Phage infection reinstates antibiotic sensitivity in MDR Pseudomonas aeruginosa: A study on phage and bacterial evolution

The emergence of antibiotic resistance among bacterial pathogens is a significant public health threat affecting humans worldwide. In Europe, Pseudomonas aeruginosa contributes to almost 9% of overall multi-drug-resistant (MDR) infections. Alternative methods for controlling MDR pathogens have been explored for several decades. Bacteriophage therapy is one of the oldest and most efficient alternative solutions. The study described in this thesis began with the isolation and characterization of 25 MDR P. aeruginosa clinical strains and eigth novel lytic phages. The investigation disclosed the infection with two phage isolates, PIAS and PAPSZ1, led to the sequential appearance of phage-resistant colonies with two phenotypes (green and brown). We examined the evolutionary basis for the two types of mutants and uncovered phage mutants capable of infecting green mutants. Simultaneously we also learned that PIAS phage infected the host via the OrpM-MexXY system involved in drug efflux. Thus, the PIAS-resistant mutants decreased the minimum inhibitory concentrations (MIC) for several non-effective antibiotics. After this new insight into the evolutionary arms race between hosts and phages, we decided to use this window to comprehensively eradicate mutants by treating MDR strain with previously resistant antibiotics combined with PIAS phage. The in vitro study with PIAS phage-antibiotic combination completely prevented the formation and growth of mutants. We tested the same strategy in an in vivo rescue experiment in the mouse lung infection model, when combined with PIAS phage and fosfomycin. The combination therapy saved 75% of the animals. Later, we used PAPSZ1 phage to investigate whether phage mutants can suppress bacterial resistance. We isolated multiple PAPSZ1 mutants after a continuous infection cycle, which can block or suppress bacterial resistance and mutant formation and broaden the host specificity of the phages. Phages like PIAS and PAPSZ1 offer a unique window that can exploit to eradicate MDR bacteria. This study highlights the importance of preliminary and detailed examinations of phage-host bacterium interactions preceding the application of a given phage. The experimental data in this thesis shows that studying phage-host bacterium interactions and coevolution will help to utilize phage therapy’s full potential when treating MDR infections

Propagating Relationship of Cerebral Oximetric Volume and the Clinical Outcome of Recombinant Tissue Plasminogen Activator (r-TPA) Therapy on Acute Cerebral Ischemic Stroke Patients

Introduction: Currently, the most available treatment for acute ischemic stroke (AIS) is thrombolytic therapy with recombinant tissue plasminogen activator (r-TPA). A challenge in r-TPA therapy is the prediction of recovery in each case. Objective: The aim was to find a possible relationship between the cerebral oximetry indexes and the clinical outcome of r-TPA therapy to assess the cerebral oximetry as a non-invasive monitoring agent for therapy. Methods: The inclusion criteria were all patients with AIS who received r-TPA. The neurologic status was evaluated based on the national institutes of health stroke scale (NIHSS) score at arrival, and after a period of 24 hours. In addition, the levels of brain oxygenation in both hemispheres were measured before and continuously over the first 24 hours after r-TPA injection, using an oximetric sensor in the frontal lobes. The clinical success was defined as a 4-point improvement from the baseline NIHSS. Results: Total 44 patients with the mean age of 58.2 ± 2.18 years were enrolled, of whom 68.18% were male. Twenty-eight patients remained clinically unimproved and 16 patients were improved. A significant difference was found in the mean surface area under the brain oximetric curve in the 24 hour, in the affected hemisphere in the improved group, compared to the unimproved group (P = 0.007). There was a significant difference between the mean increase in brain oxygenation within 24 hours in the improved and unimproved groups (P = 0.002). Conclusion: The cerebral oximetry could contribute to predict the likelihood of r-TPA prognosis in patients with AIS

Effects of Listerner Knowledge on Stories Told by Aphasic and Non-Brain-Damaged Subjects

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Glycogen Synthase Kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation

Survival Comes at a Cost: A Coevolution of Phage and Its Host Leads to Phage Resistance and Antibiotic Sensitivity of Pseudomonas aeruginosa Multidrug Resistant Strains

The increasing ineffectiveness of traditional antibiotics and the rise of multidrug resistant (MDR) bacteria have necessitated the revival of bacteriophage (phage) therapy. However, bacteria might also evolve resistance against phages. Phages and their bacterial hosts coexist in nature, resulting in a continuous coevolutionary competition for survival. We have isolated several clinical strains of Pseudomonas aeruginosa and phages that infect them. Among these, the PIAS (Phage Induced Antibiotic Sensitivity) phage belonging to the Myoviridae family can induce multistep genomic deletion in drug-resistant clinical strains of P. aeruginosa, producing a compromised drug efflux system in the bacterial host. We identified two types of mutant lines in the process: green mutants with SNPs (single nucleotide polymorphisms) and smaller deletions and brown mutants with large (∼250 kbp) genomic deletion. We demonstrated that PIAS used the MexXY-OprM system to initiate the infection. P. aeruginosa clogged PIAS phage infection by either modifying or deleting these receptors. The green mutant gaining phage resistance by SNPs could be overcome by evolved PIASs (E-PIASs) with a mutation in its tail-fiber protein. Characterization of the mutant phages will provide a deeper understanding of phage-host interaction. The coevolutionary process continued with large deletions in the same regions of the bacterial genomes to block the (E-)PIAS infection. These mutants gained phage resistance via either complete loss or substantial modifications of the phage receptor, MexXY-OprM, negating its essential role in antibiotic resistance. In vitro and in vivo studies indicated that combined use of PIAS and antibiotics could effectively inhibit P. aeruginosa growth. The phage can either eradicate bacteria or induce antibiotic sensitivity in MDR-resistant clinical strains. We have explored the potential use of combination therapy as an alternative approach against MDR P. aeruginosa infection

Complete Genome Sequences of 10 Xanthomonas oryzae pv. oryzae Bacteriophages

Xanthomonas oryzae pv. oryzae is the causative agent of bacterial leaf blight of rice. The application of bacteriophages may provide an effective tool against this bacterium. Here, we report the complete genome sequences of 10 newly isolated OP2-like X. oryzae pv. oryzae bacteriophages

Identification of a novel archaea virus, detected in hydrocarbon polluted Hungarian and Canadian samples

Metagenomics is a helpful tool for the analysis of unculturable organisms and viruses. Viruses that target bacteria and archaea play important roles in the microbial diversity of various ecosystems. Here we show that Methanosarcina virus MV (MetMV), the second Methanosarcina sp. virus with a completely determined genome, is characteristic of hydrocarbon pollution in environmental (soil and water) samples. It was highly abundant in Hungarian hydrocarbon polluted samples and its genome was also present in the NCBI SRA database containing reads from hydrocarbon polluted samples collected in Canada, indicating the stability of its niche and the marker feature of this virus. MetMV, as the only currently identified marker virus for pollution in environmental samples, could contribute to the understanding of the complicated network of prokaryotes and their viruses driving the decomposition of environmental pollutants

Survival Comes at a Cost : A Coevolution of Phage and Its Host Leads to Phage Resistance and Antibiotic Sensitivity of Pseudomonas aeruginosa Multidrug Resistant Strains

The increasing ineffectiveness of traditional antibiotics and the rise of multidrug resistant (MDR) bacteria have necessitated the revival of bacteriophage (phage) therapy. However, bacteria might also evolve resistance against phages. Phages and their bacterial hosts coexist in nature, resulting in a continuous coevolutionary competition for survival. We have isolated several clinical strains of Pseudomonas aeruginosa and phages that infect them. Among these, the PIAS (Phage Induced Antibiotic Sensitivity) phage belonging to the Myoviridae family can induce multistep genomic deletion in drug-resistant clinical strains of P. aeruginosa, producing a compromised drug efflux system in the bacterial host. We identified two types of mutant lines in the process: green mutants with SNPs (single nucleotide polymorphisms) and smaller deletions and brown mutants with large (∼250 kbp) genomic deletion. We demonstrated that PIAS used the MexXY-OprM system to initiate the infection. P. aeruginosa clogged PIAS phage infection by either modifying or deleting these receptors. The green mutant gaining phage resistance by SNPs could be overcome by evolved PIASs (E-PIASs) with a mutation in its tail-fiber protein. Characterization of the mutant phages will provide a deeper understanding of phage-host interaction. The coevolutionary process continued with large deletions in the same regions of the bacterial genomes to block the (E-)PIAS infection. These mutants gained phage resistance via either complete loss or substantial modifications of the phage receptor, MexXY-OprM, negating its essential role in antibiotic resistance. In vitro and in vivo studies indicated that combined use of PIAS and antibiotics could effectively inhibit P. aeruginosa growth. The phage can either eradicate bacteria or induce antibiotic sensitivity in MDR-resistant clinical strains. We have explored the potential use of combination therapy as an alternative approach against MDR P. aeruginosa infection

The role of myosin 1c and myosin 1b in surfactant exocytosis

Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression