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Phages 2015 Bacteriophage in Medicine, Food and Biotechnology
01-02 September 2015 St Hilda’s College, Oxford, UK
Email: [email protected] Web: www.lpmhealthcare.com/phages-2015
KEYNOTE SPEAKERS
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WELCOME
A very warm welcome to the conference.
We hope very much that the conference will prove to be scientifically and socially rewarding and that the emphasis on applied science will provoke and stimulate new ideas on application of bacteriophage across the fields of medicine, food and biotechnology.
SCIENTIFIC ADVISORY BOARD
Professor Dennis Bamford, Finland Professor Douwe van Sinderen, Ireland Professor Grzegorz Wegrzyn, Poland Professor Martha Clokie, UK Professor Rob Levigne, Belgium Professor Alfonso Jaramillo, UK Dr Tatiana Lehnherr, Germany Dr Mark J van Raaij, Spain Dr Amin Hajitou, UK Dr Laurent Debarbieux, France Dr Toby Jenkins, UK Dr Brian Jones, UK Dr Brendan Gilmore, UK Dr Muhammad Sohail, UK
©2015 COPYRIGHT INFORMATION: This handbook is for use by the Phages 2015 Oxford (01-02 September 2015) participants only. Textual and graphical contents of this handbook are copyright of presenters, sponsors, instructors and/or LibPubMedia Ltd/Congress Oxford Ltd, unless explicitly stated otherwise. No part of this handbook may be reproduced, distributed or transmitted in any form or by any means, electronic or mechanical, including but not limited to, photocopy, recording, or any other information storage or retrieval system, without the prior written permission of the legal copyright owners.
TABLE OF CONTENTS
Delegate Information 003 Insurance and Liability 004 Disclaimer 004 Conference Agenda 005 Poster Listing 007 Oral Abstracts 008 Poster Abstracts 019
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DELEGATE INFORMATION
CONFERENCE DATES: 01-02 September 2015
WEBSITE: http://www.lpmhealthcare.com/phages-2015
VENUE: St Hilda’s College, Cowley Place, Oxford OX4 1DY, UK
PRESENTATIONS: All oral presentation will be held in the Edward Boyle Auditorium, Jacqueline Du Pré Music Building (JdP) and poster presentation in the JdP foyer.
We will not distribute speaker presentations . Therefore, if you interested in a particular presentation please get in touch with the speaker directly.
NAME BADGES: The College politely requests all delegates to wear name badges while on the premises.
REFRESHMENTS: Lunches and refreshments will be served in the JdP foyer or the College Dining Hall. Lunches and refreshments are open to all participants. Networking dinner is by prior booking/invitation only and entry will be via dinner tickets. Extra dinner tickets can be bought on site for £50 (cash only).
MOBILE PHONES: As a courtesy to speakers and participants, please switch off your mobile phone during oral presentations.
POSTER DISPLAY: Posters will be displayed in the JdP foyers for the full duration of the conference. Please leave your poster at the registration desk upon arrival. Presenters are encouraged to be present at their posters during the breaks to answer questions and to meet colleagues with similar research interests.
ORAL PRESENTATIONS: If you are presenting your abstract orally, please leave your presentation in a marked memory stick at the registration desk. Presentation standard will be data projection from a central PC. Macintosh will not be available , so you may bring your own, if you wish. Furthermore, as a courtesy to other presenters, please finish your talk absolutely within your allocated time slot. (Guide: For a 30 minute talk, prepare 18-20 slides maximum; for a 20 minute talk, prepare 12-14 slides maximum; allow 3-4 minutes for questions). You can find your presentation date and allocated time in the agenda below and also on the conference website.
HEALTH AND SAFETY: Please do not leave your belongings unattended and in passageway and also familiarise yourself with the emergency exits.
SMOKING: In addition to any local regulations UK no-smoking regulations apply, which prohibit smoking is in closed spaces.
INTERNET ACCESS: Please use edurom if you can. Otherwise, WiFi Code and instructions for internet access via your laptop/mobile device can be obtained at the time of registration .
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INSURANCE AND LIABILITY:
Participants are responsible for taking appropriate insurance cover (including health insurance) in connection with their attendance of this event. The event organisers and hosts are not responsible for personal accidents, any travel costs, or the loss of private property, and will not be liable for any claims. Event participants shall be responsible for compensating any loss, should they cause any damage to the host’s property or the venue.
DISCLAIMER:
The information specified in oral and poster presentations, written abstracts, biographies and exhibitions come from diverse sources and it is not in the capacity of event organisers to validate it, and is provided on an ‘as-is’ basis. Responsibility for the literary and scientific content of the abstracts and the presentations, both oral and poster, remains with the authors and the presenters. Therefore, the event organisers accept no responsibility for literary or scientific correctness of this information, and shall accept no liability of any kind, should any of the information be incorrect. The event organisers and hosts make no representation or warranty of gain of business or profits as a result of use of services or information provided in connection with the even and shall not be liable for any direct or indirect damages, loss of business, employment, profits or anticipated savings resulting from the use of the services or information provided in connection with the event, in any country or court of law. Furthermore, the materials contained in the event handbook are provided on the understanding that speakers or presenters have the right to their presentation in this manner. Therefore, event organisers and hosts shall not be liable for infringement of third party rights by an event presenter, participant, sponsor, supporter or exhibitor.
ABOUT LIBPUBMEDIA
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CONFERENCE AGENDA
(Subject to change)
Tuesday 1 st September, Jacqueline Du Pré Music Building (JdP) REGISTRATION AND WELCOME
08.30-9.30: Registration and welcome coffee 09.30: Welcome and housekeeping announcements
Tuesday 1 st September, The Edward Boyle Auditorium , Jacqueline Du Pré Music Building (JdP) PODIUM SESSION-I
SESSION 1: BACTERIOPHAGE MOLECULAR BIOLOGY, STRUCTURAL BIOLOGY AND BIOINFORMATICS
Chair: Dr Lone Brøndsted
09.35: Welcome by Professor Martha Clokie 09.40: Professor Dennis Bamford (Keynote), University of Helsinki, Finland The (prokaryotic) viral structural diversity is incredibly small 10.30: Professor Grzegorz Wegrzyn , University of Gdansk, Poland Newly discovered regulations of gene expression in lambdoid phages 11.00 Refreshments, networking and poster viewing 11.30: Professor Alfonso Jaramillo, University of Warwick, UK Engineering synthetic bacteriophages with targeted functions 12.00: Professor Gabriel Kaufmann, Tel-Aviv University, Israel The suicidal, phage excluding anticodon nucleases PrrC and RloC 12.30: Dr Paul CM Fogg, University of York, UK Serine Recombinases – Biology and Applications 12.50: Dr Daria Lavysh, Russian Academy of Sciences, Moscow, Russia Transcription strategy of a giant Bacillus phage AR9, encoding two multisubunit RNA polymerases 13.10: Group photo 13.15: Lunch and networking
Tuesday 1 st September, The Edward Boyle Auditorium , Jacqueline Du Pré Music Building (JdP) PODIUM SESSION-2
SESSION 2: BACTERIOPHAGE IN FOOD AND BIOTECHNOLOGY
Chair: Professor Martha Clokie
14.30: Dr Hansjörg Lehnherr, PTC Phage Technology Center GmbH, Germany Bacteriophages to fight Salmonella in poultry 15.00: Professor Ian Connerton, University of Nottingham, UK The bacteriophage carrier state of Campylobacter jejuni 15.30: Dr Lone Brøndsted, University of Copenhagen, Denmark Campylobacter phages: Biocontrol and resistance development 16.00: Refreshments, networking and poster viewing 16.30: Dr Martine C Holst Sørensen, University of Copenhagen, Denmark Genome sequences and comparative genomics of four bacteriophages infecting Campylobacter jejuni 16.50: Dr Tan Geok Hun, Universiti Putra Malaysia, Malaysia Phage therapy for diseases control in tomato and dragon fruit 17.10: Dr Rok Sekirnik, BiaSeparations, Slovenia Purification of Staphylococcus aureus bacteriophages VDX-10 using CIM® monolithic columns 17.30: Close of Day 1
19.00: NETWORKING DINNER (BY PRIOR BOOKING OF INVITATION)
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Tuesday 2 nd September, The Edward Boyle Auditorium , Jacqueline Du Pré Music Building (JdP) PODIUM SESSION-3
SESSION 3: BACTERIOPHAGE THERAPY AND THERAPEUTICS
Chair: Professor Dennis Bamford
9.00: Professor Mark Enright, Manchester Metropolitan University, UK S. aureus Population Structure and the Implications for Phage Therapy 9.30: Professor Martha Clokie (Keynote), University of Leicester, UK Using phage ecology, co-evolutionary dynamics, and model systems to inform phage therapy development 10.10: Dr Luisa De Sordi, INSTITUT PASTEUR, France Throwing the bad and keeping the good: a story of phage therapy in the gut 10.30 : Refreshments, networking and poster viewing 11.00: Dr Stefan Miller, Lisando GmbH, Germany Artilysins: Highly effective antimicrobial enzymes for targeted elimination of bacterial pathogens 11.30: Dr Aidan Coffey, Cork Institute of Technology, Ireland Bacteriophages and their peptidoglycan degrading enzymes for control of staphylococcus aureus 12.00: Dr Toby Jenkins, University of Bath, UK Polymer architectures for the triggered delivery of bacteriophage 12.30: Lunch and networking
Chair: Dr Toby Jenkins
13.10: Dr Ezra Yagil, Tel-Aviv University, Israel Site promiscuity of a phage integrase as a tool for human gene therapy 13.40: Dr Jinyu Shan, University of Leicester, UK Is phage therapy for Lyme disease possible? 14.00: Dr Andrey Aleshkin, Gabrichevsky Moscow Research Institute for Epidemiology & Microbiology, Russia Phage-based cocktail for control of hospital-acquired pathogens 14.20: Miss Hollie Hathaway, University of Bath, UK Poly(N-isopropylacrylamide-co-allylamine) (PNIPAM-co-AA) microspheres for thermally triggered release of bacteriophage k 14.40: General discussion and closing remarks by Professor Martha Clokie 15.00: Close of conference and departure of delegates
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POSTER LISTING
Presentation Dates: 01-02 September, The JdP Foyer
Please leave your poster at the registration desk when you register. Your poster will be displayed for the full duration of the conference.
Diana R Alves: A novel bacteriophage cocktail reduces Pseudomonas aeruginosa PAO1 biofilms Katja Becker: A T7 phage replication system for the directed evolution of tailor-made proteins Steven G Bell: Bacterial Pathogens Associated with Fresh Produce: An Innovative Solution
Sylwia Bloch: Role of the exo-xis region in development of lambdoid bacteriophages: λ and Ф24 B Prabhjyot K Dehal: Characterisation of a Bacteriophage mix against Blackleg-causing Bacteria in Potatoes Logan Donaldson: Structural investigations of proteins in the lambda exo-xis region Aleksandra Dydecka: Role of orf61 , orf73 , ea22 and ea8.5 genes from the exo-xis region in the development of recombinant lambdoid phages: λ and Ф24 B Michalina Filipiak: Influence of hydrogen peroxide on gene expression of strain MG1655(933WΔtox) Leonam Gonçalves: Mechanisms of virucidal action of alcohol and zinc ions combination against MS2 and F116 bacteriophages Hansjörg Lehnherr: In vitro assay to evaluate the efficacy of a bacteriophage in vivo Katarzyna Licznerska: The deletions in the exo-xis region affect the development of lambdoid bacteriophages with regard to λ and φ24B phages Lukas Lis: Investigation of structures influencing bacteriophage infection of Campylobacter jejuni Scarlet E Milo: Infection Responsive Surface Coatings for Urinary Catheters: Prevention of Encrustation and Blockage by Proteus mirabilis
Bożena Nejman-Faleńczyk: A microRNA-size small RNA encoded within the genome of the Φ24 B phage, one of the Shiga toxin-converting bacteriophages Sepo Nyambe: Survival of a temperate and lytic bacteriophage in soil and water Jonathan Nzakizwanayo: Use of bacteriophage to control catheter encrustation and blockage by Proteus mirabilis Eugenijus Simoliunas: Arthrobacter phage vB_ArtM-ArV1: a solitary myovirus among the phages from family Siphoviridae
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ORAL ABSTRACTS
SESSION 1: BACTERIOPHAGE MOLECULAR BIOLOGY, STRUCTURAL BIOLOGY AND BIOINFORMATICS 1st September (morning)
Chairs: Dr Lone Brøndsted
The (prokaryotic) viral structural diversity is incredibly small
Dennis H Bamford
University of Helsinki, Finland
Viruses are the most abundant living entities in the biosphere (10 31 ) outnumbering their host organisms by one to two orders of magnitude. It is conceivable that they cause the highest selective pressure their hosts encounter. What are the possible structural principles to build viruses is an open question. However, structural studies on virus capsids and coat protein folds propose that there are only a limited number of ways to construct a virion. This limitation may be based on the limited protein fold space. Consequently, relatedness of viruses is not connected to the type of cells they infect and the same architectural principle of the capsid has been observed in viruses infecting bacteria as well as humans. Using the viral capsid architecture it is possible to group viruses to several structure based lineages that may have existed before the three cellular domains of life (bacteria, archaea and eukarya) were separated. This would mean that viruses are ancient and that early cells were already infected with many different types of viruses proposing that the origin of viruses is polyphyletic opposing to the monophyletic origin of cellular life. To test the hypothesis of limited viral structure space we have collected information from globally collected environmental viruses infecting archaea and bacteria. A global search of cultivable prokaryotic viruses revealed only 27 different morphotypes distributed globally in the virosphere. This strongly disagrees about what we know about viral numbers and statistically we should never isolate very similar viruses.
Newly discovered regulations of gene expression in lambdoid phages
Grzegorz Wegrzyn 1, Sylwia Bloch 1, Aleksandra Dydecka 1, Agnieszka Felczykowska 1, Katarzyna Licznerska 1, Joanna Los 1, Bozena Nejman 1, Dariusz Nowicki 1, Agnieszka Szalewska-Palasz 1, Gracja Topka 1, Alicja Wegrzyn 2
1Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland 2Lab of Molecular Biology, Inst of Biochemistry and Biochysics, Polish Academy of Sciences, Gdansk, Poland
Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as they are carriers of genes coding for Shiga toxins. We found that in both lambda and Shiga toxin-converting (Stx) bacteriophages, patterns of phage genes’ expression were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents. Then, we indicated that RNA polyadenylation significantly modulates expression of phage genes. After prophage induction, a drastically decreased abundance of certain mRNAs was observed in the pcnB mutant cells, deficient in production of poly(A) polymerase I. Levels of mRNA derived from the pcnB gene were significantly increased shortly after prophage induction by mitomycin C, and decreased back after next 20 min, while no such changes were observed in non-lysogenic cells treated with this antibiotic. This prophage induction-dependent transient increase in pcnB transcript abundance may explain the polyadenylation-caused coordinated regulation of expression of phage genes. In the course of searching for potential regulatory RNAs modified by polyadenylation, we have discovered a microRNA-size ribonucleic acid (named 24B_1), encoded by PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 9 bacteriophage Phi24B. Expression of most of phage genes was drastically increased after infection of E. coli by the phage deficient in this small RNA. Since 24B_1 may impair expression of the d_ant gene, coding for an anti- repressor, these results may explain the mechanism of regulations of the physiological processes by this small RNA due to impaired activity of the cI repressor and changed expression of vast majority of phage genes. To our knowledge, this is the first example of functional microRNA-size molecule in bacterial cells.
Engineering synthetic bacteriophages with targeted functions
Alfonso Jaramillo
University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom
Application of synthetic biology methods is enhancing our ability to engineer phage genomes, reducing our dependence on environmental sources by allowing manipulation of phage properties to unprecedented detail. To expand their natural capabilities, we rationally engineered novel biological functions into the genomes of lytic phages by in vivo homologous recombination and CRISPR/Cas9-based selection. We have engineered the genome of phage T7 to encode E. coli genes essential for productive infection or to modify the capsid gene by displaying affinity tags for visualization and isolation. We have engineered phages with chimeric tail fibres containing heterologous receptor binding domains, which can later be evolved in continuous culture bioreactors using rationally-designed genetic circuitry to select for altered host specificity. Together, our work demonstrates how next-generation engineering and evolution of lytic phages can be used for designing novel functions.
The suicidal, phage excluding anticodon nucleases PrrC and RloC
Gabriel Kaumann
Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv 6997801, Israel
Bacteria and their viral parasites survive by constantly evolving defensive measures and countermeasures. This “Red Queen Effect” is exemplified by a survival cascade involving the conserved bacterial anticodon nuclease (ACNase) PrrC and its distant homologue RloC. PrrC is silenced in its uninfected host by a genetically- linked type I restriction-modification (RM) protein and turned on by a phage T4-encoded anti-DNA restriction factor. The resulting incision of a host tRNA species has the potential to block the synthesis of T4 late proteins. However, T4’s tRNA repair enzymes reverse the damage and rescue the infection. RloC overcomes this hurdle by damaging its tRNA substrates beyond such repair. Yet, there are reasons to assume that tRNA species encoded by the phage and targeted by RloC can counteract this tRNA repair defiant ACNase. RloC’s other salient feature is an internal, ACNase-regulating switch responsive to double stranded DNA breaks (DSB). This property could have freed RloC from reliance on an ACNase-silencing RM partner, to which RloC is rarely linked, and allowed RloC co-opt instead DSB generated by phage-encoded, host DNA-specific endonucleases.
Serine Recombinases – Biology and Applications
Paul CM Fogg and Maggie CM Smith
Biology Department, University of York, Wentworth Way, York YO10 3HE, UK
Over the past 25 years, serine integrases have rapidly become established as essential tools for synthetic biology. Serine integrases recognize two simple, compatible DNA attachment sites, attB and attP , and recombine them to produce two new sites, attL and attR . The new attachment sites are no longer a substrate for the integrase and, therefore, the reaction is strongly unidirectional. These simple requirements mean that serine integrases are amenable to a wide variety of applications, from in vitro assembly of multiple components to complex in vivo DNA rearrangements in diverse organisms. The irreversible nature of the reaction also means that recombined products or chromosomal insertions are extremely stable, even if the integrase is still present. Some applications, such as post factum modifications or biocomputing, need the
PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 10 recombination reaction to be reversed. In order for this to occur a second protein known as a recombination directionality factor (RDF) is required. The RDF binds directly to the C-terminal domain of the integrase protein and is thought to induce a conformational change that allows attL and attR to be used as recombination substrates and inhibits use of attB and attP . I will present our work on understanding the interactions between the archetypal ΦC31 serine integrase and its RDF and the importance of the 3D structure of the C-terminal domain. We are also working to discover and optimize new integrases and RDFs to allow advanced synthetic biology applications and creation of complex assemblies such as antibiotic biosynthesis pathways.
Transcription strategy of a giant Bacillus phage AR9, encoding two multisubunit RNA polymerases
Daria Lavysh 1,2 , Maria Sokolova 3,4 , and Konstantin Severinov 1,2,3,4,5
1Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia 2Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia 3Skolkovo Institute of Science and Technology, Skolkovo, Russia 4St. Petersburg State Polytechnical University, St. Petersburg, Russia 5Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ USA
AR9 (or PBS1) is a giant phage that infects Bacillus subtilis . Genome of AR9 is a linear, AU-rich double-stranded DNA with deoxyuridine instead of thymidine. AR9 complete 251,042 bp genome sequence was determined and 292 protein coding genes and one tRNA gene were predicted. 132 AR9 gene products show homology to proteins of other phages and bacteria. Phylogenetically AR9 is a member of the phiKZ-like phages. AR9 closest relative is Yersinia phage phiR1-37, with 56 homologous genes. Multiple AR9 genes are interrupted by short non-coding sequences or sequences encoding putative endonucleases. We experimentally confirmed splicing of these sequences. The AR9 genome encodes eight polypeptides that are distant homologs of fragments of bacterial β and β’ RNA polymerase subunits. Four of these polypeptides are present in AR9 virions, suggesting that they assemble as virion RNA polymerase that transcribes early mRNAs. The other set of polypeptides should form a non-virion RNA polymerase. The early promoter consensus was predicted bioinformatically; it is characterized by a highly conserved single consensus element AAATATATATTAT(6N)G, where G is a TSS and is located before 34 AR9 genes. A directional RNA-seq analysis was performed on RNA samples purified at 5, 20 and 40 minutes of infection. Analysis of all sequences 150 bp before 5’ TSS have confirmed all predicted early promoters and identified 87 late promoters, which are characterized by a AACA(6N)TA consensus, where the last A is a TSS. A non-virion multisubunit AR9 RNA polymerase was purified and was shown to be capable to synthesize RNA from late promoter containing DNA templates containing dU.
SESSION 2: BACTERIOPHAGE IN FOOD AND BIOTECHNOLOGY 1st September (afternoon)
Chairs: Professor Martha Clokie
Bacteriophages to fight Salmonella in poultry
Hansjörg Lehnherr and Tatiana Lehnherr
PTC Phage Technology Center GmbH, Siemensstrasse 42, D-59199 Bönen, Germany
Next to Campylobacter, Salmonella is the second most frequent cause of food poisoning in the European Union and the data reported by EFSA indicate that the number of unreported cases is high. Poultry, pork, eggs and fresh produce are the most frequent sources of Salmonella in our diet. In 2003 the European Parliament drafted a law in order to reduce the number of Salmonella infections. For fresh poultry meat: Salmonella, absence in 25 grams. Since the law took effect in 2010 the poultry industry is struggling to comply. That bacteriophages are suitable tools to control and reduce bacterial loads has been shown by academic and industrial laboratories countless times. Large scale applications, however, are still surprisingly rare. One important, but often ignored limitation is
PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 11 in fact scale. To run a single experiment in a standard poultry shed with 40.000 chickens, 24 litres of a highly concentrated, purified bacteriophage cocktail are required. A second major hurdle is the goal to achieve the absence of Salmonella from a poultry shed. In nature bacteria and bacteriophages are present in a stable equilibrium, neither eliminating the other. In theory it is possible to artificially shift this equilibrium in favour of the bacteriophages and thus eliminate the bacteria. However, is it possible to transfer this theory from the laboratory to the shed? In the past few years the scientists of PTC GmbH have been addressing these questions and results from both laboratory and field experiments will be presented.
The bacteriophage carrier state of Campylobacter jejuni
Ian Connerton
Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD UK
The bacterial pathogen Campylobacter jejuni is a common cause of human diarrhoeal disease worldwide. There are over 70,000 cases reported each year in the UK but due to underreporting this belies an estimated case load of ~450,000 and 9 million across the EU (European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011). Infection can arise from food and water-borne sources but is notably associated with the consumption of contaminated poultry products. Campylobacters and their bacteriophage are ubiquitous in the environment, often residing in watercourses and mixed biofilms between colonising the intestines of wild and farm animals. Amongst campylobacters recovered from biofilms after phage infection we identified isolates in which otherwise virulent phage had established a relationship with their hosts typical of a poorly understood phenomenon referred to as the carrier state life cycle (CSLC). In CSLC cultures bacteria and bacteriophages remain associated in equilibrium upon serial propagation (Siringan et al., 2014 Open Biology DOI: 10.1098/rsob.130200). The association does not prevent the release of free phage particles to explore the environment for new host bacteria. C. jejuni CSLC cultures do not efficiently colonise chickens but when administered to Campylobacter pre-colonised chickens the CSLC phage will replicate to bring about a reduction in the intestinal counts of the resident population. CSCL bacteria also have notable phenotypic changes compared to wild type bacteria that include improved aerotolerance under nutrient limited conditions, which would confer a survival advantage in extra-intestinal environments, and a lack of motility, which would account for their inability to colonise chickens. Changes in gene expression and regulation associated with C. jejuni CSLC host phenotypes will be discussed (Brathwaite et al., 2015 Res Microbiol doi.org/10.1016/j.resmic.2015.05.003).
Campylobacter phages: Biocontrol and resistance development
Athina Zampara 1, Martine C. Holst Sørensen 1, Yilmaz Emre Gencay 1, Anne Elsser-Gravesen 2 and Lone Brøndsted 1
1Dept of Veterinary Disease Biology, University of Copenhagen, Frederiksberg C, Denmark 2ISI Food Protection, Aarhus, Denmark
Campylobacter spp. are the most common cause of bacterial foodborne illness in the European Union and it has been estimated that 29% cases of campylobacteriosis are attributed to chicken meat. Campylobacter colonizes the intestinal tract of poultry and is readily transmitted from the avian cecum to poultry meat during slaughter. Currently, there are no effective control measures against Campylobacter in poultry meat production. A novel method such as the use of phages as biocontrol agents to control Campylobacter has been mainly investigated in chicken livestock. Although this approach seems appealing, phage resistance appears with relatively high frequencies and may represent a major limitation for such application. Our results suggest that resistant C. jejuni develops after phage exposure due to phase variable expression of phage receptors. In contrast, the use of phages to combat C. jejuni in food has only been reported in a limited number of studies, only including single phages. Using our previously gained knowledge about host range and phage receptors being either flagella or capsular polysaccharide (CPS), we aimed to identify phages capable of reducing C. jejuni in food. We tested the activity of single phages and phage cocktails against C. jejuni NCTC12662 in broth and
PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 12 on chicken skin at 5°C under anaerobic conditions for 24 hours . Interestingly, individual phages showed different ability to reduce C. jejuni . While phages dependent on motile flagella were inefficient, two CPS phages were able to reduce C. jejuni counts up to 1 log. The effectiveness of single phages and phage cocktails to reduce C. jejuni counts on chicken skin pieces at 5°C in modified atmosphere for 24 hours were similar our in vitro results. In conclusion, selecting phages for reduction of C. jejuni in food requires a thorough screening for the most effective phages as well as the knowledge of phage biology to further develop this approach.
Genome sequences and comparative genomics of four bacteriophages infecting Campylobacter jejuni
Martine C Holst Sørensen 1, Witold Kot 2, Amira Vitt 1, Lars H Hansen 2 and Lone Brøndsted 1
1Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg C, Denmark 2Department of Environmental Science, Aarhus University, Roskilde, Denmark
Campylobacter jejuni is a worldwide cause of human gastroenteritis with contaminated poultry meat being the main source of infection. As C. jejuni are part of the normal intestinal flora in birds without causing any apparent symptoms of disease, phage therapy of poultry has been investigated with promising results. However, to ensure that the phages applied as therapeutic agents do not carry unwanted genetic material such as virulence genes that may be transferred to surviving bacteria, genome sequencing of the phages is required. Here we present the genome sequences and the comparative sequence analyses of four phages infecting Campylobacter jejuni . The four phages belong to the Myoviridae family and were isolated throughout Denmark during the summer of 2011. Our recent analysis showed that their genomes are 180-190 kb and that the phages, according to Campylobacter phage grouping, belong to group II, CP220-type phages. The sequencing libraries were obtained from a single plaque from each of the four C. jejuni phages and sequenced using the Illumina MiSeq technology. The genomic analysis showed that all four phages had a large dsDNA genome with a size of about 180 kb coding for approximately 200 CDSs with genomic G+C content around 27%. Further analysis of the DNA sequences revealed that the overall similarities of the phages are high. Moreover, we analyzed the phage genomes with respect to homology to identify putative gene functions and unwanted genomic features as well as overall phylogeny and genome organization.
Phage therapy for diseases control in tomato and dragon fruit
G.H. Tan 1 and P.S.H. Tony 2
1Dept of Agriculture Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2All Cosmos Bio-tech Holding Corporation, Pasir Gudang Industrial Estate, Pasir Gudang, Johor, Malaysia
Bacteria plant pathogens are responsible for major economic losses in agriculture. Bacterial outbreaks are generally problematic to be controlled due to lack of effective bactericides and resistance development from the pathogen itself. These phages have recently been evaluated for controlling a number of phytobacteria. The efficacy test of phage incorporated with fertilizer for controlling bacterial wilt in tomato and soft rot in dragon fruit were determined. In tomato study, after two seasons of application, results indicated that 70-80 % of plants showed reducing in wilting symptom. The vegetative growth of plants also increased for the two seasons. The survival rate of phage-cocktail in fertilizer was determined and they can be detected after two months of incorporated process (7-Log PFU/ml). In dragon fruit, the application of phage managed to control disease spreading which caused the plants to be sustained and showed some positive impacts on vegetative growth as well.
Purification of Staphylococcus aureus bacteriophages VDX-10 using CIM® monolithic columns
Rok Sekirnik
BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia
In recent years, bacteriophages were identified as a useful tool for applications ranging from alternative antibiotics, detection of pathogenic bacteria, delivery vehicles for protein and DNA vaccines, to gene therapy delivery vehicles. PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 13
For all applications, it is important that phages are highly purified with preserved biological activity. Phage purification has traditionally been carried out using CsCl density gradient ultracentrifugation, which is time- consuming, requires tedious preparation of CsCl density gradients, and is challenging to scale-up. Chromatography has become an efficient alternative for separation and purification of many virus types, including phages. Methacrylate monoliths (CIM Convective Interaction Media® monolithic columns) were designed for fast and scalable purification of bionanoparticles with high efficiency for concentration and purification of several plant and human viruses (influenza A, influenza B, adenovirus type 5, hepatitis A and others). Due to convective mass transfer, monoliths facilitate flow-rate independent chromatographic purifications with high binding capacity for large biomolecules, such as phages. The talk will focus on our investigations into the use of CIM® methacrylate monolithic columns for purification of Staphylococcus aureus phage VDX-10. Screening of different chromatographic support chemistries and buffer/pH combinations led to the development of a purification method on CIM® QA strong anion exchange monolith. Optimised single-step purification method developed for S. aureus VDX-10 phage on 0.34 mL CIM® QA monolithic column was successfully scaled-up to 8 mL CIM® QA, demonstrating the scalability of the method. Efficient removal of host cell DNA and proteins with high recovery of viable phage was achieved, identifying this method as a faster and more efficient alternative to CsCl centrifugation, which is compatible with cGMP production of phages for therapeutic applications.
SESSION 3: Bacteriophage therapy and therapeutics 2nd September (morning)
Chairs: Professor Dennis Bamford
Staphylococcus aureus Population Structure and the Implications for Phage Therapy
Elliot Whittard 1, D R Alves 2, A T A Jenkins 2 and Mark C Enright 1
1School of Healthcare Science, Manchester Metropolitan University, Chester Street, Manchester, UK 2Department of Chemistry, University of Bath, Bath, UK
Staphylococcus aureus has a largely clonal population structure with variation in core genes occurring predominantly by point mutation. Horizontal gene transfer (HGT) via plasmids, transposons and bacteriophage has had a large impact on the accessory genome of the species with large variations, for example in toxin and antibiotic resistance gene repertoire between lineages. The major barriers to horizontal gene transfer between other species and S. aureus and between S. aureus lineages are the phylogenetically conserved restriction modification systems such as sau1 . Such systems are thought to be the major barrier to phage infection in the species. We have tested this hypothesis by examining the differences in sensitivity of 120 isolates of S. aureus including multiple members of the same MRSA lineages to a panel of 16 lytic podoviruses and Twort-Like myoviruses. Preliminary results show strong associations between host genotype and lytic phage susceptibility. Some phage from our collection can infect >70% of isolates and we have employed a series of methods to overcome barriers to recombination to increase phage host range against a collection of genetically characterized S. aureus . Further preclinical data and our work on engineering suitable host systems will be presented in an effort to support our view, that of the major bacterial targets available for phage therapy, Staphylococcus aureus appears the most attractive due to its population structure and the availability of malleable, extremely wide host range phage with excellent in vitro characteristics.
Using phage ecology, co-evolutionary dynamics, and model systems to inform phage therapy development
Martha RJ Clokie , Jinyu Shan, Ananthi Ramachandran, Tolis Panayi, Anisha Thanki, Janet Y Nale
Department of Infection, Immunity and Inflammation, Medical Sciences Building, University of Leicester, Leicester, UK
This year, we as phage biologists, celebrate 100 years since the first bacteriophages were isolated. It was clear to these early researchers that phages could be useful as therapeutic agents, and over the ensuing few PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 14 decades they were used widely to treat many topical and internal disorders in humans. Research, and the practice of using phages continued in some parts of the world, such as the former USSR, Poland and France but was largely neglected elsewhere until recently. The appreciation of lack of new antimicrobials, and the diversity and abundance of phages, has reinvigorated the research field of phage therapy, which is receiving renewed attention from academics and industry. In my laboratory we have worked with clinicians, chemists and engineers in order to gather experimental data to underpin the development of phages as therapeutic agents. Our research has focused on phages that target Clostridium difficile and Pseudomonas aeruginosa but we have also worked collaboratively to characterise phages that target Spirochetes and other respiratory and enteric pathogens. Our understanding of the roles that phages play in the environment and the way in which they influence the population dynamics of bacteria has significantly increased in the last century, and there is much data now to build into our studies of phage therapy development. For example, our understanding of phage ecology can inform our strategies to isolate sets of phages that target relevant organisms. I will show how an understanding of the ecology of C. difficile and other bacterial species has informed phage isolation practices in my laboratory. It is also appreciated that in order to use phages in humans their safety and efficacy must be tested in relevant models. We have studied the dynamic interactions of phages with bacteria in vitro, and developed ex-situ cell line models, and insect and animal models in which to study such interactions further. We have also been observing how phages coevolve with their hosts. We have had to establish, or adapt many of these models for phages. We have also studied the cost of phage resistance to bacteria by examining phage resistant mutants in some of these models. I will show highlights from these data sets, in order to illustrate how information from phage ecology and model systems can be useful to develop novel phage based therapeutics.
Throwing the bad and keeping the good: a story of phage therapy in the gut
Luisa De Sordi and Laurent Debarbieux
Department of Microbiology, Institut Pasteur, Molecular Biology of Gene in Extremophiles, Paris, France
Both commensal and pathogenic Escherichia coli strains reside in the human gut and years of antibiotics pressure led to selecting for drug-resistant strains. Using mouse models, our team both studies the therapeutic potential of bacteriophages and the consequences of their use on evolution. We recently focused our attention on Adherent-Invasive E. coli (AIEC) strains which are associated to Inflammatory Bowel Disease (IBD), being recovered from more than 30% of Crohn’s disease (CD) patients. Using a mouse model of IBD, we showed effectiveness of a cocktail of three selected bacteriophages in targeting the AIEC reference strain LF82. By significantly decreasing both intestinal colonisation and symptoms of disease, phage therapy showed promise as specific treatment for controlling AIEC proliferation in CD patients. A long-term gut co-colonisation model using both pathogenic LF82 and commensal MG1655 strains was setup to study bacteriophage adaptation in vivo. Bacteriophage LF110_P3, which infects LF82 but is inactive towards MG1655, was introduced in such model and its behaviour was recorded over several weeks. Remarkably, bacteriophages collected over time displayed the ability to infect the ancestral MG1655 strain while keeping its infectivity towards LF82. In addition, such bacteriophage variants presented differential infectivity towards bacterial clones isolated from the gut. In parallel, experiments performed in vitro never led to obtaining such variants, suggesting a role for the gut environment in the adaptation mechanism. While the potential of phage therapy to target E.coli – associated gut infections is undisputable, the research into understanding the impact of bacteriophage-bacteria interactions on the gut ecology is still in its infancy.
Artilysins: Highly effective antimicrobial enzymes for targeted elimination of bacterial pathogens
Stefan Miller 1, Yves Briers 2, Rob Lavigne 2
1Lisando GmbH, Josef-Engert-Straße 13, D-93053 Regensburg, Germany 2Katholieke Universiteit Leuven, Laboratory of Gene Technology, Kasteelpark Arenberg, Heverlee, Belgium
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Broad use, but especially misuse of antibiotics is resulting in increasing levels of antibiotics present in the environment, leading to a growing antibiotic resistance. Thus, there is a clear need for effective antimicrobial alternatives, which ideally show a targeted mode of action, - especially addressing Pseudomonas and other Gram-negative bacteria. Artilysins constitute a novel class of efficient enzyme-based antibacterials with a new mode of action. Artilysins are recombinant fusion proteins consisting of a bacteriophage-encoded endolysin, which degrades the peptidoglycan, combined with a targeting peptide that transfers the endolysin through the outer membrane of Gram-negative bacteria. Artilysin® Art-175 is highly effective against P. aeruginosa , a Gram-negative pathogen well known for being highly resistant to antibiotics and being responsible for re- occurring infections. Art-175 passes the outer membrane and kills P. aeruginosa, including multidrug-resistant strains, in a rapid manner. Art-175 punctures the peptidoglycan layer within a minute, inducing a bulging membrane and complete lysis. Minimal inhibitory concentration (MIC) experiments show Art-175 to be highly effective on P. aeruginosa , with a MIC90 of 10µg/ml independent of the strains being highly resistant to antibiotics. Resistance development against Art-175 was not observed within 20 experimental cycles on all strains investigated, whereas resistance development against a ciprofloxacin control occurred already within 7 cycles of the MIC experiments. As Artilysins do not require an active bacterial metabolism for its antibacterial activity, they show a superior bactericidal effect against persisters of P. aeruginosa and other bacterial species. Systemic infections by P. aeruginosa was successfully treated with Art-175 in a mouse model. Preclinical data underline the broad applicability of Artilysins to combat bacterial infections. In summary, Artilysins are proteins using a novel antibacterial mode of action for targeted elimination of infections caused by difficult to treat antibiotic resistant and/or persistent bacteria like P.aeruginosa that favour the microbiom.
Bacteriophages and their peptidoglycan degrading enzymes for control of Staphylococcus aureus
A. Coffey , R. Keary, M. Sanz Gaitero, M. van Raaij, R.P. Ross, C. Hill, J. O’Mahony, O. McAuliffe
Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland
Staphylococcus aureus is a major cause of infection in humans and animals causing a wide variety of conditions from local inflammations to fatal sepsis. The bacterium is commonly multi-drug resistant and thus many front- line antibiotics have been rendered practically useless for treating human infections, thus bacteriophage technology was explored as a potential for eliminating this bacterium. The genomes of three staphylococcal phages were sequenced and their peptidoglycan-degrading enzymes cloned in E.coli . Typical domains identified in these enzymes included cysteine/histidine-dependent amido hydrolase peptidases (CHAP), amidases, a lysozyme and endolysin associated cell-wall binding domains. The latter facilitates attachment of the enzyme to the bacterial cell wall, while the other domains catalyse the degradation of the peptidoglycan, mediating bacterial cell death. Deletion analysis of one of the 3-domain endolysins, LysK, showed that full lytic activity against live antibiotic-resistant staphylococci was retained when the endolysin was truncated to a single CHAP (peptidase) domain. The latter enzyme was purified by ion-exchange chromatography and characterized in detail including elucidation of its 3-D structure. Addition of the enzyme to a turbid bacterial MRSA culture resulted in elimination of turbidity. The peptidase was used in in-vivo studies in mouse models where it successfully eliminated MRSA colonization without adverse effects on the animals; and furthermore, ex-vivo studies confirmed a low immunogenicity. X-ray crystallography studies confirmed the 3-D structure of the enzyme and also indicated the presence of zinc and calcium co-ordination atoms facilitating enzymatic activity.
Polymer architectures for the triggered delivery of bacteriophage
A. Toby A. Jenkins 1; Hollie Hathaway 2; Jessica Bean 3; Scarlet Milo 4, Diana Alves 5; Patricia P. Esteban 6; Mark Enright 7; Amber Young 8
1-6Department of Chemistry, University of Bath, Bath, BA2 7AY 7School of Healthcare Science, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, UK 8The South West UK Children's Burn Centre, Royal Bristol Hospital for Children, Bristol, BS2 8BJ, UK
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The delivery of bacteriophage to an infection site is a sometimes overlooked aspect of phage therapy. Topical delivery of phage to an external infection / wound site is superficially straight-forward, however our recent studies have shown that the time (on the bacterial growth curve) when phage are inoculated can affect their bactericidal activity. In this talk, various strategies for creating composite films (which contain both a responsive component and a phage cargo) which only release phage following an external trigger will be presented. The trigger for phage release can either be secretion enzymes from bacteria themselves, including hyaluronidase, or secondary effects from local infection such as pH and temperature change. These composite films can be applied to wound dressings, catheters and other medical devices. The advantage of this approach is that phage-enabled devices could be applied to wounds or other infections sites which are at risk of infection, with no further intervention then required, as phage will only be released as a consequence of pathological changes in the tissue / wound environment.
SESSION 3 (contd): Bacteriophage therapy and therapeutics 2nd September (afternoon)
Chairs: Professor Toby Jenkins
Site promiscuity of a phage integrase as a tool for human gene therapy
Ezra Yagil , Mikhail Kolot, Natalia Malchin, Amer Elias and Natalia Gritsenko
Department of Biochemistry and Molecular Biology, Tel-Aviv University, Tel-Aviv 69978, Israel The integrase recombinase encoded by the lambdoid coliphage HK022 (Int-HK022) targets in the chromosome of its E. coli host a singly assigned 21bp recombination site ( attB ). attB comprises two partially-inverted 7bp Int-binding sites flanking a central 7 bp crossover site known as the “overlap” (O). Int-HK022 can function as a site-specific recombinase in human cells. Interestingly, Int-mediated site-specific recombination proceeds also when O is replaced by a random 7 bp sequence provided that the O sequence of the cognate and larger phage recombination site attP features an identical sequence. We detected native sequences matching such modified attB sites (“ attB s”) that flank deleterious human mutations. The existence of these “ attB ” sequences raises the prospect of curing the human mutations they flank by Int-HK022 catalyzed recombinase-mediated cassette exchange (RMCE) reactions. Our analyses of these sites suggest a minimal 14-15 bp consensus “ attB” (instead of the 21 bp) with a reduced 3 bp palindrome, a definition that likely applies also to the well- documented site-specific recombination system of coliphage λ.
Is phage therapy for Lyme disease possible?
Dr Jinyu Shan and Professor Martha Clokie
Department of Infection, Immunity, and Inflammation, University of Leicester, LE1 6RH, UK
Lyme disease is caused by a group of spirochaetes, collectively referred to as Borrelia burgdorferi sensu lato (s.l. ). Among the Lyme disease spirochaetes, three genospecies are predominating: B. burgdorferi sensu stricto (s.s .), B. garinii and B. afzelli . In order to exploit the therapeutic use of bacteriophages to combat Borrelia infection, the fundamental biology of Borrelia phages needs to be investigated. So far, no Borrelia lytic phages have been identified. Only one temperate phage induced from B. burgdorferi has been carefully studied, although whole genome bacterial sequencing revealed a number of free plasmids resemble ‘putative phage DNA’ . In this project, systematic effort will be made to study phages that infect the Lyme Borrelia species. We aim to identify/characterise subsets of bacteriophages that are suitable for future potential phage therapy of Lyme disease. Two strategies will be adopted in parallel. Tick, environmental, and clinical samples will be collected and screened for lytic phages. Meanwhile, temperate phages will be induced from Borrelia strains using low dose of antibiotics. Isolated bacteriphages will be subjected to a series of experiments to determine morphology, host range, and genomes. Those phages with broad host ranges, large burst sizes, and short latent period will be further analysed in terms of their effectiveness at clearing representative Lyme strains
PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 17 using in intro models. This will involve testing individual phage as well as a phage cocktail on single and multiple Borrelia strains.
Phage-based cocktail for control of hospital-acquired pathogens
Andrey Aleshkin 1,2 , Nikolai Volozhantsev 3, Anastasiya Popova 2,3 , Edward Svetoch 3, Evgenii Rubal’sky 1,2 , Irina Kiseleva 1,2 , Svetlana Bochkareva 2, Stanislav Afanas'ev 2
1Bphage LLC, Moscow, Russia 2Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, Russia 3State Research Center for Applied Microbiology and Biotechnology, Russia
Nosocomial infections caused by drug-resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus pose a serious medical problem today. We have developed a new phage composition using 8 virulent bacteriophages which are able to lyse these bacteria. The safety of the bacteriophage cocktail was confirmed by the results of the investigation of its toxicity performed on white outbred mice. Therapeutic and prophylactic efficiency of the bacteriophage composition was demonstrated in the prevention and treatment of the experimental acute K. pneumoniae infection in mice. The investigations showed that the preparation possesses a high therapeutic efficiency which is highly competitive with that of ciprofloxacin. As a result of the treatment with the bacteriophage cocktail the mice were cured completely of the highly virulent strain K. pneumoniae. The unique character of the developed preparation is insured by particular properties of each bacteriophage comprising the preparation, by the range of its lytic activity towards specific bacterial pathogens, morphology of its negative colonies, cycle of its development, restriction profile of its DNA, specificity of its genome and other properties. The phage genomes represented by double stranded DNA with the length from 18 kbp (staphylophages) to 167 kbp (Klebsiella pneumonia phage) have no identified genes that code peptides similar to toxins or any other virulent factors as well as genes that determine moderate development of phage. Taxonomic position of 8 phages obtained by electron microscopy were confirmed by bioinformatic analysis of their phage DNAs: staphylococcal phages and one strain of Klebsiella pneumonia phage belong to the Podoviridae family, and the rest of phages are the representatives of the Myoviridae family. We created a new phage composition that may be used as an alternative to antibiotics to control the drug-resistant bacterial pathogens of the following species: A.baumannii , K.pneumoniae , P.aeruginosa and S. aureus .
Poly(N-isopropylacrylamide-co-allylamine) (PNIPAM-co-AA) microspheres for thermally triggered release of bacteriophage k
Hollie Hathaway 1, Diana R Alves 1, Khajida Ouadi 2, Patricia Pérez Esteban 3 Jessica Bean 1, Mark Sutton 4, A Toby A Jenkins 1
1Department of Chemistry, University of Bath, Bath, United Kingdom, BA2 7AY 2Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom, BA2 7AY 3Department of Chemical Engineering, University of Bath, Bath, United Kingdom, BA2 7AY 4Public Health England, Porton Down, UK
Bacteriophage (phage) therapy for the treatment of bacterial wound infections provides a viable and potentially sustainable alternative treatment to antibiotics. Due to the increased prevalence of resistant bacterial isolates which are no longer susceptible to antibiotic treatment, recent emphasis has been placed on finding alternative modes of treatment for wound infections. Bacteriophage have long been investigated for their antimicrobial properties, yet the utilization of phage therapy for the treatment of wound infections relies on a suitable delivery system. Alongside providing the basic conditions for phage activity, the development of a triggered release delivery system will prevent the unnecessary administration of the antimicrobial into the clinical setting. This will decrease the likelihood of promoting the selection of resistant variants. Poly(N- isopropylacrylamide) (PNIPAM) is a thermally responsive polymer that undergoes a temperature dependent phase transition at a critical solution temperature which is associated with a change in volume of the polymer
PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 18 matrix. Bacteriophage K (phage K) has been successfully incorporated into PNIPAM nanogels copolymerised with allylamine. By utilising a temperature responsive polymer it has been possible to engineer the nanospheres to collapse at an elevated temperature associated with a bacterial skin infection. The nanogels were reacted with surface deposited maleic anhydride in order to anchor the nanogels to non-woven fabric. Phage incorporated PNIPAM-co-ALA nanogels demonstrated successful bacterial lysis of a clinically relevant bacterial isolate - Staphylococcus aureus ST228 at 37°C, whilst bacterial growth was unaffected at 25°C, thus providing a thermally triggered release of phage.
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POSTER ABSTRACTS
A novel bacteriophage cocktail reduces Pseudomonas aeruginosa PAO1 biofilms
Diana R Alves 1, Mark C Enright 2 and Toby Jenkins 3
1School of Pharmacy and Biomolecular Sciences, University of Brighton, Lewes Road, Brighton, BN2 4GJ, UK 2School of Healthcare Sciences, Manchester Metropolitan University, Manchester, M1 5GD, UK 3Department of Chemistry, University of Bath, Claverton Down, BA2 7AY, UK
Pseudomonas aeruginosa is a ubiquitous bacterium in the environment and a concerning opportunistic human pathogen that is able to form biofilm communities when establishing an infection. P. aeruginosa often is cause of fatal disease and this is not only enhanced by the biofilm mode of growth, but also due to the wide antibiotic resistance profile that most isolates show. In this work, we describe the isolation and characterization of six novel lytic bacteriophages – DL52, DL54, DL60, DL64 and DL68 – able to infect and lyse a range of P. aeruginosa clinical isolates. The six phages were used to formulate a cocktail and when this was added to of planktonic cultures of P. aeruginosa PAO1 growth was inhibited. Phage cocktail concentration (MOIs: 10, 1, 0.1 and 0.01) and time of their addition to the culture (0h, 2h and 4h) were studied and an improved bacterial inhibition was observed when the cocktail was added later in the bacterial growth for all the cocktail concentrations. The same phage cocktail was after used to treat bacterial biofilms of P. aeruginosa PAO1 established under flow conditions on stainless steel disks and after 48 hours of addition of the cocktail the biofilm was greatly reduced and dispersed compared to the biofilms not receiving the phage suspension. This cocktail can have the potential to be developed as a therapeutic to control P. aeruginosa infections, mainly the biofilms related.
A T7 phage replication system for the directed evolution of tailor-made proteins
Katja Becker 1, Sven Panke 1, Andreas Meyer 2
1Bioprocess Laboratory, ETH Zurich, Switzerland 2FGen GmbH, Basel, Switzerland
Directed evolution mimics Darwinian evolution and has become a powerful tool to identify biomolecules with any given property. A wide range of directed evolution protocols for iterative cycles of diversification, expression, screening and selection have been developed. Combining the advantages of several of these methods, in vivo continuous directed evolution is highly desirable. In my present work, I investigate the possibility of a T7 phage replication system in E. coli and its potential for directed evolution. Traditionally, directed evolution approaches rely on in vitro mutagenesis to diversify the genetic element of interest. Unfortunately, in vitro mutagenesis requires repetitive, labor-intensive cloning and transformation limiting the maximal possible library size. However, in vivo mutagenesis methods like UV radiation or mutator strains have an impact on the whole genome resulting in reduced strain fitness. To overcome these issues, an orthogonal in vivo mutagenesis method, targeting just the gene of interest and leaving the host genome intact, would be highly advantageous. The desired orthogonal in vivo mutagenesis could be achieved via independent replication of a genetic element by an external error-prone DNA polymerase. Genes of the replication machinery of the T7 bacteriophage were successfully used to replicate a plasmid with a T7 phage origin of replication and the gene of interest in Escherichia coli . Furthermore, the designed T7 plasmid is an interesting in vivo system for the study of the T7 replication mechanism and initiation thereof. Currently, I am working on the validation of the essential nature of various T7 phage genes and the quantification of the error rates on the chromosome and the plasmid. The presented method couples diversification and screening/selection might allow for the continuous creation of libraries within the living cell. This will facilitate the fast evolution to new biomolecules for industrial and medical biotechnology.
Bacterial Pathogens Associated with Fresh Produce: An Innovative Solution
Steven G Bell, Prabhjyot K Dehal, Louise M Disbury, Christopher W Gallagher, Kiri L Mack & Alison Blackwell
APS Biocontrol Ltd, Prospect Business Centre, Dundee Technology Park, Dundee DD2 1TY, UK
Bagged salads are a relatively new and rapidly growing (7% p.a.) category for the UK’s retailers, with product development and innovation central to success. The industry, however, is hampered by short shelf life and bacterial rots, causing losses throughout the supply chain; on farm, in the factory and the home. There is a need to develop a solution to this problem with an innovative technology to control bacterial pathogens. Bacteriophage are naturally-occurring, highly specific and sustainable biocides which represent environmentally-friendly and safe bacterial control agents. This work describes the PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 20 development and trial of bacteriophage active against bacterial species isolated from salads, aiming to reduce supply-chain waste by controlling food spoilage. Over 500 bacterial strains were isolated from rotting lettuce leaves obtained over two years, 70 of which caused substantial rot when re-applied to salad leaves. Approximately 90% of all isolates which caused rots were identified by PCR as Pseudomona s species, 55% as P. fluorescens and 25% identified as P. putida . Over 400 bacteriophage were isolated and screened for broad-range activity against P. fluorescens isolates. A cocktail of isolated bacteriophage had activity against 23-73% of all bacterial isolates. Laboratory and factory trials have been carried out to demonstrate efficacy of isolated bacteriophage against bacterial rot on salad leaves. This is the first bacteriophage processing aid treatment to be applied to washed and packed salads, and demonstrates the potential for this innovative technology to be utilised as a safe, specific and effective bacteriocide for the prevention of rots in processed lettuces.
Role of the exo-xis region in development of lambdoid bacteriophages: λ and Ф24 B
Sylwia Bloch 1, Bozena Nejman-Falenczyk 1, Aleksandra Dydecka 1, Katarzyna Licznerska 1, Gracja Topka 1, Alicja Wegrzyn 2, Grzegorz Wegrzyn 1
1Depatment of Molecular Biology, University of Gdansk, Wita Stwosza 59, Gdansk, Poland 2Lab of Molecular Biology, Inst of Biochemistry and Biophysics of Polish Academy of Sciences, Gdansk, Poland
The family of lambdoid bacteriophages plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as thay are carries of genes coding for Shiga toxins (Stx phages). The most important is fact, that the efficient expression of stx genes requires prophage induction and multiplication of the phage genome. The consequences of these processes are bloody diarrhea with severe complications. Moreover, treatment of EHEC infection is also difficult, because antibiotics are prophage inducers which increase toxins genes expression. Stx phages, like Ф24B, are lambdoid phages - a viral family with bacteriophage λ as the best-investigated member. The mechanism of λ prophage induction has been investigated in detail, however, functions of some of its genes are not yet clear. This concerns also the region located between exo and xis genes that may be involved in the control of lambdoid phages development. Previously, we showed that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24 B prophages. Now, we demonstrate that after prophage induction, an increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region. Importantly, by using quantitative real-time reverse transcription PCR, we have investigated expression patterns of genes from exo-xis regions of phages mentioned above after infection of host cells or induction of corresponding prophages. We observed that despite homologous regulatory sequences, identified and predicted in genomes of λ and Ф24 B, gene expression patterns were significantly different between these two tested phages. Moreover, even in the same phage, considerably different patterns of gene expression were detected, depending on the nature of agent used to induce the Ф24 B prophage. This may shed a new light on our understanding of regulation of lambdoid phage development.
Characterisation of a Bacteriophage mix against Blackleg-causing Bacteria in Potatoes
Prabhjyot K Dehal, Kiri L Mack, Louise M Disbury, Christopher W Gallagher and Alison Blackwell
APS Biocontrol Ltd., Prospect Business Centre, Gemini Crescent, Dundee, UK, DD2 1TY
Bacterial pathogens of potatoes are responsible for substantial losses through disease, damage and failure to meet market specifications. Control measures are limited and an effective, sustainable solution is a priority across the UK and wider European industry. Bacteriophage, as natural, specific and sustainable bacteriocides, represent environmentally friendly and safe alternatives and this study concentrates on their application to control blackleg development in potatoes; the commonest fault observed during the growing crop inspections and associated reason for crops being downgraded/failing. Bacterial-induced blackleg is a key diseases across all sectors of the UK potato industry, particularly the high-value seed sector, in which even minimal blackleg levels can result in significant financial losses through crop downgrade or even disqualification from the seed-classification system. The project will build on proof-of-concept data from an earlier project funded by Innovate UK in which proof-of-prinicpal data were produced to demonstrate the reduction of blackleg symptoms in bacteriophage-treated crops. The current study characterises the bacteriophage mix developed in terms of its efficacy under various environmental conditions and investigates the optimal point(s) of application within the growing cycle.
Structural investigations of proteins in the lambda exo-xis region
Logan Donaldson
Department of Biology, York University, Toronto, Canada PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 21
As many as seven open reading frames collectively known as bin (blocks of initiation of DNA replication), are situated between exo and xis of the pL operon. Here, we present a completed NMR structural study of the two lambda ea8.5 homologs and preliminary structural and expression data on two additional gene products from the exo-xis region. The ea8.5 homologs both demonstrate the same overall fold that can be described as a tight fusion of a three-helix homeodomain motif and a zinc binding motif. Titrations with nucleic acids were inconclusive suggesting that ea8.5 may mediate interactions with other host or viral proteins. Overall, we hope that structural investigations of this kind will reveal functional insights that help us understand the role of exo-xis proteins in the pathogenicity of shiga toxin producing strains of E. coli .
Role of orf61 , orf73 , ea22 and ea8.5 genes from the exo-xis region in the development of recombinant lambdoid phages: λ and Ф24 B
Aleksandra Dydecka 1, Sylwia Bloch 1, Agnieszka Necel 1, Gracja Topka 1, Katarzyna Licznerska 1, Bozena Nejman-Falenczyk 1, Grzegorz Wegrzyn 1, Alicja Wegrzyn 2
1Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, Gdansk, Poland 2Lab of Molecular Biology, Inst of Biochemistry and Biophysics of Polish Academy of Sciences, Gdansk, Poland
Shiga toxins are the main factors causing the pathogenicity of enterohemorrhagic Escherichia coli (EHEC). These toxins are encoded by stx genes located in genomes of Shiga toxin-converting bacteriophages (Stx phages) and their expression is stimulated upon prophage induction. Stx phages belong to the lambdoid family of phages, of which phage λ is the best investigated member. In this report, we would like to point out the region between exo and xis genes of phages λ and Stx phage - Ф24 B, which function is not yeat clear. Our previous results indicated, that the presence of the exo-xis region on a multicopy plasmid resulted in earlier induction and impaired lysogenization of E. coli . In the light of this observation, we decided to determine the role of the deletions of orf61 , orf73 and ea22 and ea8.5 genes from the exo-xis region in the development of recombinant lambdoid phages. We observed that deletion of orf61 or orf73 results in more efficient lysogenization of E. coli by phage Ф24 B. Interestingly, higher number of cells survived after infection of phage Ф24 B bearing the deletion of orf61 or ea22 gene. Moreover, the deletion of the ea8.5 gene resulted in earlier induction, while orf61 and orf73 delayed the induction of λ prophage after treatment of lysogenic cells with mitomycin C. Under the same conditions, the deletion of orf61 caused earlier induction of recombinant Ф24 B prophage. It is worth to mention, that the lack of the ea22 gene in the genome of λ prophage did not change the time of induction with mitomycin C. Surprisingly, different results were obtained after induction of analyzed recombinant prophages with hydrogen peroxide and UV- irradiation. We conclude that the deletions of orf61 , orf73, ea22 and ea8.5 genes from the exo-xis region of lambdoid phages may have specific effects on the regulation of development of phage λ and Stx phages, like Ф24 B, especially at the stage of the lysis-vs-lysogenization decision and prophage induction.
Influence of hydrogen peroxide on gene expression of strain MG1655 (933WΔtox)
Michalina Filipiak , Marcin Łoś and Joanna M Łoś
Department of Molecular Biology, Faculty of Biology, University of Gdańsk, Wita Stwosza, Gdańsk, Poland
Shiga toxin-producing Escherichia coli strains (STEC), are responsible for bloody diarrhea and hemorrhagic colitis. Pathogenicity of these bacteria is caused by Shiga toxins which genes are located on lambdoid prophages present in STEC strains. 933WΔtox phage gene expression occurs only after prophage induction and during further lytic development of the phage. Previous studies showed that hydrogen peroxide, an agent stimulating condition of oxidative stress, can induce prophages. This study focused on transcriptomics of strain MG1655(933WΔtox) after induction with H 2O2. Total RNA was isolated from the samples collected before induction and after 1 and 3 hours after induction and send for RNA sequencing. Gene Ontology (GO) analysis showed that 13930 enriched GO in differentially expressed genes in control sample were categorized into 83 functional groups. The most abundant Gene Ontology groups (GOgs) were: “primary metabolic process”, “biological process” and “cellular biosynthetic process” and belong to biological process cluster. 7 host GOgs belonging to cellular component cluster were expressed during time of the experiment. In samples induced with H 2O2 4376 out of 13129 host enriched GO categorized into 75 functional groups of the biological process cluster were differently expressed. Most represented GOgs were: “primary metabolic and biological process”, “cellular metabolic process” and “single-organism process”. In early stage of phage induction 5202 enriched GO out of 8453 were differently expressed belonging to cellular component cluster and 190 out of 220 to molecular function cluster. Downregulated phage 933WΔtox gene is responsible for maintenance of lysogeny whereas upregulated phage genes were: antitermination gene N and MokW responsible for modulation of host cell killing. The results of RNA-seq showed that
PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015 22 gene expression of host and phage genes in strain MG1655(933WΔtox) is variable during time both in control and after prophage induction with H2O2.
Mechanisms of virucidal action of alcohol and zinc ions combination against MS2 and F116 bacteriophages
Leonam Gonçalves 1, Jean-Yves Maillard 1, Ian Fallis 1, Joseph R. Rubino 2,3 and M Khalid Ijaz 2,3
1Cardiff School of Pharmacy and Pharmaceutical Sciences, King Edward VII Avenue, Cardiff, Wales, UK 2Reckitt Benckiser R&D, Montvale, New Jersey, United States 3 The City University of New York (CUNY), Brooklyn, New York, United States
Studying mechanisms of action of biocides active against viruses is essential for the development of more efficient formulations and for gathering deeper insights about the reasons underlying virucidal activity. Alcohol based formulations in combination with other excipients are being studied to improve virucidal activity and product stability while maintaining product usage characteristics. The main goal of the present study was to understand the mechanism by which the combination of alcohol at low concentration (25-40% v/v) and zinc ions (Zn 2+ ) exhibits virucidal effects against bacteriophages as surrogates of mammalian viruses. The virucidal efficacy of formulations based on different alcohol/zinc ratios was measured against two bacteriophages, MS2 and F116, using standardised suspension (EN13610) and hard- surface carrier (ASTM E1053) tests. Transmission electron microscopy (TEM) was performed to identify specific structural damages caused by formulation against F116. Virucidal activities associated to formulations with ethanol content 25-40% v/v were less than 1 log 10 reduction (LR) and 1 LR after 5-min exposure for MS2 and F116, respectively. Activity against MS2 increased significantly at 30-min (2.5-LR and 1.5-LR without Zn 2+ ) and 1-hour (>4-LR and 2-LR without Zn 2+ ). Differences between the virucidal activities of the formulations against MS2 and F116 were significant (p<0.05). TEM studies on bacteriophage F116 showed alcoholic formulations containing zinc led to specific changes in capsid permeability presumably affecting viral nucleic acid, and capsid damage. In conclusion, the addition of zinc to formulation leads to increased virucidal activities. TEM results showed there was a damage pattern associated with ethanol/zinc- containing formulations targeting the capsid but probably the nucleic acid within the capsid, implying virucidal activity of the alcoholic formulation may present a distinct mechanism of action when zinc is present.
In vitro assay to evaluate the efficacy of a bacteriophage in vivo
Hansjörg Lehnherr, Dinah Mennigmann, Anika Faros, Jennifer Hoffman, Tatiana Lehnherr
PTC Phage Technology Center GmbH, Siemensstrasse 42, D-59199 Bönen, Germany
There are three commonly used assays to evaluate the properties of a bacteriophage. The classical titration method determines the concentration of a bacteriophage stock. A spot test analysis is used to determine the host range and a liquid titration method (Appelman) is used to check if a bacteriophage is able to outgrow its host. All three methods have in common, that the host bacteria are present in high concentrations (106-109 bacteria per ml), thus there is no diffusion- limited step in which the bacteriophages have to “search” for a host to adsorb to. In the spot test also the phage concentration is high, while in the two titration methods a single bacteriophage is able to produce the observed effect. When considering practical applications the data reported in the literature are not consistent. While some authors claim that an efficient spot test analysis is highly indicative of the efficacy in vivo, other authors find no correlation between in vitro activity and in vivo efficacy. These differences might of course result from different model systems, but they could also be linked to the fact that the in vitro assays do not reflect the conditions the bacteriophages encounter in vivo, where the density of the pathogenic bacteria might be low and the concentration of the bacteriophages artificially high. To address this problem we designed an in vitro assay called “inhibition test” that mimics these conditions. The assay shows whether a single bacteriophage or a bacteriophage cocktail is able to reduce or eradicate a specific number of bacterial host cells. Especially the latter trait is important for the potential of a bacteriophage to be effective in vivo.
The deletions in the exo-xis region affect the development of lambdoid bacteriophages with regard to λ and φ24B phages
Katarzyna Licznerska 1, Sylwia Bloch 1, Aleksandra Dydecka 1, Gracja Topka 1, Agnieszka Necel 1, Bożena Nejman-Faleńczyk 1, Grzegorz Węgrzyn 1, Alicja Węgrzyn 2
1Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
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The family of lambdoid bacteriophages, in which bacteriophage λ is the best investigated member, include Shiga toxin- converting bacteriophages (Stx phages) like φ24B (vb_EcoP_24B). These group of viruses in prophage stage can be found in the genomes of enterohaemorrhagic Escherichia coli -EHEC and after prophage induction produce toxin which is the major virulence factor of EHEC. In the central part of lambdoid bacteriophages’ genomes is located the exo-xis region which contains highly conserved genes of largely unknown functions. For bacteriophage λ identified two genes ( ea8.5 and ea22 ) and five open reading frames (ORFs) which four ORFs: orf60a , orf63 , orf 61 are highly conserved sequence in lambdoid bacteriophages’ genomes (λ and Stx phages exhibit >80% sequence similarity). This region participates in modulating host genome functions but physiological significance of the regulation in phage development has remained unknown. We present the results of the influence of deletion mutations in the exo -xis region on the development bacteriophage λ and Shiga toxin-converting bacteriophage Φ24B. We constructed recombinant phages devoid of either all genes between exo and xis (called ∆exo-xis ) or four ORFs ( orf60a , orf63 , orf 61 and orf73 ; called ∆orfs ).The absence of genes from the exo-xis region caused deleyed induction of both prophages following stimulation by various agents (mitomicyn C, hydrogen peroxide and UV irradiation). Both types of deletions in this region resulted in changes in efficienty of lysogenization by both Φ24B mutants. Moreover, survival of cells after phage infection differed between bacteriophages with ∆exo-xis and ∆orfs mutation. Our results demonstrated that the exo-xis region played an important role in the regulation of development of lambdoid bacteriophages, including Stx phages, especially at induction of prophages and the stage of the lysis-vs-lisogenization decision.
Investigation of structures influencing bacteriophage infection of Campylobacter jejuni
Lukas Lis 1, and Ian F Connerton 2
1PTC Phage Technology Center GmbH, Im Kompetenzzentrum BioSecurity, Bönen, Germany 2Division of Food Sciences, School of Biosciences, University of Nottingham, Loughborough, Leicestershire, UK
Campylobacter jejuni is a major food borne pathogen and has been target of bacteriophage based biocontrol studies. We have focused on the identification of factors that influence bacteriophage infection, and could impact biocontrol. Through generation of a monogenetic mutant library, based on universal phage propagation strain C. jejuni NCTC12662 PT14, we analysed the effects of Campylobacter surface structures and virulence related factors on bacteriophage infection by screening on a set of phages. In agreement with previous reports, this screening revealed phages with dependence on capsular polysaccharide (CPS) and flagellotropic phages. Further, we found phages independent of both structures. Notably, we have observed an increase in susceptibility to infection to bacteriophage F1 in two mutant strains: flagellin ( flaB ) and a heptosyltransferase ( waaf ), involved in lipooligosaccharide (LOS) synthesis. These mutations give rise to clear lysis and transient changes in growth kinetics of infected Campylobacter cultures relative to the wild type. Furthermore, a 10-fold increase in phage propagation was noted in both cases. Disruption of waaf results in undetectable LOS and reduced amounts of capsular polysaccharide, suggesting an involvement in synthesis of both surface polysaccharide structures. Inactivation of FlaB did not impair swarming motility in PT14, indicating no major function in this process. In silico analysis of the FlaB protein sequence in relation to major flagellin (FlaA) revealed 95 % identity, with substitutions existing predominantly in core associated terminal regions. In the surface exposed area two successive substitutions at residue 371 were found, which may have an influence on interaction with bacteriophages. Furthermore, a substitution introducing a threonine residue at position 493 was observed, which could have an influence on FlaB glycosylation pattern.
Infection Responsive Surface Coatings for Urinary Catheters: Prevention of Encrustation and Blockage by Proteus mirabilis
Scarlet E Milo and Toby A Jenkins
Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY
Abstract excluded on request from the authors.
A microRNA-size small RNA encoded within the genome of the Φ24 B phage, one of the Shiga toxin-converting bacteriophages
Bożena Nejman-Faleńczyk 1, Sylwia Bloch 1, Katarzyna Licznerska 1, Aleksandra Dydecka 1, Agnieszka Felczykowska 1, Gracja Topka 1, Alicja Węgrzyn 2, Grzegorz Węgrzyn 1
1Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland 2Lab of Molecular Biology, Inst of Biochemistry and Biophysics of Polish Academy of Sciences, Gdansk, Poland
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Genome-wide searches allowed to identify a large group of small bacterial RNAs (sRNAs). These molecules have been well studied in case of model organisms such as Escherichia coli bacteria. The range of 80-100 sRNAs have been reported for E. coli bacteria including pathogenic Shiga toxin-producing E. coli strains (STEC), an important class of diarrheagenic bacteria. These E. coli strains are highly harmful to humans as they contain lambdoid prophages bearing stx genes coding for Shiga toxins, the major agents responsible for development of severe diseases. Research on these pathogenic E. coli bacteria allowed to identify sRNAs within bacteriophage-derived regions such genomes of non-cryptic bacteriophages carrying Shiga toxin genes. Interestingly, microRNA-size small RNA fragments (15-28 nt) were also reported in recent studies on these microorganisms, however RNAs of comparable size to eukaryotic microRNAs have received little attention up to now. In this work we would like to present a new microRNA-size molecule, named by us 24B_1, which has been identified in E. coli after induction of Shiga toxin-converting bacteriophage Φ24 B.The sequence of 20-nt long 24B_1 is located in the lom -vb_24B_43 region of the phage genome, and supposedly it is produced by cleavage of a larger transcript. A phage lacking the sequence of 24B_1, revealed decreased efficiency of lysogenization, faster switch from lysogeny to lytic phage development after treatment with mitomycin C, higher efficiency of progeny phage production during the lytic cycle and less efficient adsorption on the host cells. Beside, expression of most of phage genes was increased after infection of E. coli by the Φ24 BΔ24B_1 phage. Since 24B_1 may impair expression of the d_ant gene, coding for an anti-repressor, these results may explain the mechanism of regulations of the physiological processes by this small RNA due to impaired activity of the cI repressor and changed expression of vast majority of phage genes.
Survival of a temperate and lytic bacteriophage in soil and water
Sepo Nyambe 1,2 , Catherine Burgess 1, Paul Whyte 2 and Declan Bolton 1
1Food Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland 2School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
Verocytotoxigenic E. coli (VTEC) are a group of zoonotic food-borne pathogens in humans mainly associated with food products contaminated with bovine faeces such as raw meat and milk. Symptoms range from mild to bloody diarrhoea and can lead to severe complications such as haemolytic uraemia syndrome (HUS). The main virulence factors of VTEC are the verocytotoxin ( vtx ) genes encoded on the vtx bacteriophage genome; these genes may be acquired and transferred to other E. coli strains by transduction. Cattle are the main reservoir of many human pathogenic VTEC strains which are shed in their faeces, and can survive in many environments encountered on beef farms for up to three months or more. The objective of this study was to investigate the survival of a vtx2 encoding bacteriophage labelled 24 B::kanamycin, and an anti E. coli O157:H7 lytic bacteriophage proposed as a bio-control of foodborne pathogens labelled e11/2, in four different water types and two soil types. The samples were incubated at 4ºC and 14ºC, and analysed every two days for cell free infectious bacteriophage using a direct plaque assay technique and screened for 24 B::kanamycin lysogens. The results show that both 24 B::kanamycin and e11/2 are capable of surviving in the water and soil types studied for an extended period of time. Furthermore, 24 B::kanamycin was able to form lysogens. It was concluded that ecological environments found on beef farms, such as soil and water, can act as a reservoir for cell free vtx2 bacteriophage, which will facilitate transduction and the emergence of new pathogenic VTEC strains.
Use of bacteriophage to control catheter encrustation and blockage by Proteus mirabilis
Jonathan Nzakizwanayo 1, Aurelie Hanin 2, Cormac Gahan 2, Cinzia Dedi 1, Jason Clark 3, Brendan Gilmore 4, Toby Jenkins 5, Brian Jones 1,6
1School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK 2Alimentary Pharmabiotic Centre and Dept of Microbiology, University College Cork, Ireland 3Novolytics Ltd, Daresbury Science and Innovation Campus, Warrington, UK 4School of Pharmacy, Queens University Belfast, Belfast, UK 5Dept of Chemistry, University of Bath, Bath, UK 6Queen Victoria Hospital NHS Foundation Trust, Holtye Road, East Grinstead, UK
Abstract excluded on request from the authors.
Arthrobacter phage vB_ArtM-ArV1: a solitary myovirus among the phages from family Siphoviridae
Eugenijus Simoliunas, Laura Kaliniene, Miroslav Stasilo, Lidija Truncaite, Aurelija Zajanckauskaite, Rolandas Meskys
Dept of Molecular Microbiology and Biotechnology, Inst of Biochemistry, Vilnius University, Vilnius, Lithuania
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This is the first report on a complete genome sequence, biological characterization and phylogenetic analysis of the myovirus that infects Arthrobacter . A novel virus vB_ArtM-ArV1 (ArV1) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. In total, 51 bacterial strains, including 32 Arthrobacter sp., were used to explore the host range of ArV1. It was determined that phage ArV1 infects 6 Arthrobacter sp. strains, which were shown to be phylogenetically related. The efficiency of plating (e.o.p.) test revealed that ArV1 has an optimum temperature for plating around 28ºC. Transmission electron microscopy showed its resemblance to members of the family Myoviridae : ArV1 has an isometric head (~74 nm in diameter) and an anusually contractible nonflexible tail (~192 nm). Whole-genome sequencing of ArV1 revealed a linear circularly permutated double-stranded DNA (71,200 bp) with a G+C content of 61.6 %. The genome of ArV1 contains 100 ORFs yet encodes no tRNA genes. With the amino acid identity ranging from 25 to 40%, only 41 out of 100 ArV1 ORFs have homologues in other sequenced viral genomes, while 47 % of ArV1 ORFs are unique to this phage. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 34 of ArV1 ORFs were given a functional annotation. Interestingly, while ArV1 clearly has myovirus morphology, the majority of the annotated ArV1 proteins, especially structural virion proteins, were most similar to those found in siphoviruses. Moreover, the comparative phylogenetic analysis based on the amino acid sequence alignment of conserved structural proteins placed ArV1 in a clade formed by phages from the family Siphoviridae exclusively. Thus, the data presented here advance our understanding of the genetic diversity and evolution of phages from the order Caudovirales.
PHAGES 2015 | 01-02 SEPTEMBER 2015 | OXFORD, UK | w ww.LPMHealthcare.com/phages-2015