Assessing the role of ulvan as immunonutrient in Solea senegalensis.

Immunonutrition is a promising and viable strategy for the development of prophylactic measures in aquaculture. Ulvan, a sulphated marine polysaccharide from green seaweeds, has many biological activities including the immunomodulatory ones. The aim of this study was to assess the short and long-term effects of an ulvan-rich extract obtained from U. ohnoi as immunonutrient in Senegalese sole juveniles. In this work, an ulvan-rich extract from Ulva ohnoi has been obtained by the hot water method and isolated by ethanol precipitation. The FTIR analysis revealed that the ulvan-rich extact had very similar characteristics to previously published ulvan spectra. The total sulfate and protein content was 24.85 ± 3.98 and 0.91 ± 0.04 %, respectively. In vitro assays performed in Senegalese sole (Solea senegalensis) macrophages showed that the ulvan obtained in this study did not compromise the cell viability at concentrations up to 1 mg ml-1 and expression levels of lyg, irf1, il6, il10, c7, tf and txn were significantly upregulated in a concentration dependent-manner. Finally, S. senegalensis juveniles were fed basal diets and diets supplemented with the ulvan-rich extract at ratios 1 and 2 % for 30 days and then, challenged with Photobacterium damselae subsp. piscicida (Phdp). Thereafter, ulvan was withdrawn from the diet and all juveniles were fed the basal diet for 30 days. At 30 days post withdrawal (dpw), juveniles were challenged with Phdp. The expression profiles of a set of genes related to the immune system in spleen were evaluated as well as the lysozyme, peroxidase and bactericidal activity in plasma. Dietary effects of 1 % ulvan resulted in a boost of the immune response and increased disease resistance at short-term whereas juveniles fed diets supplemented with 2 % ulvan showed a significant decrease in the bactericidal activity and lack of protection against Phdp. At long-term (30 days after the withdrawal of ulvan), an improved response was observed in juveniles previously fed 1 % ulvan.

Photobacterium pectinilyticum sp. nov., a novel bacterium isolated from surface seawater of Qingdao offshore.

A Gram-staining-negative, facultative aerobic, motile strain, designated strain ZSDE20T, was isolated from the surface seawater of Qingdao offshore. Phylogenetic analysis of the 16S rRNA gene of strain ZSDE20T, affiliated it to the genus Photobacterium. It was closely related to Photobacterium lutimaris DF-42 T (98.92% 16S rRNA gene sequence similarity). Growth occurred at 4-28ºC (optimum 28ºC), pH 1.0-7.0 (optimum 7.0) and in the presence of 1-7% (w/v) NaCl (optimum 3%). The dominant fatty acids were summed feature 3 (C16:1 ω7c or/and C16:1 ω6c, 34.23%), summed feature 8 (C18:1 ω7c and C18:1 ω6c, 10.36%) and C16:0 (20.05%). The polar lipids of strain ZSDE20T comprised phosphatidylethanolamine, phosphatidylcholine, lyso-phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol dimannoside, phosphatidylinositol mannosides and two unknown lipids. The major respiratory quinone was ubiquinone-8 (Q-8). The DNA G + C content of strain ZSDE20T was 45.6 mol%. Average nucleotide identity (ANI) values between ZSDE20T and its reference species were lower than the threshold for species delineation (95-96%); in silico DNA-DNA hybridization further showed that strain ZSDE20T had less than 70% similarity to its relatives. Based on the polyphasic evidences, strain ZSDE20T is proposed as representing a novel species of the genus Photobacterium, for which the name Photobacterium pectinilyticum sp. nov. is proposed. The type strain is ZSDE20T (= MCCC 1K06283T = KCTC 82885 T).

In vitro efficacy of aquaculture antimicrobials and genetic determinants of resistance in bacterial isolates from tropical aquaculture disease outbreaks.

Understanding the efficacy of antimicrobials against pathogens from clinical samples is critical for their responsible use. The manuscript presents in vitro efficacy and antimicrobial resistance (AMR) genes in seven species of fish pathogens from the disease outbreaks of Indian aquaculture against oxytetracycline, florfenicol, oxolinic acid, and enrofloxacin. In vitro efficacy was evaluated by minimum inhibitory concentration and minimum bactericidal concentration. The gene-specific PCR screened AMR genes against quinolones (qnrA, qnrB, and qnrS) and tetracyclines (tetM, tetS, tetA, tetC, tetB, tetD, tetE, tetH, tetJ, tetG, and tetY). The results showed that Aeromonas veronii (45%) showed the maximum resistance phenotype, followed by Streptococcus agalactiae (40%), Photobacterium damselae (15%), Vibrio parahaemolyticus (10%), and Vibrio vulnificus (5%). There was no resistance among Vibrio harveyi and Vibrio alginolyticus against the tested antimicrobials. The positive association between tetA, tetB, tetC, tetM, or a combination of these genes to oxytetracycline resistance and qnrS to quinolone resistance indicated their potential in surveillance studies. The prevalence of resistance phenotypes (16.43%) and evaluated AMR genes (2.65%) against aquaculture antimicrobials was low. The resistance phenotype pattern abundance was 0.143. All the isolates showed susceptibility to florfenicol. The results help with the appropriate drug selection against each species in aquaculture practices.

Quantification of Photobacterium swingsii and characterisation of disease progression in the New Zealand Greenshell™ mussel, Perna canaliculus.

Greenshell™ mussels (Perna canaliculus) are endemic to New Zealand and support the largest aquaculture industry in the country. Photobacterium swingsii was isolated and identified from moribund P. canaliculus mussels following a mass mortality event. In this study, a challenge experiment was used to characterise, detect, and quantify P. swingsii in adult P. canaliculus following pathogen exposure via injection into the adductor muscle. A positive control (heat-killed P. swingsii injection) was included to account for the effects of injection and inactive bacterial exposure. Survival of control and infected mussels remained 100% during 72-hour monitoring period. Haemolymph was sampled for bacterial colony counts and haemocyte flow cytometry analyses; histology sections were obtained and processed for histopathological assessments; and adductor muscle, gill, digestive gland were sampled for quantitative polymerase chain reaction (PCR) analyses, all conducted at 12, 24, 48 h post-challenge (hpc). The most profound effects of bacterial injection on mussels were seen at 48 hpc, where mussel mortality, haemocyte counts and haemolymph bacterial colony forming were the highest. The quantification of P. swingsii via qPCR showed highest levels of bacterial DNA at 12 hpc in the adductor muscle, gill, and digestive gland. Histopathological observations suggested a non-specific inflammatory response in all mussels associated with a general stress response. This study highlights the physiological effects of P. swingsii infection in P. canaliculus mussels and provides histopathological insight into the tissue injury caused by the action of injection into the adductor muscle. The multi-technique methods used in this study can be applied for use in early surveillance programs of bacterial infection on mussel farms.

Acute nitrite exposure causes gut microbiota dysbacteriosis and proliferation of pathogenic Photobacterium in shrimp.

Nitrite exposure has become a significant concern in the aquaculture industry, posing a severe threat to aquatic animals such as shrimp. While studies have reported the adverse effects of nitrite on shrimp growth, the part played by the gut microbiota in shrimp mortality resulting from nitrite exposure is poorly understood. Here, the effects of nitrite on shrimp gut bacterial community were investigated using 16S rRNA amplicon sequencing, bacterial isolation, genomic analysis, and infection experiments. Compared to the control_healthy group, changes in the bacterial composition of the nitrite_dead group were associated with reduced abundance of specific beneficial bacteria and increased abundance of certain pathogenic bacteria. Notably, members of the Photobacterium genus were found to be significantly enriched in the nitrite_dead group. Genomic analysis of a representative Photobacterium strain (LvS-8n3) revealed a variety of genes encoding bacterial toxins, including hemolysin, adhesin, and phospholipase. Furthermore, it was also found that LvS-8n3 exhibits strong pathogenicity, probably due to its high production of pathogenic factors and the ability to utilize nitrite for proliferation. Therefore, the proliferation of pathogenic Photobacterium species appears pivotal for driving shrimp mortality caused by nitrite exposure. These findings provide novel insights into the disease mechanism in shrimp under conditions of environmental change.

Cryo-EM structure of the fatty acid reductase LuxC-LuxE complex provides insights into bacterial bioluminescence.

The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC-LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy.

The effect of temperature and challenge route on in vitro hemocyte phagocytosis activation after experimental challenge of common octopus, Octopus vulgaris (Cuvier, 1797) with either Photobacterium damselae subsp. damselae or Vibrio anguillarum O1.

Infectious diseases in aquaculture could be associated with high mortalities and morbidity rates, resulting in negative impacts to fish farming industry, consumers, and the environment. Octopods are reared near marine fish farming areas, and this may represent a major risk since fish pathogens may cause pathologies to octopods. Up to date cephalopods immune defense and pathologies, are incompletely understood. Therefore, the aim of this study was to determine the effect of water temperature and challenge route on hemocyte phagocytosis in vitro after experimental challenge of common octopus with Photobacterium damselae subsp. damselae or Vibrio anguillarum O1. Hemolymph was withdrawn at various time-points post-challenge and the number of circulating hemocytes, and phagocytosis ability were determined. No mortalities were recorded irrespective of pathogen, route of challenge and temperature employed. Great variation was observed in the number of circulating hemocytes of both control and challenged specimens in both experiments (1.04 × 105 to 22.33 × 105 hemocytes/ml for the Photobacterium damselae subsp. damselae challenge and 1.35 × 105 to 24.63 × 105 hemocytes/ml for the Vibrio anguillarum O1 and at both studied temperatures). No correlation was found between circulating hemocytes and baseline control specimens body weight. Probably, the number of circulating hemocytes is affected by many extrinsic, and intrinsic factors such as size, age, maturity stage, natural fluctuations and temperature, as indicated in the literature. The hemocyte foreign particles binding ability observed in Photobacterium damselae subsp. damselae experiments, at 21 ± 0.5 °C and 24 ± 0.5 °C, was (mean ± SD) 2.26 ± 2.96 and 11.72 ± 12.36 yeast cells/hemocyte for baseline specimens and 7.84 ± 8.88 and 8.56 ± 9.89 yeast cells/hemocyte for control and challenged specimens, respectively. The corresponding values for Vibrio anguillarum O1 experiments were (mean ± SD) 6.68 ± 9.26 and 7.00 ± 8.11 yeast cells/hemocyte for baseline specimens and 8.82 ± 9.75 and 6.04 ± 7.64 yeast cells/hemocyte for control and challenged specimens, respectively. Hemocytes of the Photobacterium damselae subsp. damselae and Vibrio anguillarum O1 challenged specimens, were more activated at lower temperature. Apparently, temperature is an important factor in hemocyte activation. In addition, our results indicated that time post challenge, route of challenge and pathogen may influence phagocytosis ability.

Photobacterium obscurum sp. nov., a marine bacterium isolated from the coast of Qingdao.

A Gram-stain-negative, facultative anaerobic, rod-shaped strain, named SDRW27T, was isolated from offshore seawater collected near Qingdao. Strain SDRW27T was able to grow at 16-37 °C (optimum, 28 °C), pH 6.0-9.0 (optimum, pH 6.0) and in the presence of 1-7 % (w/v) NaCl (optimum, 3 %). Phylogenetic analysis using 16S rRNA gene sequences indicated that strain SDRW27T was most closely related to Photobacterium toruni H01100410BT (97.89 % sequence similarity), Photobacterium andalusiense H01100409BT (97.89 %) and Photobacterium leiognathi ATCC 25521T (97.82 %). The predominant fatty acids were summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The polar lipids of strain SDRW27T comprised phosphatidylglycerol, phosphatidylinositol dimannoside, phosphatidylcholine, phosphatidylethanolamine and three unidentified lipids. The major respiratory quinone was ubiquinone-8. The G+C content was 47.71 mol%. The genome size was 5.84 Mbp, including 85 contigs with an N50 value of 223 542. The average nucleotide identity (ANI) values of SDRW27T with its three most similar strains, P. toruni H01100410BT, P. andalusiense H01100409BT and P. leiognathi ATCC 25521T, were 71.36, 71.58 and 72.23 %, respectively (all lower than the 95-96 % ANI threshold), and the DNA-DNA hybridization (DDH) values were 20.4, 20.8 and 20.4 % (all lower than the 70 % DDH threshold). The obtained results of polyphasic analysis demonstrate that strain SDRW27T represents a novel species, for which the name Photobacterium obscurum sp. nov. is proposed. The type strain is SDRW27T (=MCCC 1K06286T=KCTC 82892T).

Identification and characterization of toll-like receptor genes in silver pomfret (Pampus argenteus) and their involvement in the host immune response to Photobacterium damselae subsp. Damselae and Nocardia seriolae infection.

Toll-like receptors (TLRs) are vital pattern recognition receptors that play a critical role in the innate immune response against pathogenic attack. Among the bacteria commonly found in the culture process of silver pomfret, Photobacterium damselae subsp. Damselae (PDD, gram-negative) and Nocardia seriolae (NS, gram-positive), can cause large-scale mortality in this fish species. However, there is currently no research on the role of TLRs in mediating the immune response of silver pomfret to these two bacterial infections. Therefore, in this study, we identified nine PaTLRs family members, including several fish-specific TLRs (TLR14 and TLR21). Phylogenetic analysis revealed that these PaTLRs genes could be classified into five subfamilies, namely TLR1, TLR3, TLR5, TLR7, and TLR11, indicating their evolutionary conservation. To further explore the interactions of TLR genes with immune-related mediators, protein and protein interaction network (PPI) results were generated to explain the association of TLR genes with TNF receptor-associated factor 6 (TRAF6) and other relevant genes in the MyD88-dependent pathway and NF-κb signaling pathway. Subsequently, RT-qPCR was conducted to verify the expression patterns of the nine TLR genes in the gills, skin, kidney, liver, and spleen of healthy fish, with most of the TLRs showing high expression levels in the spleen. Following infection with PDD and NS, these PaTLRs exhibited different expression patterns in the spleen, with PaTLR2, PaTLR3, PaTLR5, PaTLR7, PaTLR9, and PaTLR14 being significantly up-regulated. Furthermore, when spleen cells were treated with bacterial compositions, the majority of PaTLRs expression was up-regulated in response to Lipopolysaccharide (LPS) and lipophosphorylcholic acid (LTA) treatment, except for PaTLR21. Finally, changes in the expression levels of TLR-interacting genes were also observed under the stimulation of bacteria and bacterial compositions. The results of this study provide a preliminary reference for further understanding the mechanism of the innate immune response of the TLR gene family in silver pomfret and offer theoretical support for addressing the disease problems encountered during large-scale fish breeding.

Implication of storage conditions on luminescence response from Photobacterium phosphoreum in free and immobilized form.

Bioluminescent bacteria in the form of a cell suspension for on-site hazard analysis are not suitable as in vivo luminescence in free cells fluctuates and may lead to erroneous results. Furthermore, the culture broth cannot be stored for long durations to continue sensing analytes as the luminescence ceases over time. Factors that affect luminescence response include growth dynamism, and ambient environmental conditions. The present study investigated the effect of storage conditions such as temperature (25 ± 2°C, room temperature; 4°C; and -20°C) and ambient aqueous environment (M1: sucrose, 1.02 M; M2, bioluminescent media [tryptone, 10 g L-1 ; NaCl, 28.5 g L-1 ; MgCl2 .7H2 O, 4.5 g L-1 ; CaCl2 , 0.5 g L-1 ; KCl 0.5 g L-1 ; yeast extract, 1 g L-1 ; H2 O, 1 L]; M3, bioluminescent media and 95% glycerol, 1:1 ratio) on the luminescence emission from the calcium alginate-immobilized Photobacterium phosphoreum (Sb ) against the cells in free suspension for an extended period. The results indicated that both the parameters that were undertaken markedly affected the luminescence. In the study, Sb showed an enhanced luminescence emission than the control up to 18.5-fold and for a prolonged period which can be efficiently utilized for rapid biosensing of hazardous materials.

Characterization and Vaccine Potential of Outer Membrane Vesicles from Photobacterium damselae subsp. piscicida.

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative fish pathogen with worldwide distribution and broad host specificity that causes heavy economic losses in aquaculture. Although Phdp was first identified more than 50 years ago, its pathogenicity mechanisms are not completely understood. In this work, we report that Phdp secretes large amounts of outer membrane vesicles (OMVs) when cultured in vitro and during in vivo infection. These OMVs were morphologically characterized and the most abundant vesicle-associated proteins were identified. We also demonstrate that Phdp OMVs protect Phdp cells from the bactericidal activity of fish antimicrobial peptides, suggesting that secretion of OMVs is part of the strategy used by Phdp to evade host defense mechanisms. Importantly, the vaccination of sea bass (Dicentrarchus labrax) with adjuvant-free crude OMVs induced the production of anti-Phdp antibodies and resulted in partial protection against Phdp infection. These findings reveal new aspects of Phdp biology and may provide a basis for developing new vaccines against this pathogen.

Enzyme Inhibition-Based Assay to Estimate the Contribution of Formulants to the Effect of Commercial Pesticide Formulations.

Pesticides can affect the health of individual organisms and the function of the entire ecosystem. Therefore, thorough assessment of the risks associated with the use of pesticides is a high-priority task. An enzyme inhibition-based assay is used in this study as a convenient and quick tool to study the effects of pesticides at the molecular level. The contribution of formulants to toxicological properties of the pesticide formulations has been studied by analyzing effects of 7 active ingredients of pesticides (AIas) and 10 commercial formulations based on them (AIfs) on the function of a wide range of enzyme assay systems differing in complexity (single-, coupled, and three-enzyme assay systems). Results have been compared with the effects of AIas and AIfs on bioluminescence of the luminous bacterium Photobacterium phosphoreum. Mostly, AIfs produce a considerably stronger inhibitory effect on the activity of enzyme assay systems and bioluminescence of the luminous bacterium than AIas, which confirms the contribution of formulants to toxicological properties of the pesticide formulation. Results of the current study demonstrate that "inert" ingredients are not ecotoxicologically safe and can considerably augment the inhibitory effect of pesticide formulations; therefore, their use should be controlled more strictly. Circular dichroism and fluorescence spectra of the enzymes used for assays do not show any changes in the protein structure in the presence of commercial pesticide formulations during the assay procedure. This finding suggests that pesticides produce the inhibitory effect on enzymes through other mechanisms.

Cloning, expression, purification and functional study of low-temperature chitinase PBCHI5 gene from marine-derived photobacteria.

BACKGROUND: Chitin is the second largest carbon source on the earth, and chitosan oligosaccharides produced by its degradation have good application prospects in medicine, cosmetics, and agricultural production. OBJECTIVE: The discovery of a chitinase with high efficiency, high stability and clear degradation mechanism is of great help to promote the research of chitin derivatives and the development of the industrial chain. MATERIALS AND METHODS: In this experiment, a low-temperature chitinase-producing strain Photobacterium sp. LG-29 was isolated from deep-sea mud in the Bohai Sea, and studied by means of molecular biology, biochemistry and bioinformatics. RESULTS: Purification of chitinase yielded an enzyme solution with a concentration of 0.918 mg per mL and a specific activity of 21.036 U per mg. The optimum action temperature is 35 degree C, and it is still active at 4 degree C, showing low-temperature enzymatic activity, and also has certain thermal stability. The optimum pH is 8.0, and it maintains more than 70% of the enzyme activity at pH 11, which is very stable in an alkaline environment. Mn2+, Ca2+, and Mg2+ are the main activators of enzymes, while Fe2+, Zn2+, etc. have extremely significant inhibitory effects on enzymes. The Km and Kcat of chitinase were determined to be 269.05 µmol/L and 0.49 min-1, respectively. Chitinase PbCHI5 has both endonuclease and exonuclease activity. The theoretical pI of the enzyme is 4.16, which is a stable hydrophilic protein. CONCLUSION: This experiment laid a theoretical foundation for the development and utilization of new low-temperature chitinases. Doi.org/10.54680/fr23510110212.

The Evolution of a Specialized, Highly Virulent Fish Pathogen through Gene Loss and Acquisition of Host-Specific Survival Mechanisms.

Photobacterium damselae comprises two subspecies, P. damselae subsp. damselae and P. damselae subsp. piscicida, that contrast remarkably despite their taxonomic relationship. The former is opportunistic and free-living but can cause disease in compromised individuals from a broad diversity of taxa, while the latter is a highly specialized, primary fish pathogen. Here, we employ new closed curated genome assemblies from Australia to estimate the global phylogenetic structure of the species P. damselae. We identify genes responsible for the shift from an opportunist to a host-adapted fish pathogen, potentially via an arthropod vector as fish-to-fish transmission was not achieved in repeated cohabitation challenges despite high virulence for Seriola lalandi. Acquisition of ShdA adhesin and of thiol peroxidase may have allowed the environmental, generalist ancestor to colonize zooplankton and to occasionally enter in fish host sentinel cells. As dependence on the host has increased, P. damselae has lost nonessential genes, such as those related to nitrite and sulfite reduction, urea degradation, a type 6 secretion system (T6SS) and several toxin-antitoxin (TA) systems. Similar to the evolution of Yersinia pestis, the loss of urease may be the crucial event that allowed the pathogen to stably colonize zooplankton vectors. Acquisition of host-specific genes, such as those required to form a sialic acid capsule, was likely necessary for the emergent P. damselae subsp. piscicida to become a highly specialized, facultative intracellular fish pathogen. Processes that have shaped P. damselae subsp. piscicida from subsp. damselae are similar to those underlying evolution of Yersinia pestis from Y. pseudotuberculosis. IMPORTANCE Photobacterium damselae subsp. damselae is a ubiquitous marine bacterium and opportunistic pathogen of compromised hosts of diverse taxa. In contrast, its sister subspecies P. damselae subsp. piscicida (Pdp) is highly virulent in fish. Pdp has evolved from a single subclade of Pdd through gene loss and acquisition. We show that fish-to-fish transmission does not occur in repeated infection models in the primary host, Seriola lalandi, and present genomic evidence for vector-borne transmission, potentially via zooplankton. The broad genomic changes from generalist Pdd to specialist Pdp parallel those of the environmental opportunist Yersinia pseudotuberculosis to vector-borne plague bacterium Y. pestis and demonstrate that evolutionary processes in bacterial pathogens are universal between the terrestrial and marine biosphere.

Solonamides, a Group of Cyclodepsipeptides, Influence Motility in the Native Producer Photobacterium galatheae S2753.

The marine bacterium Photobacterium galatheae S2753 produces a group of cyclodepsipeptides, called solonamides, which impede the virulence but not the survival of Staphylococcus aureus. In addition to their invaluable antivirulence activity, little is known about the biosynthesis and physiological function of solonamides in the native producer. This study generated a solonamide-deficient (Δsol) mutant by in-frame deletion of the sol gene, thereby identifying the core gene for solonamide biosynthesis. By annotation from antiSMASH, the biosynthetic pathway of solonamides in S2753 was also proposed. Mass spectrometry analysis of cell extracts found that deficiency of solonamide production influenced the production of a group of unknown compounds but otherwise did not alter the overall secondary metabolite profile. Physiological comparison between Δsol and wild-type S2753 demonstrated that growth dynamics and biofilm formation of both strains were similar; however, the Δsol mutant displayed reduced motility rings compared to the wild type. Reintroduction of sol restored solonamide production and motility to the mutant, indicating that solonamides influence the motility behavior of P. galatheae S2753. Proteomic analysis of the Δsol and wild-type strains found that eliminating solonamides influenced many cellular processes, including swimming-related proteins and proteins adjusting the cellular cyclic di-GMP concentration. In conclusion, our results revealed the biosynthetic pathway of solonamides and their ecological benefits to P. galatheae S2753 by enhancing motility, likely by altering the motile physiology. IMPORTANCE The broad range of bioactive potentials of cyclodepsipeptides makes these compounds invaluable in the pharmaceutical industry. Recently, a few novel cyclodepsipeptides have been discovered in marine Proteobacteria; however, their biosynthetic pathways remain to be revealed. Here, we demonstrated the biosynthetic genetic basis and pathway of the antivirulence compounds known as solonamides in P. galatheae S2753. This can pave the way for the biological overproduction of solonamides on an industrial scale. Moreover, the comparison of a solonamide-deficient mutant and wild-type S2753 demonstrated that solonamides stimulate the swimming behavior of S2753 and also influence a few key physiological processes of the native producers. These results evidenced that, in addition to their importance as novel drug candidates, these compounds play a pivotal role in the physiology of the producing microorganisms and potentially provide the native producer competitive benefits for their survival in nature.

Transcriptomic analysis of the response of Photobacterium phosphoreum and Photobacterium carnosum to co-contaminants on chicken meat.

This study investigated the impact of Brochothrix (B.) thermosphacta and Pseudomonas (Ps.) fragi on the transcriptomes of Photobacterium (P.) phosphoreum and P. carnosum on chicken meat under modified atmosphere (MA) and air atmosphere (AA). P. phosphoreum TMW2.2103 responded to MA with a reduced transcript number related to cell division and an enhanced number related to oxidative stress. Concomitantly, the analysis revealed upregulation of fermentation and downregulation of respiration. It predicts enhanced substrate competition in presence of co-contaminants/MA. In contrast, the strain upregulated the respiration in AA, supposably due to improved substrate accessibility in this situation. For P. carnosum TMW2.2149 the respiration was downregulated, and the pyruvate metabolism upregulated under MA. MA/co-contaminant resulted in multiple upregulated metabolic routes. Conversely, AA/co-contaminant resulted only in minor regulations, showing inability to cope with fast growing competitors. Observations reveal different strategies of photobacteria to react to co-contaminants on meat.

Monitoring Infection and Antibiotic Treatment in the Skin Microbiota of Farmed European Seabass (Dicentrarchus Labrax) Fingerlings.

The microbiota of fish skin, the primary barrier against disease, is highly dynamic and modulated by several factors. In fish aquaculture, disease outbreaks occur mainly during early-life stages, with associated high economic losses. Antibiotic treatments sometimes remain the best option to control bacterial diseases, despite many reported negative impacts of its use on fish and associated microbiota. Notwithstanding, studies monitoring the effects of disease and antibiotic treatment on the microbiota of fingerlings are scarce. We sequenced the bacterial 16S rRNA V4 gene region using a metabarcoding approach to assess the impact of a mixed infection with Photobacterium damselae ssp. piscicida and Vibrio harveyi and subsequent antibiotic treatment with flumequine, on the skin microbiota of farmed seabass (Dicentrarchus labrax) fingerlings. Both infection and antibiotic treatment led to a significant increase in bacterial diversity and core microbial communities and impacted microbiome structure. Dysbiosis was confirmed by changes in the abundance of potential pathogenic and opportunistic bacterial taxa. Skin bacterial metabolic function was also significantly affected by flumequine administration, suggesting a detriment to fish skin health. Our results add to an increasing body of literature, showing how fish microbiome response to infection and antibiotics cannot be easily predicted.

Effects of dietary marine sulphated polysaccharides (Algimun®) on growth performance, immune responses and disease resistance of juvenile gilthead seabream (Sparus aurata) to Photobacterium damselae subsp. piscicida.

The present study evaluated the effects of a dietary mix of marine sulphated polysaccharides, named Algimun® (AL), supplementation to gilthead seabream (Sparus aurata) juveniles in terms of growth performance, immune responses, and resistance against Photobacterium damselae subsp. piscicida. A total of 240 fish (initial mean weight of 6.00 ± 0.03 g) was randomly separated into 12 tanks (400 L, 20 fish per tank) distributed in four replicates. Fish were fed three experimental diets: a basal diet (Control), and a basal diet with two inclusion rates of Algimun® as 3 g/kg (AL0.3) and 5 g/kg (AL0.5) for 30 days before bacterial infection with P. damselae subsp. piscicida. After a 30-day feeding-period, growth performance was significantly improved in AL0.3 and AL0.5 groups compared to the control group (P < 0.05). AL0.3 and AL0.5 groups showed significantly higher lysozyme activity and myeloperoxidase activity when compared to the control group (P < 0.05). The gene expression of immune mediators (IL-1ß, IL-6, IL-10, IL-18, TNF-α and COX-2) was significantly upregulated in the intestine, spleen and head kidney in AL0.3 and AL0.5 groups when compared to the control group (P < 0.05). Eight days post-challenge, the survival rate against P. damselae subsp. piscicida was numerically higher in fish within AL0.3 and AL0.5 groups compared to control (+20%). The study findings suggest that marine sulphated polysaccharides (Algimun®) could be used as an immunomodulator in gilthead seabream to support animal's health and boost resistance in case of disease outbreak.

Inhibition of lysozyme lytic activity by Ivy derived from Photobacterium damselae subsp. piscicida.

A pseudotuberculosis pathogen, Photobacterium damselae subsp. piscicida (Pdp), has caused enormous economic damage to yellowtail aquaculture in Japan. The Ivy gene has been discovered in plasmid of Pdp, and it has been proposed that it may help bacteria evade lysozyme-mediated lysis during interaction with an animal host. However, the lysozyme-inhibiting activity of Pdp-derived Ivy (Ivy-Pdp) is unknown, and it is unclear whether it acts as a virulence factor for host biophylaxis. In this study, the inhibitory effect of Ivy-Pdp on lysozyme was evaluated by expressing and purifying the recombinant Ivy-Pdp protein (rIvy-Pdp). The rIvy-Pdp protein inhibited hen egg white lysozyme activity in an rIvy-Pdp-concentration-dependent manner, and its inhibitory effect was similar under different temperature and pH conditions. The serum and skin mucus of the yellowtail (which is the host species of Pdp), Japanese flounder, and Nile tilapia showed bacteriolytic activity. In contrast, the addition of rIvy-Pdp inhibited the lytic activity in the serum of these fish species. In particular, it significantly inhibited lytic activity in the serum and skin mucus of Nile tilapia. On the basis of these results, we suggest that Ivy-Pdp is a temperature- and pH-stable lysozyme inhibitor. Additionally, Ivy-Pdp inhibited the lytic activity of lysozyme, which is involved in host biophylaxis. In summary, we inferred that Ivy-Pdp is an important factor that diminishes the sterilization ability of C-type lysozyme when Pdp infects the host.

Dietary supplementation with Gracilaria gracilis by-products modulates the immune status and oxidative stress response of gilthead seabream (Sparus aurata) stimulated with Photobacterium damselae subsp. piscicida.

This study evaluated the effects of agar waste (AW) dietary supplementation, obtained from the seaweed Gracilaria gracilis cultivated under two different spectral lights, neutral (NT) and blue (BL), on haematological parameters, inflammatory response, and antioxidant biomarkers of gilthead seabream (Sparus aurata). Three diets were prepared: i) a basal diet (CTR), ii) a diet supplemented with 2.5% NT, and iii) a diet supplemented with 2.5% BL. After 15 days of feeding, fish were injected with PBS (placebo) or inactivated Photobacterium damselae subsp. piscicida (stimulated) and sampled at 4 h and 24 h post-stimulus. Results indicated that fish fed NT and BL supplemented diets had lower Ht value and mean corpuscular volume (MCV) than fish fed the CTR diet, regardless of the stimulus and the sampling time. No differences in mean corpuscular haemoglobin (MCH) were found between fish fed the different diets, while the mean corpuscular haemoglobin concentration (MCHC) increased in fish fed AW supplemented diets compared to fish fed the CTR diet, regardless of the stimulus and the sampling time. In response to inflammation, fish fed the NT diet displayed higher neutrophils count in blood when compared to the CTR group, regardless of the stimulus and sampling time. Thrombocyte count was higher in fish fed NT and BL diets than in the CTR group, especially in the stimulated fish (Diet*injection (D*I), P = 0.004). An increase in plasma protease activity was detected in fish fed NT or BL diets in both placebo and stimulated fish regardless of the sampling time. Hepatic catalase activity was higher in fish fed the NT and BL than in the CTR group, particularly in the stimulated fish (D*I, P < 0.001). In addition, both stimulated and placebo fish that received the BL diet showed an increase in hepatic GR activity compared to the CTR group, regardless of the sampling time. Dietary supplementation with AW by-products obtained from G. gracilis cultured under NT and BL conditions showed to improve the inflammatory and antioxidant mechanisms in gilthead seabream in response to a UV-killed bacterial stimulus, having valuable applications for the sustainable use of seaweed toward improving the health and welfare of cultured fish.