Erwinia amylovora bacteriophage resistance

It has been proposed that phages can be used commercially as a biopesticide for the control of fire blight caused by the phytopathogen Erwinia amylovora. The aim of these studies was to investigate two common bacterial resistance mechanisms, lysogeny and exopolysaccharide production and their influence on phage pathogenesis. A multiplex real-time PCR protocol was designed to monitor and quantify Podoviridae and Myoviridae phages. This protocol is compatible with known E. amylovora and Pantoea agglomerans rtPCR primers/probes which allowed simultaneous study of both phage and bacterial targets. Using in vitro positive phage selection, bacteriophage insensitive derivatives were isolated within sensitive populations of E. amylovora. Prophage screening with real-time PCR and mitomycin C induction determined that the insensitive derivatives harboured the temperate Podoviridae phage ΦEaTlOO. Lysogenic conversion resulted in resistance to secondary homologous phage infections. Prophage screening of environmental samples of E. amylovora and P. agglomerans collected from various locations in Canada, United States and Europe did not demonstrate lysogeny. Therefore, lysogeny is rare or absent while these bacterial species reside on the plant. Recombineering was used to construct exopolysaccharide deficient E. amylovora mutants. The EPS amylovoran mutants became resistant to Podoviridae and certain Siphoviridae phages. Increasing amylovoran production increased phage population growth, presumably by increasing the total number of bacterial cell surface receptors which promoted increased phage infections. In contrast, amylovoran did not playa role in Myoviridae infections, nor did production of the EPS levan for any phage pathogenesis

Stronger together? Perspectives on phage-antibiotic synergy in clinical applications of phage therapy

Increasingly, clinical infections are becoming recalcitrant or completely resistant to antibiotics treatment and multidrug resistance is rising alarmingly. Patients suffering from infections that used to be treated successfully by antibiotic regimens are running out of the treatment options. Bacteriophage (phage) therapy, long practiced in parts of Eastern Europe and the states of the former Soviet Union, is now being reevaluated as a treatment option complementary to and synergistic with antibiotic treatments. We discuss some current studies that have addressed synergistic killing activity between phages and antibiotics, the issues of treatment order and antibiotic class, and point to considerations that will have to be addressed by future studies. Overall, co-treatments with phages and antibiotics promise to extend the utility of antibiotics in current use. Nevertheless, a lot of work, both basic and clinical, remains to be done before such co-treatments become routine options in the hospital setting

Human Neutrophil Response to <i>Pseudomonas</i> Bacteriophage PAK_P1, a Therapeutic Candidate

The immune system offers several mechanisms of response to harmful microbes that invade the human body. As a first line of defense, neutrophils can remove pathogens by phagocytosis, inactivate them by the release of reactive oxygen species (ROS) or immobilize them by neutrophil extracellular traps (NETs). Although recent studies have shown that bacteriophages (phages) make up a large portion of human microbiomes and are currently being explored as antibacterial therapeutics, neutrophilic responses to phages are still elusive. Here, we show that exposure of isolated human resting neutrophils to a high concentration of the Pseudomonas phage PAK_P1 led to a 2-fold increase in interleukin-8 (IL-8) secretion. Importantly, phage exposure did not induce neutrophil apoptosis or necrosis and did not further affect activation marker expression, oxidative burst, and NETs formation. Similarly, inflammatory stimuli-activated neutrophil effector responses were unaffected by phage exposure. Our work suggests that phages are unlikely to inadvertently cause excessive neutrophil responses that could damage tissues and worsen disease. Because IL-8 functions as a chemoattractant, directing immune cells to sites of infection and inflammation, phage-stimulated IL-8 production may modulate some host immune responses

Correction: Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation.

[This corrects the article DOI: 10.1371/journal.pone.0123282.]

Schematic of the isolation of the hepatocyte-like cell line, PICM-19FF, from pig embryo epiblast cells.

<p>Schematic of the isolation of the hepatocyte-like cell line, PICM-19FF, from pig embryo epiblast cells.</p

MPIO-loaded PICM-19FF cells stained for GGT activity 20 min after the addition of glucagon (100 ng/mL final) to the culture medium.

<p>Panels A and B are phase-contrast images of the same area before and after, respectively, the addition of glucagon (200x). C) The same area photographed with Hoffman modulation after histochemical staining for GGT activity (200x). Note the intense GGT histochemical staining at the apical cell membranes comprising the biliary canaliculi (arrows). Arrowheads indicate MPIO iron particles. Scale bar = 16 μm.</p

Microscopy of ppHEP and PICM-19FF cells labeled with 0.86 μm MPIO at 100 MPIO/cell after 24 h.

<p>Panels A and C are phase-contrast images of labeled ppHEP and PICM-19FF cells, respectively (200x). Panels B and D are corresponding bright-field images showing peri-nuclear MPIO particles sequestered within the cells (arrowheads). Transmission electron micrographs of MPIO-loaded PICM-19FF (Panels E and G) and ppHEP cells (Panels F and H) with arrowheads denoting the intracellular MPIO. Note that the morphology of the cells, their cellular junctions, their intimate cell-to-cell interdigitions (*), and their internal organelles appear normal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123282#pone.0123282.ref016" target="_blank">16</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123282#pone.0123282.ref019" target="_blank">19</a>]. bc = biliary canaliculus, g = Golgi apparatus, n = nucleus, m = mitochondrion, mv = microvillus, rer = rough endoplasmic reticulum, tj = tight junction-like structure.</p

Two-dimensional electrophoretic polyacrylamide gels of conditioned serum-free medium from MPIO-labeled and unlabeled PICM-19FF.

<p>Proteins are indicated by spot number and name as identified by MALDI-TOF-TOF mass spectroscopy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123282#pone.0123282.t001" target="_blank">Table 1</a>). *Previously identified; see Talbot et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123282#pone.0123282.ref043" target="_blank">43</a>].</p

Maldi-TOF/TOF mass spectrometry analysis.

<p>Maldi-TOF/TOF mass spectrometry analysis.</p

Urea production and cytochrome P450 activity of MPIO-labeled and unlabeled cells.

<p>A) Urea production with and without the addition of 5 μmol NH₄Cl to the culture medium. B) P450 EROD activity after a 48-h 3-methylcholanthrine (5 μM) induction. Total fluorescent activity is with the addition of β-glucuronidase/arylsulfatase so as to detect total (left) and conjugated (right) EROD activity products. (n = 3, bars represent SEM) **P<0.01, ***P<0.001</p