Abstract
Use of bacteriophages, which are viruses that kill bacteria, for biocontrol of pathogens and antimicrobial resistant bacteria has become increasingly important in recent years. As traditional culture-based methods are laborious and time-consuming, practicable use of bacteriophages will hinge on development of rapid and high throughput methods to analyze, characterize and screen large bacteriophage libraries. We thus established a novel method to fluorescently tag bacteriophages for virus screening and interaction studies, without the need for complicated and laborious purification procedures or genetic engineering of viruses to express fluorescent proteins. Bacteriophage PMBT14 was tagged using DNA dye Syto 13. Simply by using a membrane filter, tagged bacteriophages can be separated from non-sequestered excess dye rapidly, effortlessly, and cheaply. The procedure takes less than 30 min and makes use of simple laboratory consumables that are already commonly used for bacteriophage preparations. As proof of concept, we present here flow cytometric methods to analyze bacteriophage binding, infection and killing that are very accessible for high throughput analysis. We show that the resulting fluorescently tagged bacteriophage can be used to specifically stain its host bacterium Pseudomonas fluorescens DSM 50090. Individual fluorescent bacteriophages, their binding to and initial infection of bacteria could also be observed using confocal microscopy. The infection process was halted by the metabolic inhibitor sodium azide, suggesting a requirement of host metabolic processes for penetration by PMBT14. Flow cytometric live/dead assays was used as a complementary method to determine bacteriophage infection of its host. We made preliminary efforts to adapt the tagging method to two other bacteriophages and discuss potential pitfalls and solutions in the use of tagged phages. Fluorescent phage tagging has previously been demonstrated to facilitate analysis of bacteriophage–host interactions. The method adopted in this study makes it fast, easy as well as cost effective.
Highlights
Bacteriophages, the mortal enemies of bacteria, have been increasingly recognized as a valuable tool in the biocontrol of both food spoilage as well as pathogenic bacteria
The global annual World Health Organization estimates of deaths attributed to foodborne bacterial infections stands at 190,000, stemming from 350 million cases of foodborne bacterial infections occurring each year (Havelaar et al, 2015)
For P. aeruginosa PAO 1 DSM 22644, JG004 Syto 13 was incubated with bacteria diluted in Caso broth for 60 min before dilution in Phage acquisition medium (PAM) + 2.5 μM propidium iodide (PI) and acquired immediately with the FACSCalibur
Summary
Bacteriophages, the mortal enemies of bacteria, have been increasingly recognized as a valuable tool in the biocontrol of both food spoilage as well as pathogenic bacteria This is because they represent a natural and green technology compared to conventional heat and chemical decontamination processes for pathogen control (Moye et al, 2018). Each potential bacterial pathogen will have to be tested against a library of bacteriophages to determine suitable candidates to be included in the treatment cocktail (Chan et al, 2013) This is currently very labor and time intensive, as decades old culture-based methodologies are still being widely used in which the host bacteria are required to first grow to confluence before growth inhibition, or lack thereof, can be observed. Two other phages were tagged and we discuss possible problems and solutions that can occur when using tagged phages
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