Design study w1526455 finished

W1526455 Connor Downing

Design Study

W1526455 Connor Downing BSc. (Hons) LIBMS

MODULE: (2014) FSLS701.2 POSTGRADUATE PROJECT

Module Leader: Sterghios Moschos

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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.


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 Biofilm Development. 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: [email protected]

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.

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