The problem of antimicrobial resistance is nowadays major and the need to identify alternative methods to antibiotics to tackle bacterial pathogens is obvious. One of the potential alternatives is phage therapy, the use of bacteriophages with high specificity against the targeted pathogen, as antimicrobial agents. The interest in phage therapy has grown lately and therefore different factors that can increase or decrease the phages’ killing efficiency, are becoming more and more interesting to study.
In this project, we aim to investigate the effect of different parameters, such as oxygen levels and temperature, to the killing efficiency and infection ability of bacteriophages. It has been shown previously that both oxygen availability (1) and temperature conditions (2,3), can play a role in the infection ability of bacteriophages, as it happens in nature, when phages have to deal with for example oxygen limited environments, as well as low temperatures. Since we aim to further investigate what happens inside the human body, where the conditions are fluctuating, we will use in this project a model system previously established in Sagona lab (4,5), consisting of K1F bacteriophage (6), human cells and EV36, a K1/K12 hybrid mimicking E. coli O18:K1:H7 (7).
The specific objectives are the following:
- Examine using basic microbiology and phage biology methods (plaque assays and growth curves) the killing efficiency of bacteriophage K1F in:
- Oxygen rich environments
- Oxygen-limited environments
- High temperatures
- Low temperatures
This data will be compared with the control conditions that are used regularly in the lab.
- Investigate the effect of the above conditions in the health of human cells, using specific assays for cytotoxicity (flow cytometry) and confocal and live microscopy, to observe the tendency of human cells. For this set of experiments, we will use a variety of incubation times under the different oxygen and temperature conditions.
- Further examine using the above methodology, the interaction between K1F phage, its host EV36 and human cells, under different conditions. Again, different oxygen levels and temperatures will be tested individually and in combination for a variety of time points. We aim to understand in vitro what happens inside the human body and how phages’ efficiency is affected on different oxygen and temperature levels, to ameliorate the application of phage therapy.
- Develop methodology to determine the scale and scope of innate immune responses in human cells under the different conditions against the host and K1F phage, as well as combination of bacteria and phages, with the use of qPCR to measure the mRNA levels of the genes involved.
- Compare the different conditions/parameters described by statistical analysis to conclude in the optimal conditions, where the phage presents the highest killing efficiency and bacterial clearance along with the least innate immune response, towards establishing efficient safe phage therapy.
- As a future scope of this project, is to establish a system and gain knowledge on this, so that the same approach will be implemented on more phages and their hosts, along with a broader variety of human cells, in order to understand as mentioned above what happens inside the human body-where the oxygen and temperature levels vary- when phage therapy is applied.
A combination of molecular biology, microbiology, immunology and human cell culture methods will be implemented towards the objectives.
1. Francesca E. Hodges, Thomas Sicheritz-Pontén, and Martha R.J. Clokie
Published Online: 17 Mar 2021 The Effect of Oxygen Availability on Bacteriophage Infection: A Review https://doi.org/10.1089/phage.2020.00412.
- Shan J, Korbsrisate S, Withatanung P, Adler NL, Clokie MR, Galyov EE. Temperature dependent bacteriophages of a tropical bacterial pathogen. Front Microbiol. 2014 Nov 14;5:599. doi: 10.3389/fmicb.2014.00599. eCollection 2014.PMID: 25452746 Free PMC article.
- Egilmez HI, Morozov AY, Clokie MRJ, Shan J, Letarov A, Galyov EE. .Temperature-dependent virus lifecycle choices may reveal and predict facets of the biology of opportunistic pathogenic bacteria. Sci Rep. 2018 Jun 25;8(1):9642. doi: 10.1038/s41598-018-27716-3.PMID: 29941954
- Møller-Olsen C, Ho SFS, Shukla RD, Feher T, Sagona AP. Engineered K1F bacteriophages kill intracellular Escherichia coli K1 in human epithelial cells. Sci Rep. 2018 Dec 3;8(1):17559. doi: 10.1038/s41598-018-35859-6. PMID: 30510202; PMCID: PMC6277420.
- Møller-Olsen C, Ross T, Leppard KN, Foisor V, Smith C, Grammatopoulos DK, Sagona AP. Bacteriophage K1F targets Escherichia coli K1 in cerebral endothelial cells and influences the barrier function. Sci Rep. 2020 Jun 1;10(1):8903. doi: 10.1038/s41598-020-65867-4. PMID: 32483257; PMCID: PMC7264188.
- Scholl D et al.,. 2005. The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli. J Bacteriol 187:8499-503
- Silver RP et al.,. The K1 capsular polysaccharide of Escherichia coli. Reviews of infectious diseases 10 Suppl 2:S282-6
BBSRC Strategic Research Priority: Understanding the rules of life – Immunology, and Microbiology.
Techniques that will be undertaken during the project:
Human cell culture
Microbiology/phage biology methods