Posters and poster guidelines
Thank you for considering to present your work as a poster at Phages 2022.
Digital poster preparation and submission
- Page size: Prepare your poster as you would normally do for printing. You can prepare your poster in sizes A1 or A0, as the page size is not important for digitally presented posters.
- Naming your poster files: Name your poster files as follows: <your surname>-Phg22-Poster.pdf | <your surname>-Phg22-Poster.png | <your surname>-Phg22-Poster.jpg, etc. For example, for David Jones, name your file as Jones-Phg22-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, Phage2022, Oxford-phage-poster. Such files will be automatically rejected.
- Poster submission and deadlines: All poster presenters, whether attending virtually or in-person, are required to submit a digital version of their poster. Submit your final poster as both PDF (<5MB) and JPG/PNG (<1MB) files via the link below no later than 31st August 2022 (we must have received your poster abstracts by 15th August). Late posters may not be included in the symposium programme. Please DO NOT send your poster (or abstract) files by email. Please ensure you send us the very final version of your poster (as well as your poster abstract), as once published, it cannot be replaced.
The poster PDF files, whether the presenter is attending virtually or in-person, will be made available via the secure conference documents page to the conference participants before the meeting.
- 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 #PhgOx22, as well as the poster specific hashtag (given under each poster abstract) – do tag @PhageOxford in your tweets. Do include your Twitter handle in your poster, if you have one.
Hardcopy posters: If attending in-person, you may bring along a printed copy of your poster (maximum A1 size) to be displayed during the conference.
Any further information about the poster presentations at this digital meeting will be available in the future.
Before uploading your poster, you must make sure that you follow ALL of the instructions above!
(Presenters in Bold)
Accepted poster abstracts will be displayed below. If your abstract has been accepted for presentation but it does not appear in the list below, please let us know as soon as possible by emailing PhageOxford@gmail.com.
Enterococcal phages and prophages: Isolation and Tail-associated lysins analysis and expression
Twitter hashtags: #PhgOx22, #AAlrafaie
Alhassan Alrafaie1,2 and Graham P Stafford1
1School of Clinical Dentistry, University of Sheffield, Sheffield, United Kingdom
2Department of Medical Laboratory Sciences, College of Applied Medical Sciences in Al-Kharj, Prince Sattam Bin Abdulaziz University, Al-Kharj 11942, Saudi Arabia
Vancomycin-Resistant Enterococci (VRE) are robust bacteria that cause various diseases and are disseminated worldwide. As they are highly antibiotic resistant, one promising way to tackle these bacteria is by exploiting the natural enemy of bacteria known as phages. In our work, we successfully isolated a panel of phages targeting strains of Enterococcus faecium and E.faecalis. One of which, phiSHEF14, is a podovirus (19.3Kb) which we characterized using TEM, Host range, killing assay and genome analysis. phiSHEF14 shows a narrow host range infecting the VRE strain E1071 and an efficient killing at different multiplicity of infection (MOI). As per genome annotation, a predicted lytic protein with New Lipoprotein C/Protein of 60-kDa (NLPC/P60) domain was identified as well as cloned, expressed and purified. In a separate project, we analysed 507 phage and prophage genomes and identified in-sillico a range of proteins (mostly in tail module) with various predicted lytic activities. One protein with a predicted pectinesterase activity was successfully cloned, expressed, and purified. Our future work involves testing these proteins to determine their activity and efficiency. Enterococcal phage and prophage genomes revealed various potential lytic proteins that have the potential to better combat antibiotic resistant bacteria.
Defense mechanism and structural basis for the anti-phage and anti-conjugation activity of DISARM class 1
Twitter hashtags: #PhgOx22, #CPMaldonado
Cristian Aparicio-Maldonado1, Jack P. K. Bravo2, Gal Ofir3, Andrea Salini1, David W. Taylor2, Rotem Sorek3, Stan J.J. Brouns4,5and Franklin L. Nobrega1
1School of Biological Sciences, University of Southampton, SO17 1BJ, Southampton, United Kingdom
2Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA
3Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel
4Department of Bionanoscience, Delft University of Technology, 2629HZ, Delft, The Netherlands
5Kavli Institute of Nanoscience, Delft University of Technology, Delft, 2629HZ, The Netherlands
Under the constant pressure of mobile genetic elements, bacteria have evolved a wide arsenal of defense mechanisms in the fight for survival. Recent computational studies have predicted the presence of novel mechanisms of innate defense localized in nearby regions to known defense systems. Most of them have a molecular mechanism that remains uncharacterized. Here, we study the Class 1 DISARM (Defense Island System Associated with Restriction–Modification) system from Serratia sp. SCBI and provide a structural model for the molecular mechanism of the effector complex DrmAB. This system was able to provide broad protection against double-stranded DNA phages and reduce a population of single-stranded phages to undetectable levels. Such protection was abolished by the deletion of essential genes, like drmA and drmB, but not others like the methyltransferases. These ones, DrmMI or DrmMII, recognize and methylate the motifs ACACAG and MTCGAK, respectively, but surprisingly the absence of one or both genes do not result in autoimmunity. In addition to antiphage activity, Class 1 DISARM also limits plasmid conjugation, a protection that is linked to the number of methylase cognate sites in the invader DNA. By cryo-EM, we modeled the structure of DrmA-DrmB, a heterodimer complex. The defense activity occurs through the recognition of the invader DNA by the complex DrmAB. This complex is autoinhibited by a trigger loop within DrmA and gets dislodged by the binding of a free 5’-single-stranded DNA. This DNA binding triggers a long-range structural rearrangement that leads to the activation of the complex.
Probing the function of the highly unusual split DNA ligase encoded by the T5-like bacteriophages
Twitter hashtags: #PhgOx22, #ABasit
Abdul Basit1,Julia M. Richardson2, Stuart A. MacNeill1
1School of Biology, University of St Andrews,St Andrews, UK
2School of Biological Sciences, University of Edinburgh,Edinburgh, UK
T5 is an intriguing bacteriophage due to its large genome size (122 kb) encoding over 160 proteins, only around half of which have proposed functions based on homology. Studying T5 holds the potential to reveal novel biological or biochemical mechanisms in addition to providing potential new avenues to controlling pathogens. T5 has a linear, double-stranded DNA genomethat contains single-stranded nicks at defined positions, and which encodes a highly unusual DNA ligase that is encoded by adjacent ORFs (ligA, ligB) expressed from different promoters. This split DNA ligase is a unique defining feature of the T5-like phage family, suggesting that having a split DNA ligaseis essential or highly advantageous for the phage life cycle. To understand the split nature of T5 DNA ligase and its role in phage infection, we aim to mutate the ligase ORFs usingCRISPR-Cas9 genome editingand determine the consequences on the phage life cycle.Initially, in order to optimize the T5 genome engineering methodology, we have focused on a dnk gene, encoding deoxynucleosidemonophosphate kinase (Dnk), which was previously reported as non-essential in phage T7. The dnk gene was successfully replaced with lacZα, with the recombinant phages producing blue plaques on plates containing X-gal, showing for the first time that Dnk activity isnot required for the T5 infection cycle. Subsequently we showed that lacZαcould be removed from the dnk::LacZα phage, with recombinant phage being identified by their failure to produce blue plaques. The frequency of successful recombinant phages obtained using our method was 0.3% in both cases. We are currently expanding this workwith the aim of deleting the ligA and ligB ORFs encoding the split ligase subunits, with the mutated phage being propagated in host cells expressing the corresponding genes (wild-type or mutated)from anarabinose-inducible promoter, and will present the preliminary results of this work.
Genome Analysis and antibiofilm activity of a novel phage RDN8.1 against multi-drug resistant and extensively drug-resistant biofilm forming uropathogenic E. coli isolates
Twitter hashtags: #PhgOx22, #NChaudhary
Naveen Chaudhary1, Balvinder Mohan1, Ravimohan S. Mavuduru2, Yashwant Kumar3 Neelam TanejaI*1
1Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research,Chandigarh, India
2Department of Urology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3Central Research Institute, National Salmonella and Escherichia Centre, Kasauli, India
We aimed to study whole genome and antibiofilm activity of a novel RDN8.1 active against multi-drug- resistant (MDR) and extensively drug-resistant (XDR) biofilm-forming Uropathogenic E. coli isolates. We isolated novel phage RDN8.1, from community raw sewage water belonging to the order Caudovirales and the family Autographiviridae. It has a large burst size of 250 plaque-forming units per infected cell. The lytic ability of phage RDN8.1 was tested at MOIs 0.01, 0.1, and 1.0 in time-kill assays, and the best killing was achieved at MOI 1.0. The lytic activity started within 2 hours and was sustained for up to 18 hours. The complete genome of Escherichia phage RDN8.1 is 39.5 kb with a GC content of 51.6%, consisting of 49 ORFs, and all ORFs are present in the direct strand. RDN8.1 genome displays closest similarity (Blastn identity 86–92.97%) with four phages (Citrobacter phage CR44b, Escherichia virus Vec 13, Citrobacter phage SH3, and Enterococcus phage EFA-1).. The phage was able to inhibit biofilm formation with a clear disruption of the biofilm structure. Endolysin found in the phage RDN8.1 genome might have a role in the disruption of biofilms. The phage is stable over a wide range of temperatures and pH. We did not find any genes encoding markers of temperate bacteriophages such as integrase, recombinase, repressor, and excisionase. Therefore, the RDN8.1 phage is considered a virulent bacteriophage. We provide detailed genome and antibiofilm activity information of a novel lytic Escherichia phage RDN8.1 that has the potential for inclusion into phage cocktails being developed for the treatment of urinary tract infections (UTIs) caused by biofilm forming highly drug-resistant UPEC isolates.
The Use of Lytic Phage in the Treatment of Bacterial Urinary Tract Infections, and the Potential of Re-sensitising Bacteria to Antibiotics Using Phage Exposure
Twitter hashtags: #PhgOx22, #LADuignan
Libby A M Duignan1, Isik Kaya1, Craig Winstanley1, Rachel Floyd1 and Jo L Fothergill1, Chloe James2
1Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Ronald Ross Building, 8 West Derby Street, Liverpool L69 7BE
2University of Salford, 43 Crescent, Salford M5 4WT
Antimicrobial resistance (AMR) is a major global health issue. Escherichia coli is a common cause of urinary tract infections (UTIs) and one potential alternative with growing traction is the use of lytic phages. E. coli isolated from patients with UTIs (151 isolates), were used to test the efficacy of 56 novel lytic phages isolated from sewage. The bacterial panel were genome sequenced and extensively tested for antimicrobial resistance. This revealed a range of ST types including ST131. Antibiotic resistance testing showed many of the isolates were multidrug resistant and carried many AMR genes.The 56 new phages were used in cross-infection studies against the clinical isolates and showed that some phages displayed a broad infection range, up to 72% of the bacterial isolates tested. A subset were sequenced and bioinformatic analysis of the phage genomes confirmed they are obligate lytic phages that do not harbour toxin or virulence factor genes. Screening assays identified the emergence of in vitro bacterial resistance against some of the phages. These evolved isolates were then tested for antibiotic resistance and complex dynamics were revealed. Phage exposure led to examples of both resensitisation and increased AMR. Analysis using the in vivo Galleria infection model, showed that E.coli strains evolved in the presence of phages were less virulent than prior to phage exposure. This study highlights phages with clinical potential against a wide range of isolates from UTIs including MDR E.coli, however the need to further understand the complex interplay between phage therapy, bacterial adaptation and AMR during infection before this synergy is used in a therapeutic approach for UTIs.
Phage host range evolution in spatially structured heterogeneous bacterial populations
Twitter hashtags: #PhgOx22, #DDutta
Dibyendu Dutta, Emma Tollafield, Wolfram Moebius
Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
While bacteriophage host range is typically narrow, de novo mutations that emerge as phages replicate can expand or shift their host range. In an environment with only ‘the original’ host, such mutations are lost (due to associated fitness cost of host range modification). However, upon encountering compatible novel hosts, mutant phages have a selective advantage with the exclusive ability to infect and replicate within them. In a liquid culture environment, all virions can infect all original and novel host cells of the population, which may enhance the population fraction of the mutant virions proportional to the percentage of novel hosts. However, in a spatially structured environment, virions only encounter hosts in their vicinity, thus raising the question about how a spatially structured heterogeneous environment affects host range evolution. Towards study this question, we use the model system of bacteriophage T7 with its ‘original’ host E. coli BW25113 and novel host E. coli ΔwaaR, resistant to wildtype T7. We ask how the relative abundance and spatial organisation of original and novel host affects bacteriophage host range evolution. Experiments show rapid lysis of original host cells while regions of novel host remain unaffected. With some delay, a second plaque is observed affecting the novel host, indicating the emergence of a host range expansion or shift. We quantify this observation by collecting the phages present at the end of the experiment and determining their host ranges. Control experiments in liquid allow us to explore the effects of spatial structure. To rationalise our findings, we use an analogous mathematical model on a lattice where each lattice site can originally be occupied by either original or novel host, phage can replicate on suitable host, and mutate to expand their host range.
Transcriptomic landscape of therapeutic Kayvirus phage during infection of Staphylococcus aureus
Twitter hashtags: #PhgOx22, #AFinstrlova
Adéla Finstrlová1, Ivana Mašlaňová1, Bob G. Blasdel Reuter2, Jiří Doškař1, Friedrich Götz3 and Roman Pantůček1
1Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
2Vésale Bioscience, Vésale Pharma, Noville sur Mehaigne, Belgium
3Microbial Genetics, Interfaculty Institute of Microbiology and Infection Medicine Tübingen, University of Tübingen, Tübingen, Germany
The treatment of infections caused by human and veterinary pathogen Staphylococcus aureus is becoming worldwide healthcare concern due to the increasing resistance to antibiotics. A promising alternative to currently used drugs is represented by lytic phages from genus Kayvirus, but their use is impeded by the lack of knowledge of phage-bacterium molecular interactions. Here, we performed RNA sequencing of two S. aureus strains infected with Kayvirus bacteriophage K to decipher the transcriptomics of the phage lytic life-cycle and the host response. We found that the temporal transcriptional profile of phage K was comparable in both strains except for a few loci. The RNA-Seq data also revealed presence of phage non-coding RNAs, which may play a role in the regulation of phage and host gene expression. The response of S. aureus to phage K infection resembles a general stress response and involves upregulation of nucleotide, amino acid and energy synthesis and transporter genes and the downregulation of host transcription factors. Our results clarify the global transcriptional interaction between phage and host, which will ensure safer usage of phage therapeutics and may also serve as a basis for development of new antibacterial strategies. Acknowledgments: Supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) – Funded by the European Union – Next Generation EU and grant NU22-05-00042 from the Ministry of Health of the Czech Republic.
Polyphasic survey of A. hydrophila phages and Genomic Characterization of a novel lytic phage, Aeromonad phage B614
Twitter hashtags: #PhgOx22, #AGutierrez
Tracey Antaeus D Gutierrez1, Reuel M Bennett2,3 and Donna May D C Papa2,3
1The Graduate School
2Research Center for Natural and Applied Sciences
3Department of Biology, College of Science
University of Santo Tomas, España, Manila 1015 Philippines
Bacteriophages are considered vital in the microbial ecosystems for their role in bacterial mortality and evolution. Aside from their ecological importance, these viruses are considered as viable candidates for therapeutic applications in view of the emergence of antibiotic resistance. However, there are limited studies about phages in the country, which hinder further understanding of the nature of these viruses. This study employs a polyphasic approach in classifying bacteriophages by determining phenotypic characteristics, including the stability of phages in different environmental conditions (pH, temperature, and saline concentrations) and genotypic properties. Twenty-seven (27) previously isolated Aeromonas hydrophila phages were acquired from the UST-Bacteriophage Ecology, Aquaculture, Taxonomy, and Systematics (BEATS) Reference Collection, and were initially propagated using the host bacterium, A. hydrophila BIOTECH 10089. Physiological characterization of phages through stability testing and host range showed the phages isolated from sewage and polluted river systems exhibited the most stability and polyvalency in infection. Moreover, the diversity of the phages in the collection was established through these characteristics, resulting in 71% viable phages with varying characteristics. Initial analysis of g23 sequences, coupled with the phenotypic characteristics of the phages, suggested a highly unique set of phages isolated from four sample types (sewage, lake, river, and aquaculture pond) that infect A. hydrophila and high similarity in marker gene sequence. The results indicate a widespread distribution of related Aeromonad phages in the country, with phenotypic characteristics that cannot be limited to its environment. Finally, this paper also described the genome characteristics of phage B614, a novel lytic bacteriophage isolated from sewage water. Phage B614 may present a new species of Aeromonas phage under unclassified Biquartaviruses in the country, showing only an ANI of 84.22% to the closest Aeromonas phages in NCBI (Aeromonas phage ASFD-1). The results presented in this study may present evidence of the micro diversity of bacteriophages in local environments and their role in the abundance, control, and the diversity of bacterial communities.
Liposome-encapsulated bacteriophages for enhanced intracellular delivery of phages
Twitter hashtags: #PhgOx22, #XinyaoHe
Xinyao He, Wafa T. Al-Jamal, Timofey Skvortsov
School of Pharmacy, Queen’s University Belfast, Belfast, UK
As one of the solutions to the problem of antimicrobial resistance, phage therapy has been actively investigated to treatment of various bacterial infectious diseases through different routes of administration and substantial progress has been made in bringing phage-based approaches closer to clinical practice. However, there are still many difficulties in clinical application of phages, such as the narrow bacterial host range and the rapid elimination of phages by the host immune system. In addition, the application of phages against intracellular bacteria is hindered by the fact that phage particles must penetrate the eukaryotic cell membrane to lyse pathogenic bacteria inside host cells. Meanwhile, many important human and animal bacterial pathogens are either obligately or facultatively intracellular, so it is important to develop strategies to enhance intracellular delivery of phage particles. To improve the viability and stability of therapeutic phages in vitro and in vivo, as well as their uptake by infected cells, the use of liposomes with high biocompatibility as transport carriers is a good choice. The phospholipid bilayer of liposomes easily fuses with cell membranes, making them one of the preferred delivery methods for small molecules and biological agents. In this project, we focus on the study of phage therapy against foodborne pathogens L. monocytogenes and S. enterica, and the development of liposome formulations loaded with corresponding therapeutic phages to enhance their efficacy against intracellular pathogens. The microfluidics technology has been applied to fabrication of liposomes and the preliminary experiments have shown encouraging results, paving the way to the production of phage-loaded liposomes. We anticipate that the encapsulated phage cocktail will improve the efficacy of elimination of intracellular bacterial pathogens using relevant in vitro and in vivo infection models.
Phages as biocontrol of Pseudomonas syringae pathovars
Twitter hashtags: #PhgOx22, #JHudson
Jacob Hudson1,2, Cristian Aparicio-Maldonado1, Franklin L. Nobrega1, Mat Papp-Rupar2
1School of Biological Sciences, University of Southampton, Southampton
2Pest and Pathogen Ecology, NIAB, East Malling
Pseudomonas syringae is a highly diverse plant pathogen of over 60 pathovars (pv.), which together can infect almost all crop species. Some can infect multiple hosts, and hosts can be susceptible to multiple pathovars. In cherries, the P. syringae pv. syringae (PSS), morsprunorum race 1 (PSM1) and race 2 (PSM2) are the causative agents of bacterial canker, which kills up to 75% of young cherry trees in nurseries. Current therapeutics are ineffective. Copper compounds cause soil toxicity whilst selecting for copper and anti-microbial resistance, and antibiotics use faces similar problems. Alternatively, breeding resistant trees is a far too long process, due to the length of time to grow cherry trees and the wide range of pathovars to be tested. Here, we propose the use of bacteriophages (in short, phages) as a promising alternative treatment. We have sampled soils and roots from domesticated and wild cherry trees with and without canker symptoms, and performed isolation against PSS, PSM1 and PSM2 strains. Preliminary results show that we isolated 18 novel phages, and seven phages could lyse 88% (8/9) of P. syringae pathovars tested. Our goal is to contribute to the establishment of a well-characterised phage biobank against plant pathogens.
Diverse viral strategies of BREX defence inhibition
Twitter hashtags: #PhgOx22, #AIsaev
Alena Drobiazko1, Aleksandr Andriianov1, Mikhail Skutel1, Silvia Triguis2, Maria Selmer2, Konstantin Severinov3, Artem Isaev1
1Skolkovo Institute of Science and Technology, Moscow 143028, Russia
2Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, 751 24, Uppsala, Sweden
3Waksman Institute of Microbiology, Piscataway, NJ 08854, USA
Recent discoveries of the novel immunity systems significantly expanded the known arsenal of anti-viral defences encoded by prokaryotes. It became apparent that in natural environments phage infections often involve interaction with various immunity systems and thus phages developed different strategies to overcome or inhibit defensive barriers of the host. Here, we describe 3 unrelated mechanisms that allow phages T7, T3 and P1 to suppress BREX defence system from Escherichia coli HS. Phage T7 encodes DNA-mimic Ocr – a protein that inhibits modification and restriction complexes of the Type I Restriction-Modification systems and also suppressess BREX defence. We show that Ocr interacts with methyltransferase BrxX and efficiently competes with DNA for binding to the BrxX in vitro. The results also suggest that BrxX is responsible for the recognition of BREX sites and self non-self discrimination by the BREX system. Phage T3 carries SAM lyase – an enzyme that cleaves S-adenosyl methionine, an important co-factor of the restriction complexes in Type I R-M. Low-level expression of SAM lyase also inhibits BREX defence while supporting BREX methylation, which suggests that similar to Type I R-M complexes, BREX requires SAM as a co-factor at the defence stage. In addition, we show that SAMase has an alternative mechanism to deplete SAM pool – inhibition of SAM synthesis achieved through binding to the SAM synthase MetK. Phage P1 inhibits various Type I R-M systems in cis with the help of capsid-loaded Dar proteins. Here we show that in contrast to previous beliefs one of the Dar proteins – DarA is active in trans, and can inhibit BREX defence, once expressed from plasmid. The mechanism of DarA activity involves interaction with predicted ATPase BrxC. The study was supported by grants from RFBR (Ko_A_21-54-10001), RSF (22-14-00004 and 22-74-00126) and the Ministry of Science and Higher Education of the Russian Federation (075-10-2021-114).
Engineering Clostridioides difficile phages for therapeutic use – identifying the potential repressor gene
Twitter hashtags: #PhgOx22, #SKerr
Sarah Kerr, Terry Bilverstone and Nigel P Minton
BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UK
Clostridioides difficile infection is one of the leading causes of nosocomial diarrhoea in the developed world. The bacteria capitalise on gut dysbiosis from antibiotic use to colonise free niches in the colon, creating secondary infections in patients being treated with antibiotics for other reasons. Antibiotic use is therefore a pre-disposing factor to infection with C. difficile, and finding an alternative treatment is a necessity. Bacteriophage therapy is one alternative however this relies on obligatory lytic phages to be available. All published phages isolated for C. difficile so far have been temperate, implying that lytic C. difficile phages, if they exist, may be rare. We therefore attempted to engineer an existing temperate phage (φCD2301) to make it suitable for therapeutic use. RNA sequencing was used to identify the expression levels of phage genes while stably integrated into a bacterial host, in order to identify genes which may be involved in maintaining lysogeny. Four genes were identified as showing activity during integration, including a gene encoding for a hypothetical protein with a significant degree of expression and no previously identified function. Comparisons to C. difficile phage genomes (both in-house and published) identified several phages containing a homologous gene consistently located between the DNA polymerase III and parA genes, suggesting a degree of conservation. This gene has now been selected as a target to establish whether its removal through genetic engineering inhibits the ability of the phage to form a lysogen. If successful, it would mark a key milestone in the development of C. difficile phages that are suitable for therapeutic use.
Repurposing CRISPR against Salmonella spp.
Twitter hashtags: #PhgOx22, #SKKushwaha
Simran K Kushwaha¹,2, Sandhya A Marathe¹, Franklin L Nobrega²
¹Department of Biological Sciences, Birla Institute of Technology and Science, Pilani Campus, India
²School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Southampton, UK
Salmonellosis is a food-borne disease caused by Salmonella that annually affects 14.3 million people with 136 thousand deaths worldwide (PMID 30792131). The infections can lead to gastroenteritis and/or enteric fever with systemic disease, and with the increasing incidence of antibiotic-resistant Salmonella, most infections can be fatal with hardly any treatment options (PMID 30136920). CRISPR-Cas is a broadly distributed adaptive immune mechanism in bacteria and archaea (PMID 20556198). Our analysis on 16,349 Salmonella strains shows that 89% of the strains contain a type I-E system. Studies demonstrated that this system is generally silenced by the global regulator H-NS (PMID 21398529). However, Song et al. 2022 suggest that the system in E. coli is active against cryptic prophages. Here, we propose activating and exploiting the endogenous I-E system of Salmonella entericasubsp. enterica serovars Paratyphi A, Typhi, Typhimurium, and Welterveden for self-targeting. For this, we induced the expression of the cas operon by the expression of the positive transcriptional regulator, LeuO, in trans. Semiquantitative RT-PCR of eight cas genes, confirm the activation of the cas operon. To confirm the system’s functional activity, a plasmid loss assay (PMID 30107450, PMID 30691655), was performed by expressing a plasmid containing LeuO, and a synthetic CRISPR array, targeting a 3.1kb pTarget (p15A origin) having Kanamycin resistance. Within 24h, we observed 95% of CRISPR-induced loss of the target plasmid, and 100% after 48h. We speculate that this self-targeting strategy might be an effective alternative therapy to selectively eradicate bacterial pathogens.
Understanding phage resistance for efficient formulation of phage treatment in multidrug-resistance Klebsiella pneumoniae
Twitter hashtags: #PhgOx22, #JHLiew
Jun Hao Liew1,2, Ayuni Norman1, Pablo Bifani1,2
1A*STAR Infectious Diseases Lab (A*STAR ID Labs), Agency for Science, Technology and Research (A*STAR), Singapore
2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Klebsiella pneumoniae (Kp) has been gaining attention as a global health threat due to rising multidrug resistance (MDR) and virulence. Since limited treatments are available for MDR strains, phage therapy is being explored to treat severe Kp infections with reports of a few successful cases. To date, they are primarily limited to a compassionate basis in life-threatening infections with the administration of personalised therapeutic phage cocktail. As phage resistance is one of the challenges in phage therapy, it is crucial to design cocktails that can anticipate and target phage-resistant bacteria within a population. This could involve combining phages that target different receptors to reduce the probability of mutations in multiple genes. Through understanding phage resistance, strategies can be explored and used in formulating phage combinations and ascertaining the need for phage editing when lysogeny is of concern in phage resistance. Thus, this study aims to investigate the therapeutic application of the selected phage combination by evaluating the phage defence mechanisms of Kp and its phage-resistant mutants. Phage combinations of ΦBZ, ΦFAJ16 and ΦSP21 were selected against an ST23/KL1 carbapenem-resistant hvKp strain, and in vitro assays of the phage cocktail showed efficient phage killing. The isolated phage-resistant mutants showed various gene mutations that could potentially confer phage resistance and suggests that different defence mechanisms may be involved against the three phages. Verification of these mutations conferring phage resistance may lead to key insights about phage resistance mechanisms in Kp and lay down a more effective workflow in designing phage cocktails to combat MDR-hvKp. Future work will involve the validation of the effective phage combination in a mouse model and exploring the efficacy of the phage combination in other strains as a proof of concept that similar phage combinations can be used against similar Kp strains.
Search for therapeutic Mycobacterium abscessus mycobacteriophages – The Melbourne Experience
Twitter hashtags: #PhgOx22, #BBLin
Belinda B Lin1, Jessica L. Porter1, Dieter Bulach2, Ian R. Monk1, Sacha J. Pidot1, Justin Denholm3,5, Rebekah M. Dedrick4, Graham F. Hatfull4, Koen Vandelannoote5, Katherine Bond1,6,7,8, Maria Globan8, Deborah Williamson6,8,9, Timothy P Stinear1
1Department of Microbiology and Immunology, The University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Level 1, Melbourne, Victoria, Australia
2The Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, The University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Level 1, Melbourne, Victoria, Australia
3Victorian Tuberculosis Program, Melbourne Health, Melbourne, VIC, Australia; Department of Infectious Diseases, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
4Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, USA
5Medical Biology Laboratory, Institut Pasteur du Cambodge, Phnom Penh, Cambodia
6Department of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Australia
7Department of Microbiology, Royal Melbourne Hospital, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Australia
8Victorian Infectious Diseases Reference Laboratory at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Australia
9The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
Mycobacteriophages are bacteriophages that infect mycobacteria. Non-tuberculous mycobacterial infections are difficult to treat and the average rate of treatment success is estimated at 45.6%. Lytic mycobacteriophages were notably used in a 2019 report for treatment of a patient with disseminated drug-resistant Mycobacterium abscessus infection. They have since been safely utilised in a further 21 patients. However, relatively few active lytic phages are available and they are often strain-specific. We describe a mycobacteriophage discovery campaign using Mycobacterium abscessus TPS8830 cultured from a patient with a lung infection and other serious comorbidities. We also used M. smegmatis mc2155. We enriched 250 soil and 18 water samples from metropolitan, coastal and regional areas in south eastern Australia with M. smegmatis mc2155 and M. abscessus TPS8830. A total of 65 phages were isolated from 55 samples. Of these, 25 phages were genome sequenced. Analysis showed they belonged to genera Fromanvirus, Pipefishvirus, Bixzunavirus, Kostyavirus, Cheoctovirus, Timquatrovirus, Bongovirus and Fishburnevirus, from phylogenetic clusters A, B, C, E, F, K, M and P respectively. These are non-enveloped double-stranded DNA viruses, predominantly from family Siphoviridae, with exception of the Bixzunavirus which is from family Myoviridae. All phages plaqued on M. smegmatis mc2155. Two showed lysogenic plaque morphology on the clinical M. abscessus GD206 isolate. Genome sequencing showed that these phage were closely related Timquatrovirus, from cluster K, with efficiency of plaquing (EOP) of 10-5 compared to M. smegmatis mc2155. Bacteriophage recombineering (BRED) was used to delete the integrase repressor gene from one of these phages. This modification markedly increased the EOP to 1. Testing is in progress to further confirm the phenotypes of the engineered phage.
Gel preparations containing phages (S. aureus) for specific local use
Twitter hashtags: #PhgOx22, #LMateju
Ludmila Mateju, Sona Necadova, Marie Vavrova, Lenka Zakova, Marek Vronka, Lubos Jakub, and Juraj Vronka
AUMED a.s., Komoranska 326/63, 143 145 Praha 4 – Modrany, Czech Republic
AUMED deals with immunobiological preparations in general. The issue of the use of phages in human medicine is one of the main topics of the company. In this context, AUMED is contacted by three types of domestic specialized clinical workplaces: gynecological clinics, clinics that work with implants (breast, joint) and cardio surgical fields. The topic in surgical workplaces is the urgent need to eliminate primarily nosocomial bacterial infections, showing signs of resistance to antibiotics and possibly biofilm formation. Implant manufacturers have provided samples of materials for research on the suppression of biofilm formation using phages (on animal models). The gynecological topic is primarily focused on bacterial contamination, which in connection with childbirth poses a risk of infecting the newborn. The product also has potential use in preventive medicine, as an alternative to overuse of antibiotics. A hydrogel was developed for the mentioned three medical specialties, which, thanks to its balanced composition, preserves the viability of the contained phage particles and at the same time does not lose the physical properties of the gel. Bacteriophage subtype S 10/57 (host strain S. aureus 1137) specific for S. aureus was used as a model phage. A gel based on PEG 35,000 was developed as a carrier for this phage model. This system has been tested in animal models (pig) for safety and efficacy. Based on the results of the cumulative irritation index, the system was evaluated as a product with a negligible reaction (application period of 10 days). We have proven that the bacteriophage is long-term stable and effective in this gel base. In all three mentioned medical fields, we try to qualify the product for practical use as a medical device. Medicinal products containing phages are currently unregisterable in the Czech Republic due to the fact that the EMA has not yet published competent guidelines. AUMED is open for cooperation in mentioned activities.
Bioinformatic characterization of 7 members the Munster Technological University’s mycobacteriophage collection
Twitter hashtags: #PhgOx22, #LOConnell
Laura M O’Connell1, Colin Buttimer2, Francesca Bottacini1, Lorraine Endersen1, Aidan Coffey1 and Jim M O’Mahony1
1Department of Biological Sciences, Munster Technological University, Rossa Avenue, Bishopstown, Cork, Ireland, T12 P928
2APC Microbiome Ireland, University College Cork, Cork, Ireland, T12 YT20
Munster Technological University (MTU) has a long history of bacterio(phage) research. The adventure into mycobacteriophage (MP) research began over 10 years ago, with several PhDs, masters students and even undergraduates attempting to isolate novel viruses with potential diagnostic and therapeutic purposes. Such applications of phage have garnered renewed interest given the increasingly frequent incidences of antimicrobial resistance presenting in both community- and hospital-acquired infections. In the case of mycobacterial infections, there is the potential for MP to be used in therapies for multidrug-resistant tuberculosis infections, as well as chronic resistant infections caused by non-tuberculosis mycobacteria, such as Mycobacterium abscessus and Mycobacterium avium sbsp. paratubercuolosis. The advent of the genomics and proteomic era has made the detailed characterisation of phage possible at a nucleotide and protein level, which could improve the design of phage therapies, as carefully annotated phage may be selected (or disqualified) from consideration based on their gene content. Bioinformatic charactersation also paves the way for genetic engineering of phage to suit specific requirements and likewise adds to the existing knowledge of phage evolutionary dynamics. Regarding the MTU collection of MP, 7 MP (6 from archival stocks and one isolated from soil) from the were recently sequenced and characterised to aid in phage selection for ongoing studies within MTU, as well as contribute to the ever-increasing bank of genomic information regarding MP. It was determined that 6 of the phage are closely related and belong to the genus Fishburnevirus, and the seventh phage is a novel species of Cheoctovirus.
Staphylococcus aureus prophage-accessory gene encoding immunity to myovirus by abortive infection
Twitter hashtags: #PhgOx22, #RPantcek
Ivana Maslanova1, Lucie Kuntova1, Adela Indrakova1, Radka Oborilova2, Hana Simeckova1, Pavol Bardy1,3, Marta Siborova2, Tibor Botka1, Zdenek Farka2, Jiri Doskar1, Roman Pantcek1
1Department of Experimental Biology, Faculty of Science, Masaryk University, 611 37, Brno, Czech Republic
2Central European Institute of Technology, Masaryk University, 625 00, Brno, Czech Republic
3Department of Chemistry, York Structural Biology Laboratory, University of York, York, UK
Prophages play an important role in the virulence, pathogenesis or host preference, as well as in horizontal gene transfer in staphylococci. On the other hand, broad-host-range lytic staphylococcal kayviruses lyse most Staphylococcus aureus strains. Lysogenic S. aureus strains become immune to infection by closely related phages, but interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a novel resistance mechanism towards lytic phages of the genus Kayvirus, mediated by S. aureus prophage accessory gene. Based on the predicted structure of the prophage-encoded protein, we assume its transmembrane localization. We demonstrated that the mechanism of action does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed, which lead to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a spontaneous host-range Kayvirus mutant. We conclude that the defence mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behaviour of infected cells, thus ensuring the survival of the host population that is unable to produce phage progeny. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for development of phage therapy.This work was supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) funded by the European Union – Next Generation EU and grants (NU22-05-00042 and NU21J-05-00035) from the Ministry of Health of the Czech Republic.
Molecular characteristic of PhiKo lytic enzyme of bacteriophage origin sheds new light on the search for antimicrobial peptides hidden inside primary sequence of large proteins
Twitter hashtags: #PhgOx22, #MPlotka
Monika Szadkowska1, Aleksandra Kocot1, Dariusz Wyrzykowski2, Joanna Makowska2, Magdalena Plotka1
1Laboratory of Extremophiles Biology, Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
2Department of General and Inorganic Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland
The growing problem of antibiotic resistance forces societies to look for new antimicrobial agents. One of the current trend is the use of lytic enzymes from bacteriophage origin. Phage phiKo infects the extremophilic bacterium Thermus thermophilus HB27 and uses the PhiKo endolysin for the release of its progeny. The purified enzyme shows lytic activity toward thermophiles, i.e., T. thermophilus (100%), Thermus flavus (88%), Thermus parvatiensis (42%), and Thermus scotoductus (35%). PhiKo endolysin was found to be highly thermostable with melting temperature 91.89 ± 0.10°C. The optimum temperature for the enzyme lytic activity against T. thermophilus HB8 was 60°C, but at 37°C the relative activity was shown at the level above 82%. The enzyme exhibited lytic activity in the pH range of 5.5 to 8.8 (maximum at pH 8.0) and was active in the presence of up to 800 mM NaCl. The bactericidal activity of PhiKo endolysin against Gram-positive and Gram-negative mesophilic bacteria was low with CFU log reduction levels of 0.02-0.82 and 0.02-2.57, respectively. We have used the bioinformatics tool to predict the presence of regions with antibacterial properties hidden in the primary sequence of PhiKo endolysin. The 29 amino acid sequence (P29) with antimicrobial potential was localized within the 191 amino acid sequence of PhiKo protein using the AMPA web software. Based on the algorithms provided by the software, P29 was shown to be the antimicrobial peptide (AMP) with high probability. P29 peptide showed a strong antibacterial effect against panel of Gram-positive and Gram-negative mesophilic bacteria. The lowest effectiveness of the P29 was observed against P. aeruginosa PAO 1 (3.67 CFU log reduction), while the highest against multidrug-resistant A. baumannii KPD 581 (7.10 CFU log reduction). Moreover, experimental data showed that bactericidal activity of P29 was caused by bacterial membrane depolarization and permeabilization.
Phage tRNAs Subvert Host Resistance
Twitter hashtags: #PhgOx22, #RQuinn
Rebecca Quinn1, Richard J. Puxty1, Andrew Millard2, Dave J. Scanlan1
1School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK
2Department of Genetics and Genome Biology, University of Leicester, Leicester, LE1 7RH, UK
Bacteriophages are an integral component of the evolution of bacteria through population control, spreading of genetic material by horizontal gene transfer, and introducing genetic novelty through the selection for resistance. Bacteriophage-host interactions have been the basis on which many biotechnological tools were discovered, such as restriction enzymes and CRISPR-Cas9. It has been known for several decades that phage T4 encodes tRNAs, but more recently, we have found that about 20% of fully sequenced phage genomes also encode tRNAs. Yet why this is the case remains unknown. The main hypothesis, the Codon Usage Bias Hypothesis (CUBH), suggests that differences between bacteriophage and host in codon choice necessitates that bacteriophage encode their own tRNAs to complement host tRNAs. Here we show that codon usage bias does not explain the presence of tRNAs in phage genomes. Instead, through experiments in T4 and E. coli, we show preliminary evidence that tRNAs are involved in host-range by reducing the rate at which spontaneous phage resistance mutants arise. We are currently investigating the mechanistic basis for the control of host-range by tRNAs in this model system.
High-throughput screening for CRISPR immunity acquisition in Pseudomonas aeruginosa
Twitter hashtags: #PhgOx22, #ARichmond
Anna K Richmond¹, Rosanna C T Wright¹, Ville-Petri Friman², Joanne L Fothergill³, Aras Kadioglu³, Edze R Westra⁴ and Michael A Brockhurst¹
1Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester M13 9PL
2Department of Biology, University of York, Heslington, York, YO10 5DD
3Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, L69 3BX
4Environment and Sustainability Institute, University of Exeter, Penryn Campus, Penryn, Cornwall, TR10 9FE
The rising threat of multidrug resistant bacteria has led the WHO to classify Pseudomonas aeruginosa as a priority pathogen for investigation. An alternative to antibiotics is the use of phages to tackle bacterial infections. However, resistance to phages can evolve within bacterial populations. Understanding how and when this resistance evolves is crucial in prolonging the efficacy of phage therapies. For instance, the P. aeruginosa strain PA14 can evade phages using the defence mechanisms of surface modification or CRISPR immunity. To understand this, we evolved PA14 populations alongside selected phages and screened the subsequent phage resistant populations for the resistance mechanism. This high-throughput method of screening for CRISPR spacer acquisition at single colony and population level reveals how frequently CRISPR acquisition occurs. This data, alongside assays for surface modification, provides a window into how multiple different forms of resistance can occur within a single bacterial population. Focusing on the method of resistance leads the way for the rational design of phage combinations that reduce probability of host resistance evolving, thereby improving the longevity of effective phage therapies.
Selection of phages for GMP-compliant production in the context of individualized medicine
Twitter hashtags: #PhgOx22, #FRieper
Finja Rieper1,2, Imke H. E. Korf1, Sarah Wienecke1, Dieter Jahn2, Holger Ziehr1
1Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Inhoffenstraße 7, 38124 Braunschweig, Germany
2Technical University of Braunschweig, Institute of Microbiology, Systems Biology Centre BRICS, Rebenring 56, 38106 Braunschweig, Germany Multidrug-resistant bacteria are a serious problem for many patients worldwide. This can lead to long-term hospitalization, costly treatments, and increased healthcare expenditure. An alternative therapeutic approach for the treatment of these infections is the use of phages.The PhagoFlow project was launched in 2019 with the goal of establishing and operating an infrastructure for magistrale phage manufacturing in Germany. The major intention is to treat wound infections of the extremities. Phagoflow is funded by the Innovation Fund of the Federal Joint Committee (G-BA) and conducted in close alignment with the Federal Institute for Drugs and Medical Devices (BfArM). Beside ITEM the project partners are the Leibniz Institute DSMZ GmbH and the Bundeswehr Hospital Berlin. The aim is to isolate characterize, manufacture and administer phages against bacteria such as P. aeruginosa, S. aureus and E. coli, where antibiotic treatment is increasingly reaching its limit.Until now, out of almost 140 phages, 3 phages against P. aeruginosa have been selected that lyse 53 % of the P. aeruginosa isolates tested, as determined by efficiency of plating (EOP) tests. Especially for some highly antibiotic resistant P. aeruginosa isolates, it turned out to be difficult to find suitable phages. Therefore, further efforts were made to isolate phages for previously uncovered strains. Actually, 82% of the P. aeruginosa isolates can be covered by 6 phages. Using 14 phages provides the actually highest coverage of 91% which clearly shows the therapeutic potential for phage therapy. Before using those phages for GMP-compliant manufacturing, further phage-host interaction investigations are ongoing.The results so far indicate that at least for some P. aeruginosa the hypothesis that a suitable phage can simply be found for each isolate is not universally valid. In order to be able to treat patients with phages in the future, r&d on potentially therapeutic phages is seen as necessary.
A catalogue of phage-encoded carbohydrate-interactive proteins in the human gut
Twitter hashtags: #PhgOx22, #DRRodriguez
Daniela Rothschild-Rodriguez, Moi Taiga Nicholas, Morgen Hedges and Franklin L. Nobrega
School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Southampton, UK
The gastrointestinal tract is lined by a mucus layer that is structurally and functionally defined by large, glycosylated mucin proteins. In unison, different mucin proteins and glycosylation patterns form a heterogeneous glycan coat that extends outwards, which not only functions as a protective barrier for the epithelia, but also provides an environmental niche for mucus-associated microbiota. Mucus-residing bacteria encode a plethora of enzymes that can cleave different mucin glycan linkages, and therefore serve as an anchor or nutritional source. Likewise, gut phages are suggested to encode glycan-binding domains that allow them to anchor to mucus mucins, and thereby co-exist with their bacterial hosts. Such is the case for T4-like phages via the Hoc protein , ES17 phage via the tail fibre protein (TFP), and strong suggestions have been made for the BACON domain in crAss-like phages. Phage-mucus interactions in the human gut are largely understudied, and thus the underlying mechanisms remain unknown. Here, we demonstrate the result of literature and computational searches of phage carbohydrate-interactive proteins. Preliminary results identified 59,076 unique proteins from phages with 55% classified as glycan-degrading enzymes, E.C. 220.127.116.11. We aim to study these proteins to help elucidate the role of phage-mucus interactions in mucosal integrity.
Pseudolysogeny in Mycobacterium smegmatis: What genetic factors contribute to the change in host resistance?
Twitter hashtags: #PhgOx22, #MMRoyam
Madhav Madurantakam Royam1, Elizabeth M H Wellington1, Martha RJ Clokie2, Richard J Puxty1
1School of Life Sciences, University of Warwick, CV4 7AL, UK
2Department of Genetics, University of Leicester, Adrian Building, University Road, Leicester, LE1 7RH, UK
Many clinical isolates of the human pathogen Mycobacterium are now multi-drug resistant. Using viruses that kill Mycobacteria (phage therapy), could be an effective alternative treatment to antibiotics. Two major phage life-history strategies exist lytic, where phage kills their host, and lysogenic, where the phage integrates into the host’s genome and is spread throughout the population. Phages suitable for phage therapy should be lytic and should cause low rates of spontaneous resistance. During the investigation of four lytic phages of Mycobacterium smegmatis, we observed a high frequency of phage-resistant mutants (PRMs). We noticed that lawns of PRMs could produce spontaneous plaques (SPs) at different rates. SPs derive from an intracellular agent, given they cannot readily be washed off. SPs can infect isolates that were previously resistant to the ancestral phage and some SPs are positive for marker genes of the ancestral phage, whilst others remain negative. SPs are rapidly lost after several generations of growth in the absence of exogenous phage. We are currently investigating the nature of these ‘pseudolysogens’ using long-read sequencing and pulsed-field gel electrophoresis. Our data raise subtle exceptions to traditional boundaries of lytic and lysogenic modes that should be considered when selecting phage for phage therapy.
Isolation and characterisation of Proteus bacteriophages (vB_PmiS-ACS) for the development of therapeutic phage cocktails
Twitter hashtags: #PhgOx22, #AShambharkar
Akash Shambharkar, Brendan F Gilmore, Timofey Skvortsov
School of Pharmacy, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom
Proteus mirabilis is a common opportunistic human pathogen mainly responsible for urinary tract infections (UTIs), including catheter-associated infections. It is characterised by several unusual features, including its swarming activity and the ability to form crystalline biofilms which cause encrustation and eventual blockage of urinary catheters. This could lead to the ascension of bacteria up the urinary tract and result in chronic infections including cystitis, pyelonephritis, and kidney failure. The treatment of P. mirabilis infections is complicated and challenging due to the biofilm forming ability. Although normally the infection can be efficiently treated with antibiotics, resistance to various antibiotics such as polymyxins, nitrofurans, tigecycline and tetracycline has been reported, and the number of drug-resistant isolates being identified has been increasing over time. If this trend continues, P. mirabilis threatens to become a dangerous multi-drug resistant pathogen, causing significant treatment difficulties. Proteus phage vB_PmiS-ACS was isolated from composted cow dung collected from a farm 6 Brackenridge, Magheraline, Armagh, Northern Ireland. This is a novel phage with broad spectrum activity. Phage vB_PmiS-ACS belongs to the Siphovirodae family, with an icosahedral head and flexible tail. The genome is 58521 bp in length and has a 46.82% GC content. This phage is stable withing a temperature range of 40 to 90 °C and a pH range of 3 to 11. This phage has been extensively characterised and added to the phage collection being created to develop phage cocktails against a range of Proteus spp. The phage has been tested in combinations with different antibiotics normally used to treat Proteus infections; although most combinations showed additive/synergistic effect, the co-application of phage vB_PmiS-ACS and levofloxacin resulted in noticeable antagonism.
Functional analysis of host recognition of the four tail spike proteins of Agtrevirus AV101
Twitter hashtags: #PhgOx22, #ANSorensen
Anders Nørgaard Sørensen, Dorottya Kalmar, Veronika Theresa Lutz, Martine Camilla Holst Sørensen, Lone Brøndsted
Department of Veterinary and Animal Sciences, University of Copenhagen, Stigbøjlen 4, 1870 Frederiksberg C, Denmark
The host recognition of phages encoding multiple receptor binding proteins is poorly understood but is essential for understanding phage biology and evolution. Moreover, well-characterized receptor binding proteins may be used for engineering phages with altered host range for biocontrol and therapy agents, but it is then crucial to obtain knowledge of the host recognition. Phages in the Ackermannviridae family express up to four tail spike proteins (TSPs) that each recognize different receptors on the bacterial host. Phages in the Agtrevirus genus in this family identified so far infects Shigella and Salmonella, however, none of their TSPs have been studied in terms of host recognition. Here we characterize a new phage AV101 belonging to the Agtrevirus genus. Host range analysis showed that the phage infects multiple Extended Spectrum Beta-lactamase (ESBL) E. coli strains. In-silico analysis revealed that AV101 encodes four TSPs where TSP1, TSP2, and TSP3, all representing unique receptor binding domains not found in any other TSP in the Ackermannviridae family. TSP4, however, showed sequence similarity to receptor binding domain of TSP1 of kuttervirus LPST94’s, implying a recombination event between phage genera not observed before in the Ackermannviridae family. By purifying all four TSPs and spotting them on bacterial lawns, we could determine the hosts of each TSPs, which were corelated to specific serotypes. TSP-resistant hosts were isolated, and whole genome sequencing revealed that O-antigens of the respective hosts were indeed TSP receptors. Thus, exchanging receptor binding domains between TSPs could serve as a mechanism for adapting to diverse O-antigens of Enterobacteriaceae. Overall, AV101 is a novel phage in the Agtrevirus genus that infect multiple ESBL E. coli strains by recognizing the O-antigens and provide new insight into the host recognition and TSP evolution in phages encoding multiple receptor binding proteins.
Oral Prophylactic Administration of Phages Reduced Salmonella Proliferation and Dissemination in Mouse
Twitter hashtags: #PhgOx22, #CSukjoi
Chutikarn Sukjoi1, Songphon Buddhasiri2, Arishabhas Tantibhadrasapa1, Panupon Monkolkarvin1, Songbo Li1,3, Preeda Phothaworn4, Janet Y. Nale5, Angela V. Lopez-Garcia6, Manal AbuOun6, Muna F. Anjum6, Danish J. Malik7, Edouard E. Galyov8, Martha R. Clokie8, Sunee Korbsrisate*4, and Parameth Thiennimitr*1,9
1Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand
2Department of Veterinary Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand
3Youjiang Medical University for Nationalities, Cheng Xiang Road No. 98, Baise, Guangxi, 533000, P.R. China
4Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
5Department of Veterinary and Animal Science, Northern Faculty Scotland’s Rural College, United Kingdom
6Department of Bacteriology, Animal and Plant Health Agency, Weybridge, United Kingdom
7Department of Chemical Engineering, Loughborough University, Loughborough, United Kingdom
8Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom
9Research Center of Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, 50200, Thailand
Salmonella enterica serovar Typhimurium (STM) is the significant causative agent of foodborne acute non-typhoidal salmonellosis (NTS). The rising of multi-drug resistance (MDR) STM strains is alarming healthcare worldwide. The alternative method, including bacteriophage (phage), is one of the most promising ways against MDR STM. In our previous studies, we found that an oral administration of two Salmonella lytic phages (ST-W77 and SE-W109 are Myovirus and Siphovirus, respectively) significantly attenuated the severity of acute NTS in mice (the manuscript is in submission). This study demonstrated that phages ST-W77 and SE-W109 could be therapeutic agents for MDR STM. However, the prophylactic phage approach will offer the proper control for Salmonella spreading, especially in the food supply chain. Here, we aimed to investigate the preventive effect of ST-W77 and SE-W109 in the mouse. Our preliminary data showed that orally fed mice with 2 x 1011 PFU ST-W77 and SE-W109 daily for seven days significantly decreased Salmonella numbers in STM-infected mouse tissues compared to the untreated control. Phage-prophylactic mice conferred markedly lower Salmonella numbers in their gut (colon, cecal, colon, and cecum) and systemic tissue (spleen). These data supported that preventive giving of both phages reduced STM colonization in mouse gut and systemic dissemination. Moreover, we detected significant numbers of phages in fecal shedding during the oral prophylactic period, indicating phages’ availability in the mammalian gastrointestinal tract even without their bacterial host. In conclusion, phages ST-W77 and SE-W109 are effective anti-Salmonella agents orally given before the infection.
Deciphering the molecular mechanism of Kiwa
Twitter hashtags: #PhgOx22, #TCTodeschini
Thomas C Todeschini, Yi Wu, Ameena Naji, Rupavidhya Mondi, and Franklin L Nobrega
School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Southampton, UK
Bacteria encode diverse genetic systems that enable them to fight against phage predation. To date, the prokaryotic antiviral arsenal consists of 110 systems with 201 subtypes, acting through different mechanisms such as Abortive infection (e.g. Thoeris) and degradation of phage nucleic acids (CRISPR-Cas, Restriction-Modification). Several of these systems have been characterised, though the molecular mechanism of many remains unknown. Doron et al. in 2018 discovered Kiwa, a prokaryotic antiviral system that is present in 1.8% of analysed genomes which are predominantly proteobacteria (79%) and its mode of action remains unknown. The Kiwa operon encodes two genes, KwaA containing transmembrane regions (TM-regions) and KwaB encoding a DUF4868 domain. We studied Kiwa by collecting all Kiwa operons present in the NCBI database, for-which phylogenetic analysis resulted in 4 distinct clades for both KwaA and KwaB genes. Interestingly, KwaB homologs all contained a DUF4868 domain, whilst KwaA homologs showed diversity in size and encoded transmembrane regions (TM-region). Next, Kiwa operons present in different clades from E. coli ECOR8, ECOR12, ECOR49, D9 and R. mannitolilytica were picked to test anti-phage activity. We found that Kiwa systems can give up to 105-fold protection against specific phage families. With anti-phage activity being lost when either KwaA or KwaB were deleted. Interestingly, reconstitution of additional TM-regions in KwaA operons resulted in previously non-existing antiphage activity. Additionally, deletion of the conserved cytoplasmatic C-terminal of KwaA abolished protection. We constructed a Kiwa immunised strain to select for phage escape mutants, which revealed defence to be activated by phage-host hijacking proteins.
Investigating a novel phage targeting Serratia marcescens in the Aedes aegypti gut microbiome for mosquito control
Twitter hashtags: #PhgOx22, #SVenkatesan
Samiksha Venkatesan1, Toby Ross2, Shivanand Hegde1, Thomas Edwards2, Grant L. Hughes1
1Departments of Vector Biology and Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK L3 5QA
2Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK L3 5QA Strategies that manipulate the mosquito microbiome are becoming more important for the control of mosquito populations. Current methods employed to alter the microbiome in the lab, such as heat shock and antibiotic treatments, are incapable of targeting distinct bacterial species. However, the use of bacteriophages, with their narrow host specificity, can overcome this issue to deplete specific bacteria from the microbiome, allowing us to uncover how individual species influence various aspects of mosquito biology. This knowledge would be invaluable in developing new phage-based tools for mosquito control, a strategy that has yet to have been exploited for this global concern. We found a novel phage in human sewage with lytic activity against Serratia marcescens Alb1 (isolated from mosquitos) but not against eight other common members of the microbiome. Certain Serratia spp., including S. marcescens, have been shown to aid arboviral replication in the Aedes gut epithelia. Hence, it will be interesting to comprehend how its depletion will impact vector competence, as well as mosquito lifespan and fertility. Plaque assays revealed that this phage is much less efficient at lysing S. marcescens Alb1 at 27-30°C than at 37°C, with efforts underway to elucidate which stage(s) of the phage lifecycle are influenced by temperature. While adult mosquitoes can fly and seek cooler temperatures around 27°C, aquatic larvae cannot, implying that an Aedes mosquito can possibly experience this broad temperature range over its life. Thus, a future goal is to understand how this phage impacts mosquito biology at different life stages. We are also working on isolating new phage that can efficiently lyse Serratia regardless of temperature. Alternatively, there is potential to engineer this phage to eliminate its temperature-dependent lifecycle, or to exploit it as a gene delivery system to incorporate novel genes into the bacteria with numerous outcomes.
Characterization of Lactococcus laudensis prophages by whole genome sequencing
Twitter hashtags: #PhgOx22, #FKVogensen
Göksen Arik1, Dorentina Humolli1, Yan Hui1, Witold Kot3, Dennis S. Nielsen1, Charles M.A.P. Franz2, Natalia Biere2, Horst Neve2, Lukasz Krych1, Finn K. Vogensen1, Josue L. Castro-Mejia1
1Department of Food Science, University of Copenhagen, Denmark
2Department of Microbiology and Biotechnology, Max Rubner-Institut, Kiel, Germany
3Department of Plant and Environmental Science, University of Copenhagen, Denmark
Lactococcus laudensis is a newly described Lactococcus species, that has been associated with Italian raw cow’s milk (Meucci et al. 2015). Profiling of a traditional Danish dairy starter (DL-starter) allowed isolation of 61 isolates dominated by Lactococcus and Leuconostoc members, amongst which 9 isolates could be identified as Lactococcus laudensis (ANI = 0.97), as determined by whole genome sequencing based on Oxford Nanopore GridIon technology. Prediction of prophage regions (VIBRANT) on complete bacterial chromosomes, indicated the likely presence of prophage regions in 5 isolates. Clustering analysis of the viral-associated proteins (open reading frames – ORF) revealed low similarity (50% cut-off identity) to those found in the P335 quasi species. The genomes of the 5 putative prophages were approx. 35 kbp. Alignment of the 5 prophage genomes indicated that they belonged to the same phage species. The 5 isolates with likely prophage regions were then subjected to prophage induction with mitomycin C. Using epifluorescence microscopy, we quantified >108 viral-like particles per ml of induced lysate (VLP/ml) in isolates with positive induction. Electron microscopy images, sequencing data from induced prophages will be presented at PHG22.
Defence systems cooperate to increase anti-phage activity in E. coli
Twitter hashtags: #PhgOx22, #YiWu
Yi Wu, and Franklin L Nobrega
School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Southampton, UK
Anti-phage defence systems are abundant and diverse in bacterial genomes, but the interactions established between defences are mostly unknown. We investigated cooperation between defence systems present in genomes of Escherichia coli and found that certain combinations of defences provide synergistic protection from phages infection.