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The commercialization of processed fish products is rising in restaurants and small to medium enterprises. However, there is a lack of data related to the microbiological safety of such products. In this study total aerobic colony count and Enterobacteriaceae, as proxy of process hygiene criteria, and detection of Listeria monocytogenes and concentration of histamine, as food safety criteria, were investigated in Salmo salar (salmon), Xiphias gladius (swordfish) and Thunnus albacares (yellowfin tuna), before, during, and at the end of a dry-curing process, performed in a dedicated cabinet, at controlled temperature, relative humidity and ventilation, up to 240 h. The microbiological parameters were investigated in the tested fish products by culture methods and shotgun metagenomic, while the presence of histamine, and other biogenic amines, was quantified by High Performance Liquid Chromatography. In the raw material, and up to the end of the dry curing process, the concentration of Enterobacteriaceae was always lower than 10 CFU/g, while total aerobic colony counts ranged between 3.9 and 5.4 Log CFU/g in salmon; 5.5 and 5.9 Log CFU/g in swordfish; 4.4 and 4.8 Log CFU/g in tuna. The pH values were significantly different between fish species, in the raw materials and during processing except for T4, occurring 70 h after the start of the process for salmon and after 114 h for swordfish and tuna. Water activity was different at specific sampling points and at the end of processing. Overall, 79 % of the sequences identified in the tested fish samples were assigned to y bacteria. The most abundant phyla were Pseudomonadota, Bacillota and Mycoplasmatota. The microbial populations identified by shotgun metagenomic in the tested fish species clustered well separated one from the other. Moreover, the microbial richness was significantly higher in salmon and tuna in comparison to swordfish. Listeria monocytogenes was not detected in the raw material by using the reference cultural method and very few reads (relative abundance <0.007) were detected in swordfish and tuna by shotgun metagenomic. Histamine producing bacteria, belonging to the genera Vibrio, Morganella, Photobacterium and Klebsiella, were identified primarily in swordfish. However, histamine and other biogenic amines were not detected in any sample. To the best of our knowledge this is the first paper reporting time point determinations of microbiological quality and safety parameters in salmon, swordfish and tuna, before, during and at the end of a dry-curing process. The data collected in this paper can help to predict the risk profile of ready to eat dry-cured fish products during storage before consumption., Competing Interests: Declaration of competing interest None of the authors declare to have financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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.)

Seafood is one of the essential sources of nutrients for the human diet. However, they can be subject to contamination and can cause foodborne illnesses, including scombroid fish poisoning caused by histamine. Many microorganisms can produce enzymes that eventually decompose endogenous histidine to histamine in postmortem fish muscles and tissues. One of these is histamine-forming bacteria (HFB), primarily found in the gills, gut, and skin of fishes. Previous studies linked a plethora of Gram-negative HFB including Morganella spp. and Photobacterium spp. to scombroid fish poisoning from many types of seafood, especially the Scombridae family. These bacteria possess the hdc gene to produce histidine decarboxylase enzyme. It was reported that Gram-negative HFB produced 6345 ppm in tuna and 1223 ppm in Spanish mackerel. Interestingly, Gram-positive HFB have been isolated in the seafood samples with lower histamine levels. It suggests that Gram-negative HFB are the major contributor to the accumulation of histamine in seafood. Several analytical methods are available to detect and identify HFB and their histamine metabolites from seafood substrates. Rapid test kits can be used in food production settings for early detection of histamine to avoid food intoxication. Furthermore, high hydrostatic pressure and irradiation treatment could prevent the proliferation of HFB and inactivate the existing histidine decarboxylase (HDC) activity. As demonstrated in different seafood model systems, the HDC activity was deactivated at a maximum high hydrostatic pressure level of 400 MPa. The complete inactivation of HFB was achieved by gamma irradiation at a dose of 4.0 kGy. Other postharvest treatments, like enzymatic degradation and electrolyzed oxidizing water, were studied as sustainable methods for bacterial growth prevention and enzyme inactivation. However, other HFB react differently to these treatment conditions, and further studies are recommended., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

Scombridae fish (tuna, bonito and mackerel) have significant ecological and economic values. They are very appreciated by consumers worldwide for their high-quality flesh and for their high nutritional value. However, consumption of Scombridae fish is potentially hazardous. Indeed, several cases of infections and toxicity linked to the consumption of Scombridae fish as raw, or processed food products have been reported worldwide. In this review, we presented the most common health risks associated with Scombridae fish consumption. Diseases associated with the consumption of these fish are generally infectious or toxic and are caused by biological hazards, such as bacteria, viruses, parasites or chemicals hazards that enter the body through contaminated fish (polycyclic aromatic hydrocarbons, histamine) or by physical contaminants, such as heavy metals. The risks of contamination exist throughout the food chain, from primary production to the preparation of products for consumption., (© 2022 Society for Applied Microbiology.)

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.

Bacteria release membrane vesicles into the extracellular environment but which activity is unclear. We investigated the applications of extracellular vesicles (EVs) isolated from probiotic Lactobacillus plantarum to protect tuna fish against spoilage and quality loss in this study. A significant difference was found in EVs size obtained from L. plantarum after 8, 24, and 48 hr incubation. The L. plantarum-derived EVs were collected and used to confirm the anti-bacterial activity versus Shewanella putrefaciens. Finally, the tuna fish was stored at 4 °C for 5 days after coating with EVs or sodium erythorbate, and the quality indexes were assayed. Results indicated that EVs markedly inhibited oxidation reaction, total volatile base nitrogen (TVBN), peroxide value (PV), malondialdehyde (MDA), and bacteria levels. These results finding out that EVs from L. plantarum may have potential for application in food storage technology. Overall, we indicated this new material may be developed as an anti-bacterial agent for prolonging the shelf life of tuna fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 (scombroid) poisoning is a foodborne illness caused by ingestion of histamine-contaminated seafood; therefore, inhibition of the growth of histamine-producing bacteria is key for it prevention. Infection of pathogenic bacteria by bacteriophages (phages) is being developed to prevent multiple foodborne illnesses. Here, we describe the inhibitory effect of a phage mixture on growth and histamine accumulation of Morganella morganii subsp. morganii, the primary causative agent of histamine poisoning in fish meat. We isolated novel two phages, ΦMV-1 and ΦMV-4, which infected M. morganii subsp. morganii strains tested in this study. ΦMV-1 and ΦMV-4 belong to family Myoviridae. Pulsed-field gel electrophoresis revealed that these phages are jumbo bacteriophages with large genomes. The latent period, rise period and burst size of ΦMV-1 were 30 min, 60 min, and 224 PFU per infected cell, respectively, and those of ΦMV-4 were 60 min, 50 min, and 62 PFU per infected cell, respectively. A mixture of ΦMV-1 and ΦMV-4 effectively prevented regrowth of M. morganii subsp. morganii after phage treatment, suggesting that the phage mixture treatment is more effective for inhibition of growth and histamine accumulation by M. morganii subsp. morganii than single phage treatment. Treatment with phage mixture inhibited growth and histamine accumulation by M. morganii subsp. morganii in canned and fresh tuna. The phage mixture might be an effective way to prevent growth of the histamine producer and accumulation of histamine in seafood., Competing Interests: Declaration of competing interest None declared., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

On 20 April 2017, an outbreak of histamine food poisoning occurred in a French military unit located near Paris. A total of 40 cases were identified (attack rate: 16.6%). We conducted a case-control study on 31 cases and 63 controls. Multivariate analysis pointed to cooked yellowfin tuna fillet as the very likely source of food poisoning (odds ratio = 156.8; 95% confidence interval: 18.4-1,338.4). The fresh yellowfin tuna was from Reunion Island and was supplied vacuum-sealed and packed with ice at the principal food market of Paris. No cold chain issues could be established in the upstream and downstream supply chains. Histamine concentration was found to be 1,720 mg/kg in leftover raw tuna, and 3,720 mg/kg in control cooked tuna, well above the threshold limit values defined by European regulations (200 mg/kg). The presence of Klebsiella variicola and Pantoea agglomerans, microorganisms of the Enterobacterales order that have been reported to produce histamine, was confirmed in the leftover raw tuna. This type of food poisoning is rarely recognised and confirmed. We describe the outbreak to highlight the specific key points of this type of investigation.

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.

Bioo Scientific Corp. has developed a rapid enzymatic quantitative assay for the determination of histamine in seafood. Fresh/frozen tuna, canned tuna, pouched tuna, and frozen mahi mahi samples were used for the validation study under the specific guidelines of the AOAC Research Institute Performance Tested MethodsSM program. Recoveries ranged from 82 to 107% at concentrations ranging from 6 to 72 ppm, with RSDr values between 0.8 and 6.5% (6-72 ppm). The linearity of the assay ranged from 0 to 108 ppm, with R2 values exceeding 0.99. The LOD was 0.9 ppm and the LOQ was 2.6 ppm for frozen tuna, which gave the lowest background level of contaminant. Cross-reactivity of the assay was tested against 14 other biogenic amines and was found to be minimal for all (<0.5%), except for agmatine (4.1%) and putrescine (0.9%). There was no observable interference from any tested biogenic amines. Product consistency was verified by validating lot-to-lot variations and variations within the same lot. Overall recoveries for all tested matrixes were within the acceptable range (80-120%). A 1-year claimed shelf life of the kit at 4°C was verified by accelerated stability study data collected on days 1, 15, and 32 at 25°C and by real-time stability testing at 1-month, 6-month, and 1-year at 4°C. No difference in histamine detection was observed in ruggedness testing, in which minor changes were introduced to the assay protocol. Good agreement was observed between AOAC Official MethodSM 977.13 and the MaxSignal® Histamine Enzymatic Assay method. Independent laboratory testing demonstrated that the MaxSignal method works with the same precision in the hands of minimally trained technicians as with the expert method developers. This study validates the performance of Bioo Scientific's rapid enzymatic method.

The MC-Media Pad ACplus™ is a dry, rehydratable film medium for the enumeration of aerobic bacterial colonies. The performance of the method in a variety of foods was compared to that of U.S. reference methods: U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) Chapter 3.02 "Quantitative Analysis of Bacteria in Foods as Sanitary Indicators" (USDA/FSIS MLG 3.02); Standard Methods for the Examination of Dairy Products (SMEDP) Chapter 6 "Microbiological Count Methods, Standard Plate Count Method" (SMEDP 6); AOAC Official MethodSM 966.23 Microbiological Methods; and ISO 4833-1:2013 "Microbiology of the food chain-Horizontal method for the enumeration of microorganisms-Part 1: Colony count at 30 degrees C by the pour plate technique." The validated matrixes included raw chicken breast and raw ground pork for USDA/FSIS MLG 3.02; cream cheese and yogurt drink for SMEDP 6; parsley, vegetable juice, prawns, tuna pate, sandwiches, and pasta salad for AOAC Method 966.23, and raw chicken breast, raw ground pork, cream cheese, yogurt drink, parsley, vegetable juice, prawns, tuna pate, sandwiches, and pasta salad for ISO 4833-1:2013. In each matrix study, five replicates at each of three contamination levels were tested as paired test portions. All 10 matrixes were compared to the appropriate U.S. reference methods under MC-Media Pad ACplus standard-usage conditions (35 ± 1°C for 48 ± 2 h). Across all matrixes, the difference of mean log10 values ranged from -0.43 to 0.44, within the acceptable range of -0.50 to 0.50. The candidate method repeatability SD (sr) varied from 0.03 to 0.23 log10 CFU/g, comparing favorably to the reference method SD, which ranged from 0.06 to 0.30 log10 CFU/g. Seven matrixes were compared to the appropriate U.S. reference methods under MC-Media Pad ACplus rapid-usage conditions (35 ± 1°C for 24 ± 2 h). Of the 21 matrix/concentration combinations, only three instances of difference of mean >0.5 log were observed. The ranges of sr values of the rapid-usage candidate method (0.023-0.324) and the reference method (0.013-0.236) were similar for the seven matrixes tested. All 10 matrixes were compared to the International Organization for Standardization (ISO) reference method under MC-Media Pad ACplus alternate-method conditions (30 ± 1°C for 72 ± 3 h). All 10 matrixes yielded a mean difference between methods of <0.5 log, and the ranges of sr values were similar between the candidate alternate method (0.037-0.378) and the ISO reference method (0.037-0.437). The product consistency study demonstrated no significant difference between lots of product and supported the 2-year shelf life. Robustness testing yielded no significant differences when small variations were made in sample volume, incubation temperature, and incubation time. Thus, the data show equivalent or better performance of the MC-Media Pad ACplus method compared to the relevant reference methods in support of AOAC Performance Tested MethodSM certification.

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.)

The aim of this study was to determine the time for a 3-log CFU/g outgrowth of Staphylococcus aureus and its toxin production in previously frozen precooked tuna meat (albacore [Thunnus alalunga] prepared as loin, chunk, and flake or skipjack [Katsuwonus pelamis] prepared as chunk and flake) held either at 21 or 27°C. A five-strain cocktail of enterotoxin-producing S. aureus was surface inoculated with ∼10(3) CFU/g onto tuna samples. The experimental time-temperature conditions were designed to mimic common industry holding conditions. After a 3-h incubation at 37°C, inoculated samples were individually vacuum sealed and stored at 20°C for 4 weeks. Following frozen storage, samples were thawed to the target temperature (21 or 27°C) and then incubated aerobically. Growth of S. aureus in tuna was then monitored using Baird Parker agar; simultaneously, aerobic plate counts, enterotoxin production, and sensory profile (color and odor) were determined. The results showed that the time for a 3-log CFU/g increase was >20 h at 21°C and 8 to 12 h at 27°C for albacore, with toxin production observed at 14 to 16 h at 21°C and at 8 h at 27°C. A 3-log CFU/g increase for skipjack occurred at 22 to 24 h at 21°C and at 10 to 14 h at 27°C. The toxin production in skipjack started at 20 to 22 h at 21°C and at 8 to 10 h at 27°C. Toxin production was observed before a 3-log increase was achieved in albacore samples at 21°C. Under all conditions, toxins were detected when the cell density of S. aureus was 6 log CFU/g. Overall, significantly faster S. aureus growth was observed in albacore compared with skipjack (P < 0.05), possibly owing to differences in sample composition (e.g., pH and salt content). The data developed from this study can be used by the tuna industry to model the growth and enterotoxin production of S. aureus and to design manufacturing controls that ensure food safety.

Background: Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India., Methods: Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network., Results: Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India., Conclusions: These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general., (Published by Oxford University Press for the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

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.

Scombrotoxin fish poisoning remains the primary cause of seafood poisoning outbreaks despite preventive guidelines. The purpose of this study was to investigate the use of pH for the control of growth and histamine formation by histamine-producing bacteria in fish muscle. We examined pH effects on growth and histamine formation in tuna fish infusion broth and in inoculated tuna and mahi-mahi fish muscle. Histamine production was significantly less for all bacterial strains at pH 8.5 compared to pH 5.5 in tuna fish infusion broth with no significant difference in growth. Elevated pH due to phosphate treatment of fish muscle tissues significantly reduced histamine formation with no effect on the growth of histamine-producing bacteria. This study revealed that phosphate treatment of mahi-mahi and tuna fish muscle resulted in significantly lower histamine production over 4 d of storage at 10 °C. Phosphate treatment of fish muscle may serve as a secondary barrier in addition to FDA recommended time and temperature controls for reducing public health concerns of scombrotoxin fish poisoning., (© 2015 Institute of Food Technologists®)

We report on human illness due to histamine fish poisoning outbreaks in Australia from 2001 to 2013. Histamine fish poisoning results from the ingestion of histamine contained within the flesh of certain fish species that naturally contain histidine, which has been converted to histamine by spoilage bacteria following poor handling or temperature control after harvesting. While symptoms vary, allergic symptoms such as facial flushing, headaches and rashes are frequently reported. Using the OzFoodNet outbreak register, published case reports and surveillance reports, we found data on 57 outbreaks of histamine fish poisoning, which affected 187 people, of whom 14% were hospitalised. There were no deaths reported. Outbreaks were generally small in size, with a median of 2 cases per outbreak (range 1 to 22 people), with 88% of outbreaks comprising less than 5 people. Tuna (in the family Scombridae) was the most frequently reported food vehicle, while 18 outbreaks involved non-scombridae fish. Median incubation periods among the outbreaks were short; being less than 1 hour for 22 outbreaks. The most frequently reported symptoms were diarrhoea and rash. Symptoms of facial/body flushing were reported for at least one case in 19 outbreaks and tingling, burning or swelling of the skin, especially around the lips for at least 1 case in 13 outbreaks. In 3 outbreaks, one or more cases were reported to have had respiratory distress or difficulty breathing. While the condition is often mild, improved recognition and appropriate treatment is important, as it will reduce the possibility of any severe health effects resulting from this condition. Key features of histamine fish poisoning outbreaks are the high attack rate, rapid onset, the typical symptoms and their short duration., (This work is copyright. You may download, display, print and reproduce the whole or part of this work in unaltered form for your own personal use or, if you are part of an organisation, for internal use within your organisation, but only if you or your organisation do not use the reproduction for any commercial purpose and retain this copyright notice and all disclaimer notices as part of that reproduction. Apart from rights to use as permitted by the Copyright Act 1968 or allowed by this copyright notice, all other rights are reserved and you are not allowed to reproduce the whole or any part of this work in any way (electronic or otherwise) without first being given the specific written permission from the Commonwealth to do so. Requests and inquiries concerning reproduction and rights are to be sent to the Online, Services and External Relations Branch, Department of Health, GPO Box 9848, Canberra ACT 2601, or by email to copyright@health.gov.au.)

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.)

Twenty-five tuna sausage products were purchased from retail markets in Taiwan. The rates of occurrence of biogenic amines, histamine-forming bacteria, and adulteration by pork and poultry were determined. The average content of various biogenic amines in all tested samples was less than 2.0 mg/100 g (<0.05 to 1.85 mg/100 g). Thirteen histamine-producing bacterial strains isolated from tested samples produced 12.1 to 1,261 ppm of histamine in Trypticase soy broth supplemented with 1.0% L-histidine. Among them, Raoultella ornithinolytica (one strain), Enterobacter aerogenes (one strain), and Staphylococcus pasteuri (two strains) were identified as prolific histamine formers. PCR assay revealed that the adulteration rates were 80% (20 of 25) and 4% (1 of 25) for pork and poultry, respectively, in tuna sausage. The fish species in the tuna sausage samples were identified as Thunnus albacares for 22 samples (88%), Thunnus alalunga for 1 sample (4%), and Thunnus thynnus for 1 sample (4%), whereas the remaining sample was identified as Makaira nigricans (blue marlin).

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)

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens.

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.

A high-performance liquid chromatographic method with indirect fluorescence detection has been developed for quantification of dipicolinic acid, a major constituent of bacterial endospores. After separation on a reversed-phase column, a post-column reagent of sodium acetate at 1 mol l(-1) with 50 micromol l(-1) terbium chloride was added for complexation of dipicolinic acid. Terbium monodipicolinate complexes formed were quantified by measuring the fluorescence emission maximum at 548 nm after excitation with UV light at 270 nm wavelength. Parameters of post-column complexation were optimized to achieve a detection limit of 0.5 nmol DPA l(-1), corresponding to about 10(3) Desulfosporosinus orientis endospores per ml. The method was applied to the analysis of spore contamination in tuna and for estimating the endospore numbers in marine sediments.

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.

Immunoassays were developed for the simultaneous detection of staphylococcal enterotoxin B and botulinum toxoid A in buffer, with limits of detection of 0.1 ng/ml and 20 ng/ml, respectively. The toxins were also spiked and measured in a variety of food samples, including canned tomatoes, sweet corn, green beans, mushrooms, and tuna.

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.