Development of Polymerase Chain Reaction for Rapid Identification of Paenibacillus and Cohnella with High Peracetic Acid Tolerance

Introduction: Peracetic acid is one of the bactericides used for sterilization of container in soft drink manufacturing process. Certain species of genera Paenibacillus and Cohnella were reported to have peracetic acid tolerance. Therefore it's important to detect these species rapidly in manufacturing environment for quality control of soft drink. 

Purpose: The purpose of this study was to develop a PCR assay for rapid identification of species of genera Paenibacillus and Cohnella resistant to peracetic acid.

Methods: A primer set specific to genera Paenibacillus and Cohnella and a primer set specific to P. favisporus, P. cineris, and P. chibensis with high peracetic acid tolerance were designated on the basis of 16S rDNA sequences and literatures. To test the specificity of the primers, 26 Paenibacillus species, 6 Cohnella species, 13 Bacillus species and 4 Brevibacillus species were used.

Results: DNAs from 49 strains of spore-forming bacteria were analyzed by the developed PCR method. PCR products were generated from templates of 26 Paenibacillus and 6 Cohnella species by the PCR using the primer set specific to genera Paenibacillus and Cohnella. The template DNAs only from P. favisporus, P. cineris, and P. chibensis generated PCR products by the PCR using the primer set specific to these high peracetic acid tolerant species. Ten strains isolated from manufacturing environment, 3 Paenibacillus species including 2 P. favisporus and 7 other genera, were correctly identified by the present PCR methods.

Significance: The developed PCR methods are specific to detect peracetic acid tolerant genera and the species with peracetic acid tolerance and easy to use at the manufacturing environment on site in the beverage manufacturing.