PHIDA: A High Throughput Turbidimetric Data Analytic Tool to Compare Host Range Profiles of Bacteriophages Isolated Using Different Enrichment Methods

Abstract

Bacteriophages are viruses that infect bacteria and are present in niches where bacteria thrive. In recent years, the suggested application areas of lytic bacteriophage have been expanded to include therapy, biocontrol, detection, sanitation, and remediation. However, phage application is constrained by the phage's host range-the range of bacterial hosts sensitive to the phage and the degree of infection. Even though phage isolation and enrichment techniques are straightforward protocols, the correlation between the enrichment technique and host range profile has not been evaluated. Agar-based methods such as spotting assay and efficiency of plaquing (EOP) are the most used methods to determine the phage host range. These methods, aside from being labor intensive, can lead to subjective and incomplete results as they rely on qualitative observations of the lysis/plaques, do not reflect the lytic activity in liquid culture, and can overestimate the host range. In this study, phages against three bacterial genera were isolated using three different enrichment methods. Host range profiles of the isolated phages were quantitatively determined using a high throughput turbidimetric protocol and the data were analyzed with an accessible analytic tool "PHIDA". Using this tool, the host ranges of 9 Listeria, 14 Salmonella, and 20 Pseudomonas phages isolated with different enrichment methods were quantitatively compared. A high variability in the host range index (HR_i) ranging from 0.86-0.63, 0.07-0.24, and 0.00-0.67 for Listeria, Salmonella, and Pseudomonas phages, respectively, was observed. Overall, no direct correlation was found between the phage host range breadth and the enrichment method in any of the three target bacterial genera. The high throughput method and analytics tool developed in this study can be easily adapted to any phage study and can provide a consensus for phage host range determination.

Keywords: bacteriophage; host range; isolation protocol; lytic phages; phage biocontrol; phage enrichment; phage therapy.

Figure 1

Methods for bacteriophage enrichment and isolation evaluated in this study. (1) Single strain (SS): processed sample is enriched with an overnight monoculture and incubated overnight. Mixture is then centrifuged, filtered, and presence of phage is verified by plaque assay on the same host used for enrichment. Plaques are purified and propagated for host range determination using high throughput turbidimetric assay. (2) Cocktail enrichment (CE): processed sample is enriched with equal ratios of overnight monocultures (cocktail) and incubated overnight. The mixture is then centrifuged, filtered, and presence of phage is verified by plaque assay on every host used for the enrichment. Plaques from individual hosts are purified and propagated for host range determination using high throughput turbidimetric assay. (3) Cyclic sequential (CS) was performed as previously described [31] with some modifications; processed sample is enriched with an overnight monoculture of the first host in the series and incubated overnight. Mixture is then centrifuged, filtered, and presence of phage is verified by plaque assay on the first host. All plaques are pooled and for propagation in the second host of the series. Presence of phage is verified by plaque assay on the second host. The same process is repeated with all the hosts in the series. Plaques recovered from last enrichment are purified and propagated for host range determination using high throughput turbidimetric assay. Created with BioRender.com.

Figure 2

The 384-well plate layout for the high throughput turbidimetric host range determination assay. Columns indicate the number of bacterial strains, and rows, the treatments. Phages 1–4 indicate different phages tested in technical triplicates (50 µL of bacteria dilution + 50 µL of phage dilution). Untreated control (50 µL of bacteria dilution + 50 µL of CM buffer). Sterility control (50 µL of 2 x media + 50 µL of phage dilution). Blank (50 µL of 2 x media + 50 µL of CM buffer). Created with BioRender.com.

Figure 3

Growth inhibition designations for host range characterization. (A) Complete inhibition of the bacterial growth; C: sample never reaches detection threshold for the duration of the experiment. (B) More than 5 h delay in bacterial growth compared to control; D+: time difference to reach detection threshold between sample and control is ≥5 h and <“experiment duration time–detection time of the control”. Less than 5 h delay in bacterial growth compared to control D: time difference to reach detection threshold between sample and control is ≥1 and <5 h). (C) Time difference to reach detection threshold between sample and control is <1 h. Small effect on bacterial growth endpoint; N/L: OD_max is 70–85% of control. Moderate effect on bacterial growth endpoint; N/L+: OD_max is 40–70% of control. Large effect on bacterial growth endpoint N/L++: OD_max is ≤40% of control. Created with BioRender.com.

Figure 4

Stacked bar plot showing the host range of Listeria phages isolated using different enrichment methods. Designation: C, complete inhibition, D+, >5 h delay to reach exponential phase; D, <5 h delay to reach exponential phase; N, no effect; HR_i, host range index.

Figure 5

Stacked bar plot showing the host range of Salmonella phages isolated using different enrichment methods. Designation: C, complete inhibition, D+, >5 h delay to reach exponential phase; D, <5 h delay to reach exponential phase; N, no effect; HR_i, host range index.

Figure 6

Stacked bar plot showing the host range of phages infecting Pseudomonas clinical strains isolated using different enrichment methods. Designation: C, complete inhibition, D+, >5 h delay to reach exponential phase; D, <5 h delay to reach exponential phase; N, no effect; HR_i, host range index.

Figure 7

Stacked bar plot showing the host range of phages infecting Pseudomonas environmental strains isolated using different enrichment methods. Designation: C, complete inhibition, D+, >5 h delay to reach exponential phase; D, <5 h delay to reach exponential phase; N, no effect; HR_i, host range index.