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The efficacy of bacterial viruses against multi-resistant Escherichia coli: from isolation to pharmacology
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Nilsson)
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 [en]
Phage therapy, Multiresistant E. coli, Pharmacodynamics, Pharmacokinetics, Bacterial viruses, Genomic, proteomic
National Category
Microbiology
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-126328ISBN: 978-91-7649-346-5 (print)OAI: oai:DiVA.org:su-126328DiVA: diva2:899124
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
List of papers
1. Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy
Open this publication in new window or tab >>Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3, e0118557Article in journal (Refereed) Published
Abstract [en]

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of pro-phages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.

National Category
Biological Sciences Microbiology in the medical area
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-116617 (URN)10.1371/journal.pone.0118557 (DOI)000351275000027 ()
Note

AuthorCount:2;

Available from: 2015-04-30 Created: 2015-04-22 Last updated: 2017-12-04Bibliographically approved
2. Genomic, Proteomic, Morphological, and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology
Open this publication in new window or tab >>Genomic, Proteomic, Morphological, and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology
Show others...
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.

National Category
Biological Sciences Microbiology in the medical area
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-113563 (URN)10.1371/journal.pone.0116294 (DOI)000347119100128 ()
Note

AuthorCount:5;

Available from: 2015-02-05 Created: 2015-02-04 Last updated: 2017-12-05Bibliographically approved
3. Immunogenic profiling of structurally distinct bacteriophages and their interaction with human cells
Open this publication in new window or tab >>Immunogenic profiling of structurally distinct bacteriophages and their interaction with human cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Due to a global increase in the range and number of infections caused by multi-resistant bacteria, 11 phage therapy is currently experiencing a resurgence of interest. However, there are a number of 12 well-known concerns over the use of phages to treat bacterial infections. In order to address concerns 13 over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, 14 immunological characterization is required. In the current investigation, the immunogenicity of four 15 distinct phages and their interaction with donor derived peripheral blood mononuclear cells and 16 immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using 17 standard immunological techniques. When exposed to high phage concentrations (109 PFU/well), 18 cytokine driven inflammatory responses were induced from all cell types. Although phages appeared 19 to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite 20 co-incubation with different cell types, phages maintained a high killing efficiency, reducing 21 extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to 22 untreated controls. Phages were also able to actively reproduce in the presence of human cells 23 resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. 24 Through an increased understanding of the complex pharmacokinetics of phages, it may be possible 25 to address some of the safety concerns surrounding phage preparations prior to creating new 26 therapeutic strategies.

Keyword
Pharmacokinetics, Phage therapy, Cytokines, Immune response, Multiresistant bacteria
National Category
Microbiology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-126324 (URN)
Available from: 2016-01-31 Created: 2016-01-31 Last updated: 2016-02-01Bibliographically approved
4. Infection Dynamics within a Two Phage One Bacterium System: 1 Implications for Therapy
Open this publication in new window or tab >>Infection Dynamics within a Two Phage One Bacterium System: 1 Implications for Therapy
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(English)Manuscript (preprint) (Other academic)
Keyword
Pharmacodynamics, Phage therapy, E. coli, In silico modelling
National Category
Microbiology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-126325 (URN)
Available from: 2016-01-31 Created: 2016-01-31 Last updated: 2016-02-09Bibliographically approved

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