Study of the biofilm form of vibrio cholera by RT-PCR

Abstract

BACKGROUND: Vibrio cholerae can exist in planktonic and biofilm forms. There are no unified methods for recording the formation of a biofilm and the quantitative determination of microorganisms; the known methods are laborious and do not allow an objective assessment of the concentration of V. cholerae in biofilms. AIM: This study aimed to evaluate the method for the quantitative determination of V. cholerae in biofilm and planktonic forms based on real-time PCR. MATERIALS AND METHODS: The concentration of V. cholerae in plankton was determined by the bacteriological method based on the number of colony-forming units per 1 ml; in biofilms, the method of imprint depletion on agar plates was used. Real-time PCR was performed using the primers and probes described in the literature for detecting the hlyA and ctx genes. V. cholerae were quantified using built-in software and standard preparations with a known concentration of bacterial cells. The results were processed in Microsoft Office Excel 2016 spreadsheets using the decimal logarithm; statistical analysis was performed using the Statistica 13.3 program. RESULTS: During the observation period, the concentration of V. cholerae in biofilms on chitin and plastic increases as the incubation period increases. The amount of V. cholerae in the composition of biofilms and plankton on/above chitin exceeded those when used as a plastic substrate. On the 30th day, the difference was two or more orders of magnitude. The results of the two methods were reproducible and comparable; at the same stages, the concentration of V. cholerae varied within the same order of magnitude, which indicated the reliability of the PCR-RT results. CONCLUSION: The bacteriological method is informative in the qualitative assessment of biofilms, in determining the viability of cholera vibrios. However, due to its complexity, with the impossibility of quickly determining the concentration of V. cholerae in a biofilm on chitin, it is preferable to use real-time PCR, which allows you to assess the concentration of V. cholerae in plankton and biofilm accurately and quickly.