Posters and poster guidelines
Thank you for considering to present your work as a poster at Phages 2023.
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>-Phg23-Poster.pdf.For example, for David Jones, name your file as Jones-Phg23-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, poster2023, 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 30th August 2023 (we must have received your poster abstracts by 15th 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.
- Digital posters: The poster PDF files, whether a presenter is attending virtually or in-person, are required for presentation, and will be made available via the secure ‘PHGOx23 Documents’ page to the conference participants before the meeting. The participants will be able to ask questions via the Zoom chatbox during the conference. There is no specific time for presenting digital posters.
- Hardcopy posters: If attending in-person, you may bring along a printed copy of your poster (maximum A1 size, portrait format) to be displayed during the conference.
- You may be assigned a specific day to display your poster.
Any further information about the poster presentations at this digital meeting will be available in the future.
Before uploading your poster, you must make sure that you follow ALL of the instructions above!
(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.
Development of a phage-based deodorant: novel phages targeting Staphylococcus species efficiently destroy bacterial biofilms
Abdullah A AlAhmadi1, Andrew Millard3, Edouard Galyov2
Department of Genetics and Genome Biology, University of Leicester, Leicester LE1 7RH, UK
Several Gram-positive bacterial species are implicated in the development of armpit malodour. Compounds such as volatile fatty acids and thioalcohols are products of microbial metabolism of odorless substrates secreted predominantly from the sebaceous, eccrine, and apocrine glands of the human axilla. Members of the Genus Staphylococcus are believed to be significant contributors to malodour. Malodour-associated compounds have traditionally been masked by the application of deodorant-based products. However, fragrant-associated compounds break down relatively quickly leading to the re-emergence of body odour. The removal of the causative microbial agents may lead to the elimination of the distinctive smells associated with human body odour. Increasingly, many deodorant products are formulated to contain antimicrobial compounds that whilst extending the amount of ‘protection’ provided to the consumer, can be viewed as indiscriminate in their modes of action (broad-spectrum killing). This is deemed to be an unwanted effect due to the important roles many microorganisms perform in maintaining skin homeostasis. The application of lytic bacteriophages (phages) that show specificity towards malodour-producing species could therefore be selectively used to remove malodourous bacteria. In a cosmetic context, phages offer a natural solution towards controlling the levels of target bacteria in the human axilla. The highly specific nature of lytic phages can be employed to eliminate/control malodour-producing microbes whilst leaving the remaining axilla-associated microbiota intact. This project is focused on the isolation and characterisation of bacterial species colonising human skin and phages targeting odor-producing Staphylococcus genera. Several phages were isolated and their genomes sequenced. Many of the isolated Staphylococcal strains showed the ability to form biofilms. We showed the ability of two isolated bacteriophages to efficiently destroy biofilm formed by S. haemolyticus and S. lugdunensis which is one of the main bacterial species associated with production of body malodour.
Identifying skin bacteriophages of Staphylococcus to determine population and community dynamics
Samah Alsaadi, Hanshuo Lu, Heather E Allison and Malcolm J Horsburgh
Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 7ZB, UK
Human skin is colonised by diverse microbial communities that are important for skin health. Species of coagulase-negative Staphylococcus are highly abundant and vary across the different skin regions, with S. epidermidis being the most frequent. We seek to investigate how the skin virome influences the dynamics of staphylococcal populations in bacterial communities of the skin microbiome, with focus on skin bacteriophages infecting Staphylococcus spp. We collected swabs from >80 healthy volunteers across different skin sites to isolate cutaneous phages that infect different Staphylococcus spp. From the swabs we isolated, purified and sequenced the genomes of 42 phages that infect 8 different Staphylococcus species, including multiple isolates of a novel phage. We assessed qualitatively the degree of phage resistance using a wide host range of 138 Staphylococcus strains of the 8 species. Among the coagulase-negative staphylococci tested, S. hominis exhibits broad resistance to phage infection. We hypothesise that S. hominis encodes pathways to limit phage infection and have explored its mechanisms using experimental evolution by selecting for phage resistance. We also determined that S. aureus was resistant to every phage isolated from the skin that infects coagulase-negative staphylococci. The phages that infect staphylococci will inform our studies of their potential contributions to skin population dynamics and dysbiosis and will enable interrogation of metagenomic datasets to explore relationships with skin health.
Isolation and characterization of bacteriophages with lytic activity against S. enteritidis as potential bio-control agents
Shahad A Alsalman1, Fahad M Alreshoodi1, Khawlah K Alotaibi2, Saleh I Alaqeel1, Mohammed Imam3 , Nada F Alosaimi4 , Abdulmohsen L Alharbi1 , Elaf A Alshodokhi1, Ashwaq S Alhamed1, Suliman M Alajel1
1Reference Laboratory for Microbiology, Saudi Food and Drug Authority, Riyadh, Kingdom of Saudi Arabia
2Microbiology Department, Collage of science , King Saud University, Riyadh, Kingdom of Saudi Arabia
3Infection and Immunity Department, College of Medicine, Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia
4Advanced Diagnostic And Therapeutic Institute, King Abdulaziz City for Science and Technology, Riyadh, Kingdom of Saudi Arabia
Salmonella Enterica serotype Enteritdis is a common foodborne pathogen cause severe gastrointestinal illness in humans. Poultry products are a major source of S.Enteritidis, and conventional control measures such as antibiotics and vaccination has limited effectiveness were has been reported in recent years, and become a major public health concern. Bacteriophages have been explored as promising alternatives to conventional methods for Salmonella biocontrol in poultry and production systems. Moreover, phages are generally considered safe for human consumption and non-target organisms, making phages a potentially environmentally friendly approach to controlling bacterial pathogens. In this study, we develop new strategies for preventing and controlling S.Enteritidis which is essential to restrict the use of antibiotics in poultry in Saudi Arabia. Nine different phages were isolated from poultry farms in Riyadh, Saudi Arabia, and chosen for further purification, propagation, and characterization using transmission electron microscopy, whole genome sequencing, and evaluation the efficiency of isolated phages against Salmonella strains resistant to many antibiotics.
Use of the bacteriophage product Custus®YRS for biological control of Yersinia ruckeri and disease prevention in Atlantic salmon
Eirik Bårdsen1, Ingrid Støtvig1, Cyril Frantzen1, Jakob Mo2, Bjørn Gillund2, Hans Petter Kleppen1
1ACD Pharma, Storeidøya 60, 8370 Leknes, Norway
2SinkabergHansen AS, Marøya, 7900 Rørvik, Norway
After incidents of yersiniosis-induced mortality and reduced fish welfare shortly after sea transfer of Atlantic salmon at two Sinkaberg-Hansen locations, the bacteriophage product Custus®YRS was included in an extensive plan for disease control. Custus®YRS was used to control the infection pressure of Yersinia ruckeri in well boat water during transport and freshwater de-licing, operations known to trigger yersiniosis outbreaks. Water sample analyses showed that the bacteriophages efficiently controlled the infection pressure of Y. ruckeri during the operations. No yersiniosis related mortality was observed after the operations. A comparison study was performed, in which two fish groups, both sub-clinical carriers of Yersinia ruckeri, were de-liced with the freshwater treatment “Freshwell”, one group with Custus®YRS and the other without. The one group de-liced without Custus®YRS experienced a typical yersiniosis outbreak. The group de-liced with Custus®YRS-bacteriophages present in the well boat water had no signs of yersiniosis outbreak and performed similar to no-yersiniosis reference groups at the same location. This study show that bacteriophages can be used to control bacterial infection pressure in fish holding water, and through it prevent triggering of disease outbreaks.
Identification of new endolysins and depolymerases in FAGOFARMA‘s microbial collection
Tomáš Bartejs1,2, Kristina Čuprová1,2, Martin Benešík1,2,3, Kateřina Plachá1,2, Simona Košiarčiková1,2, Tibor Botka3, Marek Moša1,2,4
1MB Pharma s.r.o., Prague, Czech Republic
2FAGOFARMA s.r.o., Prague, Czech Republic
3Masaryk University, Faculty of Science, Department of Experimental Biology, Brno, Czech Republic
4Charles University, Faculty of Science, Prague, Czech Republic
Privately owned company FAGOFARMA in association with company MB Pharma curates and constantly expands their collection of microbes and bacteriophages. Most microbes in collection are pathogens (A. baumanni, K. pneumoniae, P. aeruginosa, S. aureus etc.) with multiple resistances and have proven as a great source for new bacteriophages isolation and identification. Huge potential of this collection still lies in its genomic data – genomes of both bacteriophages and bacteria can be used for novel enzybiotic identification. In this study, over 40 genomes of bacteria/bacteriophage were analyzed. Many different prediction tools were tested, but in the end two main approaches emerged. Firstly, genomes were subjected to automatic annotation, potential lytic enzymes were then further analyzed with domain prediction software and compared to already annotated enzybiotics. Some of the identified enzymes were completely new. For the second approach, depolymerase prediction software was used. This approach promised identification of enzymes with depolymerase-like activity. Although this identification is still highly experimental due to current lack of deeper understanding of depolymerase structure and lack of prediction software, few more depolymerase-like enzymes were identified. These enzymes could manifest depolymerase-like activity, which could prove extremely helpful against biofilm producing bacteria. Degradation of biofilm should in turn help to increase lytic activity of other enzybiotics. Although all of these are just predictions, which will need to be experimentally proven. But the advantages of collecting and curation of not only the microbial/phage physical collection, but also its bioinformatic data are already clear.
Virulent Salmonella phages can overcome phenotypic resistance conferred by O-antigen phase variation in the gut
Anouk Bertola1*, Nicolas Wenner1*, Nahimi Amare Bekele1, Louise Larsson1,2, Valentin Druelle1, Andrea Rocker1, Camilo Perez1, Alexander Harms1,2 and Médéric Diard1
1Biozentrum, University of Basel, Basel, Switzerland
2D-HEST, ETH Zürich, Zürich, Switzerland
*These authors contributed equally
The enteropathogenic bacterium Salmonella enterica serovar Typhimurium (S.Tm) causes enteric disease in a wide range of hosts. Bacteriophages (phages) have been proposed as potent control agents to control Salmonella. Here, we address the role of phenotypic resistance in the interactions between S.Tm and phages, and its impact on their co-existence and co-evolution. We have observed that the T5-like phage φ37 fails to eradicate and co-exists with S. Tm during multiple passages of in vitro culture and during intestinal colonization in mice. This co-existence is dependent on the presence of at least one of two epigenetically controlled O-antigen modifying systems (GtrABC and OpvAB), leading to the presence of a phage-resistant subpopulation of bacteria. This prevents the fixation of mutations in phage receptor, BtuB. Double inactivation of GtrABC and OpvAB prevents co-existence in vitro and in vivo. During long-term infections in mice we observed the evolution of phage φ37 and rise of btuB mutants in the bacterial population. The phages accumulate mutations in the L-shaped fiber, which allow the infection of previously resistant subpopulations of opvAB or gtrABC-expressing bacteria. This increased the selective pressure and drove the bacteria towards fixation of btuB mutations. Our data demonstrated that virulent phages can evolve to overcome phenotypic resistance conferred by O-antigen phase variation, leading to the fixation of phage receptor mutations, potentially costly for Salmonella.
Characterization of the Contributions a Detoxified Stx-Prophage Makes to the Fitness of Its Bacterial Host
Yueyi Cai, Heather E. Allison
Department Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, UK
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 research focuses on identifying the function of a hypothetical gene, vb_24B_13c, from Stx-phages Φ24 towards the fitness of its host. Multiple approaches including RNA-seq, NanoString datasets, bioinformatics analyses, 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 motility of MC1061 pBAD_13c induced by arabinose was enhanced at 37℃ compared to the naïve cell, indicating prophage gene vb_24B_13c can contribute to the motility of naïve cells even without the presence of other prophage genes. However, there was no enhanced motility of MC1061 pBAD_13c at 30℃, suggesting that temperature is a factor that affects the host’s motility. RNA-seq data, as validated by an amylase assay, showed that α-amylase activity is upregulated in the lysogen. Moreover, NanoString data revealed that overexpression of prophage repressor gene, cI, could potentially inhibit the expression of other prophage genes, including vb_24B_13c. This indicates that the lysogen’s expression of cI is not high enough to repress these prophage genes. In conclusion, the expression of prophage vb_24B_13c can enhance the motility of its host cell by upregulating genes involved in flagellar synthesis and rotation, and it can inhibit the expression of some its downstream genes. Moreover, these results confirm that the prophage plays a significant role in reprogramming the metabolic function of the host, indicating a profound influence on the host’s phenotype.
Determining the Relationship between Growth Media and T1 Phage Growth Rate
Eric R I Chapman1, Aidan T Brown1, Teuta Pilizota2
1School of Physics and Astronomy, University of Edinburgh, UK
2School of Biological Sciences, University of Edinburgh, UK
Bacteria play a huge role in the human and natural world, for richer and for poorer. Their most well-known role being a pathogen. With the gradual reduction in antimicrobial efficacy, the promise of phage as a potential solution has gathered attention. Bacteria are also able to rapidly develop resistances to bacteriophage through numerous pathways. Here I have investigated how the bacteria-bacteriophage system dynamics depends on the carbon source being used by the bacteria. Escherichia coli, grown in a range of media with various carbon sources, were exposed to T1 bacteriophage. In all cases the bacteria developed a resistance to the phage at long times with long-read and short-read sequencing of surviving mutant bacteria. However, the population dynamics of the system both post and during infection varied with both carbon source and the number of phage added. Through initial fitting of the phage dynamics, it appeared that the growth rate of phage was uncorrelated with the growth rate of the bacteria. A more sophisticated model has then been created to probe which factors are dominant in the growth of a phage population. These results suggest a coupling between carbon source and the progression of a bacterial populations response. These results contribute to the understanding of the dominant factors in the growth of phage which could result in significant differences between the rates of death and resistance development in vivo and in vitro due to differing environmental conditions.
Enhancing phage therapy safety: reliable and sensitive phage genome annotation with rTOOLS2 high-throughput pipeline
Antoine Culot, Guillaume Abriat
Rime Bioinformatics SAS, Palaiseau, France
Phage therapy is an exciting and promising approach to fight bacterial infections. However, ensuring the safety and efficiency of phages for therapeutic use requires a thorough understanding of their genomic properties. This process enables detection of genes that make phages potentially harmful for the subject of the therapy or the environment, such as antibiotic resistance, lysogeny, and virulence genes. Traditional bioinformatics tools designed for bacterial genomes are not well-suited for phage genomes due to their unique structure, leading to poor gene calling and function annotation. Recently, phage-focused tools have been released, such as Pharokka and rTOOLS2. rTOOLS2 is a multi-hypothesis, phage-focused annotation pipeline: its advanced algorithm uses the output produced by widely-used annotation tools to find more gene functions, with high evidence thresholds to avoid false positives. In this study, 135 phage genomes published in Genbank were annotated using Pharokka and rTOOLS2’s high-thoughput version, and the results were compared. Pharokka was able to improve the average published annotation, as the average number of genes functionally annotated grew from 29.5% to 35.9%. On the other hand, rTOOLS2’s high-throughput version was able to significantly increase the rate of annotated genes, reaching 54.6%. To promote the safest possible use of phages for patients and for the environment, it is key to use thoroughly characterized phages. rTOOLS2’s high-throughput version can rapidly provide a strong basis for genome characterization. The use of curated databases ensure that meaningful annotations are provided, and results can be published with low risk of public database poisoning. Moreover, rTOOLS2 is able to produce more information, as it nearly doubled the number of annotated genes in the initially published genomes.
Recombinant exopolysaccharide-depolymerases of the three broad-host-range phages, infecting phytopathogenic bacteria
Yuliia Faidiuk1,2,3, Maryna Zlatohurska2,4, Natalia Shenderovska2,5, Tetiana Gorb2, Liudmyla Romaniuk2, Maja Muszer6, Alla Kushkina1,2, Fedir Tovkach2
1Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
2Institute of Microbiology and Virology of the National Academy of Science of Ukraine, Kyiv, Ukraine
3ESC “Institute of Biology”, Taras Shevchenko Kyiv National University, Kyiv, Ukraine
4Central European Institute of Technology, Masaryk University, Brno, Czech Republic
5YuriaPharm LLC, Kyiv, Ukraine
6Wroclaw University of Science and Technology, Wroclaw, Poland
Phage exopolysaccharide depolymerases (EPSDs) are often represented by tail spike proteins that harbour lytic domain, capable of degrading bacterial exopolysaccharide (EPS). Recombinant EPSDs are of specific interest for structural studies as a basis for further crystal structure obtaining, as well as are regarded as promising tools for pathogen biocontrol. EPS, a major component of a bacterial capsule and biofilm matrix, functions as a shield against environmental factors, antibiotics, as well as host defense mechanisms. Application of EPSDs for reducing the amount of EPS in natural conditions can lead to a decrease of the pathogen virulence and resistance. Recently we have sequenced and characterized the genomes of three broad-host-range bacteriophages E105, TT10-27, and KEY, active against plant-infecting bacteria of Erwinia and Pantoea genus, including fire-blight disease-causing agent, Erwinia amylovora. In all three genomes EPSD genes were detected, coding for proteins of 93.50, 95.5, and 111.5 kDa, respectively. Relevant genes were cloned into pET22b(+) vector. Due to the large size of the proteins, soluble forms were obtained only after testing a large set of expression conditions. These eventually were selected to be: cotransformation with pGro7 chaperone plasmid, induction with 0.05 mM IPTG at OD 0.4, 20°C for 17 hours; autoinduction media, and\or OverExpress(tm)C41(DE3) expression strain. When applied on the lawn of host bacteria in conditions that stimulate EPS-overproduction, all three EPSD proteins were efficiently degrading the EPS of E. billingae and P.agglomerans, however, did not reveal a substrate specificity against E.amylovora EPS. Obtained results indicate that in natural conditions, when EPS is overproduced, EPSD action is required for efficient infection, while in case of its absence phages may possess another mechanism or receptor-binding protein that allows them to infect a more wide spectrum of bacteria.
The Citizen Phage Library: rapid isolation of phages for the treatment of antibiotic resistant infections in the UK
Julie Fletcher1, Robyn Manley1, Christian Fitch1, Christina Bugert1, Karen Moore1, Audrey Farbos1, Michelle Michelsen1, Shayma Alathari1, Nicola Senior1, Alice Mills3, Natalie Whitehead3, James Soothill2, Stephen Michell1, Ben Temperton1
1Biosciences, University of Exeter, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK
2Microbiology, Virology and Infection Control, Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street, London, WC1N 3JH, UK
3Exeter Science Centre, Kaleider Studios, 45 Preston Street, Exeter, EX1 1DF, UK
Antimicrobial resistance poses one of the greatest threats to global health and there is an urgent need for new therapeutic options. Phages are viruses that infect and kill bacteria and phage therapy could provide a valuable tool for the treatment of multi-drug resistant infections. In this study water samples collected by citizen scientists as part of the Citizen Phage Library (CPL) project and wastewater samples from the Environment Agency yielded phages with activity against clinical strains K. pneumoniae BPRG1484 and E. cloacae BPRG1482. A total of 169 and 163 phages were found for K. pneumoniae and E. cloacae respectively within 4 days of receiving the strains. A third strain (E. coli BPRG1486) demonstrated cross-reactivity with 42 E. coli phages already held in the CPL collection. Seed lots were prepared for four K. pneumoniae phages and a cocktail combining these phages was found to reduce melanisation in a Galleria mellonella infection model. The resources and protocols utilised by the Citizen Phage Library enabled rapid isolation and characterisation of phages targeted against multiple strains and in future, within a clearly defined regulatory framework, phage therapy could be made available on a named-patient basis within the UK.
Engineering a Rapid Phage Modification Platform for Phages Targeting Salmonella Enterica Serovars
Nicolas E Fournier1,2, Marielou Tamayo1, Henrik Almblad1, Deborah Court2, Steven Theriault1,2
1Cytophage Technologies Inc., 26 Henlow Bay, Winnipeg, MB R3Y 1G4, Canada
2University of Manitoba, Department of Microbiology, Winnipeg, MB R3T 2N2 Canada
In the industrial agriculture sector, Salmonella Enterica presents a major biosecurity concern through opportunistic infections leading to disease-causing transmission. As conventional treatment methods are not always effective in disease mitigation, one area that has gained recent interest is the use of bacteriophages (phages). Selection of therapeutic phage candidates relies on thorough characterization to determine lifestyle, define genetic safety signatures, and host range. Ideal candidates will have a strict host range such that disease-related serovars are specific targets. Current methods for expanding the host range of phages can be time consuming, laborious, with low success rates making them impractical for rapid therapeutic development. The synthetic modification of phages is a potential avenue to circumvent these shortcomings, however the inability to utilize antibiotic or auxotrophic counter selection limits the ability of synthetic phage recovery. Recently, the incorporation of an RNA-based counter-selection system during the recombination of synthetic phages has improved efficiency and recovery of modified phages. Although these systems have seen success in model organisms such as E. coli, they have yet to be used in the modification of phages that infect S. Enterica. This work looks to engineer a Cas13a based counterselection system to expedite the modification of S. Enterica-infecting phages to improve their efficacy and increase their host range. Four genetically distinct phages with genomes ranging between 42-220 kb have been isolated and identified as candidates to evaluate the limits of modification using this platform. Finally, modified phages will be assessed against their native counterparts through growth kinetic, virulence, and host range assays to determine if modifications increased host susceptibility. This research highlights the importance of developing rapid and flexible platforms for synthetic phage construction.
Identification of cross-reacting IgG hotspots to prevent immune evasion of SARS-CoV-2 variants using phage display technology
Katarzyna Gembara1,2, Marek Harhala1,2, Krzysztof Baniecki3, Aleksandra Pikies3, Artur Nahorecki3, Natalia Jędruchniewicz1, Zuzanna Kaźmierczak1,2, Izabela Rybicka2, Tomasz Klimek1, Wojciech Witkiewicz1, Kamil Barczyk3, Marlena Kłak1, Krystyna Dąbrowska1,2
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
3Healthcare Centre in Bolesławiec, Jeleniogórska 4, Bolesławiec, Poland
The major factor that shapes the global perspective for increase or diminution of successive pandemic waves of COVID-19 is the immunological protection. The SARS-CoV-2 virus constantly develops new variants, and capability of immune evasion is among the major factors that promote variant spreading in the human population. After two years of the pandemic and virus evolution, it is almost impossible to explain effects of all possible combinations different viral strains, a few types of vaccinations or new variants infecting an individual patient. Instead of variant-to-variant comparisons, identification of key protein regions linked to immune evasion could be efficient. Here we report an approach using phage display technology for experimental identification of SARS-CoV-2 protein regions that (i) have characteristics of cross-reacting IgG hot-spots, and (ii) are highly immunogenic. Cross-reacting IgG hot spots are regions of protein frequently recognized in many variants by cross-reacting antibodies. Immunogenic regions efficiently induce specific IgG production in SARS-CoV-2 infected patients. We determined four regions that demonstrate both significant immunogenicity and the activity of a cross-reacting IgG hot-spot in protein S, and two such regions in protein N. Their distribution within the proteins suggests that they may be useful in vaccine design and in serological diagnostics of COVID‑19.
Phage against intracellular methicillin-resistant Staphylococcus aureus
Juliet Curry1, Liam Good2, Shannon Stockdale3, Jan Kim3, Andreas Chrysanthou3, Shan Goh1
1Department of Clinical, Pharmaceutical and Biological Sciences, School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield AL10 9AB, UK
2Tecrea Ltd, London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK
3 School of Physics, Engineering, and Computer Science, University of Hertfordshire, College Lane, Hatfield AL10 9AB, UK
Methicillin-resistant Staphyloccus aureus (MRSA) is capable of invading and persisting within human host cells that become reservoirs for recurrent and chronic infections, because antimicrobials are ineffective in eliminating intracellular bacteria. There are conflicting reports on intracellular activity of strictly lytic S. aureus phages in vitro. NanocinTM is a transfection agent capable of delivering nanoscale particles into mammalian cells. This study aimed to test intracellular uptake of MRSA phages with NanocinTM in an in vitro double infection model using human keratinocytes HaCaT cells. A virulent phage fSW1-40 active against EMRSA-15, EMRSA-16 and USA300 strains was isolated. It is a Twort-like myovirus with a genome of 142 kb. NanocinTM at 0.125 and 0.5 mg/mL resulted in £2.7 ´ increase in particle size and £3.7 ´ decrease in phage titre. MRSA at 106 cfu/mL within HaCaT cells were treated either with 108 pfu/mL phage, phage- NanocinTM (105-107 pfu/mL+ 0.5 mg/mL), or NanocinTM (0.5 mg/mL). Intracellular MRSA survival after 18h was 18% when treated with phage alone, 25% with phage- NanocinTM, and 45% with NanocinTM alone. Interestingly, intracellular phage titre 18h post-infection was 2.3 ´ greater with NanocinTM than without, despite 10-100 ´ lower starting phage titres with Nanocin, suggesting intracellular phage uptake or accumulation mediated by NanocinTM. The double infection HaCaT cell model showed phage killing of intracellular MRSA, similar to a previous study using MAC-T bovine mammary alveolar cells but in contrast to another study on MG-63 osteosarcoma cells. NanocinTM at low concentrations had marginal effects on phage particle size, but aided intracellular phage uptake, which was inefficient. Nevertheless, intracellular MRSA survival was reduced by phage, indicating their potential in effective elimination of intracellular bacteria.
Phase-Variable Capsular Polysaccharides of Bacteroides Regulate Bacterial Phage Susceptibility via the Adsorption Phase of Infection
Ekaterina Goldobina, Niamh A. Madden, Andrey N. Shkoporov
APC Microbiome Ireland, University College Cork, Cork, Ireland
Members of the Bacteroidetes phylum, the main inhabitants of the human gastrointestinal tract, produce various types of capsular polysaccharides (CPS) and can switch their expression on and off in a process called phase variation. Previous studies have demonstrated the significant role of these surface structures in virus-host interactions. Bacteroides thetaiotaomicron VPI-5482 and Bacteroides intestinalis APC919/174 can modulate the expression of multiple CPS loci to evade phage infections.The objective of this research was to establish a connection between the ability of crAss-like phages (DAC15, DAC17, and crAss001) to infect different CPS variants of their hosts, B. thetaiotaomicron VPI-5482 and B. intestinalis APC919/174, and the capability of these CPS variants to adsorb the phages. We tested three bacteriophages against ten CPS variants of B. thetaiotaomicron and three CPS variants of B. intestinalis in various combinations. The adsorption of phages to bacteria was assessed using plaque assays.The expression of phase-variable CPS in Bacteroides species, specifically B. intestinalis and B. thetaiotaomicron, significantly influences the susceptibility of bacteria to crAss-like phages. The results of this study demonstrate that this regulatory effect occurs during the adsorption stage of infection.
Phage-mediated horizontal gene transfer: exploring the network
Bianca Govi, Andrey Shkoporov
APC Microbiome Ireland, University College Cork, Cork City, Ireland
Bacteriophages are biological entities evolved to package and transfer genetic material from one bacterial cell to another. As viruses, they do so to the effect of propagating their own genomes by parasitizing bacterial cells’ biochemical machinery. There are instances in which phages and phage-adjacent elements can form viral-like particles (VLP) carrying fragments of host genome, providing a route for horizontal gene transfer in the microbial community. The Horizon2020 PhaGeNet project aims to investigate the prevalence and function of this phenomenon, with a special focus on the human gut microbiome. We are investigating how transducing bacteriophages, phages capable of packaging and transferring non-own DNA, can impact microbial community dynamics, the spread of functional genes, and modulate microbiome evolution. The experimental approach taken involves propagation of human faecal microbiomes by continuous anaerobic fermentation in a cell-fee VLP-recirculation system, the creation of transposon libraries carrying marker genes, and pre-clinical in vivo experiments. Long read and shotgun VLP and total metagenomic sequencing will be used to track the packaging and transduction of DNA fragments under different environmental conditions and in different microbial assemblages. The project seeks to identify patterns in phage-mediated transduction, to answer question such as: how common is non-own DNA packaging versus successful transduction? Is it possible to predict a phage’s transduction ability from genome features? The project will also deliver a collection of phage-host pairs and investigate the compatibility of phage and hosts from sympatric and allopatric gut environments to assess the extent of bacteriophage host specificity.
Tangential flow filtration as a modern tool for a quality control of phage lysate – story of deoxycholate
Rostislav HALOUZKA1,2, Dominik CHMELÍK1,2, Kateřina PLACHÁ1,2, Martin BENEŠÍK1,2,3, Tomáš BARTEJS1,2, Marek MOŠA1,2,4
1MB PHARMA s.r.o., Prague, Czech Republic
2FAGOFARMA s.r.o., Prague, Czech Republic
3Masaryk University, Faculty of Science, Department of Experimental Biology, Brno, Czech Republic
4Charles University, Faculty of Science, Prague, Czech Republic
TFF (Tangential Flow Filtration) is a pressure controlled membrane separation process based on a molecular size. In general, this is a remarkably effective method for concentrating and desalting the sample. It can be used to separate biomolecules by size, capture cell suspensions, clarify fermentation broths or phage lysates. Here, we present an optimized purification procedure and liquid chromatography (HPLC) detection method that provides relevant data and enables process quality control of phage lysate during and even after the product purification by TFF. Moreover, the workflow is set in accordance with the guidelines of the GMP. We focused on determining the amount of sodium deoxycholate (DOC), which can negatively affect the lifespan of some bacteriophages. Commonly used buffers, stabilizers or detergents were completely removed during the purification process by TFF. It mainly involves the removal of bacterial residues and impurities. The highest working concentration in purification protocol of DOC did not exceed 1 %. Some phages reacted negatively to its presence. A significant decrease was observed after exposure (≥ 1 h) in the titer value by several orders of magnitude. It is important to mention that DOC is a necessary component for successful purification. Without its use, the final sample has shown a higher endotoxin level and total protein content. The introduction of new control elements for determining the quality of phage preparations is the key to successfully guarantee their safety, effectiveness, identity and purity by using suitable modern analytical tools and methods, not only to the public, but also to customers.
Isolation and characterization of bacteriophages infecting Serratia marcescens as a novel treatment for antibiotic resistant infections
Mia Horton, Lesley Hoyles, David Negus
Department of Biosciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK
Serratia marcescens has been responsible for a notable increase in multidrug resistant infections in both the healthcare and community setting. In the UK, the independent emergence of multidrug resistant clones suggests that future treatment of S. marcescens with antibiotics will become increasingly difficult. We aimed to isolate lytic bacteriophages specific to S. marcescens and to identify encoded depolymerases. Such depolymerases could be used as anti-virulence agents and therefore therapeutic alternatives to antibiotics. Phages were isolated from river water sampled from around Nottingham using three clinical strains of Serratia marcescens as hosts. 11 morphologically distinct plaques from different water sources were picked to purity. Genomic DNA was purified and sequenced in-house using the Illumina MiSeq platform. Genome assembly and comparative genomics revealed four genetically distinct phages; 1a, 6a, 10b1 and 12. Host range analysis of the four phages was performed using a range of 15 clinically relevant Serratia strains. Phage morphology was visualised by transmission electron microscopy. Three of the phages displayed halos on host lawns indicative of capsule-degrading enzymes. Four candidate depolymerase genes were identified using the machine learning depolymerase prediction tools PhageDPO and DePP. Candidate depolymerase genes were cloned and expressed within E. coli. A halo on spot tests indicated the soluble fraction of the crude lysate containing the expressed minor tail protein from phage 12 possessed potential depolymerase activity. This research contributes to the limited range of S. marcescens phage genomes already published. Furthermore, the phage encoded depolymerases have the potential to be used in future therapy to mitigate the effects of multidrug resistance in S. marcescens.
Predicted endolysins from phages with broad lytic activity against Shiga toxin producing Escherichia coli of diverse serogroups
Ana E Juárez1,2, Victoria A Rodríguez1,2, Stefanía B Pascal1,2, Melany Dualde1,2, Alejandra Krüger1,2, Paula M A Lucchesi1,2
1Facultad de Ciencias Veterinarias, CISAPA, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Tandil, Argentina.
2Centro de Investigación Veterinaria de Tandil (CIVETAN), UNCPBA-CICPBA-CONICET, Tandil, Argentina.
Shiga toxin-producing Escherichia coli are important causative agents of foodborne outbreaks. They can cause serious illnesses such as haemolytic uraemic syndrome. The use of phages and their enzymes are novel strategies which show a high antibacterial potential. Among these enzymes are the endolysins, used by double-stranded DNA phages to degrade the cell wall at the end of their virulent cycle. Endolysins can be classified according to their capacity to cleave linkages in the peptidoglycan layer as glycosidases, amidases, and endopeptidases. In this work, phages isolated from the dairy environment and minced meat were chosen for genome characterization. Previous studies have shown the potential of these phages to lyse strains belonging to different relevant STEC serogroups. DNA was purified with a commercial kit from high-titer stocks, and sequenced using Illumina technology. Ten contigs containing complete phage genomes were characterized. Virus genera were assigned using the software Kraken. Genomes were annotated with RAST and putative endolysins were screened with BLAST, Interpro, Uniprot and HHpred. A molecular phylogenetic analysis of the predicted endolysins was performed by maximum likelihood method conducted in Mega 7. Seven different genomes were further characterized corresponding to different genera: Tequatrovirus (3), Vequintavirus (2), Mosigvirus (1) and Gamaleyavirus (1). Predicted endolysins were analysed and showed to encode six different amino acid sequences. The enzymatically active domains belonged to the glycoside hydrolase family 24 (IPR002196) except one that corresponded to the glycoside hydrolase family 108 (IPR008565). The phylogenetic tree grouped endolysins in accordance to the phage genus. In conclusion, this work showed diversity in the phages we isolated against STEC and also in the endolysins they encode. Further studies will be performed to continue their characterization as antimicrobial agents against this pathogen.
Unravelling Phage-Mediated Gene Transfer in Complex Ecosystems: A Single-Cell Perspective on Gut Microbiome-like Dynamics
Sara Haghayegh-Khorasani1, Marc G Aucoin1, Brian P Ingalls2
1Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada
2Department of Applied Mathematics, University of Waterloo, Waterloo, Ontario, Canada
The mysterious nature of phage-mediated gene transfer across diverse ecosystems, including within the intricate environment of the gut microbiome, has persisted due to several factors, including limited insights into behaviours of individual cells. Our study aimed to target this gap by investigating the infection process of the M13 bacteriophage within a bacterial population, while also exploring the intricate dynamics of single-cell behaviours over an extended period. Utilizing microfluidic devices designed to resemble the gut microbiome’s structure, we aimed to introduce a level of similarity to the complex gut environment. To enable precise tracking of the infection process, we engineered the M13 phage to carry a fluorescence gene. Through gene transfer, the phage transferred the fluorescence gene to their bacterial hosts, allowing us to monitor infections with accuracy. Subsequently, using fluorescence microscopy, we captured a sequence of time-lapse images. These images were then segmented and subjected to analysis, facilitating the extraction of individual cellular data from the recorded frames. Exploring the heterogeneity within populations, we then proceeded to quantify a noticeable delay time between the introduction of phage and the detection of a fluorescent protein signal. This delay aligns with our understanding about how fluorescent proteins mature and the intricate steps involved in starting an infection. Our data analysis further covers a range of aspects, including elongation rate, the distribution of cell lengths, and the impact on cell health following infection. As we move forward, this research can be extended by investigating situations that more closely resemble the gut microbiome’s complexities, including employing multiple cultures or replicating specific environmental conditions. By doing so, we aim to gain a deeper understanding of the infection process and its relevance to the gut microbiome’s intricate dynamics.
Steering evolution of pathogenic K1 E. coli in the gut with bacteriophages
Louise Larsson1,2, Anouk Bertola2, Nicolas Wenner2, Jasmine Kaur1, Emma Slack1, Médéric Diard1
1Institute of Food, Nutrition and Health, ETH Zurich, Switzerland
2Biozentrum, University of Basel, Switzerland
The gut can serve as a reservoir for opportunistic pathogens, such as multidrug resistant Escherichia coli (E. coli). While harmless in the gut, these strains can cause severe extraintestinal infections, including recurring urinary tract infections and life-threatening sepsis in patients at risk. Prevalent sepsis strains are protected by the polysaccharide capsule K1. This capsule type promotes resistance to the host’s humoral immune response by biological mimicry and thus prevents vaccination strategies. It is therefore crucial to develop new approaches to specifically target the K1 capsule and exclude these pathogens from the gut microbiota. While the capsule is an important virulence factor, it can also be a target for bacteriophages (phages) that attach to it in order to infect and kill the bacteria. In this study, we have isolated a cocktail of K1 capsule-specific phages that can infect a range of K1 E. coli patient isolates. We show that our phage cocktail exerts a strong selective pressure against encapsulated bacteria in the murine gut, hence rapidly driving the emergence and fixation of capsule-less mutants. As a result, the surface antigens of the mutant population are now exposed, and the strain is disabled from systemic survival. Overall, we show that we can rationally steer the evolutionary trajectory of pathogenic strains in the gut to become less virulent and disabled from extraintestinal replication, thus reducing the risk of sepsis.
Prediction of virus-host associations using protein language models and multiple instance learning
Dan Liu1, Francesca Young1, David L Robertson1, Ke Yuan2
1MRC-University of Glasgow Center for Virus Research, Glasgow, UK
2School of Computing Science, University of Glasgow, Glasgow, UK
Predicting virus-host associations is essential to determine the specific host species that viruses interact with, and discover if new viruses infect humans and animals. Currently, the host of the majority of viruses is unknown, particularly in microbiomes. To address this challenge, we introduce EvoMIL, a deep learning method that predicts the host species for viruses from viral sequences only. It also identifies important viral proteins that significantly contribute to host prediction. The method combines a pre-trained large protein language model (ESM) and attention-based multiple instance learning to allow protein-orientated predictions. Our results show that protein embeddings capture stronger predictive signals than sequence composition features, including amino acids, physiochemical properties, and DNA k-mers. In multi-host prediction tasks, EvoMIL achieves median F1 score improvements of 8.6%, 12.3%, and 4.1% in prokaryotic hosts, and 0.5%, 1.8% and 3% in eukaryotic hosts. EvoMIL binary classifiers achieve impressive AUC values of over 0.95 for all prokaryotic and ranging from roughly 0.8 to 0.9 for eukaryotic hosts. Furthermore, EvoMIL estimates the importance of single proteins in the prediction task and maps them to an embedding landscape of all viral proteins, where proteins with similar functions are distinctly clustered together, highlighting EvoMIL’s ability to capture key proteins in virus-host specificity.
PhageLimp: An interventional study using bacteriophages to combat the AMR dissemination in hospitals from Brazil
Willames M. B. S. Martins1,2, Aghata C. Ribeiro2, Michael H. Lenzi2, Camila Siqueira2, Ikechukwu Moses2, Fernanda Santos2, Kirsty Sands1, Aditya Lankapalli1, Tazio Vanni3, Leticia Sudrack3, Julival Ribeiro3, Ricardo D. Guzman3, Priscila Dantas4, Eduardo Medeiros5, Luiz Felipe V. de Oliveira6, Ana P.Christoff6, Bianca L. Texeira6, Timothy Walsh1, Ana C. Gales2
1Ineos Oxford Institute, Department of Biology, University of Oxford, Oxford, UK
2Division of Infectious Diseases, Department of Internal Medicine. Escola Paulista de Medicina/Universidade Federal de São Paulo – UNIFESP, São Paulo, Brazil
3Hospital de Base de Brasília, Brasília, Brazil
4Hospital Municipal do Campo Limpo, São Paulo, Brazil
5Department of Internal Medicine, Division of Infectious Diseases, Hospital São Paulo, Universidade Federal de São Paulo, Unifesp, SP, Department of Hospital Epidemiology and Antimicrobial Stewardship of Hospital São Paulo, Universidade Federal de São Paulo, Unifesp, SP, Brazil
6BiomeHub, Florianopolis, Brazil
PhageLimp is a prospective, interventional, single-blinded clinical study that is starting to be performed in Brazil, aiming to reduce/stop the transmission of MDR K. pneumoniae in intensive care units (ICU) using phage cocktails. The study is constituted by three major phases: i) Standardisation of an in vitro protocol of surface decontamination; ii) Epidemiological approach; iii) Interventional step. For this purpose, two ICUs of four hospitals in Brazil were selected based on K. pneumoniae infections rates before the study. For the standardization step, different surfaces (glass, plastic, stainless steel) were contaminated with an MDR K. pneumoniae ST16 strain and cleaned with a phage cocktail. The epidemiological approach aims to understand the circulation of different clones of K. pneumoniae strains (before, during, and after intervention) in each ICU during the study (in total, nine months of epidemiologic follow-up). Based on K. pneumoniae epidemiology, we will design phage cocktails and use them as bio disinfectants, applying them in the ICUs every day for 3 months (interventional period). The impact of phage cocktails use will be assessed by i) collecting/analysing K. pneumoniae colonisation rates, density of infection and antimicrobial consumption before, during, and after the intervention; ii) environmental microbiology cultures looking for K. pneumoniae across the ICUs; iii) Microbiome analysis. The first step of this study was concluded since we were able to demonstrate that phages can reduce the abundance of bacterial cells on different surfaces after our phage cocktail application. In the following months, we will be starting the epidemiological investigation of K. pneumoniae in each hospital. The study is under development, but much remains to be done and improved. The proposal is to present a pilot project and adapt it for future interventions in other hospitals and other countries.
Screening and development of effective phage cocktail against Acinetobacter baumannii
Lalit Mohan1,2, Pablo Bifani1,2,3*
1A*STAR Infectious Diseases Labs (A*STAR ID Labs), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos #05-13, Singapore 138648, Singapore
2Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
3Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom
A. baumannii, an opportunistic pathogen of increasing concern worldwide due to the outbreaks caused by its multidrug-resistant (MDR) strains. Amongst all the prevailing sequence types (ST), ST1 and ST2 are most prevalent in Asia. Bacteriophage therapy is now re-emerging as an alternative therapy to treat bacterial infections especially the ones caused by MDR strains. The objective of the present study is to isolate bacteriophages against selected ST1 and ST2 A. baumannii and to study the spectrum of action of the identified bacteriophages. To access the effectiveness of single phage v/s phage cocktail against selected bacterial strains. Bacteriophage hunting was carried out using selected sequence type ST1 and ST2 from sewage samples obtained from wastewater treatment plant. Upon isolation, the phages were amplified to obtain high titer for further application. The isolated phages were then tested against 101 clinical isolates from Singapore of Acinetobacter spp. The phages showing lytic activity against majority of the tested strains were then used to perform bacterial killing assay as to access the effectiveness of single phage vs phage cocktail. 30 distinct bacteriophages targeting A. baumannii ST 1 and 2 were isolated. Phages AbauΦ 5, AbauΦ 8 and AbauΦ 9 were found to be showing high lytic activity against almost 76% of the tested bacterial species. The results from bacterial killing assay depicted that phage cocktails were more effective in reducing bacterial load as compared to single phage treatment. These phage cocktails will enable potential treatment of both ST1 and ST2 A. baumannii infections.
THE VIABILITY OF PHAGES IN ANTISEPTIC SOLUTIONS
Michaela Nováková1,2, Kateřina Plachá1,2, Tereza Šopíková1,2, Dana Štveráková1,2, Martin Benešík1,2,3, Marek Moša1,2,4
1MB PHARMA s.r.o., Lužická 1893/9, 120 00 Vinohrady, Praque, Czech Republic
2FAGOFARMA s.r.o., Lodýnská 730/59, 120 00 Vinohrady, Praque, Czech Republic
3Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
4 Charles University, Faculty of Science, Praque, Czech Republic The viability of phages is significantly affected by the physical and chemical conditions of their environment. For this reason, before using phage therapy, it is necessary to find out what other medicines they may come into direct contact with. Since superficial infections are the most common complication of wound healing, we focused this experiment mainly on the interaction between phages and products for the disinfection and cleansing of superficial wounds. For the treatment of superficial infections with phage products, it is important to know what can and cannot be used to cleanse an infected wound without compromising the effectiveness of the phage treatment. In this experiment, we focused on phages against S. aureus and P. aeruginosa. These bacteria cause a wide range of infections (e.g. in superficial wounds, burns, chronic wounds, etc.). In this experiment, commercial antiseptic products with different active substances were selected. These products could be used as rinsing solutions on infected skin, in the mouth and in the eyes. The aim of the experiment was to assess the viability of phages in selected antiseptic solutions. This was determined by a double agar overlay assay after 10 minutes, 3 hours and 24 hours. We kindly acknowledge the financial support of the project: CZ.01.1.02/0.0/0.0/15_019/0004353, VB01000025.
Characterisation of a new lytic Klebsiella phage isolated from Thai sewage
Claudia Orbegozo-Rubio1, Willames M B S Martins1 2 3, Michael Lenzi3, Jordan Mathias2, Kirsty M Sands1 2, Edward A R Portal1 2, Dann Turner4, Mark A Toleman2, Ana C Gales3, Timothy R Walsh1
1Ineos Oxford Institute, Department of Biology, University of Oxford, Oxford, UK
2Department of Medical Microbiology, Division of Infection and Immunity, Cardiff University, Cardiff, UK
3Division of Infectious Diseases, Department of Internal Medicine. Escola Paulista de Medicina/Universidade Federal de São Paulo – UNIFESP, São Paulo, Brazil
4Department of Applied Sciences, University of the West of England, Bristol BS16 1QY, UK
Phages are ubiquitous and the most abundant biological entities on the Earth. With the advancement of Whole Genome Sequencing tools, more accurate classification related to the genomic characteristics of phages is gaining prevalence to unlock their potential to treat bacterial infections. Here, we have searched for new phages against K. pneumoniae ST16 using sewage samples from Bangladesh, Brazil, and Thailand. Among our identified phages, we detected a double-strand linear DNA phage (PWKp21) in the Thai sewage with a genome size of 41,230bp, 40% of GC content, and 61 codified ORF. Although multiple attempts at phage genomic classification, this phage did not match any phage family previously described. Phylogenetic analysis confirmed this phage as representing a new family and genera. PWKp21 belongs to the newly assigned Cruzviridae family, the sub-family as Costavirinae and the genus as Zehvirus. Besides the new genetic features, the phage was stable at high temperatures (1h at 70°C) and has 30oC as the optimal infection temperature, although no statistical difference was observed between 30°C and 37°C. Following our host range assays where 300 microorganisms belonging to different species and lineages were tested, a narrow spectrum activity against only K. pneumoniae ST16 was observed. Inhibition curves demonstrated the emergence of PWKp21 resistance after 4-6 hours of incubation, however, the Galleria mellonella infection model demonstrated an increase of the survival rate up to 73% (Kpn31/MOI100). Based on these finds, we believe this new phage could be used to treat Klebsiella infections in clinical settings.
Phages Targeting Danish Soft Rot Isolates Display Difference In Host Range Even Within Species Level And Could Be Promising Biocontrol Agents
Julie Stenberg Pedersen, Alexander Byth Carstens, Magnus Mulbjerg Rothgardt, Anouk Viry, Witold Kot, Lars Hestbjerg Hansen
Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg 1871, Denmark
The bacterial diseases black leg and soft rot in potatoes causes heavy losses of potatoes worldwide. Bacteria within the genus Pectobacteriacea are the causative agents of black leg and soft rot in Potatoes. The use of antibiotics in agriculture is heavily regulated and no other effective treatment currently exists, but phages have shown promise as a potential biocontrol agent. In this study we aimed to isolate phages targeting Danish soft rot bacteria isolated from tubers and plants symptomatic with soft rot or black leg disease. Using organic waste we isolated 19 phages targeting different species within Pectobacterium. Here we focus on seven of these phages representing a new genus targeting P. brasiliense; phage Ymer, phage Amona, phage Sabo, phage Abuela, phage Koroua, phage Taid and phage Poppous. TEM image of phage Ymer showed siphovirus morphology, and phages belong to the class Caudoviricetes, with double-stranded DNA genomes varying from 40kb to 42kb. A hostrange experiment was done with 59 bacterial isolates from Danish tubers and plants symptomatic with soft rot or black leg disease. Interestingly the seven phages displayed difference in hostrange even within species level, with two of the phages being able to infect two or more species of Pectobacterium. All phages were able infect 8, or more, out of 17 isolates of P. brasiliense. In situ analysis of the P. brasiliense genomes showed difference in genes encoding antiphage systems as well as in genes involved in the outer membrane, even in closely related isolates, which correlated with host range results. None of the phages encodes any integrase or other genes typically associated with lysogeny. Based on the genome analysis together with the host range results these phages could have potential as biocontrol agents against soft rot and black leg in potatoes and should be tested further.
Comparison of different animal-free media for propagation of bacteriophage SA 402
Kateřina Plachá1, 2, Martin Benešík1, 2, 3, Michaela Nováková1, 2, Tereza Šopíková1, 2, Marek Moša1, 2, 4
1FAGOFARMA, Londýnská 730/59, Prague, Czech Republic
2MB PHARMA, Lužická 1893/9, Prague, Czech Republic
3Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
4 Faculty of Science, Charles University, Prague, Czech Republic
Cultivation of phages on standard media for therapeutic purposes may carry disadvantages and risks. Animal-based media can be a source of Bovine Spongiform Encephalopathy (BSE), Transmissible Spongiform Encephalopathy (TSE) or foot-and-mouth disease. Furthermore, their production leaves a larger CO2 footprint as well as they are also less ethically acceptable. One option how to avoid these problems is using animal-free media, in which animal components are replaced by plant components. Substitution of animal components for plant components can affect the dynamics of bacterial growth and phage multiplication. The aim of the study was to test the multiplication of antistaphylococcal bacteriophage SA 402 on selected animal-free media from Hi Media company. The vitality of overnight cultures was tested as well as the growth time of working cultures and the achieved titre in individual multiplied volumes. The results were compared with the commonly used SA medium. The most advantageous variant of the animal-free medium was determined based on these data, while also considering the cost of media per litre. Following this pilot study, further studies on the feasibility of multiplying other phages of different bacteria can be performed. We kindly acknowledge the financial support of the research projects: FX04030008, FW01010350.
Evaluation of Host Immune Responses to Mycobacteriophages Administered for Reduced Transmission of Mycobacterium tuberculosis
Thomas Smytheman1, Tiffany Pecor1, Dana Miller1, Debora Ferede1, Suhavi Kaur1, Hazem Abdelaal1, Carlos A. Guerrero-Bustamante2, Krista G. Freeman2, Graham F. Hatfull2, Rhea N. Coler3, Sasha E. Larsen1
1Seattle Children’s Research Institute, Center for Global Infectious Disease Research, Seattle, WA, USA
2Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA
3Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA
Tuberculosis (TB) kills more individuals globally than any other single infectious disease. An active case of TB disease can result in infection of 10-15 other individuals in one year. Those most at risk are health care professionals and household contacts with routine repeated exposures. Focused intervention strategies to block transmission in these populations would help to significantly reduce the global health burden of TB disease. Mycobacteriophage (phage) are a sorely underutilized biologic intervention for the pathogen Mycobacterium tuberculosis (M.tb) and here we aim to address outstanding questions about their utility as a therapy. Specifically, we aim to determine the impact of repeated mucosal or I.V. exposures on host anti-phage immunity in a preclinical mouse model, using representative phages Muddy HRMN0052-1 (Muddy) and FionnbharthΔ45Δ47 (Fionnbharth). We hypothesize that repeated phage delivery via the aerosol route will not induce high titers of functionally neutralizing anti-phage antibodies. To evaluate the impact of repeat exposures on host anti-phage immunity, male and female mice were dosed 6 times weekly with Fionnbharth I.V. or via aerosol. Mice were euthanized after repeat doses for collection of serum and bronchoalveolar lavage fluid (BALf). Weekly I.V. phage dosing generated a significantly higher magnitude of anti-phage humoral responses than the same phages delivered via aerosol, samples from the aerosol cohorts were often below the limit of detection for ELISA. Additionally, we observed that these anti-phage antibodies were functionally neutralizing ex vivo. Aerosol delivery of phage is a viable approach for therapy because it does not induce a robust neutralizing anti-phage response and should be advanced through the in vivo preclinical pipeline for efficacy evaluation.
Finding bacteriophages to combat skin ulcers in Atlantic salmon
Karoline Sveinsson1, Snorre Gulla1, Hans Petter Kleppen2, Stephen Mutoloki3
1Norwegian Veterinary Institute, Elizabeth Stephansens vei 1, 1433 Ås, Norway
2ACD Pharma, Christian Magnus Falsens vei 18, 1430 Ås, Norway
3Norwegian University of Life Sciences, Elizabeth Stephansens vei 15, 1433 Ås, Norway
Winter ulcer disease is a bacterial disease found in farmed salmonids. It is caused by the bacterium Moritella viscosa and in its classic form it is easily recognized by deep skin wounds. Systemically, the disease affects several organ systems with extensive bleeding of the liver generally acknowledged as a common feature. In addition to being painful, the lesions also predispose the animals to secondary infections. As antibiotic therapy is seldom successful in these patients, and as a rule should be avoided when possible, bacteriophages could prove useful. Field observations indicate that stress and excessive handling is a key component to developing the disease. Sorting, transportation and delousing methods can be tough on the skin barrier, leaving it weakened or injured creating port of entry for the bacterium. A suitable phage cocktail administered during these critical time points could reduce or even eliminate the infection pressure. In order to find suitable phages, we need to know how diverse the target bacterium population is. From whole genome sequencing it is evident that M. viscosa consists of more than one distinct genetic clonal cluster. Unsurprisingly, there appears to be a strong correlation between which bacterial cluster is historically and currently dominant and how easy it is to find specific bacteriophages. Using plaque assays, phages isolated from enrichments using a more obscure M. viscosa have, with very few exceptions, little to no effect on the more commonly occurring bacterial clonal complex CC1. Phages isolated from enrichments using CC1 isolates tend to display stronger lytic activity and broader host range, but so far only within its own clonal complex. Our findings indicate that careful tailoring of phage cocktails is required to achieve desired preventive and/or therapeutic effect against the relevant diversity of clinical M. viscosa strains.
LES Phage 5: length, encapsidation, amplification, and relationship with other Pseudomonas phages
Reem A Talat1,2, Enrique González-Tortuero 3,4, Revathy Krishnamurthi1, Jo L Fothergill1, Chloe E. James3, Heather E Allison1
1Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, L69 7ZB, UK
2College of Environment sciences and Technology, University of Mosul, Iraq
3School of Science, Engineering and Environment (SEE), University of Salford, M5 4WT, UK
4European Virus Bioinformatics Center, Leutragraben 1, 07743 Jena, Germany
aeruginosa LES is an opportunistic pathogen that is transmissible between CF patients. LES is also capable of infecting the lungs of non-CF patients and superinfecting patients with other P. aeruginosa strains. Within the P. aeruginosa LES genome lie 20 genomic islands, including the presence of five active prophages and cryptic, prophage-like element. LES φ5 is a temperate and active Pseudomonas prophage that plays an essential role in host’s competitiveness. Seventy-six ORFs are predicted in LES Pseudomonas phage 5 genome. Following careful re-annotation, only 20 ORFs lack a functional prediction (Hypothetical protein). We proved that the total length of LES phage 5 genome is 50,235 bp, and the attL and attR regions were located at 2,690,327 – 2,690,341 and 2,740,547 – 2,740,561 in the P. aeruginosa LESB58 genome, respectively. It was proved that LES phage 5 is a complete, inducible phage because it encapsidates its DNA. There are 4 additional LES prophages in LESB58. They LESB58 prophages 2,3 and 4 all share some degree of homology with LES phage 5. Moreover, LES phage 2,3,4 and 5 have significant spontaneous induction rates in the log and stationery growth phases of their lysogens. Differences between the replication outcomes of all LES phages were detected, Work is ongoing to identify a P. aeruginosa host that will support LES phage 5 infection for further characterisation.
Studying the Transcriptional Organization and Infection Process in A. baumannii phage model using Differential RNA-Sequencing in order to discover novel phage based antibacterials
Kawtar Tiamani¹˒², Xue Peng¹˒², Jinlong Ru¹˒², Mohammadali Khan Mirzaei¹˒², Li Deng¹˒²
¹Institute of Virology, Helmholtz Centre Munich, Neuherberg, Germany
²Chair of Microbial Disease Prevention, School of Life Sciences, Technical University of Munich, Freising, Germany
The past decades have witnessed an explosion of high-throughput genome sequencing, and as a consequence numerous phage genomes have been sequenced. Before it can be possible to make use of phage-derived products in order to manufacture novel antibacterials, it is necessary to understand the phage infection process from all angles possible, namely, the transcriptome architecture. Recent years have witnessed a soaring interest in genome-wide transcription start site (TSS) mapping in both phages and their hosts, in order to track the early infection phase. Differential RNA-seq (dRNA-seq) has been universally applied for TSS mapping in different organisms. Global TSS knowledge allows the annotation of 5’UTR and promoter regions as well as Ribosome Binding Sites (RBS) and non-coding genes, skipped by DNA-based genome annotations. TSS mapping by dRNA-seq allows the discrimination between transcripts, based on the presence of a 5’-triphosphate (5’-PPP) end in primary transcripts, while processed RNA transcripts mostly possess a 5’-monophosphate (5’-P) group. Here, we apply dRNA-seq to a phage infection model, comprising of A. baumannii and its lytic phage vB_AbaS_HMGU1 to uncover specific enrichment patterns based on the time post phage infection, and to understand the transcriptional layout and organization in our phage model. This would enable our understanding of the infection process and how the phage manages to take over the bacteria’s metabolism while navigating the synergistic or antagonistic effect of antibiotics, which would ultimately lead to the discovery of novel phage derived antibacterial agents.
Measuring the kinetics of phage infection steps with single-cell time lapse microscopy
Charlie Wedd1, Aaron Smith2, Ruizhe Li1, Michael Hunter2, Georgeos Hardo1, Racha Majed2, Temur Yunusov2, Diana Fusco2, Somenath Bakshi1
1Department of Engineering, University of Cambridge, Trumpington Street, Cambridge, CB2 1PZ, UK
2Department of Physics, University of Cambridge, JJ Thomson Avenue, Cambridge, CB3 0HE, UK
Phage therapy is a promising avenue for the treatment of antibiotic resistant bacterial infections. However, as with antibiotic treatment, a bacterial population infected by phages gives rise to a distribution of phenotypes. Developing a better understanding of this distribution and its impact on population level behaviour is important for the prediction of clinical outcomes. A key characteristic that determines the efficacy of lytic phages is the lysis time. We have developed simulations which suggest that the ability of a phage to lyse a population of bacteria is influenced not only by the mean lysis time, but also by the variance and overall shape of the lysis time distribution. However, current experimental methods such as lysis curves are not sufficient to accurately measure the lysis time distribution. Here, we present a microfluidics enabled, single-cell time lapse microscopy-based assay to accurately estimate the distribution of lysis times across individual infection events. The assay works by using time lapse images to detect different events in the phage infection cycle, such as the adsorption of individual phages to bacterial cells, the arrest of bacterial growth, and transcription of the phage genome. We first demonstrate an optimisation of the microfluidic device, which allows us to study phage infection in a spatiotemporally uniform environment. We then present a novel lineage tracking algorithm which allows us to track phage infection events over time. Finally, we present experimentally measured lysis time distributions of wild type and engineered T7 bacteriophages obtained from high-throughput imaging experiments. We believe that this assay presents new opportunities to study the phage life cycle at the single-cell level, offering insights and details into phage-host interactions which are lost when using population level assays, but are of critical importance to predict population level outcomes of infection.