Development of a high throughput assay for indirectly measuring phage growth using the OmniLog(TM) system

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 OmniLog(TM) system. The OmniLog(TM) 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 OmniLog(TM) system could be used reliably for indirectly measuring phage growth in high throughput host range and phage and antibiotics combination studies.

Figure 1. Electron micrographs of bacteriophages used in this study and viewed at 150,000x magnification: (A) Giraffe, (B) Gamma, (C) BA21, (D) BA28, (E) BA39 and (F) BA51.

Figure 2. Kinetics of bacterial growth in the presence of phage Giraffe. B. anthracis strain 7702 was grown at 37°C in tryptic soy media and 1% (v/v) of tetrazolium dye. The wells designed to receive bacteria were seeded with 10⁶ cells. Microtiter plates for the phage assay were prepared as described in materials and methods. The tests were done with (A) vegetative cells, (B) spores and (C) Giraffe-resistant B. anthracis Sterne strain 8009, a derivative of the 7702 strain.

Figure 3. Kinetics of bacterial growth in the presence of phage Gamma. B. anthracis strain 7702 was grown at 37°C in tryptic soy media and 1% (v/v) of tetrazolium dye. The wells designed to receive bacteria were seeded with 10⁶ cells. Microtiter plates for the phage assay were prepared as described in materials and methods. The tests were done with (A) vegetative cells, (B) spores and (C) gamma-resistant B. anthracis Sterne strain 8030, a derivative of the 7702 strain.

Figure 4. Kinetics of bacterial growth in the presence of T4 like phages. B. anthracis strain 7702 was grown at 37°C in tryptic soy media and 1% (v/v) of tetrazolium dye. The wells designed to receive bacteria were seeded with 10⁶ cells. Microtiter plates for the phage assay were prepared as described in materials and methods. These experiments were performed with vegetative cells and using the phages: (A) BA21, (B) BA28, (C) BA39, (D) BA51 and (E) a mixture of Gamma, Giraffe, BA21, BA28, BA39 and BA51.

Figure 5. Kinetics of bacterial growth in the presence of phage Giraffe or ciprofloxacin. B. anthracis HS2–7, a ciprofloxacin resistant strain, was grown at 37°C in tryptic soy media and 1% (v/v) of tetrazolium dye. The wells designed to receive bacteria were seeded with 10⁶ cells. The Giraffe phage PFU ranged from 10⁸ PFU to 10² PFU and ciprofloxacin concentrations ranged from 32 µg/ml to 0.5 µg/ml. One well was used as a phage only control and another as a bacterial positive control. The experiments were performed with (A) B. anthracis Sterne 7702 plus ciprofloxacin, (B) HS2–7 plus ciprofloxacin. (C) HS2–7 plus ciprofloxacin and Giraffe phage and (D) HS2–7 plus Giraffe.