A colorimetric method for detecting virulent bacteriophage to Vibrio cholerae in fecal and environmental samples.

BACKGROUND: Vibrio cholerae are often infected with pathogen-specific virulent bacteriophages (phages) that may be found associated with V. cholerae or independently from the bacteria, especially in environmental water samples. When detected, these phages can serve as a surrogate for detection of V. cholerae and they also have an important role in the ecology of V. cholerae. Vibriophages can be detected using plaque or molecular (PCR) assays, but these methods are time-consuming and require specialized laboratory resources. METHODOLOGY: To increase the accessibility of phage detection for epidemiologic applications, we developed a simple, rapid, and inexpensive colorimetric assay to detect vibriophage that can be scaled quickly to screen large numbers of samples. The assay uses resazurin and V. cholerae AC6169 that is susceptible to vibriophages ICP1, 2, and 3. Resazurin is a dye that turns from blue to pink when added to a culture broth with growing bacteria. We hypothesized that when a bacteria-free test sample, such as filtered wastewater containing vibriophage is added to culture broth with AC6169 and resazurin, the phages will lyse the bacteria preventing their growth, and the color of the broth will remain blue. However, if there are no vibriophages in the sample, the bacteria will grow rapidly, and the culture broth will turn pink. RESULTS: We developed the assay using ICP1 spiked samples of environmental water, stool and frozen bile peptone and found it to be sensitive with a limit of detection of 4-40 plaque forming units/ml. CONCLUSION: This colorimetric assay provides a convenient method to detect vibriophages on a larger scale than was possible earlier. Its use should help to better understand the role of vibriophage as a surrogate for detecting V. cholerae and better understand their role in the pathogenesis, ecology, and epidemiology of cholera.