Posters and poster guidelines
Thank you for considering to present your work as a poster at Phages 2021.
Digital poster submission deadline: Prepare your poster as you would normally do for printing, and submit your final poster as both PDF (maximum file size 10MB) and JPG/PNG (maximum size 2MB) files via the link below no later than 03 September 2021. 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>-PHG21-Poster.pdf | <your surname>-PHG21-Poster.png | <your surname>-PHG21-Poster.jpg, etc. For example, for David Jones, name your file as Jones-PHG21-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, Phages2021, Oxford-phages-poster. Such files will be automatically rejected. Also, don’t forget to include your own Twitter handle, if you have one, in your poster.
Poster presentation: Posters will be made available via a secure page to the participants several days before the conference. The posters will also be posted on Twitter on the first day of the conference.
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 #PhgOx21, as well as the poster specific hashtag (given under each poster abstract) – do tag @PhageOxford in your tweets.
Any further information about the poster presentations at this digital meeting will be available in the future.
(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.
Using Bacteriophages to Reduce Salmonella prevalence in Chickens: A Systematic Review
(hashtags: #PhgOx21, #MAlmutairi)
Malak Almutairi1, Mohammed Imam2, Nouf Alammari1, Radwan Hafiz1 and Suliman Alajel1*
1Saudi Food and Drug Authority, Riyadh, Saudi Arabia
2College of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia
Salmonellosis is an infection that significantly impact chickens, thus acting as a public health burden and a contributor to commercial losses in the chicken’s industry worldwide. To tackle chickens related bacterial infections, a great amount of antibiotics along with several infection prevention measures are used worldwide. However, using different chemical additives such as organic acids, and chlorine-based interventions have different limitations, including feed refusal due to a change of taste, incompatibility between organic acids and other inoculated preservative agents such as antimicrobial agents. Bacteriophages are host-specific viruses which interact with bacteria in a specific manner. Therefore, they possess unique biological and therapeutic features that can be used to reduce bacterial contamination, leading to improved food safety and quality. This systematic review aims to summarize the current evidence regarding the effectiveness of various bacteriophages on Salmonella colonization in chickens and chicken meat. 17 reviewed studies were conducted in-vitro and in all these experiments similar conditions (temperature and incubation parameters) were used in order to test the efficacy of isolated and commercially available phages on chicken’s raw meat samples. The current evidence showed that most of the in-vitro studies used phages as a biocontrol tool. Phages are designed to eradicate Salmonella contamination in chickens, and the results of these studies have generally produced positive outcomes. This indicates that phages is a promising solution worldwide for tackling foodborne bacteria, including Salmonella.
Stability of Novel Non-Typhoidal Salmonella Phages in Simulated Gastric Fluid and In Vitro Efficacy of Silica Vesicle to Protect Phages
(hashtags: #PhgOx21, #AMhone)
Amos L. Mhone, Angela Makumi, Linda Guantai, Josiah Odaba, Anna Lacasta, Nicholas Svitek
Animal and Human Health Program, International Livestock Research Institute (ILRI), Nairobi, Kenya
The zoonotic Multi-Drug Resistant (MDR) non-typhoidal Salmonella (NTS) enterica serovar Enteritidis is one of the major causes of foodborne infections worldwide. Current methods of controlling Salmonella infections at the farm level include the use of antibiotics, particularly in poultry farming. An estimated 75% of antibiotics administered to poultry are released in the environment and contribute to the emergence of antimicrobial resistance (AMR). Bacteriophages are a potential alternative to fight MDR NTS. Phages stable at low pH and high temperatures would render them more suitable for the control of Salmonella in poultry as they have a higher chance to survive the harsh gastrointestinal environment. This study tested the thermal and pH stability of 13 different S. Enteritidis specific phages, previously selected from a cohort of phages based on Restriction Fragment Length Polymorphism (RFLP) patterns. Three novel silica vesicles (SV 100, SV 140, and SV 100 C18) were used to test whether they increased the survival of phages in simulated gastric fluid (SGF). All 13 phages were relatedly stable from pH 4 to 12 after 24 hours of incubation with an average titre of 8.1 x109 PFU/ml, while they all lost their viability within 3 hours at pH 2-3. The thirteen phages were relatively stables from temperatures ranging from 25℃ to 42℃ after 12 hours of incubation but started losing their viability at 50℃. All three Novel Silica Vesicles demonstrated a low but longer rate of phage release upon adsorption for 96 hours. Preliminary data indicate that SV 140 C18 nanoparticles shown the ability to protect phages longer, with an average titer of 6.4 x 106 PFU/ml at 60 minutes compared to SV 100 (12.6 X 104 PFU/ml) and SV 140 (6.3 X104 PFU/ml). In contrast, free phages in SGF had an average concentration of 3.7 x103 PFU/ml after 60 minutes of incubation. Future experiments are underway to evaluate the stability of these phages in vivo in chickens with and without SV.
Isolation, characterization and genetic modification of novel Autographiviridae phages to construct highly diverse cocktails against antibiotic resistant clinical isolates
(hashtags: #PhgOx21, #GApjok)
Gábor Apjok1,2, Tóbiás Sári1,2, Mihály Koncz1, Bálint Eszenyi1, Bálint Kintses1
1HCEMM-BRC Translational Microbiology Research Group, Szeged, Hungary
2Doctoral School of Biology, University of Szeged, 6726 Szeged, Hungary
Therapeutic application of bacterial viruses (phages) is an exceptionally promising, albeit unrefined approach to battle against the spread of antibiotic resistant pathogenic bacteria. Clinical usefulness of phages is gravely constrained by their innate narrow-host specificity. A possible avenue to mitigate this issue is to deploy mixtures of various eligible phages, so-called phage cocktails. Unfortunately, creating (and constantly updating) phage cocktails is a time- and labour-intensive process. Our aim is to establish a genome engineering method to rapidly create phage cocktails of high diversity. We isolated novel phages of the Autographiviridae family from lakes and hospital sewage against antibiotic resistant clinical isolates (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli species) and characterized those with therapeutic potential. Then, we are going to apply DIvERGE, a site-specific genome engineering technology to induce high-frequency randomized mutagenesis on phage tail fiber regions to modify the ability of isolated phages to target variable bacterial cell-surface structures, thus alter their host-specificity and diversify their mode of action to eliminate bacteria. As a preliminary experiment we have managed to generate T7 phage mutants that are able to infect both wild type and evolved phage resistant pathogenic E. coli NCTC13351 strains. By optimizing the method we expect that phage cocktails constructed in this manner will be able to target a wider range of pathogenic species, compared with their non-mutagenized counterparts and to be less susceptible against potentially emerging bacterial resistance. Our next goal is to generate bacterial strains wherein the newly isolated phages are able to reboot efficiently, and thus make them amenable to diversification by our approach.
Investigation of in vitro phage therapy with free and encapsulated phage targeting S. aureus infection in human cells
(hashtags: #PhgOx21, #NBurton)
Nathan Burton1, Antonia Sagona1, Danish Malik2
1School of Life Sciences, Gibbet Hill Campus, University of Warwick, Coventry CV4 7AL, UK
2Chemical Engineering Department, Loughborough University, Loughborough LE11 3TU, UK
With the advent of antibiotic resistance, bacterial infections may be treated with bacteriophage therapy. Bacteriophages show great specificity towards their bacterial host and due to their small genomes, can be easily genetically modified for different applications. A “Trojan Horse” approach employing liposome encapsulated phages may facilitate access to phagocytic cells infected with intracellular pathogens e.g., Listeria, Salmonella, and Staphylococcus. Orally delivered phages tend to have short residence times in the gastrointestinal tract due to clinical symptoms such as diarrhea; this may be addressed through mucoadhesion of liposomes. Here, using homologous recombineering, we have successfully engineered a fluorescent bacteriophage specific for S. aureus, a primarily nosocomial pathogen responsible for urinary tract, soft tissue and skin/wound infections. By confocal imaging, we demonstrate the fluorescence of our engineered K phage and show how unencapsulated phage enters human cells by phagocytosis, but not endocytosis, and is then targeted for lysosomal degradation. We show whether unencapsulated bacteriophage K-GFP efficiently kills intracellular S. aureus in T24 human urinary bladder epithelial cells and RAW 264.7 Mouse macrophages. Finally, we investigate the size and aggregation of our K-GFP phage as a marker for the efficiency of phage encapsulation. We then aim to encapsulate our fluorescent bacteriophage in varying formulations of liposomes and test the delivery efficacy of the encapsulated phages compared to free phages.
Generating phenotypically distinct E. coli responses to T4 by varying carbon source
(hashtags: #PhgOx21, #EChapman)
Eric R I Chapman1, Aidan T Brown1, Teuta Pilizota2
1School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK
2School of Biological Sciences, University of Edinburgh, Edinburgh, UK
Bacteria can rapidly evolve resistance to bacteriophages through numerous pathways. Here, we investigate how the mutational pathway depends on the environmental conditions. Escherichia coli, grown in a range of media with various carbon sources, were exposed to T4 bacteriophage. In all cases the bacteria developed either partial or total resistance at long times. However, the characteristic population dynamics of this resistant regrowth varied with the carbon source, and the mutant bacteria consistently showed different phenotypes under optical microscopy. These results suggest a coupling between the environment and the pathway by which phage resistance evolves in E. coli. This may have profound implications for phage therapy, as there could be significant differences between the mechanism and rate of resistance development in vivo and in vitro due to different environmental conditions.
Pseudotyping bacteriophage P2 tail fibers extends the host range for biomedical applications
(hashtags: #PhgOx21, #TCunliffe)
Tabitha G. Cunliffe1, Alan L. Parker1, Alfonso Jaramillo2
1Division of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK
2School of Life Sciences, University of Warwick, Gibbet Hill Campus, Coventry, CV4 7AL, UK
Antibiotic resistant bacteria are one of the biggest threats to global health currently, with an estimated 70% of infection-causing bacteria being resistant to at least one antibiotic. The vital development of new antibiotics is time-consuming and bacteria can quickly become resistant to new agents. However, bacteriophage may hold the key to future antibiotic treatment. Bacteriophages have proven useful agents for treating antibiotics resistant bacterial infections previously, can be incredibly specific thus removing the chance of other bacteria developing resistance to the treatment, and eliminates side effects like beneficial bacterial death.Here we present a new method of using chimeric tail fibers in bacteriophage to re-target towards pathogenic bacteria without the need to culture the pathogen directly. Chimeric tail fibers fusing P2 and S16 genes were designed and generated through two approaches. By presenting a chimeric P2:S16 fiber on the P2 particle, were able to demonstrate that the resultant phage was retargeted away from the native infection target, LPS, and now able to infect via ompC, the natural receptor for S16 expressed on Salmonella. Our work provides proof of feasibility that pseudotyping P2 is both possible and can extend the host range to alternative receptors. Extension of this work could produce other tail fiber chimeras that target other pathogenic bacterial treats.
Investigation of the Localisation of FtsZ in Pathogenic E. coli Upon Bacteriophage Addition in a Human Cell Model as a Biomarker for Antibacterial Agents
(hashtags: #PhgOx21, #GDhanoa)
Gurneet K Dhanoa 1, Inbar Kushnir 1, Udi Qimron 2, David I Roper 1 and Antonia P Sagona 1
1School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, UK, CV4 7AL
2Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
Antimicrobial resistance is a growing problem worldwide and has created a need for novel antibacterial agents and strategies. Escherichia coli is one of the most common Gram-negative pathogens and is responsible for infection leading to neonatal meningitis and sepsis. The FtsZ protein is a bacterial tubulin homolog required for cell division in most species, including E. coli. Agents that block cell division have been shown to mis-localise FtsZ, including the bacteriophage l encoded Kil peptide, resulting in defective cell division and a filamentous phenotype and therefore FtsZ may be an attractive target for new antimicrobials. In this project, we are interested in studying the localisation of FtsZ in pathogenic E. coli in the presence and absence of human cell cultures, in order to establish how and if this localisation changes upon infection. We are also interested in investigating whether bacteriophages specifically attacking pathogenic E. coli have an effect on the localisation of FtsZ in a human cell environment and want to study the mechanism of this process. We have observed E. coli FtsZ localising to the cell midbody as a ring in a commensal K12 strain and in the K1 strain EV36 using confocal microscopy. These strains were used to infect human cerebral microvascular endothelial cells (hCMEC/D3) to create a meningitis model. We will present our results showing the effect of the Kil peptide and bacteriophages on this localisation within the model system, along with results using mutant bacteriophages, in an effort to validate FtsZ as a potential biomarker for antibacterial agents.
The Use of Lytic Phage as Therapy to Treat Bacterial Urinary Tract Infections
(hashtags: #PhgOx21, #LDuignan)
Libby A M Duignan1, Jo L Fothergill1, Rachel Floyd1, Craig Winstanley1
1 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Ronald Ross Building, 8 West Derby Street, Liverpool L69 7BE
The aim of this study was to identify new lytic phages from environmental sources to target Escherichia coli (E.coli) isolated from patients with UTIs. Over 150 E.coli isolates were collected from patients in the North West of England, which were then used to test the efficacy of 56 lytic phages isolated from a water-treatment centre water sample. Phenotypic tests were carried out on the E.coli isolates, such as antibiotic resistance testing and biofilm analysis. The genomes of the isolates and the promising phages (24) were sequenced for bioinformatic analysis. Results from the initial cross-infection showed that a number of the phages were effective against a high proportion of the isolates, with the highest effectivity being 72%. Only 4 isolates showed resistance to all phages. The phage infection profiles were analysed to streamline the number of phages used for sequencing, discarding phages with the same infection profile. The phenotypic tests showed many of the isolates are MDR, therefore showing alternative therapy is needed against these isolates. Biofilm analysis showed that a proportion of the isolates formed strong and moderate biofilms, highlighting the importance of testing the efficacy of the lytic phages to these isolates in a biofilm. The bioinformatic analysis of the E.coli genomes supported the results that many are MDR as they carried AMR genes when compared to CARD. Future work will analyse the phage genomes to confirm that they are obligate lytic phages, and they do not harbour toxin or virulence factor genes. The virulence index of the phages can then be measured against biofilms to establish the best cocktail of phages against the E.coli isolates in the North West. This study shows the promise that lytic phage therapy has for UTIs, with activity against a wide range of isolates from UTIs, including MDR E.coli seen, with the aim of developing a phage cocktail with wide-ranging anti-bacterial efficacy take forward to clinical trials.
Phage cocktails to reduce the antibiotic usage in Kenyan chicken farms
(hashtags: #PhgOx21, #MGunathilake)
M. Damitha Gunathilake1, Stephanie Loignon1, Angela Makumi2, Clement Fage1, Francoise B. Leblanc1,
Josiah Odaba2, Linda Guantai2, Denise Tremblay1, Nicholas Lemire1, Nicholas Svitek2, and Sylvain Moineau1
1Université Laval, QC, Canada
2International Livestock Research Institute, Kenya
The overall goal of this collaborative international project is to use phages as sustainable alternatives to antibiotics in order to reduce the prevalence of Salmonella in Kenyan chicken farms. Here, we describe our initial phage-host interaction studies to develop a phage cocktail. A total of 16 Salmonella strains and 68 phages were isolated from chicken faeces and water samples from Kenyan chicken farms. Bacterial strains were identified as Salmonella using several markers and the serotypes were confirmed through sequencing of their conserved CRISPR arrays. Considering CRISPR profiling and phage host range data, these 16 Salmonella strains were categorized into 5 groups. The phage sensitivity of these 16 Salmonella strains was also checked with an additional set of 32 Salmonella phages, which were conserved at Félix d’Hérelle Reference Centre for Bacterial Viruses (www.phage.ulaval.ca). Eight Centre phages including S16 had wider host ranges than any of the Kenyan phages. Three Kenyan Salmonella strains (Sal 172, Sal 181 and Sal 182, group 3) were particularly resistant to the d’Hérelle Centre phages, except for phage S16 which is a T4-like phage. Only one type of the Kenyan phages could infect these group 3 strains. The CRISPR systems in Salmonella are inactive to most of our knowledge and the only known phage to infect group 3 Kenyan Salmonella is a T4-like phage with a modified genome which is resistant to restriction enzyme cleavage. Hence, there is a high possibility that these Salmonella strains are protected by an efficient restriction-modification system. However, sequencing these Salmonella strains is underway to confirm the exact type of defence mechanism in them.
Observations of Cytoplasmic Volume Dynamics of E. coli During T4 Infection
(hashtags: #PhgOx21, #JHocking)
Jack Hocking1, Teuta Pilizota2, Aidan Brown1
1School of Physics and Astronomy, University of Edinburgh, King’s Buildings, Edinburgh, EH9 3FD
2School of Biological Sciences, University of Edinburgh, Edinburgh, UK
Much of the work surrounding the T4-E. coli interaction has taken place with a focus on the phage in regards to production, activation, diffusion, and killing. Turning to the other side, understanding the bacterial response to infection and how the host physiology affects the infection process can deepen our understanding of the interaction and allow us to make certain predictions based on nutrient availability. We show the reduction and subsequent recovery of the cytoplasmic volume at time of infection, and present a comparison of the host growth rate pre- and post-infection versus cells not exposed to phage. The reduction is likely representative of a cytoplasmic leak due to puncture by the phage, and the mode of recovery is as yet unknown. We plan to observe the event in media containing different amounts of certain osmolytes that may be taken up by the bacteria in response to pinpoint the mechanism of recovery.
Use of 2, 3, 5-Triphenyltetrazolium chloride (TTC) to assay the kinetics of phage and host interactions
(hashtags: #PhgOx21, #AHuynh)
Albert Huynh1, Philip J. Hill1, Benjamin M. C. Swift2, and Catherine E. D. Rees1
1School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK (United Kingdom)
2The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire, AL9 7TA, UK (United Kingdom)
Emerging interest in the specificity and sensitivity of phage therapy and diagnostics has prompted an increasing need to further understand the physical interactions between phage and host. Whether using phage for therapy or diagnostics, one of the key steps is determining the multiplicity of infection (MOI). Therefore, in this study a method was developed to determine the effect of different MOI on the lysis of slow growing mycobacteria by its cognate phage, using a microtiter plate format and a water-soluble compound 2, 3, 5- triphenyl tetrazolium chloride (TTC) as an indicator of viable mycobacteria. This method utilises the reduction of TTC into the dark red compound triphenyl formazan (TPF). It is a rapid, inexpensive, and simple method, which yields results that can be both quantified and qualitatively observed. It is noted that the fast and slow growing species of Mycobacterium tested (M. smegmatis, M. avium subsp. paratuberculosis and M. bovis BCG) reduce TTC to TPF at varying rates and have varying tolerances to the concentration levels of TTC and therefore the assay needs to be optimised to accommodate variations in host cell biology. The colorimetric reduction of TTC to TPF yields qualitative information on phage kinetics and host viability that can also be quantified by colorimetric absorbance at 540 nm that can be monitored using standard microplate spectrophotometers. The method was used to monitor the interactions between the mycobacteriophage D29 and host mycobacteria: M. smegmatis, M. avium subsp. paratuberculosis and M. bovis BCG and was utilised to determine the minimum MOI or minimum inhibitory concentration of phage required to ablate TTC reduction to TPF. Here, we observed the ablation of TCC reduction by M. smegmatis exposed to high phage titres (MOI= ≤ 1), and at lower titres TTC reduction increases, demonstrating the useability of TTC to study phage host interactions.
Rapid detection of phage killing through bacterial membrane potentials
(hashtags: #PhgOx21, #EJameson)
Eduardo Goicoechea Serrano1, Conor Edwards2, James Stratford2, Eleanor Jameson1
1School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom
2Cytecom Ltd, Cytecom Ltd. Unit 27f The Venture Centre Sir William Lyons Road Coventry CV4 7EZ, United Kingdom
Through electrical stimulation of bacteria their health can be established. This property of bacterial membranes paved the way to developing a novel method to rapidly measure phage killing. Working with Cytecom we have developed a method to rapidly monitor infection and killing of bacteria by phages. This method allows the user to detect phage killing in less than 2 hours. Bacteria and phages are applied to agar pads and electrically shocked. This enables fluorophores present in the agar to cross the bacterial membrane, and light up the bacteria, which is measured by the machine over the course of the assay. Live cells light up over the course of the assay, and the Cytecount records a composite image of the agar pad and any bright individual bacterial cells. The signal is measured across the agar pad in response to the electrical shock, with healthy cells giving off a high, increasing signal. Our testing has shown that this Cytecount method of monitioring bacterial membrane potentials can be successfully applied to detect phage killing. Using the Cytecount is much faster than traditional phage plaque assays and compared to optical density measurements for virulence assays it does not rely on bacterial division and growth, but rather directly detects bacterial killing. This method has great potential to speed up phage efficacy testing for the application of phages in clinical settings and to verify phage activity in slow growing bacteria.
First report of phages isolated from dairy farms in Argentina to control Shiga toxin producing Escherichia coli
(hashtags: #PhgOx21, #AJuarez)
Ana E Juárez, Stefanía B Pascal, Alejandra Krüger, Paula M A Lucchesi
Centro de Investigación Veterinaria de Tandil (CIVETAN, CONICET-CIC-UNCPBA), Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Buenos Aires, Argentina
Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen of global concern. The main reservoir for STEC is the bovine cattle, from which food, water and the environment can become contaminated. Lytic phages represent a promising alternative to control this threat throughout the food production chain. In this study, phages were isolated from bovine manure and effluents from two dairy farms. Samples were added to LB broth an incubated overnight for phage enrichment with the endogenous bacteria present in the samples. Then, culture supernatants were tested by the spot test for the presence of phages that could lyse STEC strains and, afterwards, phages were isolated and purified from the positive samples by the double agar method with E. coli DH5a as host strain. Nine native STEC strains representing different serotypes were used for analysis of phage host ranges. Phages were also preliminary evaluated for virulence traits performing a PCR screening for the gene encoding Shiga toxin 2 (stx2). Several phages that initially produced a lytic effect on O145:H- and O157:H7 STEC strains were either lost during purification stages on E. coli DH5a or discarded because they rendered a positive PCR result for stx2. One of the purified phages, designated as “L7.3”, was found to be lytic against O157:H7 STEC, rendering clear plaques on the STEC lawn, and was negative for stx2. This phage showed a higher EOP on E. coli DH5a than on the native O157:H7 STEC strains tested and stability in SM buffer for at least 103 days at 4°C. Although more studies are needed to evaluate its performance and safety, the present results suggest that L7.3 phage could be a good preliminary candidate to use in STEC biocontrol.
An optimised phage cocktail targeting pathogenic Klebsiella in an animal model
(hashtags: #PhgOx21, #LKelly)
Lucy Kelly1, Antonia Sagona1, Meera Unnikrishnan2, Eleanor Jameson1
1School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom
2Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom
Antibiotic-resistant Klebsiella are increasingly becoming a threat to human health, causing diseases such as pneumonia, urinary tract infections and meningitis. Bacteriophage offer a promising alternative to antibiotic treatment of Klebsiella infections. A cocktail of 7 Klebsiella phage was produced to target a wide range of Klebsiella strains. In vitro culture studies were used to first evaluate the efficacy of the phage cocktail against 4 Klebsiella strains, using the virulence index as a quantitative measure of phage cocktail virulence. Both 96-well plate and 10 mL MicrobeMeter based methods were used to analyse the optimal dosing concentration for administration of the phage cocktail. A range of doses of phage cocktail were then applied to a Galleria mellonella animal model to determine how the phage cocktail treatment performs in vivo. By studying the survival along with changes of bacterial and phage numbers in G. mellonella infected with Klebsiella, we can begin to understand how effective the phage cocktail is when applied to an animal with an innate immune system and a complex microbiome. The phage cocktail inhibited the growth of all the strains of Klebsiella tested, with a high virulence index that varied dependent on the Klebsiella strain. The survival of Klebsiella-infected G. mellonella was also increased upon treatment with the phage cocktail. These results demonstrate the efficacy of the optimised phage cocktail, suggesting such a treatment could be used as an accompaniment, or alternative, to antibiotic treatment in Klebsiella infection.
Phage prevalence in chicken internal organs and during experimental phage therapy against Salmonella Typhimurium
(hashtags: #PhgOx21, #KKwasnicka)
Katarzyna Kosznik-Kwaśnicka1, Łukasz Grabowski1, Magdalena Podlacha2, Dorota Myślińska3, Jagoda Mantej2, Karolina Ciemińska2, Karolina Zdrojewska2, Alicja Nowak-Zaleska2, Zuzanna Cyske2, Lidia Gaffke2, Karolina Pierzynowska2, Gracja Topka-Bielecka2, Agnieszka Necel2, Aleksandra Dydecka2, Grzegorz Węgrzyn2, Alicja Węgrzyn1
1 Laboratory of Phage Therapy, Institute of Biochemistry and Biophysics, PAS, Kładki 24 Gdańsk
2 Department of Molecular Biology, University of Gdansk, Wita Stwosza 59 Gdańsk
3 Department of Animal and Human Physiology, University of Gdansk, Wita Stwosza 59 Gdańsk
One of the main problems of contemporary medicine is growing numbers of antibiotic resistant strains of bacteria. The use of antibiotics in farming as mean of prevention of infections was identified as one of the ways of the resistance spreads. Thus, restrictions are being placed on farmers regarding the use of medications, but no alternative is given by the governments. Bacteriophages, used successfully to treat bacterial infections in human can be used in animals as well. However, concerns regarding the use of phages in farming are being raised as the influence on animal organism is not fully studied. In our work, we used a chicken model to analyse the spread of phages in internal organs during and after phage therapy against Salmonella Typhimurium, a common foodborne pathogen. Phages were administered at different time points during S. Typhimurium infection and were given daily for two weeks. At 5th and 14th day of the treatment, as well as 14 days after the last dose of the phage cocktail, the animals were sacrificed and their organs were analysed for bacteria and phage presence. We have observed that bacteriophages were present in samples taken from gastrointestinal tract, such as stomach, large intestine or liver. In some cases, phages were also isolated from brain, heart or kidney samples. Phage penetration seemed to be highly dependent on individual features of animals, as in some cases phages were present in every sample, while in some – only in gastrointestinal tract. The PFU/g also varied and seemed to be individual-dependent. We did not isolate S. Typhimurium from organs and no active infection was detected by scanning of feces samples. We have also analysed the level of inactivation of phages by sera obtained from chicken blood. These features also seem to be individual-specific and also phage-specific. This suggests that the use of phage therapy, though successful, was different for individual cases, pointing for large intrapopulation variability.
Topical bacteriophage application – a promising treatment against dog otitis externa
(hashtags: #PhgOx21, #JKwon)
Jun Kwon, Sang Guen Kim, Sang Wha Kim, Sung Bin Lee, Won Joon Jeong, Jeong Woo Kang, Sib Sankar Giri, Se Chang Park
Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea
Otitis externa (OE) is common disease occurred in the ear canal in the dogs. The causative agents are various, including fungi, yeast, and mainly bacteria. Various genus of bacteria, such as Staphylococci, Pseudomonas, Escherichia, Proteus, Streptococcus, and Enterococcus, have been isolated from dogs with OE. To date, main therapeutic strategies are especially based on systemic or topical antibiotic applications. However, the prolonged OE treatment is not rare complication. Secondary and perpetuating factors such as microbes, epidermal and sebaceous hyperplasia, and ulcers are responsible for its chronicity. Therefore, the antibiotic resistances can be induced as mentioned at several previous studies. In this study, we utilized bacteriophages (phages) as an alternative to antibiotics. The phage is a promising living anti-microbial agent. However, only a single phage administration cannot decimate bacteria colonized ear canals. For the breakthrough, we mixed several components for topicals and tested in vitro the capacity to eliminate planktonic and colonized bacteria. We used Clinical isolated, and multi-drug resistant Staphylococcus pseudintermedius, and Pseudomonas aeruginosa strain and the bacterial strain-targeting bacteriophages, pSp_J and pPa_SNUABM_DT01, which were studied at previous studies. And, we used glycerol, surfactant, and glycine as topical components. The clearance efficacy was tested against planktonic cells and prepared biofilm on 96-well polystyrene plates. The mixture topicals showed enhanced lysis activity on planktonic cells. Towards biofilm on 96-well plate, the topical also showed enhanced biomass degradation capacity (CFU reduction of 1-2 logs). We suggest bacteriophage topicals to treat dogs with otitis externa which are likely to involve antibiotic resistant bacteria.
Overexpression and isolation of recombinant, thermostable TP-84 bacteriophage depolymerase-glicosylase and development of functional enzymatic assay for removal of bacterial capsules
(hashtags: #PhgOx21, #BLubkowska)
Beata Łubkowska1,2, Ireneusz Sobolewski, Katarzyna Adamowicz, Edyta Czajkowska, Natalia Krawczun, Agnieszka Zylicz-Stachula and Piotr M. Skowron
1Department of Molecular Biotechnology, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland
2The High School of Health in Gdansk, Pelplinska 7, 80-335 Gdansk, Poland
TP-84 bacteriophage, infecting thermophilic bacteria – Geobacillus stearothermophilus exhibits unique features, such as very wide the bacteriophage growth range of 43-76oC, unidirectional genes arrangments and robust infection course, yielding very high titer, up to 1013/ml. These features make TP-84 an interesting target for the research on biology as an emerging model of a thermophilic bacteriophage and its applications in biotechnology (PLOS ONE 2018, 13: e0195449). We are studying a number of TP-84 proteins, one of those is bacteriophage enzyme depolymerase-glycosylase (TP84_26), degrading the bacterial capsule. Research on depolymerases is developed mainly in the medical aspects of pathogenic bacteria elimination (Front. Microbiol. 2019, 10: 2768). Besides potential medical applications we are aiming at basic research to determine the enzyme properties and its biological role in TP-84 infection as well as biotechnological applications, including industrial and scientific/pharmaceutical. These include: (i) removal of bacterial biofilms from industrial installations; (ii) antibiotics alternatives or supplements helping combat multi-drug resistant bacteria; (iii) the development of a new generation thermophilic phage display system as a research tool and peptide drugs development. Important aspect of such system is development of TP-84 bacterial host capabilities to uptake DNA, such as engineered TP-84 genomes and plasmids via transfection and transformation. For that purpose the glycosylase-depolymerase may be necessary tool, as Geobacillus stearothermophilus envelopes comprize 50 times higher volume than bacterial cells, thus being effective barrier for DNA transfer. Our studies on TP84_26 have shown: (i) TP-84 plaques on Geobacillus stearothermophilus lawn produce semi-transparent ‘halo’, apparently a result of stripping live cells from capsules; (ii) the ‘halo’ size increases with incubation time, caused by a factor diffusing faster through agar than the TP-84 particles. Likely it is an enzyme that degrades the capsule, but not the bacterial cell wall; (iii) the bioinformatics analysis has reveiled that TP84_26 gene encodes a glycosylase of 112 kDa, as further confirmed by LC-MS analysis; (iv) the native enzyme was purified and characterised; (v) its multimeric composition has been determined; (v) the TP84_26 gene was cloned in Escherichia coli, expressed at high level, the glycosylase-depolymerase purified and was shown to be enzymatically active in stripping host Geobacillus strearothermophilus from capsules; (vi) functional assay for evaluation of stripping of bacteria from capsules has been developed. Acknowledgements: Work supported by grant: TECHMATSTRATEG2/410747/11/NCBR/2019.
Utilising metatranscriptomics to investigate phage-bacteria dynamics in an agricultural soil ecosystem
(hashtags: #PhgOx21, #GMuscatt)
George Muscatt1, Eleanor Jameson1, Sally Hilton1, Sebastien Raguideau2, Emma Picot1, Ian Lidbury3, Elizabeth Wellington1, Christopher Quince4, Gary Bending1, Andrew Millard5
1School of Life Sciences, University of Warwick, Coventry, UK
2Warwick Medical School, University of Warwick, Coventry, UK
3Department of Animal and Plant Sciences, University of Sheffield, Sheffield, UK
4Earlham Institute, Norwich Research Park, Norwich, UK
5Department of Genetics and Genome Biology, University of Leicester, Leicester, UK
The “kill the winner” hypothesis states that phage infection prevents the domination of single bacterial populations within a community by targeting the fastest growing species. Thus, phage lytic activity is predicted to regulate bacterial community composition and maintain high community diversity. There has been little research to test these predictions, particularly in soils, where the roles of phages are less understood than in marine ecosystems. Typically, soil viromics studies overlook phage activity, which has the potential to reveal phage-host dynamics. Utilising metatranscriptomics, we simultaneously described phage activity and bacterial community diversity in an agricultural soil ecosystem. Both novel and previously discovered phages were found to be active. The predicted host range of active phages included dominant soil bacterial phyla, across a range of genera. Significant linear relationships were identified between the number of active phages detected and bacterial community diversity, providing support for the “kill the winner” hypothesis. Furthermore, active phages were associated with the relative abundance of bacterial groups, indicating the potential for phage activity to shape bacterial community composition. Evidence of phage-mediated diversification of soil bacterial communities has potential implications on plant-microbe interactions and terrestrial biogeochemical cycling.
Comparative analysis of rainforest Salmonella phages
(hashtags: #PhgOx21, #PMutusamy)
Prasanna Mutusamy1, Su Yin Lee1,2, Bent Petersen3,1, Thomas Sicheritz-Ponten3,1, Martha Clokie4, Andrew Millard4, Stella Loke5, Sivachandran Parimannan1, Heraa Rajandas1
1Centre of Excellence for Omics-Driven Computational Biodiscovery (COMBio), AIMST University, Kedah, Malaysia
2 Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Semeling, Kedah, Malaysia.
3Section for Evolutionary Genomics, The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
4Department of Genetics and Genome Biology, University of Leicester, Leicester, UK
5Deakin Genomics Centre, School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds Campus, Victoria 3216, Australia
With the rise in antimicrobial resistance (AMR) among bacterial strains, phage therapy has gained popularity as an alternative method for biocontrol of human pathogens. Most of the phages studied to date are from human derived environments and there are limited reports of phage isolation from tropical rainforests. Considering the diversity of microenvironments offered by the rainforest, we expected the presence of novel phages with different characteristics. Hence, we characterized and performed comparative analysis of 30 lytic Salmonella phages isolated from common environments (sewage, contaminated water) and soil and water samples from the Penang National Park, a conserved rainforest. The isolated phages belonged to four different phage families (Siphoviridae, Autographviridae, Myoviridae and Demerecviridae). Among the 30 phages, 23 were new species; 11 of these were from rainforest environments. The phages were able to infect a wide range of tested Salmonella enterica strains with different serovars and two of the rainforest phages also infected E.coli. Some of the rainforest phages exhibit a much shorter latent period (10 mins) and larger burst size (276 pfu/cell) compared to the other phages, which is a preferred criteria for a phage to be used therapeutically. Host challenge tests revealed that three of the rainforest phages were able to suppress the growth of Salmonella for up to 18 hours, while phage resistant mutants appeared as early as 4 hours with the other phages. Results suggest that rainforest phages are suitable candidates for phage therapy as they have a broad host range, high virulence, short killing period and they are free from any lysogenic or antimicrobial resistance genes. As the rainforest is a promising source of novel phages with superior characteristics, we can use this to our advantage to isolate more phages as alternative antimicrobial agents for curbing the growing AMR problem worldwide.
The challenges of phage therapy to treat the multi-drug resistant Escherichia coli EC958 (ST131)
(hashtags: #PhgOx21, #APark)
Alba Park-de-la-Torriente, Alison Low, Marianne Keith, Sean McAteer, David L Gally
Roslin Institute and the Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh UK
Urinary tract infections caused by multi-drug resistant (MDR) Escherichia coli are a common problem in humans and dogs that can lead to recurrent infections or more severe complications like pyelonephritis and sepsis. Phage therapy is considered a potential solution to treat MDR infections but to maximise the probabilities of success it is important to be aware of the possible challenges. We worked with E. coli strain EC958 from the ST131, a clonal group of special concern due to its global distribution, high pathogenicity and multi-drug resistance. We screened 30 phages against EC958 in LB and urine and saw differing phage activity in the different growth conditions: 60% of the phages tested in LB could effectively lyse EC958, whilst only 20% showed lysing activity in urine. We searched for a combination of phages that could inhibit the culture for an 18-hour period but, but with limited active phages in urine, no combination showed a significantly better result than the best single phage alone, which invariably ended in the resurge of the bacterial population. Our initial data suggests that EC958 is able to resist phage infection under in vivo-like conditions (i.e. in urine media), which led us to further investigate the mechanism of resistance. By recovering the surviving population and challenging them to a second phage infection we found that at least two different resistance mechanisms are involved: a transitional one in which a reversible change leads the bacteria to become susceptible to phage again, and a permanent one, mostly caused by mutations in the phage receptor. It is important to highlight that we have observed very different results when these assays are done in LB, which emphasises the need to study phage-host interactions in conditions that can mimic the real environment of the animal host to make these studies more translatable to clinical settings.
A different toolbox: isolation and characterisation of bacteriophages with activity against multidrug resistant invasive non-typhoidal Salmonella
(hashtags: #PhgOx21, #BSepulveda)
Ella V. Rodwell1, Nicolas Wenner1, Caisey V. Pulford1, Yueyi Cai1, Arthur Bowers-Barnard1, Alison Beckett2, Jonathan Rigby3,4, David M. Picton5, Tim R. Blower5, Nicholas A. Feasey3,4, Jay C. D. Hinton1, Blanca M. Perez-Sepulveda1
1Institute of Infection, Ecological and Veterinary Sciences, University of Liverpool, Liverpool, UK
2Biomedical Electron Microscopy Facility, University of Liverpool, Liverpool, UK
3Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK
4Malawi Liverpool Wellcome Trust Clinical Research Programme, University of Malawi, Blantyre, Malawi
5Department of Biosciences, Durham University, Durham, UK
The high prevalence of immunosuppressive illness in sub-Saharan Africa predisposes individuals to iNTS disease, which has led to the identification of new lineages of Salmonella enterica serovars Typhimurium and Enteritidis. These lineages are characterised by genomic degradation, altered prophage repertoires and novel multidrug resistant plasmids. Here, we isolated and characterised phages capable of lysing S. Typhimurium and S. Enteritidis associated with bloodstream infection. This pool of 32 phages were isolated from water samples collected from different locations in the UK and Malawi. Individual plaques with different morphologies were repeatedly purified by plaque assay, and phage DNA was extracted and sequenced. The isolated phages were further characterised based on morphology, host range, genome structure, and phylogenetic analysis, and classified into three major geographically distributed clusters. Cluster 1 phages were isolated from the UK, and were able to infect all bacterial hosts tested, whereas phages from Clusters 2 and 3 presented a more restricted host range profile. Sub-cluster 3.a phages were isolated from the UK, and 3.b from Malawi. Seven phages were unable to infect S. Enteritidis, and only one phage was able to lyse all of the tested bacterial hosts. We used a genetic approach to delete the prophages carried by wild-type S. Typhimurium ST19 and ST313 strains, and observed differences in phage infection profiles. Our findings are consistent with the existence of prophage-encoded “anti-phage” mechanisms in S. Typhimurium. This study represents the first exploration of the potential for phages to target the lineages of Salmonella that are responsible for bloodstream infections in Sub-Saharan Africa. This phage collection has the potential for wider applications and could have utility against multidrug resistant iNTS infections in clinical and food preparation settings.
How do temperate bacteriophage affect the fitness of Pseudomonas aeruginosa?
(hashtags: #PhgOx21, #GPlahe)
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.
Excursion in Staphylococcus aureus during bacteriophage infection
(hashtags: #PhgOx21, #MProchazkova)
Michaela Procházková1, Lenka Šmerdová1, Pavol Bárdy2, Roman Pantůček2, Pavel Plevka1
1CEITEC Masaryk University, Kamenice 753/5, 625 00 Brno, CZ
2Faculty of Science, Masaryk University, 753/5, 625 00 Brno, CZ
Antibiotic-resistance in bacteria is a global issue perpetuated by overuse of antibiotics in medicine and animal production. Gram-positive Staphylococci are a natural part of our skin flora and are also a common cause of infections both in the community and in healthcare facilities. Methicillin-resistant Staphylococcus aureus (MRSA) causes 10-30% of hospital-acquired infections and forms persistent biofilms on hospital instruments, thus complicating post-transplant healing. Untreated MRSA progresses in chronic infections leading to endocarditis, pneumonia, and metastases to other tissues. Patients with MRSA infections are 64% more likely to die than people with drug-sensitive infections. Bacteriophages are viruses that infect exclusively bacteria and can be used to eliminate bacterial infections, even those resistant to antibiotics. Utilizing cryo-electron tomography and light-sheet fluorescence microscopy, we follow the progress of Myoviridae bacteriophage φ812 from attachment to the bacterial cell wall, through the production of phage particles in the cytosol, to the cell lysis and release of progeny phages. With light-sheet fluorescence microscopy, we show that the growth of biofilm in an ultrathin tube is concluded in 6 – 8 hours post-seeding and that the metabolic activity of S. aureus cells gradually decreases. In 24 hours, biofilm releases from support and disperse with concurrent boost in metabolic activity and re-appearance of fluorescence. Tomography on thin sections produced by focused ion beam milling provides not only a snapshot of the phage-producing bacteria in a near-native state but also the high-resolution structures of phage particles in individual assembly stages. This insight into the mechanism and kinetics of phage infection is instrumental for an assessment of the phage therapy for population-wide use as an alternative to antibiotics.
Efficiency of encapsulated bacteriophage preparation BAFASAL+G on Salmonella Enteritidis reduction in broilers
(hashtags: #PhgOx21, #MRzepkowska)
Magdalena Rzepkowska, Monika Sakosik, Wojciech Kropiwnicki, Paulina Wigner, Elżbieta Górecka, Jarosław Dastych
Proteon Pharmaceuticals S.A., Łódź, Poland
Bacteriophage preparations used as feed additives are an effective alternative method of preventing and treating poultry infections caused by Salmonella bacteria. These types of poultry-to-human infections are the second most common cause of the epidemic of foodborne diseases, so preventing them is extremely important from a public health and economic perspective. The six-component bacteriophage preparation BAFASAL + G shows effective specific activity against 92% of tested Salmonella strains from PP bacterial strain collection. The preparation in a dose of 1*106 PFU/bird is used as an additive to water, along with it, it reaches the bird’s intestines through successive elements of the digestive system and there it infects Salmonella rods. Passage through the digestive system causes losses in the titre of bacteriophages, therefore an attempt was made to protect the bacteriophages through the use of encapsulation in a polymer, polysaccharide capsule. The introduction of such modification may reduce the concentration of bacteriophages (2*104 PFU/bird) necessary to inhibit Salmonella infection, it is achieved by protecting the preparation with a capsule against the adverse environmental influences and the release of bacteriophages at the target site. This work describes an in vivo experiment carried out on broiler hens infected with an environmental strain of S. Enteritidis, followed by supplementation with the liquid and encapsulated BAFASAL + G preparation. The experiment compared parameters such as: average consumption of feed and water, feed conversion rate, mortality and the Salmonella loads in the intestines in two bacteriophage treatments, in relation to the two control groups: one not infected with bacteria and without supplementation and the second one group infected with the S. Enteritidis strain and not treated with bacteriophages.
Diversity of bacteriophages from sulfate-type gypsum karst lake of Northern Lithuania
(hashtags: #PhgOx21, #ESimoliunas)
Monika Šimoliūnienė1, Patricija Petrikonytė1, Sigitas Šulčius2, Rolandas Meškys1, Eugenijus Šimoliūnas1
1Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio av. 7, Vilnius, Lithuania
2Laboratory of Algology and Microbial Ecology, Nature Research Centre, Akademijos str. 2, Vilnius, Lithuania
In this study we present nine bacteriophages isolated from water samples of sulfate-type gypsum karst Lake Kirkilai (Lithuania). The host range determination experiments showed that three bacteriophages (vB_BauS_KLEB27-1, vB_BauM_KLEB27-3 and vB_BceS_KLEB30-3S) are active against bacteria from the genus Bacillus, two phages (vB_PsoM_KLER1-1 and vB_PsoM_KLER1-2) infect Pararheinheimera, while the host of vB_AveS_KLEA5, vB_PmaP_KLEP18-1, vB_PpeM_KLEP7 and vB_PcuM_KLEP17-4 are bacteria from the genera Aeromonas, Paracoccus, Pseudaeromonas and Pseudomonas, respectively. TEM analysis demonstrated that most of the phages (KLEB27-3, KLER1-1, KLER1-2, KLEP7 and KLEP17-4) are myoviruses. Phages KLEA5, KLEB27-1 and KLEB30-3S share the morphology of siphoviruses, while KLEP18-1 is a podovirus. Efficiency of plating (EOP) tests revealed that all phages can form plaques in the temperature of 22°C. Complete genome sequences of all aforementioned bacteriophages were identified, and it was demonstrated that phages contain linear, double-stranded DNA genomes ranging from 37, 134 bp (KLEB30-3S) to 216, 748 bp (KLEB27-3). Phylogenetic analysis, based on the comparison of essential structural and functional genes, as well as total proteome and overall nucleotide identity, revealed that the vast majority of the phages (except KLEB30-3S), have no close phylogenetic relatedness to other viruses published to date and potentially represent new genera within the order Caudovirales. Moreover, to our knowledge, none of genomes of Pararheinheimera or Pseudaeromonas phages have been published to date. Thus, the results of this study not either lead for a better understanding of almost unexplored diversity of bacteriophages in the unique sulfate-type gypsum karst lakes, but also expand our knowledge of bacterial viruses in general.
Pathways to synthetic bacteriophages for phage therapy: Do tRNAs control host-range and virulence?
(hashtags: #PhgOx21, #ARoyam)
Madhav Madurantakam Royam1, Elizabeth M Wellington1, Martha RJ Clokie2, Richard J Puxty1
1Gibbert Hill Campus, School of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom
2Department of Genetics and Genome Biology, University of Leicester, University Road, LE1 7RH, United Kingdom
The rising global infectious diseases are of great concern, and it is challenging for clinicians and scientists to develop a strategy in curbing them. The genus Mycobacterium contains many pathogenic members. Among them, tuberculosis caused by M. tuberculosis is among the top 10 causes of death globally. Both innate and emerging antibiotic resistance in the mycobacteria is a concern, which limits therapeutic options. Phage therapy can be effective as a drug of last resort for some mycobacterial infections, and there is a promise for this technique throughout hospitals and veterinary clinics. However, once a pathogenic agent is identified, a trial and error approach for finding an effective phage is required. This can slow down treatment and reduce the adoption of phage therapy more broadly. Improvements in our understanding of the genetic factors underpinning host range and phage virulence are required. Ultimately, this may lead to better predictions of host-range from phage genetic sequence or the genetic engineering of phage to better target selected pathogens. One particular genetic factor that may be important for host range and virulence in the presence of tRNAs in phage genomes. Despite many hypotheses, the exact role(s) for tRNAs in phage infection remain enigmatic. Here, I focus on the cluster C1 mycobacteriophages, which encode an average of 30 tRNAs per genome. I discuss current reverse genetics approaches to inactivate tRNAs to attempt to understand their role in host range and virulence and future-forward genetic screens to identify genetic determinants of host range.
Bioinformatic and phenotypic characterization of bacteriophages encoded within and infecting Klebsiella michiganensis
(hashtags: #PhgOx21, #TZaitlik)
Thomas Smith-Zaitlik1, Preetha Shibu2, Anne L. McCartney3, Geoffrey Foster4, Lesley Hoyles1, David Negus1
1 Nottingham Trent University, Nottingham, NG11 8NS
2 University of Westminster, London, W1B 2HW; Frimley Health NHS Trust, Camberley GU16 7UJ
3 University of Reading, Reading, RG6 6AH
4 SRUC Veterinary Services, Inverness, IV2 5NA
Klebsiella michiganensis is frequently misidentified as K. oxytoca in clinical and veterinary laboratories, resulting in under-recognition of this emerging pathogen. It is predicted to encode an extensive array of virulence factors as well as a thick capsule likely to shield it from the host immune system. Bacteriophages (phages) are viruses which exclusively infect bacteria, often with a narrow host range. Phages are promising therapeutics for treating antibiotic-resistant K. michiganensis infections. This study looked to fully characterise six lytic phages isolated from sewage water against three multidrug-resistant K. michiganensis host strains and to induce prophages identified within the host genomes as potential therapeutics. A collection of 59 clinical (KO) and 52 veterinary (GFKO) isolates, putatively identified as K. oxytoca, were reanalysed by sequencing and alignment of the rpoB gene to Klebsiella spp. reference alleles. Sequence alignment revealed 26 KO and 21 GFKO isolates were K. michiganensis. The host range of the six phages was determined against the K. michiganensis isolates and revealed that 24 KO and 17 GFKO isolates were lysed by at least one of the six phages. Further characterisation of the phages was achieved by a combination of transmission electron microscopy and genome sequencing. Prophages were identified within all three K. michiganensis host genomes using PHASTER and induced using mitomycin C. Successful induction was confirmed by amplification of prophage-specific DNA sequences following digestion of host genomic DNA in mitomycin C-treated cultures. These results highlight that phages present a potential alternative to antibiotics for the treatment of K. michiganensis infections in both humans and animals. Moreover, K. michiganensis itself may provide a rich source of prophages which are potentially lytic on related strains.
Isolation and characterisation of bacteriophage CSP3 for the development of a Burkholderia sp. biosensor
(hashtags: #PhgOx21, #Stanton)
Cassandra R Stanton1, Michael Beer2, Steven Batinovic1, Steve Petrovski1
1Department of Physiology, Anatomy & Microbiology, La Trobe University, Victoria 3086, Australia
2Department of Defence Science and Technology, Fishermans Bend Victoria 3207, Australia
The Burkholderia cepacia complex (Bcc) contains 24 species of opportunistic pathogens that cause detrimental pulmonary infections in immunocompromised individuals. Due to their phenotypic similarities, current biochemical and culture-based assays for diagnosis are often unable to accurately differentiate the Bcc. Bacteriophages (phages) are an attractive option to use in place of traditional assays due to their host specificity and rapid infection. Combined with genetic engineering techniques, phages have the potential to be developed into biosensors, able to rapidly detect live bacteria in various environments. Four putative phages infective for two clinical isolates of Bcc were isolated and characterised. One phage, CSP3, was chosen as the candidate for the development as a biosensor as it displayed a narrow host range. Phenotypic and genomic characterisation revealed CSP3 has a Podoviridae morphology and a 63 kb genome. VIRIDIC analysis of the four closest BLASTn matches shows CSP3 to have 78.8% intergenomic similarity with DC1, a phage isolated from Canada. Along with DC1, CSP3 can be classified as a novel member of the Lessievirus genus and the first Australian isolated phage of this group. However, the CSP3 genome contains an integrase gene and repressor-like gene, making it unfavourable for clinical use due to its potential impact on its lytic capabilities. We then aimed to genetically engineer CSP3 to create a more advantageous phage for clinical use. A CRISPR-cas9 vector was designed to knock out the CSP3 integrase and insert red fluorescent protein (RFP) in its place. As the recombinant CPS3 begins to infect and replicate in other bacteria cells, the RFP will be transcribed and expressed, resulting in a fluorescent signal. Since integrase gene will no longer be present, the lysogenic life cycle will not occur. This study will provide proof of concept results for phage biosensor production that can be used in industry and clinical settings.
A new, natural bacteriophage gel preparation preventing E. coli and S. aureus udder infections in cows
(hashtags: #PhgOx21, #MSzymanska)
Marta Szymańska, Marta Krzyżaniak, Daria Królikowska, Arkadiusz Guziński, Przemysław Szewczyk, Ewelina Wójcik, Agnieszka Maszewska
Proteon Pharmaceuticals S.A., Łódź, Poland
Bovine mastitis is the inflammation of the mammary gland most frequently caused by bacteria, especially Staphylococcus aureus and Escherichia coli. Mastitis is one of the largest production concerns in the dairy industry worldwide. Due to antibiotic resistance problem and the limitation of the use of antibiotics in dairy industry, it is necessary to develop alternative methods of controlling mastitis. The aim of this study was to develop a broad spectrum gel bacteriophage preparation for the prevention of S. aureus and E.coli udder infections. In the development of phage preparation the following steps were involved: isolation of phages specific to E.coli and S.aureus causing mastitis, their lytic activity evaluation, differentiation by RFLP or RAPD, bioinformatic analysis of phage genomes to determine their taxonomy and virulent character. Next, selection of the strictly virulent phages for cocktail with the broadest antibacterial activity spectrum was performed. The maintenance of lytic activity against variants of bacteria resistant to single phages included in the preparation, activity in milk and ability to prevent biofilm formation were also tested. Technology of biological material immobilization in natural polymer used in pharmacy and food industry was used to formulation of preparation. The developed phage cocktail showed specificity for 98% and 100% of different mastitis causing strains of E.coli and S. aureus, respectively. Phage cocktail antibacterial activity in liquid culture, ability to prevent biofilm formation and activity in milk were proved. The gel phage preparation showed 95% stability for at least 1 year. The research resulted in obtaining the natural gel bacteriophage preparation in easy-to-application form and presenting a high potential for controlling mastitis caused by E.coli and S.aureus. Research carried out under project no POIR.01.01.01-00-0016/16, co-financed by NCRD, “Smart Growth Operational Programme 2014-2020″
Ride the filament! – A hunting strategy of P. aeruginosa infecting phage JBD30 revealed by cryo-electron tomography
(hashtags: #PhgOx21, #Valentova)
Lucie Valentová, Tibor Füzik and Pavel Plevka
Central European Institute of Technology, Masaryk University, Kamenice 735/5, 625 00 Brno, Czech Republic
Pseudomonas aeruginosa is an opportunistic human pathogen that causes acute and chronic infections, which can lead to life threatening septic shock. Treatment of these infections is complicated by frequent antibiotic resistance of this bacterium. Here, we present bacteriophage JBD30 that infects P. aeruginosa and is a potential candidate for phage therapy. Using cryo-electron tomography we followed the infection of P. aeruginosa by bacteriophage JBD30 from attachment to the bacterial cell, to the cell lysis and production of new phage progeny. Bacteriophage JBD30 belongs to the family Siphoviridae. Its virion is composed of icosahedral head connected via dodecameric connector complex with long flexible non-contractile tail. JBD30 tail is terminated with a baseplate decorated with three long and three short tail fibres. Virions of bacteriophage JBD30 bind to P. aeruginosa type IV pili protruding from a bacterial cell pole in the close neighbourhood of a flagellum. Pili type IV are involved in twitching motility, adherence to the surfaces and biofilm formation. Bacteriophages JBD30 bind to pili (type IV) by their long tail fibres. Then they are pulled towards the cell surface by pili retraction, where they irreversibly bind with short tail fibers to their secondary receptor. Finally, the bacteriophages puncture the outer cellular membrane, degrade the peptidoglycan layer and inject their DNA into the host cell. Our results revealed the strategy that bacteriophage JBD30 uses to attack and infect bacterium P. aeruginosa. Combined with the information gained from our wet-lab experiments it enable us to propose the model of phage (JBD30) – bacterium (P. aeruginosa) interaction.