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Bacterial viruses targeting multiresistant Klebsiella pneumoniae and Escherichia coli Harald Eriksson ©Harald Eriksson, Stockholm University 2015 ISBN 978-91-7649-123-2 Published papers are reproduced with the permission of the publisher. Printed in Sweden by Holmbergs, Malmö 2015 Distributor: Department of Molecular Biosciences, the Wenner-Gren Institute Populärvetenskaplig sammanfattning Ett växande problem i dagens samhälle är den ökade resistensen hos bakterier mot antibiotika, vilket i det långa loppet kan leda till att tidigare milda infektioner blir svårbehandlade och i vissa fall dödliga. Forskning pågår på flera fronter för att lösa detta problem; från att minska användningen av antibiotika och endast behandla svåra infektioner, till att ny- eller vidareutveckla ny antibiotika. Dessutom sker det forskning på att utveckla alternativa behandlingsmetoder mot bakteriella infektioner, som inte är lika tidsoch kostnadsintensiva som utvecklingen av ny traditionell antibiotika. Fokus i denna avhandling har varit bakteriens naturliga fiende, bakterieviruset, som i vissa fall kan nyttjas i terapeutiskt syfte för avdödandet av bakterier i infektioner hos människan. Genom att isolera bakterievirus från miljöer där det sker en kontinuerlig kamp om överlevnad mellan bakterierna och dess virus, kan vi ta del av den naturliga genetiska variation som det finns mellan dessa. Studierna i denna avhandling har lett till att vi isolerat ett flertal nya bakterievirus, primärt mot multiresistenta Klebsiella pneumoniae men även mot Escherichia coli. De isolerade bakterievirusen har sedan karaktäriserats, från evolutionära och taxonomiska aspekter (artikel I, II), till ingående analys av arvsmassa, strukturella proteiner och infektionsförlopp (artikel II). Vidare har vi utvärderat hur en sammansatt blandning av bakterievirus lyckas avdöda en större mängd kliniska isolat av multiresistenta K. pneumoniae (artikel III). Slutligen har vi studerat den biologiska effekten av predation på den bakteriella värden, genom att studera ett kliniskt isolat av multiresistent K. pneumoniae och deras förmåga att producera biofilm, deras tillväxthastighet och virulens (artikel IV) efter att ha utvecklat resistens mot antagonistiska bakterievirus. Abstract 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 multiresistant 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. List of papers This doctoral thesis is based on the papers listed below, referred to in the text by their associated Roman numerals. I. Eriksson, H., Maciejewska, B., Latka, A., MajkowskaSkrobek, G., Hellstrand, M., Melefors, Ö., Wang, JT., Kropinski, A.M., Drulis-Kawa, Z. and Nilsson, A.S. A suggested new bacteriophage genus, “Kp34likevirus”, within the Autographivirinae subfamily of Podoviridae. Viruses 2015, 7, 1804-1822; doi:10.3390/v7041804 II. Khan Mirzaei, M., Eriksson, H., Kasuga, K., HaggårdLjungqvist, E., Nilsson, A.S. Genomic and proteomic analysis of ECORp10, a newly characterized Podoviridae phage with C3 morphology. PLoS ONE 2014, 9(12): e116294; doi:10.1371/journal.pone.0116294 III. Eriksson, H., Berta, D., Örmälä-Odegrip, A., Giske, G. C., Nilsson, A.S. A novel phage cocktail inhibiting the growth of 99 βlactamase carrying Klebsiella pneumoniae clinical isolates in vitro Manuscript. IV. Örmälä-Odegrip, A., Eriksson, H., Mikonranta, L., Ruotsalainen, P., Mattila, S., Hoikkala, V., Nilsson, A.S., Bamford, J.K.H. and Laakso, J. Evolution of virulence in Klebsiella pneumoniae treated with phage cocktails Manuscript. Table of contents Introduction .................................................................................................. 9 Phages ......................................................................................................................... 9 Phage classification .............................................................................................. 9 Caudovirales taxonomy .................................................................................... 10 Phage infection ................................................................................................... 12 Phage evolution .................................................................................................. 13 Phage impact on bacterial hosts ........................................................................... 15 Phage–bacteria coevolution ............................................................................. 15 Bacterial defense systems ................................................................................ 16 Phage therapy .......................................................................................................... 22 The rise of antibiotic resistance....................................................................... 22 The history of phage therapy........................................................................... 23 Phage therapy today ......................................................................................... 24 Proteins as antimicrobial compounds ............................................................. 26 Enterobacteriaceae ................................................................................................. 27 Klebsiella spp. ..................................................................................................... 27 Escherichia coli ................................................................................................... 28 Aims.............................................................................................................. 29 Results and discussion ............................................................................. 30 Paper I ....................................................................................................................... 30 Paper II ..................................................................................................................... 32 Paper III .................................................................................................................... 33 Paper IV .................................................................................................................... 34 Concluding remarks and future perspectives ..................................................... 35 Acknowledgments ..................................................................................... 38 References .................................................................................................. 40 Abbreviations Abi carba Cas CRISPR DNA ECOR ESBL HGT Kpn LO LPS LIN MS MOI PEG RNA RM SEM Sie TA TEM abortive infection carbapenem CRISPR associated genes Clustered Regularly Interspaced Short Palindromic Repeats deoxyribonucleic acid Escherichia coli reference collection extended spectrum β-lactamase horizontal gene transfer Klebsiella pneumoniae lysis from without lipopolysaccharide lysis inhibition mass spectrometry multiplicity of infection polyethylene glycol ribonucleic acid restriction modification scanning electron microscopy superinfection exclusion toxin-antitoxin transmission electron microscopy Introduction Phages Bacteriophages, or phages for short, are viruses that infect and propagate in bacteria. Phages are the most abundant biological entity on the planet, with estimated amounts of up to 1031 in the biosphere (Hendrix et al., 1999). Out of all isolated phages, 96 % are tailed phages that belong to the order Caudovirales (Ackermann, 2001). Phages are ancient molecular machines that are believed to have existed by the time bacteria, archaea and eukarya diverged in the tree of life, based on the folding patterns of their proteins (Ackermann, 1998). They are highly specific for their target bacterial strain; this specificity is often as narrow as individual serotypes of a bacterial species. The large biodiversity of viruses is reflected in but also driven by their hosts, the prokaryotes and archaea. Phages can carry either RNA or DNA genomes that are either single or double stranded, and they can be enveloped or nonenveloped and have varied morphologies. Phage classification The relatively simple and straight forward method of classifying prokaryotes by their 16S rRNA (Woese and Fox, 1977) has no analogue in the phage world (Hendrix et al., 1999). Phages traditionally have been taxonomically classified based on the visualization of the free virion structure. With advances in whole genome sequencing, attempts have been made to classify phages in more detail. The mosaic-like structure of phage genomes requires the use of more intricate methods to classify them, not only by comparing the identities of individual genes but also the spatial placement of the genes in the genome and the gene order (Hendrix et al., 1999). The crystal structures of phage capsid proteins have revealed a conserved three dimensional (3-D) structure, even though there is no or very low amino acid sequence similarity between phage families. These tertiary structures recur throughout the viral kingdom, e.g., between the capsid folds of Podoviridae HK97, Myoviridae T4 and even in the eukaryote Herpes simplex virus (Cardone et al., 2013). This structural conservation of phage proteins is evident in several parts of the phage virion, from the assembly of the phage 9 head by the capsid proteins to the joining of the head and tail assembly by the head to tail connector proteins and also in the setup and assembly of phage tails. The phage tails consist of tape measure proteins, tail tube proteins, tail terminator proteins and, in the case of Myoviridae, the tail sheath protein. At the distal end, the host adsorption apparatus can be either a relatively simple tail spike or a more complex baseplate with tail fibers, or both components (Veesler and Cambillau, 2011). The head to tail connector proteins exhibit large size and sequence variations between phages, as with the 37-kDa Φ29 portal protein compared to its 83-kDa P22 counterpart. Regardless of size variation, these compared portal proteins assemble into an overall similar structure (Veesler and Cambillau, 2011). The similarity of the tertiary structures of these viral proteins might either be an indication of a common archaea/eukarya ancestry for all viruses or evolutionary convergence (Veesler and Cambillau, 2011). Caudovirales taxonomy The phages of Caudovirales are classified into three distinct families (fig. 1) based on their tail morphology. While the archetypical phage has an icosahedral and symmetrical head, there is a variation with an elongated morphology for the head structure, often referred to by a letter followed by a numeral; as shown, A represents Myoviridae phages (fig. 1A), B represents Siphoviridae (fig. 1B) and C is Podoviridae (fig. 1C). The numeral can range from 1 to 3: 1 is a symmetrical head, 2 an elongated head with a length-towidth ratio ranging between 1 and 2, and 3 has a length-to-width ratio greater than 2 (Bradley, 1967). Podoviridae have a short and non-flexible receptor binding complex for cell adhesion and DNA injection into the host cell, and their genome is encoded by double-stranded DNA. Further subdivision of the Podoviridae family comprises the Autographivirinae and the Picovirinae subfamilies; the Autographivirinae-type phages carry a gene for a single-subunit RNA polymerase and thus have the ability to transcribe their own genes, while the Picovirinae are classified on the basis of their small genome size, special tail vertex structure and DNA polymerase (Adriaenssens et al., 2015, Lavigne et al., 2008). From recent classification studies, it has been shown that while horizontal gene transfer (HGT) mechanisms are a contributing factor to the genetic diversity and exchange of genetic material between phages, evolutionary relationships can be readily observed between different phage subfamilies and genera (Adriaenssens et al., 2015, Lavigne et al., 2009, Lavigne et al., 2008). 10 The second Caudovirales family are the Siphoviridae, which have a double-stranded DNA genome, and a long, flexible and non-contractile tail (Adriaenssens et al., 2015). Approximately 61 % of all isolated and visualized tailed phages belong to this morphotype (Ackermann, 2001). Figure 1. Graphical representation of the virion structure of the tailed phages. A) Myoviridae, with elongated head and neck whiskers (phage T4), B) Siphoviridae with flexible tail and tail fibers (phage T5) and C) Podoviridae with icosahedral head and tail fibers (phage T7). The third family of the order Caudovirales is the Myoviridae, whose members have a complex and contractile tail and encode their genomes using double-stranded DNA. The Myoviridae adhesion process to the bacterial cell is a two-step process. Reversible adhesion occurs through interactions between the long tail fibers of the phage with a bacterial cell receptor, which in turn prompts a conformational change in the phage that allows for irreversible binding to the cell through the short tail fibers (Thomassen et al., 2003). Binding leads to a contraction of the outer tail sheath, which exposes the inner tube, leading to penetration of the cell wall and injection of the phage DNA into the cell. There are currently four subfamilies described in the Myoviridae family: the Tevenvirinae, Spounavirinae, Peduovirinae and Eucampyvirinae. These subfamilies have been classified and established based on sequence similarity and morphological features of the contained phages, but there are few shared characteristics that are conserved throughout these phage subfamilies (Lavigne et al., 2009, Javed et al., 2014). 11 Phage infection The main concern of phages is temporal: to ensure that their genes are transcribed in the correct order so that the host cell is not lysed before progeny virions are assembled. This process is commonly called a genetic cascade, where each set of genes that gets transcribed during the infection opens up another set of genes for expression, leading to a step-wise activation of each set of genes that drives the infection. This is mediated through several different strategies, from utilizing the host's own transcriptional machinery with phage-encoded transcription factors, activators, repressors and antiterminators or through phage-encoded polymerases that are utilized to drive the infection and open up the next operon for transcription (Krebs et al., 2012). The organization of genes into functional modules is a conserved feature among phages and can often be used to predict functions of de novo sequenced genes through the placement of their genome (Veesler and Cambillau, 2011). Phages can exhibit three different types of lifecycles that are dependent on their genetic heritage: virulent, temperate or persistent (fig. 2). A virulent phage that infects a bacterial cell progresses through the lytic cycle (fig. 2A, left side), in which the phage genome is replicated and capsids are assembled, followed by host cell lysis to release the progeny phages. Figure 2. A) Phages that have the genetic prerequisites can enter both the lytic and lysogenic cycles (both left and right cycle), while others can only follow the lytic cycle (leftmost cycle). These are called temperate and virulent (or lytic) phages, respectively. B) Persistent infection. Host chromosome depicted in blue, phage genome in red. A temperate phage can coexist with a bacterial cell via the integration of its genome either into the host or through a plasmid. When a temperate phage infects a bacterium, there is a competition between phage expressed proteins to either repress or initiate the lytic cycle versus the lysogenic cycle (Bertani, 1951). The outcome of the competition results in the phage entering the lytic cycle or integrating into the bacterial genome. The prophage will be maintained and inherited as part of the bacterial genome until a de-repression event. While the factors that cause de-repression of the lysogenic state are dependent on the prophage in question, DNA damage, heat shock or chemical 12 induction have been identified in phage lambda as causing excision of the prophage and induction of the lytic pathway (Little and Michalowski, 2010). Some phages can enter a temporary and quasi-stable state of pseudolysogeny, sometimes called a carrier state, during cell starvation and nutrient depletion of the bacterial cell that is being infected (Los and Wegrzyn, 2012). In this pseudolysogenic state, the infection process will stall, providing the bacterium more time to live and potentially a more promising environment for the phage to propagate in the future. This pseudolysogenic state can persist (Cenens et al., 2013a), however, even after the bacterial cell enters a high-nutrition environment, and their genomes are asymmetrically inherited in the daughter cells as demonstrated with the temperate phage P22 in Salmonella typhimurium (Cenens et al., 2013b). A third type of life style has been described in certain filamentous phages, e.g., phage M13, in which the infection is persistent and progeny phages are continuously released by excretion (fig. 2B). In this type of infection, the cells remain unharmed albeit with decreased cellular growth (Chopin et al., 2002). Phage evolution There are several mechanisms that contribute to the evolution of phages, such as DNA replication errors, environmental mutagens and HGTmechanisms (Abedon, 2009, Duffy et al., 2008). Single point mutations occur at a low frequency during genome replication. These mutations are then incorporated into progeny phages, which creates a large pool of diversity among the phages. Due to the fast generation times of bacteria and phages, together they constitute a good model system for insight into evolutionary processes (Abedon, 2009). This natural plasticity of phages to adapt through single point mutations has been demonstrated using phage T7, which uses lipopolysaccharides (LPS) as binding receptors. The phage was subjected to multiple passes through its wild type E. coli host and various LPS mutants, which led to the expansion of the phage host range across these mutants through point mutations in its tail fiber gene (Qimron et al., 2006). However, HGT mechanisms open up more rapid evolutionary forces that operate not only in the same bacterial species but also over species boundaries (Ochman et al., 2000). The integration of DNA into genomes is often facilitated through a process termed recombination, which is responsible for the exchange or rearrangement of genetic material. In prokaryotic cells, recombination comprises some of the DNA repair mechanisms that maintain the integrity of the genome but also part of the exchange of genetic material through different HGT mechanisms. In general, two types of recombination have been described: homologous and 13 site-specific. The requirement for homologous recombination is the presence of two similar or identical nucleotide strands, which can be joined. In E. coli, the RecF pathway is primarily responsible for repairing single-stranded breaks but can also repair double-stranded breaks, while the RecBCD pathway is responsible for repairing double-stranded breaks. In general, for both pathways, helicases are recruited to the identified DNA break to unwind the DNA helix and nucleases degrade one of the DNA strands, which in turn recruits the protein RecA to the free 3´ single-stranded end of the DNA. RecA then takes part in the joining of a homologous DNA strand and a Holliday junction is formed, followed by branch migration and strand separation after the recombination is complete. For a comprehensive review of these mechanisms, see references (Kowalczykowski et al., 1994, Kowalczykowski, 2000). Site-specific recombination can be classified into two general categories: transpositional recombination, which is further subdivided into replicative or non-replicative transposition, or conservative site-specific recombination. Transpositional recombination is mediated by a site-specific recombinase protein designated transposase, which is often encoded in the mobile genetic element. The transposase, depending on the type, can either insert the mobile element through excision and integration, utilizing double-stranded breaks of both the target and recipient sequences, or through replicative recombination, in which the donor sequence is joined to the recipient, replicated and then disjoined (Hallet and Sherratt, 1997). Conservative site-specific recombination is a common method for temperate phages to integrate themselves into bacterial genomes, in which site-specific recombination proteins termed integrases mediate the insertion of the phage genome at specific sites in the bacterial genome. Integrases are categorized into two families: serine recombinases, which insert the DNA by creating a double-stranded break, or tyrosine recombinases, which cleave the DNA in two constitutive steps via single-strand breaks (Hallet and Sherratt, 1997). For reviews of these mechanisms, see references (Hallet and Sherratt, 1997, Groth and Calos, 2004). 14 Phage impact on bacterial hosts In contrast to their lytic counterparts, temperate phages can confer advantages via phage-encoded genes that are beneficial to the bacterial host (Brabban et al., 2005). There are several examples in which the prophage encodes for exotoxins, which directly promote virulence of the bacterial host cell. For example, Vibrio cholera lysogenized by the temperate phage CTXφ introduces cholera toxins ctxA and ctxB (McLeod et al., 2005), which cause severe diarrhea in the host, increasing the transmission of the bacterial host into the environment and to other hosts. There are also more discrete methods to increase virulence gained by infecting phages, such as regulatory factors that increase the expression of native bacterial virulence genes or phageencoded enzymes that change the expression profiles of a bacterium to increase its virulence (Wagner and Waldor, 2002). Salmonella enterica phage phage SopEφ encodes a type three secretion system effector protein SopE, that promotes the invasion capability of its bacterial host (Mirold et al., 1999), and Vibrio cholera phage CTXφ also encodes for a toxin co-regulated type IV pilus (Karaolis et al., 1999). Other notable examples of phages that improve bacterial host fitness include the S. enterica phage Gifsy-2, which encodes a superoxide dismutase that enables the bacteria to survive the oxidative stress inside phagocytes by catalyzing innate superoxide and hydrogen peroxide in the environment into molecular oxygen and hydrogen peroxide. Another example is the Staphylococcus aureus phage φPVL (Kaneko et al., 1998), which encodes a human phagocyte cytotoxin that is released into the surrounding environment and damages phagocytes (Wagner and Waldor, 2002). These examples illustrate the balance and tradeoff between the decreased fitness of the bacteria via increased genome size by the integrated prophage and the increased fitness conferred by the phage. Phage–bacteria coevolution Bacteria have numerous predators, which include phages, protozoans and protists. It is estimated that a large portion of bacterial mortality, approximately 50 %, is due to phage predation (Diaz-Munoz and Koskella, 2014). There are two main explanatory models to describe the complex relationship between predator and prey, phage and bacteria. The “kill the winner” hypothesis argues that any abundant host in the environment will become a target for predation due to its abundance, and subsequently lead to an increase in the predators of this prey. Thus, any successful phenotype in the prey would eventually lead to a negative frequency selection of the population density, where the actual benefits of a phenotype with high fitness would be selected against when becoming abundant in the biosphere. A 15 possible parallel driving force behind the biodiversity of phages and bacteria is the niche differentiation hypothesis, where rather than competition between phages towards the same abundant host, a selective pressure exists to drive differentiation towards different hosts, thereby eliminating the need for competition and extinction of the less fit predator (Diaz-Munoz and Koskella, 2014). Lytic phages prey on susceptible bacteria in an antagonistic manner, where the phage goal is to produce progeny and the goal of the bacterium is to propagate. This drives the evolution and diversification of both bacteria and phage, and each in turn responds to an evolutionary adaptation or circumvention of the adaptions of the other, which drives biodiversity (Buckling and Brockhurst, 2012). Bacterial defense systems The close and intricate relationship between bacteria and phage is obvious when investigating bacterial defense against phages and how phages have counter-evolved against these defenses (Emond et al., 1998). Bacteria have evolved strategies to counter each step of the phage infection process, from the first step of cell adhesion to the last step in which progeny are assembled and released into the environment. This interaction and defense against phage predation can be divided into three discrete categories: adsorption inhibition, restriction and abortive infections (Hyman and Abedon, 2010). Adsorption inhibition Adsorption prevention is the first line of defense of the bacterial cell where the phage is prevented from attaching to and injecting its genome into the host (fig. 3A). Phages have been shown to bind a multitude of different surface structures of the bacterial cell wall prior to adsorption. The bacterial cell wall component adhered to is often a vital and conserved part of the bacterial cell, such as ferrichrome outer membrane transport protein, FhuA, which phage T5 binds to (Flayhan et al., 2012). Phage attachment to the bacterial cell is often a two-part process, as with the Myoviridae phage T4, where the long tail fibers interact and reversibly bind with outer membrane porin protein C (ompC) of the bacterial cell surface, leading to a conformational change in the short tail fibers of the phage, which irreversibly bind to the core region of the bacterial LPS layer, leading to injection of its genome into the bacterial cell (Thomassen et al., 2003). 16 Bacterial surface receptors The blockage of phage receptors (fig. 3A) not only enables bacteria to be protected against infection but can also be used by phages to prevent superinfection in a process called superinfection exclusion (Sie). Sie has been demonstrated with phage T5, a virulent phage that infects E. coli and expresses a lipoprotein early during the infection process that blocks the cell receptor that it originally adhered to, thus hindering superinfection by other T5 phages. This masking process is also beneficial to the phage after host cell lysis and progeny release as it stops progeny phages from adhering to lysed cell debris (Labrie et al., 2010). An example of a competitive inhibitor produced by bacteria is an antimicrobial protein designated J25, which binds to the iron transporter channel FhuA in E. coli (Labrie et al., 2010). FhuA is also used by phages T1, T5 and φ80 as an adsorption receptor with which it competes. Antimicrobial protein J25 is produced by E. coli in low-nutrition environments when competition for resources is high to promote its own growth (Destoumieux-Garzon et al., 2005). Capsule Both E. coli and K. pneumoniae can produce an exopolysaccharide capsule layer around the cell. This capsular layer is often considered a virulence factor because it prevents the immune system from recognizing immunogenic components of the bacterial cell wall, as is the case with some strains of E. coli (Jann and Jann, 1987) and with K. pneumoniae (Podschun and Ullmann, 1998) infections. The capsule also protects the bacterium from phagocytosis. Phages have evolved the ability to degrade these polysaccharide capsule layers, as demonstrated by phages K1E and K1-5 (Leiman et al., 2007), which carry virion-associated glycosidases that enable the phages to degrade and penetrate the capsular layer to reach the cellular wall (fig. 3B). 17 Figure 3. Bacterial phage defense mechanisms. A) Adsorption inhibition, B) Restriction, C) Abortive infection. 18 Biofilm The ability to produce an alternative external protective barrier, or biofilm, is a highly successful defense mechanism for bacteria (Hall-Stoodley et al., 2004). These biofilms consists of secreted proteins, lipids, DNA and polysaccharides that mediate the aggregation of bacteria into micro-colonies with their own microclimates that protect the bacteria from environmental threats such as phages and antibiotics (Bjarnsholt, 2013). Biofilms are produced by some bacteria when they cross a threshold in terms of cell numbers via quorum sensing mechanisms. Through the production of biofilms, bacteria have been found to survive for prolonged amounts of time on surfaces such as catheters, artificial implants and contact lenses, which is problematic in health care settings (Kostakioti et al., 2013). As with capsules, biofilms are highly efficient in masking bacteria. Biofilms decrease the amount of active compound that reaches the bacteria during antibiotic treatments and also prevent phage tail fibers from finding their surface receptors on the bacterial cell (fig. 3A). Although bacterial cells remain sensitive to an administered antibiotic, a biofilm hinders successful antibiotic treatment, as the antibiotic is prevented from being taken up by the bacteria and metabolized (Bjarnsholt, 2013). Some phages have evolved a response to biofilms by encoding tailassociated biofilm degrading enzymes called depolymerases (Cornelissen et al., 2011). These phage-associated enzymes can disrupt the protective biofilm that shields the bacterium. Subsequently, by infecting and replicating in host cells, phages spread their progeny further into the biofilm-shielded microcolony and step-wise destroy the protective barrier of the biofilm. Restriction The second line of bacterial defense is the restriction of invading foreign DNA. Several types of systems exist that work towards the same goal: the digestion of foreign DNA and the protection of the host DNA (fig. 3B). Restriction modification systems Restriction modification (RM) systems target invading and un-methylated DNA, while the host DNA is protected through methylation. These two component systems are divided into different categories dependent on protein complexity, recognition sites and their mode of action. The type II RM system is the most disseminated of the groups and has been found in 80 % of all sequenced bacterial genomes (Wilson and Murray, 1991). RM systems are composed of the restriction endonuclease (REase) and the methyltransferase (MTase), which work in concert. The REase specifically targets and cleaves unmethylated DNA strands at specific nucleotide 19 sequences, while the MTase methylates these same nucleotides. Both proteins exist in the cell at the same time, albeit at different concentrations, where the REase is in abundance. Phages have evolved several different strategies to evade digestion by RM systems, e.g., base modification to prevent recognition, DNA masking and endonuclease blocking (Samson et al., 2013). An example of endonuclease blocking occurs with phage T7, which co-injects its genome with an accessory protein designated Ocr that mimics the structure of the EcoKI REase-recognized site. Ocr has a higher affinity for the REase, preventing digestion of the phage genome. This gives the MTase time to methylate the phage genome, which is then protected from REase digestion, and the infection can proceed without interruption (Atanasiu et al., 2002). Subsequent phage progenies will have methylated genomes that will protect them from similar RM systems until they infect a susceptible bacterial cell that does not contain an RM system. Progeny phages from these bacterial cells will not have methylated genomes and will be susceptible to digestion by the same RM system. Another escape strategy of note is the lambda-encoded antirestriction protein Ral, which enhances the MTase activity of the infected cell (Loenen and Murray, 1986). This changes the balance between the REase and MTase in favor of the unprotected foreign DNA, which allows the invading and replicating genome to be methylated and protected from REase degradation in the cell. CRISPR-Cas The CRISPR-Cas system consists of genes encoding protein components, the Cas proteins, and a nucleotide array of clustered regularly interspaced short palindromic repeats (CRISPR). In this CRISPR array, short nucleotide sequences called spacers are stacked between repeats. The spacers have been acquired from previous invading foreign DNA and confer immunity against subsequent infection of the same origin. The CRISPR-Cas system has been denoted as an adaptive immune system of prokaryotes (Westra et al., 2012a, Wiedenheft et al., 2012) and is a defense system that works in two steps: an adaption/acquisition phase (Westra et al., 2012b) in which foreign DNA is identified, cleaved, and inserted into the CRISPR array and an interference phase (Swarts et al., 2012) in which the acquired DNA confers immunity against a subsequent infection by a phage with an identical sequence to the acquired DNA. In the type I CRISPR-Cas system, the acquired DNA is transcribed and inserted into a ribonucleoprotein complex called the Cascade complex. This Cascade complex consists of proteins encoded by the Cas genes (casABCDE), which are assembled together with transcribed, processed and matured RNA transcripts from the 20 CRISPR array, now called crRNA. It is this ribonucleoprotein complex that targets specific DNA strands using its antisense RNA strand and then recruits Cas3 for nucleolytic degradation (Yosef et al., 2012). Three major types of CRISPR-Cas systems have been described, based on the homology of the Cas proteins (Fineran and Charpentier, 2012). Several evasion strategies exist for phages to overcome restriction by the CRISPR-Cas system, for example through point mutations in the recognized region. Another evasion strategy that has been described with Pseudomonas aeruginosa temperate phages is a phage-encoded small protein that disrupts the Cas and crRNA complex and thus inactivates the entire CRISPR-Cas defense system (Bondy-Denomy et al., 2013). Interestingly, these genes are placed in an operon that is associated with late gene expression, during which the phage capsid genes are transcribed and translated, which is the final stage of the genetic cascade of the infecting phage. This suggest that the antiCRISPR protein is packaged inside the virion and injected with the DNA to immediately hinder the CRISPR acquisition process during the infection process. Abortive infection Abortive infection (Abi) either drives the infected cell to arrest the phage infection cycle or to induce bacterial autolysis. This type of phage exclusion system has mostly been studied in Lactococcus lactis, due to its extensive use in the dairy fermentation industry, but has also been identified in E. coli (Chopin et al., 2005). The Abi systems characteristically allow a successful phage infection to progresses normally until it is suddenly aborted, often just prior to the release of progeny phages (fig. 3C). Most Abi systems that have been identified are residents on plasmids; however, there are some exceptions in which the Abi system is part of the bacterial genome (Samson et al., 2013). Several types of Abi systems have been identified and characterized, and most are dependent on a single gene for their mode of action. The targets of the Abi systems are as diverse as the systems themselves, and hindrance of a cell’s DNA replication process, destabilization of RNA transcripts and arresting protein synthesis have been identified (Emond et al., 1998). Some of these identified Abi systems are constitutively expressed in the cell, while others are induced only after a phage infection. However, in general, all Abi gene products are toxic for the cell when artificially induced at high levels (Chopin et al., 2005). An example of an Abi system is the cryptic phage e14, which is part of the Escherichia coli K12 genome. E14 encodes for the superinfection exclusion protein Lit, which arrests all cellular translation upon T4 infection, aborting the infection and inducing cellular suicide of the infected cell. This cascade of events is started by the transcription of the major 21 capsid protein of T4, which interacts with the major host translation factor EFTu in its host, which in turn becomes susceptible to proteolytic cleavage by the metalloprotease Lit. This cascade of events leads to suspension of the translation of all proteins in the bacterial cell, and cellular death is ensured (Bingham et al., 2000). Another type of abortive infection of phages in bacteria is facilitated by toxin-antitoxin (TA) systems, which are encoded on plasmids that require the constant expression of both the toxin and the antitoxin in the host cell to ensure its survival. Phage infection disrupts this homeostasis, and cell death follows. In a recent study of phage infection and TA system coevolution in Erwinia carotovora, the ToxIN system was investigated for its mode of action during phage infection by examining escape mutants of surviving phages (Fineran et al., 2009). These Abi escape mutant phages were subsequently sequenced, and an identified mutated sequence was similar to the RNA antitoxin transcript of the TA system, which mimicked the function of the original antitoxin in the host cell, thus preventing the inhibition of phage infection (Blower et al., 2012). Phage therapy The rise of antibiotic resistance The spread of multidrug-resistant bacteria is of increasing concern to both the medical sector and to society as a whole. Since the 1970s, only four new classes of antibiotics have been approved, and due to the high cost associated with the development of new antibiotics, as well as the rapid acquisition of resistance, few new antibiotics are in the pipeline (Cooper and Shlaes, 2011). However, over the last couple years, two new classes of antibiotics have been approved: fidaxomicin, a narrow spectrum antibiotic that has been approved for the treatment of Gram-positive Clostridium difficile, and bedaquiline, a narrow spectrum antibiotic against Mycobacterium tuberculosis (Butler et al., 2013). The acquisition and dissemination of resistance towards novel antibiotics is a rapid process and can pass between bacterial species (Davies, 1994). For most novel types of antibiotics, resistant bacteria can be isolated within months if not immediately after introduction. The ease of this rapid dissemination of resistance between bacterial species is a major health concern. This alarming trend is visible everywhere, with a 424 % increase in reported multi-resistant ESBL-carrying bacterial infections in Sweden during the years 2007-2014 (Anonymous, 2013b). This type of increase is a 22 worldwide trend, though it occurs at different levels. Countries that have generous antibiotic prescription routines or over-the-counter distribution of antibiotics have the highest incidences of multi-resistant bacteria in the world. Several actions have been launched from government bodies since 2004 to combat this problem; one example is the EU-wide ban in 2006 of the use of antibiotics in animal food, where antibiotics were added to promote growth in livestock (Anonymous, 2005). Another action that has been taken is the creation of the ReAct network in 2004, a global network of scientists to promote debate and increase awareness of growing resistance against antibiotics (Cars and Hällström, 2004). The major focus of all efforts is to decrease the spread of multi-resistant bacteria by decreasing the usage of antibiotics in society. This is made possible by discontinuing the use of antibiotics for minor infections. Due to the pressing need for alternatives to conventional antibiotics, research into bacteria- and bacteriophage-derived antimicrobial compounds has been greatly expanded. Traditionally, ESBL is carried on plasmids and is disseminated among the Enterobacteriaceae family of prokaryotes, and the conferred resistance has evolved rapidly over time. Since the isolation of the first ESBL-resistant isolate, clinical isolates with larger resistance profiles have been characterized, from the original characterization of resistance against aminoglycosides in the 1970s, followed by resistance to extended spectrum cephalosporins and to ceftazidime (Podschun and Ullmann, 1998). Resistant clinical isolates of K. pneumoniae and E. coli have emerged against what was previously the last-resort class of antibiotics, the carbapenems. This highlights the importance of not only decreasing the widespread misuse of antibiotics in today’s society but also in finding alternative treatment methods for bacterial infections. The history of phage therapy Phage therapy was originally pioneered by Felix d´Herelle (D´Herelle, 1917, Schultz, 1927) as a remedy against dysentery caused by Shigella infection during 1930s. This sparked great interest in the scientific and medical community during that time, and many phage mixes and treatment remedies were launched against various infections and ailments. However, the lack of scientific understanding of phage biology, the high level of phage specificity and the discovery of antibiotics led to the quick abandonment of phage therapy in the West. In the following decades, only a few institutes continued to research and use phages as a treatment method, most notably the Eliava Institute in Georgia and the Wroclaw Institute of Experimental Therapy in Poland (Miedzybrodzki et al., 2012). These institutes are actively involved 23 in the development and administration of phages to patients. However, the validity of such therapies is still treated with skepticism in the West due to a lack of adequately controlled clinical trials (Sulakvelidze et al., 2001). Phage therapy today Due to the high specificity and narrow host range of phages, treatment is often more complex when compared to traditional antibiotics. A reason behind the success of antibiotic treatment is the availability of broad spectrum antibiotics, which only require the identification of the class of bacterium before therapy can be administered. As both a blessing and a curse, bacteriophage specificity requires the identification of the infecting bacterial species before treatment can be administered. The specificity of bacteriophages also reduces unwanted damage to the patient microflora during therapy compared to treatment with antibiotics. There are several commercial entities currently working within this field, with phage products for food processing (Intralytix Inc., Micreos BV) and human phage therapy product investigations (Ampliphi Biosciences corp., Novolytics ltd.). Clinical trials for phage therapy are underway; for example, in 2009 a successful double blind, placebo and randomized phase II clinical trial was performed using a phage cocktail consisting of six different phages. This study investigated the safety and efficacy of using phages as a treatment method for multi-resistant Pseudomonas aeruginosa in chronic otitis patients (Wright et al., 2009). The treated group showed a 76 % decrease in bacterial counts, while the placebo group showed a 9 % increase in bacterial cell density in the place of infection. A phage therapy study funded by Nestlé has been running extensive trials of phage cocktails in Bangladesh (Sarker et al., 2012). Nestlé’s main bacterium of interest is E. coli, which contaminates many sources of drinking water, causing severe dehydration due to diarrhea in local populations. Safety trials have been performed in adults, but the project was terminated in 2013 without notice. In 2013, the PhagoBurn project received funding from the European Union 7th framework program to evaluate phage therapy treatment of E. coli and P. aeruginosa infections in burn wound infections in clinical trials (Ravat and Chatard, 2013). There are solutions to address the narrow host ranges of phages; one is to isolate phages with broad host ranges that utilize conserved receptors. Another solution is to combine multiple phages with similar virulence characteristics but with different specificities for bacterial surface receptors to produce a phage cocktail. Ideally this cocktail would decrease the risk of bacterial resistance developing. Bacteria acquire resistance towards bacteriophages 24 rapidly, as they do towards antibiotics, but the ease of isolation of new phages and the low cost creates a lower threshold for investigating and replacing phages that have become ineffective against the bacteria. Treating untyped bacteria using bacteriophages creates novel challenges, and strategies are to either use a pre-made phage cocktail with phages specific against a multitude of serotypes of the same bacterial species or to tailor a phage cocktail selection with several phages specific for the patient serotype (Pirnay et al.). The first method requires only the isolation and typing of the infecting species before treatment starts, while the second tailored treatment requires not only isolation and typing of the bacteria but also susceptibility testing of phages using an existing phage library. After susceptibility tests have been performed, a selection of effective phages can be put into a cocktail and administered to the patient. Susceptibility tests to ascertain the host ranges of bacterial viruses can give false positives or negatives for various reasons. One parameter of a phage cocktail that must be monitored is its multiplicity of infection (MOI). This number describes the number of phages added per bacterial cell, and thus how many phages are expected to infect an individual bacterial cell. Problems arise when using too high a multiplicity of infection, creating phenomena such as lysis from without (LO) (Abedon, 2011) or lysis inhibition (LIN) (Bode, 1967). Both phenomena have been observed and investigated for phage T4, one of the most studied phages in the Myoviridae family. LO occurs when a bacterial cell membrane is destabilized by a multitude of phages adsorbing to its membrane, with phage-associated and enzymatically active domains that degrade the integrity of the cell membrane without specificity for the bacterium (Abedon, 2011). This process leads to the premature lysis of the host cell with no release of phage progeny. A passive phage treatment consists of using a high MOI phage cocktail that utilizes the ability of phages to induce lysis from without, for which a productive infection is not needed for pathogen clearance and treatment success. This ability of phages to non-specifically lyse bacteria can be utilized to create passive treatment regimens, in which the outcome of the treatment is not dependent on the successful infection and replication of the phages. LIN has been identified and studied for phage T4 and occurs when a successful T4 infection is underway, followed by a superinfecting T4. During the normal progression of a T4 infection, the transmembrane T4 holin is expressed and accumulates in the inner membrane during the late phase of infection. It stays dormant while structural proteins are assembled and the phage genome is packaged into the progeny capsids. At a critical concentration, the holin oligomerizes and creates pores in the inner membrane that allow the phage endolysin to escape the cytoplasm and degrade the outer 25 cell membrane, causing cell lysis. LIN has been demonstrated from superinfecting T4 phages, where the protein RI interacts with the transmembrane holin and delays oligomerization and bacterial cell lysis. If the infected cell is challenged by a superinfection phage every 10 minutes, LIN can be maintained indefinitely (Tran et al., 2005). Proteins as antimicrobial compounds Commercial interest in phage therapy is tentative because the use of natural proteins or viruses are not patentable, and the regulatory framework for development and testing in clinical trials creates a high threshold for academic groups to overcome (Verbeken et al., 2012). The main difficulty in using proteins as antimicrobial agents concerns their possible rapid inactivation through proteolytic degradation, renal clearance or immunological blockage. These obstacles can, however, be overcome by screening for inherently robust and non-immunogenic proteins (Resch et al., 2011). In summary, several frontiers in bacteria- and phage-encoded antimicrobial proteins exist; for example, endolysins (Schmelcher et al., 2012, Walmagh et al., 2012), holins (Ludwig et al., 2008), colicins (Hecht et al., 2012) and other protein products have been isolated from phages (Liu et al., 2004). Large substrate screenings are underway to identify compounds with antimicrobial activity, both originating from phage (Liu et al., 2004) and bacteria (Haney and Hancock, 2013). Proteins as antimicrobial compounds have the advantage of being less immunogenic compared to whole phages, and recombinant proteins are patentable and thus marketable. Lysins are proteins that are expressed late in phage infections when phage progeny have already been assembled in the infected cell. The lysins mediate the degradation of the peptidoglycan layer of the bacterial cell wall, which eventually leads to cellular lysis. They are effective in lysing Gram-positive bacteria that have an exposed peptidoglycan layer, while Gram-negative bacteria are protected due to their peptidoglycan cell wall layer being sandwiched between an outer and inner cell membrane. Another example of active research for bacteria-derived antimicrobial compounds is the investigation of bacteriocins, such as colicins (Hecht et al., 2012). Colicins are small protein molecules that can kill susceptible bacteria by creating pores in the cell membrane or by inhibiting peptidoglycan synthesis, thus hindering cell growth. These proteins are isolated from bacteria, which encode for these proteins to increase their competitive advantage in their habitat by inhibiting the growth of closely related bacterial strains. Work is ongoing to screen, identify and characterize this class of proteins for possible use as antimicrobial compounds (Hecht et al., 2012). 26 Recent advances in creating a hybrid bacteriocin-lysin show promise, and a recombinant lysin has been shown to successfully target and kill Yersinia pestis in vitro (Lukacik et al., 2012). Enterobacteriaceae In this thesis, we have isolated bacterial viruses targeting E. coli and K. pneumoniae, which are both found in the bacterial Enterobacteriaceae family. E. coli and K. pneumoniae are the most commonly isolated multi-resistant bacterial species in the European Union; their isolation rates exceed that of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycinresistant enterococcus (VRE) (Anonymous, 2013a). Enterobacteriaceae constitute a large family of rod-shaped, Gram-negative and facultative anaerobic bacteria. This family of bacteria includes a wide range of bacterial species from commensal organisms to potential human pathogens, including E. coli, Klebsiella, Salmonella, Shigella and Y. pestis. Several bacterial species of this family live in the intestines of warm-blooded animals, while others live in soil or water. Klebsiella spp. Klebsiella is a rod shaped, non-motile, encapsulated and facultative anaerobic Gram-negative bacterium. The genus Klebsiella contains species that have a wide range of habitats, soil and plants, including mammalian intestine, mucosal surfaces and skin lesions. Most Klebsiella species have the ability to produce a thick and protective capsule around the bacterial cell, which protects the cell from phagocytosis and other host defense mechanisms. This capsule is composed of a diverse range of acidic polysaccharides, of which 77 different serotypes have been described. Some of these capsular antigens, termed K-antigens, have been associated with higher pathogenicity in both animal models and isolated clinical samples (Simoons-Smit et al., 1984). Serotyping of the LPS layer has revealed eight different compositions of its outer polysaccharide layer, designated O-antigen, whereas the O1 antigen is the predominant variation in clinical isolates. This highly immunogenic and endotoxic LPS layer is mostly concealed by the outer capsule. K. pneumoniae is an opportunistic and nosocomial pathogen that infects immunocompromised patients, infants and the elderly. Patients who are already hospitalized for other afflictions are especially vulnerable to K. pneumonia infections, and the risk of being colonized is directly correlated 27 with duration of hospital stay (Podschun and Ullmann, 1998). While in normal situations an infection by K. pneumoniae would be easily treatable, the increased spread and dissemination of the ESBL resistance plasmid throughout Enterobacteriaceae is increasing the severity and problems in treating these infections. This increases the need for alternative treatment methods and the need to lower the use of antibiotics in minor infections to decrease the dissemination of antibiotic resistance in bacteria. Escherichia coli E. coli is a rod-shaped, Gram-negative and facultative anaerobic bacterium that is a commensal species often found in the gastrointestinal tract of warmblooded animals. The majority of E. coli species are flagellated and are encapsulated or produce biofilms. Due to its intimate relationship with humans and our ability to easily cultivate this bacterium, E. coli has been one of the most well-characterized bacterial species since its identification in 1885. E. coli is the most routinely used laboratory organism, due to its ease of cultivation, its ability to easily accept foreign DNA and the ease of genetic manipulation of this bacterial species. There have been described over 145 different O-antigens and over 80 different K-antigens in E. coli species (Davis et al., 1973). The majority of E. coli species are commensal and assist the host with the breakdown of food and production of vitamin K (Bentley and Meganathan, 1982), but several pathogenic serotypes have been described that cause diarrheal diseases or colonize extra-intestinal sites, such as the urinary tract (Lai et al., 2013). 28 Aims The purpose of this thesis project has been to isolate, purify, and characterize phages and to test different hypotheses regarding phage therapy efficacy, in vitro and in vivo, for single phages and on phage mixtures. The long term goal is to establish a phage collection and strategies for curing multiresistant Klebsiella pneumoniae infections in humans. These aims have been realized in the papers of this thesis as follows: Isolation and characterization of phages against multi-resistant Klebsiella pneumoniae and Escherichia coli (Paper I and Paper II) Characterization and annotation of phage genomes to identify possible toxins (Paper I, II) Creation of novel phage cocktails and test them against a selection of Klebsiella pneumoniae clinical isolates (Paper III) Testing treatment efficacies in vitro and in vivo (Paper IV) 29 Results and discussion Paper I A suggested new bacteriophage genus, “Kp34likevirus”, within the Autographivirinae subfamily of Podoviridae Paper I is a product of our continuous work to isolate and sequence novel phages from waste water effluent plants in Stockholm, Sweden. The decreased cost of whole genome sequencing has resulted in an abundance of phage genomes being submitted to NCBI GenBank, but there is limited work being done on the taxonomical classification of these phages. Newly sequenced phages are often loosely associated into large super-genera based on similarity to the closest available phage genome, as with several recent cases with Phikmvlikevirus (Drulis-Kawa et al., 2011, Lin et al., 2014). There are some ongoing efforts to classify and categorize phages into new families and genera, for example with Podoviridae (Lavigne et al., 2008), Myoviridae (Lavigne et al., 2009) and Siphoviridae (Adriaenssens et al., 2014). Within the Podoviridae subfamily of Autographivirinae there are currently three established genera: the T7likevirus, Sp6likevirus and Phikmvlikevirus (Lavigne et al., 2008). The results of this study increased the taxonomical resolution of the Autographivirinae subfamily of phages. Based on sequence homology to Klebsiella phages KP34 (Drulis-Kawa et al., 2011), NTUH-K2044-K1-1 (Lin et al., 2014), and F19 (KF765493), newly isolated phages SU503 and SU552A were characterized and compared to each other and the Phikmvlikevirus genus. These five phages demonstrate a high nucleotide pairwise identity to each other, between 75.1 – 75.9 %, with 29 conserved genes between themselves and similar gene arrangements. In comparison to members of the taxonomically close Phikmvlikevirus, 18 conserved genes are shared across the genus boundaries. Within the subfamily of Autographivirinae, a total of nine genes are conserved between the type phages of the genera T7likevirus, Sp6likevirus, Phikmvlikevirus and the proposed “Kp34likevirus”. Furthermore, the phages of the proposed phage genus “Kp34likevirus” show distinct differences compared to the phages in the closest relative phage genus, the Phikmvlikevirus. The setup and gene variants of the lysis cassette genes differ between the genera: the KP34-like 30 phages have a unimolecular spanin (u-spanin) and a spanin-holin-endolysin setup, while the φKMV lysis cassette has the gene arrangement holinendolysin-spanin with a dual protein spanin (i-spanin/o-spanin) complex similar to that of phage lambda. The Autographivirinae obligatory RNA polymerase also shows divergence between the KP34-like phages and the Phikmvlikevirus group members. When comparing RNA polymerase specificity and recognition loops, they are highly conserved between the member phages of the proposed “Kp34likevirus” and divergent from the phages in the Phikmlikevirus genus group. Among the KP34-like phages, the recognition loops are identical, while the specificity loops show a minor divergence of four amino acids out of 25, just after the DNA interacting basepairs of the loop. The conservation of the DNA interacting regions of the RNA polymerase suggests the use of a genusspecific promoter sequence within the proposed “Kp34likevirus” phages. This suggests that a conserved genus of phages that is widespread in the biosphere has been identified. 31 Paper II Genomic, Proteomic, Morphological, and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology Paper II describes vB_EcoP_SU10 (SU10) as an E. coli phage with the rare C3 morphology that has an elongated head with a length-to-width ratio of over 2.5. Phages with this rare phenotype have only been fully described in a few cases (Ackermann, 2001). Morphological analysis using transmission electron microscopy (TEM), ultra-thin section electron microscopy, and scanning electron microscopy (SEM) was performed on the phage and on infected bacterial cultures at different time points in the infection cycle. Thin section micrographs showed that phage capsids are arranged in a honeycomb-like structure before and during the DNA packaging process in the host cell. No relationship between the genome size and head length was observed in comparison to φEco32 (Pavlova et al., 2012), Serratia marcenses phage KSP100 (Matsushita et al., 2009), Salmonella enterica phage 7-11 (Kropinski et al., 2011) and E. coli phage NJ01 (Li et al., 2012), all of which are C3 morphology Podoviridae phages. Comparison of the tail lengths between free and adsorbed virions revealed more than a two-fold difference in length. Two possible explanations exist for this observation: either there is a post-adsorption change in the virion tail structure or the observed difference is an artifact of the two different preparation methods for the ultra-thin sectioning TEM and regular TEM. Genome analysis and annotation of SU10 revealed over 40 % nucleotide sequence similarity and also a gene arrangement in common with phage φEco32 (Pavlova et al., 2012). Based on these similarities, a phage-encoded sigma factor and a sigma-70 inhibitor were identified. Transcriptional in silico analysis suggested that several structural genes are transcribed early in the infection process through host sigma-70 promoters. Ultra-thin sectioning micrographs confirm these structural proteins as early as five minutes after adsorption to host cells. Structural proteins were investigated using mass spectrometry (MS) from mature and PEG-precipitated virions, where 34 proteins out of 125 predicted coding sequences were identified and confirmed. The phages contain a ribosomal slippage site that can create an alternative major capsid gene variant, which has also been confirmed through MS. Phylogenetic analysis was performed with C1 and C3 Podoviridae phages that demonstrated sequence similarity with the major capsid and scaffolding protein of SU10 to investigate the evolutionary divergence of the elongated head morphology. Based on these phylogenetic analysis, the C1 and C3 morphology phages diverged approximately 280 million years ago and 32 adapted to different bacterial family hosts. The C3 morphology phages infect members of the Enterobacteriaceae family, while the C1 morphology phages infect members of Pseudomonadaceae bacteria. Paper III A novel phage cocktail inhibiting the growth of 99 β-lactamase-carrying Klebsiella pneumoniae clinical isolates in vitro The antibiotic resistance of Enterobacteriaceae is often conferred through a plasmid-mediated enzyme that has the ability to hydrolyze and inactivate βlactam antibiotics, which includes penicillins and third-generation cephalosporins (Doern, 1995). Additional resistance to carbapenem antibiotics (ESBLcarba) has emerged (Jacoby, 1997), further reducing the number of effective treatments. A phage cocktail consisting of six phages that target K. pneumoniae isolates was assembled from a collection of 60 isolated phages. The phages were selected for their ability to prey on 24 extended spectrum β-lactamase (ESBL)carrying K. pneumoniae isolates. The cocktail was then assessed against a total of 125 K. pneumoniae isolates with different resistance profiles, including carbapenem-hydrolyzing ESBLcarba isolates. As part of this study, host range screenings of the phage cocktail and the individual phages were performed using standard materials and methods adapted for antibiotic resistance screenings in clinical laboratories to determine the ease of adaptation of phage screenings to these labs. The use of standard protocols and equipment only required slight adjustments to determine phage susceptibility in clinical laboratories. The phage cocktail inhibited the growth of 99 of the 125 (79 %) clinical isolates. However, two phages out of the six were responsible for the majority of the bacterial inhibition across the isolates, and productive infection was not confirmed to be the cause of the bacterial growth inhibition. 33 Paper IV Evolution of virulence in Klebsiella pneumoniae treated with phage cocktails Paper IV investigates the impact of phage predation on bacterial virulence. While resistance is a major problem with traditional antibiotics, the short generation times and ongoing co-evolutionary battle between bacteria and phage have created a large natural reservoir of phages that can be used to overcome bacterial phage resistance. The assembly of phage cocktails that adsorb to different bacterial receptors decreases the risk of resistance developing during treatment (Levin and Bull, 2004, Chan and Abedon, 2012). Selecting phages that utilize conserved bacterial receptors that are also virulence factors has previously been shown to attenuate the pathogenicity of phage-resistant bacteria (Filippov et al., 2011, Laanto et al., 2012). The study sought to isolate phage-resistant bacterial clones that had grown resistant to one or more phages and to characterize these bacterial clones in terms of growth characteristics, biofilm production and virulence in a eukaryotic host. K. pneumoniae isolate 07RAFM-KPN-524 (CTX-M9 ESBLharboring) was isolated from a patient in Sweden in 2007 and chosen as a routine bacterial host. Four strictly lytic phages that produced halo plaques were isolated for this study, suggesting the presence of biofilm-degrading depolymerases (Hughes et al., 1998). In order to obtain phage-resistant mutants, several microcosms were set up with combinations of the four phages to facilitate predation on 07RAFMKPN-524 continuously for one week. Phage-resistant bacteria were isolated, growth rate characteristics and biofilm production determined for each of the bacterial clones. Clones were then used to infect Galleria mellonella, a model organism for virulence testing in eukaryote infections (McLaughlin et al., 2014). Three out of the four treatments that contained a single phage showed an increased bacterial growth rate (phages B, H and K; p = < 0.001), while a single multi-phage combination (BHX) showed a decrease in maximum growth rate compared to an untreated control (p < 0.001). Biofilm production was lowered in the phage resistant bacteria from multi phage treatments compared to the single phage treatments (p < 0.001). Individually, two phages decreased biofilm production (phages K and X) in comparison to the untreated control (p = 0.001 and p = 0.002). While these results were surprising, and our hypothesis was that phage predation would decrease the virulence of the bacteria in the in vivo model, it has been previously reported that increased growth rate could be a plastic response to phage predation in the bacteria. These results suggest that terminal 34 investment (Poisot, 2012) plays some role in the increased growth rates observed; however, further investigation is required. Concluding remarks and future perspectives The aim of this thesis has been to gain a better understanding and increase our knowledge of lytic bacteriophages that infect and kill multi-resistant bacteria and to sequence, annotate and characterize these phages. One goal has also been to test phage cocktails against a selection of multi-resistant bacteria. We have isolated bacteriophages against ESBL-carrying Enterobacteriaceae with a special focus on K. pneumoniae. The long term goal has been to create a phage cocktail that is effective against a range of K. pneumoniae isolates that would effectively lyse a large selection of clinical isolates. Two phages that were isolated during the study exhibited large sequence similarities to each other and also to already-sequenced Klebsiella phages KP34, F19 and NTUH-K2044-K1-1. From our analysis, we suggest the creation of a new Podoviridae genus called “Kp34likevirus” within the Autographivirinae subfamily (Paper I). However, in contrast to the member phages of the proposed “Kp34likevirus” genus, the existing Phikmvlikevirus member phages φKMV, LKA1, LIMElight and LIMEzero have demonstrated distinct differences between themselves, and their relationships to each other should be re-examined (Adriaenssens et al., 2011, Ceyssens et al., 2006). Based on phylogenetic analysis of the conserved genes of the phages that were compared in this study, the monophyletic clade of Pseudomonas phages φKMV, PT2, PT5, MPK6, MPK7, LUZ19, LKD16 and φkF77 comprises candidates for a study to revise the Phikmvlikevirus genus. In paper II, we characterized the polyvalent Podoviridae phage vB_KpnP_SU10 (SU10) with the rare C3 morphology, a phage that infects a broad range of the E. coli reference collection (ECOR) (Ochman and Selander, 1984) but also two clinical ESBL-harboring E. coli isolates. From our study, we suspect that the process of phage infection and assembly are not as straightforward as first thought. SU10 has both host and phage promoters upstream of some structural genes, for example the capsid gene, scaffold gene and portal protein gene. The classical model of a phage infection process can be divided into three parts: 1) transcription of early phage genes and commandeering of the host transcriptional apparatus, 2) transcription of middle genes and replication of the phage genome and 3) transcription of late genes, e.g., structural genes and cell lysis. This set of events suggest that DNA packaging is one of the last steps of the infection cycle; however, with 35 observations from our in silico analysis of transcriptional starts combined with ultra-thin TEM micrographs, we observed the assembly of capsids early in the infection process. Further studies of the head elongation process and the mechanisms underlying size determination of the elongated head morphology are needed to determine if, for example, the scaffolding protein or a unidentified tape measure protein is responsible for the capsid length. In paper III, new phages were isolated and assembled into an experimental phage cocktail, which was assessed for in vitro activity against 125 clinical ESBL-harboring K. pneumoniae isolates. These isolates were collected worldwide and included different types of ESBL-resistant strains, including metallo-β-lactamase ESBLcarba resistant strains. This pilot study was to determine the feasibility of a pre-made phage therapy cocktail against a wide selection of unknown isolates. Phages that possessed good growth characteristics and a wide host range were selected and assembled into a phage cocktail. Further characterization of the phages in the cocktail is recommended, such as their average burst size, latent period and adsorption rate. When compared, these characteristics could improve the selection of phages for future cocktails. By adding or replacing phages in the current cocktail, we can extend the host range to include, for example, capsular serotype K2. Optimization can be performed by both identifying the epitope that the phage receptor recognizes and by selecting phages that use essential surface proteins as receptors, thus decreasing the probability of resistance by receptor mutation/variation in the bacteria. However, to ascertain the receptors that phages use for adsorption, deletion mutants must be constructed in a susceptible host or through whole genome sequencing of the wild type bacterial host and subsequent phage resistant strains. The phage cocktail could next be evaluated in pre-clinical in vivo trials using mice in a lung infection model (Debarbieux et al., 2010). However, most animal models do have drawbacks that must be considered, as the infecting bacterial isolate must be optimized for disease progression in the animal. This optimization consists of selecting pathogenic strains of the bacterium that result in quick disease progression and 100 % lethality. Another drawback of current phage therapy studies is that phage inoculation occurs immediately prior to or after the administration of pathogenic bacteria, and thus these results should be viewed with some caution. These protocols should be adjusted for more realistic disease progression, i.e., with a slow infection and no phage treatment until symptoms arise that would simulate a real-world infection scenario. Another aspect of phage therapy that must be addressed is the current legislation that regulates medicinal products, which has been written with 36 traditional antibiotics and medicinal products in mind, and future phage therapy products and their standing in these regulations must be investigated (Verbeken et al., 2014). Furthermore, the possibility of using engineered phages must be addressed, both legally and biologically. The template for this engineered phage would be a fully characterized, sequenced and annotated phage, selected for its high burst size and short latency period. By replacing the recognition region of the tail fibers, one could switch the specificity of the phage and thus create a fast track for pharmaceutical use, lessening the legislative burden for phage therapy. By using one model phage for each bacterial genus, co-evolutionary aspects of the phage bacteria could be exploited, such as promoter and codon usage adaption. The aspect of acceptance from society in using genetically modified bacterial viruses as therapeutic agents must also be considered before their use as medicinal products. Paper IV explores the relationship between phages and bacteria and the effects that phage predation has on the bacterium, whether it be with a single phage or several phages simultaneously. Interestingly, we discovered that the selection of the phage used in therapy is important, some of the resulting phage-resistant bacteria in our study exhibited an increased growth rate and lethality to the eukaryotic host. To further understand the mechanism underlying the increased virulence effects in the bacterium, further characterization of phages as well as phage-bacteria interactions should be performed. This is in contrast to studies that have shown attenuated bacterial virulence in phage-resistant bacteria from phages that adsorb using bacterial receptors that also serve as virulence factors. Phages H and X in the study (Paper IV) demonstrate two opposite effects on the host bacterium, and their difference in gene content, receptor usage and growth characteristics must be investigated to understand and predict which phages are suitable for phage therapy. 37 Acknowledgments First and foremost I would like to thank my wife Joanna and my family for the support and encouragement over these years. I would also like to thank my supervisor Dr. Anders S. Nilsson for taking me in as PhD student, and my cosupervisor Professor emerita Elisabeth Haggård-Ljungquist for all her suggestions, help and support during these years. For making my time at Stockholm University more enjoyable, I would like to especially thank Jaroslav Belotserkovsky, Sidney Carter and Bo Lindberg. Good friends and great colleagues! I would also like to mention and thank previous members of the group: Lina Sylwan, Hanna Nilsson, Richard Odegrip, Sridhar Mandali, Marios Hellstrand, David Berta and Andreas Bertilsson. It wouldn’t have been the same without you! From the corridor, with shared moments and lunches together: Asal, Alicia, Ali, Fredrik, Kun, Mattias, Melanie, Mohea, Noushin and Sara. For bringing new perspectives to old problems, sharing a lab bench and projects together, Anni-Maria Örmälä-Odegrip. Your refreshing energy, optimism and “can do” attitude really made my last year go so much faster! Many thanks to my international collaborations Andrew Kropinski, Zuzanna Drulis-Kawa and her group for the joint work with paper I. Thank you for the great teamwork and collaboration! For assistance with the thesis preparation I would like to mention Asem Abd El Monem Fatom (figure 1), Susanna Hua (figure 3), Callum Cooper and American Journal Experts for editing and proof-reading the thesis. 38 For making my days at Stockholm University more fun and engaging, I would like to thank the early morning gym group at the SU staff gym: Peter Reinhed and Wilhelm Widmark for all those mornings together, the afternoon coffee club: Ainars Bajinskis and Sara Shakeri Manesh for all the nice talks, shared moments and coffee together, and the technical staff for all the help I received from Inga-Britt Olausson, Görel Lindberg, Eva Eyton and Eva Pettersson during these years. My grateful and cordial thanks go to Stiftelsen Olle Engquist Byggmästare, for their funding of the project over the years and to the foundation Sven and Lilly Lawskis fond for their stipend and travelling grants during my first two years. 39 References ABEDON, S. T. 2009. Phage evolution and ecology. 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