title: Development of molecular-based methods for determination of high histamine producing bacteria in fish.
abstract: Histamine (or scombroid) fish poisoning is a significant cause of food borne disease in the United States. In this study, we describe the development of a molecular-based technique which uses digoxigenin (DIG) labeled DNA probes for the detection of gram negative bacteria producing high amounts of histamine (>1000 ppm). A cocktail of PCR amplification fragments corresponding to a 709 bp fragment of the histidine decarboxylase (hdc) gene of four high producing bacteria (Morganella morganii, Enterobacter aerogenes, Raoultella planticola and Photobacterium damselae) was DIG-labeled and screened against a strain bank of 152 gram negative bacteria isolated from scrombroid fish and their harvest environment. The probe cocktail reacted specifically (100%) with the high histamine producing strains but failed to react with low histamine producers and non-producers. To further evaluate the feasibility of the approach, fish homogenate inoculated with known concentrations of four high histamine producing bacterial strains was plated on modified Niven's medium (culture method) and trypticase soy agar supplemented with 2% NaCl (for colony lift hybridization). The colony lift hybridization counts did not differ significantly from the level of the initial inoculum (p>0.05), while the modified Niven's counts were significantly lower (p<0.05) than either inoculum or colony lift counts. The use of digoxigenin (DIG) labeled DNA probes with colony lift hybridization shows promise for accurate and specific enumeration of histamine producing bacteria in scombroid fish.
authors.affiliation: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Center for Marine Sciences and Technology, 303 College Circle, Morehead City, North Carolina 28557, United States.
authors.forename: Kristin
authors.initials: K
authors.lastname: Björnsdóttir-Butler
authors.forename: Gregory E
authors.initials: GE
authors.lastname: Bolton
authors.forename: Lee-Ann
authors.initials: LA
authors.lastname: Jaykus
authors.forename: Patricia D
authors.initials: PD
authors.lastname: McClellan-Green
authors.forename: David P
authors.initials: DP
authors.lastname: Green
mesh_terms: D000362:Agar / Q000032:analysis; D000818:Animals; D015169:Colony Count, Microbial; D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D004076:Digoxigenin / Q000737:chemistry; D005506:Food Contamination; D005516:Food Microbiology; D005517:Foodborne Diseases / Q000382:microbiology*; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification*; D006632:Histamine / Q000032:analysis; D006640:Histidine Decarboxylase / Q000235:genetics; D016133:Polymerase Chain Reaction; D017747:Seafood / Q000382:microbiology*
chemical_list: D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial; D006632:Histamine; D000362:Agar; D006640:Histidine Decarboxylase; D004076:Digoxigenin