Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence

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.

Evaluation of Commercial Prototype Bacteriophage Intervention Designed for Reducing O157 and Non-O157 Shiga-Toxigenic Escherichia coli (STEC) on Beef Cattle Hide

“…While STEC-inoculated plates were solidifying, phages (individual or pre-mixed cocktail) were serially diluted 10 −1 to 10 −7 . Efficiency of plating (EOP) assays were performed to determine how well individual phages or the mixed cocktail of phages infected each STEC serogroup by published methods [ 27 , 28 ]. Evaluation of STEC isolate and phage spot-tests was completed to determine EOP outcomes for each STEC host/phage pairing (individual phages or phage cocktail).…”

“…Evaluation of STEC isolate and phage spot-tests was completed to determine EOP outcomes for each STEC host/phage pairing (individual phages or phage cocktail). The spot-test reports the number of phage infecting a host bacterium per mL of applied phage suspension, and can be impacted by variations in host susceptibility between host isolates [ 27 , 28 ]. The E. coli O157:H7 isolate was used as the reference STEC for EOP assignment for experiments using the cocktailed phage intervention.…”

Quantifying relative virulence: When μmaxfails and AUC alone just isn’t enough

“…Given limitations of μ max in determining V R in non-lytic viral infections, an alternative approach is to determine a percent inhibition (PI) based on area-under-the-curve (AUC) [13–16]. Specifically, determining AUC for infected host growth (AUC infected ) and uninfected control (AUC CTL ) provides a calculated PI AUC on non-transformed data such that: …”

The Novel Enterococcus Phage vB_EfaS_HEf13 Has Broad Lytic Activity Against Clinical Isolates of Enterococcus faecalis

“…Among the 17 strains of E. faecalis tested in the host range assay, 12 strains sensitive to the purified phages based on the results of host range analysis were chosen for the bacterial challenge assay (Xie et al, 2018). One percent of an overnight culture of each E. faecalis strain was inoculated in triplicate on a 96-well plate and the plate was cultured at 37 • C with shaking until the optical density at 600 nm (OD 600 ) reached 0.2 (∼1 × 10 8 CFU/mL).…”