Posters and poster guidelines

Thank you for considering to present your work as a poster at this conference.

Digital poster preparation and submission

• Page size: Prepare your poster as you would normally do for printing. You can prepare your poster in sizes A1 or A0, as the page size is not important if only presenting digitally. However, print hardcopy posters in A1 portrait format. Larger posters and those in landscape format may not be displayed due to space constraints.
• Naming your poster files: Name your poster files as follows:<your surname>-Phg24-Poster.pdf. For example, for David Jones, name your file as Jones-Phg24-Poster.pdf. DO NOT name your poster files as, e.g.,  Oxford-poster, poster2024, Oxford-phage-poster. Such files will be automatically rejected.
• Poster submission and deadlines: All poster presenters, whether attending virtually or in-person, are required to submit a digital version of their poster so that your poster is accessible to virtual attendees. Submit your final poster as PDF (<5MB) and via the link below no later than 26th August 2024 (we must have received your poster abstracts by 17th August). Late posters may not be included in the conference programme. Please DO NOT send your poster (or abstract) files by email. Please ensure you send us the very final version of your poster (as well as your poster abstract), as once published, it cannot be replaced.

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

Upload your poster

Poster presentation

• Poster PDF files (required): Whether the presenter is attending virtually or in-person, poster PDF files are required, which will be made available via the secure conference documents page to the conference participants. The participants will be able to ask questions via the Zoom chatbox during the conference. There is no specific time for presenting digital posters.
• Flash-talk videos (optional, but strongly encouraged): We are pleased to offer poster presenters the opportunity to prepare a short video presentation about their poster and send it before the conference. The videos will be made available on the LPMHealthcare YouTube channel. Below is further information for sending your video presentation.
  • Download the opening slide (PhgOx24 posters first slide) and use it as the first slide of your presentation (see example: https://youtu.be/XatqenCd_IU?si=Yu1PooCD4JmSLAiz).
  • Give your presentation (no longer than 5 minutes) using Zoom or another platform of your choice.
  • Convert the video into a format compatible with YouTube (e.g., MP4).
  • Send your video to PhageOxford@gmail.com using a file transfer program, such as MailBigFile or WeTransfer.
• Hardcopy posters (optional): If attending in-person, you may bring along a printed copy of your poster (maximum A1 size) to be displayed during the conference. You may be assigned a specific day to display your poster.
• The participants can post their questions on X at any time using the meeting hashtag #PhgOx24, as well as the poster specific hashtag (given under each poster abstract) – do tag ~PhageOxford and @LPMHealthcare in your tweets.

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

Upload your poster

Accepted posters

(Presenters 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.

The Effect of Phage Cocktails on Biofilms In Catheters Sourced From Long-Term Catheterised Patients With Urinary Tract Infections

Karen Adler¹, Paul Wilson², Pravisha Ravindra³, Andrew Millard¹, Melissa Haines^1,3, Martha Clokie¹

¹Leicester Centre for Phage Research, Department of Genetics, Genomics and Cancer Sciences, University of Leicester, University Road, Leicester, LE1 7RH, UK

²Centre for Imaging, Metrology, and Additive Technologies, WMG, University of Warwick, International Manufacturing Centre, University Road, CV4 7AL

³University Hospitals of Leicester NHS Trust, Leicester, United Kingdom

E. coli and K. pneumoniae are collectively responsible for the majority of cases of both urinary tract (UTIs) and bloodstream infections (BSIs). UTIs which spread into the bloodstream causing BSIs – known as urosepsis – account for 25% of all BSIs in adults and 5% of severe BSIs and septic shock. In the UK, the mortality rate for BSIs with treatment is over 20% – #1 cause of in-hospital death. The emergence of antibiotic-resistant strains of these bacteria has become a growing concern in healthcare, prompting the search for alternative treatments; bacteriophages, viruses which infect and kill bacteria, are one such alternative. In order to test the effect of phages on robust, patient-derived biofilms, a phage cocktail was created, based on in vitro assays, which was designed to cover a broad spectrum of E. coli isolates. Catheters were sourced from long-term catheterised patients (>8 weeks) with symptoms of UTIs, and cultured to identify those containing E. coli within the polymicrobial infections (n = 4). Each catheter was cut into two, with one segment placed in the fridge to act as an untreated baseline, and the other segment used for the ex vivo assay. The latter segments were treated in an Artificial Urinary Tract model developed to simulate a 5-day IV administration of phages that then pass to the urine and flow naturally out, where the catheters simulate biofilms within the urinary tract (e.g. in the kidneys, ureters, etc.). After the phage administration, the baseline and treated segments were scanned in a high-resolution micro-CT. The results show that the phage cocktail had a profound effect on the catheter biofilms: all catheters displayed a considerable decrease in biofilm volume, with a >97% reduction in three of the four; all four also had a considerable decrease in biofilm thickness, with two displaying a >92% reduction in average thickness and >93% reduction in maximum thickness. These findings demonstrate the substantial effect of a phage cocktail on polymicrobial, partially crystalline biofilms. With further work , this could contribute to the development of more effective treatments for antibiotic-resistant bacterial infections, understanding of the selectivity of the phages and their effect on the rest of the polymicrobial biofilm, and ultimately form the basis for creating phage cocktails for clinical use.

Phage-based deodorant: isolation and characterisation of a bacteriophage targeting odour-generating Staphylococcus hominis and expression of recombinant phage endolysins

Abdullah A AlAhmadi, Ugokwe Nzubechukwu Innocent, Andrew Millard, Edouard Galyov

Department of Genetics, Genomics and Cancer Sciences., University of Leicester, Leicester LE1 7RH, UK

Several gram-positive bacteria are thought to contribute to armpit malodour. Substances secreted by the sebaceous, eccrine, and apocrine glands in the human axilla are metabolised by bacteria into volatile fatty acids, thioalcohols, and steroids, which are then converted into odorant compounds. Treatment of malodour targets the root cause by eliminating these bacteria, particularly those from the Staphylococcus genus, such as Staphylococcus hominis, which is known for producing pungent thioalcohols via enzyme-mediated biotransformation. Many deodorants contain broad-spectrum antimicrobials to extend odour protection; however, this approach can disrupt skin homeostasis. A more selective method could potentially involve using lytic phages or phage-derived lysins, which target malodour-producing microbes while preserving the rest of the axilla microbiota. This project focuses on the isolation and characterisation of phages and phage-derived lysins that target odour-producing Staphylococcus species. Among the isolated phages, one named AA002 demonstrated the ability to infect several strains of S. hominis, although it had a narrow host range. Genomic analysis of AA002 revealed that it possesses two distinct endolysins, named AaeA and AaeB. Both recombinant AA002 endolysins, AaeA and AaeB, could lyse all tested strains of S. hominis as well as other Staphylococcus species involved in malodour production, such as S. haemolyticus, S. lugdunensis, and S. aureus and thus can be developed as active ingredients of novel deodorants.

Characterisation of LES prophage 6 from the Liverpool Epidemic strain of Pseudomonas aeruginosa

Lamyaa Alqarni¹, Jo Fothergill¹, Chloe James² Heather E. Allison¹

¹Department Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, United Kingdom

²School of Science Engineering and Environment, The University of Salford, The Crescent, Salford M5 4WT, UK

The Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa, is known to cause chronic infections in the lungs of people with  cystic fibrosis that are highly transmissible and difficult to treat. Notably, the genome of clinical isolate LESB58 harbours five prophages (LESφ2-6) that have been suggested to contribute to LES competitiveness. Most of the LES phages are unique and have been extensively characterised. However, less attention has been paid to LESφ6, which shares considerable similarities with the filamentous phage pf4 that is integrated into the PA01 chromosome and has been strongly associated with biofilm formation. The purpose of this study was to employ a molecular biology approach to investigate the role that LES φ6 plays in promoting biofilm formation and antibiotic resistance in P. aeruginosa. The first stages of this study used bioinformatic tools to identify the start and end points of LESφ6, and design outward facing primers to confirm the prophage ends by amplifying and sequencing the circular form.  The next steps are to create a clean integration site in PA01 to replace Pf4. This will enable construction of an isogenic version of PA01 that carries LES prophage 6 in place of Pf4 to enable empirical comparisons. This work will ensure that the functions of LESφ6 can be reliably determined in further studies to unveil how filamentous phages influence key traits such as biofilm formation and antimicrobial resistance of their bacterial hosts.

Human skin bacteriophages infecting coagulase-negative Staphylococcus identify barriers to phage infection in S. hominis

Samah E. Alsaadi^1,2, Heather E. Allison¹, and Malcolm J. Horsburgh¹

¹Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, UK

²Department of Biology, Faculty of Science, Taif University, Taif, Saudi Arabia

Human skin is colonised by Staphylococcus species that have varying abundance in different regions of the body. Previous studies have shown that the relative abundance of Staphylococcus spp. differs across skin sites and the skin virome influences the dynamics of bacterial populations in the skin microbiome. This study aimed to investigate the diversity of skin bacteriophages infecting major skin coagulase-negative Staphylococcus spp (CoNS) and identify host range and barriers. Skin swabs were collected from 80 healthy volunteers at different body sites to isolate cutaneous phages that infect 5 selected Staphylococcus spp. A total of 40 phages were isolated and genome sequenced, corresponding to six genetic clusters with two clusters representing novel phages. Phage infection was qualitatively assessed using a wide host range of 140 strains across 8 different Staphylococcus species. We found that one novel phage, named øAlsa, had a greater ability to infect S. hominis, which was otherwise infected much less than other CoNS species using the 40 identified phages, indicating the presence of a defence barrier to limit phage infection. To further investigate the resistance pattern observed in S. hominis strains, a co-evolution experiment was conducted using S. hominis LIV1218 as a representative strain with the novel phage øAlsa_1. Phage-resistant mutants were isolated and sequenced, revealing different single nucleotide variants in the spoVG gene, which encodes a transcriptional regulator. The S. hominis spoVG mutant phenotypes showed increased biofilm formation and increased autolysis. In addition, the host range analysis of spoVG mutants showed discrete patterns of phage susceptibility compared to the wild type. Our study identifies a link between the SpoVG regulator, biofilm formation and phage resistance in S. hominis.

Expanding the chemical diversity of the M13 phage system

Paula Mendes¹, Tim Dafforn², Marcos Fernandez-Villamarin¹, Sona Babayeva¹

¹School of Chemical Engineering, University of Birmingham, S W Campus, Birmingham B15 2TT, UK

²School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

M13 bacteriophage is the versatile viral scaffold which allows both chemically and genetically modification on its capsid, enabling further clinical applications from tissue regeneration to cancer detection. Both chemical modification and genetic engineering have been employed to diversify the functional groups presented on the surface of virions which are limited to the standard set of 20 amino acids of L-chirality. However, challenges exist in the chemical functionalization of M13 bacteriophage such as incorporating functional groups at desired active sites while maintaining the phage’s stability and controlling the attachment of synthetic functional groups to bind exclusively to desired sites, without unintended reactions. This study focuses on enhancing the phage by incorporating different chemical groups, such as boronic acids, benzoxaboroles, to bind unconventional targets. In this study, we employed the M13 bacteriophage, specifically the commercial Ph.D.-7 library, which consists of a combinatorial collection of random heptapeptides fused to the N-terminus minor coat protein (pIII), allowing for versatile modifications. Our initial objective was to modify this peptide library with benzoxaborole (BOB) via maleimide conjugation to cysteine residues. To achieve this aim, maleimide-BOB was synthesized and purified, followed by the amplification and spectral characterization of the commercial Ph.D.-7 library. We performed reactions between the phage (Ph.D.-7 as the target; M13KE as the control) and maleimide-BOB, with control experiments using maleimide-Bodipy and Bodipy fluorophores, to identify binding effectiveness and confirm the specificity of maleimide conjugation. Post-conjugation and characterization, the phage was amplified in E. coli, confirming that the modification did not compromise phage infectivity. Additionally, we explored optimal reaction conditions to refine the modification process.

Characterization of the Contributions a Detoxified Stx-Prophage Makes to the Fitness of Its Bacterial Host

Yueyi Cai¹, Heather E. Allison¹

¹Department Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, United Kingdom

Stx-phages can horizontally transfer Shiga toxin-encoding genes between bacteria, and can enter the lysogenic cycle in their hosts. Stx-phages prophages can carry and introduce additional genetic material to the host cell and confer traits on their bacterial lysogen. This project studied the effects of a hypothetical gene, vb_24B_13c from Stx-phages Φ24, as well as the well-known phage-encoded regulators CI and CII co-expression of CIII on their host. By overexpressing these phage regulators in both naïve and lysogenic cells, at 37 or 30℃, their functions were determined, while excluding the effects from phage genes involved in self-induced lysis. Multiple approaches including RNA-seq, NanoString nCounter analysis, growth curve measurement and motility tests were used. Results from motility assay showed that motility of MC1061/Φ24B::Cat was enhanced compared to MC1061 naïve cell, at both 37 and 30℃, suggesting prophage Φ24B contributes to the motility of its host. The prophage gene vb_24B_13c can independently increase the motility of MC1061 naive cells at 30°C even at a low expression level without the need for other phage gene expressions. It was found that at 30°C, overexpressed CI can enhance motility in both naïve cells and lysogens. At both 37°C and 30°C, CI can promote the TCA cycle by upregulating TCA cycle genes, while CII and CIII can inhibit the expression of genes related to maltose transport and metabolism, the TCA cycle, and glycolysis. In addition, this project demonstrated temperature-determined phage-bacteria interaction, finding that temperature can affect lysogen flagella assembly and motility, as well as their metabolic pathways. Overall, this project demonstrated that prophage DNA, which constitutes only about 1.1% of the E. coli genome, is powerful enough to reprogram the host’s expression profile and behaviour.

Antibiotic-resistant Acinetobacter baumannii can be killed by a combination of phages and complement

Carmen Chen^1,2, Eva Krzyżewska-Dudek^1,3, Sheetal Patpatia^4,5, Vinaya Dulipati¹, Sarah Natalia Mapelli⁶, Aycan Meral⁶, Juha Kotimaa^1,7, Saija Kiljunen⁴, Seppo Meri^1,8

¹Translational Immunology Research Program, Department of Bacteriology and Immunology, Faculty of Medicine, University of Helsinki, Finland

²Department of Biomedical Sciences, Humanitas University, Milan, Italy

³Department of Immunology of Infectious Diseases, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland

⁴Human Microbiome Research Program, Department of Bacteriology and Immunology, Faculty of Medicine, University of Helsinki, Finland

⁵Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institutet, Stockholm, Sweden

⁶Department of Research in Inflammation and Immunology, IRCCS Humanitas Research Hospital, Milan, Italy

⁷VTT Technical Research Centre of Finland, Espoo, Finland

⁸Helsinki University Hospital, Diagnostic Center (HUSLAB), Helsinki, Finland

Infections caused by multidrug-resistant Acinetobacter baumannii are an emerging global health threat. Currently, case studies have shown promising results on treating infections caused by A. baumannii. However, due to the concern of phage clearance by our immune system and the development of phage resistance, further studies on the mechanisms of the combined effect of phages and innate immunity on clearing A. baumannii are needed to further improve phage therapy. Here, we report a synergistic effect of the complement system and phages on clearing multidrug-resistant A. baumannii. After incubating A. baumannii with a phage, the bacteria rapidly became resistant to the phage. This was due to downregulation of capsule synthesis and production of truncated lipooligosaccharide (LOS), which led to the loss of the bacteriophage receptor. Although the phage could no longer infect and kill the non-encapsulated A. baumannii, the bacteria simultaneously became susceptible to complement killing by the membrane attack complex (MAC). Thus, the capsule and longer LOS were indispensable for the phage to recognize A. baumannii, but also served as an immunoevasion mechanism by shielding MAC to insert efficiently on the bacterial outer membrane. Multiple sequence comparison of the genomes of the different bacterial phenotypes revealed that A. baumannii can regulate capsule synthesis through transposon mutagenesis at the K-locus. By insertion of a mobile element within a central gene in the K-locus, a premature stop codon was formed, which led to downregulation of the mutated gene at the translational level. Together, our study highlights the combination of phage and the complement system acting in concert to kill multidrug-resistant A. baumannii. These results support a combination of phage and antibody-targeted complement therapy as a means to combat antibiotic-resistant bacteria.

Evolution of Klebsiella pneumonia resistance to phage therapy: from one receptor to multiple receptors

Xiang-Dang Du, Wenbo Zhao

College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, P.R. China

Phage therapy, as an alternative or complementary therapy to antibiotics, is considered one of the important solutions for the treatment of systemic infections caused by multidrug-resistant Klebsiella pneumoniae. However, K. pneumoniae also undergoes resistance mutations against bacteriophages during the process of phage lysis, which limits the application of phage therapy. In this study, a phage that can lyse the epidemic K64 K. pneumoniae was selected from our phage library. We sequentially induced phage-resistant mutant bacteria. Phage growth and lysis assay were used to verify phenotypic changes of mutant strains. Once resistant, sequentially to select another effective phage from the phage library until all bacteriophages become ineffective. Conformation of key mutation genes is performed by whole genome sequencing and gene complementary experiment. Differences in phage-resistant strains are mainly reflected in the narrowing of the phage cleavage spectrum. WGS analyses showed that K. pneumoniae gradually developed tolerance to multiple phages, with the tolerance factors being the mutations in wcaJ, waaH and loss of CPS gene clusters, respectively. Our study revealed the progressive phage resistance in K. pneumoniae, identifying key genetic factors, such as the mutations in wcaJ, waaH and loss of CPS gene clusters that contribute to phage resistance. These findings highlight the need to use cocktail phages mixed from multiple receptor types in clinical phage therapy to counteract the evolution of bacterial defense mechanisms.

Harnessing Phage Receptor Binding Proteins as Bio-Probes for Detecting High-Risk Pathogens

Matthew Dunne^1,2,3, Leonie Reetz¹, Oliver Dietrich¹, Sabine Suppmann¹, Gregor Grass⁴, Peter Braun^1,2

¹Fraunhofer Institute for Translational Medicine and Pharmacology ITMP – Immunology, Infection and Pandemic Research IIP, Penzberg, Germany

²Institute of Infectious Diseases and Tropical Medicine, University Hospital Ludwig-Maximilian University Munich, Munich, Germany

³Micreos GmbH, Wädenswil, Switzerland (primary address)

⁴Bundeswehr Institute of Microbiology, Munich, Germany

Bacteriophages, nature’s bacterial predators, are renowned for their extraordinary precision in binding to bacterial targets, which is achieved through specialized receptor binding protein (RBP) complexes on the phage particle. RBPs are typically categorized into two distinct groups: tail fibers (TFs) and tailspike proteins (TSPs), each with unique morphology. TFs are elongated and slender fibrous proteins, while TSPs are short, stocky, and often equipped with enzymatic capabilities targeting specific surface structures. As the initial point of contact with a bacterial host, RBPs serve as critical gatekeepers for all phage infections, fundamentally determining the phage’s host range. Compared to antibodies, phage RBPs have evolved over millennia to recognize their bacterial targets in diverse environments and under challenging conditions, making them ideal candidates for bio-probe engineering. While significant advancements have been made in rapid diagnostics, such as PCR-based detection of specific genes, the presence of intact bacteria can usually only be verified after one to several days, typically by culture-based methods. Our consortium is dedicated to developing an RBP identification and engineering platform that can be harnessed for different bacterial pathogens, enabling the rapid development of RBP bio-probes and associated diagnostic assays. Our initial focus is on highly pathogenic bacteria, such as Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei and Brucella spp.. Developing new assays for these pathogens is of high priority as these so-called biothreat agents pose a major biosecurity risk due to their potential misuse in biological warfare or bioterrorism. To hear about our progress, Dr. Peter Braun and Sebastian Kachel will present some of the outputs from our work on RBPs and reporter phages in the Molecular and In Silico Tools session on Monday afternoon.

Leveraging temperate phages to enhance antibiotic effectiveness

Rabia Fatima¹, Alexander P. Hynes²

¹Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4L8, Canada

²Department of Medicine, McMaster University, Hamilton, Ontario L8S 4L8, Canada

With a decline in antibiotic effectiveness, there is a renewed interest in bacteriophage (phage) therapy. Phages are bacterial-specific viruses that can be used alone or in combination with antibiotics to reduce bacterial load. Most phages are unsuitable for therapy because they are ‘temperate’ and can integrate into the host genome, protecting it from subsequent phage infections. However, the prophage can be awakened by stressors such as DNA-damaging antibiotics. Parallel work from the lab uncovered a strong synergy between a model temperate phage and sublethal ciprofloxacin, a fluoroquinolone, and gentamicin, an aminoglycoside, eradicating the phage’s Escherichia coli host. Here we explore the potential of this synergy against a clinically relevant drug resistant pathogen, Pseudomonas aeruginosa. Temperate phages isolated from clinical strains were screened for synergy with six antibiotics (ciprofloxacin, levofloxacin, meropenem, piperacillin, tobramycin, polymyxin B), using checkerboard assays. Interestingly, our screen identified phages that can synergize with each antibiotic, despite their widely differing targets. When meropenem and tobramycin were effective, it did not reduce the frequency of lysogens. In contrast, ciprofloxacin and piperacillin were able to reduce the frequency of phage dormancy events; by 70% and 25%, respectively. Ciprofloxacin also worked in combination with multiple phages, even in antibiotic resistant clinical host, re-sensitizing the bacteria. In a Caenorhabditis elegans model, temperate phage-ciprofloxacin pairing increased the lifespan of P. aeruginosa infected worms to that comparable to the uninfected control. Similar rescue was also observed for the lysogen treated with the antibiotic, supporting that the phage even in its prophage form can enhance antibiotic effectiveness. This is the first in vivo testing of temperate phage-antibiotic synergy and highlights the therapeutic potential of temperate phage-antibiotic combination.

Bacteriophage RBP and Human IgG1 Fc Fusion Protein Enhances Yersinia enterocolitica Phagocytosis by THP-1 Macrophages

Karolina Filik-Matyjaszczyk¹, Marzena Ciesielska¹, Irwin Matyjaszczyk², Bożena Szermer-Olearnik¹_, Andrzej Gamian¹

¹Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolf Weigl Street 12, 53-114 Wroclaw, Poland

²Department of Biochemistry, Selvita S.A., Podole Street 79, 30-394 Cracow, Poland

Receptor binding proteins (RBPs), or adhesins, are bacteriophage proteins that determine phage infectivity by binding to a receptor on the host surface in the first step of infection. The group consists of two types of proteins based on their morphology: Tail fiber proteins (TFP) and Tailspikes (TS). RBPs recognize a variety of receptors, including porins, flagella, bacterial outer membranes, or LPS. In our previous study by Filik et al. 2022, we investigated TFPgp17 RBP and demonstrated that this protein from the Yersinia phage φYeO3-12 recognizes the O:3 serotype of this pathogen with high specificity. Based on these results we engineered a bispecific protein: TFPgp17 fused with the Fc fragment of IgG1 to evaluate its efficiency in promoting bacterial phagocytosis in its presence. Since TFPs naturally recognize bacteria, their use to create  Fc fusion proteins simulating monoclonal antibodies which act as opsonin is highly rational and promising. We used the THP-1 cell line, differentiated into macrophages, as our phagocytosis model. We proved binding to both,  bacteria and macrophages. Furthermore, we demonstrated that phagocytosis of Y. enterocolitica was more effective and significantly enhanced in the presence of Fc_TFPgp17 compared to non-opsonized bacteria. Additionally, we used the Gentamicin Protection Assay to determine the number of surviving bacteria inside the phagocytic cells. We observed a lower number of surviving bacteria in samples pre-opsonized with Fc_TFPgp17  compared to non-opsonized bacteria. Obtained results indicate reduced intracellular survival and lead to the conclusion that in the Fc_TFPgp17 treated samples, more bacterial cells are inactivated by the macrophages.  This is the first attempt to use the phage TFPs to design molecules that enhance pathogen elimination by opsonophagocytosis. This work was supported by the National Science Center, Poland (DEC-2023/07/X/NZ6/00118).

Investigation of the molecular specificity of phage-neutralizing antibodies using λ (Lambda) phage, a model component of the human microbiome

Katarzyna Gembara^1,2, Marek Harhala^1,2, Izabela Rybicka², Aleksandra Wilczak², Krystyna Dąbrowska^1,2

¹Regional Specialist Hospital in Wrocław, Research and Development Center, Kamieńskiego 73a st., Wrocław, Poland

²Hirszfeld Institute of Immunology and Experimental Therapy, Weigla 12 st., Wrocław, Poland

Phages, known to be immunogenic, induce specific immune responses, including the production of phage-specific antibodies. This phenomenon is observed in both naturally occurring phages within the human body (the phageome) and those introduced through phage therapy. Understanding the interactions between phage antigens and human antibodies is crucial, as these antibodies can potentially reduce or block the therapeutic efficacy of phages. This study aims to elucidate the molecular specificity of antibodies that neutralize phages, using the model bacteriophage λ (Lambda), a component of the human microbiome. Specifically, we sought to identify which proteins and domains of phage λ elicit phage-specific antibody responses. We employed phage display technology to experimentally identify immunogenic oligopeptide regions within Lambda phage. A library of 480,000 phage epitopes was screened using immunoprecipitation, followed by next-generation sequencing (NGS) to identify the epitope-coding sequences. Our findings revealed that certain phages, including Lambda, contain immunogenic oligopeptides derived from various proteins, such as those that make up the phage tail. In future studies, we plan to use phage λ to further understand which phage proteins and their domains trigger antibody responses that alter phage bioavailability in vivo. Chemically synthesized sequences of the identified immunogenic oligopeptides will be tested in animal models to induce specific immune responses against individual immunogenic elements. We will then assess the impact of these responses on the biological activity of the phage in vivo. This approach will allow us to pinpoint the exact molecular targets on the phage, which, as a complex multi-antigenic structure, are critical for phage neutralization and pharmacokinetic alterations in vivo. This work was funded by the Polish National Science Centre under grant MINIATURA-7 (2023/07/X/NZ6/00853).

Unravelling Phage-Host Attachment and Recognition Modalities (PHARM) in the dairy bacterium Streptococcus thermophilus

Zoe Kampff, Douwe van Sinderen, Jennifer Mahony

School of Microbiology and APC Microbiome Ireland, University College Cork, Cork, Ireland

The dairy fermentation industry depends on reliable, technologically favourable and robust starter cultures. Streptococcus thermophilus is a lactic acid bacterium that has long been associated with the production of fermented dairy products. Bacteriophages (or phages) represent a significant and consistent threat to food fermentation processes. S. thermophilus infecting phages have recently been classified into five genetically distinct groups termed the Moineauvirus, Brussowvirus, Vansinderenvirus and the 987 and P738 genera of the Aliceevansviridae family. Several phages capable of infecting S. thermophilus recognize and bind to cell wall polysaccharides presented on the cell surface of the host. Rhamnose glucose polysaccharides (RGP) and exopolysaccharides (EPS) have been implicated in phage-host interactions, acting as the receptor for many of their infecting phages. These phages typically exhibit a narrow host range suggesting there are diverse receptor moieties (RGP and/or EPS structures) being presented on the cell surface of different S. thermophilus strains. In this study, efforts have been made to expand the current knowledge on the specific phage-host interactions in S. thermophilus using a model strain, which is host to members of the Brussowvirus, 987 and P738 genera. The generation of spontaneous bacteriophage-insensitive mutants of the host strain generated in this study was applied to aid in the identification of specific host-encoded receptor(s) of the 987 and P738 phage groups, which are poorly defined to date. Additionally, to explore the specific interactions of phage-encoded RBPs with their cognate receptors, green fluorescent protein-tagged derivatives of selected RBPs were constructed and applied in binding studies. Furthermore, analysis of the partial and complete RGP structures may provide insight into phage binding and subsequent infection profiles of certain phage groups, enhancing our understanding of S. thermophilus phage-host dynamics.

Prophage activation by Staphylococcus aureus Pathogenicity Islands

Melissa-Jane Chu Yuan Kee, Yin Ning Chiang and John Chen

Department of Microbiology and Immunology, Infectious Diseases Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore, Singapore

Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements that encode superantigens and toxins. They are parasites that exploit bacteriophages for their reproduction, packaging and dissemination. Normally, SaPIs reside stably in the host chromosome until they are induced to excise by anti-repressor proteins from helper phages they parasitize. Recently, we have identified a phenomenon where a SaPI-encoded protein (inducer) can induce a prophage to enter the lytic cycle in an SOS-independent manner. Using genetic and biochemical analyses, we showed that the SaPI determinant is a small protein, while the phage-encoded target is the putative CI repressor protein. Structural prediction using Alphafold3 revealed that the SaPI inducer is an alpha-helical protein which is predicted to form a homodimer. To investigate if the SaPI inducer and the prophage repressor interact, the two proteins were His- and S-tagged, respectively, and the fusions were co-expressed in E. coli. Pull-down assays and western blot analyses demonstrated direct interaction between the wild-type SaPI inducer and wild-type prophage repressor proteins. SaPI inducer mutant D11A, identified previously through a genetic screen, did not bind to wild-type prophage repressor; however, the interaction was restored between the SaPI inducer mutant D11A and a prophage repressor S6F mutant. Further work elucidating the structural basis of prophage activation by SaPI-encoded inducers will shed light on this new SaPI-prophage interaction. This, in turn, will explain how SaPIs promote their own survival and high-frequency transfer among Staphylococcal strains.

Structural Characterisation of Lysogenic Phage from the Liverpool Epidemic Strain of Pseudomonas aeruginosa and their Influence on Type Six Secretion Systems

Andrew Martin¹, Enrique González- Tortuero¹, Revathy Krishnamurthi², Reem Almeran², Ian B. Goodhead¹, Jo L. Fothergill², Heather E. Allison², Chloë E. James¹

¹School of Science, Engineering and Environment, University of Salford, UK

²Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, UK

The Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa is a key opportunistic pathogen and major cause of respiratory morbidity and mortality in cystic fibrosis (CF) patients. A set of active prophages have been associated with fitness advantages of LES. Transcriptomic studies revealed that each LES phage affects the expression of Pseudomonas host genes differently during polylysogeny of the well characterised PAO1 strain. A range of virulence-associated genes were affected, including several genes belonging to Type Six Secretion Systems (T6SS). This study carried out structural analysis of LES phages 2, 3 and 4 by transmission electron microscopy (TEM) confirming Siphoviridae morphology with ichosahedral capsid heads (50-60 nm diameter) and long flexible tails (~200 nm long). Tail fibre structures were clearly visible at the end of LES phage 4 tails. Proteomic analysis of purified LES phage suspensions by SDS page detected 26, 13 and 13 structural proteins for LES phages 2, 3 and 4 respectively, which is more than could be identified using genome annotation tools. Functional competition assays can be used to investigate whether prophage-associated changes in T6SS genes affected PAO1 killing of Escherichia coli in co-culture. Preliminary results suggest that carriage of LES phages 3 or 4 individually or combination affect the rate of killing of E. coli in co-culture. However, overall numbers were too low for robust statistical analysis and further optimisation of the method is required.  Overall, this study has used a combination of genomic, proteomic, TEM and functional analyses to improve our understanding of LES phage biology.

Rational Development of Innovative Bacteriophage Delivery Systems for Local Tissue Infection Therapy

Johannes Most, Viktoria Planz and Maike Windbergs

Institute of Pharmaceutical Technology, Goethe University Frankfurt, Max-von-Laue-Straße 9, Frankfurt am Main, 60438, Germany

In the post-antibiotic era, effective management of persistent tissue infections poses a major therapeutic challenge in clinical practice due to the presence of multidrug-resistant and hypervirulent bacterial strains known as “ESKAPE” pathogens. Among them, Pseudomonas aeruginosa accounts for a broad range of hospital-acquired tissue infections such as chronic wounds. Thus, novel therapeutic options combating these intractable pathogens are urgently needed. The combined usage of bacteriophages and antibiotics recently gained attention, with promising in vitro results demonstrating superior antibacterial efficacy as opposed to monotherapies. However their clinical translation is hampered by the lack of adequate infection-targeted drug delivery systems and predictive analytical models. To address this issue, we developed a novel analysis method for identification and evaluation of synergistic and antagonistic effects across various antibiotics and bacteriophages. Synergistic effects were macroscopically visualized through semi-automatic plaque analysis, verified by scanning electron microscopy analysis, and validated by key bacteriophage infection parameters, such as burst size. Using these findings, we developed electrospun fibers for the release of bacteriophage JG004 and sub-lethal concentrations of Aztreonam for the treatment of local tissue infections caused by Pseudomonas aeruginosa. Benefitting from the effects of Aztreonam on the bacteria, phage infection counts were significantly increased, culminating in higher rates of progeny release, and ultimately, bacterial eradication. Based on a novel computer-aided analytical approach, promising candidates for phage-antibiotic therapy of wound infections could be identified. Electrospun fibers emerged as versatile drug carrier platforms, combining the therapeutic advantages of both substance classes in one formulation.

Attachment sites that affect prophage stability

Muhammad Zulfadhly Bin Mohammad Muzaki and John Yu-Shen Chen

Department of Microbiology and Immunology, Infectious Diseases Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore

Bacteriophage attachment sites (attB) are specific sequences in bacterial genomes where temperate phages integrate their genomes. Most commonly occupied attB sites in bacterial species such as Staphylococcus aureus and Salmonella typhimurium are known to cluster in certain regions in the chromosome and exhibit polarity. These observations suggest that there has been evolutionary pressure for stable prophage integration into attB sites of certain localisation and orientation in the host chromosome. Here, we investigated how the localisation and orientation of attB sites influence prophage stability in S. aureus lysogens. This was done by shifting the attB site to a region in the chromosome where natural attB sites are scarce. Our findings reveal that prophages integrated into these attB sites were unstable, resulting in lower lysogenisation rates. Additionally, we found that prophages integrated into attB sites in their natural orientation were significantly more stable than those integrated in the reverse orientation. These findings improve our understanding of phage-bacteria interactions and may reveal further insights into how phages and bacteria co-evolve.

Pathogenicity Islands Induce Prophage Activation in Staphylococcus aureus

Franda Ng and John Chen

Department of Microbiology & Immunology, Infectious Diseases Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore, Singapore

Staphylococcus aureus is a versatile and opportunistic pathogen responsible for a wide range of infections, from minor skin conditions to severe systemic diseases. Its virulence is significantly enhanced by pathogenicity islands (SaPIs), which are mobile genetic elements that carry genes for toxins and other virulence factors. SaPIs are known for their ability to hijack the machinery of certain temperate bacteriophages, facilitating their transfer between bacterial cells. Recently, SaPI1 was found to encode a small protein that causes the induction of resident prophages into the lytic cycle. In this study, we aim to show that SaPI1 inducer homologues can activate a different subset of prophages to enter the lytic cycle. Plasmids encoding potential inducers were transduced into clinical isolates of S. aureus and induction of resident prophages was evidenced by a significant drop in the transduction frequency of the plasmids. Repressors targeted by the inducers were identified and their regulated promoters were measured for transcription activation via a reporter assay. These results highlight novel interactions between pathogenicity islands and phages that likely play a significant role in pathogen evolution.

Utilising bioinformatic tools to identify and characterise subtypes of the anti-phage system Shield

Jude KJ Salaymeh, Stephen Garrett and Giuseppina Mariano

School of Biosciences and Medicine, University of Surrey, Stag Hill Campus, Guildford GU2 7XH, UK

The continuous arms race between bacteria and their viruses (bacteriophages) has led bacteria to the evolution of a plethora of phage defence mechanisms. In the last 5 years, ~200 distinct anti-phage systems have been discovered, with some showing evolutionary links to the innate immunity components of human and plants. As many were only recently discovered, for many newly-discovered anti-phage systems, their full diversity and their mechanism remains unknown. In the lab, we previously discovered a novel anti-phage system, named Shield, that is exclusive to Pseudomonas spp. In Pseudomonas, Shield exists in four subtypes, all sharing the core component ShdA, a promiscuous nuclease that damages chromosomal, phage and plasmid DNA. In this study, to find more instances of Shield in other bacterial species, we searched for structural homologues of ShdA using Delta-BLAST. The genomic neighbourhood of the resulting hits was screened to determine their minimal operon structure and their proximity to known defence islands. With this approach we identified 20 novel putative Shield subtypes wherein ShdA is associated with proteins that harbour a domain that was previously associated with phage defence. We further provide a more in-depth description of 4 selected operons, showing their domain composition, structural prediction, and phylogenetic distribution across bacterial species. Our findings only expand the known diversity of the Shield system but also provide a new framework for exploring novel anti-phage defence mechanisms across diverse bacterial species.

Biological activity of a hydrophobically stabilized F8 bacteriophage preparation against Pseudomonas aeruginosa in the presence of human serum

Bożena Szermer-Olearnik, Karolina Filik-Matyjaszczyk, Jarosław Ciekot, Andrzej Gamian, Janusz Boratyński, Tomasz Goszczyński

Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland

Bacteriophage F8 belongs to the Myoviridae group and is a pathogen of Pseudomonas aeruginosa. P. aeruginosa causes healthcare-associated infections and some types of multidrug-resistant P. aeruginosa are resistant to almost all antibiotics. Phage therapy is becoming popular as an alternative in treating antibiotic-resistant infections but there is still a lack of standardized protocols that health organizations will approve for its possible use in clinics. In our studies, we focus on the properties of hydrophobic 1-octanol as a possible additive for long-term storage of purified phage liquid preparations. 1-octanol is a fatty alcohol that occurs naturally in citrus oils and is used successfully in the perfume industry. It has been approved for human use as a food additive by the Food and Drug Administration and the Council of Europe. During our experiments, we observed the protective effect of 1-octanol on long-term storage of purified F8 phage preparation. Additionally, as an introduction to in vivo conditions, we decided to evaluate the activity of the purified and stabilized bacteriophage F8 preparation in the presence of human pooled serum. This study was performed to assess the influence of serum components on the activity of phage particles. The lowest number of bacteria was detected in preparations preincubated with F8 phage and then treated with active serum, according to that, it can be initially concluded that preincubation with a purified and stabilized F8 bacteriophage preparation may support serum activity in the killing and elimination of P. aeruginosa. In summary, a purified preparation of bacteriophage F8 with higher stability in the presence of 1-octanol was obtained and revealed increased activity in biofilm reduction. Moreover, it was demonstrated that the antibacterial activity of the obtained preparation was not inhibited in the presence of human serum. This work was supported by the National Science Center, Poland (UMO-2019/03/X/NZ6/01710).

Single-molecule insights into Qβ phage infection of the E. coli host

Sammi Ta^1,2, Hafez El Sayyed^1,2, Oliver N. Pambos^1,2, Henrik Pedersen³, Charlotte R. Knudsen³, Achillefs N. Kapanidis^1,2

¹Department of Physics, University of Oxford, Parks Road, Oxford, OX1 3PJ, United Kingdom

²Kavli Institute for Nanoscience Discovery, University of Oxford, South Parks Road, Oxford, OX1 3QU,United Kingdom

³Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.

RNA viruses use their viral genome as a direct template for both replication and translation, yet how these dual processes occur without clashing remains poorly understood. Our study explores the interplay between the bacteriophage Qbeta (Qβ) and the E. coli host as a simplified model to understand RNA virus dynamics. During Qβ infection, the ribosomal protein S1 is recruited to the Qβ replicase complex. S1 may therefore function as a regulatory unit determining the fate of the viral RNA for replication by the replicase or translation by the ribosome. To understand how S1 dynamically interacts with translation and replicase complexes, we used photoactivated localisation microscopy (PALM) and single-molecule fluorescence tracking to study the in vivo diffusive properties of S1 and the viral B-subunit, the polymerase component of the Qβ replicase. Our findings provide insights into S1 and B-subunit dynamics, revealing that S1 exists in three diffusive subpopulations: 30% bound to RNA, 40% slowly diffusing within a complex, and 30% freely diffusing. The lack of change in these proportions upon infection indicates that S1 mobility is similar whether it is a component of the Qβ replicase or the ribosome, and is likely a reflection of its high copy number. Notably, Qβ infection also induced significant changes in cell morphology, including bloating and filamentation, which correlated with viral load. Timeseries imaging showed that Qβ infection reduces the rate of cell division and disrupts normal septum formation, leading to elongated cells. This disruption mirrors the effects seen with certain antibiotics, suggesting that Qβ infection may employ similar mechanisms, or could represent a novel mechanism yet to be explored. Ultimately, these findings highlight the complex virus-host dynamics occurring between Qβ and the E. coli host at the single-molecule level.

Bio-informatic and Molecular Characterisation of the Elusive 5^th Prophage of Pseudomonas aeruginosa LESB58

Reem A. Talat^1,2*, Revathy Krishnamurthi³, Enrique González-Tortuero ⁴,⁵, Chloe E. James⁵, Jo L. Fothergill¹, Heather E. Allison¹

¹Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, L69 7ZB. United Kingdom

²College of Environmental sciences, University of Mosul, Iraq

³Quadram Institute of Biosciences, Norwich, NR4 7UQ, United Kingdom

⁴Faculty of Health and Life Sciences, Northumbria University, NE1 8ST, United Kingdom

⁵School of Science, Engineering and Environment (SEE), University of Salford, M5 4WT, United Kingdom

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa causes transmissible chronic respiratory infections that negatively impact the functions of the cystic fibrosis lung and decrease the effectiveness of antibiotic therapies. LES is also capable of infecting the lungs of non-CF patients and superinfecting patients with other P. aeruginosa strains. Several prophages have been detected in the accessory genome of LES and have been suggested to play a role in the competitiveness of their host. Although the infective properties of LES phages φ2-4 have been characterised, LES φ5 is yet to be isolated..Through bioinformatic and PCR techniques, we determined the total length of the LESφ5 prophage to be 50,235 bp, with the attL and attR regions located at 2,690,327 – 2,690,341 and 2,740,547 – 2,740,561 in the P. aeruginosa LESB58 genome, respectively. We identified several new putative gene functions and proved that LES φ5 is a complete, inducible phage through detection of encapsidated LES φ5 DNA in induced and DNAse-treated LESB58 supernatants.Plaque assay and spot assays were used in attempted to isolate active LES φ5 from filtered supernatants of LESB58 under a range of culture conditions. Screening of 34 potential P. aeruginosa host strains yielded no clearing or plaques that could be attributed to LESφ5 lysis. It was thus speculated that common phage defence systems may be preventing productive infection.Work is ongoing to synthesise LESφ5 and to identify a P. aeruginosa host that will support lytic LESφ5 infection for further characterisation.

The bully phage: A Shiga toxin-encoding phage suppresses host-sharing prophages

Tamara Wellins*, Rachel Hashuel*, Maayan Rachimi, Eran Gutman, Reut Melki, Linoy Taktuk, Pe’era Pash Wasserzug, Shahar Silverman, Karina Roitman, and Yael Litvak

*Equal contributors

Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus Givat-Ram, Jerusalem 9190401, Israel

Gut bacteria frequently encode multiple prophages, yet only a subset or a singular prophage produce virions upon induction. We show that during intestinal infection, a shiga toxin-producing prophage suppresses the induction of other prophages carried in the pathogen’s genome. We demonstrate that the shiga toxin phage lysogenizes various commensal E. coli strains in the gut during infection. In all the lysogen strains that we tested the Shiga toxin phage could be induced by DNA-damaging agents, however, it suppressed the replication of co-hosted prophages that were active in the parental strain. Suppression of host-sharing prophages by the Shiga toxin phage promoted pathogen fitness by preventing excessive host cell lysis. We further found that the mechanism of phage-phage cross-inhibition is common among Shiga toxin phages and in other temperate E. coli phages. Our work presents a novel mechanism that governs interactions between host-sharing temperate phages and plays a significant role in regulating polylysogeny and virulence.Top of Form

Shift in dominant bacterial species exhibit host-driven viral community diversity

Caroline S. Winther-Have¹, Jacob Agerbo Rasmussen¹, Thomas Sicheritz-Pontén^1,2, Morten Tønsberg Limborg¹

¹Center for Evolutionary Hologenomics, Globe Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

²Centre of Excellence for Omics-Driven Computational Biodiscovery (COMBio), Faculty of Applied Sciences, AIMST University, Kedah, Malaysia Understanding host-specific phage diversity is crucial for deciphering the complex dynamics and arms races that govern the ecology and evolution of microbial communities. A major challenge in elucidating these dynamics is the overwhelming complexity of natural systems and the limitations of in vitro studies. Metagenomics is a powerful tool for studying viruses in their natural settings. However, it is limited by the fact that uncultivated viruses lack an inherent host association, making it difficult to link viruses to their hosts. This limitation becomes more pronounced in complex environments, hindering our ability to understand the drivers of natural diversity and the interplay between viruses and their prokaryotic hosts. In this study, we utilized a simple system from a study on farmed Atlantic salmon (Salmo salar). The study found that the microbial community in the distal gut of these salmon consisted of just two dominant bacteria: the salmonid-associated Mycoplasma sp., observed in wild or healthy salmon, and the salmon dysbiosis-associated Aliivibrio sp. Benefitting from the simplicity of this system, we extracted the viral community using virus-like particle fractionated shotgun metagenomes. Fascinatingly, we observed a pronounced difference between the viral communities present, depending on the bacterial composition. Samples dominated by Mycoplasma sp. had few to no viruses recovered, whereas samples with Aliivibrio sp. present or dominant had viral communities comprising up to 22 viral taxonomic operational units. The two bacteria occupy vastly different niches in the environment: Mycoplasma is host-associated, with a reduced genome, intracellular, and lacking cell walls, whereas Aliivibrio is ubiquitous in the marine environment, motile, and has large genomes of ~4.5 Mb. This study provides thrilling insight into the significance of bacterial trade-offs in an ecological setting and how these shape the associated viral communities.