Detection and control of histamine-producing bacteria in fish

Histamine (or scombroid) fish poisoning is the most frequently reported human illness associated with consumption of fish despite considerable control efforts. Lack of reliable detection methods and limited measures for control of histamine-producing bacteria (HPB) are partially responsible for the high incidence of this disease. To address these concerns, this dissertation focused on development of a rapid and reliable detection method for quantification of histamine-producing bacteria (HPB) in fish. Objectives were: (i) to compare available methods for use in detection of HPB, (ii) to develop DNA-based probe(s) to detect the histidine decarboxylase (hdc) gene in HPB, (iii) to apply the DNA probe(s) with colony-lift hybridization for quantification of HPB, and (iv) to evaluate the effect of pH for control of histamine formation using the colony lift hybridization method and DNA probe(s). In the first study, 152 histamine- and non-HPB were collected and examined for their histamine-forming capabilities. Strains were divided into high-, low- and non-histamine producing strains based on production of >1000 ppm, 125-500 ppm and <125 ppm of histamine in histidine broth. Histamine production was compared to results produced using Niven's medium, impedance by a potentiometric method and a PCR-based method. Of 152 strains examined, 73 were classified as high-histamine producers, 6 low histamine producers and 73 were non-detectable for histamine. Results demonstrated the potentiometric and PCR methods detected high-HPB but were not able to detect the low histamine-producers. The culture-based Niven's medium was able to detect high and low histamine-producers, but resulted in a large number (38%) of false positive responses.;Next DNA-based probe(s) were evaluated for detection of the histidine decarboxylase (hdc) gene from HPB. Two types of dioxigenin (DIG) labeled probes were examined using DNA dot-blot hybridizations: (i) six degenerate probes obtained by comparison of 17 hdc genes cloned and sequenced from HPB and (ii) 249 bp and 709 bp PCR DIG-labeled probes generated from four high histamine-producing bacteria. Detection of HPB using the degenerate probes resulted in weak responses from high-HPB. Detection of HPB using the 249 bp and 709 bp probes applied individually did not result in positive response for known histamine-producing strains. However, when 709 bp probes were examined as a mixture of equal proportions from the 4 high histamine-producing bacteria, positive responses were obtained for all high-histamine producing bacteria examined. The third study applied the hdc-probes to colony lift hybridization for rapid detection and quantification of HPB in fish. First, labeled DNA probes were evaluated for detection specificity when applied to 152 histamine- and non-histamine producing bacteria using dot-blot hybridization. The assay was converted to colony lift hybridization format and the efficiency was compared to Niven's agar medium and validated using artificially inoculated fish samples. The hdc-probe mix detected all 73 high-histamine producing bacteria. Six low and 73 non-histamine producing bacteria were not detected. The colony lift hybridization method with DNA probes was found to be accurate for quantification of HPB in inoculated fish samples. The final study evaluated pH for potential control of HPB and histamine formation by phosphate treatment of fish muscle. First, phosphate treatment effects on fish muscle pH were examined. Then histamine-producing bacteria inoculated mahi-mahi and tuna samples were vacuumed-packed with 7% trisodiumphospate (TSP) and stored under slightly abusive temperature (10°C) for 4 days. Samples were analyzed for surface pH, drip loss, growth of HPB and histamine-production. Phosphate treatment significantly increased fish muscle pH and reduced histamine formation in the inoculated fish samples. However, growth of HPB was not significantly reduced by phosphate treatments.;These studies have resulted in a new colony lift hybridization method for quantification of HPB in fish. The new method can be used to evaluate control measures for prevention of histamine fish poisoning and help in study of the microbial ecology in fish and fishery products leading to histamine fish poisoning.