Development of a Method for Detecting Viable Cholera Vibrios by Determining the Increase in the Titer of a Specific Bacteriophage Using RT-PCR

The aim of the work was to develop an indirect method for identifying viable cholera vibrios by evaluating the titer increase of a specific bacteriophage detected in RT-PCR.
Materials and methods. The cholera bacteriophage Rostov M3 was used for the study (myovirus class Caudoviricetes; GenBank: MN379460.1-MN379463.1). The study of biological properties was carried out using conventional methods with minor modifications. Primers for phage amplification were designed using https://bioinfo.ut.ee/primer3-0.4.0. Cultivation of samples containing viable and non-viable Vibrio cholerae with bacteriophage was carried out in 1 % peptone water for times Т₀ and Т_n. The PCR result was expressed as the number of phage particles per ml of sample or as the Cp value.
Results and discussion. Bacteriophage Rostov M3 has a high adsorption rate and yield, and also has a broad spectrum of lytic activity against V. cholerae O1 Classical and El Tor. During the accumulation of Rostov M3 phage particles, a decrease in the Cp value was recorded when the sample was incubated for at least two hours. In this case, a conclusion is made about the presence of viable V. cholerae in the sample. Using this method, it is possible to identify bacterial cells that are in a living but non-culturable state, since phages retain the ability to reproduce in this cell phenotype. The application of the proposed method to inactivated cultures (negative control) of V. cholerae did not show an increase in the number of phage particles relative to the zero point, therefore a conclusion is made about the absence of viable cells in the sample. The authors put forward the method that allows one to establish the difference in the levels of accumulation of phage particles when studying samples containing live and non-live V. cholera O1 bacteria in contact with the Rostov M3 bacteriophage for a certain period of time Т₀ and Т_n. The developed method allows for expanding the possibilities of indirect detection of viable V. cholerae O1 Classical and El Tor in potentially contaminated objects.

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