Phages 2015 - Lpmhealthcarelpmhealthcare.com/wp-content/uploads/2015/08/PHG15-Book.pdf · 10.30: Refreshments, networking and poster viewing 11.00: Dr Stefan Miller, Lisando GmbH,

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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

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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.

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participant, sponsor, supporter or exhibitor.

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CONFERENCE AGENDA

(Subject to change)

Tuesday 1st

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 1st

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 1st


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 2nd


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 Ф24B

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 Ф24B

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 Φ24B 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 1

st 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 (1031

) 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 Wegrzyn1, Sylwia Bloch

1, Aleksandra Dydecka

1, Agnieszka Felczykowska

1, Katarzyna Licznerska

1,

Joanna Los1, 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

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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

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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 Lavysh1,2

, Maria Sokolova3,4

, and Konstantin Severinov1,2,3,4,5

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 1

st 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

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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 Zampara1, Martine C. Holst Sørensen

1, Yilmaz Emre Gencay

1, Anne Elsser-Gravesen

2 and Lone

Brøndsted1

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

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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 Sørensen1, 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. Tan1 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.

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 2

nd September (morning)

Chairs: Professor Dennis Bamford

Staphylococcus aureus Population Structure and the Implications for Phage Therapy

Elliot Whittard1, 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

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 Miller1, 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

15


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. Jenkins1; Hollie Hathaway

2; Jessica Bean

3; Scarlet Milo

4, Diana Alves

5; Patricia P. Esteban

6; Mark

Enright7; Amber Young

8

1-6

Department 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

16


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 2

nd 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 (“attBs”) 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

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 Aleshkin1,2

, Nikolai Volozhantsev3, Anastasiya Popova

2,3, Edward Svetoch

3, Evgenii Rubal’sky

1,2, Irina

Kiseleva1,2

, Svetlana Bochkareva2, 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 Hathaway1, Diana R Alves

1, Khajida Ouadi

2, Patricia Pérez Esteban

3 Jessica Bean

1, Mark Sutton

4, A Toby

A Jenkins1

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

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.

19


POSTER ABSTRACTS

A novel bacteriophage cocktail reduces Pseudomonas aeruginosa PAO1 biofilms

Diana R Alves1, 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 Becker1, 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

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 Pseudomonas 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 Ф24B

Sylwia Bloch1, Bozena Nejman-Falenczyk



1, Gracja Topka

1, Alicja Wegrzyn

2,

Grzegorz Wegrzyn1

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 Ф24B 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 Ф24B, 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 Ф24B 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

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 Ф24B

Aleksandra Dydecka1, Sylwia Bloch

1, Agnieszka Necel

1, Gracja Topka


1, Bozena Nejman-Falenczyk

1,

Grzegorz Wegrzyn1, Alicja Wegrzyn

2

1Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 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 - Ф24B, 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 Ф24B. Interestingly, higher number of cells survived after infection of phage Ф24B

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 Ф24B 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 Ф24B, 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 H2O2. 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 H2O2 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

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 Gonçalves1, 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 (Zn2+

) 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 log10 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 Zn2+

) and 1-hour (>4-LR and 2-LR without Zn2+

).

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 Licznerska1, Sylwia Bloch


1, Gracja Topka

1, Agnieszka Necel

1, Bożena Nejman-Faleńczyk

1,

Grzegorz Węgrzyn1, 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

23


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 Lis1, 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 Φ24B phage, one of the Shiga toxin-converting

bacteriophages

Bożena Nejman-Faleńczyk1, Sylwia Bloch



1, Agnieszka Felczykowska

1, Gracja

Topka1, Alicja Węgrzyn

2, Grzegorz Węgrzyn

1

1Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland


24


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 Φ24B.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 Φ24BΔ24B_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 Nyambe1,2

, Catherine Burgess1, 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 24B::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 24B::kanamycin lysogens. The

results show that both 24B::kanamycin and e11/2 are capable of surviving in the water and soil types studied for an

extended period of time. Furthermore, 24B::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 Nzakizwanayo1, Aurelie Hanin

2, Cormac Gahan

2, Cinzia Dedi

1, Jason Clark

3, Brendan Gilmore

4, Toby Jenkins

5,

Brian Jones1,6

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

25


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.

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