Quantification of total and specific gram-negative histamine-producing bacteria species in fish using an MPN real-time PCR method

• Published in: Food microbiology 2011-10, Vol.28 (7), p.1284-1292
• Main Authors: Bjornsdottir-Butler, Kristin, Jones, Jessica L., Benner, Ronald A., Burkhardt, William
• Format: Article
• Language: English
• Subjects: Animals; Bacteria; Bacteria - growth & development; Bacteria - metabolism; Bacterial Load - methods; Biological and medical sciences; Coryphaena hippurus; Enterobacter aerogenes; Fish; Fishes - microbiology; Food industries; Food microbiology; Fundamental and applied biological sciences. Psychology; Gram-negative bacteria; Histamine; Histamine - biosynthesis; Histidine decarboxylase; mackerel; Marine; Morganella morganii; MPN; Photobacterium damselae; polymerase chain reaction; Raoultella planticola; Real-time PCR; Real-Time Polymerase Chain Reaction - methods; risk reduction; Scomber; Scomberomorus; Scomberomorus maculates; Species Specificity; Thunnus albacares; tuna
• ISSN: 0740-0020; 1095-9998
• DOI: 10.1016/j.fm.2011.05.006

Quantification of histamine-producing bacteria (HPB) is necessary in order to elucidate the role that HPB play in scombrotoxin (histamine) fish poisoning. We report here the evaluation of a real-time PCR method for the quantification of total and specific Gram-negative HPB species in fish using a most probable number (MPN) format. The species-specific real-time PCR assay was 100% inclusive for independently detecting Morganella morganii, Enterobacter aerogenes, Raoultella planticola/ornithinolytica and Photobacterium damselae and did not cross react with other histamine- or non- histamine-producing bacteria. The efficiency of the reactions in the absence and presence of Spanish mackerel enrichment containing 1 × 106 CFU/ml of background microflora were 93–104 and 92–99%, respectively. The MPN-real-time PCR assay accurately quantified total and specific HPB in spiked mahi–mahi (Coryphaena hippurus) and Spanish mackerel (Scomberomorus maculates) samples. These methods were used to quantify total and specific HPB in naturally contaminated, decomposing mahi–mahi, Spanish mackerel and tuna (Thunnus albacares) samples. The results of this study indicate that MPN-real-time PCR assays can be used to accurately enumerate total and specific HPB in fish samples. These assays can be applied to assess the effectiveness of mitigation strategies and understand the relationship between HPB and histamine production in decomposing fish. ► Quantification of total and specific histamine-producing bacteria in fish by MPN RTi-PCR. ► Assay was 100% inclusive for detecting specific histamine-producing bacteria (HPB) species. ► The assays accurately quantified total and specific HPB in spiked and decomposed fish samples.