Extraction of bacteriophages from fecal samples
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR748825.fastq.gz | Fastqsanger.gz | |||
| ERR748826.fastq.gz | Fastqsanger.gz | |||
| ERR748827.fastq.gz | Fastqsanger.gz | |||
| ERR748828.fastq.gz | Fastqsanger.gz | |||
| ERR748829.fastq.gz | Fastqsanger.gz |
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Project description:The DNA content of bacteriophages from Bartonella grahamii was investigated by hybridization against cellular DNA from the same organism. Phage particles were isolated from plate grown bacteria as well as from different growth phases during culture in liquid medium.
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Project description:Virulent bacteriophages (or phages) are viruses that specifically infect and lyse a bacterial host. When multiple phages co-infect a bacterial host, the extent of lysis, dynamics of bacteria-phage and phage-phage interactions are expected to vary. The objective of this study is to identify the factors influencing the interaction of two virulent phages with different Pseudomonas aeruginosa growth states (planktonic, an infected epithelial cell line, and biofilm) by measuring the bacterial time-kill and individual phage replication kinetics. A single administration of phages effectively reduced P. aeruginosa viability in planktonic conditions and infected human lung cell cultures, but phage-resistant variants subsequently emerged. In static biofilms, the phage combination displayed initial inhibition of biofilm dispersal, but sustained control was achieved only by combining phages and meropenem antibiotic. In contrast, adherent biofilms showed tolerance to phage and/or meropenem, suggesting a spatiotemporal variation in the phage-bacterial interaction. The kinetics of adsorption of each phage to P. aeruginosa during single- or co-administration were comparable. However, the phage with the shorter lysis time depleted bacterial resources early and selected a specific nucleotide polymorphism that conferred a competitive disadvantage and cross-resistance to the second phage. The extent and strength of this phage-phage competition and genetic loci conferring phage resistance, are, however, P. aeruginosa genotype dependent. Nevertheless, adding phages sequentially resulted in their unimpeded replication with no significant increase in bacterial host lysis. These results highlight the interrelatedness of phage-phage competition, phage resistance and specific bacterial growth state (planktonic/biofilm) in shaping the interplay among P. aeruginosa and virulent phages.
2024-07-17 | GSE271537 | GEO
Project description:Phage therapy is a therapeutic approach to treat multidrug resistant infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells derived from a person with cystic fibrosis, we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.
2024-02-19 | GSE255619 | GEO
Project description:Bacteriophages are potent therapeutics against biohazardous bacteria that are rapidly acquiring multidrug resistance. However, routine administration of bacteriophage therapy is currently impeded by a lack of safe phage production methodologies and insufficient phage characterization. We thus developed a versatile cell-free platform for host-independent production of phages targeting gram-positive and gram-negative bacteria. A few microliters of a one-pot reaction produces effective doses of phages against potentially antibiotic-resistant bacteria such as enterohemorrhagic E. coli (EAEC) and Yersinia pestis, which also possibly pose threats as biological warfare agents. We also introduce a method for transient, non-genomic phage engineering to safely confer additional functions, such as a purification tag or bioluminescence for host detection, for only one replication cycle. Using high-resolution and time-resolved mass spectrometry, we validated the expression of 40 hypothetical proteins from two different phages (T7 and CLB-P3) and identified genes in the genome of phage T7 that express exceptionally late during phage replication. Our comprehensive methodology thus allows for accelerated reverse and forward phage engineering as well as for safe and customized production of clinical-grade therapeutic bacteriophages.
2022-07-20 | PXD019543 | Pride
Project description:The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile, by means of whole transcriptome analysis, of peripheral blood mononuclear cells (PBMCs) derived from a healthy human donor and stimulated with a Pseudomonas aeruginosa phage PNM lysate, or P. aeruginosa strain 573. The PBMCs were stimulated for 20 h, followed by lysis of the cells and RNA extraction. In total, three stimulations were performed: control sample (i.e. not stimulated), P. aeruginosa phage PNM lysate and P. aeruginosa strain 573. Each stimulation was conducted in triplicate. The transcriptome analysis showed that the phage induce a clear immunological responses. Both pro- and anti-inflammatory genes were up-regulated in the PBMCs in the presence of the phage or its bacterial host. Our results indicate that bacteriophages might play a bigger role in the immune response then previously described and might have a broader effect than the clearing of bacterial infections alone, such as the suppression of the immune response to benefit their own survival.
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Project description:The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. We have identified an optimal RNA extraction method of microRNAs from human plasma samples. We also report that the addition of low doses of carrier RNA before starting RNA extraction improves microRNA extraction and quantification.
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Project description:Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector’s toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr’s pivotal role in immunity.
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Project description:This study analysed the temporal transcriptional response of L. lactis UC509.9 undergoing infection with either Tuc2009 or c2, representing phages of two different species (P335 and c2, respectively) of the family Siphoviridae. For the first time, to our knowledge, both DNA microarrays of the host and high resolution tiling arrays of each phage were used provide corresponding data sets of the entire transcriptome at various points post-infection. DNA microarrays containing oligonucleotide primers representing each of the 2066 annotated genes on the genome of L. lactis UC509.9 (Genbank accession number: CP003157), in addition to complete genome tiling arrays of bacteriophages Tuc2009 NC_002703) and C2 (NC_001706) at 4 bp resolution, were designed using eArray (https://earray.chem.agilent.com/earray/) and ChipD (http://www.ncbi.nlm.nih.gov/pubmed/20529880) and obtained from Agilent Technologies (Palo Alto, CA). For sample collection, pre-warmed GM17 (30 M-BM-0C) was inoculated with 2 % of an overnight culture of L. lactis UC509.9. This was grown at 30 M-BM-0C under static conditions to an OD600 of 0.13 at which point CaCl2 was added to a final concentration of 10 mM. The culture was further incubated for 10 min to allow equilibration. At this point, the culture was split into two equal volumes. To one, phage (either C2 or Tuc2009) in TBT buffer (100 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2), was added to a final multiplicity of infection (MOI) of 5. To the other, acting as control, a corresponding amount of TBT buffer without phage was added. Samples (60 ml) were collected at 2, 5, 10, 15, 25, 35 and, in the case of Tuc2009 only, 45 min post infection (p.i.) by centrifugation. Pellets were flash frozen in a -80 M-BM-0C EtOH bath. Samples were then maintained at -80 M-BM-0C until further processing and analysis.
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