Phage Therapy Potentiates Second-Line Antibiotic Treatment against Pneumonic Plague
<p>Pharmacokinetic analysis of фA1122 in naive mice. A single dose of фA1122 phage suspension (1 × 10<sup>9</sup> PFU) was administered to naive C57BL/6J mice, either by (<b>A</b>) IP injection (0.5 mL) or (<b>B</b>) via the IN route (35 µL). For each administration route, n = 3 for each time point. Phage titration was performed using a spot assay test. Each dot represents the mean value in terms of PFU/organ (lung—blue dots, spleen—green dots, liver—black dots) or PFU/mL blood (red dots). Bars represent the standard deviations (SDs).</p> "> Figure 2
<p>Intranasal administration of a single phage dose delayed disease progression. C57BL/6J mice were intranasally infected with 10 × LD<sub>50</sub> <span class="html-italic">Y. pestis</span> Kim53 strain (<b>A</b>) or Kim53-lux luminescent strain (<b>B</b>–<b>F</b>). For phage treatment, one dose of 1 × 10<sup>9</sup> фA1122 PFU/mouse was IN administered at 5 hpi. Control mice were subjected to intranasal administration of PBS. (A) Survival curves of control mice (black line, n = 6) and phage-treated mice (blue line, n = 12). (<b>B</b>,<b>C</b>) Control (n = 3) and phage-treated mice (n = 4) were anesthetized at the indicated time points post <span class="html-italic">Y. pestis</span> infection. Lungs were harvested, and imaging was performed using IVIS as detailed in the <a href="#sec2-viruses-14-00688" class="html-sec">Section 2</a>. (<b>D</b>,<b>E</b>) The bacterial load in the lungs and blood was quantified by plating serial dilutions of tissue homogenate/blood on BHIA plates supplemented with 200 μg/mL ampicillin and counting the colonies. Black squares represent nontreated control mice and blue squares represent phage-treated mice. (<b>F</b>) Phage titration was performed by the spot assay technique described in the <a href="#sec2-viruses-14-00688" class="html-sec">Section 2</a>. Each point represents the phage load in the lungs (blue squares, total PFU/lung) or blood (black squares, PFU/mL) of an individual mouse. Horizontal bars represent median values. Dotted lines mark the limit of detection. Statistically significant differences between groups are denoted by asterisks (* <span class="html-italic">p</span> < 0.05; *** <span class="html-italic">p</span> < 0.0001; log-rank (Mantel–Cox) test). Bars indicate standard errors of the means.</p> "> Figure 3
<p>Multiple-dose фA1122 phage treatments did not improve treatment efficacy. Schematic representation of mouse treatment regimens (<b>A</b>) and survival curves (<b>B</b>). C57BL/6J mice were IN infected with 10 × LD<sub>50</sub> of <span class="html-italic">Y. pestis</span> Kim53. For IN phage treatment, mice were inoculated with 35 µL of 1 × 10<sup>9</sup> фA1122 PFU; IP injection included the administration of 0.5 mL of 1 × 10<sup>9</sup> фA1122 PFU. The treatment regimens were as follows: one dose of IN фA1122 at 5 hpi (n = 22; light blue line), two doses of IN фA1122 at 5 hpi and 24 hpi (n = 12, olive green line), one dose of IN фA1122 at 5 hpi + IP injections at 24 and 48 hpi (n = 17, green line) and one dose of IN фA1122 at 5 hpi + IP injections for 6 days, every 24 h (n = 10, purple line). Control mice: n = 17, black line. (<b>B</b>): Statistically significant differences between the control group and phage-treated groups are denoted by asterisks (*** <span class="html-italic">p</span> < 0.0001; log-rank (Mantel–Cox) test).</p> "> Figure 4
<p>PST is more potent than фA1122 in the presence of blood. Phage-based lysis assays were performed with the bioluminescent <span class="html-italic">Y. pestis</span> strain EV76-lux (10<sup>7</sup> CFU/mL) suspended in BHI broth (blue and orange lines) or in mouse whole blood (gray and red lines). The <span class="html-italic">Y. pestis</span> strain was infected with фA1122 (<b>A</b>) or PST (<b>B</b>) phages (10<sup>6</sup> PFU/mL; multiplicity of infection (MOI) = 0.01). Bioluminescence (RLU) was measured at 37 °C in 15 min intervals for 24 h using a SPARK 10M plate reader. The experiment was performed in biological duplicates (using blood pooled from three mice for each experiment), and the results are representative of one experiment. Presented values are the average results from three wells in a single triplicate experiment, and the error bars represent the standard deviations (SD). The green line represents the background from BHI broth. G.C. = growth control.</p> "> Figure 5
<p>Pharmacokinetic analysis of PST in naive mice. A single dose of PST phage suspension (1 × 109 PFU) was administered to naive C57BL/6J mice, either by (<b>A</b>) IP injection (0.5 mL) or (<b>B</b>) via the IN route (35 µL). For each administration route, n = 3 for each time point. Phage titration was performed using a spot assay test. Each dot represents the mean value in terms of PFU/organ (lung—blue dots, spleen—green dots, liver—black dots) or PFU/mL blood (red dots). Bars represent the standard deviations (SDs).</p> "> Figure 6
<p>Treatment of pneumonic plague with фA1122 or PST results in similar outcomes. (<b>A</b>) Schematic presentation of the phage treatment regimen. C57BL/6J mice were IN infected with 10 × LD<sub>50</sub> <span class="html-italic">Y. pestis</span> Kim53 (gray symbols), followed by PBS (control group) or phage administration. Each dose of PST (red phage symbol) or фA1122 (blue phage symbol) suspension contained 1 × 10<sup>9</sup> phages. (<b>B</b>) Survival curves. The mouse groups were as follows: no phage (n = 5, black line), IN phage administration at 5 hpi followed by IP injections every 24 h on days 1–7 post bacterial infection (n = 10; green line for фA1122 and red line for PST).</p> "> Figure 7
<p>Effective rescue of infected mice by the phage–antibiotic combination treatment. Schematic presentation of the treatment regimens (<b>A</b>) and survival curves (<b>B</b>) Female C57BL/6J mice were IN infected with 100 × LD<sub>50</sub> <span class="html-italic">Y. pestis</span> Kim53. Mouse groups included control nontreated (n = 4), phage-treated (n = 10), ceftriaxone-treated (n = 10) and phage–ceftriaxone combination-treated mice (n = 9). Phage treatment was performed using a phage cocktail composed of фA1122 and PST (1 × 10<sup>9</sup> each phage/dose; 35 µL for intranasal administration or 0.5 mL for IP injection). Treatment included IN administration at 5 hpi followed by IP injections at days 1–10, with 24 h intervals. Ceftriaxone was subcutaneously injected every 12 h on days 2–6 post bacterial infection. Mice were monitored for 22 days. Statistically significant differences are denoted by asterisks (*** <span class="html-italic">p</span> < 0.001; log rank (Mantel–Cox) test).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Bacteriophages and Growth Media
2.2. Animal Studies
2.3. Phage Treatment
2.4. Antibiotic Treatment
2.5. Statistical Analysis
2.6. Bioluminescence-Based Lysis Assay
2.7. Phage Preparation and Purification
3. Results
3.1. Intranasal Administration of фA1122 Leads to High Phage Titers in the Lungs
3.2. Intranasal Administration of a Single Dose of Phage Suspension Delayed Mortality in a Mouse Model of Pneumonic Plague
3.3. Multiple-Dose Phage Administration Did Not Improve Treatment Efficacy
3.4. PST Phage Shows Improved Persistence and Activity in Mouse Blood Compared with фA1122
3.5. Comparing the Protective Potential of PST and фA1122
3.6. Phage–Ceftriaxone Combination Therapy Is Highly Effective against Pneumonic Plague
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Vagima, Y.; Gur, D.; Aftalion, M.; Moses, S.; Levy, Y.; Makovitzki, A.; Holtzman, T.; Oren, Z.; Segula, Y.; Fatelevich, E.; et al. Phage Therapy Potentiates Second-Line Antibiotic Treatment against Pneumonic Plague. Viruses 2022, 14, 688. https://doi.org/10.3390/v14040688
Vagima Y, Gur D, Aftalion M, Moses S, Levy Y, Makovitzki A, Holtzman T, Oren Z, Segula Y, Fatelevich E, et al. Phage Therapy Potentiates Second-Line Antibiotic Treatment against Pneumonic Plague. Viruses. 2022; 14(4):688. https://doi.org/10.3390/v14040688
Chicago/Turabian StyleVagima, Yaron, David Gur, Moshe Aftalion, Sarit Moses, Yinon Levy, Arik Makovitzki, Tzvi Holtzman, Ziv Oren, Yaniv Segula, Ella Fatelevich, and et al. 2022. "Phage Therapy Potentiates Second-Line Antibiotic Treatment against Pneumonic Plague" Viruses 14, no. 4: 688. https://doi.org/10.3390/v14040688