Rozprawy doktorskie na temat „Bacteriophages”

The oceans cover ca. 70% of the Earth’s surface and due to their depth encompass around 300 times the habitable volume of the terrestrial environment. The exact proportion of life on Earth that exists in the oceans is unknown as many ocean species remain undiscovered; in particular this holds true for the viruses that infect marine bacterioplankton. It is currently thought that viruses that infect bacteria, bacteriophages or phages, can numerically exceed their hosts by a factor of ten, however, this abundant and diverse group of organisms is still poorly understood. This is especially true of phages that infect members of the Roseobacter clade. Globally, members of the Roseobacter lineage can comprise up to a quarter of the marine microbial community and often dominate the alga-associated bacterial community. In this study phages capable of infecting species of Roseobacter were isolated and characterised. Two Roseovarius-specific phages, RLP1 and RPP1, were isolated from UK coastal waters; morphological and sequence data identified them as belonging to the N4-like genus of Podoviridae. Comparative genomic analysis of both Roseovarius phages to other N4-like phages such as Escherichia coli phage N4 and Sulfitobacter sp. EE-36 phage EE36Φ1, revealed a number of conserved core genes involved in DNA metabolism, transcription control and virion structure. Comparison of N4-like Roseobacter phages (RLP1, RPP1, EE36Φ1 and Ruegeria pomeroyi DSS-3 phage DSS3Φ2) also revealed a number of peripheral genes which are likely to interact directly with host proteins/machinery specific to the Roseobacter group. Unusually, both RLP1 and RPP1 appeared to only infect host cells when in semi-solid agar matrix, but not in liquid culture. Comparison of the outer surface of agar-embedded and planktonic cells revealed different outer-membrane protein and lipopolysaccharide expression profiles. This suggests that some Roseobacter species (spp.) change components of their bacterial cell surface according to their physiological state: agar-embedded/sessile or planktonic and RLP1 and RPP1 exploit this by binding to (a) receptor(s) only expressed during sessile conditions. A number of prophage-like elements were also induced from three Roseobacter spp. by exposure of growing cultures to the DNA-damaging chemical Mitomycin C. These were identified by electron microscopy as belonging to the Siphoviridae family. The results of this project suggest that within the marine environment there remain many uncharacterised phages with peculiar biochemical properties and a wealth of genomic information.

The increasing incidence of infections due to antibiotic resistant bacteria has led to renewed interest in bacteriophages (= phages) and phage therapy. Although phage therapy has been applied to control bacterial diseases in plants, poultry, livestock and humans, its application in aquaculture is still relatively limited. The emergence of phage-resistant bacterial mutants has been considered to be one of the major limitations of phage therapy. This study aimed to (i) isolate and characterise phages; (ii) select phages and their bacterial hosts to set up in vivo phage therapy models with aquaculture animals, and estimate the efficiency of phage therapy; (iii) investigate the generation and characteristics of phage-resistant mutants, and thus estimate the consequence of applying phage therapy when phage-resistant mutants emerge; and (iv) discuss the prospects for application of phages in aquaculture. Two Vibrio isolates and their phages were isolated from a Scottish marine fish farm. Based on the results of conventional phenotype testing and 16S rRNA gene sequencing analysis, the two vibrios, V9 and V13, were identified as Vibrio splendidus and Vibrio cyclitrophicus, respectively. The bacterial characteristics including morphology, temperature and salinity range of growth, production of extracellular enzymes, and the possession of virulence genes were examined. According to the morphological characteristics observed using transmission electron microscopy by negative staining, phage PVS9 of V. splendidus V9 was identified as a myophage, while phage PVC13 of V. cyclitrophicus V13 was identified as a siphophage. The phages could only lyse one bacterial host strain and their genomic DNA was double stranded with a size of ~46 kb. The two Vibrio isolates were found to be non- or of low virulence to rainbow trout, goldsinny wrasse and Artemia in pathogenicity experiments. Thus an in vivo phage therapy model could not be set up using these Vibrio isolates and their phages. Two phages pAS-3 and pAS-6 were isolated using the Aeromonas salmonicida subsp. salmonicida Hooke strain as the host. Phages pAS-3 and pAS-6 had a similar genome size of ~50 kb, and the same relatively narrow host range within A. salmonicida subsp. salmonicida strains. The siphophage pAS-3 formed clear plaques and inhibited A. salmonicida Hooke growth in vitro completely for at least 18 hours when using MOI = 1,000, whereas the podophage pAS-6 formed turbid plaques and weakly inhibited Hooke growth. Rainbow trout exposed by intraperitoneal injection with 0.1 mL of the raw phage preparations at a concentration of 108 PUF mL-1 showed no adverse effects over 14 days. In the phage therapy trial, fish were firstly injected with 1 x 102 CFU fish-1 of A. salmonicida Hooke, then immediately injected with phage preparations of pAS-3 and pAS-6, respectively, using MOI = 10,000. Compared with the control group (which did not receive phage treatment), phage treated groups showed a delay in the time to death, and lower mortalities. However, the mortalities and time to death between phage treated and non-treated groups were not significantly different. Phage-resistant mutants of pathogenic A. salmonicida strain Hooke were induced by repeatedly challenging with phage pAS-3. One of the mutants, termed HM, was chosen to compare the characteristics with the parental wild-type strain Hooke. Test results including the formation of ‘smooth’ colonies on TSA, autoagglutination negative, the formation of creamy colonies on Coomassie Brilliant Blue agar, and the degradation of a thick/furry layered structure on the cell surface indicated a deficiency of the A-layer in the phage-resistant mutant HM. Therefore, it was deduced that the A-layer either directly acted as the receptor of A. salmonicida phage pAS-3, or was affected indirectly by the change of an unknown phage receptor. The greater wax moth larvae model was used to compare the virulence of the phage-resistant mutant HM and the parental wild-type strain Hooke, as it is an ethically acceptable animal model, which has the advantages of being low cost and convenient for injection, and is also a recognised alternative model for bacterial pathogens of fish. The results showed that virulence of the phage-resistant mutant HM did not decline in the greater wax moth larvae model compared with that of the parental wild-type strain Hooke. In conclusion, different approaches were used to isolate and characterise phages from different aquaculture environments for potential use in phage therapy. A rainbow trout model was set up using pathogenic A. salmonicida strain Hooke and two A. salmonicida phages pAS-3 and pAS-6. The use of phage treatment led to lower cumulative mortalities and delay to the time of death, although the differences between the groups were not significant, futher work is required to determine if these phages have potential in phage therapy. The consequence of applying phage therapy when phage-resistant mutants emerge was estimated based on their characteristics and virulence, and no decline in virulence of the phage-resistant mutant from this study indicates the importance of fully testing the virulence of phage-resistant mutants before carrying out large scale field trials of phage therapy. It appears feasible to use phage therapy as an alternative approach to control bacterial infections in aquaculture, but further studies are required to focus on improving effectiveness, and also to overcome the concrete limitations and hurdles in application and commercialisation. Moreover, a broader range of applications of phages in aquaculture should be explored.

The history of the discovery of bacteriophages began almost a century ago. In spite of the appearance of antibiotics, bacteriophages are still extremely promising. Bacterial infections are an urgent public health problem, due to the increasing of antibiotic resistance and negative health consequences. Thus, phages are currently considered as an alternative treatment, which are going to replace antibiotics.

Mass production of translationally optimized bacteriophages (hereafter referred to as phages) is the need of the hour in the application of phages to therapy. Understanding translational efficiency of phages is the major preliminary step for mass producing efficient phages. The objective of this thesis is to understand factors affecting translational efficiency of phages. In chapter two, we hypothesized that weak translation initiation efficiency is responsible for weak codon concordance of Escherichia coli lambdoid phages with that of their hosts. We measured the strength of translation initiation using two indices namely minimum folding energy (MFE) and proportion of Shine-Dalgarno sequence (PSD). Empirical results substantiate our hypothesis suggesting lack of strong selection for improving codon adaptation in these phages is due to their weak translation initiation. In chapter three, we measured codon usage concordance between GC-rich and GC-poor Aeromonas phages with their GC-rich host Aeromonas salmonicida. We found low codon usage concordance in the GC-poor Aeromonas phages. We were interested in testing for the role of tRNAs in the GC-poor phages. We observed that the GC-poor phages carry tRNAs for codons that are overused by the phages and underused by the host. These findings suggest that the GC-poor Aeromonas phages carry their own tRNAs for compensating for the compositional difference between their genomes and that of their host. Previously several studies have reported observed avoidance of stable secondary structures in start site of mRNA in a wide range of species. We probed the genomes of 422 phage species and measured their secondary structure stability using MFE. We observed strong patterns of secondary structure avoidance (less negative MFE values) in the translation initiation region (TIR) and translation termination region (TTR) of all analyzed phages. These findings imply selection is operating at these translationally important sites to control stable secondary structures in order to maintain efficient translation.

Carbohydrate recognition is a ubiquitous principle underlying many fundamental biological processes like fertilization, embryogenesis and viral infections. But how carbohydrate specificity and affinity induce a molecular event is not well understood. One of these examples is bacteriophage P22 that binds and infects three distinct Salmonella enterica (S.) hosts. It recognizes and depolymerizes repetitive carbohydrate structures of O antigen in its host´s outer membrane lipopolysaccharide molecule. This is mediated by tailspikes, mainly β helical appendages on phage P22 short non contractile tail apparatus (podovirus). The O antigen of all three Salmonella enterica hosts is built from tetrasaccharide repeating units consisting of an identical main chain with a distinguished 3,6 dideoxyhexose substituent that is crucial for P22 tailspike recognition: tyvelose in S. Enteritidis, abequose in S. Typhimurium and paratose in S. Paratyphi. In the first study the complexes of P22 tailspike with its host’s O antigen octasaccharide were characterized. S. Paratyphi octasaccharide binds less tightly (ΔΔG≈7 kJ/mol) to the tailspike than the other two hosts. Crystal structure analysis of P22 tailspike co crystallized with S. Paratyphi octasaccharides revealed different interactions than those observed before in tailspike complexes with S. Enteritidis and S. Typhimurium octasaccharides. These different interactions occur due to a structural rearrangement in the S. Paratyphi octasaccharide. It results in an unfavorable glycosidic bond Φ/Ψ angle combination that also had occurred when the S. Paratyphi octasaccharide conformation was analyzed in an aprotic environment. Contributions of individual protein surface contacts to binding affinity were analyzed showing that conserved structural waters mediate specific recognition of all three different Salmonella host O antigens. Although different O antigen structures possess distinct binding behavior on the tailspike surface, all are recognized and infected by phage P22. Hence, in a second study, binding measurements revealed that multivalent O antigen was able to bind with high avidity to P22 tailspike. Dissociation rates of the polymer were three times slower than for an octasaccharide fragment pointing towards high affinity for O antigen polysaccharide. Furthermore, when phage P22 was incubated with lipopolysaccharide aggregates before plating on S. Typhimurium cells, P22 infectivity became significantly reduced. Therefore, in a third study, the function of carbohydrate recognition on the infection process was characterized. It was shown that large S. Typhimurium lipopolysaccharide aggregates triggered DNA release from the phage capsid in vitro. This provides evidence that phage P22 does not use a second receptor on the Salmonella surface for infection. P22 tailspike binding and cleavage activity modulate DNA egress from the phage capsid. DNA release occurred more slowly when the phage possessed mutant tailspikes with less hydrolytic activity and was not induced if lipopolysaccharides contained tailspike shortened O antigen polymer. Furthermore, the onset of DNA release was delayed by tailspikes with reduced binding affinity. The results suggest a model for P22 infection induced by carbohydrate recognition: tailspikes position the phage on Salmonella enterica and their hydrolytic activity forces a central structural protein of the phage assembly, the plug protein, onto the host´s membrane surface. Upon membrane contact, a conformational change has to occur in the assembly to eject DNA and pilot proteins from the phage to establish infection. Earlier studies had investigated DNA ejection in vitro solely for viruses with long non contractile tails (siphovirus) recognizing protein receptors. Podovirus P22 in this work was therefore the first example for a short tailed phage with an LPS recognition organelle that can trigger DNA ejection in vitro. However, O antigen binding and cleaving tailspikes are widely distributed in the phage biosphere, for example in siphovirus 9NA. Crystal structure analysis of 9NA tailspike revealed a complete similar fold to P22 tailspike although they only share 36 % sequence identity. Moreover, 9NA tailspike possesses similar enzyme activity towards S. Typhimurium O antigen within conserved amino acids. These are responsible for a DNA ejection process from siphovirus 9NA triggered by lipopolysaccharide aggregates. 9NA expelled its DNA 30 times faster than podovirus P22 although the associated conformational change is controlled with a similar high activation barrier. The difference in DNA ejection velocity mirrors different tail morphologies and their efficiency to translate a carbohydrate recognition signal into action.<br>Kohlenhydraterkennung ist ein fundamentales Prinzip vieler biologischer Prozesse wie z.B. Befruchtung, Embryogenese und virale Infektionen. Wie aber Kohlenhydratspezifität und –affinität in ein molekulares Ereignis übersetzt werden, ist nicht genau verstanden. Ein Beispiel für ein solches Ereignis ist die Infektion des Bakteriophage P22, der drei verschiedene Salmonella enterica (S.) Wirte besitzt. Er erkennt und depolymerisiert die repetitiven Einheiten des O Antigens im Lipopolysaccharid, das sich in der äußeren Membran seines Wirtes befindet. Dieser Schritt wird durch die Tailspikes vermittelt, β helicale Bestandteile des kurzen, nicht kontraktilen Schwanzapparates von P22 (Podovirus). Das O Antigen aller drei Salmonella enterica Wirte besteht aus sich wiederholenden Tetrasacchariden. Sie enthalten die gleiche Hauptkette aber eine spezifische 3,6 Didesoxyhexose Seitenkette, die für die P22 Tailspikeerkennung essentiell ist: Tyvelose in S. Enteritidis, Abequose in S. Typhimurium und Paratose in S. Paratyphi. Im ersten Teil der Arbeit wurde die Komplexbildung von P22 Tailspike mit O Antigen Octasaccharidfragmenten der drei verschiedenen Wirte untersucht. S. Paratyphi Octasaccharide binden mit einer geringeren Affinität (ΔΔG≈7 kJ/mol) an den Tailspike als die beiden anderen Wirte. Die Kristallstrukturanalyse des S. Paratyphi Octasaccharides komplexiert mit P22 Tailspike offenbarten unterschiedliche Interkationen als vorher mit S. Enteritidis und S. Typhimurium Oktasaccharidkomplexen mit Tailspike beobachtet wurden. Diese unterschiedlichen Interaktionen beruhen auf einer strukturellen Änderung in den Φ/Ψ Winkeln der glykosidischen Bindung. Die Beiträge von verschiedenen Proteinoberflächenkontakten zur Affnität wurden untersucht und zeigten, dass konservierte Wasser in der Struktur die spezifische Erkennung aller drei Salmonella Wirte vermittelt. Obwohl die verschiedenen O Antigen Strukturen unterschiedliches Bindungsverhalten auf der Tailspikeoberfläche zeigen, werden alle vom Phagen P22 erkannt und infiziert. Daher wurde in einer zweiten Studie die multivalente Bindung zwischen P22 Tailspike und O Antigen charakterisiert. Die Dissoziationskonstanten des Polymers waren drei Mal langsamer als für das Oktasaccharid allein, was auf eine hohe Affinität des O Antigens schließen lässt. Zusätzlich wurde gezeigt, dass die Aggregate des Lipopolysaccharids in der Lage sind, die Infektiösität vom P22 Phagen zu reduzieren. Ausgehend davon wurde in einer dritten Studie die Bedeutung der Kohlenhydrat Erkennung auf den Infektionsprozess untersucht. Große S. Typhimurium Lipopolysaccharide Aggregate bewirkten die DNA Freisetzung vom P22 Kapsid. Dies deutet darauf, dass der P22 Phage keinen weiteren Rezeptor für die Infektion auf der Oberflächen seines Wirtes verwendet. Zusätzlich moduliert die P22 Tailspike Aktivität den Ausstoss der DNA vom P22 Phagen: Er ist langsamer, wenn der Phage Tailspikes besitzt, die weniger hydrolytisch aktiv sind und wurde nicht induziert, wenn Lipopolysaccharid eingesetzt wurde, dass zuvor mit Tailspike hydrolysiert wurde. Darüber hinaus wurde der Start der DNA Ejektion verzögert, wenn Tailspikes mit verminderter Affinität am Phagen vorhanden waren. Die Ergebnisse führten zu einem Modell für die Infektion von P22: Tailspikes positionieren den Phagen auf Salmonella enterica und ihre Aktivität drückt ein zentrales Strukturprotein des Phagen, das Stöpselprotein, auf die Membranoberfläche. Aufgrund des Membrankontaktes findet eine Konformationsänderung statt die zur Ejektion der Pilotproteine und zur Infektion führt. Vorhergehende Studien haben bisher nur die DNA Ejektion in vitro für Viren mit langen, nicht kontraktilen Schwänzen (Siphoviren) mit Proteinrezeptoren untersucht. In dieser Arbeit wurde das erste Mal die DNA Ejektion für einen Podovirus mit LPS Erkennung in vitro gezeigt. Die O Antigen Erkennung und Spaltung durch Tailspikeproteine gibt es häufig in der Phagenbiosphere, z.B. am Siphovirus 9NA. Die Kristallstrukturanalyse von 9NA Tailspike zeigt eine komplett gleiche Struktur, obwohl beide Proteine nur zu 36% Sequenzidentität besitzen. Zusätzlich hat 9NA Tailspike ähnliche enzymatische Eigenschaften. Diese ist für den DNA Ejektionsprozess im Siphovirus 9NA verantwortlich, der auch durch LPS Agreggate induziert wird. 9NA stößt dabei seine DNA 30 Mal schneller aus als Podovirus P22 obwohl die damit verbundene Konformationsänderung mit einer ähnlich hohen Aktivierungsbarriere kontrolliert wird. Daher spiegeln die Unterschiede in der DNA Ejektionsgeschwindigkeit der verschiedenen Tailmorphologien die Effezienz wieder, mit der die spezifische Kohlenhydraterkennung in ein Signal umgewandelt wird.

Mannheimia haemolytica is the principal bacterial agent associated with bovine respiratory disease (BRD). It has a significant economic impact on the beef feedlot industry. The current methods for BRD prevention and treatment have various problems and limitations, especially with reports of increased antimicrobial resistance in M. haemolytica. Bacteriophage therapy presents a novel method to mitigate M. haemolytica. This study aimed to isolate strictly lytic M. haemolytica-specific bacteriophages from bovine nasopharyngeal swabs and feedlot trough water. This was accompanied by an extensive characterization of temperate bacteriophages induced from representative strains of a M. haemolytica collection. Phage morphology, host specificity, genomic diversity, and comparative genomics were determined. Even though temperate bacteriophages are not ideal candidates for phage therapy, they can be engineered or modified to serve this function. Genome sequences of selected temperate bacteriophages also provide a foundation for future studies on the biology of these microorganisms.<br>viii, 107 leaves : ill. ; 29 cm

Members of the genus Campylobacter are a major cause of food-borne disease worldwide. They can colonize the intestinal mucosa of poultry, to high levels leading to contamination of meat, at slaughter. Their numbers can be reduced in different ways including chicken treatment with bacteriophages. For such treatments to be successful, in depth understanding of the bacteriophage that infects and kills campylobacters is vital. The work in this thesis describes: isolation and comprehensive characterisation of bacteriophage candidates for future therapy applications. In order to increase the available stocks of characterized candidate bacteriophage, a number of attempts were made to isolate bacteriophages from poultry excreta. The new isolates together with some uncharacterized phages from our laboratory stocks were characterized with respect to their host range and genomic size. Some bacteriophages preparations in previous studies showed genomes of different sizes and a number of attempts were done for their separation. This raised questions about the relationship between the two different sized genomes. Prior to this work, a co isolate pair had been successfully separated and the sequence of the larger genome, CP220, was determined. Part of the work here, was performed to extend this study by obtaining the sequence of the smaller co isolate, CPX and compare it to CP220. They did not appear to have any identifiable relationship at the genetic level, but the availability of the CPX sequence will further extend our knowledge of bacteriophage genetics and this phage has clear therapeutic potential. Attempts were also made to separate and characterize a second co-isolate pair but these were unsuccessful. The availability of the DNA sequence of CP220 allowed a much closer molecular characterisation and comparison of Campylobacter phage genomes, than had previously been possible. One area that was investigated in this study was the presence of repeat regions identified in the CP220 genome, which were amplified by PCR, but could not be cloned in E. coli. Furthermore, genes encoding potential lysins were identified in the CP220 genome and they were amplified, cloned and attempts were made to express the proteins, which may have potential therapeutic value. One gene product was successfully expressed and showed evidence of lytic activity on Campylobacter and other bacterial genera. In summary, this thesis describes a much closer examination of molecular biology of Campylobacter bacteriophage than had previously been possible, including the determination of the sequence CPX phage.

The cyanobacterium Acaryochloris marina was isolated in 1996 and solved a 50 year old mystery as to the origin of the pigment chlorophyll d, which was thought to be a pigment of red algae or a breakdown product of the universal chlorophyll, chlorophyll a. Over the next decade, new Acaryochloris spp. were isolated from all over the world as the genus received international interest from the scientific community, with the majority of research directed towards understanding the mechanisms of photosynthesis of this uniquely pigmented cyanobacterium, using A. marina as the model organism of the genus. During this project, characterisation of different aspects of photosynthesis in Acaryochloris spp. was performed including an investigation of pigment adaptation and composition and the growth and characterisation of A. marina biofilms. However, the main focus of the thesis concerns the isolation and characterisation of cyanophages A-HIS1 and AHIS2, which infect A. marina as a basis to investigate and understand the impact of phage on host physiology in this new model system. A-HIS1 and A-HIS2 were characterised by their morphology, growth behaviour and genomes. Experiments were designed and implemented to investigate interactions between the phages and host. Interestingly, an analysis of novel genes in these phages revealed a surprising evolutionary history of phages A-HIS1 and A-HIS2 providing new insights into the origin of DNA polymerase, which is found only in the mitochondria of eukaryotes.

Campylobacter jejuni is a leading cause of human bacterial enteritis worldwide. Consumption of contaminated poultry meat is considered a major source of infection. The use of virulent bacteriophages as a form of biocontrol to specifically reduce this pathogen in poultry (phage therapy) is a promising intervention that does not rely on antimicrobials and therefore circumvents the emergence of antibiotic-resistant Campylobacter strains. In order to achieve this, a better understanding of the mechanisms involved in phage-host interactions at the molecular level would assist in the development of the strategy and the selection of bacteriophages. The main objective of this study was to therefore examine such interactions between Campylobacter and its virulent phages. To achieve this, the transcriptional response of C. jejuni to phage infection was investigated, along with the role of a Type II restriction-modification system during phage infection of Campylobacter. These studies were conducted using the highly phage-sensitive Campylobacter strain, C. jejuni PT14, in conjunction with a number of group II and III bacteriophages (Eucampyvirinae). Transcriptome studies (RNA-Seq) revealed a phage-induced host response that included a demand for iron and oxygen. This was highlighted by the up-regulation of several siderophore-based iron acquisition genes and down-regulation of genes associated with a number of anaerobic electron transport pathways that utilise alternative electron acceptors to oxygen. In addition, the pattern of gene regulation also suggested apo-Fur regulation of the iron-responsive and flagellar biogenesis genes. This host response has been proposed to occur as a consequence of the reduction of ribonucleotides to form deoxyribonucleotides during phage DNA replication. This process is catalysed by the enzyme ribonucleotide reductase and requires iron and oxygen during the formation of a reactive di-iron centre within the β-subunit of the enzyme. Unusually knock-out mutants of a Type II restriction-modification system had a negative impact on phage replication. The A911_00150 mutant displayed pleiotropic changes in motility, cell based invasion and the ability to colonise chickens. Transcriptome analysis highlighted down-regulation of the genes required for the synthesis of the bacterial flagellum.

Clostridium difficile is an important hospital-acquired pathogen causing C. difficile-associated diarrhoea (CDAD) in patients exposed to antibiotics. The lack of information on bacteriophages of C. difficile, and the potential of phages as therapeutic agents for the treatment of CDAD, prompted the isolation and characterisation of phages active against clinical isolates of C. difficile in order to determine the prevalence and significance of phages of this anaerobe. Three (5.4 %) of 56 clinical C. difficile isolates induced by mitomycin C yielded dsDNA phages C2, C5, C6 and C8. The four phages differed from previously described C. difficile phages in particle morphology, burst size and host range. C2, C5 and C8 particles were members of the family Myoviridae, while C6 belonged to Siphoviridae. The burst sizes were 5 for C2, 7 for C5, 19 for C6 and 33 for C8. C8 had the broadest host range, lysing 27 out of 56 (48 %) C. difficile isolates, followed by C6 (43 %), C5 (20 %) and C2 (20 %). Superinfection experiments, restriction enzyme analysis and Southern hybridisation showed C2 and C5 to be closely related with C8 somewhat related to them, however, C6 was distantly related to the other three phages. C2 was further characterised as a representative phage. Its genome did not possess cohesive ends, and was shown to integrate chromosomally via an attP site identified within a 1.9 kb HindIII fragment. However, an integrase gene, which is typically close to the attP region, was not located. Nine of 16 HindIII fragments of C2, including the 1.9 kb fragment, were cloned into pUC18. Approximately 9 kb of the estimated 43 kb genome of C2 was sequenced and analysed. Seven of the nine translated sequences were homologous to phage structural proteins, two sequences were not homologous to any relevant protein in the Genbank and EMBL databases, and one was homologous to proteins of Clostridium species. Nucleotide homology between the C2 sequences and the recently sequenced C. difficile strain CD630 was found in three regions within CD630 genome. Seven of the nine sequences, including the 1.9 kb fragment, were clustered in one region. These data suggest that the genes constitute a phage structural gene module. The presence of C2-like sequences in CD630, and Southern hybridisation of C. difficile strains using phage probes, suggested related prophage sequences may be commonly present in this bacterial species. An investigation was carried out to determine the presence of toxin genes tcdA and tcdB, and PaLoc-associated gene tcdE, in phage DNA. In addition, the effect of phage infection on toxin production of toxigenic C. difficile strains was studied. Of the three genes, tcdE only was detected in phages C2, C5 and C8, but not in C6. Strains that maintained phages in a stable manner (lysogens) were isolated and used in toxin studies. The amount of toxin B produced was measured by cytotoxic assays using Vero cells, and toxin A production was measured by ELISA. Although phages did not encode toxin A or B genes, there was a significant increase in toxin B production in some lysogens. There was no increase in toxin A production. Transcriptional analyses of tcdA and tcdB in lysogens and parental strains was performed by real-time RT-PCR and Northern hybridisation to determine whether phage was affecting regulation of toxin transcription. Phage did not appear to affect toxin gene transcription, although results from real-time RT-PCR and Northern hybridisation were conflicting. A phage induced from the highly toxigenic reference strain VPI 10463 was also briefly characterised and investigated for its effect on toxin production in VPI 10463. The phage, ΦCV, had similar particle morphology to C2, C5 and C8, and had some HindIII bands in common with C2 and C5. Two cured variant strains produced significantly less toxin B compared to VPI 10463. In conclusion, several important properties of C. difficile phages were characterised. It appears these temperate phages may play a role in toxin production making them unsuitable as therapeutic agents for the treatment of CDAD. However, C2 phage may have potential as the basis for an integrative vector that will add to the genetic tools available for clostridia.

A lack of innovation in production strategies of (bacterio)phages—viruses parasitic to bacteria—has limited recent developments in the applications. New phage-based industries need to develop efficient, large-scale bacteriophage production technologies. In this thesis, parameters affecting the phage infective process such as adsorption, host intracellular genetic material and host cell life cycle were studied; their influence on the production of virulent phages and recombinant proteins using a lysogenized phage as the expression vector is discussed. Self-Cycling Fermentation (SCF), a cyclical, semi-continuous, automated process that maintains a bacterial culture in synchronized growth was incorporated into an industrially-relevant two-stage, recombinant protein production scheme using bacteriophages as the expression vector. By using SCF, productivity of the expressed gene product increased compared to a batch culture under similar conditions. Also, protein synthesis was influenced by the time at which the lytic phase was induced in the cell life cycle, suggesting protein production can be optimized with respect to the cell life cycle using SCF.In addition, a new model for phage adsorption is proposed and the impact of phage adsorption efficiency on large-scale production was demonstrated. The proposed model simplifies phage attachment to a single step: irreversible binding. The adsorption efficiency is used to account for unadsorbed phages. Results from phage T4 infections indicated that increasing adsorption efficiency had a similar effect on phage amplification to increasing the initial multiplicity of infection. Self-Cycling Fermentation was also used as a research tool to study how intracellular levels of host DNA and RNA influence phage productivity during T4 infection. Experiments monitoring cell burst size and lysis time for cells infected at various points during a SCF cycle revealed that the intracellular RNA level correlates with phage productivity during infection. This work suggests that SCF can be used to complement and enhance the metabolic requirements of organisms in order to maximize productivity.<br>Les récents développements dans l'application des bactériophages – des virus qui sont parasitiques aux bactéries – n'ont jusqu'ici pas été accompagnés par des innovations dans les stratégies pour leur production. Il y a certainement un besoin de développer des technologies de production efficaces et à grande échelle afin d'en faire bénéficier les industries présentes et futures utilisant les phages. Dans la présente recherche, des paramètres affectant le processus infectieux du phage, tel l'adsorption, les concentrations de matériel génétique et intracellulaire de l'hôte, et le cycle de vie de ces cellules, ont été examinés. Leur influence sur la production de phages virulents et de protéines recombinantes ayant un phage lysogénique comme vecteur d'expression a aussi été étudiée. La fermentation auto-cyclante (SCF) est un processus cyclique, en semi-continu et automatisé qui maintient une population bactérienne en croissance synchronisée. Ce système a été incorporé à un procédé industriellement pertinent pour la production, en deux étapes, de protéines recombinantes en utilisant des bactériophages comme le vecteur d'expression. Avec la SCF, la productivité du gène exprimé a augmenté comparée à une culture batch créée dans des conditions similaires. De plus, la synthèse de protéines a été influencée par le temps auquel la phase de lyse a été incitée dans le cycle de vie de la cellule, suggérant que la production de protéines pourrait être optimisée en combinant le cycle de vie de la cellule avec la SCF.En outre, un nouveau modèle pour l'adsorption de phages a été proposé et l'impact de l'efficacité d'adsorption du phage sur la production à grande échelle à été démontré. Le modèle proposé simplifie l'adsorption de phage à une étape unique : l'attachement irréversible. L'efficacité d'adsorption des phages est liée à la proportion de phages qui n'adhérait pas à l'hôte. Les résultats obtenus avec des phages T4 suggèrent que l'efficacité d'adsorption a un effet similaire à augmenter la multiplicité d'infection initiale; le nombre des phages produits pendant l'amplification diminue avec l'augmentation de l'efficacité d'adsorption.La SCF fut aussi utilisée comme outil de recherche afin d'étudier comment les niveaux intracellulaires d'ADN et d'ARN influencent la productivité des phages pendant une infection par le phage T4. Les mesures du burst size et du temps de lyse de cellules infectées à différents temps dans un cycle de SCF ont démontré que le niveau d'ARN intracellulaire était corrélé à la productivité des phages pendant une infection. Cette étude suggère que la SCF pourrait être utilisée pour identifier et contrôler les besoins métaboliques des organismes afin d'augmenter la productivité.

Clostridium difficile infection (CDI) currently affects around 20,000 people each year, in healthcare institutions and in the community, and will often follow disruption of the gut microbiome. Current treatment strategies call for the use of further antibiotics, of which there is a limited choice. There is a need for additional remedial and prophylactic solutions with greater specificity and low levels of toxicity and resistance. This thesis describes the pathogenesis of CDI, the current treatment strategies and navigates the growing body of studies investigating the potential use of phage. The project involved extensive screening including faecal samples and environmental sources in an attempt to identify novel phages of C. difficile and documents efforts to improve the therapeutic capacity of a selected phage, ФCD27, by mutagenesis. No exclusively lytic phages were isolated or obtained following mutagenesis with ethylmethane sulphonate, hydroxylamine or sodium pyrophosphate. Batch fermentation models of CDI showed that a prophylactic approach to phage therapy of CDI offers a higher efficacy than a remedial regime. A continuous model of CDI in a colon model was successfully produced and demonstrated variable efficacy rates from no apparent decrease in the burden of C. difficile to a reduction to below the limit of detection by culture, with no detrimental effect on commensal microbiota. The lysogenic capacity of ФCD27 appeared to prevent clearance of C. difficile in the models, but some strains containing the prophage exhibited reduced toxin production phenotypically. A possible mechanism of this altered phenotype included the action of ФCD27 repressor proteins on the promoter regions of C. diffiicle toxin genes or regulatory elements, but affinity of a candidate repressor, ORF44, to PaLoc constituents was not demonstrated. Studies have also demonstrated the ability of ФCD27 to prevent outgrowth of germinating C. difficile spores, thus potential as an environmental decontaminant. iii The findings of the project and the future prospects of phage therapy as an agent against CDI are discussed.

Lactobacillus helveticus est un lactobacille thermophile caracteristique des levains utilises pour fabriquer les fromages a pate cuite. Ce lactobacille est la cible de bacteriophages specifiques qui detruisent les cellules, ce qui conduit a des produits de qualite mediocre. Jusqu'a present, les phages de l. Helveticus demeuraient largement meconnus. Dans cette etude, 35 phages ont ete caracterises. Vingt-trois d'entre eux avaient ete isoles d'echantillons de lactoserum de fromagerie alors que les 12 autres, temperes, ont ete isoles a partir de surnageants de cultures traitees avec la mc. Tous ces phages possedent une tete isometrique et une queue contractile. Ils appartiennent au groupe a de bradley ou a la famille des myoviridae de l'ictv et se repartissent en 2 sous-groupes: 1) un sous-groupe de 32 phages morphologiquement identiques, dits a queue courte (21 isoles comme lytiques et 11 temperes), qui ont une queue de 150 nm et 2) un sous-groupe de 3 phages, dits a queue longue (2 isoles comme lytiques et 1 tempere), qui ont une queue de 260 nm. Les 2 types de phages ont des spectres d'hotes, des profils proteiques, des proprietes serologiques et des adns differents. Dans le sous-groupe des phages a queue courte, il existe une parente etroite entre des phages temperes, heberges par des souches lysogenes, et certains des phages isoles de lactoserums de fromagerie. On a montre que ces derniers etaient aussi des phages temperes. Par contre, d'autres phages a queue courte sont virulents. La lysogenie est repandue et la plupart des souches isolees de levains artisanaux francais possedent, integre dans leur genome, un prophage, actif ou defectif. Dans le cas des phages a queue courte, les souches lysogenes constituent une source majeure de phages dans les fromageries. L'impact pratique des phages a queue longue parait etre moindre

Thesis (Ph. D.)--University of Alabama at Birmingham, 2009.<br>Title from PDF title page (viewed Jan. 26, 2010). Additional advisors: Asim K. Bej, Gail E. Christie, Peter E. Prevelige, Jr., R. Douglas Watson. Includes bibliographical references.

Files on accompanying CD-ROM: Appendix III Phages ST64T and ST64B sequences, are in rtf format. Bibliography: leaves 279-324. System requirements for accompanying CD-ROM: IBM or compatible ; Microsoft Word or compatible to read rtf files.

Thesis (Master)--İzmir Institute of Technology, İzmir, 2005.<br>Keywords: Phage display technology, bactriophage, homologous recombination, M13, fusion protein. Includes bibliographical references (leaves. 56).

Clostridium difficile is the most common bacterial cause of infectious diarrhoea in healthcare environments and in 2014 was responsible for 13,785 infections in the UK. C. difficile infection (CDI) is spread via the faecal-oral route and by contact with contaminated surfaces. However, despite the healthcare concerns no tests are available to validate if sufficient cleaning has been conducted. In addition, Polymerase Chain Reaction (PCR) and Enzyme Immunoassays (EIAs)-based tests used to diagnose CDI lack sensitivity and specificity and hence false negative results are commonly obtained. To overcome these concerns the aim of the PhD research has been to develop the first diagnostic test that exploits the specific interactions of C. difficile bacteriophages (phages), viruses that specifically infect and kill C. difficile. In order to develop a C. difficile phage-based test, first suitable phages that can be used for the test were identified and this was conducted by screening 35 different C. difficile phages against 160 clinically relevant C. difficile isolates. Five phages were found to infect the most number of isolates and were investigated further to identify whether a phage-based diagnostic could be developed based on phages binding (adsorption) to different C. difficile subgroups. However, for all five phages, adsorption rates were not consistently high for C. difficile subgroups in comparison to other common bacteria found in similar locations to C. difficile. Therefore, to increase specificity of the phage-based diagnostic test a new approach was taken by tagging two phages with luminescence luxAB genes (reporter phages), which would be expressed once C. difficile cells were infected with the phages. To design the C. difficile reporter phages, non-essential phage genes were replaced with the luxAB genes, but this study revealed mutagenesis of C. difficile was troublesome and extensive optimisation was required. In addition, once the reporter phages had successfully been constructed the luxAB genes were unstable within the phage genome and were lost during phage replication. Despite extensive optimisation and due to time constrains the luxAB genes were not stabilised within the phages but future work will focus on stabilising the genes.

Thesis (Ph. D.)--University of California, San Diego, 2008.<br>Title from first page of PDF file (viewed March 24, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Etude des bacteriophages lytiques des bacteries lactiques. Les analyses morphologiques, de composition proteique et l'homologie adn-adn ont montre que les phages lytiques se repartissent en deux groupes, prolates et isometriques et qu'ils appartiennent a la famille des siphoviridae. L'analyse en microscopie electronique des genomes phagiques a permis d'emettre l'hypothese d'extremites cohesives. L'existence et la nature de celles-ci ont ete mise en evidence par le sequencage d'une partie du genome

Brevibacillus laterosporus (B. laterosporus) is a pathogen difficult to distinguish from Paenibacillus larvae (P. larvae), and contributes to Colony Collapse Disorder (CCD) of honeybees. To develop a biocontrol agent to limit its presence, bacteriophages were isolated from Utah County soil samples and used to infect B. laterosporus isolated from Utah County honey and larvae samples. Since CCD is prevalent in Utah beehives, bacteriophage that infect and lyse B. laterosporus may be isolated and characterized. Pathogens were isolated from soil samples, and 16S rRNA gene tests initially identified the strains as P. larvae. Bacteriophages were isolated, purified, and amplified sufficiently to obtain images by electron microscope and genome sequencing by 454 pyrosequencing. Genomes were annotated with DNA Master, a Multiple Document Interface (MDI) program. Open reading frames (ORF's) were compared to the National Center for Biotechnology Information's (NCBI) database of primary biological sequence information via the Basic Local Alignment Search Tool (BLAST) algorithm. Later testing determined the pathogen to actually be B. laterosporus. Plaques demonstrated lytic activity, and electron microscopy revealed bacteriophages of the myoviridae family. The five sequenced genomes were composed of linear dsDNA ranging from 45,552 to 58,572 base pairs in length, 92 to 100 genes per genome, and a 38.10% to 41.44% range of G + C content. Discovering and describing new bacteriophages is a reasonably reproducible process and contributes to appreciating the diverse relationships between bacteriophage, bacteria, and eukaryota. Scientific facilitation of the bacteriophages role in limiting detrimental bacteria may contribute as an adjunctive therapy for CCD.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (pages 89-103).<br>One of the major public health concerns of the modern day is the emergence and spread of extensively antibiotic-resistant pathogens. We have already seen the arrival of infections caused by bacteria resistant to all available antibiotics in the therapeutic arsenal. In addition, we have learned much of the incredible importance of the microbial communities that cohabit our bodies, and of how perturbations to these communities can lead to long-lasting health effects. Bacteriophages may provide a solution for both of these problems, in that they are narrow-spectrum and can be used to specifically kill target microbes without disrupting whole community structure through off-target effects. Here, various approaches to creating phage-based therapeutics are explored, including the isolation and application of naturally occurring wild-type phages, the conversion of temperate phages to obligately lytic phages to permit their use as a resource in phage therapeutics, and the creation of programmable, sequence-specific antimicrobials through phage-mediated genetic payload delivery. These efforts are expected to contribute to the field by expanding the approaches available to develop next-generation, phage-based antimicrobials.<br>by Robert James Citorik.<br>Ph. D.<br>Ph.D. Massachusetts Institute of Technology, Department of Biology

Seventy eight serologically distinct strains of Klebsiella bacteria are known to exist. The capsular polysaccharide surrounding the bacterial cell of these pathogenic Enterobacteria is of immunological significance. Structures of the capsular polysaccharides of nearly sixty seven strains of Klebsiella have been established, and each one found to be unique. The structures of the K antigens from Klebsiella, serotypes K67 and K80 are presented as a contribution to the continuing program of elucidation of the chemical structures of these antigens in an attempt to explain their immunological responses. Chemical methods of structural elucidation were employed and the following two structures were obtained. [formula omitted] The polysaccharide from K67 was unique among the Klebsiella K antigens in having a four-plus-two-plus-one repeating-unit (indicating a branch on a side chain), while K80 was unique as it was the first instance that a pyruvic acetal was found on a terminal rhamnose unit. The importance of bacteriophage-borne enzymes in the generation of single repeating-units containing labile substituents is demonstrated. Klebsiella K44 polysaccharide was degraded using a crude solution of Φ44 bacteriophage. The oligosaccharides obtained were crucial in the determination of the position of the 0-acetate group. In the case of the polysaccharide from Klebsiella K26, the degradation performed using a crude solution of Φ26 bacteriophage resulted in the isolation of a single repeating-unit containing a pyruvic acetal together with an oligosaccharide corresponding to a single repeating-unit devoid of its terminal pyruvate containing sugar. The structures of these compounds which are as follows, were useful in further confirmation of the structures of the original polysaccharides. [formula omitted] Escherichia coli. another pathogenic Enterobacteria possessing immunologically significant K antigens, has been found to contain capsular polysaccharides bearing a strong resemblance to those of Klebsiella. Recently it was discovered that some strains of E. coli contained K antigens comprising amino sugars. A preliminary study on the chemical behaviour of amino sugars, and chemical methods of structure elucidation of such polysaccharides have been included in an appendix as this has been a new area of research in this laboratory. An appendix containing compilations of the cross-reactions, known structures, and chemotypes of the Klebsiella K antigens has also been included.<br>Science, Faculty of<br>Chemistry, Department of<br>Graduate

Vibrio sp. 2 stationary phase cells are novel and interesting in that they are able to support phage growth in standing cultures, but not in shaken (aerated) cultures. Many physiological and morphological characteristics change when Vibrio sp. 2 stationary phase cells are removed from aeration: the relatively high levels of protein synthesis (Robb et al., 1977; 1978) decrease, with a concomitant increase in the levels of RNA synthesis; protein degradation rises from 1 %h⁻¹ to 2,9 %h⁻¹, and whilst the average cell length decreases, the range of cell lengths markedly increases. The magic spot nucleotides, ppGpp and pppGpp, which are present in stressed exponential phase Vibrio sp. 2 cells, are not detectable in stationary phase Vibrio cells. The specific proteolytic activity of shaking stationary phase cell-free extracts against the foreign protein [¹⁴C-me]globin was slightly higher than that of extracts from standing or exponential phase cells, while the specific proteolytic activity against [¹²⁵I]-insulin was slightly lower. On the basis of inhibitor studies and subcellular distribution, the proteolytic activities of the three types of extract differed. The addition of exogenous ATP to cell-free extracts either stimulated (Car & Woods, 1984) or depressed proteolytic activity depending on the procedure used to prepare the extracts. The proteolytic activity of fractions containing substantial amounts of membrane material, from all three types of extract, were markedly depressed by ATP. On preincubation of cell-free extracts from exponentially growing cells prior to assay of proteolytic activity, the activity was markedly stimulated (two- to four-fold). The stimulation,. however, varied, greatly between independently produced extracts. ATP had a much smaller stimulatory effect on preparations free of cell wall material from both types of stationary phase cells (the stimulation was less than two-fold), and the stimulation was not affected by preincubation of the extracts. Extracts prepared from starving cells, previously in exponential growth, were affected by the addition of ATP in a similar manner to that observed with stationary phase extracts (Car & Woods, 1984). Exponential and both types of stationary phase Vibrio sp. 2 cells have ATP-stimulated and ATP-depressed activities separable by ion-exchange chromatography, in addition to several other proteolytic activities. All types of Vibrio sp. 2 cells have a similar complement of proteolytic activities.