Posters

Posters and poster guidelines

Thank you for considering to present your work as a poster at Phages 2020 Virtual.

Digital poster submission deadline: Prepare your poster as you would normally do for printing, and submit your final poster as both PDF and JPG/PNG files via the link below no later than the extended deadline of 07th September 2020. Late posters may not be included in the conference programme. Please DO NOT send your poster files by email.

Naming your poster files: Name your poster files as follows: <your surname>-PHG20V-Poster.pdf | <your surname>-PHG20V-Poster.png | <your surname>-PHG20V-Poster.jpg, etc. For example, for David Jones, name your file as Jones-PHG20V-Poster.pdf. DO NOT name your poster files as, e.g.,  Oxford-poster, Phages2020v, Oxford-phages-poster. Such files will be automatically rejected.

Poster presentation: Posters will be made available via a secure page to the symposium participants before the meeting. There will be two ways to interact with the poster presenters:

• the participants will be able to ask questions via the Zoom chatbox during the mid-conference break each day; and/or
• the participants can post their questions on Twitter at any time using the meeting hashtag #PhgOx20V, as well as the poster specific hashtag (given under each poster abstract) – do tag @PhageOxford in your tweets.

The poster presenters should regularly check Twitter for any questions about their posters before, during and after the meeting, and post their answers on Twitter using appropriate hashtags, as above.

Any further information about the poster presentations at this digital meeting will be available in the future.

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.

Characterization of six related K.pneumoniae phages with divergent depolymerases

(Poster)

Twitter: #PhgOx20V & #BBeamud

Beatriz Beamud¹, Lucas Mora-Quilis², J Vienne³, Fernando González-Candelas^1,4, Rafael Sanjuán², Pilar Domingo-Calap²

¹Joint Research Unit “Infection and Public Health” Universitat de València-I2SysBIo-FISABIO, Valencia, Spain.

²Institute for Integrative Systems Biology, I2SysBio, Universitat de València-CSIC, Paterna, Spain

³College West Flanders, Howest University, Kortrijk, Belgium

⁴CIBER in Epidemiology and Public Health, Valencia, Spain

Klebsiella pneumoniae is an opportunistic bacteria included in the ESKAPE group of pathogens which is responsible for most nosocomial antibiotic resistant infections. The search for new therapies has brought bacteriophages into the spotlight. Here, we describe six new K.pneumoniae phages, πVLC1-πVLC6, isolated from environmental samples. The six phages belong to the genus Drulisvirus within the family Podoviridae. We have performed host range assays against 216 K.pneumoniae strains with diverse capsular types and, after complete genome sequencing, we have analysed their gene content and performed phylogenetic analyses. πVLC phages do not conform a monophyletic group in the Drulisvirus genus and the most relevant differences in gene content and sequence divergence are found in tail fiber proteins. All phages encode a similar tail spike protein, with putative depolymerase activity, whose enzymatic domain is only present in πVLC phages. This tail spike would explain the ability of all πVLC phages to infect K22/K37 K.pneumoniae strains. Additionally, phages πVLC5 and πVLC6 present a second tail spike with depolymerase activity next to the lysis cassette. The second tail of πVLC5 presents low identity with any phage protein described so far and could be responsible for infecting some K39 K.pneumoniae clinical strains. On the contrary, the second tail spike of phage πVLC6 is also present in four other phages of the same genus, with a high level of identity. This protein might confer the ability for this phage to also infect K13 and digest K2 capsules. Surprisingly, πVLC6 is also able to infect K3 strains whose antigenic structure overlap only partially with K22/K37 and K2/K13. Hence, both enzymes might be able to recognize the K3 capsule. In conclusion, these novel phages reveal interesting aspects of the Drulisvirus evolution and suggest that host range expansion can occur via the acquisition of divergent depolymerases.

Phage anti-biofilm activity against Staphylococcus aureus prosthetic joint infection clinical isolates

(Poster)

Twitter: #PhgOx20V & #KMCaflisch

Katherine M Caflisch¹, Robin Patel²

¹Infectious Diseases Research Laboratory, Mayo Clinic; Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 200 First St SW, Rochester, Minnesota, USA 55905

² Infectious Diseases Research Laboratory, Mayo Clinic; Division of Clinical Microbiology, Mayo Clinic; Division of Infectious Diseases, Mayo Clinic, 200 First St SW, Rochester, Minnesota, USA 55905The potential of lytic phages to treat bacterial disease, especially biofilm-associated infections, is the subject of renewed scientific interest in the wake of mounting antimicrobial resistance. In this work, we tested the in vitro activity of five American Type Culture Collection phages against biofilms of Staphylococcus aureus clinical isolates from periprosthetic joint infection cases seen at our institution between April 1999 and February 2018 (53 methicillin-susceptible, 33 methicillin-resistant, 29 unknown methicillin susceptibility). Biofilms were grown in cation-adjusted Mueller-Hinton broth from log-phase cultures (10⁴ cfu/mL) in polystyrene, flat-bottomed 96-well plates for 4 hours at 37°C in room air with shaking. Plates were rinsed and treated with 200 μL phage (MOI>10) in 10 mM MgSO₄ tryptic soy broth (TSB) or 10 mM MgSO₄ TSB (controls) per well for 24 hours under the same incubation conditions. Wells were rinsed, dried, and stained with 0.1% safranin prior to spectrophotometric measurement (OD₄₉₂ nm). All conditions were tested in technical and biological duplicates, and only isolates with agreement across duplicates were included in the analysis. Percentage of treated isolates with significant reductions in optical density versus controls ranged from 38-97% for each phage tested. Statistical analyses showed that small colony variants and oxacillin-resistant isolates demonstrated similar average OD reductions versus normal colony phenotype and oxacillin-susceptible isolates, respectively, when treated with phage. Furthermore, no correlation was observed between an isolate’s biofilm formation capacity and phage susceptibility, except between non-adherent or weak biofilms versus moderate/strong or strong biofilms treated with ATCC 23360-B1. This research lends support to the potential use of phage for the management of clinically-relevant biofilms.

Understanding the costs and mechanisms of bacteriophage resistance in Enterobacteriaceae

(Poster)

Twitter: #PhgOx20V & #LGannon

Lucy Gannon¹, Eleanor Jameson² and Andrew Millard¹

¹ Department of Genetics and Genome Biology, University of Leicester, UK

² Warwick Integrative Synthetic Biology (WISB) Centre, University of Warwick,  UK

Enterobacteriacae are a widely found family of Gram-negative bacteria commonly responsible for pathogenic infections including urinary tract and gastrointestinal infections, pneumonias and septicaemia. Multiple antimicrobial resistances have also been found accumulating in these species. Recently there has been a renewed interest in using bacteriophages as treatments for bacterial infections. Bacteriophages offer a potential therapy for treating bacterial infections and can be used alone or in conjunction with antibiotics, but bacterial resistance against bacteriophage often arises quickly in laboratory cultures. A diverse set of six lytic bacteriophage were used to generate bacteriophage resistant Escherichia coli and Salmonella typhimurium mutants from susceptible host strains to identify the diversity of resistance mechanisms utilised and any associated fitness costs. Phage resistance mutants generally had increased growth rates compared  to wild-type, however some surprisingly displayed increased  growth rate altered bacteriophage resistance profiles and genome mutations associated with phage resistance.

Development of Phage-assisted Evolution and Riboregulation Strategies

(Poster)

Twitter: #PhgOx20V & #EGoicoechea

Eduardo Goicoechea, Alfonso Jaramillo

School of Life Sciences, University of WarwickRiboswitches are short RNA sequences that modify their 3D structure in the presence of a specific molecule, regulating gene expression in the process. A few riboswitches have been described in eukaryotes, but they are mostly present in bacterial species. They are of great interest in synthetic biology for their inductive regulatory properties, which could be used in metabolic studies, or as biosensors in medical, industrial or environmental cases. However, riboswitches are very substrate-specific, creating a problem for their use with novel compounds; and current development methods suffer from issues such as being too laborious and not using in vivo conditions. By developing strategies based on Phage Assisted Continuous Evolution (PACE) and using T7 phage along a double selection system, we developed a way of obtaining riboswitches that show improvements compared to a control sequence. Once the method is tuned, it could be used to develop novel riboswitches not present in nature, and not only for the directed evolution of riboswitches, but also other types of sensors, such as protein or RNA receptors. A second inducible RNA system based on the phage Qb was also tested. These have yielded a randomised library of riboswitch sequences in phages and an inducible RNA plasmid, respectively. In the first case, using a double selection process to achieve evolution, we have shown sequence variation in phage populations decrease over time, until a single sequence was prominently represented. The sequence showed higher activation folds at a lower concentration of the activating molecule than in initial generations, indicative of evolution. As for the RNA-based plasmid system, we have shown it to be an inducible, transient expression system that could be used as a novel way to regulate gene expression and bypass CRISPR systems. These results speak of the possibilities held by these strategies, not just for their specific areas of research, but for synthetic biology at large.

Bacteriophage preparation to prevent and control poultry infections

(Poster)

Twitter: #PhgOx20V & #ProteonPharma

Anna M Pękala, Edyta M Śmigielska, Ewelina Wojda, Jolanta A Witaszewska, Joanna Kazimierczak, Ewelina A Wójcik, Agnieszka Maszewska, Arkadiusz Guziński

Proteon Pharmaceuticals S.A., Łódź, Poland

A significant etiological factor of poultry infectious diseases is Avian Pathogenic Escherichia coli (APEC), causing colibacillosis. This disease causes vast economic losses due to high poultry mortality and treatment costs. Due to the increasing number of antibiotic resistant bacteria and the limitation of the use of antibiotics in poultry, it is necessary to develop alternative methods of controlling APEC. The aim of this study was to develop a broad spectrum composition of bacteriophages for the prevention and control of poultry infections caused by APEC. The development of bacteriophage preparation included the following stages: isolation of bacteriophages specific to APEC, phage lytic activity evaluation, differentiation of bacteriophages by RFLP or RAPD, bioinformatic analysis of phage genomes to determine their identity taxonomy and virulent or temperate character. In the next step different combinations of strictly virulent bacteriophages were composed and their antibacterial activity was tested. The commercial features of the selected composition were evaluated, e.g. the maintenance of lytic activity against variants of bacteria resistant to single bacteriophages included in the preparation as well as the activity against antibiotic resistant APEC strains. This study resulted in the composition of lytic bacteriophages specific for 95% of APEC among 86 tested strains, including multidrug resistant bacteria. The cocktail has a broader activity in comparison to single phage preparations specific only for 78% of APEC strains. Furthermore, the preparation reduces the frequency of inducing phage resistant variants, at least 10.000-fold. In conclusion, the developed bacteriophage cocktail is a non-antibiotic preparation with a high potential for combating APEC infections in poultry. Research carried out under project no POIR.01.01.01-00-0149/16, co-financed by The National Centre for Research and Development from “Smart Growth Operational Programme 2014-2020.”

Genetically Engineering Bacteriophages to Combat Antibiotic Resistance

(Poster)

Twitter: #PhgOx20V & #CHarrison

Christian Harrison¹, Nick Waterfield², Martha Clokie¹, Andrew Millard¹

¹Department of Genetics and Genome Biology, University of Leicester, Leicester, LE1 7RH, UK

²Warwick Medical School, University of Warwick, Coventry, CV4 7HL, UK

In light of the rising levels of antimicrobial resistance, phages have been proposed as an alternative to antibiotics for the treatment of bacterial infections. Among the numerous advantages of phages for therapeutic treatments is their ability to be genetically engineered to alter their host range and carry payloads to enhance their potential as therapeutics. Colicins are antibacterial proteins produced by E. coli to kill other susceptible E. coli strains with two common mechanisms of activity: nuclease and pore-forming. The aim of this project is to genetically engineer a phage to produce a colicin in an attempt to increase the virulence of the phage and reduce the development of bacterial resistance to the phage. Colicin E9 (DNase) and its immunity gene were cloned into a plasmid vector between homologous recombination arms designed to create insertions in the genome of T4-like phage SLUR96. The colicin was confirmed to be functional by performing a colicin activity assay which also identified multiple susceptible isolates of E. coli from bacteraemia and urine samples. Recombination was performed by propagating the phage on the strain harbouring the recombination plasmid and recombinants were selected for by a modified plaque assay. Future work will include exchanging the promoters driving the expression of the colicin and immunity gene in the phage to optimise colicin production while limiting the impact on phage replication. The efficacy of the engineered phage will be examined by the virulence index assay before performing in vivo survival studies in Galleria mellonella. The production of the colicin by the phage during infection should increase the quantity of antimicrobial agents released upon phage lysis of the cell. Furthermore, the phage and colicin differ in both the receptors to which they bind and the mechanisms through which they kill bacteria thus potentially decreasing the likelihood of bacteria developing resistance to the treatment.

Isolation and characterization of Carnobacterium bacteriophages

(Poster)

Twitter: #PhgOx20V & #Lasagabaster1

Amaia Lasagabaster¹, Katherine Miranda-Cadena¹, María Lavilla¹, Felix Amarita¹

¹AZTI, Food Research, Basque Research and Technology Alliance (BRTA). Parque Tecnológico de Bizkaia, Astondo Bidea, Edificio 609, 48160 Derio, Bizkaia, Spain

Carnobacterium divergens (CD) and Carnobacterium maltaromaticum (CM) are ubiquitous LAB that can act as food spoilage agents in several modified atmosphere packed (MAP) and vacuum-packed (VP) food products. Their biocontrol by a specific bacteriophage-based solution could reduce Carnobacterium load and increase the shelf life of this kind of products. In this work, eight CM and two CD strains were used for the isolation of phages from sea/river water as well as MAP or VP seafood, meat and dairy products. Five bacteriophages were isolated from 230 analysed samples (isolation rate of 2.2%). Their host range evaluation revealed that one phage was active against two different CM strains while the other four showed high specificity against a single CD strain. All phages showed chloroform resistance, but only three phages were stable at 4 °C over six months. A cocktail combining the three stable phages to a final concentration of 10⁶ PFU/ml allowed a broader host range than the individual application of each single phage. The cocktail was active against three hosts, including both CD and CM species, increasing the bacterial latency period of CD for 10 h at a multiplicity of infection (MOI) of 100. Moreover, the application of the cocktail at 10⁴ PFU/g (MOI of 1) on the surface of cold smoked salmon, previously autoclaved and inoculated at 10⁴ CFU/g with two host (independent assays), reduced by 0.5 – 1 log the counts of Carnobacterium. This work showed the use of Carnobacterium bacteriophages as a promising tool to fight the spoilage of MAP and VP food products related to these bacteria. However, further studies are needed before suitable food applications.

Optimization of Campylobacter specific bacteriophage isolation

(Poster)

Twitter: #PhgOx20V & #Lasagabaster2

Ibai Nafarrate¹, Estibaliz Mateo² and Amaia Lasagabaster¹

¹AZTI, Food Research, Basque Research and Technology Alliance (BRTA). Parque Tecnológico de Bizkaia, Astondo Bidea, Edificio 609, 48160 Derio, Bizkaia, Spain

²Departamento de Inmunología, Microbiología y Parasitología, UFI 11/25, Facultad de Medicina y Enfermería, Universidad Del País Vasco-Euskal Herriko Unibertsitatea (UPV/EHU), Bilbao, Spain

Campylobacter is responsible for the 70% of all the reported zoonotic cases in the European Union (UE) and poultry is considered its natural reservoir. The control strategies proposed for the control of this pathogen have not completely solved the problem. Moreover, the development and spread of antimicrobial resistant Campylobacter highlights the need of alternative strategies to reduce the burden of this pathogen. Thus, the application of specific bacteriophages is a promising tool as a complement to currently existing measures. Their isolation is the first challenge to face since their occurrence appears to be low. To optimize it, seven isolation methods were assayed on the basis of an efficient phage recovery from broiler skin samples inoculated at loads of 5.0×10¹ – 5.0×10⁶ PFU/g. The enrichment of samples in selective Bolton broth with target isolates was the most efficient procedure, showing a detection limit of 5.0×10¹ PFU/g and recovery rates up to 338%. Furthermore, this method was more effective at lower phage titres, showing its suitability for phage isolation at the low concentrations occurring in foods. When this method was applied to isolate new Campylobacter phages from retail broiler skin, a total of 280 phages were recovered achieving an isolation success rate of 257%. From the 109 samples 68 resulted phage positive (62%). The optimized method also showed its effectiveness for the isolation of phages from swine faeces. From this latter matrix, 18 new campylophages were recovered which also means an isolation success rate of 257%. In this case a 43% of bacteriophage positive samples was observed. Therefore, both broiler skin and swine faeces could be considered a rich source in Campylobacter phages when the appropriate methodology is used. This is a simple, reproducible and efficient method for the successful isolation of campylophages, which could be helpful for the enhancement of food safety via the reduction of this pathogen contamination.

DNA alteration of Φ13 and its host Staphylococcus aureus after excision from alternative chromosomal integration sites

(Poster)

Twitter: #PhgOx20V & #HLeinweber

Helena Leinweber, Hanne Ingmer

Section of Food Safety and Zoonoses, Institute of Veterinary and Animal Sciences, University of Copenhagen, Denmark

Temperate bacteriophages of the Sa3Int-family carrying virulence genes for human immune evasion, are suggested to play a crucial role for the host jump of Staphylococcus aureus from animals to humans. Those phages normally integrate into the ß-haemolysin gene hlb. In livestock-associated S. aureus, the 14bp long chromosomal core attachment site (attB) in hlb is mutated in 2bp and the phages integrate elsewhere in the chromosome. However, livestock-associated S. aureus strains have recently been found to carry those Sa3Int phages and thereby facilitating the colonization and infection of humans. Here we addressed the question of how the phage interacts with a host not harbouring its preferred integration site. For that, we challenged the model phage Φ13 with phage-cured laboratory strain S. aureus 8325-4 carrying the 2bp mutation in hlb resembling attB of the livestock strains. We find Φ13 to integrate at many alternative sites, which are widely distributed over the chromosome and show up to 70% mismatches compared to the original attB. The phages could be induced by Mitomycin C and depending on integration site formed different amounts of active phage particles, visualized by plaque assays. We discovered that non-matching attB and attP core sequences can result in non-identical attR and attL framing the phage upon integration and furthermore lead to modified attB and attP sequences after excision of the phage. Subsequent lysogenization assays with those modified phages will show, whether the phage has adapted and lysogenization frequencies will increase again. This modification in the attP site could allow the phage to quickly adapt to the alternative attB sites which potentially accelerates the spread in the livestock-associated S. aureus community.

Isolating novel Kenyan Salmonella enterica phages for the development of phage therapy in Kenyan poultry farms

(Poster)

Twitter: #PhgOx20V & #AMakumi

A. Makumi¹, A. Mhone¹, L. Guantai¹, J. Odaba¹, & S. Moineau², N. Svitek¹

¹International Livestock Research Institute, Nairobi, Kenya

²Université Laval, Département de biochimie, Faculté des sciences et de génie, Québec, Canada

Poultry farming is one of the fastest growing industries in Low- and Middle-Income Countries (LMIC) and especially in Kenya, representing about 30% of the total agriculture contribution to the Gross Domestic Product (GDP), with an estimated 75% of rural families keeping chicken. However, the presence of bacterial pathogens leads to poultry death or damage of poultry products and finally leads to huge economic losses. Salmonella sp. is among the most important bacterial disease that affect chicken in LMIC and the most responsible species for enteric disease in humans. Current methods of controlling or preventing Salmonellosis in poultry farms include the use of antibiotics but the emergence of drug resistant bacteria due to unrestricted use of antibiotics clearly indicates the need in more sustainable and environmentally friendly strategies such as bacteriophages. The main goal of the present work is to study the antibiotic susceptibility of Salmonella enterica isolated from Kenyan poultry farms and to isolate specific bacteriophages against Salmonella enterica. A total of 61 poultry farms were visited, collecting a total of 631 samples.  Sixteen Salmonella sp. strains were identified as Enteritidis, Kentucky and Heidelberg serovars while susceptibility of the identified strains to 12 antibiotics mostly used in poultry farming was performed. Additional Salmonella enterica strains [S. Braenderup, S. Typhimurium, S. Braendeburg, S. Pullorum and S.cholerasius] from the Kenya Medical Research Institute were added to the Salmonella strains isolated during this study to test the 631 crude phage lysates. Work on selection of therapeutic phage candidates for poultry farming and their detailed characterization is on-going and will be tested in a salmonella infection model in chicken. Hopefully, this will lead to the development of an alternative strategy to prevent salmonella infection in chicken in LMIC.

Roles of Cro₂   and CI₂   binding to O_R  operators in controlling lysogeny stability during prophage induction

(Poster)

Twitter: #PhgOx20V & #YPang

Yiyu Pang^1,2, Xue Lei³, Farid Manuchehrfar¹, Jie Liang¹

¹Department of Bioengineering, University of Illinois, Chicago, IL, USA

²Department of Physics, University of Illinois, Chicago, IL, USA

³Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA

The lysogeny-lysis switch in bacteriophage lambda provides a general framework for understanding cell fate decision. The molecular network that controls switching between the lysogeny and lysis states has been studied extensively experimentally. Computational modeling has subsequently provided additional insight. However, how interactions such as binding of proteins CI2 and Cro2 to operators control specifically lysogeny stability in prophage induction remains unclear. Furthermore, how component interactions of the network affect its global behavior under different physiological conditions such as UV irradiation requires further investigations. Here we study the lysogeny-lysis control network using a minimalistic model. Our approach is to construct the exact probability landscapes of the network using the Accurate Chemical Master Equation (ACME) method (bioacme.org). We examined how different binding of Cro2 to OR3/OR1 and CI2 to OR3/OR1 affect switching at different dissociation rates. We further examined how such effects change at different degradation rate of CI2, which is used to mimics different UV irradiation. Our results show that binding of Cro2 to OR3 and CI2 to OR1 regulate lysogeny stability: the former impairs the lysogeny stability and the latter aids in the maintenance of lysogeny during induction. However, we find binding of Cro2 to OR1 and CI2 to OR3 have little effects on lysogeny stability. Overall, we carried out a study modeling the exact probability landscape and examined the behavior of lysogeny-lysis control network using the ACME method. This allowed us to investigate the network behavior under a broad range of conditions that would not be feasible using methods such as stochastic simulation algorithm. Our results show how binding of protein-DNA complexes regulate lysogeny stability, and how the switching network behave globally under different conditions.

How do temperate bacteriophages affect the fitness of Pseudomonas aeruginosa?

(Poster)

Twitter: #PhgOx20V & #GIPlahe

Grace I Plahe¹, Heather E Alison², Ian Goodhead¹, Chloe E James¹.

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

²Institute of Integrative Biology, University of Liverpool, UK

The Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa is a key opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Once established in the CF lung it is harder to treat and control then other strains of P. aeruginosa. This is due to distinctive prophages within its genome, which provide fitness advantages that of yet have not been well characterised. This project explores the dynamics between the model organism P. aeruginosa PAO1 and 3 LES phage using different models of infection and treatment regimens including growth curves, plaque assays and Galleria mellonella models.