Characterization of PAK-P3 and P3-CHA bacteriophages.

<p>(<b>A</b>, <b>C</b>) Electron micrographs of PAK-P3 (<b>A</b>) and P3-CHA (<b>C</b>). Scale bar: 100 nm. (<b>B</b>) SDS-PAGE of PAK-P3 and P3-CHA proteins; only the most abundant proteins give visible signals (MW: molecular weight markers, the arrow points to the major capsid proteins). (<b>D</b>) Clustal alignment of the three major capsid proteins of PAK-P1, PAK-P3 and P3-CHA bacteriophages with their closest homologs in the database with known function (the major capsid protein of Felix 01 bacteriophage; NP_944891). Major capsid proteins of PAK-P3 and P3-CHA are 100% identical.</p

Survival curve of infected mice treated with bacteriophage PAK-P3 compared to P3-CHA.

<p>Mice were infected intranasally with 3×10⁶ cfu (CHA strain) and 2 h later were treated with either PBS (♦) or 3×10⁷ pfu of bacteriophage PAK-P3 (•), or 3×10⁷ pfu of bacteriophage P3-CHA (□) also administered intranasally (P<0.005 for both P3-CHA and PAK-P3 bacteriophage doses compared to PBS and P<0.05 between P3-CHA and PAK-P3 bacteriophage doses).</p

Histological and immunohistochemical analyses of lung sections from mice treated or pre-treated with bacteriophage P3-CHA.

<p>(<b>A–D</b>; scale bar 200 µm) Thin sections of lungs obtained after 20 h from, (<b>A</b>) mice infected with the CHA strain, (<b>B</b>) infected mice treated with bacteriophage P3-CHA, (<b>C</b>) mice pre-treated with bacteriophage P3-CHA four days before infection, and (<b>D</b>) uninfected mice were stained with hematoxylin-eosin and Gram. Histological analyses included the determination of a lesion severity score (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016963#s4" target="_blank">materials and methods</a>) to allow comparison (<b>M</b>). This score was significantly lower for mice given curative or preventive bacteriophage treatments than for untreated mice (* P<0.05). (<b>E–L</b>) Immunohistochemistry was performed with anti-<i>Pseudomonas</i> antibodies on sections obtained from the same samples as above (<b>E–H</b>; scale bar 100 µm and <b>I–L</b>; scale bar 50 µm). In lung sections from infected mice, bacteria are both intracellular and extracellular (<b>E</b> and <b>I</b>) whereas no signal can be seen in lung sections from uninfected mice (<b>H</b> and <b>L</b>). The signal was lower following either curative (<b>F</b> and <b>J</b>) or preventive (<b>G</b> and <b>K</b>) bacteriophage treatments and bacteria were only observed in macrophage cells.</p

Efficacy of plating of PAK-P3 and P3-CHA bacteriophages on CF <i>P. aeruginosa</i> strains.

<p>Efficacies of PAK-P3 and P3-CHA bacteriophages on their reference strains, respectively. PAK and CHA strains were fixed at 100%. Results are expressed as relative percentage to these references.</p

Bacteriophage P3-CHA cure and prevent lung infections caused by a clinical <i>P. aeruginosa</i> strain.

<p>(<b>A–B</b>) Survival curves of mice infected with the CHA strain and treated or pre-treated with P3-CHA bacteriophage. (<b>A</b>) PBS (♦), 3×10⁶ (•) and 3×10⁷ (□) pfu of bacteriophage were given intranasally 2 h after bacteria (3×10⁶ cfu) were administered. This curative treatment appears to be dose dependent (P<0.0001 for both bacteriophage doses compared to PBS and P<0.01 between 3×10⁶ and 3×10⁷ bacteriophage doses). (<b>B</b>) Four days before infection with 3×10⁶ bacteria, mice were given either 3×10⁷ (•), or 3×10⁸ (□) pfu of P3-CHA or 3×10⁸ pfu of heat-inactivated P3-CHA solution (♦). These survival curves indicate that the preventative treatment is dose dependent (P<0.0005 and P<0.0001 for 3×10⁷ and 3×10⁸ bacteriophage doses respectively compared to heat-inactivated bacteriophage solution and P<0.0005 between 3×10⁷ and 3×10⁸ bacteriophage doses). (<b>C–D</b>) 20 h after infection with strain CHA, mice were euthanized and BAL fluids were assayed for bacteria (<b>C</b>) and bacteriophages (<b>D</b>). In the curative treatment protocols, mice were treated with PBS or bacteriophage 2 h post infection (phage 2 h pi). In the preventative treatment protocols, mice were intranasally administered bacteriophage solution (4d phage) or heat-inactivated bacteriophage solution (4d phage 80°C) four days before infection. (<b>C</b>) Bacterial counts were significantly lower in the BAL fluids from mice that had received either curative or preventative bacteriophage treatment than the respective control treatment (* P<0.05, and ** P<0.01). (<b>D</b>) Bacteriophage counts were significantly lower in the BAL fluids from mice that had received bacteriophage treatment than the non-infected animals (** P<0.01) or the non-infected animals pre-treated four days earlier with the bacteriophage P3-CHA (* P<0.05).</p

Persistence of P3-CHA bacteriophage in lungs of uninfected mice.

<p>Three groups of five mice were given 3×10⁸ pfu of bacteriophage P3-CHA, administered intranasally. The number of bacteriophages in BAL fluids was determined 1, 4 and 5 days post-administration (n = 5).</p

2009) “Information Technology in Supply Chains: The Value of ITenabled Resources under Competition

informs doi 10.1287/isre.1080.019

MvfR-regulon inhibitors rescue PA14-macrophage cytotoxicity.

<p>PA14-induced killing of Raw264.7 macrophage cells was determined minus and plus 100 µM inhibitor. Error bars represent mean +/− SEM of at least 3 replicates. Differences between PA14 + vehicle and the samples PA14 + M64, PA14 + M62, PA14 + M59, PA14 + M51, PA14 + M50, or PA14 + M27 are statistically significant (<i>p</i><0.01, one way ANOVA, Dunnett's test). Differences between MvfR and MvfR + M64 (<i>p</i>>0.05) or vehicle and M64 (<i>p</i>>0.05) are not statistically significant (unpaired t test). Notably, M64 does not alter cytotoxicity of <i>mvfR</i> cells, and is itself non-cytotoxic.</p

The most potent inhibitors reduce 2-AA production and the formation of antibiotic tolerant persisters.

<p><b>a.</b> 2-AA levels in presence of 10 µM inhibitor. Error bars show mean +/− SD of at least 2 replicates. <b>b.</b> Observed fold change in persister cell concentrations of PA14 cultures with 10 µM inhibitor or with 0.75 mM 2-AA. Untreated PA14 cells and <i>mvfR-</i> cells were the positive and negative controls, respectively. Error bars show mean +/− SEM of at least 3 replicates. Differences between PA14 and the samples M34, M50, M62, M59, M51, M64 or mvfR- (<i>p</i><0.01) as well as between PA14 and the samples PA14 + 2-AA or M29 (<i>p</i><0.01) are statistically significant (one way ANOVA, Dunnett's test). <b>c.</b> Observed fold change in persister cell concentrations of PA14 plus 5 µM M64 in the presence of clinical antibiotics used to treat <i>P. aeruginosa</i> infections: amikacin (blue), levofloxacin (purple), ciprofloxacin (orange) and meropenem (red). All values were normalized to control cultures in 0.01% DMSO. Error bars show mean +/− SEM of at least 3 replicates. Differences between control and the samples amikacin, levofloxacin, ciprofloxacin or meropenem are statistically significant (<i>p</i><0.01, one way ANOVA, Dunnett's test).</p

M64 inhibits <i>P. aeruginosa</i> persistence in the mouse burn and infection models.

<p>PA14 CFU quantification in muscle (<b>a</b>) underlying or (<b>b</b>) adjacent to the abdominal infection site in mice infected with PA14 and treated with ciprofloxacin (10 mg/kg), and minus (green) or plus (red) M64 (4 mg/kg). Ciprofloxacin and M64 were administered by intravenous injection 6 hours post-infection and then twice a day. Ciprofloxacin was administered for 4 days as described until no CFUs were detected in the muscle samples. Ciprofloxacin administration was stopped at day 4 to allow for the potential emergence and detection of antibiotic-tolerant cells. M64 was administered for 6 days, up until antibiotic-tolerant cells were detected in the PA14 + ciprofloxacin only group. Error bars represent mean +/− SEM of at least 3 replicates. d.l., detection limit.</p