Development of a high throughput assay for indirectly measuring phage growth using the OmniLog^TMsystem

Abstract

The conventional and most accepted method of measuring the lytic activity of a phage against its bacterial host is the plaque assay. This method is laborious, time consuming and expensive, especially in high throughput analyses where multiple phage-bacterial interactions are required to be monitored simultaneously. It can also vary considerably with the experimenter and by the growth and plating conditions. Alternatively, the lytic activity can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. Here we describe an automated, high throughput, indirect liquid lysis assay to evaluate phage growth using the OmniLogTM system. The OmniLogTM system uses redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a tetrazolium dye and produces a color change that is measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color. Here we show that microtiter plate wells inoculated with Bacillus anthracis and phage show decreased or no growth, compared with the wells containing bacteria only or phage resistant bacteria plus phage. Also, we show differences in the kinetics of bacterial growth and the timing of appearance of phage resistant bacteria in the presence of individual phages or a cocktail of B. anthracis specific phages. The results of these experiments indicate that the OmniLogTM system could be used reliably for indirectly measuring phage growth in high throughput host range and phage and antibiotics combination studies.

Isolation of Lytic Bacteriophages for Nanobiocontrol of Pathogenic and Antibiotic Resistant Salmonella Present in Poultry in Ecuador

“…The literature recognizes as evidence of the lytic activity of bacteriophages, reduction in the optical density of host bacteria cocultivated with lytic phages in relation to bacterial cultures without the phage [20]. In our experiments co-cultivating filtered waste water from artisanal poultry processing plants with SE and SI we saw strong evidence of bacteriophages lytic activity by the constant decrease of bacterial concentration comparing with bacterial cultures without the filtrates.…”

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

“…This subset of 98 phages was initially selected for screening because previous work had demonstrated that, collectively, these bacteriophages are highly active against a broad range of MDR A. baumannii isolates (data not shown). Within 18 h of receipt of the first clinical isolate, selection of an appropriate therapeutic cocktail for the original isolate, named TP1, was achieved via an OmniLog-based time-kill assay system (19,20). The bacterial isolates from this study are summarized in Table 1.…”

“…Finally, small-scale phage amplifications on their corresponding host bacterium were performed to prepare the A. baumannii-specific phage library, which was subsequently stored at 4°C until required. The growth of A. baumannii clinical isolates TP1, TP2, and TP3 in the presence of phage was evaluated via spot testing and also in a Biolog imaging system (Biolog, Hayward, CA) (19,20). In this case, one covered 96-well plate was used per phage strain and incubated at 37°C for 24 h, with wells positive only for bacteria and test wells with a single phage added at a multiplicity of infection (MOI) of approximately 100.…”

Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections

“…An automated, indirect, liquid lysis assay was used to evaluate the activity of phages against A. baumannii strains (32). Briefly, an overnight culture of each strain was inoculated into the wells of a 96-well plate containing TSB mixed with 1% (vol/vol) tetrazolium dye.…”

“…An efficiency-of-plating assay showed that when a dilution series of 10 10 to 10 2 of all four individual phages was spotted on an overlay plate of AB5075P, all four phages could infect at a concentration as low as 10 3 PFU per ml. To demonstrate that phages AB-Navy1-4 were each unique, each was tested against a genetically diverse set of A. baumannii strains to compare host ranges by use of a Biolog system (32). The host range of each phage was distinct, not only when the strains infected were compared but also when the numbers of hours each phage could prevent growth of the bacterial host in liquid (hold time) were compared (Table 1).…”