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

Abstract

BackgroundV. cholerae are often infected with vibriophages and these phages may be found associated with V. cholerae bacteria or may be detected independently from the bacteria, especially in environmental water samples. When detected, vibriophages 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 assays or PCR, but these methods are time-consuming and require specialized laboratory resources. MethodologyTo address limitations of other methods, we developed a simple, rapid, and inexpensive colorimetric assay to detect vibriophage that can be scaled up to quickly and easily to screen a large number of samples. The assay uses resazurin and a bacterium, AC6169 that is susceptible to vibriophages ICP1, 2, and 3. Resazurin is a dye that turns color from blue to pink when added to a culture broth with growing bacteria. When a bacteria-free test sample, such as a Millipore filtered wastewater sample containing vibriophages, is added to a culture broth with AC6169, the bacteria will be lysed by the phages and will not grow, and the color of the broth will remain blue. However, if there are no phages in the sample, the bacteria will grow rapidly, and the culture broth will turn pink. 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 to 40 plaque forming units/ml. ConclusionThis colorimetric assay promises to provide a convenient method to detect vibriophages on a larger scale than was possible earlier to better understand their role as a surrogate for detecting V. cholerae and to better understand their role in the pathogenesis, ecology, and epidemiology of cholera. Author SummaryWe developed a simple and inexpensive colorimetric assay to detect virulent vibriophage in samples of wastewater and stool. The assay uses resazurin, a blue dye and a unique bacterium, AC6169, that is known to be susceptible to vibriophages. Bacteria-free samples (e.g. Millipore filtrates) suspected of having vibriophage are incubated for 2 or 3 hours in a broth with AC6169, following which resazurin is added. After a further 30-minute incubation, the color of the broth is observed. If the sample contains vibriophage, the broth will remain blue, but if there are no vibriophage, the broth will turn pink. The change in color from blue to pink is due to the metabolic reaction from the growing bacteria. When vibriophage are present, they lyse the bacterium AC6169, thus preventing the color change, but if vibriophage are not present, the bacteria multiply rapidly, and the metabolic reaction causes the broth to change color to pink. Because the assay procedure is inexpensive and simple, laboratories in cholera endemic areas should find it convenient for their epidemiologic and clinical surveillance activities.