Skin and stinger bacterial communities in two critically endangered rays from the South Atlantic in natural and aquarium settings.

Bacterial communities of two critically endangered rays from the South Atlantic, the butterfly ray (Gymnura altavela) and the groovebelly ray (Dasyatis hypostigma), were described using 16S rRNA gene metabarcoding. The study characterized the bacterial communities associated with (i) G. altavela in natural (in situ) and aquarium (ex situ) settings, (ii) skin and stinger of G. altavela, and D. hypostigma in aquaria, and (iii) newborns and adults of D. hypostigma. The results revealed potentially antibiotic-producing bacterial groups on the skin of rays from the natural environment, and some taxa with the potential to benefit ray health, mainly in rays from the natural environment, as well as possible pathogens to other animals, including fish and humans. Differences were observed between the G. altavela and D. hypostigma bacteria composition, as well as between the skin and stinger bacterial composition. The bacterial community associated with D. hypostigma changed with the age of the ray. The aquarium environment severely impacted the G. altavela bacteria composition, which changed from a complex bacterial community to one dominated almost exclusively by two taxa, Oceanimonas sp. and Sediminibacterium sp. on the skin and stinger, respectively.

Changes in the Microbial Community of the Mottled Skate (Beringraja pulchra) During Alkaline Fermentation.

Beringraja pulchra, Cham-hong-eo in Korean, is a mottled skate which is belonging to the cartilaginous fish. Although this species is economically valuable in South Korea as an alkalinefermented food, there are few microbial studies on such fermentation. Here, we analyzed microbial changes and pH before, during, and after fermentation and examined the effect of inoculation by a skin microbiota mixture on the skate fermentation (control vs. treatment). To analyze microbial community, the V4 regions of bacterial 16S rRNA genes from the skates were amplified, sequenced and analyzed. During the skate fermentation, pH and total number of marine bacteria increased in both groups, while microbial diversity decreased after fermentation. Pseudomonas, which was predominant in the initial skate, declined by fermentation (Day 0: 11.39 ± 5.52%; Day 20: 0.61 ± 0.9%), while the abundance of Pseudoalteromonas increased dramatically (Day 0: 1.42 ± 0.41%; Day 20: 64.92 ± 24.15%). From our co-occurrence analysis, the Pseudoalteromonas was positively correlated with Aerococcaceae (r = 0.638) and Moraxella (r = 0.474), which also increased with fermentation, and negatively correlated with Pseudomonas (r = -0.847) during fermentation. There are no critically significant differences between control and treatment. These results revealed that the alkaline fermentation of skates dramatically changed the microbiota, but the initial inoculation by a skin microbiota mixture didn't show critical changes in the final microbial community. Our results extended understanding of microbial interactions and provided the new insights of microbial changes during alkaline fermentation.

Biosynthesis of 1α-hydroxycorticosterone in the winter skate Leucoraja ocellata: evidence to suggest a novel steroidogenic route.

The present study explores the ability of intracellular bacteria within the renal-inter-renal tissue of the winter skate Leucoraja ocellata to metabolize steroids and contribute to the synthesis of the novel elasmobranch corticosteroid, 1α-hydroxycorticosterone (1α-OH-B). Despite the rarity of C1 hydroxylation noted in the original identification of 1α-OH-B, literature provides evidence for steroid C1 hydroxylation by micro-organisms. Eight ureolytic bacterial isolates were identified in the renal-inter-renal tissue of L. ocellata, the latter being the site of 1α-OH-B synthesis. From incubations of bacterial isolates with known amounts of potential 1α-OH-B precursors, one isolate UM008 of the genus Rhodococcus was seen to metabolize corticosteroids and produce novel products via HPLC analysis. Cations Zn2+ and Fe3+ altered metabolism of certain steroid precursors, suggesting inhibition of Rhodococcus steroid catabolism. Genome sequencing of UM008 identified strong sequence and structural homology to that of Rhodococcus erythropolis PR4. A complete enzymatic pathway for steroid-ring oxidation as documented within other Actinobacteria was identified within the UM008 genome. This study highlights the potential role of Rhodococcus bacteria in steroid metabolism and proposes a novel alternative pathway for 1α-OH-B synthesis, suggesting a unique form of mutualism between intracellular bacteria and their elasmobranch host.

The skin microbiome of cow-nose rays (Rhinoptera bonasus) in an aquarium touch-tank exhibit.

Public aquaria offer numerous educational opportunities for visitors while touch-tank exhibits offer guests the ability to directly interact with marine life via physical contact. Despite the popularity of touch-tanks, there is a paucity of research about animal health in these exhibits and, in particular, there is little research on the microbial communities in these highly interactive exhibits. Microbial community structure can have implications for both host health and habitat function. To better understand the microbiome of a touch-tank we used high-throughput sequencing of the 16S rRNA gene to analyze the microbial community on the dorsal and ventral surfaces of cow-nose rays (Rhinoptera bonasus) as well as their environment in a frequently visited touch-tank exhibit at the New England Aquarium. Our analyses revealed a distinct microbial community associated with the skin of the ray that had lower diversity than the surrounding habitat. The ray skin was dominated by three orders: Burkholderiales (∼55%), Flavobacteriales (∼19%), and Pseudomonadales (∼12%), taxonomic groups commonly associated with other fish species. Our results provide a survey of ray-associated bacterial communities in a touch-tank environment, thereby laying the foundation for future studies examining the role of potential challenges to ray microbiota and their associated health.

Prokaryotic community composition in alkaline-fermented skate (Raja pulchra).

Prokaryotes were extracted from skates and fermented skates purchased from fish markets and a local manufacturer in South Korea. The prokaryotic community composition of skates and fermented skates was investigated using 16S rRNA pyrosequencing. The ranges for pH and salinity of the grinded tissue extract from fermented skates were 8.4-8.9 and 1.6-6.6%, respectively. Urea and ammonia concentrations were markedly low and high, respectively, in fermented skates compared to skates. Species richness was increased in fermented skates compared to skates. Dominant and predominant bacterial groups present in the fermented skates belonged to the phylum Firmicutes, whereas those in skates belonged to Gammaproteobacteria. The major taxa found in Firmicutes were Atopostipes (Carnobacteriaceae, Lactobacillales) and/or Tissierella (Tissierellaceae, Tissierellales). A combination of RT-PCR and pyrosequencing for active bacterial composition showed that the dominant taxa i.e., Atopostipes and Tissierella, were active in fermented skate. Those dominant taxa are possibly marine lactic acid bacteria. Marine bacteria of the taxa Lactobacillales and/or Clostridia seem to be important in alkaline fermentation of skates.

Isolation of a novel 'atypical' Brucella strain from a bluespotted ribbontail ray (Taeniura lymma).

A pleomorphic Gram-negative, motile coccobacillus was isolated from the gills of a wild-caught bluespotted ribbontail ray after its sudden death during quarantine. Strain 141012304 was observed to grow aerobically, to be clearly positive for cytochrome oxidase, catalase, urease and was initially identified as "Brucella melitensis" or "Ochrobactrum anthropi" by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and VITEK2-compact®, respectively. Affiliation to the genus Brucella was confirmed by bcsp31 and IS711 PCR as well as by Brucella species-specific multiplex PCR, therein displaying a characteristic banding pattern recently described for Brucella strains obtained from amphibian hosts. Likewise, based on recA sequencing, strain 141012304 was found to form a separate lineage, within the so called 'atypical' Brucella, consisting of genetically more distantly related strains. The closest similarity was detected to brucellae, which have recently been isolated from edible bull frogs. Subsequent next generation genome sequencing and phylogenetic analysis confirmed that the ray strain represents a novel Brucella lineage within the atypical group of Brucella and in vicinity to Brucella inopinata and Brucella strain BO2, both isolated from human patients. This is the first report of a natural Brucella infection in a saltwater fish extending the host range of this medically important genus.

Diversity of bacteria cultured from the blood of lesser electric rays caught in the northern gulf of Mexico.

The prevalence and taxonomic diversity of bacteria cultured from the blood of apparently healthy Lesser Electric Rays Narcine bancroftii captured from open beach habitat in the north-central Gulf of Mexico are reported herein. The blood of 9 out of 10 Lesser Electric Rays was positive for bacteria, and bacterial isolates (n = 83) were identified by 16S rRNA gene sequencing. The majority of the isolates belonged to the phylum Proteobacteria (91.5%). Vibrio spp. comprised 53% of all isolates and were recovered from all Lesser Electric Rays with culture-positive blood. Among them, V. harveyi (n = 14) and V. campbellii (n = 11) were most common, followed by a group of unidentified Vibrio sp. (n = 10) related to V. nigripulchritudo. Isolates representing other species of Proteobacteria included Pseudoalteromonas (n = 13), Shewanella (n = 5), Amphritea (n = 3), Nautella (n = 3), and Arenibacter (n = 1). Higher bacterial diversity was observed in blood cultured on marine agar relative to blood agar, but gram-positive bacteria were isolated from the latter only. The 16S rRNA gene sequences of bacterial isolates were compared phylogenetically to those from related type strains. Most isolates were identified to the level of species, but some clustered independently from reference strains, likely representing new species of Vibrio, Amphritea, Shewanella, and Tenacibaculum. The present study is the first record of any bacterium from this ray species and reveals a taxonomically and phylogenetically diverse microbiota associated with its blood. Moreover, these data document that the presence of bacteria in elasmobranch blood is not coincident with clinical signs of disease, thereby rejecting the paradigm of septicemia indicating a disease condition in aquatic vertebrates.

Bacterial succession during curing process of a skate (Dipturus batis) and isolation of novel strains.

AIMS: To study the succession of cultivated and uncultivated microbes during the traditional curing process of skate. METHODS AND RESULTS: The microbial diversity was evaluated by sequencing 16Sr RNA clone libraries and cultivation in variety of media from skate samples taken periodically during a 9-day curing process. A pH shift was observed (pH 6·64-9·27) with increasing trimethylamine (2·6 up to 75·6 mg N per 100 g) and total volatile nitrogen (TVN) (from 58·5 to 705·8 mg N per 100 g) but with relatively slow bacterial growth. Uncured skate was dominated by Oceanisphaera and Pseudoalteromonas genera but was substituted after curing by Photobacterium and Aliivibrio in the flesh and Pseudomonas on the skin. Almost 50% of the clone library is derived from putative undiscovered species. Cultivation and enrichment strategies resulted in isolation of putatively new species belonging to the genera Idiomarina, Rheinheimera, Oceanisphaera, Providencia and Pseudomonas. The most abundant genera able to hydrolyse urea to ammonia were Oceanisphaera, Psychrobacter, Pseudoalteromonas and isolates within the Pseudomonas genus. CONCLUSIONS: The curing process of skate is controlled and achieved by a dynamic bacterial community where the key players belong to Oceanisphaera, Pseudoalteromonas, Photobacterium, Aliivibrio and Pseudomonas. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the bacterial population developments in the curing process of skate are presented and demonstrate a reservoir of many yet undiscovered bacterial species.

The influence of environmental bacteria in freshwater stingray wound-healing.

Invasion by bacteria can influence the course of healing of wounds acquired in aquatic environment. In this study, the bacteria present in Potamotrygon motoro stingray mucus and in the Alto Paraná river water were identified, and their ability to induce tissue injury and resist antibiotics was determined. Biochemical identification analysis showed that 97% of all bacterial isolates were Gram negative, Aeromonas spp., Enterobacter cloacae and Citrobacter freundii being the species most prevalent. Gelatinase and caseinase were produced by Aeromonas hydrophila, Aeromonas sobria and Pseudomonas aeruginosa strains. Erythrocyte hemolysis assay showed that A. sobria, A. hydrophila and to a lesser extent, other Gram-negative bacteria produced hemolysin. It was also observed that molecules released in culture by these bacteria were toxic to human epithelial cells. Antibiogram results showed that 68% of all bacterial isolates were resistant to at least one type of antibiotic, mainly B-lactams. Finally, it was demonstrated that although P. motoro venom was toxic to epithelial cells it did not influence bacterial proliferation. In summary, the results obtained in this work indicate that during the accident, the mucus of P. motoro and the environmental water may transfer into the wound pathogenic multi-resistant bacteria with the potential to cause severe secondary infections.

Blood culture results from healthy captive and free-ranging elasmobranchs.

Blood culture is a diagnostic tool used in confirming bacterial disease in teleostean and elasmobranch fishes. Unlike teleosts, elasmobranchs have a normal microflora in multiple organs, but their blood has generally been considered to be sterile. In regular exams of elasmobranchs conducted at a public aquarium, occasional blood samples have tested positive on culture. This finding prompted a blood culture survey of healthy captive and wild elasmobranchs (sharks and stingrays), which showed that 26.7% of all animals were positive. Stingrays alone showed a 50% occurrence of positive blood cultures, although the total number of animals was low and freshwater species were included in this number. When elasmobranchs other than stingrays were evaluated according to metabolic category, pelagic animals had a higher percentage of positive cultures than nonpelagic animals (38.7% versus 13.9%). These results indicate that a single positive blood culture without other corroborating diagnostics is not sufficient to confirm septicemia in elasmobranchs.

Salinicoccus salsiraiae sp. nov.: a new moderately halophilic gram-positive bacterium isolated from salted skate.

Two moderately halophilic low G + C Gram-positive bacteria were isolated from a sample of salted skate (Class Chondrychthyes, Genus Raja). Phylogenetic analysis of the 16S rRNA gene sequence of strains RH1(T) and RH4 showed that these organisms represented a novel species of the genus Salinicoccus. The new isolates formed pink-red colonies and flocculated in liquid media, with optimum growth in media containing 4% NaCl and pH of about 8.0. These organisms are aerobic but reduce nitrate to nitrite under anaerobic conditions. Acid is produced from several carbohydrates. Oxidase and catalase were detected. Menaquinone 6 was the major respiratory quinone. The major fatty acids of strains RH1(T) and RH4 were 15:0 anteiso and 15:0 iso. The G + C contents of DNA were 46.2 and 46.0 mol%, respectively. The peptidoglycan was of A3alpha L-Lys-Gly(5-6) type. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we suggest that strain RH1(T) (=LMG 22840 = CIP 108576) represents a new species of the genus Salinicoccus, for which we propose the name Salinicoccus salsiraiae.

Nutritional composition and microflora of the fresh and fermented skate (Raja Kenojei) skins.

The proximate compositions of fresh and fermented skate skin were each 75.95% and 74.5% moisture, 22.7% and 21.8% protein, 0.5% and 0.7% lipid and 0.6% and 0.9% ash, respectively. The predominant minerals were potassium and phosphorus (i.e. 53.5 and 33.0 mg/100 g in fresh skin, and 10.46 and 10.51 mg/100 g in fermented skin, respectively). Amino acid concentrations were lower in the fermented skin compared with the fresh skin. Histidine, glycine, alanine and glutamic acid were the major free amino acids in both skins. Palmitic acid (C16:0) was the major fatty acid in both fresh (16.68%) and fermented (20.38%) skate skin. Omega-3 polyunsaturated fatty acids were higher in fresh skin (22.17%) and fermented skin (24.54%) compared with omega-6 polyunsaturated fatty acids. The predominant microflora present in the both fresh and fermented skin were Photobacterium sp. and Vibrio sp. Total plate counts for the fresh and fermented skin were 2.4x10(4) CFU/g and 7.7x10(7) CFU/g, respectively.