Microbiota profile of filleted gilthead seabream (Sparus aurata) during storage at various conditions by 16S rRNA metabarcoding analysis.

The aim of the present work was to study the microbiota profile of gilthead seabream (Sparus aurata) fillets stored either aerobically or under Modified Atmosphere Packaging (MAP) conditions at 0, 4, 8 and 12 °C, via 16S rRNA metabarcoding sequencing. Throughout storage, sensory assessment was also applied to estimate fillets' end of shelf-life. Results indicated that storage conditions strongly influenced the shelf-life of the fillets, since the sensorial attributes of air-stored samples deteriorated earlier than that of MAP-stored fillets, while higher temperatures also contributed to a more rapid products' end of shelf-life. Metataxonomic analysis indicated that Pseudomonas was by far the dominant genus at the end of fillet's shelf-life, in the vast majority of treatments, even though a sporadic but noteworthy presence of other genera (e.g, Shewanella, Carnobacterium, Brochothrix etc.) at the middle stages of MAP-stored fillets is also worth mentioning. On the other hand, a completely different profile as well as a more abundant bacterial diversity was observed at the end of shelf-life of MAP-stored fillets at 12 °C, in which Serratia was the most dominant bacterium, followed by Kluyvera, Hafnia, Rahnella and Raoultella, while Pseudomonas was detected in traces. The findings of the present work are very important, providing useful information about the spoilage status of gilthead seabream fillets during several storage conditions, triggering in parallel the need for further studies to enrich the current knowledge and help stakeholders develop innovative strategies that delay the growth of key spoiler players and consequently, retard spoilage course.

Bacterial Communities and Antibiotic Resistance of Potential Pathogens Involved in Food Safety and Public Health in Fish and Water of Lake Karla, Thessaly, Greece.

Bacterial communities, microbial populations, and antibiotic resistance of potential pathogens in the water and fish (Cyprinus carpio, flesh and gut) from different areas (A1, A2 and A3-A1 was linked with river water, A2 with cattle activity, and A3 with waters of a spring after heavy rains) of Lake Karla (Thessaly, Central Greece) were investigated. The isolated bacteria were identified using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and were tested for resistance in 21 antibiotics. The microbiota composition of fish flesh was also studied using 16S amplicon-based sequencing Serratia fonticola and several species of Aeromonas (e.g., Aeromonas salmonicida, Aeromonas bestiarium, Aeromonas veronii, etc.) exhibited the highest abundances in all studied samples, while the microbiota profile between the three studied areas was similar, according to the culture-dependent analysis. Of them, S. fonticola was found to be resistant in the majority of the antibiotics for the water and fish (gut and flesh), mainly of the areas A1 and A2. Regarding 16S metabarcoding, the presence of Serratia and Aeromonas at genus level was confirmed, but they found at very lower abundances than those reported using the culture-dependent analysis. Finally, the TVC and the rest of the studied microbiological parameters were found at acceptable levels (4 log cfu/mL or cfu/g and 2-4 log cfu/mL or cfu/g, extremely low levels of E. coli/coliforms) in both water and fish flesh. Based on our findings, the water of Lake Karla would be used for activities such as irrigation, recreation and fishing, however, the development and implementation of a quality management tool for Lake Karla, to ensure environmental hygiene and prevention of zoonosis during the whole year, is imperative.

Microbiota Succession of Whole and Filleted European Sea Bass (Dicentrarchus labrax) during Storage under Aerobic and MAP Conditions via 16S rRNA Gene High-Throughput Sequencing Approach.

In the present work, the profiles of bacterial communities of whole and filleted European sea bass (Dicentrarchus labrax), during several storage temperatures (0, 4, 8 and 12 °C) under aerobic and Modified Atmosphere Packaging (MAP) conditions, were examined via the 16S rRNA High-Throughput Sequencing (HTS) approach. Sensorial attributes were also assessed to determine products' shelf-life. Results indicated that shelf-life was strongly dependent on handling, as well as on temperature and atmosphere conditions. HTS revealed the undisputed dominance of Pseudomonas from the very beginning and throughout storage period in the majority of treatments. However, a slightly different microbiota profile was recorded in MAP-stored fillets at the middle stages of storage, which mainly referred to the sporadic appearance of some bacteria (e.g., Carnobacterium, Shewanella, etc.) that followed the dominance of Pseudomonas. It is noticeable that a major difference was observed at the end of shelf-life of MAP-stored fillets at 12 °C, where the dominant microbiota was constituted by the genus Serratia, while the relative abundance of Pseudomonas and Brochothrix was more limited. Furthermore, at the same temperature under aerobic storage of both whole and filleted fish, Pseudomonas almost co-existed with Acinetobacter, while the presence of both Erwinia and Serratia in whole fish was noteworthy. Overall, the present study provides useful information regarding the storage fate and spoilage status of whole and filleted European sea bass, suggesting that different handling and storage conditions influence the shelf-life of sea bass by favoring or delaying the dominance of Specific Spoilage Organisms (SSOs), affecting in parallel to some extent the formation of their consortium that is responsible for products' sensorial deterioration. Such findings enrich the current knowledge and should be used as a benchmark to develop specific strategies aiming to delay spoilage and thus increase the products' added value.

Volatile Organic Compounds and 16S Metabarcoding in Ice-Stored Red Seabream Pagrus major.

The profiles of bacterial communities and volatile organic compounds (VOCs) of farmed red seabream (Pagrus major) from two batches during ice storage were studied using 16S metabarcoding (culture independent approach) and headspace Solid Phase Micro-Extraction-Gas Chromatography/Mass Spectrometry (SPME-GC/MS) analysis, respectively. Sensory attributes and microbiological parameters were also evaluated. At Day 12 (shelf-life for both batches based on sensory evaluation), using classical microbiological analysis, Total Viable Counts (TVC) were found at the levels of 7-8 log cfu/g, and Pseudomonas and/or H2S producing bacteria dominated. On the other hand, the culture independent 16S metabarcoding analysis showed that Psychrobacter were the most abundant bacteria in fish tissue from batch 1, while Pseudomonas and Psychrobacter (at lower abundance) were the most abundant in fish from batch 2. Differences were also observed in VOC profiles between the two batches. However, combining the VOC results of the two batches, 15 compounds were found to present a similar trend during fish storage. Of them, 2-methylbutanal, 3-methylbutanal, 3-methyl-1-butanol, ethanol, 2,4 octadiene (2 isomers), ethyl lactate, acetaldehyde and (E)-2-penten-1-ol could be used as potential spoilage markers of red seabream because they increased during storage, mainly due to Psychrobacter and/or Pseudomonas activity and/or chemical activity (e.g., oxidation). Additionally, VOCs such as propanoic acid, nonanoic acid, decanoic acid, 1-propanol, 3,4-hexanediol and hexane decreased gradually with time, so they could be proposed as freshness markers of red seabream. Such information will be used to develop intelligent approaches for the rapid evaluation of spoilage course in red seabream during ice storage.

Spoilage Investigation of Chill Stored Meagre (Argyrosomus regius) Using Modern Microbiological and Analytical Techniques.

Spoilage status of whole and filleted chill-stored meagre caught in January and July was evaluated using sensory, microbiological, 16S metabarcoding and Volatile Organic Compounds (VOCs) analysis. Based on the sensory analysis, shelf-life was 15 and 12 days for the whole fish taken in January and July, respectively, while 7 days for fish fillets of both months. For the whole fish, Total Viable Counts (TVC) at the beginning of storage was 2.90 and 4.73 log cfu/g for fish caught in January and July respectively, while it was found about 3 log cfu/g in fish fillets of both months. The 16S metabarcoding analysis showed different profiles between the two seasons throughout the storage. Pseudomonas (47%) and Psychrobacter (42.5%) dominated in whole meagre of January, while Pseudomonas (66.6%) and Shewanella (10.5%) dominated in fish of July, at the end of shelf-life. Regarding the fillets, Pseudomonas clearly dominated at the end of shelf-life for both months. The volatile profile of meagre was predominated by alcohols and carbonyl compounds. After univariate and multivariate testing, we observed one group of compounds (trimethylamine, 3-methylbutanoic acid, 3-methyl-1-butanol) positively correlating with time of storage and another group with a declining trend (such as heptanal and octanal). Furthermore, the volatile profile seemed to be affected by the fish culturing season. Our findings provide insights into the spoilage mechanism and give information that helps stakeholders to supply meagre products of a high-quality level in national and international commerce.

Primary Processing and Storage Affect the Dominant Microbiota of Fresh and Chill-Stored Sea Bass Products.

The cultivable microbiota isolated from three sea bass products (whole, gutted, and filleted fish from the same batch) during chilled storage and the effect of primary processing on microbial communities in gutted and filleted fish were studied. Microbiological and sensory changes were also monitored. A total of 200 colonies were collected from TSA plates at the beginning and the end of fish shelf-life, differentiated by High Resolution Sequencing (HRM) and identified by sequencing analysis of the V3-V4 region of the 16S rRNA gene. Pseudomonas spp. followed by potential pathogenic bacteria were initially found, while Pseudomonasgessardii followed by other Pseudomonas or Shewanella species dominated at the end of fish shelf-life. P. gessardii was the most dominant phylotype in the whole sea bass, P. gessardii and S. baltica in gutted fish, while P. gessardii and P. fluorescens were the most dominant bacteria in sea bass fillets. To conclude, primary processing and storage affect microbial communities of gutted and filleted fish compared to the whole fish. HRM analysis can easily differentiate bacteria isolated from fish products and reveal the contamination due to handling and/or processing, and so help stakeholders to immediately tackle problems related with microbial quality or safety of fish.

HRM and 16S rRNA gene sequencing reveal the cultivable microbiota of the European sea bass during ice storage.

The total cultivable microbiota of the ice-stored European sea bass (Dicentrarchus labrax), the most important commercial fish species of the Mediterranean aquaculture, was determined using 16S rRNA gene sequence analysis. High Resolution Melting (HRM) curve profiles and sequencing analysis (V3-V4 region of the 16S rRNA gene) were used respectively for the differentiation and identification of the collected isolates from six time intervals (day 0, 4, 8, 12, 14 and 16) while fish were stored in ice. HRM analysis differentiated the unknown microbiota in ten groups (208 isolates) and in two single isolates based on their HRM curve profiles. The isolates with HRM profiles which were >91% similar within each group were found to belong to the same species using sequencing analysis. Thus, the ten groups consist of representatives of Psychrobacter glacincola, Ps. alimentarius, Ps. cryohalolentis, Ps. maritimus, Ps. fozii, Pseudomonas sp., Paeniglutamicibacter sp., Carnobacterium sp., Leucobacter aridicolis and Bacillus thuringiensis. Based on this approach, Ps. cryohalolentis was found to be the most dominant phylotype at the beginning of fish shelf-life compared to other species. The abundance of this bacterium decreased throughout storage, while Ps. glacincola increased and dominated at the time of the sensory minimum acceptability (day 14) and rejection (day 16). To conclude, HRM could be used for the rapid determination of sea bass microbiota, using the representatives of each group as reference bacterial strains, in order for scientists to solve rapidly stakeholders problems related with microbial quality or safety of fish.