug treatment groups to the results for the untreated and infected control group (which was treated with PBS) or the indicated group.

FIG 3

Survival analysis of Y. pestis CO92-infected mice treated with TFP, AXPN, or DXP alone or in combination with levofloxacin (Levo) at 48 h p.i. in a model of bubonic plague. Mice were challenged by the s.c. route with 12 LD₅₀ (1 LD₅₀ = 50 CFU) of WT strain CO92 (n = 5 to 7 per group) and administered TFP, AXPN, or DXP alone or in combination with levofloxacin (5 mg/kg) by the i.p. route at the indicated staggered time points p.i. (levofloxacin was administered 3 h after treatment with the other drugs). The animals were dosed for up to 3 days (at 24-h intervals) beginning at 48 h p.i. (as described in the Fig. 1 legend). Untreated and infected mice (which were treated with PBS) served as the control group. Mice were monitored for signs of morbidity and mortality for 24 days. The data were analyzed for significance by the use of Kaplan-Meier survival estimates. The P values were determined on the basis of a comparison of the results for each of the drug treatment groups to the results for the untreated and infected control group (which was treated with PBS) or the indicated group.

FIG 4

Y. pestis CO92 proliferation in lungs following DXP treatment in a model of pneumonic plague. At each indicated time point, three animals per group that had been infected with 8 LD₅₀ of WT strain CO92 by the i.n. route and treated with DXP at 24 h p.i. (DXP-24) or left untreated (but treated with PBS) were euthanized, and the lungs were harvested 6 h after the administration of each dose. The numbers of CFU were determined by homogenization of the lungs, followed by serial dilution and plating on SBA plates. Statistical analysis was performed by Student's t test, with a P value of <0.05 being considered statistically significant. d, day.

FIG 5

Survival analysis of C. difficile- or K. pneumoniae-infected and treated mice. (A) For the model of C. difficile infection, C57BL/6 mice were infected by oral gavage with 10⁵ C. difficile (VPI 10463) spores (n = 5 to 7 per group) and administered TFP, AXPN, or DXP alone or in combination with vancomycin (Vanco; 20 mg/kg) by the i.p. route at 24 h p.i. (vancomycin was administered 3 h after treatment with the other drugs). The drugs were administered at 24-h intervals, with TFP being administered once, AXPN and DXP being administered 3 times, and vancomycin being administered 5 times. The mice were monitored for signs of morbidity and mortality for 8 days. The data were analyzed for significance by the use of Kaplan-Meier survival estimates. P values were determined on the basis of a comparison of the results for each of the drug treatment groups to the results for the group treated with vancomycin alone. P was 0.009 for all groups treated with drug combinations in comparison to the untreated and infected control group (which was treated with PBS). (B) For K. pneumoniae infection, C57BL/6 mice were infected with 5 × 10⁵ CFU by the i.n. route and administered AXPN (3 mg/kg) up to 3 times by the i.p. route at the indicated time points at 24-h intervals. Untreated and infected animals (which were treated with PBS) served as a control. The data were analyzed by the chi-square test, with P values being based on a comparison of the results for each drug treatment group to the results for the untreated and infected group (which was treated with PBS).

FIG 6

Drugs identified posttreatment to be tier 1 or tier 2 drugs using a host cell-based screen to evaluate RAW 264.7 murine macrophage viability by the MTT assay following infection with A. baumannii strain BAA-1797 for 18 h. For tier 1 drugs, the drug-treated (33 μM) and infected macrophages exhibited viability equivalent to that of uninfected macrophages (no bacteria, dashed line) and viability significantly greater than that of drug-untreated and infected macrophages. *, a tier 1 drug. For tier 2 drugs, the drug-treated and infected macrophages exhibited viability values that were significantly greater than those for A. baumannii-infected and drug-untreated macrophages but that were not equivalent to those for uninfected macrophages (dashed line). Two independent experiments in which each drug was tested in duplicate were performed. The data were plotted as percentages of the values for uninfected macrophages and were analyzed by 2-way ANOVA with Tukey's post hoc test. All data presented were significant.

FIG 7

Drugs identified posttreatment to be tier 1 or tier 2 drugs using a host cell-based screen to evaluate RAW 264.7 murine macrophage viability by the MTT assay following infection with A. baumannii strain BAA-1799 for 18 h. (A) For tier 1 drugs, the drug-treated (33 μM) and infected macrophages exhibited viability equivalent to that of uninfected macrophages (no bacteria, dashed line) and viability significantly greater than that of untreated and infected macrophages. (B) For tier 2 drugs, the drug-treated and infected macrophages exhibited viability values that were significantly greater than those for A. baumannii-infected and untreated macrophages but that were not equivalent to those for uninfected macrophages (dashed line). Two independent experiments in which each drug was tested in duplicate were performed. The data were plotted as percentages of the values for uninfected macrophages and were analyzed by 2-way ANOVA with Tukey's post hoc test. All data presented were significant.

FIG 8

Quantification of intracellular survival of A. baumannii strain 8, BAA-1797 (97), or BAA-1799 (99) in RAW 264.7 murine macrophages. RAW 264.7 macrophages were infected with one of the A. baumannii strains for 2 h at an MOI of 100. The cell monolayers were then treated with gentamicin at a concentration of 200 to 500 μg/ml for 2 h, washed twice with PBS, and incubated with the same concentration of gentamicin. At this time, each drug at a concentration of 33 μM or PBS as a control was added to the cell monolayers and the cells were incubated for 4 h. At this point, the macrophages were lysed and the number of CFU was determined by serial dilution and plating on SBA plates. Two independent experiments in which each drug was tested in duplicate were performed. The data were analyzed by comparison of the results for untreated and infected controls (which were treated with PBS) to those for the treatment groups by using a one-way ANOVA with Tukey's post hoc test. *, P < 0.01.