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Contents
Page 1 Title page
Page 2 Contents
Page 3 Acknowledgements
Page 4 Introduction
Page 5 Research plan
Page 6 Research plan, flowchart
Page 7 Experimental approach
Page 8 Experimental approach (Continued)
Page 9 Experimental approach (Continued)
Page 10 GANNT chart
Page 11 Tasks and Milestones timetable
Page 12 Data Analysis
Page 13 Health and Safety
Page 14 References
Page 15 Appendix, Ethics statement
Page 16 Appendix, Letter of sampling
Page 17 Appendix, Letter of sampling (Continued)
Page 18 Appendix, COSHH chemical
Page 19 Appendix, COSHH chemical (Continued)
Page 20 Appendix, COSHH microbial
Page 21 Appendix, COSHH microbial (Continued)
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Acknowledgements
Thanks are given to:
Lesley Hoyles as her assistance has been crucial in study
design
Alexander Shulgin as his pioneering work in the field of
biochemistry has influenced this piece of work and promoted
many other fields of understudied research
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Introduction
In modern times increasing antimicrobial resistance due to usage of classical
antibiotics has become so much of a concern, bodies such as the WHO (World
Health Organisation) and BSAC (British Society for Antimicrobial Chemotherapy) are
issuing warnings across various pathogenic bacteria. This is exemplified by
Klebsiella pneumoniae which is a significant source of nosocomial infections in
addition to its ever increasing resistance to classical antibiotics in use. Thus a new
demand for alternative strategies against infectious agents has arisen. (Conlan et al.
2011)
A new strategy involving the natural predators of these pathogenic bacteria
has been proposed, namely the usage of bacteriophages (which are viruses) in
clinical treatment of infectious disease which has shown promise in clearing infection
(Hung et al. 2011). Lytic bacteriophage act by infecting and bursting(effectively killing)
bacteria Therefore work involving the isolation of these bacteriophages from
environmental samples), and their screening against potentially infectious strains of
K. pneumoniae may yield very valuable information to applied clinical medicine.
(Parasion et al. 2014)
Furthermore to assess their (the bacteriophage’s) characteristics and mode of
action, various studies need to be completed such as establishing bacteriophage
restriction enzyme profiles, bacteriophage morphology, sequencing of the genome,
pathogenesis (in respect to the bacteria) and testing their chemical robustness. All of
which are fundamental to assessing their practicality of possible further clinical work.
(Hung et al. 2011)
The hypothesis of this study is that lytic bacteriophages against Klebsiella
pneumoniae subsp. pneumoniae can be isolated from canal water taken from
Regent’s Canal, London.
The aim (more broadly) of this study is to investigate various characteristics of
these bacteriophage extracted from canal-water samples, including (but not limited
to) their genome, burst sizes, morphology and chemical resistance to various
stressors.
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Research plan
A flowchart illustrating the proposed research steps is shown in figure 1.
Notably “decision” boxes here signify alternative plans for variable outcomes. For
example if no bacteriophage is isolated on day 1 sample the replicate sampling of
day 1 shall be used and its absence noted as a “non-detected” rather than “absent
from sample” due to the limitations in this procedure of isolation.
Furthermore if several bacteriophages are isolated on lawns by indicative
differing plaque morphology, some may be eliminated from further investigation
based on an undesirable quality their family of bacteriophage possess (e.g.
degrades quickly in solution).
Likewise single plaques after initial screening are diluted and re-inoculated to
ensure only a single phage type is responsible for a plaque, as mixed bacteriophage
inoculation may confound further investigation. Filtration quality control can be
ensured by microscopy examination of filtrate to ensure no particulates are present
in suspension in the filtrate. (Hoyles et al. 2014)
A GANNT chart which details the projects timetable is shown in Figure 2.
Milestones are listed in Table 1. These include the detection and isolation of
bacteriophages, their characterisation using bio-molecular methods, generation of
data for statistical analyses and final submission of the purified bacteriophage.
These are deadlines that are meant to ensure the expedient progress is made on the
project. Some of these correlate to the “outputs” of in Figure 1 and are considered
final work product. Ample time has been allocated to these tasks allowing a final
week in which uncompleted or unsuccessful investigations can be resolved to a
reasonable degree.
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Figure 1. Flowchart, providing an overview of research project. Start (Green), Process
(Red), Decision (Blue), Output (Yellow).
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Experimental approach
The initial stage of research is to identify a suitable site for sample collection. Due
to the biological diversity of the Klebsiella strains used for screening (various animal
isolates) a similar mixed composition of faeces from a variety of animals should be
used. A stretch of Regent’s canal (London) between London Zoo aviary and the
main complex should contain a mixture of animal faeces from run-off into the canal.
To ensure ethical integrity of this study the appropriate authority (Canal and
River Trust) was located and contacted for permission to obtain samples from a
stretch of canal, including the exact collection procedure and geographic co-
ordinates (Appendix).
Transportation of samples is directly by foot to Cavendish campus and samples
will be stored refrigerated at between 1°C and 4°C. Initial processing of the sample
includes placing them on ice for 2 hours to enable some of the virus to desorb from
solid material. After this there are 2 rounds of centrifugation in order to remove
substances with a larger Svedberg co-efficient than that of desired bacteriophages.
This supernatant is then passed through 0.45µm pore-sized acetate filters and
collected into a sterile container. (Hoyles et al. 2014)
At this point a Nanospot™ chip fluorescent transporter assay is used to detect
proteins in this filtrate for to detect bacteriophage and the samples viability for use. If
no bacteriophage are detected the replicate is used in its place. This ensures no
unnecessary screening work be carried out on poor samples.
This “filtrate” water is then used to be inoculated onto bacterial lawns of differing
strains of K.pneumoniae. Important here are the capsular types of K.pneumoniae
which determine entry of the types of phage. By cross matching the various capsule
types against different isolated bacteriophages, increasing the chances of
discovering a useful lytic bacteriophage. For this project 6 strains of K.pneumoniae
will be screened. The lawns are then observed for “plaque” formation. These are
clear area of lysed cells caused by a bacteriophage able to attach and infect the cells
causing them to burst. It is at this point multiple phages or none at all may be
detected dictating the safeguards such as the taking of additional samples (10 in
total, 2 replicates of five days).
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Also at this point negative control of distilled water and a positive control of a
known lytic bacteriophage (named KLPN1 type) against Klebsiella pneumoniae
subsp. pneumoniae strains which possess the K2 capsular type will be used. This is
in order to assess non-bacteriophage causes (e.g. residual antibiotics) plaques and
the resistance of bacteria to phage infection.
Plaques in samples are removed from agar overlays, diluted and suspended in a
sterile medium which is again filtered. A dilution series is made from this filtrate, and
then propagated to increase bacteriophage numbers. This is repeated three times to
ensure purity in the bacteriophage stock. (Jones and Johns 2009)
Addition of this stock (100 µl) to 100ml of culture (with the same host bacterial
strain K.pneumoniae), which is in mid-exponential growth phase. Once lysis has
occurred (i.e. the bacteriophage has replicated itself) PEGylation is used to
precipitate the bacteriophage in the lysate (Jones and Johns 2009). Then a DNA
extraction procedure using a chemical technique from Murphy et al. (2013) from the
pelleted precipitate is used. This extracted DNA will go on to be used for restriction
enzyme profiling.
Restriction enzyme profiling entails additions of bacterial enzymes that break the
phosophodiester bond at varying sites along the DNA strand depending on the
enzymes specificity and target presence in the genome, creating variable lengths of
DNA. These can then have their DNA dyed and electrophoretically separated in
agarose gel for visualisation. Unique profiles (with no counterpart in different strains
using the same restriction enzyme), will be characterised further (with sequencing) in
future studies as their ability to be recognised is crucial. (Kęsik-Szeloch et al. 2013)
Nucleotide sequencing of this extracted DNA by Next Generation Sequencing
(NGS) will create genome data used for comparison of other organisms. For
example a match of percentage similarity using BLAST may reveal similar
bacteriophages with matching stretches of genome.
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Burst sizes and growth behavior is determined by altering concentration of
bacteriophage and bacteria in broth then enumerating plaque forming units on agar
from single phage assays at differing times (and thus phases) and can ultimately
determine how many bacteriophage on average it takes to burst a single microbial
cell and the replicative growth of the phage. (Hyman & Abedon 2009)
Tests adding chloroform varying in concentration to bacteriophage suspension
and subsequent ability to infect (i.e. form plaques) on a bacterial lawn determines its
chemical robustness. Likewise with adjusting pH with acidic through to basic
conditions and assessing viability. (Kęsik-Szeloch et al. 2013)
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Figure 2. GANNT chart showing illustrating tasks, with their respective tasks illustrated in the table below. . H & S (Blue), Sample
collection (Purple), Filtration etc. (Beige), PEGylation and DNA analysis (Green), Characterisation + replicates (Yellow), Growth and
Burst size (Purple) and Miscellaneous task (Grey). Milestones are in Red
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Table 1. Table of tasks illustrating tasks and milestones of
proposed research project totalling 8 weeks.
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Data Analysis
Host range tables of a simple presence (+) or absence (-) of isolated
bacteriophage against bacterial strain will be produced and interpreted as-is.
Nanospot™ assays to confirm bacteriophage are also used. This will in some
way support a preliminary hypothesis of simple presence of lytic bacteriophage
against Klebsiella pneumoniae
For statistical tests of burst sizes and growth the choice of non-
parametric tests are used considering that the predicted result will not follow a
normal distribution. This is due to the number of replicates being used for each
isolated bacteriophage (five in this case) not being sufficient to merit full
parametric tests. Interpreting the information it must be kept in mind that, since
this does not contain a sufficient amount of data it will not follow a Bayesian bell
curve, thus non-parametric tests of Mann-Whitney U or Kolmogorov–Smirnov
tests are appropriate. In the event of six or more replicates per bacteriophage,
the independent Student T-test (parametric) may be used. (Hyman & Abedon
2009)
Image data taken for electron microscopy can be interpreted semi-
quantitatively in regards to its morphology and features of bacteriophage family
such as Siphoviridae which can infect K. pneumoniae. Of note here is possible
resemblance to T-7 type phages or others which have been documented to
infect K. pneumoniae. (Kęsik-Szeloch et al. 2013)
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Health and Safety
All other procedures will be pursuant to University of Westminster’s guidelines of:
Laboratory procedures for research
COSHH form guidance notes
Use of micro-organisms form
See Appendix for completed microbial and chemical COSHH forms
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References
Conlan S, Deming C, Tsai YC, Lau AF, Dekker JP, Korlach J, Segre JA.. (2011).
Complete Genome Sequence of a Klebsiella pneumoniae Isolate with
Chromosomally Encoded Carbapenem Resistance and Colibactin Synthesis Loci.
Genome Announcements. 2 (6), 1-2.
Hoyles L, McCartney AL, Neve H, Gibson GR, Sanderson JD, Heller KJ, van
Sinderen D. (2014). Characterization of virus-like particles associated with the
human faecal and caecal microbiota. Research in Microbiology. 165 (10), 803-12.
Hung CH, Kuo CF, Wang CH, Wu CM, Tsao N. (2011). Experimental Phage Therapy
in Treating Klebsiella pneumoniae-Mediated Liver Abscesses and Bacteraemia in
Mice. Antimicrobial agents and chemotherapy. 11 (5), 211-219.
Hyman P and Abedon ST (2009). Practical methods for determining phage growth
parameters. Methods in Molecular biology. 501 (1), 175-202.
Jones TH and Johns MW (2009). Improved detection of F-specific RNA coliphages
in fecal material by extraction and polyethylene glycol precipitation. Applied and
Environmental Microbiology. 75, 6142-6146.
Kęsik-Szeloch A, Drulis-Kawa Z, Weber-Dąbrowska B, Kassner J, Majkowska-
Skrobek G, Augustyniak D, Lusiak-Szelachowska M, Zaczek M, Górski A, Kropinski
AM. (2013). Characterising the biology of novel lytic bacteriophages infecting
multidrug resistant Klebsiella pneumoniae. Virology Journal. 10 (100), 1-12.
Murphy J, Royer B, Mahony J, Hoyles L, Heller K, Neve H, Bonestroo M, Nauta A
and van Sinderen D (2013). Biodiversity of lactococcal bacteriophages isolated
from 3 Gouda-type cheese-producing plants. Journal of Dairy Science. 96 (8),
4945-4957.
Parasion S, Kwiatek M, Gryko R, Mizak L, Malm A (2014) Bacteriophages as an
Alternative Strategy for Fighting BiofilmDevelopment. Polish Journal of
Microbiology. 63 (2), 137-145.
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Appendix
Ethics statement
Pursuant with University of Westminster ethical guidelines concerning research,
permission was sought for obtaining samples from Regents Canal, Borough of
Westminster from the Canal & River Trust (Appendix).
Map Co-ordinates of sampling:
51.536388 Latitude
-0.157118 Longitude
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Dr Lesley Hoyles CBiol MSB
Lecturer in Microbiology
Department of Biomedical Sciences
University of Westminster
115 New Cavendish Street
London W1W 6UW
United Kingdom
Email: l.hoyles@westm inster.ac.uk
Telephone: +44 (0) 20 7911 5000 ext. 65480
17 February 2015
To whom it may concern,
I am contacting you on behalf of my student, Mr Connor Downing, who will
shortly be undertaking a research project in my laboratory. Connor is studying for an
MSc in Biomedical Science at the University of Westminster. His research project
involves isolating viruses that infect and kill clinically relevant bacteria. These viruses
are known as bacteriophages, and are present in numerous environments, including
water samples. As part of his project, Connor would like to take water samples from
Regent’s canal and see whether he can isolate bacteriophages from these. The
University’s research and ethical guidelines state that, where possible, permission
should be sought to obtain environmental samples. In light of this, Connor and I
believe you to be the relevant body from which to seek permission to collect water
samples from Regent’s canal and ask for permission to collect the samples Connor
requires, or to be directed to any by-laws the Canal & River Trust has concerning
conducting scientific studies on British waterways.
Connor will take no more than 10× 250 ml of water from Regent’s canal in
sterile containers over the course of 5 days during late May/early June 2015. Care
will be taken not to disturb any flora and fauna along the towpath during sample
collection, and we believe the local ecology will not be affected by removal of these
small amounts of water. Connor’s proposed location for sampling is between the
London Zoo aviary and main complex (Figure 1).
Thank you in advance for considering this request. I look forward to hearing
from you. Please do not hesitate to contact me should you require any further
information.
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Yours sincerely,
Dr Lesley Hoyles CBiol MSB
Figure 1. Proposed sampling site for Mr Connor Downing’s research project.
