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Objective: This study aimed to explore the abundance and diversity of antibiotic resistance genes (ARGs) in seahorses (Hippocampus barbouri and Hippocampus comes) and their surrounding environment., Methods: A combination of shotgun metagenomics and bioinformatics was used to investigate the resistome of both seahorse species., Results: The analyses demonstrated a higher abundance of ARGs in seahorse-associated microbiomes, particularly in skin and gut samples, compared to those from water and sediment. Interestingly, genes conferring multidrug resistance (e.g., acrB, acrF, cpxA, msbA, and oqxB) were highly prevalent in all samples, especially in skin and gut samples. High levels of genes conferring resistance to fluoroquinolones (e.g., mfd and emrB), β-lactam (e.g., bla CMY-71 , bla OXA-55 , and penA), aminocoumarin (e.g., mdtB and mdtC), and peptide antibiotics (arnA, pmrE, and rosA) were also observed in skin and gut samples. An enrichment of mobile genetic elements (MGEs) was also observed in the analysed samples, highlighting their potential role in facilitating the acquisition and spread of ARGs. In fact, the abundance of mobilisation (MOB) relaxases (e.g., MOBF, MOBP, MOBT, and MOBV) in gut and skin samples suggests a high potential for conjugation events., Conclusions: The occurrence of ARGs and MGEs in seahorses and the surrounding environment raises concerns about their transmission to humans, either through direct contact or the consumption of contaminated seafood. To the best of our knowledge, this study represents the first comprehensive analysis of ARGs in seahorse-associated microbiomes, and its results emphasise the need for monitoring and controlling the spread of ARGs in environmental settings., (Copyright © 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Gaining insight into the diversity, structure, and metabolic functions of microbial communities is essential for understanding their roles in host health and ecosystem dynamics. However, research on the seahorse-associated microbiome remains limited, despite these threatened fish facing increasing human pressures worldwide. Here, we explored the microbial diversity and metabolic functions of the skin and gut of the tiger tail seahorse ( Hippocampus comes ) and its surrounding environment using shotgun metagenomics and bioinformatics. Members of the Pseudomonadota phylum were dominant in the skin microbiome, whereas Bacteroidota was dominant in the gut. Bacillota, Actinomycetota, and Planctomycetota were also detected in the seahorse-associated microbiome. Statistical analysis revealed significant differences ( P < 0.01) in species diversity between skin and gut microbiomes, with members belonging to the Moraxellaceae family being dominant on the skin and the Bacteroidaceae family in the gut. Moreover, the surrounding environment (water or sediment) did not have a direct effect on the seahorse microbiome composition. The skin microbiome exhibited a higher abundance of functional genes related to energy, lipid, and amino acid metabolism as well as terpenoids and polyketides metabolism, xenobiotics biodegradation, and metabolism compared with the gut. Despite differences among classes, the total abundance of bacteriocins was similar in both gut and skin microbiomes, which is significant in shaping microbial communities due to their antimicrobial properties. A better knowledge of seahorse microbiomes benefits conservation and sustainable aquaculture efforts, offering insights into habitat protection, disease management, and optimizing aquaculture environments, thereby promoting seahorse health and welfare while minimizing environmental impact and enhancing aquaculture sustainability. NEW & NOTEWORTHY To the best of our knowledge, this study represents the first comprehensive examination of the taxonomic and functional patterns of the skin and gut microbiome in the tiger tail seahorse. These findings have the potential to significantly enhance our understanding of the seahorse-associated microbiome, thereby contributing to the prediction and control of bacterial infections in seahorses, which are a leading cause of high mass mortality rates in seahorse aquaculture and other fish species.

This study aimed to investigate the taxonomic and functional patterns of the microbiome associated with Barbour's seahorse (Hippocampus barbouri) using a combination of shotgun metagenomics and bioinformatics. The analyses revealed that Pseudomonadota and Bacillota were the dominant phyla in the seahorse skin microbiome, whereas Pseudomonadota and, to a lesser extent, Bacillota and Bacteroidota were the dominant phyla in the seahorse gut microbiome. Several metabolic pathway categories were found to be enriched in the skin microbiome, including amino acid metabolism, carbohydrate metabolism, cofactor and vitamin metabolism, energy metabolism, nucleotide metabolism, as well as membrane transport, signal transduction, and cellular community-prokaryotes. In contrast, the gut microbiome exhibited enrichment in metabolic pathways associated with the metabolism of terpenoids and polyketides, biosynthesis of other secondary metabolites, xenobiotics biodegradation and metabolism, and quorum sensing. Additionally, although the relative abundance of bacteriocins in the skin and gut was slightly similar, notable differences were observed at the class level. Specifically, class I bacteriocins were found to be more abundant in the skin microbiome, whereas class III bacteriocins were more abundant in the gut microbiome. To the best of our knowledge, this study represents the first comprehensive examination of the taxonomic and functional patterns of the skin and gut microbiome in Barbour's seahorse. These findings can greatly contribute to a deeper understanding of the seahorse-associated microbiome, which can play a pivotal role in predicting and controlling bacterial infections, thereby contributing to the success of aquaculture and health-promoting initiatives., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The effect of nanoemulsions prepared with grape seed and cinnamon essential oils on the shelf-life of flathead mullet (Mugil cephalus) fillets was evaluated by determining physicochemical (pH, free fatty acids, peroxide value, total volatile base nitrogen (TVB-N), and thiobarbituric acid reactive substances (TBARs)), sensory and microbiological (mesophilic aerobic bacteria, total psychrophilic bacteria, and Enterobacteriaceae counts) properties during 14 day storage at 2 °C., Results: The nanoemulsions showed good stability and low average droplet size. The results indicated that nanoemulsion treatments significantly prolonged the shelf-life of the fillets. Treatment inhibited increases in pH and TVB-N, and retarded lipid oxidation and hydrolysis. Sensory assessment revealed that treatment induced shelf-life extension from 10 to 14 days, compared with controls. Microbiological analyses showed nanoemulsion treatment caused shelf-life extension from 10 to 12 days with reduction of microbiological contamination by up to 1 log cfu g -1 in mesophilic and 1.5 log cfu g -1 in psychrotrophic bacteria., Conclusion: Considering the results, grape seed and cinnamon essential oil nanoemulsions could be considered as novel antimicrobial and antioxidant materials for shelf-life extension of flathead mullet fillets during cold storage. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Chemical analysis of an M1 agar plate cultivation of a marine fish-gut-derived fungus, Chrysosporium sp. CMB-F214, revealed the known chrysosporazines A-D ( 11 - 14 ) in addition to a suite of very minor aza analogues 1 - 6 . A microbioreactor (MATRIX) cultivation profiling analysis failed to deliver cultivation conditions that significantly improved the yields of 1 - 6 ; however, it did reveal that M2 agar cultivation produced the new natural product 15 . A precursor-directed biosynthesis strategy adopting supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible azachrysposorazines A1 ( 1 ), A2 ( 2 ), B1 ( 3 ), C1 ( 4 ), C2 ( 5 ) and D1 ( 6 ), in addition to four new chrysosporazines; chrysosporazines N-P ( 7 - 9 ) and spirochrysosporazine A ( 10 ). Structures inclusive of absolute configurations were assigned to 1 - 15 based on detailed spectroscopic and chemical analyses, and biosynthetic considerations. Non-cytotoxic to human carcinoma cells, azachrysosporazies 1 - 5 were capable of reversing doxorubicin resistance in P-glycoprotein (P-gp)-overexpressing human colon carcinoma cells (SW620 Ad300), with optimum activity exhibited by the C-2' substituted analogues 3 - 5 .

Commensal microbial communities have immense effects on their vertebrate hosts, contributing to a number of physiological functions, as well as host fitness. In particular, host immunity is strongly linked to microbiota composition through poorly understood bi-directional links. Gene expression may be a potential mediator of these links between microbial communities and host function. However, few studies have investigated connections between microbiota composition and expression of host immune genes in complex systems. Here, we leverage a large study of laboratory-raised fish from the species Gasterosteus aculeatus (three-spined stickleback) to document correlations between gene expression and microbiome composition. First, we examined correlations between microbiome alpha diversity and gene expression. Our results demonstrate robust positive associations between microbial alpha diversity and expression of host immune genes. Next, we examined correlations between host gene expression and abundance of microbial taxa. We identified 15 microbial families that were highly correlated with host gene expression. These families were all tightly correlated with host expression of immune genes and processes, falling into one of three categories-those positively correlated, negatively correlated, and neutrally related to immune processes. Furthermore, we highlight several important immune processes that are commonly associated with the abundance of these taxa, including both macrophage and B cell functions. Further functional characterization of microbial taxa will help disentangle the mechanisms of the correlations described here. In sum, our study supports prevailing hypotheses of intimate links between host immunity and gut microbiome composition. IMPORTANCE Here, we document associations between host gene expression and gut microbiome composition in a nonmammalian vertebrate species. We highlight associations between expression of immune genes and both microbiome diversity and abundance of specific microbial taxa. These findings support other findings from model systems which have suggested that gut microbiome composition and host immunity are intimately linked. Furthermore, we demonstrate that these correlations are truly systemic; the gene expression detailed here was collected from an important fish immune organ (the head kidney) that is anatomically distant from the gut. This emphasizes the systemic impact of connections between gut microbiota and host immune function. Our work is a significant advancement in the understanding of immune-microbiome links in nonmodel, natural systems., (Copyright © 2021 Fuess et al.)

Using a molecular networking guided strategy, chemical analysis of the Australian mullet fish gastrointestinal tract-derived fungus Amauroascus sp. CMB-F713 yielded a family of polyketide pyrones, amaurones A-I ( 1 - 9 ), featuring an unprecedented carbon skeleton. Structures were assigned to 1 - 9 by detailed spectroscopic analysis (including X-ray analysis of 1 ), biosynthetic considerations, and chemical interconversions. For example, the orthoacetate 5 was unstable when stored dry at room temperature, transforming to the monoacetates 2 and 3 , while mild heating (40 °C) prompted quantitative conversion of 3 to 2 , via an intramolecular trans -acetylation. Likewise, during handling, the monoacetate 1 was prone to intramolecular trans -acetylation, leading to an equilibrium mixture with the isomeric monoacetate amaurone J ( 10 ), confirmed when partial hydrolysis of the diacetate 2 yielded the monoacetates 1 and 10 and the triol amaurone K ( 11 ).

Vertebrates' gut microbial communities can be altered by the hosts' parasites. Helminths inhabiting the gut lumen can interact directly with their host's microbiota via physical contact, chemical products, or competition for nutrients. Indirect interactions can also occur, for instance when helminths induce or suppress host immunity in ways that have collateral effects on the microbiota. If there is genetic variation in host immune responses to parasites, we would expect such indirect effects to be conditional on host genotype. To test for such genotype by infection interactions, we experimentally exposed Gasterosteus aculeatus to their naturally co-evolved parasite, Schistocephalus solidus. The host microbiota differed in response to parasite exposure, and between infected and uninfected fish. The magnitude and direction of microbial responses to infection differed between host sexes, and also differed between variants at autosomal quantitative trait loci. These results indicate that host genotype and sex regulate the effect of helminth infection on a vertebrate gut microbiota. If this result holds in other taxa, especially humans, then helminth-based therapeutics for dysbiosis might need to be tailored to host genotype and sex.

Enteritis comprises one of the most common diseases affecting the survival of farmed yellow seahorse (Hippocampus kuda), an important economic fish species cultured worldwide. Although there are several studies describing bacteria associated with seahorse, the microbial alternations associated with enteritis in seahorse has not been extensively investigated. In the present study, high-throughput 16S rRNA gene sequencing was used to explore the changes in the intestinal microbiota of seahorse suffering from enteritis. The results showed that the diversity, structure, and function of intestinal microbiota were significantly different between healthy and diseased seahorse. Particularly, significant increase was observed in Brevinema, Mycobacterium, and Vibrio, as well as significant decrease in Psychrobacter, Bacillus, and Shewanella in diseased seahorse (P < 0.05). In addition, PICRUSt predictions revealed that the intestinal microbiota significantly changed the specific metabolic pathways (related to metabolic diseases, replication and repair, transport and catabolism, infectious diseases and immune system) in diseased seahorse (P < 0.05). Altogether, our findings point out the association between changes of the intestinal microbiota and enteritis in seahorse, which provide basic information useful for optimization of breeding regimes and improvements in the health of this endangered species in captivity.

Tail rot disease is associated with major economic losses in the seahorse aquaculture in China. This study aimed to isolate and identify the pathogen causing tail rot disease in seahorses. Three culturable intestinal bacteria strains were isolated from Hippocampus kuda specimens with tail rot disease. Strain HL11, HL12, and HL13 were identified as Pseudoalteromonas spongiae, Bacillus subtilis and Photobacterium ganghwense based on its morphological characteristics, physiological and biochemical properties, through 16S rRNA and gyrB sequencing, respectively. Challenge experiments using these strains on healthy H. kuda and bacterial re-isolation from challenged diseased seahorses showed that the bacteria strain named HL11 induced identical pathological symptoms, indicating that it is the causative pathogen of the disease. Antibiotic-resistance tests against of 32 antibiotics revealed that HL11 was highly sensitive to 13 kinds, while exhibited intermediate susceptibility to 6, and resistance to 13 kinds. Antibacterial tests of the bioactive agents showed that HL11 was susceptible to five kinds, including tea polyphenols, lactic acid, gallic acid, allicin, and polylysine; however, it was not susceptible to the other 13 kinds of bioactive agents. The results demonstrate the potential of using bioactive agents to replace antibiotics to generate an environmentally friendly mode of culturing seahorses., Competing Interests: The authors have declared that no competing interests exist.

The processes of local adaptation and ecological speciation are often strongly shaped by biotic interactions such as competition and predation. One of the strongest lines of evidence that biotic interactions drive evolution comes from the repeated divergence of lineages in association with repeated changes in the community of interacting species. Yet relatively little is known about the repeatability of changes in gut microbial communities and their role in adaptation and divergence of host populations in nature. Here we use three cases of rapid, parallel adaptation and speciation in freshwater threespine stickleback to test for parallel changes in associated gut microbiomes. We find that features of the gut microbial communities have shifted repeatedly in the same direction in association with parallel divergence and speciation of stickleback hosts. These results suggest that changes to gut microbiomes can occur rapidly and predictably in conjunction with host evolution, and that host-microbe interactions might play an important role in host adaptation and diversification.

This study addressed the bacteriocin production in 116 lactic acid bacteria isolated from 143 fish and seafood samples. The screening for the production of antibacterial substances allowed for the selection of 16 LAB isolates endowed with inhibitory capability. Bacteriocins (bacLP17 and bacLP18) of two strains, Enterococcus mundtii LP17 and Enterococcus mundtii LP18, respectively, isolated from red mullet and sardine samples, determined large inhibition zones against all the Listeria species. Virulence traits and antibiotic resistances of all producers were verified, and no isolates presented dangerous characteristics, including the two best bacteriocin producers E. mundtii LP17 and E. mundtii LP18, which were subsequently investigated for their potential use in fish and seafood products biopreservation. For both strains, the highest level of bacteriocin production (1280 AU/ml) was recorded when cells were grown at 30 °C in MRS broth at pH ranging from 6.0 to 9.0, and high levels of adsorption of bacteriocins, bacLP17 and bacLP18, to the target cells Listeria monocytogenes were also observed. The results obtained in this study revealed that two strains of E. mundtii originating from seafood exhibited a strong inhibitory activity against L. monocytogenes and may be useful in controlling the growth of this pathogen in the same food products.

Soiny mullet (Liza haematocheila) is an important economic fish species in China, but stress and diseases have seriously restricted its culture. There are no effective methods including vaccines to prevent or control these diseases. Alternative methods should be employed, such as using novel immunostimulant poly-β-hydroxybutyrate (PHB). The present study aimed to evaluate effects of dietary PHB supplementation on the growth, antioxidant enzymes activity, immune-related genes expression and intestinal microbiota in soiny mullet. The fish was fed for 30 or 60 days with six diets at different PHB supplementation of 0, 0.5, 1, 2, 4 and 8%, named as groups P0, P0.5, P1, P2, P4 and P8. The results showed that the weight gain and specific growth rate of fish in P2 and P0.5 groups were significantly higher than those in control P0 group at 30 and 60 days, respectively (P < 0.05). The antioxidant enzymes activity of catalase and superoxide dismutase in serum were significantly increased in P0.5/P1/P2 groups after 30 days. The transcriptional levels of penicillin-binding protein A and interleukin-8 analyzed by qRT-PCR were significantly upregulated in P2 and P4 groups compared to those in P0/P0.5/P1/P8 groups at 30 days. The transcriptional level of major histocompatibility complex class II in P2 group was significantly upregulated, and aldehyde oxidase downregulated compared to P0 group. Intestinal microbiota analysis by Illumina high-throughput sequencing showed that the microbiota diversity was not changed significantly, but the microbiota structure shifted significantly post PHB treatment. At the phyla level, Firmicutes and Proteobacteria were predominant in both P0 and P2 groups. At the genus level, the relative abundance of Bacillus spp. in P2 group increased significantly, and abundance of Achromobacter spp. decreased significantly. KEGG pathway analysis by PICRUSt showed that oral administration PHB significantly upregulated abundances of genes responsible for 10 pathways and downregulated genes involved in 17 pathways. In conclusion, soiny mullet fed with 2% PHB supplemental diets for 30 days showed better growth performance, higher antioxidant enzymes activity and immune-related genes expression. Their regulation of growth and immunity might be related with the intestinal microbiota change post PHB supplementation. It will provide very useful basic information to study the regulation mechanism of PHB in aquatic animals, and provide good green method to prevent disease in soiny mullet., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Much of animal-associated microbiome research has been conducted in&nbsp;species&nbsp;for which little is known of their natural ecology and evolution.&nbsp;Microbiome studies that combine population genetic, environment, and geographic data for wild organisms can be very informative, especially in situations where host genetic variation and the environment both influence microbiome variation.&nbsp;The few studies that have&nbsp;related&nbsp;population genetic&nbsp;and microbiome variation in wild populations&nbsp;have been constrained by observation-based kinship data or&nbsp;incomplete genomic information. Here we&nbsp;integrate population genomic&nbsp;and microbiome analyses&nbsp;in wild threespine stickleback fish distributed throughout western Oregon, USA. We found that gut microbiome diversity and composition partitioned more among than within wild host populations and was better explained by host population genetic divergence than&nbsp;by environment and geography. We also identified gut microbial taxa that were most differentially abundant across environments and across genetically divergent populations. Our findings highlight the benefits of studies that investigate host-associated microbiomes in wild organisms., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; nor in the decision to publish the results.

TLR7 subfamily members are important pattern recognition receptors participating in the recognition of pathogen-associated molecular patterns. In this study, we successfully identified 3 members of TLR7 subfamily from the spiny eel Mastacembelus armatus (MaTLR7, MaTLR8 and MaTLR9). The amino acid sequence identities of MaTLR7 and MaTLR8 with Monopterus albus TLR7 were 87.2% and 76.5%, respectively and the identity of MaTLR9 with Seriola lalandi TLR9 was 74.7%. The phylogenetic analysis revealed MaTLRs showed close relationship to other species in Synbranchiformes or Perciformes. Quantitative real-time PCR analysis revealed that they were expressed in all tested tissues and higher expression was found in spleen or gill. After infection with Aeromonas veronii, expression of MaTLR7, MaTLR8 and MaTLR9 were all significantly downregulated in spleen and kidney. Evolutionary analysis suggested that the ancestral lineages of teleost TLR8 and TLR9 had been subject to positive selection pressures and multiple Maximum likelihood methods recovered 3 positively selected sites in teleost TLR7, 4 in TLR8 and 8 in TLR9. Domain distribution revealed most positively selected sites were located in leucine-rich repeat domain. Our results will contribute to better understanding the antibacterial mechanism of TLRs and their co-evolution with pathogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The structural and evolutionary linkage between tumor necrosis factor (TNF) and the globular C1q (gC1q) domain defines the C1q and TNF-related proteins (CTRPs), which are involved in diverse functions such as immune defense, inflammation, apoptosis, autoimmunity, and cell differentiation. In this study, red-lip mullet (Liza haematocheila) CTRP4-like (MuCTRP4-like), CTRP5 (MuCTRP5), CTRP6 (MuCTRP6), and CTRP7 (MuCTRP7) were identified from the red-lip mullet transcriptome database and molecularly characterized. According to in silico analysis, coding sequences of MuCTRP4-like, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of 1128, 753, 729, and 888 bp open reading frames (ORF), respectively and encoded 375, 250, 242, and 295 amino acids, respectively. All CTRPs possessed a putative C1q domain. Additionally, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of a collagen region. Phylogenetic analysis exemplified that MuCTRPs were distinctly clustered with the respective CTRP orthologs. Tissue-specific expression analysis demonstrated that MuCTRP4-like was mostly expressed in the blood and intestine. Moreover, MuCTRP6 was highly expressed in the blood, whereas MuCTRP5 and MuCTRP7 were predominantly expressed in the muscle and stomach, respectively. According to the temporal expression in blood, all MuCTRPs exhibited significant modulations in response to polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). MuCTRP4-like, MuCTRP5, and MuCTRP6 showed significant upregulation in response to lipopolysaccharides (LPS). The results of this study suggest the potential involvement of Mullet CTRPs in post-immune responses., (Copyright © 2018. Published by Elsevier Ltd.)

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant metalloenzyme. The main function of this enzyme is to dismutase the toxic superoxide anion (O 2 -) into less toxic hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 ). Structural analysis of mullet MnSOD (MuMnSOD) was performed using different bioinformatics tools. Pairwise alignment revealed that the protein sequence matched to that derived from Larimichthys crocea with a 95.2% sequence identity. Phylogenetic tree analysis showed that the MuMnSOD was included in the category of teleosts. Multiple sequence alignment showed that a SOD Fe-N domain, SOD Fe-C domain, and Mn/Fe SOD signature were highly conserved among the other examined MnSOD orthologs. Quantitative real-time PCR showed that the highest MuMnSOD mRNA expression level was in blood cells. The highest expression level of MuMnSOD was observed in response to treatment with both Lactococcus garvieae and lipopolysaccharide (LPS) at 6 h post treatment in the head kidney and blood. Potential ROS-scavenging ability of the purified recombinant protein (rMuMnSOD) was examined by the xanthine oxidase assay (XOD assay). The optimum temperature and pH for XOD activity were found to be 25 °C and pH 7, respectively. Relative XOD activity was significantly increased with the dose of rMuMnSOD, revealing its dose dependency. Activity of rMuMnSOD was inhibited by potassium cyanide (KCN) and N-N'-diethyl-dithiocarbamate (DDC). Moreover, expression of MuMnSOD resulted in considerable growth retardation of both gram-positive and gram-negative bacteria. Results of the current study suggest that MuMnSOD acts as an antioxidant enzyme and participates in the immune response in mullet., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Two closely related isolates, 27335 T and 24999, of rapidly growing, non-pigmented mycobacteria, were cultured from two clinically ill fish of the family Syngnathidae. Whole genome sequencing of the two isolates revealed low sequence homology to documented mycobacteria within public databases such as the NCBI. Evaluation of targeted housekeeping genes, including 16S rRNA, ITS, rpoB and hsp65, related the two bacteria distantly to Mycobacterium senegalense CK2 M4421 and Mycobacterium farcinogenes DSM 43637. Phenotypic, biochemical and dDNA-DNA hybridization tests demonstrated that Mycobacterium syngnathidarum is a new species distinct from other recognized rapidly growing mycobacterial species. Phenotypic, chemotaxonomic and phylogenetic data evaluation provided evidence that the two strains represent one novel species. We propose the formal recognition of Mycobacterium syngnathidarum sp. nov., with isolate 27335 T as the type strain (=ATCC TSD-89 T ,=DSM 105112 T ).

Aims: Aeromonas hydrophila has been isolated from various fish species in Egypt and is known to carry virulence and antimicrobial resistance genes, which pose a risk for public health. The aim of the present study is to report, for the first time, the infection of mullet (Mugil cephalus) with A. hydrophila and to clarify the potential association between antimicrobial resistance and virulence traits encoded in A. hydrophila., Methods and Results: In this study, the occurrence of A. hydrophila in marketed mullet and the antimicrobial resistance phenotypes of these isolates were determined. Aeromonas hydrophila isolates were screened for the presence of virulence and β-lactam resistance genes; the correlation between both gene groups was also investigated. The infection rate of examined mullet with A. hydrophila was 37% (50/135). The highest antimicrobial resistance was detected to cefoxitin (100%), followed by ampicillin (84%), ceftazidime (56%) and cefotaxime (40%). Only 4% of the isolates were resistant to erythromycin; 6% were resistant to both gentamicin and kanamycin with no resistance to ciprofloxacin. Variable frequencies of virulence and β-lactam resistance genes were evident from PCR, where aerA and bla TEM predominated. The study also indicated a general weak positive correlation (R = 0·3) between both virulence and β-lactam resistance genes. Some of the studied virulence genes (e.g. aerA:hlyA and hlyA:ast) were found to correlate positively., Conclusions: The presence of virulence and resistance genes in A. hydrophila from food sources poses a serious threat to public health. To our knowledge, this is the first report describing the occurrence of A. hydrophila in mullet and highlighting the coexistence of virulence and β-lactam resistance genes encoded by these bacteria., Significance and Impact of the Study: These data provide insights into the potential association of antimicrobial resistance and virulence genes in A. hydrophila from marketed mullet in Egypt, which could pose threats to humans even if a weak positive correlation exists between both genes., (© 2018 The Society for Applied Microbiology.)

Glutathione S-transferases (GSTs, EC 2.5.1.18) are important Phase II detoxifying enzymes that catalyze hydrophobic, electrophilic xenobiotic substance with the conjugation of reduced glutathione (GSH). In this study, GSTμ and GSTρ paralogs of GST in the big belly seahorse (Hippocampus abdominalis; HaGSTρ, HaGSTμ) were biochemically, molecularly, functionally characterized to determine their detoxification range and protective capacities upon different pathogenic stresses. HaGSTρ and HaGSTμ are composed of coding sequences of 681bp and 654bp, which encode proteins 225 and 217 amino acids, with predicted molecular masses of 26.06kDa and 25.74kDa respectively. Sequence analysis revealed that both HaGSTs comprise the characteristic GSH-binding site in the thioredoxin-like N-terminal domain and substrate binding site in the C-terminal domain. The recombinant HaGSTρ and HaGSTμ proteins catalyzed the model GST substrate 1-chloro-2, 4-dinitrobenzene (CDNB). Enzyme kinetic analysis revealed different K m and V max values for each rHaGST, suggesting that they have different conjugation rates. The optimum conditions (pH, temperature) and inhibitory assays of each protein demonstrated different optimal ranges. However, HaGSTμ was highly expressed in the ovary and gill, whereas HaGSTρ was highly expressed in the gill and pouch. mRNA expression of HaGSTρ and HaGSTμ was significantly elevated upon lipopolysaccharide, Poly (I:C), and Edwardsiella tarda challenges in liver and in blood cells as well as with Streptococcus iniae challenge in blood cells. From these collective experimental results, we propose that HaGSTρ and HaGSTμ are effective in detoxifying xenobiotic toxic agents, and importantly, their mRNA expression could be stimulated by immunological stress signals in the aquatic environment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recent studies of interactions between hosts and their resident microbes have revealed important ecological and evolutionary consequences that emerge from these complex interspecies relationships, including diseases that occur when the interactions go awry. Given the preponderance of these interactions, we hypothesized that effects of the microbiota on gene expression in the developing gut-an important aspect of host biology-would be pervasive, and that these effects would be both comparable in magnitude to and contingent on effects of the host genetic background. To evaluate the effects of the microbiota, host genotype, and their interaction on gene expression in the gut of a genetically diverse, gnotobiotic host model, the threespine stickleback (Gasterosteus aculeatus), we compared RNA-seq data among 84 larval fish. Surprisingly, we found that stickleback population and family differences explained substantially more gene expression variation than the presence of microbes. Expression levels of 72 genes, however, were affected by our microbiota treatment. These genes, including many associated with innate immunity, comprise a tractable subset of host genetic factors for precise, systems-level study of host-microbe interactions in the future. Importantly, our data also suggest subtle signatures of a statistical interaction between host genotype and the microbiota on expression patterns of genetic pathways associated with innate immunity, coagulation and complement cascades, focal adhesion, cancer, and peroxisomes. These genotype-by-environment interactions may prove to be important leads to the understanding of host genetic mechanisms commonly at the root of sometimes complex molecular relationships between hosts and their resident microbes., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Phenotypic changes in response to environmental influences can persist from one generation into the next. In many systems parental parasite experience influences offspring immune responses, known as transgenerational immune priming (TGIP). TGIP in vertebrates is mainly maternal and short-term, supporting the adaptive immune system of the offspring during its maturation. However, if fathers and offspring have a close physical connection, evolution of additional paternal immune priming can be adaptive. Biparental TGIP may result in maximized immunological protection. Here, we investigate multigenerational biparental TGIP in the sex-role reversed pipefish Syngnathus typhle by exposing grandparents to an immune challenge with heat-killed bacteria and assessing gene expression (44 target genes) of the F2-generation., Results: Grandparental immune challenge induced gene expression of immune genes in one-week-old grandoffspring. Similarly, genes mediating epigenetic regulation including DNA-methylation and histone modifications were involved in grandparental immune priming. While grand-maternal impact was strong on genes of the complement component system, grand-paternal exposure changed expression patterns of genes mediating innate immune defense., Conclusion: In a system with male pregnancy, grandparents influenced the immune system of their grandoffspring in a sex-specific manner, demonstrating multigenerational biparental TGIP. The involvement of epigenetic effects suggests that TGIP via the paternal line may not be limited to the pipefish system that displays male pregnancy. While the benefits and costs of grandparental TGIP depend on the temporal heterogeneity of environmental conditions, multigenerational TGIP may affect host-parasite coevolution by dampening the amplitude of Red Queen Dynamics.

Four soybean meal-based diets containing increasing levels of an enzyme complex (E50, E100, E150 and E200 at 50, 100, 150 and 200 g ton-1, respectively) and one soybean meal-based diet without the enzyme complex (E0) were fed in triplicate to M. liza juveniles in a semi-static flow system with 20 fish per tank for 75 days. There were no differences between the treatments for animal performance parameters, but fish fed the enzyme complex treatment exhibited significantly (P<0.05) higher values of calcium bone retention compared with control fish. Although there was no relationship between bacterial counts in different sections of the gastrointestinal tract or enzyme levels, filamentous bacteria were increased in E50 compared with E150. All of the treatments resulted in higher bacterial counts in the stomach than in intestinal segments. Histological screening showed serious to moderate infiltration of inflammatory cells, modification in villus morphology and necrosis in some cases in fish fed the E0 diet. In addition, fish from the E0 treatment exhibited significantly (P<0.05) lower lipid deposition in the peritoneal cavity. Therefore, the use of low levels of exogenous enzyme is recommended in diets for M. liza when soybean meal is used as the main source of protein.

Beta-defensins are important part of innate immunity of fish, which are the first defense line against invading pathogens. In this study, the β-defensin (Lhβ-defensin) gene was cloned from spleen tissue of soiny mullet (Liza haematocheila). Lhβ-defensin cDNA was 747 bp in length, encoding 63 amino acids. Sequence alignment revealed that Lhβ-defensin contained six conserved cysteine residues and shared 97.5% sequence identities with grouper (Epinephelus coioides) β-defensin. Realtime PCR revealed that Lhβ-defensin was highest expressed in the immune related organs, such as spleen, kidney and gut of healthy fish. Following Streptococcus dysgalactiae infection, Lhβ-defensin was up-regulated in immune related organs, e.g. 17.6-fold in spleen and 10.87-fold in gut at 24 h post infection (hpi). Lhβ-defensin possessed a monomeric structure of a three-stranded anti-parallel β-sheet and an α-helix stabilized by three disulfide bonds formed by Cys30-Cys58, Cys36-Cys52, and Cys40-Cys59. In addition to the experimental work, computer simulation was also carried out to determine the possible conformation of β-defensin and its interaction with palmitoyloleoylphosphatidylglycerol (POPG), a model of bacteria membrane. The Lhβ-defensin was found to form dimeric structure stabilized by the van der Waals contacts of Leu35 and Cys37 in two anti-parallel β1-strands and the cation-π interaction between Tyr32 and Arg54 respectively in the two β1-strands. The most important interactions between β-defensin and membrane are the electrostatic interactions between Arg residues in β-defensin and head group of POPG bilayer as well as hydrogen bond interactions between them. Our results were useful for further understanding the potential mechanism of antimicrobial property of fish β-defensins.

Soiny mullet (Liza haematocheila) is becoming an economically important aquaculture mugilid species in China and other Asian countries. However, increasing incidences of bacterial pathogenic diseases has greatly hampered the production of the soiny mullet. Deeper understanding of the soiny mullet immune system and its related genes in response to bacterial infections are necessary for disease control in this species. In this study, the transcriptomic profile of spleen from soiny mullet challenged with Streptococcus dysgalactiae was analyzed by Illumina-based paired-end sequencing method. After assembly, 86,884 unique transcript fragments (unigenes) were assembled, with an average length of 991 bp. Approximately 41,795 (48.1%) unigenes were annotated in the nr NCBI database and 57.9% of the unigenes were similar to that of the Nile tilapia. A total of 24,299 unigenes were categorized into three Gene Ontology (GO) categories (molecular function, cellular component and biological process), 13,570 unigenes into 25 functional Clusters of Orthologous Groups of proteins (COG) categories, and 30,547 unigenes were grouped into 258 known pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Following S. dysgalactiae infection, 11,461 differentially expressed unigenes were identified including 4658 up-regulated unigenes and 6803 down-regulated unigenes. Significant enrichment analysis of these differentially expressed unigenes identified major immune related pathways, including the Toll-like receptor, complement and coagulation cascades, T cell receptor signaling pathway and B cell receptor signaling pathway. In addition, 24,813 simple sequence repeats (SSRs) and 127,503 candidate single nucleotide polymorphisms (SNPs) were identified from the mullet spleen transcriptome. To this date, this study has globally analyzed the transcriptome profile from the spleen of L. haematocheila after S. dysgalactiae infection. Therefore, the results of our study contributes to better on the immune system and defense mechanisms of soiny mullet in response to bacterial infection, and provides valuable references for related studies in mugilidae species which currently lack genomic reference., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).

An orange-pigmented, Gram-staining-negative, rod-shaped bacterium, designated 96&#95;Hippo&#95;TS&#95;3/13(T) was isolated from the brood pouch of a diseased seahorse male of the species Hippocampus barbouri from the animal facility of the University of Giessen, Germany. Phylogenetic analyses based on the nearly full-length 16S rRNA gene sequence placed strain 96&#95;Hippo&#95;TS&#95;3/13(T) into the monophyletic cluster of the genus Mesonia within the family Flavobacteriaceae. However, the strain shared only 92.2-93.8% sequence similarity to type strains of species of the genus Mesonia, with highest sequence similarity to the type strain of Mesonia aquimarina. Cellular fatty acid analysis showed a Mesonia-typical fatty acid profile including several branched and hydroxyl fatty acids with highest amounts of iso-C15 : 0 (40.9%) followed by iso-C17 : 0 3-OH (14.8%). In the polyamine pattern, sym-homospermidine was predominant. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The quinone system contained exclusively menaquinone MK-6. The only identified compound in the polar lipid profile was phosphatidylethanolamine present in major amounts. Additionally, major amounts of an unidentified aminolipid and two unidentified lipids not containing a phosphate group, an amino group or a sugar residue were detected. The genomic G+C content of strain 96&#95;Hippo&#95;TS&#95;3/13(T) was 30 mol%. Based on genotypic, chemotaxonomic and physiological characterizations we propose a novel species of the genus Mesonia, Mesonia hippocampi sp. nov., with strain 96&#95;Hippo&#95;TS&#95;3/13(T) ( = CIP 110839T =  LMG 28572(T) = CCM 8557(T)) as the type strain. An emended description of the genus Mesonia is also provided.

In the Mediterranean area, salted and dried roe from the gray Mugil cephalus "bottarga" represent a speciality food with great commercial value. Bottarga is currently produced by a traditional handmade process and, the risk of human bacterial contamination during its manufacturing is still unknown; in this perspective the foodborne pathogen Staphylococcus aureus could potentially contaminate this product due to poor sanitation or bad handling during processing. The aim of this work is: to evaluate the contamination level of foodborne pathogens at different product manufacturing stages and, in addition, to describe a fast and realizable method for the rapid detection of S. aureus in bottarga samples in the field. A cultural procedure was initially used to investigate the occurrence of S. aureus and the other main foodborne pathogens in bottarga samples at the different manufacturing stages (from roe to final product). In addition, a molecular approach was used to rapidly determine the presence of total bacteria, S. aureus, and its potential toxigenicity. Of the 194 specimens analyzed, we identified: Clostridium perfringens, Enterococcus spp. and Enterobacteriaceae. However, some samples resulted as being contaminated with S. aureus (4% in roe and 8.7% in the final product). During the bottarga manufacturing process, we observed an increase in pathogen levels (from 10(2) to 10(5) CFU/g) in contaminated samples, and entA and entB genotypes were identified. Reconstruction experiments suggest that the fresh roe and the bottarga (not completely dried) could represent a risk for the contamination and growth of pathogen bacteria., (© 2015 Institute of Food Technologists®)

Seahorses, pipefish and seadragons are fish of the Family Syngnathidae. From 1998 to 2010, 172 syngnathid cases from the Toronto Zoo were submitted for post-mortem diagnostics and retrospectively examined. Among the submitted species were yellow seahorses Hippocampus kuda Bleeker (n=133), pot-bellied seahorses Hippocampus abdominalis Lesson (n=35) and weedy seadragons Phyllopteryx taeniolatus (Lacépède; n=4). The three most common causes of morbidity and mortality in this population were bacterial dermatitis, bilaterally symmetrical myopathy and mycobacteriosis, accounting for 24%, 17% and 15% of cases, respectively. Inflammatory processes were the most common diagnoses, present in 117 cases. Seven neoplasms were diagnosed, environmental aetiologies were identified in 46 cases, and two congenital defects were identified., (© 2014 John Wiley & Sons Ltd.)

The microsporidium Glugea gasterostei from the three-spined stickleback Gasterosteus aculeatus was described as an independent species basing upon morphological and ecological traits of the parasite (Voronin, 1974), further supported by ultrastructural characters of its spores (Voronin, 1983). During the revision of microsporidia of the genus Glugea (Canning, Lom, 1986; Lom, 2002), the validity of this species was doubted and it was synonymized with G. anomala. Nevertheless, the molecular phylogenetic analysis performed in the present study showed the unique molecular haplotype of small subunit rRNA gene of G. gasterostei (Genbank accession number KM977990) and its close relatedness to G. anomala, G. atherinae and G. hertwigi (sequence similarity of 99.7 %). One of typical characters of G. gasterostei, as opposed to G. anomala, is the formation of xenomas on inner tissues and not on the surface of infected fishes. This feature is retained even after the infection of different host species. Taken together, these data confirm the validity of G. gasterostei as a separate species among closely related taxa that had diverged comparatively recently.

Animals maintain complex microbial communities within their guts that fill important roles in the health and development of the host. To what degree a host's genetic background influences the establishment and maintenance of its gut microbial communities is still an open question. We know from studies in mice and humans that external factors, such as diet and environmental sources of microbes, and host immune factors play an important role in shaping the microbial communities (Costello et al. ). In this issue of Molecular Ecology, Bolnick et al. (2014a) sample the gut microbial community from 150 genetically diverse stickleback isolated from a single lake to provide evidence that another part of the adaptive immune response, the major histocompatibility complex class II (MHCII) receptors of antigen-presenting cells, may play a role in shaping the gut microbiota of the threespine stickleback, Gasterosteus aculeatus (Bolnick et al. 2014a). Bolnick et al. (2014a) provide insight into natural, interindividual variation in the diversity of both stickleback MHCII alleles and their gut microbial communities and correlate changes in the diversity of MHCII receptor alleles with changes in the microbiota., (© 2014 John Wiley & Sons Ltd.)

Animals harbour diverse communities of symbiotic bacteria, which differ dramatically among host individuals. This heterogeneity poses an immunological challenge: distinguishing between mutualistic and pathogenic members of diverse and host-specific microbial communities. We propose that Major Histocompatibility class II (MHC) genotypes contribute to recognition and regulation of gut microbes, and thus, MHC polymorphism contributes to microbial variation among hosts. Here, we show that MHC IIb polymorphism is associated with among-individual variation in gut microbiota within a single wild vertebrate population of a small fish, the threespine stickleback. We sampled stickleback from Cedar Lake, on Vancouver Island, and used next-generation sequencing to genotype the sticklebacks' gut microbiota (16S sequencing) and their MHC class IIb exon 2 sequences. The presence of certain MHC motifs was associated with altered relative abundance (increase or decrease) of some microbial Families. The effect sizes are modest and entail a minority of microbial taxa, but these results represent the first indication that MHC genotype may affect gut microbiota composition in natural populations (MHC-microbe associations have also been found in a few studies of lab mice). Surprisingly, these MHC effects were frequently sex-dependent. Finally, hosts with more diverse MHC motifs had less diverse gut microbiota. One implication is that MHC might influence the efficacy of therapeutic strategies to treat dysbiosis-associated disease, including the outcome of microbial transplants between healthy and diseased patients. We also speculate that macroparasite-driven selection on MHC has the potential to indirectly alter the host gut microbiota, and vice versa., (© 2014 John Wiley & Sons Ltd.)

A Gram-positive, aerobic, non-motile, non-sporulating, acid-fast, and rod-shaped bacterium (BFLP-6(T)), previously isolated from a seahorse (Hippocampus guttulatus) with tail rot, was studied using a polyphasic taxonomic approach. Growth occurred at 15-35 °C (optimum 25 °C), at pH 5.0-10.0 (optimum pH 7.0) and at NaCl concentrations between 0 and 6 % (w/v). The G+C content of DNA was 66.7 mol%. The predominant fatty acids were C(18:1) ω9c, C(16:0) and C(16:1) ω6c. A mycolic acid pattern of alpha-mycolates and keto-mycolates was detected. Analysis of concatenated sequences (16S rRNA, rpoB, ssrA and tuf genes), and chemotaxonomic and phenotypic features indicated that strain BFLP-6(T) represents a novel species within the genus Mycobacterium, for which the name Mycobacterium hippocampi sp. nov. is proposed. The type strain is BFLP-6(T) (=DSM 45391(T) =LMG 25372(T)).

Vertebrates harbour diverse communities of symbiotic gut microbes. Host diet is known to alter microbiota composition, implying that dietary treatments might alleviate diseases arising from altered microbial composition ('dysbiosis'). However, it remains unclear whether diet effects are general or depend on host genotype. Here we show that gut microbiota composition depends on interactions between host diet and sex within populations of wild and laboratory fish, laboratory mice and humans. Within each of two natural fish populations (threespine stickleback and Eurasian perch), among-individual diet variation is correlated with individual differences in gut microbiota. However, these diet-microbiota associations are sex dependent. We document similar sex-specific diet-microbiota correlations in humans. Experimental diet manipulations in laboratory stickleback and mice confirmed that diet affects microbiota differently in males versus females. The prevalence of such genotype by environment (sex by diet) interactions implies that therapies to treat dysbiosis might have sex-specific effects.

A taxonomic study was carried out on strain HYC-10(T), which was isolated from the intestinal tract contents of a flathead mullet, Mugil cephalus, captured from the sea off Xiamen Island, China. The bacterium was observed to be Gram positive, oxidase and catalase positive, rod shaped, and motile by subpolar flagella. The bacterium was found to grow at salinities of 0-12 % and at temperatures of 8-45 °C. The isolate was found to hydrolyze aesculin and gelatin, but was unable to reduce nitrate to nitrite. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HYC-10(T) belongs to the genus Bacillus, with highest sequence similarity (99.3 %) to Bacillus aerophilus 28K(T), Bacillus stratosphericus 41KF2a(T) and Bacillus altitudinis DSM 21631(T), followed by Bacillus safensis DSM 19292(T) (99.5 %) and Bacillus pumilus DSM 27(T) (99.5 %), while the sequence similarities to others were all below 97.6 %. The genomic ANIm values between strain HYC-10(T) and three type strains (B. altitudinis DSM 21631(T), B. safensis DSM 19292(T) and B. pumilus DSM 27(T)) were determined to range from 89.11 to 91.53 %. The DNA-DNA hybridization estimate values between strain HYC-10(T) and the three type strains were from 36.60 to 44.00 %. The principal fatty acids identified were iso-C15:0 (39.1 %), anteiso-C15:0 (22.7 %), iso-C17:0 (13.1 %), C16:0 (6.1 %), anteiso-C17:0 (5.8 %) and iso-C16:0 (5.1 %). The G+C content of the chromosomal DNA was determined from the draft genome sequence to be 41.3 mol%. The respiratory quinone was determined to be MK-7 (100 %). Phosphatidylglycerol, diphosphatidylglycerol, aminoglycolipid, two glycolipids and two unknown phospholipids were found to be present. The combined genotypic and phenotypic data show that strain HYC-10(T) represents a novel species of the genus Bacillus, for which the name Bacillus xiamenensis sp. nov. is proposed, with the type strain HYC-10(T) (=CGMCC NO.1.12326(T) = LMG 27143(T) = MCCC 1A00008(T)).

Chlamydia are obligate intracellular bacteria and important pathogens of humans and animals. Chlamydia-related bacteria are also major fish pathogens, infecting epithelial cells of the gills and skin to cause the disease epitheliocystis. Given the wide distribution, ancient origins and spectacular diversity of bony fishes, this group offers a rich resource for the identification and isolation of novel Chlamydia. The broad-nosed pipefish (Syngnathus typhle) is a widely distributed and genetically diverse temperate fish species, susceptible to epitheliocystis across much of its range. We describe here a new bacterial species, Candidatus Syngnamydia venezia; epitheliocystis agent of S. typhle and close relative to other chlamydial pathogens which are known to infect diverse hosts ranging from invertebrates to humans.

A retrospective study of the pathologic findings in weedy (Phyllopteryx taeniolatus) and leafy (Phycodurus eques) seadragons was performed on specimens submitted to 2 reference laboratories from 1994 to 2012 to determine the range and occurrence of diseases affecting aquarium-held populations. One hundred two and 94 total diagnoses were recorded in weedy and leafy seadragons, respectively. Two of the more common etiologic diagnoses in both species were mycobacteriosis and scuticociliatosis, whereas myxozoanosis was common in weedy seadragons. Metazoan parasite infections were less common etiologic diagnoses. There were no correlations between mycobacteriosis and ciliate protozoan infections in either species. Myxozoanosis was usually found in combination with other diseases and, except for 1 case, was restricted to weedy seadragons. Phaeohyphomycosis, nonmycobacterial bacterial infections, and trauma were also important but less frequent diagnoses. Intestinal coccidiosis was found in weedy but not leafy seadragons. Mineralization of the swim bladder was detected in 26 of 197 leafy seadragons and only 2 of 257 weedy seadragons. Although weedy and leafy seadragons share certain diseases of significance to exhibit populations, there are diseases unique to each species about which the veterinary pathologist, clinician, or diagnostician should be aware.

Aim: To isolate and characterize bacteriocin-like inhibitory substance (BLIS)-producing lactic acid bacteria from the intestine of grey mullet., Methods and Results: Inhibitory activity against at least one or more indicator strains was observed in one Enterococcus thailandicus, one Enterococcus faecium and two Lactococcus garvieae strains. Enterococcus faecium B3-8 and Ent. thailandicus B3-22 showed the greatest inhibitory activities against Listeria monocytogenes ATCC 19111 and were therefore further characterized. The results suggested that the inhibitory substances from the two strains showed similar characteristics with respect to sensitivity to heat and proteolytic enzymes. BLIS from Ent. thailandicus B3-22 was characterized by a broader inhibitory spectrum than that from Ent. faecium B3-8. SDS-PAGE revealed that the molecular size of partially purified BLISs from Ent. faecium B3-8 and Ent. thailandicus B3-22 was c. 5 and 3 kDa, respectively. The molecular mass of purified bacteriocin from Ent. thailandicus B3-22 was further determined to be 6319 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results indicated that BLIS from Ent. thailandicus B3-22 can effectively inhibit the growth of all tested L. garvieae strains., Conclusions: The findings obtained in this study suggest the potential use of Ent. thailandicus B3-22 as a biocontrol agent against pathogenic L. garvieae in the aquaculture., Significance and Impact of the Study: This is the first report describing the characteristics of BLIS from Ent. thailandicus that showed potential for use as a biocontrol agent in the aquaculture., (© 2012 The Society for Applied Microbiology.)

Bacillus sp. strain HYC-10 was isolated with intestinal tract content of a fish, Mugil cephalus, captured from the sea close to Xiamen Island, China. Here, we present the draft genome of strain HYC-10, which contains 3,611,918 bp with a G+C content of 41.30% and contains 3,687 protein-coding genes and 33 tRNA genes.

Microparasites have a higher evolutionary potential than their hosts due to an increased mutation rate and a shorter generation time that usually results in parasites being locally adapted to their sympatric hosts. This pattern may not apply to generalist pathogens as adaptation to sympatric host genotypes is disadvantageous due to a narrowing of the host range, in particular under strong gene flow among host populations. Under this scenario, we predict that the immune defense of hosts reveals adaptation to locally common pathogen phylotypes. This was tested in four host populations of the pipefish Syngnathus typhle and associated bacteria of the genus Vibrio. We investigated the population divergence among host and bacteria populations and verified that gene flow is higher among host populations than among parasite populations. Next, we experimentally assessed the strength of innate immune defense of pipefish hosts using in vitro assays that measured antimicrobial activity of blood plasma against sympatric and allopatric Vibrio phylotypes. Pipefish plasma displays stronger antimicrobial activity against sympatric Vibrio phylotypes compared to allopatric ones. This suggests that host defense is genetically adapted against local bacteria with a broad and unspecialized host spectrum, a situation that is typical for marine systems with weak host population structure., (© 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.)

A Gram-negative, aerobic, motile and slightly curved rod-shaped bacterium (BFLP-8(T)) was isolated from cutaneous mucus of wild long-snouted seahorses (Hippocampus guttulatus) captured in northwest Spain (Toralla, Galicia). Strain BFLP-8(T) grew at 10-35 °C and pH 5-9 (optimally at 25 °C and pH 7.0) and with 1-6 % (w/v) NaCl (optimally with 2 % NaCl). The predominant respiratory quinone (90 %) was ubiquinone with ten isoprene units (Q-10) and the major fatty acids identified were C(18:1) ω7c (54.8 % of the total), C(19:0) cyclo ω8c (11.6 %), C(16:0) (9.5 %), C(18:1) 2-OH (7.1 %) and C(16:1) ω11c (6.7 %). The G+C content of the DNA was 57.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BFLP-8(T) formed a distinct clade within the family Sneathiellaceae but is not specifically associated with any species in the family. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-8(T) represents a novel species within a new genus, for which the name Oceanibacterium hippocampi gen. nov., sp. nov. is proposed. The type strain is BFLP-8(T) (=CECT 7691(T) = DSM 23444(T)).

Strain BFLP-10(T), isolated from faeces of wild long-snouted seahorses (Hippocampus guttulatus), is a Gram-negative, motile and facultatively anaerobic rod. This bacterium produces inhibitory activity against Vibrio species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BFLP-10(T) was a member of the genus Vibrio and was most closely related to Vibrio owensii (99%), Vibrio communis (98.9%), Vibrio sagamiensis (98.9%) and Vibrio rotiferianus (98.4%). However, multilocus sequence analysis using gyrB, pyrH, recA and topA genes revealed low levels of sequence similarity (<91.2%) with these closely related species. In addition, strain BFLP-10(T) could be readily differentiated from other closely related species by several phenotypic properties and fatty acid profiles. The G+C content of the DNA was 45.6 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-10(T) represents a novel species within the genus Vibrio, for which the name Vibrio inhibens sp. nov. is proposed. The type strain is BFLP-10(T) (=CECT 7692(T)=DSM 23440(T)).

Background: Salted fi sh products are popular in many countries around the world. Salting is one of the oldest techniques for fi sh preservation, and is essentially intended to increase the shelf-life of the product depressing water activity by means of dehydration and salt uptake by the fi sh muscle. However, the current demand for salted fi sh is driven more by the flavour of the product than for preservation purposes. Vacuum-packaging represents a static form of hypobaric storage. It is widely used in the food industry because of its effectiveness in reducing oxidative reactions in the product at relatively low cost. Low temperature storage is one of the primary methods to maintain fi sh quality, based on the reduction in the rates of microbiological, chemical and biochemical changes., Material and Methods: Fresh Golden mullets were rapidly beheaded, scaled, gutted and immediately washed with tap water then, samples were taken to the laboratory in ice box for chemical and microbial analysis of fresh fish, other samples were put in the brine (6 liter water and 2160 g salt was used for brine solution). After 14 days of brining, fish were taken out of brine solution and drained, then they were Vacuum Packed and labelled (each pack contained two fish about 1500 g weight). All the packs were stored in a refrigerator 4°C. Some quality aspects including Total Volatile Nitrogen (TVN), Peroxide Value (PV), Thiobarbituric Acid (TBA), Total Viable Count (TVC), Halophilic Bacteria (HB) and presence of Clostridium Botulinum were determined in fresh mullets, fresh brined mullets after 14 days of brining, and in (Vacuum Packed) VP samples stored at 4°C at intervals of 30, 60 and 90 days., Results: TVN increased from ten mg/100 g in fresh brined after 14 days to 30.80 mg/100 g in VP brined Golden mullet after 90 days of storage at 4°C, PV increased after brining from 1.50 meq/kg in fresh brined to 28.90 meq/kg in VP brined Golden mullet after 90 days of storage at 4°C, TBA increased from 0.07 mg MDA/kg in fresh brined to 0.10 after 60 days and then, decreased to 0.09 mg MDA/kg in VP brined Golden mullet after 90 days of storage and TVC decreased from 4.70 log CFU/gr in fresh brined to 4.40 log CFU/ gr after 30 days and then, increased to 5.70 log CFU/gr in VP brined Golden mullet after 90 days of storage at 4°C, HB increased from 4.55 log CFU/gr in fresh brined to 6.30 log CFU/gr after 90 days of storage period at 4°C and exceeded the permissible level. Clostridium botulinum toxin was not detected in any of the samples throughout the storage., Conclusions: The results from this study clearly suggested that a combination of brining, vacuum packaging and storage at refrigerated temperature prolongs the shelf-life of Golden mullet to a great extent. Our findings revealed that the longest shelf-life was for VP brined Golden mullet stored at 4°C is 30 days.

A bacteriocin producer strain MC13 was isolated from the gut of Mugil cephalus (grey mullet) and identified as Enterococcus faecium. The bacteriocin of E. faecium MC13 was purified to homogeneity, as confirmed by Tricine sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE). Reverse-phase high-performance liquid chromatography (HPLC) analysis showed a single active fraction eluted at 26 min, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry analysis showed the molecular mass to be 2.148 kDa. The clear zone in native PAGE corresponding to enterocin MC13 band further substantiated its molecular mass. A dialyzed sample (semicrude preparation) of enterocin MC13 was broad spectrum in its action and inhibited important seafood-borne pathogens: Listeria monocytogenes , Vibrio parahaemolyticus, and Vibrio vulnificus. This antibacterial substance was sensitive to proteolytic enzymes: trypsin, protease, and chymotrypsin but insensitive to catalase and lipase, confirming that inhibition was due to the proteinaceous molecule, i.e., bacteriocin, and not due to hydrogen peroxide. Enterocin MC13 tolerated heat treatment (up to 90 °C for 20 min). Enterococcus faecium MC13 was effective in bile salt tolerance, acid tolerance, and adhesion to the HT-29 cell line. These properties reveal the potential of E. faecium MC13 to be a probiotic bacterium. Enterococcus faecium MC13 could be used as potential fish probiotic against pathogens such as V. parahaemolyticus, Vibrio harveyi, and Aeromonas hydrophila in fisheries. Also, this could be a valuable seafood biopreservative against L. monocytogenes.

The Greek grey-mullet roe is produced from the fully developed gonads of the female mullet (Mugil cephalus) couth in lagoons during their reproductive migration. The traditional processing method of the roe includes, air drying, salting, shape formation and covering with multiple layers of natural beeswax for preservation and distribution. Fish Roe brands have been a staple in local diet and is increasingly becoming popular in the international market. As a ready-to-eat food it's microbial quality should be of concern for the protection of consumers health. In this study, 48 samples of fish roe, just before waxing, were collected from various local processors for microbiological examination by using selective media and incubated under aerobic and anaerobic conditions. The identification of the bacteria was carried out according to the Bergey's manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify. V. parahaemolyticus, Vibrio spp., Salmonella spp., and Aeromonas hydrophila were detected in one sample (2%). Shigella spp., and Flavobacterium spp. in two samples (4%), Clotriduim perfringens (vegetative forms), E. coli, and spores of Bacillus spp., were detected in three samples (6%), Staphylococcus aureus in four samples (8%). Various Micrococcus spp., and spores of C. perfringens in 16% and 35% of the samples respectively. From the Listeria genus, only the species Listeria innocua, Listeria welshimeri, Listeria seeligeri Listeria ivanovii and Listeria grayi were recovered from 2 to 10% of the samples. Microbiological analyses revealed the presence of a small number of pathogens in grey-mullet roe samples which are in accordance with the findings of similar studies. Traditional processing of the fish roe, seems inadequate to ensure the food safety and even waxing isn't expected to fully protect them against facultative anaerobes with salt tolerance. Therefore, additional measures should be taken during processing and marketing of fish roe to minimize potential health risks for the consumers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aims: The aim of this study was to investigate the presence of Vibrio vulnificus and potentially pathogenic strains of Vibrio parahaemolyticus in mullets collected from estuarine environment in Italy., Methods and Results: Two hundred and ninety-five mullets were analysed by culture using the selective medium thiosulfate citrate bile salt sucrose agar, during a monitoring period of 2 years (2008-2009). Presumptive Vibrio colonies were initially identified by using biochemical tests, and strains identified as V. parahaemolyticus and V. vulnificus were subsequently examined by PCR for the presence of species-specific and virulence genes (toxR, trh, tdh and vvh). V. parahaemolyticus was found in 55% (162/295) of fishes and V. vulnificus in 1% (3/295) with a higher presence in summer months. The trh+/tdh- strains were detected in 16% (47/295) of samples and only one strain resulted trh+/tdh+. One of the V. parahaemolyticus trh+ strains isolated belonged to the O1:KUT (K untypeable), a serotype recently associated to gastroenteritis in Italy., Conclusions: This is the first report demonstrating a high percentage of potential pathogenic V. parahaemolyticus trh+ strains in estuarine fishes of the Mediterranean area., Significance and Impact of the Study: These findings indicate the potential human health risk associated with the presence of pathogenic Vibrio spp. in wild fishes., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Enterococci have emerged as important nosocomial and community-acquired pathogens in humans. The presence of vanA-enterococci was investigated in 103 fecal samples recovered from mullets fish (Liza ramada). All fecal samples were inoculated in Slanetz-Bartley agar plates supplemented with 4 mg/L of vancomycin for vancomycin-resistant enterococci (VRE) recovery and two isolates/sample were characterized. Antibiotic susceptibility was tested for 11 antibiotics by disk diffusion and agar dilution methods. VRE identification was performed by biochemical and molecular methods. Additionally, the mechanisms of resistance to glycopeptides (vanA, vanB, vanC1, vanC2, and vanD) and other antibiotics [erm(A), erm(B), tet(L), tet(M), aph(2'')-aac(6'), aph(3')-IIIa, ant(6'), vat(D), vat(E)] as well as the presence of enterococcal surface protein (esp) and hyl virulence factors were investigated. vanA-Enterococcus faecium isolates were recovered from 4 of 103 tested samples, and they showed glycopeptide and erythromycin resistances. Three of them were also ampicillin resistant, two showed resistance to tetracycline, ciprofloxacin, and kanamycin, and one showed resistance to gentamicin. The tet(M) and erm(B) genes were found in all tetracycline- and erythromycin-resistant strains, respectively. The aph(3')-III and aph(2'')-aac(6') genes were identified in the kanamycin- and gentamicin-resistant isolates, respectively. The IS1216 element was identified within vanX-vanY region of Tn1546 in two vanA isolates. The hyl and esp virulence genes were found in four and two isolates, respectively. vanA-strains were ascribed to sequence types ST280 (two isolates) and ST273 (two isolates), including both lineages into the clonal complex CC17. Mullets fish can excrete VRE in their feces and may be a reservoir for such resistant bacteria that can be transmitted to other animals including humans.