PCR Detection and Identification of Histamine-Forming Bacteria in Filleted Tuna Fish Samples

Total of 14 filleted yellowfin tuna fish (Thunnus albacares) sold in wholesale fish market and super markets in Milan, Italy, were purchased and tested to deter mine microbial count, histamine level, histamine-for ming bacter ia, and their ability to produce histamine in culture broth. Although histamine level was less than 10 ppm, many samples showed high total viable bacter ial and enterobacter ial counts that reached dangerous levels after temperature abuse for short periods of time. A PCR assay targeting a 709-bp fragment of the histidine decarboxylase gene (hdc ) revealed that 30.5% of the 141 enter ic bacter ia isolated from samples were positive and potentially able to produce histamine. The hdc positive strains were mainly isolated from fish bought at wholesale fish market, where we observed several possible risk factors, such as handling in poor and non-refr igerated conditions dur ing fillet preparation. These positive strains were identified as Citrobacter koser i/Enterobacter spp. and Morganella morganii, by 16S/23S rRNA inter nal transcr ibed spacer amplification and 16S rRNA sequence analysis. The strains showed a var iable ability of histamine production, with Morganella morganii being the most active histamine-producing species. A direct DNA extraction from fish and a PCR targeting the hdc gene showed a high degree of concordance with the results obtained through microbiological and chemical analyses, and could aid in the prompt detection of potentially contaminated fish products, before histamine accumulates.