Gram-positive bacteria are part of the normal flora of fish from different aquatic environments. They are mesophilic bacteria and demonstrate optimum growth at ambient temperature. In the sub-tropics, marine fish are caught from seas at temperatures of 16 to 34C, they are usually not iced and are handled at ambient temperature. It was hypothesized that under these conditions Gram-positive bacteria will be abundant in sub-tropical marine fish and will have roles in the spoilage of fish. A review of literature showed that there is a gap in understanding the Gram-positive bacterial populations in sub-tropical marine fish. This is partly due to the fact that the selective media used for isolating Gram-positive bacteria have limitations. Ecological and speciation studies have revealed that the ecology and speciation of many Gram-positive bacteria have not been clearly elucidated. The effect of ambient storage on the individual genera and species of Gram-positive bacteria in fish has been rarely studied. The spoilage potential of Gram-positive bacteria of marine fish origin has not been clearly determined. Therefore, the main aims of this study were to isolate Gram-positive bacteria from fresh and ambient-temperature-stored sub-tropical marine fish, speciate the isolates and study the spoilage potential of the isolates. The practical components of this study were conducted in four parts. The first part dealt with validation of tryptone soya agar with 0.25% phenylethyl alcohol (PEA-TSA) to enumerate Gram-positive bacteria. The second part enumerated Gram-positive bacteria from the muscles, gills and gut of Pseudocaranx dentex (Silver Trevally), Pagrus auratus (Snapper) and Mugil cephalus (Sea Mullet) stored at 25C for 15 hours using PEA-TSA. The third part dealt with the speciation of the isolates using appropriate methods such as polymerase chain reaction, 16S rRNA gene sequence, the VITEK JR system and conventional biochemical methods. In the fourth part, the isolates were assayed qualitatively for their ability to produce volatile sulphur compounds (VSC), reduce trimethylamine oxide and decarboxylate histidine, lysine and ornithine at mesophilic temperature, 32C. Initial studies indicated that PEA-TSA significantly (P< 0.05) reduced the total aerobic bacterial count of fish whereas control Gram-positive bacteria were not affected (P> 0.05). Gram-positive aerobic bacterial counts (GABC) significantly (P< 0.05) increased in the muscles and gills during ambient storage for 15 hours. Within each species, no significant (P> 0.05) differences were found in GABC between muscles and gills. Moreover, there were no significant differences (P> 0.05) in GABC between fish species during storage. In total, 390 bacteria were isolated from the fresh and stored fish; 339 isolates (87%) were found to be Gram-positive. Two hundred and sixty-six isolates (78%) of Gram-positive bacteria were identified to fall into 13 genera, namely Staphylococcus, Micrococcus, Bacillus, Virgibacillus, Brevibacillus, Corynebacterium, Streptococcus, Enterococcus, Aerococcus, Exiguobacterium, Carnobacterium, Vagococcus and Sporosarcina and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. The effect of storage at 25C for 15 hours resulted in a change of Gram-positive bacterial populations; while S. epidermidis, S. xylosus and Bacillus megaterium were no longer present, S. warneri, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased. Three species, E. faecium, Str. uberis and B. sphaericus, were the most prevalent at the end of storage. Micrococcus luteus and S. warneri were the most prevalent isolates from Pseudocaranx dentex, but E. faecium and Str. uberis were the most frequently isolated from Pagrus auratus and Mugil cephalus. With respect to different parts of the fish body, E. faecium, Str. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Str. uberis from the gills and M. luteus from the gut. Among the 228 isolates examined, Br. borstelensis 73, Br. borstelensis 291, Str. uberis 339, Vagococcus fluvialis 31 and Vag. fluvialis 132 produced VSC from sodium thiosulphate, cysteine and methionine. However, strains varied in sulphur source utilization. Exiguobacterium acetylicum 5, Exiguobacterium spp. 191, Carnobacterium spp. 338, Br. borstelensis 73, Br. borstelensis 291, Str. uberis 30, Str. uberis 339, Vag. fluvialis 31 and Vag. fluvialis 132 reduced TMAO. No histidine decarboxylase activity was found in the Gram-positive bacterial species tested. Lysine and ornithine were decarboxylated mainly by different strains of S. warneri, S. epidermidis and M. luteus. During ambient storage of fish, the frequency of lysine-decarboxylating bacteria increased and became more diverse after 5 hours of storage. Among fish species examined, the frequencies of lysine- and ornithine-decarboxylating bacteria were higher and more diverse in Pseudocaranx dentex than in Pagrus auratus and Mugil cephalus. This study found that Gram-positive bacteria were abundant and diverse in sub-tropical marine fish; however, their frequencies were affected by fish habitat and fish body part. Ambient temperature storage determined which Gram-positive bacterial species were dominant. With the exception of one isolate of S. aureus, Gram-positive bacteria isolated from sub-tropical marine fish caught from unpolluted water were not potential pathogens. The study also showed that Gram-positive bacteria had greater ability to decarboxylate lysine and ornithine than to produce VSC or reduce TMAO, and the spoilage potential of a bacterial species was a strain-dependent behaviour. This is a significant study as it is the first study on this aspect sub-tropical marine fish. It validated a selective medium that can be used to enumerate most Gram-positive bacteria from a marine environment. Most of the Gram-positive bacterial species from sub-tropical marine fish identified in this study were documented for the first time. The effects of ambient storage and the spoilage potential of Gram-positive bacteria from sub-tropical marine were clearly elucidated.

Le poisson est un produit très périssable dont l’altération résulte essentiellement de la croissance bactérienne. Comparé aux régions tempérées, peu d’études portent sur le microbiote d’altération des poissons tropicaux. En Martinique, le thon jaune (Thunnus albacares) et l’ombrine ocellée (Sciaenops ocellatus) représentent des poissons d’intérêt pour les filières pêche et aquaculture. Dans le but de mieux connaître le microbiote d’altération de ces poissons, des analyses culturales et aculturales (séquençage de nouvelle génération des amplicons d’ARNr 16S, Illumina MiSeq) ont été réalisées.Une grande diversité d’espèces bactériennes a été retrouvée dans le thon et l’ombrine fraîchement pêchés (104 et 887 OTUs, respectivement) et la plupart d’entre elles sont communément isolées des poissons (Chryseobacterium, Burkholderia, Flavobacterium, Psychrobacter, Arthrobacter, Staphylococcus). Certaines, comme Ralstonia sp. et Rhodanobacter terrae, en quantité importante dans le thon frais, sont plus atypiques. Au cours de l’entreposage du thon sous-glace, Pseudomonas et Brochothrix deviennent dominants. L’emballage sous atmosphère modifiée (MAP) ou sous vide (VP) entraine clairement la sélection de Brochothrix dans un cas et d’un mélange de Brochothrix, bactéries lactiques (Lactococcus piscium, Carnobacterium maltaromaticum) et d’entérobactéries (Hafnia paralvei) dans l’autre, et ne permet pas une augmentation significative de la durée de conservation. Pour les filets d’ombrine, peu de différences sont observées entre MAP et VP dont le microbiote se compose essentiellement de bactéries lactiques (Carnobacterium spp., Vagococcus spp., Lactococcus spp., Leuconostoc spp.). La durée de conservation est étendue de 15 jours par rapport au poisson entier sous air.L’inoculation de différentes espèces bactériennes dans de la chair pauci-microbienne de thon ou d’ombrine a montré que Hafnia paralvei et Serratia spp. sont les espèces les plus altérantes. Brochothrix thermosphacta et Carnobacterium spp. produisent aussi des odeurs indésirables mais de façon plus modérée. Chez Pseudomonas, les espèces ne sont pas toutes altérantes et présentent même parfois des capacités à empêcher le développement des mauvaises odeurs induites par d’autres bactéries (Pseudomonas psychrophila/fragi) et à dégrader l’histamine (Pseudomonas cedrina, Pseudomonas plecoglossicida/monteilii). En parallèle, des tests sensoriels et des dosages physico-chimiques ont également été réalisés pour comprendre les conséquences de la croissance bactérienne et identifier des indicateurs fiables pour l’évaluation du degré d’altération des produits. / Fish is a highly perishable product and spoilage is mainly due to the bacterial growth. Compared to temperate regions, few studies examined the spoilage microbiota of tropical fish. In Martinique, yellowfin tuna (Thunnus albacares) and red drum (Sciaenops ocellatus) are essential fish of fisheries and aquaculture sectors. For a better characterization of the microbial ecosystem, culture-dependent and culture-independent (next-generation sequencing of 16S rRNA amplicons, Illumina MiSeq) methods were carried out.A wide diversity of species was found in freshly caught tuna and red drum (104 and 887 OTUs, respectively) and most of them are commonly isolated from fish (Chryseobacterium, Burkholderia, Flavobacterium, Psychrobacter, Arthrobacter, Staphylococcus). Others, such as Ralstonia sp. and Rhodanobacter terrae, largely present in fresh tuna, are less familiar. During the ice-storage of tuna, Pseudomonas and Brochothrix became dominant. The modified atmosphere packaging (MAP) and vacuum packaging (VP) clearly leaded to the selection of Brochothrix in one case and to a mixture of Brochothrix, lactic acid bacteria (Lactococcus piscium, Carnobacterium maltaromaticum) and enterobacteria (Hafnia paralvei) in the other case, and not conduct to a significant increase of the shelf-life. For red drum fillets, few differences were observed between MAP and VP with a microbiota essentially composed by lactic acid bacteria (Carnobacterium spp., Vagococcus spp., Lactococcus spp., Leuconostoc spp.). The shelf-life was extended by 15 days compared to the whole fish ice-stored.The inoculation of different bacterial species into the pauci-microbial flesh of tuna or red drum showed that Hafnia paralvei and Serratia spp. were the most spoiling bacteria. Brochothrix thermosphacta and Carnobacterium spp. produced more moderate undesirable odors. Among the Pseudomonas genus, not all species induced spoiling effects and some of them are even able to prevent the development of unpleasant odors from other bacteria (Pseudomonas psychrophila/fragi) and to degrade histamine (Pseudomonas cedrina, Pseudomonas plecoglossicida/monteilii).At the same time, sensory tests and physico-chemical assays were performed to understand the consequences of the bacterial growth and to identify reliable indices for the evaluation of the spoilage degree of the products.