Dietary Salicornia ramosissima improves the European seabass (Dicentrarchus labrax) inflammatory response against Photobacterium damselae piscicida.

Introduction: Modern fish farming faces challenges in sourcing feed ingredients, most related with their prices, 21 availability, and specifically for plant protein sources, competition for the limited cultivation space for 22 vegetable crops. In that sense, halophytes have the added value of being rich in valuable bioactive compounds and salt tolerant. This study assessed the inclusion of non-food fractions of S. ramosissima in European seabass diets. Methods: Different levels (2.5%, 5%, and 10%) were incorporated into seabass diets, replacing wheat meal (diets ST2.5, ST5, and ST10) or without inclusion (CTRL). Experimental diets were administered to seabass juveniles (8.62 ± 0.63 g) for 34 and 62 days and subsequent inflammatory responses to a heat-inactivated Photobacterium damselae subsp. piscicida (Phdp) were evaluated in a time-course manner (4, 24, 48, and 72 h after the challenge). At each sampling point, seabass haematological profile, plasma immune parameters, and head-kidney immune-related gene expression were evaluated. Results: After both feeding periods, most parameters remained unaltered by S. ramosissima inclusion; nonetheless, seabass fed ST10 showed an upregulation of macrophage colony-stimulating factor 1 receptor 1 (mcsf1r1) and cluster of differentiation 8 (cd8ß) compared with those fed CTRL after 62 days of feeding. Regarding the inflammatory response, seabass fed ST10 showed lower plasma lysozyme levels than their counterparts fed ST2.5 and ST5 at 24 h following injection, while 4 h after the inflammatory stimulus, seabass fed ST10 presented higher numbers of peritoneal leucocytes than fish fed CTRL. Moreover, at 4 h, fish fed ST2.5, ST5, and ST10 showed a higher expression of interleukin 1ß (il1ß), while fish fed ST5 showed higher levels of ornithine decarboxylase (odc) than those fed CTRL. An upregulation of macrophage colony-stimulating factor 1 receptor 1 (mcsf1r1) and glutathione peroxidase (gpx) was also observed at 72 h in fish fed ST10 or ST5 and ST10 compared with CTRL, respectively. Discussion: In conclusion, incorporating up to 10% of the non-food fraction S. ramosissima in feed did not compromise seabass growth or immune status after 62 days, aligning with circular economy principles. However, S. ramosissima inclusion improved the leucocyte response and upregulated key immune-related genes in seabass challenged with an inactivated pathogen.

Uncovering the microbiome landscape in sashimi delicacies.

It is widely believed that a significant portion of the gut microbiota, which play crucial roles in overall health and disease, originates from the food we consume. Sashimi is a type of popular raw seafood cuisine. Its microbiome, however, remained to be thoroughly explored. The objective of this study is to explore the microbiome composition in sashimi at the time when it is served and ready to be eaten. Specifically, our tasks include investigating the diversity and characteristics of microbial profiles in sashimi with respect to the fish types. We utilized the Sanger-sequencing based DNA barcoding technology for fish species authentication and next-generation sequencing for sashimi microbiome profiling. We investigated the microbiome profiles of amberjack, cobia, salmon, tuna and tilapia sashimi, which were all identified using the MT-CO1 DNA sequences regardless of their menu offering names. Chao1 and Shannon indexes, as well as Bray-Curtis dissimilarity index were used to evaluate the alpha and beta diversities of sashimi microbiome. We successfully validated our previous observation that tilapia sashimi has a significantly higher proportions of Pseudomonas compared to other fish sashimi, using independent samples (P = 0.0010). Salmon sashimi exhibited a notably higher Chao1 index in its microbiome in contrast to other fish species (P = 0.0031), indicating a richer and more diverse microbial ecosystem. Non-Metric Multidimensional Scaling (NMDS) based on Bray-Curtis dissimilarity index revealed distinct clusters of microbiome profiles with respect to fish types. Microbiome similarity was notably observed between amberjack and tuna, as well as cobia and salmon. The relationship of microbiome similarity can be depicted as a tree which resembles partly the phylogenetic tree of host species, emphasizing the close relationship between host evolution and microbial composition. Moreover, salmon exhibited a pronounced relative abundance of the Photobacterium genus, significantly surpassing tuna (P = 0.0079), observed consistently across various restaurant sources. In conclusion, microbiome composition of Pseudomonas is significantly higher in tilapia sashimi than in other fish sashimi. Salmon sashimi has the highest diversity of microbiome among all fish sashimi that we analyzed. The level of Photobacterium is significantly higher in salmon than in tuna across all the restaurants we surveyed. These findings provide critical insights into the intricate relationship between the host evolution and the microbial composition. These discoveries deepen our understanding of sashimi microbiota, facilitating our decision in selecting raw seafood.

Interactive effects of elevated temperature and Photobacterium swingsii infection on the survival and immune response of marine mussels (Perna canaliculus): A summer mortality scenario.

The New Zealand Greenshell™ mussel (Perna canaliculus) is an economically important aquaculture species. Prolonged increases in seawater temperature above mussel thermotolerance ranges pose a significant threat to mussel survival and health, potentially increasing susceptibility to bacterial infections. Using challenge experiments, this study examined the combined effects of increased seawater temperature and bacterial (Photobacterium swingsii) infection on animal survival, haemocyte and biochemical responses of adult mussels. Mussels maintained at three temperatures (16, 20 and 24 °C) for seven days were either not injected (control), injected with sterile marine broth (injection control) or P. swingsii (challenged with medium and high doses) and monitored daily for five days. Haemolymph and tissue samples were collected at 24, 48, 72, 96, 120 h post-challenge and analysed to quantify bacterial colonies, haemocyte responses and biochemical responses. Mussels infected with P. swingsii exhibited mortalities at 20 and 24 °C, likely due to a compromised immune system, but no mortalities were observed when temperature was the only stressor. Bacterial colony counts in haemolymph decreased over time, suggesting bacterial clearance followed by the activation of immune signalling pathways. Total haemocyte counts and viability data supports haemocyte defence functions being stimulated in the presence of high pathogen loads at 24 °C. In the gill tissue, oxidative stress responses, measured as total antioxidant capacity and malondialdehyde (MDA) levels, were higher in infected mussels (compared to the controls) after 24h and 120h post-challenge at the lowest (16 °C) and highest temperatures (24 °C), indicating the presence of oxidative stress due to temperature and pathogen stressors. Overall, this work confirms that Photobacterium swingsii is pathogenic to P. canaliculus and indicates that mussels may be more vulnerable to bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.

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.

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.

Functional groups effect on the toxicity of modified ZIF-90 to Photobacterium phosphoreum.

Zeolitic imidazolate framework (ZIF) is of wide interest in biomedical applications due to its extraordinary properties such as high storage capacity, functionality and favorable biocompatibility. However, more comprehensive safety assessments are still essential before ZIF is broadly used in biomedicine. Using the characteristic that aldehyde groups on the surface of ZIF-90 can be modified with other functional groups, a series of ZIF-90s modified with different functional groups (oxime group, carboxyl group, amino group and sulfhydryl group) were synthesized to investigate the effect of functionalization on the toxicity of ZIF-90. ZIF-90 series showed concentration-dependent toxic effects on Photobacterium phosphoreum T3 and the functionalized ZIF-90s are more toxic than pristine ZIF-90, with the ZIF-90 modified with amino group (ZIF-90-NH2) showing the strongest toxicity (IC50 = 23.06 mg/L). Based on the results of the cellular assay and stability exploration, we concluded that corresponding imidazole-ligand release and the property of positively charged are responsible for the elevated toxicity of ZIF-90-NH2. Cell membrane damage, oxidative damage and luminescence damage are the main contributors to the toxic effects of ZIF-90 series. This study explored the effect of surface functionalization on the toxicity of ZIF and proposed mechanistic clues for the safety application of ZIF.

Insight to the diversity of Photobacterium spp. isolated from European plaice (Pleuronectes platessa) based on phylogenetic analysis, phenotypic characterisation and spoilage potential.

This study aimed to explore the diversity of fifty-four Photobacterium strains isolated from muscle tissue of European plaice (Pleuronectes platessa) caught at different fishing seasons and stored 14-days under various conditions. Single phylogenetic markers (16S rRNA, gapA, gyrB and recA) and multilocus sequence analysis (MLSA) were employed to classify isolates at species level. Furthermore, intra- and interspecies variability in the phenotypic traits, maximum specific growth rate (µmax) and spoilage potential of the Photobacterium isolates were investigated. The isolates were classified into the P. iliopiscarium (53.7 %), P. phosphoreum (40.7 %) and P. piscicola (5.6 %) clades using MLSA. Two housekeeping genes, gyrB and recA, exhibited a consistent phylogenetic relationship with MLSA, suggesting that they might be used as individual phylogenetic markers for the Photobacterium genus. Intra- and interspecies variability in the expression of phenotypic characteristics and the production of trimethylamine (TMA), inosine (HxR), and hypoxanthine (Hx) were observed. A growth optimum temperature for P. iliopiscarium was approximately 20 °C, while those for P. phosphoreum and P. piscicola were closer to 15 °C. All isolates exhibited the highest growth density at 1.5 % NaCl, followed by 0.5 %, 3 %, and 6 % NaCl. However, P. phosphoreum demonstrated a higher NaCl tolerance than the other two species. Although, the high CO2 atmosphere significantly inhibited the growth of all strains at 4 °C, P. phosphoreum and P. piscicola showed higher growth density at 15 °C than P. iliopiscarium. Notably, all strains demonstrated H2S production. The µmax varied considerably within each species, highlighting the significance of strain-level variability. This study demonstrates that P. iliopiscarium and P. piscicola, alongside P. phosphoreum, are efficient TMA-, HxR-, Hx-, and H2S-producers, suggesting their potential contribution to synergistic off-odour generation and spoilage. Moreover, the Photobacterium isolates seem to exhibit diverse adaptations to their environments, resulting in fluctuated growth and spoilage potential. Understanding intra- and interspecies variability will facilitate modelling seafood spoilage in microbial risk assessments and developing targeted hurdles to prolong products' shelf-life.

Anti-Bacterial and Immunostimulatory Properties of Ulvan-Loaded Chitosan Nanoparticles for Use in Aquaculture.

Alternative prophylactic strategies to limit farm animal infection are needed in order to avoid the use of antibiotics. Anti-bacterial and immunostimulatory properties of bioactive compounds are of great interest in aquaculture. Marine derived polysaccharides, such as chitosan and ulvan, together with nanotechnology, have become the focus of attention in the scientific community due to their wide range of biological properties. In this work, chitosan and ulvan-loaded chitosan nanoparticles (referred as CS-TPP NPs and CS-UL-TPP NPs, respectively), obtained by the ionotropic gelation method, had round shape, and the mean sizes were 137.00 ± 5.44 and 325.50 ± 4.95 nm, respectively. No study about the anti-bacterial activity of both types of NPs against Photobacterium damselae subsp. piscicida, an important fish pathogen, has been reported so far. Furthermore, the potential immunostimulatory effects of CS-UL-TPP NPs after oral administration in fish have not yet been evaluated. The percentage of bacterial inhibition against P. damselae subsp. piscicida was determined through in vitro assays, and it was significantly higher in CS-UL-TPP NPs than in CS-TPP NPs at concentrations below 0.03 mg mL-1. The effects on the immune system of CS-TPP and CS-UL-TPP NPs were evaluated in Solea senegalensis juveniles at 30 days after oral administration. Lysozyme activity as well as gene expression levels of il1b, il6, hamp1, tf and c3 was significantly higher in CS-UL-TPP NP-treated groups than in the controls, and no significant differences were observed in CS-TPP NP-treated groups. Thus, ulvan extracted from the macroalgae Ulva ohnoi could improve anti-bacterial and immunostimulant properties of CS-TPP NPs thereby making them suitable to be used as vaccine adjuvant or as immunostimulant.

Comparative genomics of symbiotic Photobacterium using highly contiguous genome assemblies from long read sequences.

This study presents the assembly and comparative genomic analysis of luminous Photobacterium strains isolated from the light organs of 12 fish species using Oxford Nanopore Technologies (ONT) sequencing. The majority of assemblies achieved chromosome-level continuity, consisting of one large (>3 Mbp) and one small (~1.5 Mbp) contig, with near complete BUSCO scores along with varying plasmid sequences. Leveraging this dataset, this study significantly expanded the available genomes for P. leiognathi and its subspecies P. 'mandapamensis', enabling a comparative genomic analysis between the two lineages. An analysis of the large and small chromosomes unveiled distinct patterns of core and accessory genes, with a larger fraction of the core genes residing on the large chromosome, supporting the hypothesis of secondary chromosome evolution from megaplasmids in Vibrionaceae. In addition, we discovered a proposed new species, Photobacterium acropomis sp. nov., isolated from an acropomatid host, with an average nucleotide identify (ANI) of 93 % compared to the P. leiognathi and P. 'mandapamensis' strains. A comparison of the P. leiognathi and P. 'mandapamensis' lineages revealed minimal differences in gene content, yet highlighted the former's larger genome size and potential for horizontal gene transfer. An investigation of the lux-rib operon, responsible for light production, indicated congruence between the presence of luxF and host family, challenging its role in differentiating P. 'mandapamensis' from P. leiognathi. Further insights were derived from the identification of metabolic differences, such as the presence of the NADH:quinone oxidoreductase respiratory complex I in P. leiognathi as well as variations in the type II secretion system (T2S) genes between the lineages, potentially impacting protein secretion and symbiosis. In summary, this study advances our understanding of Photobacterium genome evolution, highlighting subtle differences between closely related lineages, specifically P. leiognathi and P. 'mandapamensis'. These findings highlight the benefit of long read sequencing for bacterial genome assembly and pangenome analysis and provide a foundation for exploring early bacterial speciation processes of these facultative light organ symbionts.

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.

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.

Transforming the untransformable with knockout minicircles: High-efficiency transformation and vector-free allelic exchange knockout in the fish pathogen Photobacterium damselae.

Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange via homologous recombination is a generic method of targeted gene editing in bacteria. However, generally applicable protocols are lacking, and suboptimal approaches are often used for nonstandard but epidemiologically important species. Photobacterium damselae subsp. piscicida (Pdp) is a primary pathogen of fish in aquaculture and has been considered hard to transform since the mid-1990s. Consequently, conjugative transfer of RK2/RP4 suicide vectors from Escherichia coli S17-1/SM10 donor strains, a system prone to off-target mutagenesis, was used to deliver the allelic exchange DNA in previous studies. Here we have achieved efficient electrotransformation in Pdp using a salt-free highly concentrated sucrose solution, which performs as a hypertonic wash buffer, cryoprotectant, and electroporation buffer. High-efficiency transformation has enabled vector-free mutagenesis for which we have employed circular minimalistic constructs (knockout minicircles) containing only allelic exchange essentials that were generated by Gibson assembly. Preparation of competent cells using sucrose and electroporation/integration of minicircles had virtually no detectable off-target promutagenic effect. In contrast, a downstream sacB selection apparently induced several large deletions via mobilization of transposable elements. Electroporation of minicircles into sucrose-treated cells is a versatile broadly applicable approach that may facilitate allelic exchange in a wide range of microbial species. The method permitted inactivation of a primary virulence factor unique to Pdp, apoptogenic toxin AIP56, demonstrating the efficacy of minicircles for difficult KO targets located on the high copy number of small plasmids.

Deciphering the growth responses and genotypic diversity of bioluminescent Photobacterium phosphoreum on chicken meat during aerobic refrigerated storage.

The advent of high-throughput sequencing technologies in recent years has revealed the unexpected presence of genus Photobacterium within the chicken meat spoilage ecosystem. This study was undertaken to decipher the occurrence, the growth patterns and the genotypic biodiversity of Photobacterium phosphoreum on chicken breast fillets stored aerobically at 4 °C through conventional microbiological methods and molecular techniques. Samples were periodically cultured on marine broth agar (MA; supplemented with meat extract and vancomycin) for the enumeration of presumptive bioluminescent Photobacterium spp. In total, 90 bioluminescent bacteria were recovered from the initial (time of first appearance), middle and end stages of storage. Concomitantly, 95 total psychrotrophic/psychrophilic bacteria were isolated from the same medium to assess the presence and diversity of non-luminous photobacteria. Genetic diversity between bioluminescent isolates was assessed with two PCR-based DNA fingerprinting methods, i.e. RAPD and rep-PCR. Moreover, the characterization of selected bacterial isolates at the genus and/or species level was performed by sequencing of the 16S rRNA and/or gyrB gene. Bioluminescent bacteria were scarcely encountered in fresh samples at population levels of ca. 2.0 log CFU/g, whilst total psychrotrophic/psychrophilic bacteria were found at levels of ca. 4.4 log CFU/g. As time proceeded and close to shelf-life end, bioluminescent bacteria were encountered at higher populations, and were found at levels of 5.3 and 7.0 log CFU/g in samples from the second and third batch, respectively. In the first batch their presence was occasional and at levels up to 3.9 log CFU/g. Accordingly, total psychrotrophic/psychrophilic bacteria exceeded 8.4 log CFU/g at the end of storage, suggesting the possible underestimation of bioluminescent populations following the specific cultivation conditions. Sequence analysis assigned bioluminescent isolates to Photobacterium phosphoreum, while genetic fingerprinting revealed high intra-species variability. Respectively, total psychrotrophs/psychrophiles were assigned to genera Pseudomonas, Shewanella, Psychrobacter, Acinetobacter, Vibrio and Photobacterium. Non-luminous photobacteria were not identified within the psychrotrophs/psychrophiles. Results of the present study reveal the intra- and inter-batch variability on the occurrence and growth responses of P. phosphoreum and highlight its potential role in the chicken meat spoilage consortium.

Low salinity activates a virulence program in the generalist marine pathogen Photobacterium damselae subsp. damselae.

Facultative marine bacterial pathogens sense environmental signals so that the expression of virulence factors is upregulated on entry into hosts and downregulated during the free-living lifestyle in the environment. In this study, we utilized transcriptome sequencing to compare the transcriptional profiles of Photobacterium damselae subsp. damselae, a generalist pathogen that causes disease in diverse marine animals and fatal infections in humans at NaCl concentrations that mimic the free-living lifestyle or host internal milieu, respectively. We here show that NaCl concentration constitutes a major regulatory signal that shapes the transcriptome and uncover 1,808 differentially expressed genes (888 upregulated and 920 downregulated in response to low-salt conditions). Growth at 3% NaCl, a salinity that mimics the free-living lifestyle, upregulated genes involved in energy production, nitrogen metabolism, transport of compatible solutes, utilization of trehalose and fructose, and carbohydrate and amino acid metabolism with strong upregulation of the arginine deiminase system (ADS). In addition, we observed a marked increase in resistance to antibiotics at 3% NaCl. On the contrary, the low salinity conditions (1% NaCl) that mimic those encountered in the host triggered a virulence gene expression profile that maximized the production of the type 2 secretion system (T2SS)-dependent cytotoxins damselysin, phobalysin P, and a putative PirAB-like toxin, observations that were corroborated by the analysis of the secretome. Low salinity also upregulated the expression of iron-acquisition systems, efflux pumps, and other functions related to stress response and virulence. The results of this study greatly expand our knowledge of the salinity-responsive adaptations of a generalist and versatile marine pathogen. IMPORTANCE Pathogenic Vibrionaceae species experience continuous shifts of NaCl concentration in their life cycles. However, the impact of salinity changes in gene regulation has been studied in a small number of Vibrio species. In this study, we analyzed the transcriptional response of Photobacterium damselae subsp. damselae (Pdd), a generalist and facultative pathogen, to changes in salinity, and demonstrate that growth at 1% NaCl in comparison to 3% NaCl triggers a virulence program of gene expression, with a major impact in the T2SS-dependent secretome. The decrease in NaCl concentration encountered by bacteria on entry into a host is proposed to constitute a regulatory signal that upregulates a genetic program involved in host invasion and tissue damage, nutrient scavenging (notably iron), and stress responses. This study will surely inspire new research on Pdd pathobiology, as well as on other important pathogens of the family Vibrionaceae and related taxa whose salinity regulons still await investigation.

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.

Photobacterium predominate the microbial communities of muscle of European plaice (Pleuronectes platessa) caught in the Norwegian sea independent of skin and gills microbiota, fishing season, and storage conditions.

The aim of this study was to investigate seasonal variations (September, December and April) in the initial microbial communities of skin and gills' external mucosal tissues (EMT) and muscle of European plaice (Pleuronectes platessa). Moreover, a potential relationship between EMT and fresh muscle microbiota was examined. The microbial community succession in plaice muscle as a function of fishing season and storage conditions was also investigated. The selected seasons for the storage experiment were September and April. Investigated storage conditions were; fillets packaged in either vacuum or modified atmosphere (70 % CO2, 20 % N2, 10 % O2) and chilled/refrigerated conditions (4 °C). Whole fish stored on ice (0 °C) was selected as a commercial standard. Seasonal variations were detected in the initial microbial communities of EMT and plaice muscle. The highest microbial diversity was found in EMT and muscle of April-caught plaice, followed by December and September catch indicating the important role of environmental factors in shaping the initial EMT and muscle microbial communities. The EMT microbial communities were more diverse than fresh muscle samples. The low number of shared taxa between EMT and initial muscle microbial communities indicates that only a minor part of the muscle microbiota came from the EMT. Psychrobacter and Photobacterium were the predominant genera in the EMT microbial communities in all seasons. Photobacterium dominated the initial muscle microbial communities with a gradual seasonal reduction of its abundance from September to April. Storage time and storage conditions shaped a less diverse and distinct community compared to the fresh muscle. However, no clear separation was seen between the communities at the middle and end of storage time. Regardless of EMT microbiota, fishing season and storage conditions, Photobacterium dominated the microbial communities of stored muscle samples. The Photobacterium prevalence as the primary specific spoilage organism (SSO) could be attributed to its high relative abundance in the initial microbiota of muscle and its CO2-tolerance. The findings of this study indicate the important contribution of Photobacterium to the microbial spoilage of plaice. Thus, the development of innovative preservation techniques addressing the rapid growth of Photobacterium could contribute to the production of high-quality and shelf-stable convenient retail plaice products.

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.

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.