Poster preparation and presentation information

Thank you for considering presenting your work as a poster at this conference. Below are the key details and submission guidelines.

Deadlines

• Poster abstracts (as MS Word) must be submitted by 15th August.
• Digital posters (as PDF) and flash-talk videos must be submitted by 20th August.

Poster preparation & format

Poster size

Digital-only presenters

• Save your poster in A1 or A0, landscape or portrait format.
• Page size is flexible if presenting only digitally.

In-person presenters

• Print your poster in A1 portrait format only
• Larger or landscape posters may not be displayed due to space constraints

File naming

For submission, name your digital poster files as follows:

• <your surname>-Phg26-Poster.
• Example: for David Jones, name your file as Jones-Phg26-Poster.
• Do not use generic names such as: Oxford-poster, phages2026, Oxford-phage-poster.

Digital poster submission

All poster presenters, whether attending virtually or in person, are required to submit a digital version (as PDF) of their poster via the designated upload link. Posters will be made accessible to all conference participants via the secure ‘Download PHG26 Documents’ page, ensuring visibility across both virtual and in-person audiences.

• Submit your final PDF poster (<5MB) via the designated upload link
• Do not send posters or abstracts by email
• Ensure your submission is final—once published, it cannot be replaced
• Late submissions may not be included in the programme

Poster presentation

Presentation timing

Presenting digitally only

• Currently, there is no scheduled time for presenting digital posters. Instead, virtual attendees will be able to interact with poster presenters via the Zoom chatbox during the conference.
• A specific poster presentation session may be announced later.

Presenting in-person

• Bring a printed A1 portrait poster for display
• Larger or landscape posters may not be displayed due to space constraints
• You are responsible for printing and transporting your poster
• You may be assigned a specific day and time for display

Presentation via a flash-talk video

To maximise visibility, exposure and engagement, we encourage all poster presenters, whether presenting digitally or in-person, to submit a short video (max 5 minutes) introducing their work. Videos will be featured on the LPMHealthcare YouTube channel.

Instructions

• Download the official opening slide (PhgOx26 opening slide) and use it as the first slide of your presentation (see example: https://youtu.be/XatqenCd_IU?si=Yu1PooCD4JmSLAiz).
• Record your presentation using Zoom or any preferred platform. Keep it under 5 minutes.
• Save your video in a YouTube-compatible format (e.g., MP4)
• Send your video via a file transfer service such as MailBigFile or WeTransfer to: PhageOxford@gmail.com

Any further information about the poster presentations will be available in the future on this page.

Before uploading your poster, you must make sure that you follow ALL of the instructions above!

Upload your poster

Accepted posters

(Unedited: V = Virtual-only posters; presenters are shown in Bold)

Accepted poster abstracts will be displayed below. If your abstract has been accepted for presentation but it does not appear in the list below, please let us know as soon as possible by emailing PhageOxford@gmail.com.

Knowledge and Attitudes Toward Phage Therapy Among Medical Students in Jordan (V)

Sireen B Alsabatien¹, Mahde Q Sabbagh¹, Hala A Abukhamseh¹, Jana M Khraisat¹, Jouri O Abulghanam¹, Marah J Zurgan¹, Ahmed Salman²

¹Faculty of Medicine, The University of Jordan, Amman, Jordan

²Department of Anatomy and Histology, Faculty of Medicine, The University of Jordan, Amman, Jordan

Antimicrobial resistance (AMR) is a rising public health concern, urging a search for alternatives such as phage therapy, but no data exists on knowledge and attitudes toward phage therapy among trainees in the Middle East. We conducted a cross-sectional survey of second- through sixth-year medical students at the University of Jordan using stratified sampling, distributed via lectures, hospital rotations, and WhatsApp. The four-part questionnaire assessed demographics, AMR, phage therapy knowledge, and attitudes/perceptions; it was piloted and reviewed for content validity. A composite knowledge score was categorized as low, moderate, or high. Spearman’s correlation assessed the relationship between AMR concern and perceived need for antibiotic alternatives; chi-square tests evaluated associations between knowledge level and demographic variables (gender, academic year, GPA). Among 520 respondents (mean age 21.8±1.66 years; 61.7% female; 67.5% clinical-year), AMR awareness was 96.7%, and 85.4% were moderately-to-extremely concerned about it. Concern correlated positively with perceived importance of alternatives (ρ=0.423, p<0.001). Despite this, composite knowledge remained low-to-moderate (median 3, IQR 1-4): 46.5% low, 50.6% moderate, 2.9% high. Male gender (p=0.017), clinical stage (p=0.040), and higher GPA (p=0.001) were associated with greater knowledge. Attitudes were largely favourable: 51.4% supported combining phage therapy with antibiotics, 94.6% wanted to learn more, and 82.1% supported licensing pending clinical evidence. Leading barriers were limited research (77.7%), cost (66.7%), and low awareness (62.0%). These findings are the first from the Middle East showing, that despite strong AMR awareness and favourable attitudes toward phage therapy, Jordanian medical students’ knowledge remains limited, highlighting a curricular gap and the need for targeted education.

Engineering and Functional Characterisation of Novel Bacteriophage Endolysins Targeting Staphylococcus aureus

Sami Asiri, John E Pearl, Edouard Galyov

Becky Mayer Centre for Phage Research, Division of Microbiology and Infection, University of Leicester, Leicester, United Kingdom

Antimicrobial resistance in Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), has increased the need for alternative antimicrobial strategies. Bacteriophage-derived endolysins are peptidoglycan hydrolases that rapidly lyse Gram-positive bacteria and represent promising protein therapeutics. In this study, ten S. aureus bacteriophages were isolated, and their genomes were sequenced, assembled and taxonomically classified. Four endolysins from these phages were selected for further characterization. Three variants of each endolysin, comprising a full-length protein and two C-terminal truncations, were engineered and cloned as His₆-SUMO fusion proteins. Recombinant protein expression conditions were optimized. All twelve constructs were successfully expressed in the soluble fraction and screened for antibacterial activity against three MRSA and three methicillin-susceptible S. aureus (MSSA) isolates. Of the twelve constructs, only the full-length E1 endolysin demonstrated reproducible lytic activity against all six strains tested. In addition, the soluble E1 lysate exhibited a broader antibacterial spectrum than its parental bacteriophage. The remaining constructs showed no detectable activity as soluble His₆-SUMO fusion proteins. Ongoing work will purify the recombinant proteins, remove the His₆-SUMO fusion tag and reassess antibacterial activity to determine whether the fusion partner influences endolysin function.

Leading the future of Phage Therapy: Targeting Microbiome in Gastrointestinal Cancer

Birhanu Ayelign^1,2,3, Shoukat Afshar-Sterle^1,2, Ryan O’Keefe^1,2, Mwila Kabwe⁴, Annalisa Carli^{1, 2,} Bushra Amin⁵, Joseph Tucci⁴, Michael Buchert^1,2

¹Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia

²School of Cancer Medicine, La Trobe University, Victoria, Australia

³Department of Immunology and Molecular Biology, School of Biomedical and Laboratory Science, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia

⁴Department of Pharmacy and Biomedical Sciences, La Trobe Institute for Molecular Science, La Trobe University, Victoria, Australia

⁵Mass Spectrometry research and infrastructure, la Trobe university, Australia

The study of the cancer microbiota is an emerging research area that has gained attention through discoveries of the tumour-associated bacterial species that can be tumourigenic. Fusobacterium nucleatum (F. nucleatum) is a recognised cancer-associated oral microbiome linked to tumour growth, immune suppression, and resistance to therapy. Clinical use of antibiotics targeting F. nucleatum has failed, leading to worse patient outcomes. Thus, there remains a significant gap in understanding tumour-promoting mechanisms and efficiently targeting the bacterium. We aimed to address a critical unmet need by understanding how tumour-associated bacteria contribute to cancer progression and discovering a world-first novel FNU1 bacteriophage capable of eliminating these bacteria. Cancer cells were infected with F. nucleatum and subsequently treated with FNU1 bacteriophage. Functional assays demonstrated that F. nucleatum infection significantly increased cellular migration, proliferation and viability in infected cells. While FNU1 bacteriophage treatment blocks F. nucleatum-induced cancer cell proliferation and migration. Flow cytometry was used to quantify the expression of major histocompatibility complex class I (MHC-I). Infected cells showed significant downregulation of MHC-I expression, as confirmed by Western blot analysis, which revealed reduced levels of MHC-I and associated chaperone proteins involved in MHC-I maturation and translocation. Treatment with FNU1 reversed these changes, restoring MHC-I expression and chaperone protein levels. Label-free quantitative proteomics employing high-resolution LC–MS/MS identified distinct infection-associated proteomic signatures enriched in pro-tumorigenic pathways. Differential expression analysis demonstrated that these alterations were significantly reduced following bacteriophage treatment, indicating extensive reprogramming of infection-driven cellular pathways.

How Mycobacterial Physiological State Influences Mycobacteriophage Infectivity

Stephen Emencheta¹, Pranabashis Haldar², Galina Mukamolova¹, Natalie Garton¹

¹Division of Microbiology and Infection, University of Leicester, Leicester, United Kingdom

²Division of Respiratory Sciences, School of Medical Sciences, University of Leicester, Leicester, Leicestershire, UK

With the rising incidence of mycobacterial diseases and the challenges of their treatment, mycobacteriophages are becoming an attractive alternative; however, many barriers remain, and few have been tested against Mycobacterium tuberculosis. There is also a lack of understanding of whether mycobacteriophages can infect mycobacteria in different phenotypic states relevant to infection. This study aims to evaluate the infectivity of mycobacteriophages (D29 and TM4) against Mycobacterium species, including Mycobacterium bovis (BCG) strains (Glaxo and Pasteur) and M. tuberculosis mc²6030, under different physiological conditions, including in the presence and absence of Tween 80, during the exponential and stationary growth phases of mycobacteria, and under varying oxygen availability. Tween negatively impacted the infectivity of D29 and TM4. Across the conditions (growth phases and oxygen tensions) tested, both infected best during the exponential phases of BCG and M. tuberculosis mc²6030; however, TM4 infected more robustly and more effectively against non-growing and stressed mycobacteria than D29. These findings lay the foundation for developing and prioritising choice mycobacteriophage(s) and show that physiologically relevant models are essential for evaluating therapeutic mycobacteriophages.

Phenotypic and Genotypic Characterisation of Novel Coliphages with Therapeutic Potential Targeting Multi-Drug Resistant E. coli Clinical Isolates

Shauna K Hurley, Peter Myintzaw, Aidan Coffey, Michael Callanan and Laura M O’Connell

Munster Technological University, Bishopstown, Cork, T12 P928

Given the concerning rise of antibiotic resistance, alternative therapeutic strategies are required for treating enteric pathogens such as Escherichia coli. One such strategy is the application of lytic (bacterio)phages or their components. As routine application of phage-based therapies is typically limited by narrow host ranges and variable stability, isolation of novel lytic phages remains an important feature of this research field. This study examined the therapeutic potential of 4 novel coliphages that were recently isolated from water sources in Cork, Ireland. Phenotypic analysis revealed that all phages remained functionally stable between pH 3 and 11 and up to 70°C and in silico analysis revealed that the phages are closely related to T4-like coliphages. Pharokka-based genomic analysis confirmed their strictly lytic lifestyle (between 110-168 kbp, lacks integrases) and the absence of toxin-encoding genes, which are considered desirable characteristics of therapeutic phages. The novel coliphages were subsequently tested in vitro to determine their host range against a panel of 20 clinical E. coli isolates obtained from the Bons Secours Hospital, Cork. The E. coli isolates were examined for their susceptibility to antibiotics. Isolates were found to be most resistant to beta-lactams and showed resistance to 14 of 16 antibiotics tested. However, the novel coliphages V2, V3, V7 and KB4 lysed 55%, 60%, 50%, and 40% of 20 E. coli isolates, respectively, with only two isolates resistant to all phages. Overall, the results demonstrate the therapeutic potential of these phages and support further experimental validation. Future work will focus on cloning and characterising the phage endolysins as potential antibacterial agents independent of the whole phage particles, as well as their targeted delivery to the gut.

Characterisation of Bacillus anthracis Bacteriophages

Catriona Matthews^1,2, Ertelt Moritz³, Georgie Metters¹, Chris Jenkins¹, Peter Braun³, Izzy Norville¹, Joanne L. Fothergill⁴, Viviana Manzulli⁵, Domenico Galante⁵, Les Baillie², Philip Ireland¹

¹Defence Science and Technology Laboratory (Dstl), Porton Down, Salisbury, UK

²Cardiff School of Pharmacy and Pharmaceutical Sciences, King Edward VII Avenue, Cardiff University, Cardiff CF10 3NB, UK

³Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Immunology, Infection and Pandemic Research, 80799 Munich, Germany

⁴Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK

⁵Istituto Zooprofilattico Sperimentale of Puglia and Basilicata, Anthrax Reference Institute of Italy,71121 Foggia, Italy

Bacillus anthracis, a Gram-positive, spore-forming bacterium, is the causative agent of anthrax, a zoonotic disease found worldwide with significant implications for both animal and human health. The disease primarily affects livestock and wild animals but can also infect humans, with the infection route influencing disease progression and severity. While tools exist for B. anthracis detection, treatment and decontamination, bacteriophages present a promising alternative and complement to these methods. Their high specificity, limited disruption to existing microbial flora, ability to replicate during treatment and environmental sustainability make them an attractive option for enhancing existing strategies. This project aims to characterise and develop bacteriophages as novel medical countermeasures against B. anthracis. A collection of 15 B. anthracis bacteriophages was assembled from soil samples sourced globally. This includes three Basilisk-like phages and one Gamma-like phage from Wales, three previously characterised phages from Poland, and eight novel phages isolated from regions of Bangladesh associated with anthrax outbreaks in humans and animals during 2010-2011. Whole genome sequencing revealed significant diversity within the collection, including representatives of the families Myoviridae, Tectiviradae and Siphoviridae. Notably, two phages, 3Ban and 6Ban, exhibit a high degree of novelty, with no close relatives identified. Ongoing work includes host range testing and investigation of phage resistance. So far, this has revealed broad susceptibility to phages LC1H911 and 3B6, and clusters of closely related phage resistant strains have been identified. Phage resistant mutants arising following phage exposure were isolated and sequenced providing an early indication of the surface properties potentially required for host cell interaction. © Crown Copyright 2026, Dstl. This material is licensed under the terms of the Open Government Licence.

Gene engineering of a capsid protein as a first step in creating bionanoparticles (V)

Jakub Mazur, Agnieszka Żylicz-Stachula, Joanna Żebrowska, Piotr Skowron

University of Gdańsk, Faculty of Chemistry, Department of Molecular Biotechnology, Wita Stwosza 63, 80-308, Gdańsk, Poland

Bacteriophages are ubiquitous in nature and represent promising sources ofnew protein for the development of novel bionanotechnological platforms. Increasing attention has been focused on phage capsid proteins due to their ability to self-organise into multimeric structures, such as portal proteins. Portal proteins form a characteristic ring-like structure, which phage DNA can pass through during infection. Importantly, such structures do not contain genetic material, they could be used e.g. as a scaffolds for protein delivery system. Their functionality can be expanded using genetic engineering. In this study, we employed SpyTag/SpyCatcher system, a pair of protein tags that, can connect to each other by creating a spontaneous covalent bond between the tags. Fusing SpyTag on one protein and SpyCatcher on second allows for connecting such recombinant proteins together. The aim of this study was to design and develop new generation bionanoparticles platform based on: (i) Portal protein derived from thermophilic bacteriophage TP-84, fused to a SpyTag motif (ii) Superfolder GFP, used as a reporter protein, fused to SpyCatcher. This approach allows for portal protein multimeric structure to couple with recombinant sfGFP. Both recombinant proteins were overproduced in E. coli and then purified. Protein-protein interaction tests were evaluated using immunodetection techniques. This study represents a first step in creating new generation bionanoparticles based on thermophilic bacteriophage capsid proteins. Future studies will focus on replacing sfGFP with biologically active peptides/proteins fused with SpyCatcher motif, expanding the potencial applications in biotechnology and biomedicine.

Breaking or Building Biofilms? The complex role of a lytic phage in Pseudomonas aeruginosa

Johanna Pukall¹, Hannah Sadrich¹, Rabea Schlüter², Susanne Sievers¹

¹Department of Microbial Physiology and Molecular Biology, University of Greifswald, Germany

²Imaging Center of the Department of Biology, University of Greifswald, Germany

Bacterial biofilms are highly complex and exhibit increased virulence due to their profound resistance to antibiotics. Bacteriophages can combat these structures by producing biofilm-degrading enzymes or by infecting persistent cells. In this study, we isolated a novel bacteriophage, vB_PaeP_HGW_003, from wastewater in Greifswald, Germany, targeting Pseudomonas aeruginosa. Sequencing and microscopic analyses revealed a strictly lytic phage belonging to the Autographiviridae with podovirus morphology. We characterized its impact on established monoculture biofilms and, to investigate the role of bacterial communication, we also tested a deletion mutant lacking two major quorum-sensing (QS) systems. Scanning electron microscopy (SEM) of phage-infected biofilms revealed severe disruption in the wild-type strain. Surprisingly, the QS-deficient mutant maintained a relatively intact biofilm structure under phage-mediated stress. Proteomic analyses revealed distinct expression changes, including the induction of the biofilm-associated protein PslJ. These findings suggest a unique survival strategy, and future studies aim to unravel the molecular mechanisms behind the competitive advantage of QS deficiency in the emergent pathogen P. aeruginosa.

Efficacy of an experimental bacteriophage preparation to support disinfection and improve the welfare of poultry housing systems in the elimination of pathogenic Escherichia coli strains in vitro

Ewelina Pyzik¹, Urban-Chmiel Renata¹, Korona-Głowniak Izabela², Suśniak Katarzyna², Ciesielka Marzanna³, Całka Paulina³, Dec Marta¹, Herman-Ostrzyżek Klaudia¹

¹Department of Veterinary Prevention and Avian Diseases, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Poland

²Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Lublin, Poland

³Department of Forensic Medicine, Faculty of Medicine, Medical University of Lublin, Poland

The high global consumption of poultry raises concerns about the risks associated with the prevalence of APEC (Avian Pathogenic E. coli) strains, which cause colibacillosis in poultry. A major challenge in controlling these infections is the limited effectiveness of antibiotic therapy due to bacterial resistance associated with biofilm formation. The aim of this study was to develop and evaluate the effectiveness of an experimental bacteriophage preparation intended to improve the welfare of poultry housing systems by eliminating APEC strains in vitro. Bacteriophages were isolated from the feces of ducks, turkeys, and broiler chickens collected from litter. Purification and propagation of the isolated phages, determination of lytic spectra, and titer were carried out using the double-layer agar method on 0.7% LB agar. Morphological characterization of phages was performed by TEM microscopy (Xie et al., 2005). The experimental preparation consisted of a cocktail of lysate of 9 bacteriophages specific for APEC, concentrated in TM buffer to a final titer ≥10⁹ PFU/mL. The in vitro activity of phage cocktail was evaluated based on its ability to eradicate biofilms formed in microtiter plates containing LB medium. Analysis enabled the isolation of phages exhibiting lytic activity against the tested APEC strains. Morphological examination in TEM revealed virions indicating that they belong to the class Caudoviricetes, Myophages, based on their virion morphology. In vitro evaluation of phage kinetics, based on their ability to eradicate biofilms formed by APEC strains, demonstrated an antibacterial efficacy exceeding 70%, which can be considered substantial. Moreover, the developed preparation showed high effectiveness in in vivo reaching 82.6%. The most effective control of APEC strains is achieved using bacteriophages isolated from the specific poultry species targeted. Combining different groups of phages can substantially enhance the antibacterial activity of phage preparations. The preparation developed from  bacteriophages isolated from various poultry species may offer a straightforward strategy to broaden the application of phages for improving the welfare of poultry. This research was funded by the National Science Centre, Poland, OPUS27 project No 2024/53/B/NZ6/00046.

CRISPRi-mediated regulation in multicellular genetic circuits

Abhinav Pujar¹, Anchita Sharma¹, Hadrien Hadi Barras¹, Guillermo Rodrigo², Manish Kushwaha¹

¹Université Paris-Saclay, INRAe, AgroParisTech, Micalis Institute, 78352 Jouy-en-Josas, France

²Institute for Integrative Systems Biology (I2SysBio), CSIC-University of Valencia, 76980 Paterna, Spain

As synthetic genetic circuits grow more complex, distributing computation across multiple cell populations is important to reduce metabolic burden and improve system robustness. For these multicellular systems to work effectively, cells need reliable and scalable ways to communicate. Small-molecule signals, such as quorum sensing molecules, are often used, but they are limited in the number of distinct messages they can carry and the amount of information they can transmit. DNA-based communication through bacteriophage transduction offers a versatile, information-rich alternative that can encode complex instructions and enable modular, programmable circuit design. Here, we present a library of five M13 phagemid variants with distinct replication origins, including those based on the Standard European Vector Architecture (SEVA) family, designed to tune the growth and secretion dynamics of sender strains. We paired these phagemids with an intercellular CRISPRi system for precise quantification of genetic payload delivery. Together, these strategies lay the foundation for programmable, DNA-mediated communication in multicellular synthetic consortia and open exciting possibilities for distributed, multicellular genetic computation.

Phylogenetic analysis and characterization of the activity of three bacteriophages isolated from poultry living environments, ECBR1, ECBR3 and ECBR4, against pathogenic APEC strains

Renata Urban-Chmiel¹, Pyzik Ewelina¹, Ciesielka Marzanna², Całka Paulina², Korona-Głowniak Izabela³, Suśniak Katarzyna³, Andrzej Krajka⁴, Dec Marta¹, Herman-Ostrzyżek Klaudia¹

¹Department of Veterinary Prevention and Avian Diseases, Faculty of Veterinary Medicine, University of Life  Sciences in Lublin, Poland

²Department of Forensic Medicine, Faculty of Medicine, Medical University of Lublin, Poland

³Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Lublin, Poland

⁴Department of Forensic Medicine, Faculty of Medicine, Medical University of Lublin; Institute of Computer Science and Mathematics, Department of Fundamentals of Computer Science, Poland

High poultry consumption is one of the factors contributing to the common occurrence of pathogenic APEC (avian pathogenic E.coli) strains inducing colibacillosis in poultry. The global problem of drug-resistance among pathogens has also led to a number of measures taken to reduce the use of antibiotics in poultry. The limited possibilities for fighting bacterial infections have prompted the search for alternative methods to antibiotics with using bacteriophages. The aim of the study was to characterize APEC-specific phages isolated from poultry as potential tools for controlling bacteria. The study was conducted using faecal samples from broiler chickens kept in free-range farming systems. Phenotypic characterization of phages was based on TEM microscopy, the range of lytic activity against APEC strains, and the stability of titres in standard and altered pH conditions. Genetic analysis of phages was based on genome analysis and comparison with the genomes of other phages. The aim of the analysis was to determine the taxonomic identity of the phages and to confirm their strictly lytic nature by analysing their complete genome. Three bacteriophages, ECBR1, ECBR3, ECBR4, exhibiting lytic properties against APEC strains, were isolated. Morphological analysis (TEM) showed that all phages belonged to Caudoviricetes. The lytic titre was 10¹⁰ PFU/ml. Lytic activity against the APEC ranged from 43.5% to 76.1%. The study confirmed that phages were lytic, and lytic genes were mapped in their genome, including RIIA and RIIB lysis, LTs, and LIN; no AMR genes were detected. The phages showed low phylogenetic similarity to one another, at a maximum level of 30%. The results indicate that APEC-specific phages have significant antibacterial potential. This creates the potential for their use as alternatives to antibiotics to control infections, especially given that costs of developing phage preparations is much lower than the cost of producing antibiotics. Due to the lack of full knowledge of the metabolism, kinetics and their effects on eukaryotic cells, further research is essential. This research was fully funded by the National Science Centre, Poland, OPUS27 project No 2024/53/B/NZ6/00046.

Identifying loci in Clostridioides difficile bacteriophage ΦCD27 for therapeutic cargo insertion

Samiksha Venkatesan¹, Ernesto Abel-Santos^2,3, Rachel McMullan⁴, Ilias Kounatidis⁴, Terry Bilverstone¹

¹Anaerobes in Medicine (AiM Lab), School of Life, Health and Chemical Sciences, The Open University, Milton Keynes, United Kingdom

²Department of Chemistry and Biochemistry, University of Nevada–Las Vegas, Las Vegas, USA

³Nevada Institute of Personalized Medicine, University of Nevada–Las Vegas, Las Vegas, USA

⁴School of Life, Health and Chemical Sciences, The Open University, Milton Keynes, United Kingdom

Clostridioides difficile is the leading cause of hospital-associated diarrhoea in the Western world. This bacterial pathogen is resistant to several antibiotics, and those that are routinely used as treatment worsen the causative gut microbiome dysbiosis. Bacteriophage (phage) therapy is an ideal alternative due to their species-specific activity, which should facilitate the restoration of a diverse microbiome and prevent recurrent infection. However, all known C. difficile phages are temperate, whereby they can integrate into the host genome and form lysogens, which reduces their therapeutic potential. Thus, this project seeks to engineer a C. difficile phage ΦCD27 with ‘therapeutic cargo’ to improve its host killing capability and prevent lysogen formation. Three putative sites along the ΦCD27 genome were determined for knocking-in the therapeutic cargo in two inserts of 679 and 807 bp respectively. These loci were predicted to have minimal polar effects on adjacent genes. CRISPR-Cas9 plasmids containing the first insert were assembled and conjugated into a lysogenised strain of the host. Colony PCR screening indicated that the first insert had integrated into two of the three loci. Subsequently, CRISPR-Cas9 plasmids containing the second insert were assembled and conjugated into mutant lysogens containing the first insert. Colony PCR screening revealed that the second insert had integrated into one of the two loci, indicating that the full sequence of the therapeutic cargo (1486 bp) had successfully been knocked into one locus along the ΦCD27 genome. These results show promise at obtaining engineered C. difficile phages, which has only been demonstrated once in the published literature. Future work will involve comparing wild-type and engineered ΦCD27 in their ability to infect and control host growth in various in vitro assays. These findings will help advance the field of C. difficile phage therapy, bringing us closer to a much-needed alterative treatment.