Polyinfection in Fish Aeromoniasis: A Study of Co-Isolated Aeromonas Species in Aeromonas veronii Outbreaks.

We studied the phenotypic and genomic characteristics related to the virulence and antibiotic resistance of two Aeromonas strains, which were co-isolated before an outbreak of Aeromonas veronii among diseased seabass on Agathonisi Island, Greece, in April 2015. The first strain, AG2.13.2, is a potentially pathogenic mesophilic variant of Aeromonas salmonicida, and the second, AG2.13.5, corresponds to an Aeromonas rivipollensis related to A. rivipollensis KN-Mc-11N1 with an ANI value of 97.32%. AG2.13.2 lacks the type III secretion system just like other mesophilic strains of A. salmonicida. This characteristic has been associated with lower virulence. However, the genome of AG2.13.2 contains other important virulence factors such as type II and type VI secretion systems, and toxins such as rtxA, aerolysin aer/act, and different types of hemolysins. The strain also carries several genes associated with antibiotic resistance such as the tetE efflux pump, and exhibits resistance to tetracycline, ampicillin, and oxolinic acid. In an in vivo challenge test with gilthead seabream larvae, the A. veronii bv sobria strain AG5.28.6 exhibited the highest virulence among all tested strains. Conversely, both A. salmonicida and A. rivipollensis showed minimal virulence when administered alone. Interestingly, when A. veronii bv sobria AG5.28.6 was co-administered with A. rivipollensis, the larvae survival probability increased compared to those exposed to A. veronii bv sobria AG5.28.6 alone. This finding indicates an antagonistic interaction between A. veronii bv sobria AG5.28.6 and A. rivipollensis AG2.13.5. The co-administration of A. veronii bv sobria AG5.28.6 with Aeromonas salmonicida did not yield distinct survival probabilities. Our results validate that the primary pathogen responsible for European seabass aeromoniasis is Aeromonas veronii bv sobria.

Pathogenic and Opportunistic Vibrio spp. Associated with Vibriosis Incidences in the Greek Aquaculture: The Role of Vibrio harveyi as the Principal Cause of Vibriosis.

A monitoring program to follow vibriosis incidents in the Greek marine aquaculture was implemented over the past 13 years. 273 isolates, from various cases originating from eight regions and nine hosts, were collected and characterized. The main aquaculture species of the survey were the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata). Various species of Vibrionaceae were associated with vibriosis. Vibrio harveyi had the highest prevalence and was isolated throughout the year from all hosts. During the warm months, Vibrio harveyi prevailed with frequent co-isolations of Photobacterium damselae subsp. damselae and Vibrio alginolyticus, while during spring, other Vibrio species were more abundant, such as Vibrio lentus, Vibrio cyclitrophicus, and Vibrio gigantis. Phylogenetic analysis using the mreB gene and the metabolic fingerprint of the isolates showed great variability within the species of the collection. The severity of the disease and the frequency of outbreaks make vibriosis (that is, mainly attributed to V. harveyi) an important concern for the regional aquaculture sector.

Isolation and characterization of a novel Tenacibaculum species and a corresponding bacteriophage from a Mediterranean fish hatchery: Description of Tenacibaculum larymnensis sp. nov. and Tenacibaculum phage Larrie.

Tenacibaculum larymnensis sp. nov., a novel species of the Tenacibaculum genus was isolated from a commercial fish hatchery in Greece. The novel species is phylogenetically close to T. discolor and was biochemically and genetically characterized. The genome of T. larymnensis has 3.66 Mbps length, 31.83% GC content and the genomic analysis demonstrated that it harbors a wide enzymatic repertoire suggestive of increased degrading capacity but also several virulence factors including hemolysins, secretion systems, transporters, siderophores, pili and extracellular proteins. Using the novel strain, a virulent bacteriophage designated as Tenacibaculum phage Larrie was isolated and characterized. Larrie is a novel Siphovirus with relatively large genome, 77.5 kbps with 111 ORFs, a GC content of 33.7% and an exclusively lytic lifestyle. The new phage-host system can serve as an efficient model to study microbial interactions in the aquatic environment which contribute to the nutrient cycling.

Evaluation of absorption and depletion of florfenicol in European seabass Dicentrarchus labrax.

The pharmacokinetic properties and residue elimination of florfenicol (FLO) and its amine were investigated in European seabass Dicentrarchus labrax at 24°C. The trial mainly included analysis of FLO in plasma after a single dose dietary administration of 10 mg/kg and in muscle plus skin following a multiple dosing (10 mg kg-1  day-1 for 7 days) to estimate pharmacokinetics and residue depletion, respectively. The maximum plasma concentration of FLO was measured to be 1.64 µg/ml, 4 hr post administration. The elimination half-life (t1/2b ) and the area under the concentration-time curve extrapolated to infinity (AUC0-∞ ) were calculated to be 13.0 hr and 34.7 µg h-1  ml-1 , respectively. Withdrawal times of FLO and its amine were calculated to be 46.3 degree-days, indicating a fast removal from the edible tissues of treated European seabass. Overall, FLO can be considered as a potentially efficient antibacterial agent for farmed European seabass provided that additional efforts will be devoted towards its in vitro and clinical efficacy.

Comparative Study on A Novel Pathogen of European Seabass. Diversity of Aeromonas veronii in the Aegean Sea.

Aeromonas veronii is an emerging pathogen causing severe pathology and mortalities in European seabass aquaculture in the Aegean Sea, Mediterranean. More than 50 strains of the pathogen were characterized biochemically and genetically in order to study the epidemiology of the disease, as well as the phylogeny and virulence of the bacterium. Based on the phenotypic characteristics, the isolates form three groups consisting of: (a) the West Aegean Sea, non-motile, non-pigment-producing strains, (b) the West Aegean Sea, motile, and pigment-producing strains and (c) the East Aegean Sea motile strains that produce minute amounts of pigment. All strains were highly similar at the genomic level; however, the pattern of West/East geographic origin was reflected in biochemical properties, in general genomic level comparison and in the putative virulent factors studied. Type VI secretion system was not detected in the western strains. The outer membrane protein (OMP) profile which contains proteins that are putative antigenic factors, was very similar between strains from the different areas. Although most of the OMPs were detected in all strains with great sequence similarity, diversification according to geographic origin was evident in known antigenic factors such as the maltoporin LamB. A systematic comparative analysis of the strains is presented and discussed in view of the emergence of A. veronii as a significant pathogen for the Mediterranean aquaculture.