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Genetic engineering of bacteriophages to be used for biofilm clearance and prevention in waste water treatment


Project Description

Project Highlights
• Mutagenesis of bacterial global transcription factors, such as CRP (cyclic AMP receptor protein) and testing of their effectiveness on biofilm clearance and prevention
• Genetic engineering of bacteriophages to increase their effectiveness towards biofilms in waste water treatment settings
• Combination of both above methods in river samples
• Construction of genetically modified bacteriophages with the ability to clear water based biofilms in pipes and water waste treatment settings

Overview
Antimicrobial resistance is a serious problem worldwide and new ways to prevent it and tackle it are necessary (Piddock, 2012). One of the ways via which antibiotic resistant bacteria spread and affect the water environments is via the dissemination of integrons (Amos et al., 2018). Sewage effluent can affect the structure of microbial community as well as the integron prevalence in water biofilms (Lehmann et al., 2016). This results in water biofilms that are able to survive exposure even to very efficient biocides, such as quaternary ammonium compounds (QACs) (Partridge et al., 2009), resulting in both chemical and microbial pollution of the environment. In this project, we propose an alternative method to clear biofilms, via the use of genetically modified bacteriophages. Bacteriophages are viruses that have the ability to kill bacteria efficiently and with high specificity. They also have some unique characteristics that make them ideal targets for genetic engineering: they have relatively small genomes and can thus be very easily genetically modified, which enables the development of phages with preferred characteristics making them ideal for widespread applications (Sagona et al., 2016). Interestingly, lytic phages are excellent anti-biofilm agents (Donlan, 2009). In order to enhance the anti-biofilm action of lytic bacteriophages, we will screen for mutations in CRP transcription factors, which according to preliminary data these appear to regulate biofilm formation (Rossiter et al., 2015, Hufnagel et al., 2016). According to recent reports, bacteriophages can been used efficiently for bacterial control in water treatment (Mathieu et al., 2019) and with this project we provide a combination of innovative methodologies to engineer phages that are able to tackle in an even more targeted manner biofilms in waste water treatment plants.

Methodology
We will initially select water samples and perform metagenomics to detect target pathogens (Wellington lab). We will further do mutagenesis in CRP transcription factors in the selected pathogens and test their effect in biofilm clearance compared to control samples (Busby lab).
We will also perform simple phage –based assays to test the phage specificity and ability on clearing biofilms present in water samples (Sagona lab).
We will further engineer a bacteriophage able to encode and express mutant CRP Proteins. The engineered bacteriophage will be tested on its ability to efficiently clear biofilms present in water samples (Sagona lab). Our long term plan is to immobilize this bacteriophage on water pipes (in collaboration with Fixed-Phage) as a methodology of water disinfection using natural sources rather than chemicals.
Via bacterial mutagenesis, metagenomics, synthetic biology and genetic engineering, we aim to enhance the lytic and killing efficiency of these phages and use them as anti-biofilm agents.

Training and skills
CENTA students are required to complete 45 days training throughout their PhD including a 10 day placement. In the first year, students will be trained as a single cohort on environmental science, research methods and core skills. Throughout the PhD, training will progress from core skills sets to master classes specific to CENTA research themes.
The PhD student will be trained in 3 labs:
In the Busby lab on CRP mutagenesis and biofilms, in the Wellington lab in metagenomics and sample collection and in the Sagona lab on genetic engineering of phages and phage biology.

Partners and collaboration
The project is a collaboration between the following labs:
Dr Antonia Sagona, University of Warwick
Professor Stephen Busby, University of Birmingham
Professor Liz Wellington, University of Warwick
Fixed-Phage Ltd (for the immobilization of the phage in pipes)

Possible timeline
Year 1: Sample collection and metagenomics of the identification of the target pathogens
Year 2: CRP mutagenesis screening and test on biofilms, phage screening and test on biofilms
Year 3: Genetic engineering of phage and test on biofilms, immobilization of phage and test of its killing efficiency on biofilms present in water samples.

Funding Notes

This funding provides full tuition fees at the Home/EU rate and pays an annual stipend in line with UK Research Councils (currently £14,777).

References

Further reading
AMOS, G. C. A., PLOUMAKIS, S., ZHANG, L., HAWKEY, P. M., GAZE, W. H. & WELLINGTON, E. M. H. 2018. The widespread dissemination of integrons throughout bacterial communities in a riverine system. ISME J, 12, 681-691.
DONLAN, R. M. 2009. Preventing biofilms of clinically relevant organisms using bacteriophage. Trends Microbiol, 17, 66-72.
HUFNAGEL, D. A., EVANS, M. L., GREENE, S. E., PINKNER, J. S., HULTGREN, S. J. & CHAPMAN, M. R. 2016. The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli. J Bacteriol, 198, 3329-3334.
LEHMANN, K., BELL, T., BOWES, M. J., AMOS, G. C. A., GAZE, W. H., WELLINGTON, E. M. H. & SINGER, A. C. 2016. Trace levels of sewage effluent are sufficient to increase class 1 integron prevalence in freshwater biofilms without changing the core community. Water Res, 106, 163-170.
MATHIEU, J., YU, P., ZUO, P., DA SILVA, M. L. B. & ALVAREZ, P. J. J. 2019. Going Viral: Emerging Opportunities for Phage-Based Bacterial Control in Water Treatment and Reuse. Acc Chem Res.
PARTRIDGE, S. R., TSAFNAT, G., COIERA, E. & IREDELL, J. R. 2009. Gene cassettes and cassette arrays in mobile resistance integrons. FEMS Microbiol Rev, 33, 757-84.
PIDDOCK, L. J. 2012. The crisis of no new antibiotics--what is the way forward? Lancet Infect Dis, 12, 249-53.
ROSSITER, A. E., GODFREY, R. E., CONNOLLY, J. A., BUSBY, S. J., HENDERSON, I. R. & BROWNING, D. F. 2015. Expression of different bacterial cytotoxins is controlled by two global transcription factors, CRP and Fis, that co-operate in a shared-recruitment mechanism. Biochem J, 466, 323-35.
SAGONA, A. P., GRIGONYTE, A. M., MACDONALD, P. R. & JARAMILLO, A. 2016. Genetically modified bacteriophages. Integr Biol (Camb), 8, 465-74.

How good is research at University of Warwick in Agriculture, Veterinary and Food Science?

FTE Category A staff submitted: 12.60

Research output data provided by the Research Excellence Framework (REF)

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