Abstract
Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.
Highlights
The growing threat of antibiotic resistance has led to increased calls for new antimicrobials to control bacterial pathogens and treat infectious diseases [1,2,3]
Phages adjusted to a consistent routine test dilution (RTD) were spotted to agar overlays inoculated with each bacterial test strain and observed for the formation of plaques
The majority of host range results produced by two methods agreed with one another and the microtiter host range assay was generally able to determine the phage host range at the same level of sensitivity as the conventional agar overlay spot method. This result indicated that the microtiter plate method developed here could serve as an alternative to the conventional agar overlay spot method for determining the phage host range
Summary
The growing threat of antibiotic resistance has led to increased calls for new antimicrobials to control bacterial pathogens and treat infectious diseases [1,2,3]. A liquid culture-based host range method is developed, which continuously monitors bacterial growth in the presence of phage in a standard 96-well format and this method is compared to the results from conventional agar overlay spot assays. The intent of this methodology is to determine the phage host range, virulence, and bacterial resistance development in a single high-throughput format by using the features of an automated plate reader to monitor the culture optical density over time in an incubating, aerated environment. This microtiter plate host range assay is expected to serve as an alternative host range method and can potentially be a more sensitive predictor of virulence of phages by providing more information on bacterial inhibition with high resolution between bacterial strains
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