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Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria

View ORCID ProfileMelissa EK Haines, View ORCID ProfileFrancesca E Hodges, View ORCID ProfileJanet Y Nale, Jennifer Mahony, View ORCID ProfileDouwe van Sinderen, Joanna Kaczorowska, Bandar Alrashid, View ORCID ProfileMahmuda Akter, View ORCID ProfileNathan Brown, View ORCID ProfileDominic Sauvageau, View ORCID ProfileThomas Sicheritz-Ponten, Anisha M Thanki, Andrew D Millard, View ORCID ProfileEdouard E Galyov, Martha RJ Clokie
doi: https://doi.org/10.1101/2020.10.13.337345
Melissa EK Haines
1Department of Genetics and Genome Biology, University of Leicester, UK
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  • For correspondence: mh508@leicester.ac.uk
Francesca E Hodges
1Department of Genetics and Genome Biology, University of Leicester, UK
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Janet Y Nale
1Department of Genetics and Genome Biology, University of Leicester, UK
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Jennifer Mahony
2School of Microbiology & APC Microbiome Ireland, University College Cork, Ireland
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Douwe van Sinderen
2School of Microbiology & APC Microbiome Ireland, University College Cork, Ireland
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Joanna Kaczorowska
2School of Microbiology & APC Microbiome Ireland, University College Cork, Ireland
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Bandar Alrashid
1Department of Genetics and Genome Biology, University of Leicester, UK
3King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
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Mahmuda Akter
4International Centre for Diarrheal Diseases Research, Bangladesh
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Nathan Brown
1Department of Genetics and Genome Biology, University of Leicester, UK
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Dominic Sauvageau
5Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Canada
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Thomas Sicheritz-Ponten
6Department of Bio and Health Informatics, Technical University of Denmark, Kgs Lyngby, Denmark
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Anisha M Thanki
1Department of Genetics and Genome Biology, University of Leicester, UK
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Andrew D Millard
1Department of Genetics and Genome Biology, University of Leicester, UK
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Edouard E Galyov
1Department of Genetics and Genome Biology, University of Leicester, UK
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Martha RJ Clokie
1Department of Genetics and Genome Biology, University of Leicester, UK
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  • For correspondence: mh508@leicester.ac.uk
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Abstract

Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as ‘critical’ for research. Bacteriophages (phages) as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains.

There is currently no ‘gold standard’ for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in UK hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species.

A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay and the biofilm assay.

The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%) for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the planktonic killing assay data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that planktonic killing assays are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows ‘phage synergy’ to inform cocktail design.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria
Melissa EK Haines, Francesca E Hodges, Janet Y Nale, Jennifer Mahony, Douwe van Sinderen, Joanna Kaczorowska, Bandar Alrashid, Mahmuda Akter, Nathan Brown, Dominic Sauvageau, Thomas Sicheritz-Ponten, Anisha M Thanki, Andrew D Millard, Edouard E Galyov, Martha RJ Clokie
bioRxiv 2020.10.13.337345; doi: https://doi.org/10.1101/2020.10.13.337345
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Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria
Melissa EK Haines, Francesca E Hodges, Janet Y Nale, Jennifer Mahony, Douwe van Sinderen, Joanna Kaczorowska, Bandar Alrashid, Mahmuda Akter, Nathan Brown, Dominic Sauvageau, Thomas Sicheritz-Ponten, Anisha M Thanki, Andrew D Millard, Edouard E Galyov, Martha RJ Clokie
bioRxiv 2020.10.13.337345; doi: https://doi.org/10.1101/2020.10.13.337345

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