Development of the genus and species determination method for histamine producing bacteria isolated from fishery product with high-resolution melting analysis

Histamine in foods with a high histidine content may be produced by bacteria with histidine decarboxylase activity. Consumption of food enriched in histamine can produce symptoms of histamine poisoning that include flushing, headache, and urticaria. The number of histamine poisoning cases in Japan has decreased with developments in food hygiene management technology. However, approximately 10 cases are still reported each year. In addition, there have been cases where histamine was detected in the end products, prompting large product recalls. To prevent and identify causes of histamine toxicity, manufacturers must identify the bacteria causing the illness. A simple method of identification is needed, since sequence-based identification is complicated to perform and the analysis takes a long time. High-Resolution Melting Analysis (HRMA) is a method that detects differences in the base sequences of PCR products manifested as varied melting temperatures of double-stranded DNA. The present study was intended to develop a rapid identification method for major histamine-producing bacteria using HRMA. Species-specific HRMA primers were designed that specifically targeted the hdcA gene of 20 Gram-negative histamine-producing bacterial strains. The designed primers were used for HRM analysis of the 20 histamine-producing bacterial strains. The strains were divided into three groups (A, B, and C) based on differences in melting temperature values obtained by Tsub(m) Calling analysis program. Group A comprised terrestrial bacteria, such as Morganella, Enterobacter, and Raoultella, while Groups B and C comprised marine bacteria, such as those belonging to the genera Vibrio and Photobacterium. The melting profiles obtained in Group A by HRMA were used to identify the aforementioned terrestrial bacteria. The findings indicated that HRMA can easily identify the major gram-negative histamine-producing bacteria. A flow chart was created to identify histamine-producing bacterial species. This method enables the identification of histamine-producing bacterial species more quickly and easily than conventional sequence-based methods. Therefore, the method could be valuable for food companies to screen raw materials and products and track the source of contamination, which will in turn contribute to the prevention of histamine-food poisoning and investigation of its causes.