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Genomic, Proteomic, Morphological, and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, e116294Article in journal (Refereed) Published
Abstract [en]

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia - Salmonella genera.

Place, publisher, year, edition, pages
2014. Vol. 9, no 12, e116294
National Category
Biological Sciences Microbiology in the medical area
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-113563DOI: 10.1371/journal.pone.0116294ISI: 000347119100128OAI: oai:DiVA.org:su-113563DiVA: diva2:786532
Note

AuthorCount:5;

Available from: 2015-02-05 Created: 2015-02-04 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Bacterial viruses targeting multi-resistant Klebsiella pneumoniae and Escherichia coli
Open this publication in new window or tab >>Bacterial viruses targeting multi-resistant Klebsiella pneumoniae and Escherichia coli
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The global increase in antibiotic resistance levels in bacteria is a growing concern to our society and highlights the need for alternative strategies to combat bacterial infections. Bacterial viruses (phages) are the natural predators of bacteria and are as diverse as their hosts, but our understanding of them is limited. The current levels of knowledge regarding the role that phage play in the control of bacterial populations are poor, despite the use of phage therapy as a clinical therapy in Eastern Europe.

The aim of this doctoral thesis is to increase knowledge of the diversity and characteristics of bacterial viruses and to assess their potential as therapeutic agents towards multi-resistant bacteria.

Paper I is the product of de novo sequencing of newly isolated phages that infect and kill multi-resistant Klebsiella pneumoniae. Based on similarities in gene arrangement, lysis cassette type and conserved RNA polymerase, the creation of a new phage genus within Autographivirinae is proposed.

Paper II describes the genomic and proteomic analysis of a phage of the rare C3 morphotype, a Podoviridae phage with an elongated head that uses multi-resistant Escherichia coli as its host.

Paper III describes the study of a pre-made phage cocktail against 125 clinical K. pneumoniae isolates. The phage cocktail inhibited the growth of 99 (79 %) of the bacterial isolates tested. This study also demonstrates the need for common methodologies in the scientific community to determine how to assess phages that infect multiple serotypes to avoid false positive results.

Paper IV studies the effects of phage predation on bacterial virulence: phages were first allowed to prey on a clinical K. pneumoniae isolate, followed by the isolation of phage-resistant bacteria. The phage resistant bacteria were then assessed for their growth rate, biofilm production in vitro. The virulence of the phage resistant bacteria was then assessed in Galleria mellonella. In the single phage treatments, two out of four phages showed an increased virulence in the in G. mellonella, which was also linked to an increased growth rate of the phage resistant bacteria. In multi-phage treatments however, three out of five phage cocktails decreased the bacterial virulence in G. mellonella compared to an untreated control.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2015. 49 p.
Keyword
Bacterial viruses, Bacteriophage, Phage, Phage therapy, multi-resistant bacteria, Klebsiella pneumoniae
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-116711 (URN)978-91-7649-123-2 (ISBN)
Public defence
2015-05-29, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2015-05-07 Created: 2015-04-23 Last updated: 2015-06-23Bibliographically approved
2. The efficacy of bacterial viruses against multi-resistant Escherichia coli: from isolation to pharmacology
Open this publication in new window or tab >>The efficacy of bacterial viruses against multi-resistant Escherichia coli: from isolation to pharmacology
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The increase of multi-resistant bacteria highlights that the golden era of antibiotics is ending and that alternative treatmentsare urgently needed. Phages have been historically used to treat bacterial infections prior to the discovery of antibiotics and have gained renewed interest in the past decade. Despite the advantages of phage therapy over traditional antibiotic usage, a number of concerns persist over their clinical application centring on their efficacy and safety. This thesis presents four papers that focus on the isolation and characterization of phages that target reference strains and drug-resistant strains of E. coli as well as their infection dynamics and kinetics. In Paper I, six of thirty isolated phages were selected to be characterized for their growth parameters and host range using two commonly used methods. The study showed that the host range (an important selection criteria for phages) of the phages can change based on the assessment method and that the lysis efficiency of phages is host-dependent. The study suggests that standardised methods to assess the host range and lytic activity of phages are required to reduce result variability between research groups. Paper II investigated a rare phage with C3 morphotype from the Podoviridae family and characterised it via genomic, proteomic, morphologic and phylogenetic analysis. The study revealed previously unseen aspects including the formation of a honeycomb structure comprised of phage head during DNA packaging, the possible contractile nature of the tail and the 280 million year co-evolution between the major head protein and the scaffolding protein. Paper III highlights the need to take the immune system into consideration when designing phage therapeutics. In the study, four purified structurally distinct phages (selected from the three main phage families) were exposed to human cells (HT-29 and Caco-2 immortalised intestinal epithelial cell lines and donor-derived peripheral blood mononuclear cells) and the immunogenicity of the phages determined. Phage immunogenicity was shown to vary in a concentration and phage dependent manner with SU63 (a Myoviridae) being the most immunogenic phage and SU32 (a Siphoviridae) the least immunogenic. In the presence of human cells and a suitable host, phages were shown to maintain their killing efficacy as well as the ability to proliferate. Paper IV studies the infection dynamics of an experimental two-phage cocktail against a single bacterial host in vitro and in silico. However, in silico analysis and in vitro analysis produced conflicting results, in which mathematical modelling predicted the complete clearance of bacteria for all treatment scenarios whereas experimental results showed a 1-3log10 reduction in bacterial content. Practical experiments also showed increased anti-bacterial activity when the time between the additions of each phage was varied. This discrepancy suggests that the current mathematical model is unsuitable due to the inability to account for discrete variables such as interference.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2016. 56 p.
Keyword
Phage therapy, Multiresistant E. coli, Pharmacodynamics, Pharmacokinetics, Bacterial viruses, Genomic, proteomic
National Category
Microbiology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-126328 (URN)978-91-7649-346-5 (ISBN)
Public defence
2016-03-14, P216, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

 

Available from: 2016-02-18 Created: 2016-01-31 Last updated: 2017-02-20Bibliographically approved

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Khan Mirzaei, MohammadaliEriksson, HaraldHaggård-Ljungquist, ElisabethNilsson, Anders S.
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