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  • 1.
    Khan Mirzaei, Mohammadali
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Study of phages for phage therapy: An Escherichia coli experience2014Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The world is rapidly moving toward a post-antibiotic era as effective antibiotics are becoming fewer. phage therapy is a potentially possible method for treating infectious diseases and counts as an alternative method for antibiotic therapy. Phages' number on earth is estimated to be 1031 which ensures a great variety for these entities, and a never ending source for new phages. However, phages vary in their morphology, life cycle and virulent ability, and thus phage therapy will be unsuccessful without enough knowledge in phage biology. Only phages with a good virulence and broad host ranges are fitted for phage therapy applications, host range being one the most important factors that needs a careful study. There is no common method for studying the host range of phages and host range measurements are consequently dependent on the method that was used to study it. In our study, six polyvalent phages were selected from thirty virulent phages isolated using strains from E. coli reference collection (ECOR) as hostes. The selected phages were tested for their host range across 234 strains of E.coli (both sensetive and resistant to antibiotics) and Salmonella (reference collection SARA and SARB) using both spot test and efficiency of plating (EOP) method. Spot test results show no correlation to results of EOP analyses. One way to explain why spot test results are uncorrelated to EOP is the effects of resistant mechanisms of the host. In spot test, phages of a high concentration lystae might absorb, degrade the cell wall and kill the bacteria but the intrinsic resistant mechanisms of the host can recognize and degrade the genome, and stop phage replication and reproduction, which is visualized in the EOP analysis. Based on our studies, it is possible that prophages can contribute to bacterial defence against phages. Phages cannot in most cases produce a high EOP on those E. coli strains that have a P2 prophage in their genome.

    The phage SU10 had the highest EOP/spot test ratio among six selected phages. Transmission electron microscopy images revealed a very rare morphology for SU10, a short tail of 19 nm long and a 137 elongated head, and it was classified as a member of Podoviridae family. Genomic analysis of the phage show a genome size of 77,327 kb, 123 ORF encodes 38 proteins with known function. Twenty-two of theses proteins were identified using a mass-spectrometry based proteomics. The size of the tail was measured to be longer, 41 nm, in ultra-thin sectioning transmission electron microscopy images. A honeycomb structure is formed by the phage capsids during structural assembly, which is very rare for bacteriophages. The honeycomb structure of SU10 capsids might work as an aid or and external scaffolding protein during morphogenesis. The SU10 capsid is elongated before its genome is packed into the head according to the ultra-thin sectioning transmission electrographs which indicates that the genome packaging process has no role in elongation of the head. Based on our phylogenetic analysis, the scaffolding protein and major head protein have coevolved and the scaffolding protein in SU10 is not a recent attachment to the major head protein. According to the phylogenetic analysis of two proteins, the C1 and C3 morphotypes diverged 280 million years.

  • 2.
    Khan Mirzaei, Mohammadali
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The efficacy of bacterial viruses against multi-resistant Escherichia coli: from isolation to pharmacology2016Doctoral 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.

  • 3.
    Khan Mirzaei, Mohammadali
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ek Blom, Linnea
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cooper, Callum J.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Weiss, Howard
    Udekwu, Klas I.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Infection Dynamics within a Two Phage One Bacterium System: 1 Implications for TherapyManuscript (preprint) (Other academic)
  • 4.
    Khan Mirzaei, Mohammadali
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Eriksson, Harald
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kasuga, Kie
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Genomic, Proteomic, Morphological, and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, article id e116294Article in journal (Refereed)
    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.

  • 5.
    Khan Mirzaei, Mohammadali
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Haileselassie, Yeneneh
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Navis, Marit
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cooper, Callum
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sverremark-Ekström, Eva
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Immunogenic profiling of structurally distinct bacteriophages and their interaction with human cellsManuscript (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.

  • 6.
    Khan Mirzaei, Mohammadali
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Haileselassie, Yeneneh
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Navis, Marit
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cooper, Callum
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sverremark-Ekström, Eva
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 437Article in journal (Refereed)
    Abstract [en]

    Due to a global increase in the range and number of infections caused by multi resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum betalactamase-producing Escherichia colinumbers by 1-4 log(10) compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

  • 7.
    Khan Mirzaei, Mohammadali
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3, article id e0118557Article in journal (Refereed)
    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.

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