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Although tunas represent a significant part of the global fish economy and a major nutritional resource worldwide, their microbiome still remains poorly documented. Here, we conducted an analysis of the taxonomic composition of the bacterial communities inhabiting the gut, skin, and liver of two most consumed tropical tuna species (skipjack and yellowfin), from individuals caught in the Atlantic and Indian oceans. We hypothesized that each organ harbors a specific microbial assemblage whose composition might vary according to different biotic (sex, species) and/or abiotic (environmental) factors. Our results revealed that the composition of the tuna microbiome was totally independent of fish sex, regardless of the species and ocean considered. Instead, the main determinants of observed diversity were (i) tuna species for the gut and (ii) sampling site for the skin mucus layer and (iii) a combination of both parameters for the liver. Interestingly, 4.5% of all amplicon sequence variants (ASV) were shared by the three organs, highlighting the presence of a core-microbiota whose most abundant representatives belonged to the genera Mycoplasma, Cutibacterium, and Photobacterium. Our study also revealed the presence of a unique and diversified bacterial assemblage within the tuna liver, comprising a substantial proportion of potential histamine-producing bacteria, well known for their pathogenicity and their contribution to fish poisoning cases. These results indicate that this organ is an unexplored microbial niche whose role in the health of both the host and consumers remains to be elucidated., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Like other seafood products, tuna is highly perishable and sensitive to microbial spoilage. Its consumption, whether fresh or canned, can lead to severe food poisoning due to the activity of specific microorganisms, including histamine-producing bacteria. Yet, many grey areas persist regarding their ecology, conditions of emergence, and proliferation in fish. In this study, we used 16S rRNA barcoding to investigate postmortem changes in the bacteriome of fresh and brine-frozen yellowfin tuna (Thunnus albacares), until late stages of decomposition (i.e. 120 h). The results revealed that despite standard refrigeration storage conditions (i.e. 4°C), a diverse and complex spoilage bacteriome developed in the gut and liver. The relative abundance of spoilage bacterial taxa increased rapidly in both organs, representing 82% of the bacterial communities in fresh yellowfin tuna, and less than 30% in brine-frozen tuna. Photobacterium was identified as one of the dominant bacterial genera, and its temporal dynamics were positively correlated with histamine concentration in both gut and liver samples, which ultimately exceeded the recommended sanitary threshold of 50 ppm in edible parts of tuna. The results from this study show that the sanitary risks associated with the consumption of this widely eaten fish are strongly influenced by postcapture storage conditions., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

A mild-flavored soup stock made from katsuobushi is an important element of traditional Japanese cuisine and is the basic seasoning responsible for the taste. Fermented and ripened katsuobushi, known as karebushi, is manufactured by simmering skipjack tuna that is then smoke-dried, fermented, and ripened in a repeated molding process by five dominant Aspergillus species. Here, our aim was to characterize and identify the lipolytic enzymes secreted by the dominant Aspergillus species, especially A. chevalieri and A. pseudoglaucus, which are involved in hydrolyzing lipids during the molding process. The crude enzyme preparations from the five Aspergillus spp. cultivated on katsuobushi solid medium hydrolyzed triglycerides in fish oil, and more saturated and unsaturated fatty acids (C16:0, C16:1, C18:0, C18:1) were produced than major polyunsaturated fatty acids (C20:5, C22:6). On the basis of ion exchange chromatograms, the composition of the lipolytic enzymes was different in the five species. There was at least one active fraction with high hydrolytic activity toward fish oil in four of the Aspergillus spp., but not A. sydowii; the lipolytic enzyme secreted by A. sydowii had quite high activity toward the artificial substrate p-nitrophenyl butyrate, but low activity toward the natural oil. The lipolytic fractions from A. chevalieri and A. pseudoglaucus were further purified by hydrophobic interaction chromatography then gel-filtration chromatography; LC-MS-MS Mascot analysis identified a variety of lipolytic enzymes, including cutinase, esterase, phospholipase, and carboxyl esterase in the lipolytic fractions from these species. The identified enzymes had 30%-70% identity to previously reported or manually annotated lipases or esterases from taxa other than Aspergillus. The different lipolytic enzymes likely acted on triglycerides in the katsuobushi fish oil. Furthermore, catalase B and Cu/Zn superoxide dismutase, which limit oxidative damage of lipids, were also identified. These antioxidant enzymes may prevent lipid oxidation and rancidity as the lipolytic enzymes hydrolyze lipids during the long fermentation and ripening process. Umami and richness tastes tended to increase in extracts from culture of protease- and peptidase-producing A. sydowii. Our results will aid in the selection and application of desirable strains of Aspergillus species as starter cultures to improve the storage and quality of fermented and ripened karebushi., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fish and other marine animals have a unique and intimate interaction with their surrounding environment. Diet type or trophic level play significant roles in modulating species digestive physiology. However, little is known about how the trophic niche influences digestive activity and gut microbiota in scombrids species. The aim of the present study was to analyse and describe the digestive physiology of the skipjack tuna (Katsuwonus pelamis) and the Atlantic bonito (Sarda sarda) as bioindicator of the feeding ecology and trophic niche for both species in the Gulf of Cádiz (Spain). For that, fish proximate composition, pH of digestive organs and digestive enzymes activity levels were analysed in 10 individuals of each species to gain insight into the digestive physiological adaptations of the two species of scombrids. In addition, intestinal microbiota composition was determined for the skipjack tuna. The integration of the results suggested a clear trophic niche segregation between both species. Stomach pH was associated in both species with infrequent feeding events. Body proximate composition and digestive lipases activity patterns pointed to a high predominance of lipids in the Atlantic bonito diet, suggesting oily fish as main prey. On the other hand, results supported the skipjack tuna as a highly opportunistic feeder with a more varied diet, including fish but also crustaceans as preys. The gut microbial community in the latter species is dominated by Firmicutes and Tenericutes at phylum level, and by Mycoplasma, Blautia and Dorea at genus level. The present study contributes to advance the knowledge on the feeding behaviour and physiology diversity in fish species as a result of adaptation to a particular habitat., Competing Interests: The authors have declared that no competing interests exist.

A. johnsonii and P. fluorescens are the well-known specific spoilage organisms in aquatic products and the study of the interactions between A. johnsonii and P. fluorescens are limited. This study aims to evaluate the growth kinetics, spoilage potential and interactions of A. johnsonii and P. fluorescens isolated from spoiled bigeye tuna (Thunnus obesus) by inoculating into sterile fish slices and stored at 4 °C for 6 days. The growth kinetics of A. johnsonii and P. fluorescens were fitted with Baranyi and Roberts model. The chemical indexes (total volatile base nitrogen (TVB-N), trimethylamine (TMA), pH, proteolytic activity and protein content) of each inoculated block of bigeye tuna were increased during refrigerated storage. Moreover, the higher contents of chemical indexes were observed in co-culture with A. johnsonii and P. fluorescens compared with single culture of A. johnsonii and P. fluorescens. In addition, atomic force microscopy (AFM) observation of co-culturing A. johnsonii and P. fluorescens inoculation into sterile fish slices revealed damage of myofibrillar protein structures and the protein degradation. Based on these parameters, a rapid method to evaluate spoilage potential of A. johnsonii and P. fluorescens was positively correlated with TVB-N value, TMA value and pH value (P < 0.05) by the correlation coefficient. Consequently, spoilage potential of microorganisms became stronger evaluated in a mixed culture than single culture. This paper provides insight for a detection method of interactions of A. johnsonii and P. fluorescens during refrigerated storage.

Histamine food poisoning is a major safety concern related to seafood consumption worldwide. Morganella psychrotolerans is a novel psychrotolerant histamine-producer. In this study, the histamine production behaviors of M. psychrotolerans and two other major histamine-producers, mesophilic Morganella morganii and psychrotrophic Photobacterium phosphoreum, were compared in seafood products, and histamine accumulation by M. psychrotolerans was characterized at various pH and temperature levels in culture broth. The growth of M. psychrotolerans and P. phosphoreum increased similarly at 4 °C in canned tuna, but M. psychrotolerans produced much higher levels of histamine than P. phosphoreum. Histamine accumulation by M. psychrotolerans was induced at lower environmental pH condition at 4 and 20 °C. The optimal temperature and pH for producing histamine by crude histidine decarboxylase of M. psychrotolerans were 30 °C and pH 7, respectively. The activity of the crude HDC extracted from M. psychrotolerans cells at 10 °C retained 45% of the activity at 30 °C. Histidine decarboxylase gene expression of M. psychrotolerans was induced by low pH conditions. These results suggest that M. psychrotolerans are also a very important histamine-producer leading to histamine poisoning associated with seafood below the refrigeration temperature.

Microbial (colony counts, 16S rRNA gene amplification), chemical (pH, 1 H NMR spectroscopy) and sensory changes in raw Atlantic Salmon (Salmo salar) and tuna (Thunnus albacares) fillets stored under vacuum at 3 °C were evaluated over a period of 12 days. Both species of fish are globally important and among the ten most consumed fishes in the world. Although the sensory analyses showed a decrease in the quality of both fish species, only the salmon fillets were considered spoiled at the end of the storage period. In salmon, trimethylamine was the main spoilage product and bacterial colony counts reached an average of 7.3 log10 cfu/g. The concentration of glucose decreased and the concentration of organic acids increased during storage revealing glucose fermentation. Photobacterium was the dominating genus in the salmon studied. In the tuna studied, the bacterial colony counts reached only an average of 4.6 log10 cfu/g. The dominating bacteria in tuna were Pseudomonas spp. Glucose levels did not decrease, suggesting that amino acids and lactate most likely acted as carbon sources for bacteria in tuna. In conclusion, the study revealed that salmon was clearly a more perishable fish than tuna., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Properties of skipjack tuna loins subjected to high-pressure processing (HPP) at 150 to 600 MPa for 1 to 5 min were compared with those of loin that underwent steam cooking for 10 min. Protein denaturation in HPP-treated loins increased with increasing pressure level, but these loins retained between 1.1% and 2.4% more water than steam-cooked loin. Water holding capacity decreased from 57% to 44% when the loins were treated at 600 MPa. ΔE value of HPP loins was between 5.8 and 26.3 when treated at 150 to 600 MPa, whereas it was 34.1 for steam-cooked sample. Hardness of HPP loins increased from 648 to 1,019, 1,918, 5,249, and 4,092 g and springiness changed from 85.2% to 79.7%, 78.2%, 91.7%, and 90.7%, respectively, when treated at 150, 300, 450, and 600 MPa. Protein fibers of HPP loins had a more irregular shape than those of steam-treated loin. Histamine levels of HPP-treated loins were in the range of 3.08 to 3.35 μg/g, identical to that of steam-treated loin. Thiobarbituric acid assay demonstrated that the level of lipid oxidation increment in HPP-treated loins was twice as high as that in steam-cooked one. Undesirable volatile compound contents in tuna loins decreased with increasing degree of protein denaturation. Steam cooking and HPP at 150, 300, 450, and 600 MPa decreased the total aerobic counts by 4.75, 0.12, 1.20, 4.69, and 6.08 log CFU/g, respectively. These results suggest that HPP at 450 MPa and above has the potential to be used as an alternative to the tuna precooking process., Practical Application: Information presented here can serve as a guideline for the selection of appropriate conditions for HPP of tuna loins. Our results show that HPP has a potential to replace the highly energy-intensive steam precooking step, which is traditionally required in a canned tuna production process., (© 2018 Institute of Food Technologists®.)

Foodborne non-typhoidal salmonellosis causes approximately 1 million illnesses annually in the USA. In April 2015, we investigated a multistate outbreak of 65 Salmonella Paratyphi B variant L(+) tartrate(+) infections associated with frozen raw tuna imported from Indonesia, which was consumed raw in sushi. Forty-six (92%) of 50 case-patients interviewed ate sushi during the week before illness onset, and 44 (98%) of 45 who specified ate sushi containing raw tuna. Two outbreak strains were isolated from the samples of frozen raw tuna. Traceback identified a single importer as a common source of tuna consumed by case-patients; this importer issued three voluntary recalls of tuna sourced from one Indonesian processor. Four Salmonella Weltevreden infections were also linked to this outbreak. Whole-genome sequencing was useful in establishing a link between Salmonella isolated from ill people and tuna. This outbreak highlights the continuing foodborne illness risk associated with raw seafood consumption, the importance of processing seafood in a manner that minimises contamination with pathogenic microorganisms and the continuing need to ensure imported foods are safe to eat. People at higher risk for foodborne illness should not consume undercooked animal products, such as raw seafood.

Aim: The spoilage potential of 28 bacterial strains isolated from spoiled raw yellowfin tuna was evaluated., Methods and Results: Bacterial species were inoculated in irradiated tuna matrix. Chemical changes, bacterial growth and sensory quality were monitored during aerobic storage at 8°C. Pseudomonas spp., Enterobacter spp. and Escherichia hermanii had no spoiling effect. Brochothrix thermosphacta and Carnobacterium divergens/maltaromaticum developed moderate unpleasant odours. Hafnia paralvei and Serratia spp. released strong off-odours (pyrrolidine, sulphur/cabbage). No bacterial group (except H. paralvei) combined with Pseudomonas spp. deteriorated the sensory quality of tuna. When C. divergens/maltaromaticum was associated with H. paralvei or B. thermosphacta, the odour is close to the naturally contaminated tuna stored on the same conditions. The pH, total volatile basic nitrogen (TVBN) and trimethylamine (TMA) were not correlated with the spoilage., Conclusions: The bacterial species had a different impact on the sensory quality of the fish. The bacterial interactions lead to an enhancement or an inhibition of the spoilage potential and the bacterial growth., Significance and Impact of Study: The specific spoilage organism (SSO) appears to be an association of lactic acid bacteria (LAB) with Enterobacteriaceae or B. thermosphacta. Pseudomonas, often dominant at the sensory rejection time, is not a good quality indicator., (© 2017 The Society for Applied Microbiology.)

Metagenomic, microbial, chemical and sensory analyses of Thunnus albacares from Martinique stored in ice (AIR - 0 °C), vacuum (VP - 4/8 °C) and modified atmosphere packaging (MAP - 4/8 °C) (70% CO2 - 30% O2) were carried out. The organoleptic rejection of AIR tuna was observed at day 13 when total bacterial counts equaled 10(6)-10(7) CFU g(-1). No extension of shelf-life was provided by VP and MAP. According to 16S rRNA gene sequence analyzed by Illumina MiSeq and PCR-TTGE, Rhodanobacter terrae was the main species of the freshly caught tuna. At the sensory rejection time, Brochothrix thermosphacta and Pseudomonas dominated the AIR products while B. thermosphacta alone or a mix of B. thermosphacta, Enterobacteriaceae and lactic acid bacteria (LAB) dominated the microbiota of MAP and VP products, respectively. The pH value remained stable in all trials, ranging from 5.77 to 5.97. Total volatile basic nitrogen (TVBN) and trimethylamine (TMA-N) concentrations were weak and not significantly different between batches. Lipid oxidation increased in the samples containing O2 (MAP > AIR). The initial concentration of histamine was high (75-78 mg kg(-1)) and stable up to 8 days but then significantly decreased in all trials to reach 25-30 mg kg(-1), probably due to the presence of histamine-decomposing bacteria., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Scombrotoxin fish poisoning (SFP) remains the main contributor of fish poisoning incidents in the United States, despite efforts to control its spread. Psychrotrophic histamine-producing bacteria (HPB) indigenous to scombrotoxin-forming fish may contribute to the incidence of SFP. We examined the gills, skin, and anal vents of yellowfin (n = 3), skipjack (n = 1), and albacore (n = 6) tuna for the presence of indigenous HPB. Thirteen HPB strains were isolated from the anal vent samples from albacore (n = 3) and yellowfin (n = 2) tuna. Four of these isolates were identified as Photobacterium kishitanii and nine isolates as Photobacterium angustum; these isolates produced 560 to 603 and 1,582 to 2,338 ppm histamine in marine broth containing 1% histidine (25°C for 48 h), respectively. The optimum growth temperatures and salt concentrations were 26 to 27°C and 1% salt for P. kishitanii and 30 to 32°C and 2% salt for P. angustum in Luria 70% seawater (LSW-70). The optimum activity of the HDC enzyme was at 15 to 30°C for both species. At 5°C, P. kishitanii and P. angustum had growth rates of 0.1 and 0.2 h(-1), respectively, and the activities of histidine decarboxylase (HDC) enzymes were 71% and 63%, respectively. These results show that indigenous HPB in tuna are capable of growing at elevated and refrigeration temperatures. These findings demonstrate the need to examine the relationships between the rate of histamine production at refrigeration temperatures, seafood shelf life, and regulatory limits., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Total mercury (Hg), cadmium (Cd) and lead (Pb) concentrations were determined in canned fish on the Ghanaian market. Total mercury was determined using an automatic mercury analyzer while cadmium and lead levels were determined by flame atomic absorption spectrophotometry. The metal contents in the samples, expressed in μg g(-1) (wet weight), varied from <0.01 to 0.20 with an average value of 0.03 for mercury, from <0.01 to 0.45 with an average value of 0.34 for cadmium, and from <0.01 to 1.44 with an average value of 0.72 for lead. The results indicate that canned fish from the Ghanaian market have concentrations well below the permissible FAO/WHO for these toxic metals. Thus considering the Provisional Tolerable Weekly Intake (PTWI) of Hg, Pb and Cd the levels obtained in this study are unlikely to constitute a significant exposure to the public through consumption of moderate amounts., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Scombrotoxin fish poisoning (SFP), also known as histamine (Hst) poisoning, has been associated with consumption of scombroid-type fish, including tuna and tuna fish products. Preparation of commercial tuna salad contaminated with Hstproducing bacteria (HPB), combined with time-temperature abuse, can present a food safety hazard. A potential source of HPB is raw ingredients, such as celery and onions. The objectives of this study were to determine whether raw ingredients can be a source of HPB and to ascertain the effects of storage time (up to 4 days or 4 weeks) and temperature (4, 10, 18, 25, 30°C) on growth and Hst production by high-HPB (>1,000 ppm of Hst) in tuna salad preparations. Pantoea-Erwinia, Erwinia persicinus, Erwinia spp., and Enterobacter pyrinus isolated from celery in this study were used to inoculate tuna salad and tuna salad with celery or onion. HPB numbers were 0.7 to 4.3 log most probable number per g higher in the presence of celery or onion versus plain tuna salad (3:1 tuna:mayonnaise). E. pyrinus-inoculated plain tuna salad and tuna salad with celery and onion had >500 ppm of Hst after 2 days at 30°C and 4 days at 25°C. E. pyrinus-inoculated salad with celery and onion had >500 ppm of Hst after 4 days at 18°C and 2 weeks at 10°C. Raw celery can introduce HPB into tuna salad, which can cause SFP if the product is time-temperature abused. Tuna salad products must be refrigerated at ≤4°C to prevent growth and Hst production by the HPB used in this study, to protect consumers from potential SFP.

This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (-20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin-producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum-packed samples were stored in a freezer (-20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (<0.7 log CFU/g) after 4 wk of storage at -20 ± 2 °C, but increased rapidly once the samples were thawed and held at 35 to 37 °C. Total S. aureus counts in albacore and skipjack samples increased by greater than 3 log CFU/g after 6 and 8 h of exposure to 35 to 37 °C, respectively. All samples became spoiled after 10 h of exposure to 35 to 37 °C, while no enterotoxin was detected in any samples. However, enterotoxins were detected in albacore loin and other samples after 12 and 24 h of incubation at 35 to 37 °C, respectively. Frozen precooked tuna meat should be used for producing canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat., (© 2014 Institute of Food Technologists®)

Aims: The Australian tuna industry is based on the ranching of wild southern bluefin tuna (SBT, Thunnus maccoyii). Within this industry, only opportunistic pathogens have been reported infecting external wounds of fish. This study aimed to identify different culturable bacteria present in three cohorts of SBT and to determine normal bacteria and potential pathogens in isolates from harvest fish and moribund/dead fish. Post-mortem changes in the microbiota were also studied., Methods and Results: Moribund/dead showed a greater proportion of members from the family Vibrionaceae than harvested fish; the latter presented mainly non-Vibrio species. In harvested fish spleens, Vibrio splendidus I complex was the most commonly identified group among Vibrio isolates, while most groups from the family Vibrionaceae were isolated from gills. For moribund/dead, Vibrio chagasii and Photobacterium damselae subsp. damselae were common in gill, spleen and kidney samples. Non-Vibrio isolates from gills were characterized using 16S rRNA sequencing as Flavobacteriaceae and classes Gammaproteobacteria and Alphaproteobacteria, mainly from the genera Winogradskyella and Tenacibaculum. Post-mortem changes showed dynamic shifts in bacterial dominance in gills, with Vibrionaceae and non-Vibrio spp. found in similar proportions initially and types related to Pseudoalteromonas ruthenica prevailing after 27 h. Spleen samples showed little bacterial growth until 5 h post-mortem, while various Vibrio-associated species were isolated 27 h post-mortem., Conclusions: Bacterial isolates found include a range of potentially pathogenic bacteria that should be monitored though most of them have yet to be associated with disease in tuna., Significance and Impact of the Study: This study forms a foundation for future research into the bacterial population dynamics under different culture conditions of SBT. An understanding of the bacterial compositions in SBT is necessary to evaluate the effects of some bacterial species on their health., (© 2013 The Society for Applied Microbiology.)

Consumption of foods high in biogenic amines leads to an illness known as histamine, or scombrotoxin, poisoning. The illness is commonly associated with consumption of fish with high levels of histamine ( $ 500 ppm). The objective of this study was to determine and compare the heat resistance of five histamine-producing bacteria in irradiated albacore tuna loins. Heat-resistance parameters (D- and z-values) were determined for Morganella morganii, Raoultella planticola, Hafnia alvei, and Enterobacter aerogenes. D- or z-values were not determined for Photobacterium damselae, which was the most heat-sensitive organism in this study. P. damselae declined > 5.9 log CFU/g after a heat treatment of 50°C for 10 min, 54°C for 3 min, and 56°C for 0.5 min. M. morganii was the most heat-resistant histamine-producing bacteria in albacore tuna loins, followed by E. aerogenes, H. alvei, and R. planticola. M. morganii and E. aerogenes had the highest D(50°C), 49.7 ± 17.57 and 51.8 ± 17.38 min, respectively. In addition, M. morganii had the highest D-values for all other temperatures (54, 56, and 58°C) tested. D- and zvalues were also determined for M. morganii in skipjack tuna. While no significant (P > 0.05) difference was observed between D(54°C) and D(56°C) of M. morganii in either albacore or skipjack tuna, the D(58°C) (0.4 ± 0.17 min) was significantly lower (P < 0.05) in skipjack than in albacore (0.9 ± 0.24 min). The z-values for all organisms tested were in the range of 3.2 to 3.8°C. This study suggests that heat treatment designed to control M. morganii in tuna loins is sufficient for controlling histamine-producing bacteria in canned-tuna processing environments.

It has been suggested that anaerobic histamine-producing bacteria (HPB) are important contributors to scombrotoxin fish poisoning (SFP). In order to assess the role of Clostridium perfringens in SFP, we developed a real-time PCR method for rapid detection of histamine-producing (HP) C. perfringens. The real-time PCR assay was 100% inclusive for detecting 23 HP C. perfringens and did not detect any of the other 116 HP or non-HP isolates examined. The efficiency of the assay with or without internal amplification control DNA was 102%; in the presence of background flora and inhibitory matrices, it was 90 to 99%. To investigate the importance of HP C. perfringens in SFP, we examined histamine production by C. perfringens in inoculated fish samples incubated under anaerobic conditions. C. perfringens produced low histamine levels in tuna (19 ppm) and Spanish mackerel (3 ppm), whereas gram-negative HPB produced high histamine levels (6,345 ppm in tuna; 1,223 ppm in Spanish mackerel) under the same conditions. When one bonito, two bigeye tuna, nine mahi-mahi, and five yellowfin tuna were examined for the presence of HPB, none (0 of 17) of the samples contained HP C. perfringens or other gram-positive HPB, whereas 86% of the samples contained gram-negative HPB. Our study indicates that histamine production by C. perfringens in scombrotoxin-forming fish was minimal compared with that by gram-negative HPB and that C. perfringens may not be an important bacterial species associated with SFP.

Temperature is known to influence inflammatory signalling in mammals, but far less understood in fish. The aim of the present study was to explore the potential effects of temperature on innate immune signalling in head kidney and leukocyte populations of the economically important southern bluefin tuna through the identification and utilization of gene expression targets in vitro. Here, we identified the mRNA sequences of five potential inflammatory mediators - TNFα (1 and 2), IL-1β, IL-8, and Cox2 - and demonstrate induction of four - TNFα (2), IL-1β, IL-8, and Cox2 - following LPS stimulation of both peripheral blood leukocytes and head kidney homogenates in vitro by real-time quantitative PCR. Comparison of transcriptional expression in cultures held at 18 and 25 °C (both within the presumed natural temperature range of this heterothermic species) showed accelerated transcription of cytokines TNFα, IL-1β and IL-8 following LPS stimulation at 25 °C in both tissue types. Peak induction reached comparable levels for each transcript at both temperatures during the 24 h test period with only limited (if any) protraction in expression resulting from cold temperature (18 °C) incubation. Partial mRNA sequences were also identified for both the constitutively expressed and heat inducible chaperone proteins Hsc70 and Hsp70, and 24 h incubation at 25 °C was sufficient to induce Hsp70 transcription in leukocyte but not in head kidney cell populations. Taken together these findings suggest that temperature exerts influence in the timing but not the degree of an innate inflammatory response in bluefin tuna and that different cell populations have differential responsiveness to heat shock in this heterothermic species. Further, LPS stimulation failed to induce Hsp70 at either incubation temperature in leukocytes; whereas 25 °C incubation caused Hsp70 up-regulation in leukocytes with or without the presence of LPS. This suggests that Hsp70 does not play a direct role in immune responsiveness for this species and that an environmental temperature of 25 °C in excess of 24 h initiates a cellular stress response in blood cells of this organism. Lastly, a strong correlation between Hsp70 and IL-8 transcriptional expression was observed following LPS/heat shock stimulation of leukocytes and five potential heat shock response elements were subsequently identified on the gene promoter region of IL-8 indicating that heat shock co-activation of this chemokine previously identified in mammals is also likely present in fish., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aims: To develop a SYBR Green quantitative PCR assay (qPCR) for the specific detection of Morganella morganii, a fish pathogen responsible for the Histamine Fish Poisoning., Methods and Results: A new primer set, amplifying a 179-bp fragment of the 16S rRNA gene, was selected for specificity, and 14 M. morganii strains and 32 non-Morganella strains were evaluated. The melting temperature of 84°C was consistently specific for the amplicon. Two standard curves were constructed: the minimum detection sensitivity was 0·563 pg of pure DNA, corresponding to DNA extracted from nine cells of M. morganii. The qPCR assay was evaluated in experiments with seeded fish samples, and the regression coefficient values were calculated., Conclusions: A highly specific and rapid assay was developed for the detection of M. morganii in tuna fish samples., Significance and Impact of the Study: This method represents the first study about the quantification of pathogenic M. morganii in fish products. This approach can be utilized to prevent the presence of this undesirable species in the food chain., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

Unlabelled: Total of 14 filleted yellowfin tuna fish (Thunnus albacares) sold in wholesale fish market and supermarkets in Milan, Italy, were purchased and tested to determine microbial count, histamine level, histamine-forming bacteria, and their ability to produce histamine in culture broth. Although histamine level was less than 10 ppm, many samples showed high total viable bacterial and enterobacterial 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 enteric bacteria 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-refrigerated conditions during fillet preparation. These positive strains were identified as Citrobacter koseri/Enterobacter spp. and Morganella morganii, by 16S/23S rRNA internal transcribed spacer amplification and 16S rRNA sequence analysis. The strains showed a variable 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., Practical Application: The use of methods for the early and rapid detection of bacteria producing biogenic amines is important for preventing accumulation of these toxic substances in food products. In this study, we used a molecular approach for the detection of histamine-forming bacteria in fish. PCR-based methods require expensive equipment and a high degree of training for the user, but are fast (< 24 h) and reliable. They now represent the best predictive methods to identify a potential risk factor in fish products during processing, storage, and marketing and can be used in the investigation of risk reduction strategies., (© 2012 Institute of Food Technologists®)

Several vomiting episodes were reported in December 2007 by the management of a beach club in Calvià. The subsequent case investigation confirmed tuna fish consumption a few hours before onset of emesis in all cases. Microbiological analyses detected high bacterial levels in ready-to-eat fish samples, indicating inappropriate cooking procedures. More important, elevated levels of Bacillus cereus were present both in raw and cooked fish. No other pathogens were detected, indicating B. cereus as the etiological agent. To our knowledge, this is the first case of emetic disease by B. cereus likely to be associated with fish consumption.

Background and Objectives: Prepared ready-to-eat salads and ready-to-eat delicatessen-style meats present a high risk for Listeria contamination. Because no foodborne illness risk management guidelines exist specifically for US hospitals, a survey of New York City (NYC) hospitals was conducted to characterize policies and practices after a listeriosis outbreak occurred in a NYC hospital., Methods: From August through October 2008, a listeriosis outbreak in a NYC hospital was investigated. From February through April 2009, NYC's 61 acute-care hospitals were asked to participate in a telephone survey regarding food safety practices and policies, specifically service of high-risk foods to patients at increased risk for listeriosis., Results: Five patients with medical conditions that put them at high risk for listeriosis had laboratory-confirmed Listeria monocytogenes infection. The Listeria outbreak strain was isolated from tuna salad prepared in the hospital. Fifty-four (89%) of 61 hospitals responded to the survey. Overall, 81% of respondents reported serving ready-to-eat deli meats to patients, and 100% reported serving prepared ready-to-eat salads. Pregnant women, patients receiving immunosuppressive drugs, and patients undergoing chemotherapy were served ready-to-eat deli meats at 77%, 59%, and 49% of hospitals, respectively, and were served prepared ready-to-eat salads at 94%, 89%, and 73% of hospitals, respectively. Only 4 (25%) of 16 respondents reported having a policy that ready-to-eat deli meats must be heated until steaming hot before serving., Conclusions: Despite the potential for severe outcomes of Listeria infection among hospitalized patients, the majority of NYC hospitals had no food preparation policies to minimize risk. Hospitals should implement policies to avoid serving high-risk foods to patients at risk for listeriosis.

The growth kinetics of Listeria monocytogenes and natural flora (NF) in minced tuna from 2 to 30 °C were examined, and a simultaneous growth model was developed. The inhibiting effect of the NF on the growth of L. monocytogenes was examined by inoculating different levels of NF isolated from the minced tuna. The kinetic data were fitted to the Baranyi model and estimated the growth parameters such as specific growth rate (μ(max)), maximum population density (N(max)), and lag time. The temperature and inoculated NF dependency on the μ(max) of L. monocytogenes and NF were described by modified Ratkowsky's square-root model. As the initial NF level increased, the slopes of the square-root models were decreased for both L. monocytogenes and NF. The N(max) of L. monocytogenes was described as a function of temperature and inoculated NF level. Simultaneous growth prediction of L. monocytogenes and NF under constant temperature conditions was examined by using the differential equations based on the Baranyi model with the effect of interspecies competition substituted into the developed μ(max) and N(max) models. The root mean square errors between the model prediction and the observation for L. monocytogenes and NF were 0.42 and 0.34, respectively. Predictive simulation under fluctuating temperature conditions also demonstrated a high accuracy of simultaneous prediction for both L. monocytogenes and NF, representing the root mean square errors of 0.19 and 0.34, respectively. These results illustrate that the developed model permits accurate estimation of the behavior of L. monocytogenes in minced tuna under real temperature history until consumption., (Copyright ©, International Association for Food Protection)

A mathematical model was developed to predict growth and histamine formation by Morganella psychrotolerans depending on temperature (0-20 degrees C), atmosphere (0-100% CO2), NaCl (0.0-6.0%) and pH (5.4-6.5). Data from experiments with both sterile tuna meat and Luria Bertani broth was used to develop the mathematical growth and histamine formation model. The expanded Logistic model with a growth dampening parameter (m) of 0.7 was used as primary growth model. A primary model for histamine formation during storage was obtained by combining the expanded Logistic growth model with a yield factor (YHis/CFU). 120 maximum specific growth rate (micromax)-values were generated for M. psychrotolerans and used to model the combined effect of the studied environmental parameters. A simple cardinal parameter type secondary model was used to model the effect of the four parameters on micro(max). The maximum population density (log Nmax) was correlated with log (YHis/CFU) and a simple constrained polynomial (quadratic) secondary model was developed for the effect of the environmental conditions on these model parameters. The developed model describes the effect of initial cell concentrations, storage conditions and product characteristics on histamine formation. This is a significant progress compared to previously available models for the effect of storage temperature only.

The objective of the study was to establish a validated kinetic model for growth of spoilage bacteria on vacuum packed tuna slices in the temperature range of 0 to 15 degrees C and to evaluate the applicability of the TTI (Time Temperature Integrators) based SMAS (Safety Monitoring and Assurance System) system to improve tuna product quality at the time of consumption in comparison to the conventional First In First Out (FIFO) approach. The overall measurements of total flora and lactic acid bacteria (LAB) on the tuna samples used in a laboratory simulated field test were in close agreement with the predictions of the developed kinetic model. The spoilage profile of the TTI bearing products, handled with SMAS, was improved. Three out of the thirty products that were handled randomly, according to the FIFO approach, were already spoiled at the time of consumption (logN(LAB)>6.5) compared to no spoiled products when handled with the SMAS approach.

The main objective of this study was to evaluate the effect of two commonly used essential oils after treatment with anodic electrolyzed NaCl solution in order to extend the shelf life of coated semifried tuna slices. Samples of tuna slices were treated with 100-fold electrolyzed water and coated with an edible solution containing 1% essential oil (0.5% eugenol [E] plus 0.5% linalool [L]). The coated slices were fried at 170 degrees C for 1 min and then stored at 5 and 25 degrees C. Total volatile basic nitrogen of slices treated with electrolyzed water and 1% (E+L) decreased from 15.5 for the nontreated control slices to 9.7 immediately after treatment and remained at a low level (<30) until the end of the storage period (day 20). Treatment with 1% (E+L) significantly suppressed lipid oxidation in coated semifried tuna. Sensory evaluation and microbiological assays showed that treatment with electrolyzed water and 1% (E+L) extended the shelf life of coated semifried slices to 15 and 2 days compared with 5 and 1 days for control slices during storage at 5 and 25 degrees C, respectively.

Temperature abuse of tuna (Thunnus alalunga) was carried out in order to assess the histamine buildup in fish-processing facilities where fish can be exposed to high temperatures for short periods of time. Histamine production was studied in tuna loins under different storage and abuse conditions. Tuna was stored at 0-2 degrees C, 3-4 degrees C, and 6-7 degrees C, and abused for 2 h daily at 20 degrees C and 30 degrees C for 7-12 days. Loins abused at 30 degrees C for 2 h daily contained potentially toxic histamine concentrations (67-382 mg kg(-1)) when stored at a low refrigeration temperature (0-2 degrees C), whereas when stored at 6-7 degrees C, the loins contained highly toxic histamine concentrations (544.5-4156.6 mg kg(-1)). Lower histamine concentrations (23-48 mg kg(-1) in loins stored at 0-2 degrees C and 124.7-2435.8 mg kg(-1) in loins stored at 6-7 degrees C) were observed in temperature-abused loins that were initially frozen. An increase over time was observed in most microbial counts tested. Bacteria isolated from the temperature-abused loins showed a varied ability of histamine production, with Morganella morganii, Klebsiella oxytoca, Staphylococcus hominis, and Enterococcus hirae being the most active histamine-producing bacteria.

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.

Mesophilic Morganella morganii (n=6) and psychrotolerant M. morganii-like isolates from various seafoods (n=13), as well as clinical M. morganii isolates (n=3), were characterized by using a polyphasic approach including multi-locus sequencing. Based on the phylogenetic analysis, the 22 strains were divided into two distinct groups comprising mesophilic and psychrotolerant isolates, respectively. This classification was supported by DNA-DNA hybridization studies, whereby a psychrotolerant isolate (strain U2/3(T)) showed 41.0 and 17.8 % relatedness to the type strains of the mesophilic species Morganella morganii subsp. morganii (strain LMG 7874(T)) and Morganella morganii subsp. sibonii (strain DSM 14850(T)), respectively. Analysis of the 16S rRNA gene sequences showed a similarity of 98.6 % between mesophilic and psychrotolerant isolates. However, fragments of seven protein-encoding housekeeping genes (atpD, dnaN, gyrB, hdc, infB, rpoB and tuf) all showed less than 90.9 % sequence similarity between the two groups. The psychrotolerant isolates grew at 0-2 degrees C and also differed from the mesophilic M. morganii isolates with respect to growth at 37 degrees C and in 8.5 % (w/v) NaCl and fermentation of d-galactose. The psychrotolerant strains appear to represent a novel species, for which the name Morganella psychrotolerans sp. nov. is proposed. The type strain is U2/3(T) (=LMG 23374(T)=DSM 17886(T)).

Two outbreaks and a single case of histamine fish poisoning associated with cold-smoked tuna (CST) were reported in Denmark during 2004. The bacteria most likely responsible for histamine formation in CST implicated in histamine fish poisoning was identified for the first time in this study. Product characteristics and profiles of biogenic amines in the implicated products were also recorded. In the single poisoning case, psychrotolerant Morganella morganii-like bacteria most likely was responsible for the histamine production in CST with 2.2% +/- 0.6% NaCl in the water phase (WPS). In outbreak 1, Photobacterium phosphoreum most likely formed the histamine in CST with 1.3% +/- 0.1% WPS. In outbreak 2, which involved 10 persons, the bacteria responsible for histamine formation could not be determined. The measured concentrations of WPS were very low compared with those of randomly collected commercial samples of CST and cold-smoked blue marlin (4.1 to 12.7% WPS). Challenge tests at 5 degrees C with psychrotolerant M. morganii and P. phosphoreum in CST with 4.4% WPS revealed growth and toxic histamine formation by the psychrotolerant M. morganii-like bacteria but not by P. phosphoreum. In a storage trial with naturally contaminated CST containing 6.9% WPS, lactic acid bacteria dominated the microbiota, and no significant histamine formation was observed during the shelf life of about 40 days at 5 degrees C and of about 16 days at 10 degrees C. To prevent toxic histamine formation, CST should be produced with >5% WPS and distributed with a declared 5 degrees C shelf life of 3 to 4 weeks or less.

Histamine poisoning is one of the most common chemically induced seafoodborne illnesses reported in the United States today. The causative agents are biogenic amines, commonly produced by gram-negative bacteria. The purpose of this study was to detect and identify histamine-producing bacteria associated with standard industry practices during the harvesting, receiving, and processing of mahimahi and yellowfin tuna in North Carolina. Twenty-nine composite samples were obtained from 18 mahimahi and 11 yellowfin tuna and analyzed for their histamine content. No sample analyzed exceeded 2 ppm histamine, the lower detection limit. Composite fish muscle and environmental samples were screened (n = 386) for the presence of histamine-producing bacteria. Twenty-six percent (145) of 549 isolates selected on the basis of their morphological characteristics tested positive on Niven's media. Sixty-three Niven-positive isolates were gram negative, and 58 were gram positive. Of the 43 isolates tested further, 5 were confirmed as histamine producers, and all 5 produced at low levels (< 250 ppm in 48 h at > 15 degrees C). Three gram-negative and two gram-positive isolates were identified as Enterobacter cloacae and Staphylococcus kloosii, respectively. This study revealed that gram-negative bacteria might not be solely responsible for histamine production in at-risk fish. The confirmation of histamine-producing bacteria demonstrates the potential risk for histamine production. However, no detectable levels were found in the composite fish muscle samples analyzed even though 60% of the yellowfin tuna harvested did not meet the U.S. Food and Drug Administration's regulatory hazard analysis critical control point guidelines for temperature reduction. Therefore, no seafood safety risks were found under the standard industry practices observed in this study.

Occurrence and importance of psychrotolerant histamine producing bacteria in chilled fresh tuna were demonstrated in the present study. The objective was to evaluate microbial formation of histamine and biogenic amines in chilled fresh tuna from the Indian Ocean and stored either vacuum-packed (VP) or modified atmosphere-packed (MAP). Firstly, biogenic amines and the dominating microbiota were determined in VP tuna involved in an outbreak of histamine fish poisoning in Denmark. Secondly, the microbiota of fresh MAP tuna was evaluated at the time of processing in Sri Lanka and chemical, microbial and sensory changes were evaluated during storage at 1-3 degrees C. To explain the results obtained with naturally contaminated tuna the effect of VP and MAP on biogenic amine formation by psychrotolerant bacteria was evaluated in challenge tests at 2 degrees C and 10 degrees C. The VP tuna that caused histamine fish poisoning had a histamine concentration of >7000 mg/kg and this high concentration was most likely produced by psychrotolerant Morganella morganii-like bacteria or by Photobacterium phosphoreum. Similar psychrotolerant M. morganii-like bacteria dominated the spoilage microbiota of fresh MAP tuna with 60% CO2/40% N2 and formed >5000 mg/kg of histamine after 24 days at 1.7 degrees C. These psychrotolerant bacteria were biochemically similar to M. morganii subsp. morganii and their 16S rDNA (1495 bp) showed >98% sequence similarity to the type strain of this species. Toxic concentrations of histamine were produced at 2.1 degrees C in inoculated VP tuna by both the psychrotolerant M. morganii-like bacteria (7400+/-1050 mg/kg) and P. phosphoreum (4250+/-2050 mg/kg). Interestingly, MAP with 40% CO2/60% O2, in challenge tests, had a strong inhibitory effect on growth and histamine formation by both the psychrotolerant M. morganii-like bacteria and P. phosphoreum. In agreement with this, no formation of histamine was found in naturally contaminated fresh MAP tuna with 40% CO2/60% O2 during 28 days of storage at 1.0 degrees C. To reduce current problems with histamine fish poisoning due to VP tuna it is suggested, for lean tuna loins, to replace vacuum packaging with MAP containing approximately 40% CO2 and approximately 60% O2.

Consumer illnesses by scombroid poisonings have been a continuing problem for many years. The intoxications follow the ingestion of fish such as tuna and mahimahi that have undergone bacterial decomposition, leading to the formation of biogenic amines. Research studies have concluded that histamine is one of the indicators of scombrotoxic fish and that other amines, such as cadaverine, could be involved in the illnesses. Guidance for the handling of fish on board fishing vessels to prevent the production of scombrotoxic fish has been limited by a lack of data addressing changes that occur in fish from the water to delivery at dockside. In this study, the changes in selected biogenic amines were determined in mahimahi and tuna, which were captured and held in seawater at 25 to 35 degrees C for incubation times up to 18 h. The fillets from the treated fish were sectioned by transverse cuts and analyzed for histamine, cadaverine, and putrescine. Results showed that at 26 degrees C, more than 12 h of incubation were required before a histamine concentration of 50 ppm was reached in mahimahi. At 35 degrees C, 50 ppm histamine formed within 9 h. Similar results were found for skipjack and yellowfin tuna. Histamine concentrations exceeded 500 ppm within an additional 3 h of incubation in mahimahi. At both temperatures, an increase in the concentration of cadaverine preceded an increase in histamine levels. Changes in putrescine concentrations in the fish were less pronounced. The study also demonstrated that histidine decarboxylase activity was retained in some frozen samples of fish and could result in further increases in histamine on thawing.

Dried and salted roe, obtained from the reproductive organs of female tuna (Thunnus tynnus L.), is a typical fish-based food in the Mediterranean area of Spain. In the present study, we monitored the formation of volatile amines (trimethyamine nitrogen [TMA-N] and total basic volatile nitrogen [TBVN]) and nonvolatile amines (biogenic amines) in dried and salted tuna roe after processing and storage for 8 weeks at 4, 20, and 30 degrees C. The salting and drying process significantly increased the TBVN, cadaverine, tyramine, phenylethylamine, and tryptamine contents, and bacteria with histamine decarboxylase activity were detected both in raw and in dried and salted tuna roes. During storage of tuna roe, TMA-N and TBVN levels increased significantly after the fourth week of storage at 30 degrees C, whereas biogenic amine contents remained more or less constant. However, samples stored at 30 degrees C showed histamine formation after the first week of storage, with a concentration of < 50 ppm. The volatile and nonvolatile amine concentrations in tuna roe were below the consumer safety limit, with the exception of the total biogenic amine level in roe stored at 30 degrees C, which exceeded the European Community's recommended limit (300 ppm). These results indicate that in properly stored tuna roe, histamine formation will not represent a serious health risk to consumers unless the tuna roe has previously been mishandled.

Microbiological assessment, sensory evaluation, and electronic nose (AromaScan) analysis were performed on yellowfin tuna stored at 0, 4, 10, and 22 degrees C for 0, 1, 3, 5, and 9 days. Fish color, texture, appearance, and odor were evaluated by a trained sensory panel, while aroma-odor properties were evaluated using an AromaScan. Bacterial enumeration was performed using plate count agar containing 1.5% NaCl. Tuna fillets stored at 22 degrees C for 3 days or longer had a bacterial load of over 10(7) CFU/g and were rated not acceptable for consumption (grade C) by the sensory panel. Tuna fillets stored at 4 degrees C for 9 days or 10 degrees C for over 5 days were rated as grade C products and also had a bacterial load of over 10(7) CFU/g. The change in fish quality as determined by AromaScan followed increases in microbiological counts in tuna fillets, indicating that bacterial load can serve as a useful and objective indicator of gross spoilage. Electronic nose devices can be used in conjunction with microbial counts and sensory panels to evaluate the degree of decomposition in tuna during storage.

Eight cases of allergy-like food poisoning resulting from the ingestion of yellowfin tuna, which had been kept in stock for 10 days prior to being cooked, are described. The main symptoms were headaches, facial flushing and palpitation. Samples of the ingested fish were analyzed for histamine content, and a high level of histamine was confirmed (310 mg/100 g of fish). Corticosteroids were given to 3 patients who exhibited dyspnea or persistent symptoms, while the remaining patients improved without medication. In situations where allergy-like clinical features are present after the ingestion of food, the possibility of allergy-like food poisoning should be recognized and included in a differential diagnosis.

Two hundred twenty-seven bacterial strains were isolated from fresh and frozen albacore stored either at -18 or -25 degrees C and investigated for their abilities to produce biogenic amines. As a preliminary screening, all 227 strains were tested in either Niven or Niven modified medium, which allowed the selection of 25 presumptive histamine-producing strains. High-pressure liquid chromatography revealed that only 10 of the 25 strains selected were able to produce low histamine concentrations (<25 ppm) in tryptic soy broth medium supplemented with 2% histidine. None of the 25 strains tested produced putrescine or spermine, whereas 6 strains produced spermidine. Histamine production by Stenotrophomonas maltophilia strain 25MC6 was not prevented at 4 degrees C, and the levels of this amine reached concentrations of 25.8 ppm after 6 days. Three S. maltophilia strains showed strong lysine-decarboxylating activity. Their cadaverine formation capacity was determined by high-pressure liquid chromatography in tryptic soy broth supplemented with 1% lysine; this revealed that the three S. maltophilia strains tested produced more than 700 ppm of cadaverine during the first 24 h of incubation at 37 degrees C. S. maltophilia strain 15MF, initially obtained from fresh albacore tuna, produced up to 2,399 ppm and 4,820 ppm of cadaverine after 24 and 48 h of incubation at 37 degrees C, respectively. To our knowledge, this is the first report on histamine and cadaverine production by strains of the species S. maltophilia, previously known as Pseudomonas and Xanthomonas maltophilia, isolated from fresh and frozen albacore tuna.

Lean tuna meat suspensions (LEAN), with a fat content of 0.006%, and fatty tuna meat suspension (FATTY), with a fat content of 3.0% were inoculated with four strains of Vibrio parahaemolyticus and wasabi (Wasabia japonica Matsumura) or allyl isothiocyanate (AIT) was added before incubation at 37 degrees C. During the incubation, viable Vibrio counts were determined on TCBS agar plates. Both LEAN and FATTY suspensions were inoculated with V. parahaemolyticus AOTO-81, (1.28+/-0.20) x 10(2) CFU/ml, followed by addition of 20 mg wasabi/ml, and incubation for 8 h. The viable Vibrio counts were (7.76+/-5.93) x 10(5) CFU/ml in LEAN and (3.50+/-2.65) x 10(1) CFU/ml in FATTY. When the same strain, at (1.18+/-0.22) x 10(2) CFU/ml, was incubated for 8 h with 50.9 microg AIT/ml, viable Vibrio counts were (4.79+/-1.78) x 10(4) CFU/ml in LEAN and (1.80+/-1.30) x 10(1) CFU/ml in FATTY. Growth of the other three strains with wasabi or AIT was shown to be less in FATTY than in LEAN. These results indicate that growth of V. parahaemolyticus is inhibited more in FATTY than in LEAN by wasabi and allyl isothiocyanate.

We report a foodborne outbreak of Campylobacter jejuni infection in a summer camp. Outbreak-related cases occurred in 79 persons including 3 secondary cases in campers. Campylobacter jejuni was isolated from stool specimens from 16 of 21 patients who submitted a sample; 13 viable isolates were serotyped and all were serotype O:33 (somatic O scheme) or HL:18 (heat-labile scheme), and biotype III (Lior scheme). This serotype is widely distributed geographically but rarely isolated from humans. Samples of water from the wells supplying the camp were negative for faecal coliforms, and raw milk had not been served in the camp. A matched (1:1) case-control study identified tuna salad served for lunch on 19 July as the likely food item associated with illness (matched odds ratio=22; 95% confidence intervals (CI)=3.6-908). Swimming in the camp pool and other recreational water use in area lakes by the campers were not statistically associated with illness. The precise mechanism of introduction of the organism into the tuna salad remains unknown; contamination most likely occurred through cross-contamination with another food product, the hands of a food handler, or a work surface. Several deficiencies in the operation of the camp kitchen were identified. In Wisconsin, kitchens of such camps are subject to different inspection rules than restaurants. Camp staff, administrators, counselors, food managers, and infirmary staff, should fulfil important roles in their respective areas to prevent future outbreaks.

Between 1987 and 1996 fresh tuna and canned tuna contaminated after processing became increasingly associated with incidents of scombrotoxic fish poisoning. Fish not commonly associated with this syndrome were implicated in several incidents. Awareness of bacterial contamination after processing has highlighted the need for submitted samples to be examined both microbiologically and toxicologically and for the potential role of other toxins in this syndrome to be considered. The overall rate of reported incidents of scombrotoxic fish poisoning remained stable.

Albacore specimens of extra quality were analyzed for their biogenic amine contents after 1, 3, 6, and 9 months of frozen storage at -18 degrees C or -25 degrees C. A high-performance liquid chromatography method involving a linear elution gradient was optimized for the identification and determination of putrescine, cadaverine, histamine, spermidine, and spermine in albacore tuna. Putrescine was the biogenic amine that showed the highest increase, reaching concentrations of 59.04 ppm (815% of the initial level) and 68.26 ppm (942% of the initial level) in the white muscle of albacore after 9 months of frozen storage at -18 and -25 degrees C, respectively. Cadaverine, histamine, and spermidine concentrations were below 3, 5, and 11 ppm, respectively, after 9 months of frozen storage, while spermidine underwent a significant decrease at both storage temperatures. Microbiological analysis confirmed the absence of species of Enterobacteriaceae in 75% of the albacore specimens after 9 months of frozen storage; coliforms were always below 3 CFU/g. The survival rate of the psychrotrophic microorganisms after 9 months of frozen storage at -25 degrees C was 4.6%, while 38.9 and 92.1% of the aerobic mesophiles present in the white muscle of albacore before freezing survived 9 months of storage at -18 and -25 degrees C, respectively.

A histamine-producing strain of Klebsiella pneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges-Proskauer tests.

Introducing the authors report on cases of scombroid poisoning episodes in food serving establishments. These cases were related to the formation of histamine in tuna meat due to Proteus morganii although the meat was sound when taken from cans. It is demonstrated experimentally (Table 1) and shown in a critical literature review (Table 2) that among Proteus species Proteus morganii is the only species being able of producing histamine levels high enough to cause food poisoning in humans. Multiplication and histamine formation by Proteus morganii at different temperatures show (Fig. 1 and 2) that the concentration of histamine increases rapidly once the formation has started and the level of 1000 mg/kg considered as critical may be exceeded within a short period of time. These results confirm our observations obtained during epidemiological studies of food poisoning cases that improper handling and storage of tuna meat from cans at food serving establishments may bear the risk of causing scombroid poisoning. It should be emphasized that certain cultures of Proteus morganii are able to form histamine at levels of more than 1000 mg/kg even at a temperature of 7 degree C although prolonged incubation is necessary (Fig. 1). This fact should be taken into consideration in the evaluation of results when a prolonged period of time has elapsed between sampling or consumption of the suspect food and its examination.

Spoilage in skipjack tuna (Katsuwonus pelamis) was studied under controlled conditions by incubating whole, fresh fish in seawater at 38 degrees C, the optimum temperature for histamine formation. Bacterial isolates were obtained from the loin tissue of a decomposing tuna containing 134 mg of histamine per 100 g and a total anaerobic count of 3.5 x 10(5)/g after incubation for 24 h. Over 92% of the 134 isolates obtained were facultatively or obligately anaerobic bacteria. Eighteen isolates produced histamine in culture media containing histidine, and these were identified as Clostridium perfringens, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, and Vibrio alginolyticus. Histidine decarboxylase activity of several isolates was measured in a tuna broth medium and with resting cells suspended in a buffered histidine solution.