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Analysis of Selection Methods to Develop Novel Phage Therapy Cocktails Against Antimicrobial Resistant Clinical

Melissa E K Haines¹, Francesca E Hodges¹, Janet Y Nale¹

¹Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom.

Frontiers in Microbiology

|April 19, 2021

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 United Kingdom 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 (PKA) 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 PKA 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 PKAs 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.

Insights

Bacteriophages offer a promising solution to combat antimicrobial resistance (AMR) in urinary tract infections. This study developed a novel phage cocktail effective against ESBL-producing E. coli and Klebsiella, identifying planktonic killing assays as a robust screening method.

Area of Science:

Microbiology and Virology
Antimicrobial Resistance (AMR) Research
Bacteriophage Therapy Development

Background:

Antimicrobial resistance (AMR) is a critical global health threat, particularly from Extended Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae like E. coli and Klebsiella.
These ESBL-producing bacteria are major causes of urinary tract infection (UTI)-associated sepsis and are listed as priority pathogens by the WHO.
Bacteriophages (phages), viruses that infect bacteria, represent a potential alternative strategy to combat AMR.

Purpose of the Study:

To develop a novel phage cocktail targeting ESBL-producing E. coli and Klebsiella strains commonly found in UK hospital urinary tract infections.
To compare different phage efficacy assays to identify the most robust and efficient methods for phage cocktail development.
To establish an efficient screening cascade for creating phage cocktails against other AMR bacterial species.

Main Methods:

A panel of 38 ESBL-producing clinical strains of E. coli and Klebsiella from urine samples was curated.
Sixty-eight phages were initially screened, with six selected for further testing against the 38 strains.
Four methods were used to assess phage efficacy: Direct Spot Test (DST), Efficiency of Plating (EOP), Planktonic Killing Assay (PKA), and biofilm assay.

Main Results:

The final six-phage cocktail demonstrated significant efficacy, killing 61% of the target strains (23/38) based on PKA data.
The cocktail was effective against 68% (13/19) of Klebsiella and 53% (10/19) of E. coli strains.
Planktonic Killing Assays (PKAs) were found to be as effective as Efficiency of Plating (EOPs) but less time-consuming, correlating well with EOP data.

Conclusions:

A six-phage cocktail was successfully developed, showing substantial killing activity against a panel of ESBL-producing E. coli and Klebsiella.
Planktonic Killing Assays (PKAs) are a reliable and efficient proxy for assessing phage virulence and informing phage cocktail design.
The study proposes an efficient screening cascade for developing phage cocktails against other priority AMR bacterial pathogens.

Read full analysis

Keywords:

E. coli ESBL Klebsiella antibiotic resistance antimicrobial resistance bacteriophage phage therapy urinary tract infection

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