Development and validation of a miniaturized bacteriophage host range screening assay against antibiotic resistant Pseudomonas aeruginosa

Renee Nicole Ng; Lucinda Jane Grey; Andrew Vaitekenas; Samantha Abagail McLean; Jack Dylan Rudrum; Daniel Rodolfo Laucirica; Matthew Wee-Peng Poh; Jessica Hillas; Scott Glenn Winslow; Joshua James Iszatt; Thomas Iosifidis; Anna Sze Tai; Patricia Agudelo-Romero; Barbara Jane Chang; Stephen Michael Stick; Anthony Kicic; AREST CF

doi:10.1016/j.mimet.2021.106346

Development and validation of a miniaturized bacteriophage host range screening assay against antibiotic resistant Pseudomonas aeruginosa

J Microbiol Methods. 2021 Nov:190:106346. doi: 10.1016/j.mimet.2021.106346. Epub 2021 Oct 9.

Authors

Renee Nicole Ng¹, Lucinda Jane Grey¹, Andrew Vaitekenas², Samantha Abagail McLean³, Jack Dylan Rudrum¹, Daniel Rodolfo Laucirica⁴, Matthew Wee-Peng Poh⁴, Jessica Hillas³, Scott Glenn Winslow³, Joshua James Iszatt², Thomas Iosifidis⁵, Anna Sze Tai⁶, Patricia Agudelo-Romero³, Barbara Jane Chang⁷, Stephen Michael Stick⁸, Anthony Kicic⁹; AREST CF¹⁰

Affiliations

¹ School of Biomedical Sciences, The University of Western Australia, Perth, Western Australia, Australia; Wal-Yan Respiratory Research Center, Telethon Kids Institute, Perth, Western Australia, Australia.
² Wal-Yan Respiratory Research Center, Telethon Kids Institute, Perth, Western Australia, Australia; Occupation, Environment and Safety, School of Population Health, Curtin University, Perth, Western Australia, Australia.
³ Wal-Yan Respiratory Research Center, Telethon Kids Institute, Perth, Western Australia, Australia.
⁴ Wal-Yan Respiratory Research Center, Telethon Kids Institute, Perth, Western Australia, Australia; Medical School, The University of Western Australia, Perth, Western Australia, Australia.
⁵ Wal-Yan Respiratory Research Center, Telethon Kids Institute, Perth, Western Australia, Australia; Occupation, Environment and Safety, School of Population Health, Curtin University, Perth, Western Australia, Australia; Center for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia.
⁶ Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia; Institute for Respiratory Health, School of Medicine, The University of Western Australia, Perth, Western Australia, Australia.
⁷ The Marshall Center for Infectious Diseases Research and Training, School of Biomedical Sciences, The University of Western Australia, Perth, Western Australia, Australia.
⁸ Wal-Yan Respiratory Research Center, Telethon Kids Institute, Perth, Western Australia, Australia; Department of Respiratory and Sleep Medicine, Perth Children's Hospital, Perth, Western Australia, Australia; Center for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia.
⁹ Wal-Yan Respiratory Research Center, Telethon Kids Institute, Perth, Western Australia, Australia; Occupation, Environment and Safety, School of Population Health, Curtin University, Perth, Western Australia, Australia; Department of Respiratory and Sleep Medicine, Perth Children's Hospital, Perth, Western Australia, Australia; Center for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia. Electronic address: anthony.kicic@telethonkids.org.au.
¹⁰ Wal-Yan Respiratory Research Center, Telethon Kids Institute, Perth, Western Australia, Australia; Department of Respiratory and Sleep Medicine, Perth Children's Hospital, Perth, Western Australia, Australia; Murdoch Children's Research Institute, Melbourne, Victoria, Australia; Department of Pediatrics, University of Melbourne, Melbourne, Victoria, Australia.

PMID: 34637818
DOI: 10.1016/j.mimet.2021.106346

Abstract

Antimicrobial resistance is a current global health crisis, and the increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Prior to clinical application, phage suitability is assessed, via susceptibility testing and breadth of host range to bacteriophage, however, these are both large-scale manual processes and labor-intensive. The aim of the study was to establish and validate a scaled down methodology for high-throughput screening to reduce procedural footprint. In this paper, we describe a scaled-down adapted methodology that can successfully screen bacteriophages, isolated and purified from wastewater samples. Furthermore, we describe a miniaturized host range assay against clinical Pseudomonas aeruginosa isolates using a spot test (2 μL/ drop) that was found to be both sensitive (94.6%) and specific (94.7%). It also demonstrated a positive predictive value (PPV) of 86.4% and negative predictive value (NPV) of 98%. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates was corroborated using the scaled down assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.

Keywords: Bacteriophages; Host range; Miniaturization; Phage therapy; Pseudomonas aeruginosa.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Anti-Bacterial Agents
Bacteriophages / isolation & purification*
Bacteriophages / physiology*
DNA, Viral
Drug Resistance, Multiple, Bacterial
High-Throughput Screening Assays / methods*
Host Specificity*
Humans
Phage Therapy
Pseudomonas Infections / microbiology
Pseudomonas aeruginosa / virology*
Sensitivity and Specificity
Wastewater / virology*

Substances

Anti-Bacterial Agents
DNA, Viral
Waste Water