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Background: Bacteriophages are known modulators of community composition and activity in environmental and host-associated microbiomes. However, the impact single phages have on bacterial community dynamics under viral predation, the extent and duration of their effect, are not completely understood. In this study, we combine morphological and genomic characterization of a novel marine phage, isolated from the Baltic sponge Halichondria panicea, and report on first attempts of controlled phage-manipulation of natural sponge-associated microbiomes. Results: We used culture-based and culture-independent (16S rRNA gene amplicon sequencing) methods to investigate bacterial community composition and dynamics in sponge microbiomes with and without the addition of phages. Upon application of a novel Maribacter specialist phage Panino under controlled conditions, we were able to detect community-wide shifts in the microbiome composition and load after 72 h. While bacterial community composition became more dissimilar over time in the presence of phages, species evenness and richness were maintained. Upon phage exposure, we observed the loss of several low-abundance constituent taxa of the resident microbiota, while other originally underrepresented taxa increased. Virulent phages likely induce community-wide disturbances, evident in changes in the total sponge microbial profile by specific elimination of constituent taxa, which leads to an increase in bacterial abundance of opportunistic taxa, such as the genera Vibrio, Pseudoalteromonas, and Photobacterium. Conclusions: Our findings suggest that sponge microbiome diversity and, by extension, its resilience depend on the maintenance of resident bacterial community members, irrespective of their abundance. Phage-induced disturbances can significantly alter community structure by promoting the growth of opportunistic bacteria like Vibrio and shifting the microbiome to a dysbiotic state. These insights highlight the role of bacteriophages in shaping microbiome dynamics and underscore the potential for phage application in managing bacterial community composition in marine host-associated environments. [ABSTRACT FROM AUTHOR]

Asian seabass (Lates calcarifer) is an economically important fish species that is widely cultivated in Thailand. However, aquaculture of Asian seabass is limited by infectious diseases. One of the most serious diseases is photobacteriosis, caused by Photobacterium damselae. Vaccination is recognized as an efficient disease prevention and pathogen control method for strengthening the aquaculture industry. To promote vaccine development, the characterization of pathogenic bacteria and their pathogenesis is required. In this study, isolates of P. damselae were obtained from commercial aquaculture farms in Thailand during 2019–2021. Analyses of 16S rRNA and the urease subunit alpha genes identified the isolates as P. damselae subsp. damselae (Phdd). Antibiotic susceptibility analyses showed that all Phdd isolates were resistant to amoxicillin (10 μg). Haemolysis and phospholipase activities were used to categorize P. damselae into three groups based on their biological activities. The pathogenicity of four candidates (SK136, PD001, PD002 and T11L) was tested in Asian seabass. Isolate SK136 showed the highest virulence, with a lethal dose (LD50) of 1.47 × 105 CFU/fish, whereas isolate PD001 did not show any virulence. Genotypic characterization, based on multi‐locus sequence typing analysis, demonstrated that all candidates were novel strains with new sequence types (64, 65, 66 and 67). Preliminary vaccination using formalin‐killed cells (FKCs) protected Asian seabass from artificial challenges. Taken together, these results provide fundamental knowledge for vaccine development against Phdd infection in Asian seabass. [ABSTRACT FROM AUTHOR]

The symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, is a tractable and well-studied model of bacteria-animal mutualism. Here, we developed a method to transiently colonize E. scolopes using D-alanine (D-ala) auxotrophy of the symbiont, controlling the persistence of viable infection by supplying or withholding D-ala. We generated alanine racemase (alr) mutants of V. fischeri that lack avenues for mutational suppression of auxotrophy or reversion to prototrophy. Surprisingly, an alr mutant did not require D-ala to grow in a minimal medium, a phenomenon requiring metC, which encodes cystathionine ß-lyase. Likewise, overexpression of metC suppressed D-ala auxotrophy in a rich medium. To block potential mechanisms of suppression, we combined the alr mutation with deletions of metC and/or bsrF, which encodes a broad-spectrum racemase and investigated the suppression rates of four D-ala auxotrophic strains. We then focused on alr bsrF mutant MC13, which has a suppression rate of <10-9. When D-ala was removed from a growing culture of MC13, cells rounded and lysed within 40 minutes. Transient colonization of E. scolopes was achieved by inoculating squid in seawater containing MC13 and D-ala, and then transferring the squid into water lacking D-ala, which resulted in loss of viable symbionts within hours. Interestingly, the symbionts within crypt 3 persisted longer than those of crypt 1, suggesting a difference in bacterial growth rate in distinct crypt environments. Our study highlights a new approach for inducing transient colonization and provides insight into the biogeography of the E. scolopes light organ. IMPORTANCE The importance of this study is multi-faceted, providing a valuable methodological tool and insight into the biology of the symbiosis between Vibrio fischeri and Euprymna scolopes. First, the study sheds light on the critical role of D-ala for bacterial growth, and the underpinnings of D-ala synthesis. Our observations that metC obviates the need for D-ala supplementation of an alr mutant in minimal medium and that MetC-dependent growth correlates with D-ala in peptidoglycan, corroborate and extend previous findings in Escherichia coli regarding a role of MetC in D-ala production. Second, our isolation of robust D-ala auxotrophs led us to a novel method for studying the squid-Vibrio symbiosis, allowing for transient colonization without the use of antibiotics, and revealed intriguing differences in symbiont growth parameters in distinct light organ crypts. This work and the methodology developed will contribute to our understanding of the persistence and dynamics of V. fischeri within its host. [ABSTRACT FROM AUTHOR]

Pale shrimp disease is a serious emerging disease affecting juvenile Pacific white shrimp (Litopenaeus vannamei) in the low salinity areas of Southern Thailand. The most prominent clinical sign is a uniformly pale body coloration, with a mortality rate of nearly 100 % in severe cases. This study aimed to identify the causative agents of this disease. The affected shrimp from the natural outbreak ponds were collected for disease investigation. The disease was proven to be transmissible, as feeding-challenged shrimp showed pale body characteristics. To demonstrate the bacterial etiology of the disease, bacteria were isolated from naturally diseased shrimp and identified using 16S rRNA gene sequencing. Pure cultures were inoculated into healthy shrimp. Among the different bacterial isolates (mainly Vibrio spp.), only Photobacterium damselae subsp. damselae (PDD) induced clinical signs of a pale body. Thus, PDD was examined for pathogenicity via immersion (105 CFU/mL) and oral gavage (107 CFU/shrimp) at 10 ppt. On day 7, the challenged shrimp showed pale body coloration, and the survival rates of both groups were 53 % and 10 %, respectively, whereas no mortality was observed in the control shrimp. Histologically, the hepatopancreatic tubules of the PDD-infected shrimp were atrophic, and lymphoid organ spheroids, myopathy, and hemocytic infiltration in the muscle were also evident. These histopathological changes are generally consistent with those observed in natural outbreaks. Consequently, our findings revealed that the enigmatic pale shrimp disease is caused by PDD, a hitherto unknown pathogen that causes massive shrimp mortality.

Stony Coral Tissue Loss Disease (SCTLD) has caused widespread coral mortality in the Caribbean Region. However, how the disease presence alters the microbiome community, their structure, composition, and metabolic functionality is still poorly understood. In this study, we characterized the microbial communities of the tissues of apparently healthy and diseased SCTLD colonies of the species Siderastrea siderea, Orbicella faveolata, and Montastraea cavernosa to explore putative changes related to the presence of SCTLD. Gammaproteobacteria, Alphaproteobacteria, and Bacteroidia were the best represented classes in the healthy tissues of all coral species, and alpha diversity did not show significant differences among the species. The microbial community structure between coral species was significantly different (PERMANOVA: F = 3.46, p = 0.001), and enriched genera were detected for each species: Vibrio and Photobacterium in S. siderea, Spirochaeta2 and Marivivens in O. faveolata and SAR202_clade and Nitrospira in M. cavernosa. Evidence of SCTLD in the microbial communities was more substantial in S. siderea, where differences in alpha diversity, beta diversity, and functional profiles were observed. In O. faveolata, differences were detected only in the community structure, while M. cavernosa samples showed no significant difference. Several microbial groups were found to have enriched abundances in tissue from SCTLD lesions from S. siderea and O. faveolata, but no dominant bacterial group was detected. Our results contribute to understanding microbial diversity associated with three scleractinian coral species and the shifts in their microbiomes associated with SCTLD in the Mexican Caribbean. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the prevalence of Vibrionaceae family in retail seafood products available in the Qidong market during the summer of 2023 and to characterize Vibrio parahaemolyticus isolates, given that this bacterium is the leading cause of seafood-associated food poisoning. We successfully isolated a total of 240 Vibrionaceae strains from a pool of 718 seafood samples. The breakdown of the isolates included 146 Photobacterium damselae, 59 V. parahaemolyticus, 18 V. campbellii, and 11 V. alginolyticus. Among these, P. damselae and V. parahaemolyticus were the predominant species, with respective prevalence rates of 20.3% and 8.2%. Interestingly, all 59 isolates of V. parahaemolyticus were identified as non-pathogenic. They demonstrated proficiency in swimming and swarming motility and were capable of forming biofilms across a range of temperatures. In terms of antibiotic resistance, the V. parahaemolyticus isolates showed high resistance to ampicillin, intermediate resistance to cefuroxime and cefazolin, and were sensitive to the other antibiotics evaluated. The findings of this study may offer valuable insights and theoretical support for enhancing seafood safety measures in Qidong City. [ABSTRACT FROM AUTHOR]

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes enormous economic losses in the food and feed industries. Simple, rapid, low-cost, and quantitative analysis of ZEN is particularly urgent in the fields of food safety and animal husbandry. Using the bioluminescent bacterium Photobacterium phosphoreum T3, we propose a bioluminescence inhibition assay to evaluate ZEN levels quickly. The limit of detection (LOD), limit of quantification (LOQ), and quantitative working range of this bioluminescence inhibition assay were 0.1 µg/mL, 5 µg/mL, and 5-100 µg/mL, respectively. The concentration-response curve of the bioluminescence inhibition rate and ZEN concentration was plotted within the range 5 to 100 μg/mL, as follows: y = 0.0069x2 – 0.0190x + 7.9907 (R2 = 0.9943, y is luminescence inhibition rate, x is ZEN concentration). First, we used the bioluminescence inhibition assay to detect the remaining ZEN in samples treated with purified lactonohydrolase ZHD101. The bioluminescence inhibition assay results showed a strong correlation with the HPLC analysis. Furthermore, we successfully evaluated the overall toxicity of samples treated with purified peroxidase Prx and H2O2 using the P. phosphoreum T3 bioluminescence inhibition assay. The results indicate that the degradation products of ZEN created by purified peroxidase Prx and H2O2 showed little toxicity to P. phosphoreum T3. In this study, a simple, rapid, and low-cost assay method of zearalenone by bioluminescent P. phosphoreum T3 was developed. The bioluminescence inhibition assay could be used to estimate the efficiency of enzymatic degradation of ZEN. [ABSTRACT FROM AUTHOR]

ABSTRACT Bioluminescence in Vibrio fischeri is regulated by a quorum-dependent signaling system composed of LuxI and LuxR. LuxI generates N-3-oxohexanoyl homoserine lactone (3OC6-HSL), which triggers LuxR to activate transcription of the luxICDABEG operon responsible for bioluminescence. Surprisingly, a ∆luxI mutant produced more bioluminescence than the wild type in culture. In contrast, a 4 bp duplication within luxI, resulting in a frameshift mutation and null allele, decreased luminescence tenfold. A second signaling system encoded by ainSR affects bioluminescence by increasing levels of LuxR, via the transcriptional activator LitR, and the N-octanoyl homoserine lactone (C8-HSL) signal produced by AinS is considered only a weak activator of LuxR. However, ainS is required for the bright phenotype of the ∆luxI mutant in culture. When 3OC6-HSL was provided either in the medium or by expression of luxI in trans, all cultures were brighter, but the ∆luxI mutant remained significantly brighter than the luxI frameshift mutant. Taken together, these data suggest that the enhanced bioluminescence due to the LuxI product 3OC6-HSL counteracts a negative cis-acting regulatory element within the luxI gene and that when luxI is absent the C8-HSL signal is sufficient to induce luminescence.IMPORTANCEThe regulation of bioluminescence by Vibrio fischeri is a textbook example of bacterial quorum-dependent pheromone signaling. The canonical regulatory model is that an autoinducer pheromone produced by LuxI accumulates as cells achieve a high density, and this LuxI-generated signal stimulates LuxR to activate transcription of the lux operon that underlies bioluminescence. The surprising observation that LuxI is dispensable for inducing bioluminescence forces a re-evaluation of the role of luxI. More broadly, the results underscore the potential deceptiveness of complex regulatory circuits, particularly those in which bacteria produce multiple related signaling molecules.

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.

Climate change is now considered one of the greatest challenges and is expected to have a drastic impact on mariculture. The present study aimed to evaluate the impact of climatic changes on the emergence of bacterial pathogens among cultured marine fish in northern Egyptian provinces. 135 samples of three marine fish species, represented as 45 of Dicentrarchus labrax (700 ±25 g), 45 of Sparus aurata (350 ±25 g), and 45 Argyrosomus regius (1 kg ±50 g) were collected from private marine fish farms located in Deeba Triangle, Shataa Damietta (Damietta governorate) and Ismailia province, Egypt. Moribund fishes exhibited erratic swimming behaviour, haemorrhage, erosion and ulcers on the skin. Necropsy findings of infected fish revealed congested liver or pale with engorged gall bladder, congested kidney and spleen. With the presence of serous to hemorrhagic ascetic fluid. Vibrio alginolyticus, Vibrio parahaemolyticus, and Photobacterium damselae subspecies piscicida were the most retrievable bacterial strains from moribund fish. V. alginolyticus was the most prevalent isolated bacterial strain and represented 50%, 50% and 40% of the total isolates recovered from Sparus aurata, Dicentrarchus labrax and Argyrosomus regius, respectively. Retrieved isolates were morphologically and biochemically identified using the API 20E system, followed by further confirmation by sequencing of 16S rRNA genes. The histopathological examination revealed severe inflammatory reactions together with melanomacrophage center alterations within the examined splenic, hepatic, and renal tissues. Data analysis has shown that poor water quality and severe climatic change, especially during the summer, were implicated in the emergence of bacterial infections among cultured marine fish. [ABSTRACT FROM AUTHOR]

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 (cd8β) 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 1β (il1β), 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. [ABSTRACT FROM AUTHOR]

Spotted sea bass (Lateolabrax maculatus) is a high-economic-value aquacultural fish widely distributed in the coastal and estuarine areas of East Asia. In August 2020, a sudden outbreak of disease accompanied by significant mortality was documented in L. maculatus reared in marine cage cultures located in Nanhuang island, Yantai, China. Two coinfected bacterial strains, namely, NH-LM1 and NH-LM2, were isolated from the diseased L. maculatus for the first time. Through phylogenetic tree analysis, biochemical characterization, and genomic investigation, the isolated bacterial strains were identified as Vibrio harveyi and Photobacterium damselae subsp. piscicida, respectively. The genomic analysis revealed that V. harveyi possesses two circular chromosomes and six plasmids, while P. damselae subsp. piscicida possesses two circular chromosomes and two plasmids. Furthermore, pathogenic genes analysis identified 587 and 484 genes in V. harveyi and P. damselae subsp. piscicida, respectively. Additionally, drug-sensitivity testing demonstrated both V. harveyi and P. damselae subsp. piscicida exhibited sensitivity to chloramphenicol, ciprofloxacin, ofloxacin, orfloxacin, minocycline, doxycycline, tetracycline, and ceftriaxone. Moreover, antibiotic resistance genes were detected in the plasmids of both strains. Extracellular product (ECP) analysis demonstrated that both V. harveyi and P. damselae subsp. piscicida can produce hemolysin and amylase, while V. harveyi additionally can produce caseinase and esterase. Furthermore, infected fish displayed severe histopathological alterations, including infiltration of lymphocytes, cellular degeneration and necrosis, and loose aggregation of cells. Artificial infection assays determined that the LD50 of P. damselae subsp. piscicida was 3 × 105 CFU/g, while the LD50 of V. harveyi was too low to be accurately evaluated. Furthermore, the dual infection of V. harveyi and P. damselae subsp. piscicida elicits a more rapid and pronounced mortality rate compared to single challenge, thereby potentially exacerbating the severity of the disease through synergistic effects. Ultimately, our findings offer compelling evidence for the occurrence of coinfections involving V. harveyi and P. damselae subsp. piscicida in L. maculatus, thereby contributing to the advancement of diagnostic and preventative measures for the associated disease. [ABSTRACT FROM AUTHOR]

The mesopelagic calanoid copepod Cephalophanes reflugens is characterized by its large eyes with semi-parabolic reflectors. It is hypothesized that the eyes aid in finding crustacean carcasses as food in poor deep-sea light and oligotrophic conditions. Here, the crustacean carcasses-associated luminous, chitin-degrading bacteria may play a vital role in detecting and digesting foods by the copepod. In this study, we collected adult female C. reflugens (n  = 9) from the mesopelagic layers off southwestern Japan in the Pacific Ocean and obtained a total of 47 bacterial isolates from the gut contents. None of the isolates were luminescent, but almost half of the isolates were chitinolytic. Based on the 16S rRNA gene sequences, these chitinolytic bacteria were identified as members of the genus Vibrio , Photobacterium and Pseudoalteromonas. In culture experiments at 15°C, most of the chitinolytic isolates efficiently utilized colloidal chitin as the sole carbon source and degraded the carapace strip of shrimp (Litopenaeus vannamei). Some isolates showed these chitinoclastic activities even at low temperatures (<5°C), equivalent to that of the deep-sea waters. The present results imply that chitin-degrading bacteria significantly contribute to chitin digestion in the gut of C. reflugens , whereas the involvement of luminous bacteria in the feeding mechanism remains unidentified. [ABSTRACT FROM AUTHOR]

Marine ecosystems are full of unique microbial phenomena, among them bioluminescence that could be harnessed for different ecological functions. This study was conducted to isolate luminous bacteria and utilize their glowing properties as a potential biosensor for toxicity assessment designs. It is worth noting that this study is the first report on the isolation of an Octopus associated Photobacterium species from Alexandria, Egypt. Based on light intensity, strain Photobacterium sp. EAH3 was selected and identified using 16S-rRNA gene sequencing. The luminescence production was further optimized using experimental. Among eight tested variables, the Plackett-Burman design showed that yeast extract, glycerol, peptone, and NaCl were the most important factors affecting luminescence. The central composite design calculated the optimum concentration of these variables in the optimized medium, which enhanced the bioluminescence activity by 1.39 folds. The employment of Photobacterium sp. EAH3 as a biosensor for the toxicity assessment of some solvents, hydrocarbons and heavy metals was examined using bioluminescence inhibition assay. According to the IC50 results, the highest toxicity values recorded were 0.004 M, 4.15 ppm and 1.4 ppm for isoamyl alcohol, Hg2+ and catechol, respectively. Moreover, our study sheds light on futuristic methodology for monitoring toxicity in real polluted environmental and effluents' samples reflecting its promising efficiency.

The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are produced by a hybrid polyketide/fatty acid synthase-encoded by the pfa genes-also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.

Understanding how microbes interact with each other is key to revealing the underlying role that microorganisms play in the host or environment and to identifying microorganisms as an agent that can potentially alter the host or environment. For example, understanding how the microbial interactions associate with parasitic infection can help resolve potential drug or diagnostic test for parasitic infection. To unravel the microbial interactions, existing tools often rely on graphical models to infer the conditional dependence of microbial abundances to represent their interactions. However, current methods do not simultaneously account for the discreteness, compositionality, and heterogeneity inherent to microbiome data. Thus, we build a new approach called "compositional graphical lasso" upon existing tools by incorporating the above characteristics into the graphical model explicitly. We illustrate the advantage of compositional graphical lasso over current methods under a variety of simulation scenarios and on a benchmark study, the Tara Oceans Project. Moreover, we present our results from the analysis of a dataset from the Zebrafish Parasite Infection Study, which aims to gain insight into how the gut microbiome and parasite burden covary during infection, thus, uncovering novel putative methods of disrupting parasite success. Our approach identifies changes in interaction degree between infected and uninfected individuals for three taxa, Photobacterium, Gemmobacter, and Paucibacter, which are inversely predicted by other methods. Further investigation of these method-specific taxa interaction changes reveals their biological plausibility. In particular, we speculate on the potential pathobiotic roles of Photobacterium and Gemmobacter in the zebrafish gut, and the potential probiotic role of Paucibacter. Collectively, our analyses demonstrate that compositional graphical lasso provides a powerful means of accurately resolving interactions between microbiota and can thus drive novel biological discovery. for this article are available online. [ABSTRACT FROM AUTHOR]

The presence of various heavy metals may influence the transformation of many organics by anaerobes under anaerobic conditions. Sometimes, the coexistence of both heavy metals and volatile fatty acids (VFAs) may cause an inhibitory impact on microbial species involved in such biotransformation. These toxic mixtures create disturbances in the bioreactors by inhibiting the ability of the microorganism to degrade the waste. To keep the anaerobic process horizontal, it is essential to know the concentrations of the heavy metals and VFAs during the wastewater treatment operations, otherwise, it would result in halting various operations. Other methods are time-consuming. However, the Microtox analysis provides quick and reliable information about the toxic concentrations of the mixtures by calculating the IC50 15 min luminosity inhibition through Photobacterium phosphoreum T3S. The current research work aimed to assess the impacts of Pb, Cd, Cu, and VFAs on luminescent anaerobic microbial species P. phosphoreum under laboratory conditions. The individual and combined acute toxicity assays were conducted following standard protocols. The results revealed that the combined treatments of individual heavy metals along VFAs caused partly additive toxic effects. The effects of two heavy metals along VFAs resulted in the synergistic type of toxic effects. The application of tri-mixtures of metals along VFAs also caused synergistic effects. The current data would be quite handy to conclude P. phosphoreum as a potential biosensor for metals and volatile fatty acids toxicity and to optimize various bioreactors against different heavy metals and VFA shocks by taking timely corrective measures. [ABSTRACT FROM AUTHOR]

Broomcorn millet Huangjiu brewing is usually divided into primary fermentation and post-fermentation. Microbial succession is the major factor influencing the development of the typical Huangjiu flavor. Here, we report the changes in flavor substances and microbial community during the primary fermentation of broomcorn millet Huangjiu. Results indicated that a total of 161 volatile flavor compounds were measured during primary fermentation, and estragole was detected for the first time in broomcorn millet Huangjiu. A total of 82 bacteria genera were identified. Pediococcus, Pantoea, and Weissella were the dominant genera. Saccharomyces and Rhizopus were dominant among the 30 fungal genera. Correlation analysis showed that 102 microorganisms were involved in major flavor substance production during primary fermentation, Lactobacillus, Photobacterium, Hyphodontia, Aquicella, Erysipelothrix, Idiomarina, Paraphaeosphaeria, and Sulfuritalea were most associated with flavoring substances. Four bacteria, Lactobacillus (R1), Photobacterium (R2), Idiomarina (R3), and Pediococcus (R4), were isolated and identified from wheat Qu, which were added to wine Qu to prepare four kinds of fortified Qu (QR1, QR2, QR3, QR4). QR1 and QR2 fermentation can enhance the quality of Huangjiu. This work reveals the correlation between microorganisms and volatile flavor compounds and is beneficial for regulating the micro-ecosystem and flavor of the broomcorn millet Huangjiu. [ABSTRACT FROM AUTHOR]

Some of the characteristics of probiotics used in aquaculture are the antagonistic activity against pathogens and the activation of the immune response. Moreover, some probiotics with common antigens to pathogens can produce antibodies that react with these pathogens. One of those potential probiotics is Vibrio proteolyticus DCF12.2, a strain with the capacity to produce antibodies in soles (Solea senegalensis) that react with Photobacterium damselae subsp. piscicida and Vibrio harveyi antigens. Therefore, the objective of the work was to determine the capacity of DCF12.2 to activate the immune response in the sole, providing protection against both pathogens. For this purpose, DCF12.2 was administered to cultured soles by intraperitoneal injection, bath, and diet. The DCF12.2 strain activated the gene expression related to the fish immune response and increased the antibody production with cross-reaction to P. damselae subsp. piscicida and V. harveyi cells. Challenges with P. damselae subsp. piscicida resulted in a 30–40% relative percent survival (RPS) through DCF12.2 treatments. After challenge with V. harveyi, only intraperitoneal treatment protected the fish (67% RPS). In conclusion, the DCF12.2 strain showed great potential to be used to prevent diseases caused by both pathogens, and indicates a way to use probiotics as live vaccines. [ABSTRACT FROM AUTHOR]

Although tunas represent a significant part of the global fish economy and a major nutritional resource worldwide, their microbiome still remains poorly documented. Here, we conducted an analysis of the taxonomic composition of the bacterial communities inhabiting the gut, skin, and liver of two most consumed tropical tuna species (skipjack and yellowfin), from individuals caught in the Atlantic and Indian oceans. We hypothesized that each organ harbors a specific microbial assemblage whose composition might vary according to different biotic (sex, species) and/or abiotic (environmental) factors. Our results revealed that the composition of the tuna microbiome was totally independent of fish sex, regardless of the species and ocean considered. Instead, the main determinants of observed diversity were (i) tuna species for the gut and (ii) sampling site for the skin mucus layer and (iii) a combination of both parameters for the liver. Interestingly, 4.5% of all amplicon sequence variants (ASV) were shared by the three organs, highlighting the presence of a core-microbiota whose most abundant representatives belonged to the genera Mycoplasma, Cutibacterium, and Photobacterium. Our study also revealed the presence of a unique and diversified bacterial assemblage within the tuna liver, comprising a substantial proportion of potential histamine-producing bacteria, well known for their pathogenicity and their contribution to fish poisoning cases. These results indicate that this organ is an unexplored microbial niche whose role in the health of both the host and consumers remains to be elucidated. [ABSTRACT FROM AUTHOR]

Prokaryotic α–carbonic anhydrases (α-CA) are metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and proton. We had reported the first crystal structure of a pyschrohalophilic α–CA from a deep-sea bacterium, Photobacterium profundum SS9. In this manuscript, we report the first biochemical characterization of P. profundum α–CA (PprCA) which revealed several catalytic properties that are atypical for this class of CA's. Purified PprCA exhibited maximal catalytic activity at psychrophilic temperatures with substantial decrease in activity at mesophilic and thermophilic range. Similar to other α–CA's, Ppr9A showed peak activity at alkaline pH (pH 11), although, PprCA retained 88% of its activity even at acidic pH (pH 5). Exposing PprCA to varying concentrations of oxidizing and reducing agents revealed that N-terminal cysteine residues in PprCA may play a role in the structural stability of the enzyme. Although inefficient in CO2 hydration activity under mesophilic and thermophilic temperatures, PprCA exhibited salt-dependent thermotolerance and catalytic activity under extreme halophilic conditions. Similar to other well-characterized α–CA's, PprCA is also inhibited by monovalent anions even at low concentrations. Finally, we demonstrate that PprCA accelerates CO2 biomineralization to calcium carbonate under alkaline conditions. [ABSTRACT FROM AUTHOR]

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Simple Summary: Traditionally, enzymes have been studied in standardized buffers, but in real life, inside cells, they have to function under conditions far from "ideal". The intracellular milieu is crowded by numerous molecules with various physical and chemical properties. What consequences does it have for enzymatic reactions? How do enzymes deal with disturbing neighbors? To examine this issue, we studied the mechanisms of influence of seven model substances with different molecular sizes on the reaction catalyzed by bacterial luciferase. This reaction passes through several stages and ends with light emission. We analyzed the responses rate in different stages to the addition of alcohols, saccharides, and biopolymers and found the counteraction of two effects. On the one hand, increased viscosity slows down many of the stages, both associative and dissociative. On the other hand, it is accompanied by an increase in the catalytic constant of luciferase, which compensates for kinetic disadvantages. We assumed that both effects could be caused by the same property of the added substances—their ability to interact with water. Our findings serve not only to shed light on the organization of the cellular metabolism, but also to improve enzyme-based biotechnologies using the recipes of nature. A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water–cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site. [ABSTRACT FROM AUTHOR]

Marine ecosystems are full of unique microbial phenomena, among them bioluminescence that could be harnessed for different ecological functions. This study was conducted to isolate luminous bacteria and utilize their glowing properties as a potential biosensor for toxicity assessment designs. It is worth noting that this study is the first report on the isolation of an Octopus associated Photobacterium species from Alexandria, Egypt. Based on light intensity, strain Photobacterium sp. EAH3 was selected and identified using 16S-rRNA gene sequencing. The luminescence production was further optimized using experimental. Among eight tested variables, the Plackett-Burman design showed that yeast extract, glycerol, peptone, and NaCl were the most important factors affecting luminescence. The central composite design calculated the optimum concentration of these variables in the optimized medium, which enhanced the bioluminescence activity by 1.39 folds. The employment of Photobacterium sp. EAH3 as a biosensor for the toxicity assessment of some solvents, hydrocarbons and heavy metals was examined using bioluminescence inhibition assay. According to the IC 50 results, the highest toxicity values recorded were 0.004 M, 4.15 ppm and 1.4 ppm for isoamyl alcohol, Hg2+ and catechol, respectively. Moreover, our study sheds light on futuristic methodology for monitoring toxicity in real polluted environmental and effluents' samples reflecting its promising efficiency. [ABSTRACT FROM AUTHOR]

Bioluminescence is a common phenomenon in nature, especially in the deep ocean. The physiological role of bacterial bioluminescence involves protection against oxidative and UV stresses. Yet, it remains unclear if bioluminescence contributes to deep-sea bacterial adaptation to high hydrostatic pressure (HHP). In this study, we constructed a non-luminescent mutant of ΔluxA and its complementary strain c-ΔluxA of Photobacterium phosphoreum ANT-2200, a deep-sea piezophilic bioluminescent bacterium. The wild-type strain, mutant and complementary strain were compared from aspects of pressure tolerance, intracellular reactive oxygen species (ROS) level and expression of ROS-scavenging enzymes. The results showed that, despite similar growth profiles, HHP induced the accumulation of intracellular ROS and up-regulated the expression of ROS-scavenging enzymes such as dyp, katE and katG, specifically in the non-luminescent mutant. Collectively, our results suggested that bioluminescence functions as the primary antioxidant system in strain ANT-2200, in addition to the well-known ROS-scavenging enzymes. Bioluminescence contributes to bacterial adaptation to the deep-sea environment by coping with oxidative stress generated from HHP. These results further expanded our understanding of the physiological significance of bioluminescence as well as a novel strategy for microbial adaptation to a deep-sea environment. [ABSTRACT FROM AUTHOR]

GDSL esterase is designated as a member of Family II of lipolytic enzymes known to catalyse the synthesis and hydrolysis of ester bonds. The enzyme possesses a highly conserved motif Ser-Gly-Asn-His in the four conserved blocks I, II, III and V respectively. The enzyme characteristics, such as region-, chemo-, and enantioselectivity, help in resolving the racemic mixture of single-isomer chiral drugs. Recently, crystal structure of GDSL esterase from Photobacterium J15 has been reported (PDB ID: 5XTU) but not in complex with substrate. Therefore, GDSL in complex with substrate could provide insights into the binding mode of substrate towards inactive form of GDSL esterase (S12A) and identify the hot spot residues for the designing of a better binding pocket. Insight into molecular mechanisms is limited due to the lack of crystal structure of GDSL esterase–substrate complex. In this paper, the crystallization of mutant GDSL esterase (S12A) (PDB ID: 8HWO) and its complex with butyric acid (PDB ID: 8HWP) are reported. The optimized structure would be vital in determining hot spot residue for GDSL esterase. This preliminary study provides an understanding of the interactions between enzymes and hydrolysed p-nitro-phenyl butyrate. The information could guide in the rational design of GDSL esterase in overcoming the medical limitations associated with racemic mixture. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lumazine protein from marine luminescent bacteria of Photobacterium species bind with very high affinity to the fluorescent chromophore 6,7-dimethyl-8-ribitylumazine. The light emission of bacterial luminescent systems is used as a sensitive, rapid, and safe assay for an ever-increasing number of biological systems. Plasmid pRFN4, containing the genes encoding riboflavin from the rib operon of Bacillus subtilis, was designed for the overproduction of lumazine. To construct fluorescent bacteria for use as microbial sensors, novel recombinant plasmids (pRFN4-Pp N-lumP and pRFN4-Pp luxLP N-lumP) were constructed by amplifying the DNA encoding the N-lumP gene (luxL) from P. phosphoreum and the promoter region (luxLP) present upstream of the lux operon of the gene by PCR and ligating into the pRFN4-Pp N-lumP plasmid. A new recombinant plasmid, pRFN4-Pp luxLP-N-lumP, was constructed with the expectation that the fluorescence intensity would be further increased when transformed into Escherichia coli. When this plasmid was transformed into E. coli 43R, the fluorescence intensity of transformants was 500 times greater than that of E. coli alone. As a result, the recombinant plasmid in which the gene encoding N-LumP and DNA containing the lux promoter exhibited expression that was so high as to show fluorescence in single E. coli cells. The fluorescent bacterial systems developed in the present study using lux and riboflavin genes can be utilized in the future as biosensors with high sensitivity and rapid analysis times. [ABSTRACT FROM AUTHOR]

The nursehound Scyliorhinus stellaris is a threatened shark species and its population in the Mediterranean Sea is declining. Programs for captive breeding and repopulation in marine protected areas (MPA) are being carried out. Unfortunately, pathogens may hinder conservation plans for this species. An impactful disease of marine animals, caused by the bacteria Photobacterium damselae, has been detected with increased frequency in recent decades in both farmed and marine animals. The aim of this work was to determine the cause of a disease outbreak in eight captive nursehounds that died after 18 months of captivity. Gross necropsy observations were indicative of a presumptive diagnosis of hemorrhagic septicemia. Histological and molecular techniques were performed, to diagnose the etiological agents that could be involved in their mortality. Phylogenetic analysis indicated the presence of P. damselae, identified as subsp. damselae by PCR-duplex, and Photobacterium swingsii in the analyzed captive nursehound Scyliorhinus stellaris. [ABSTRACT FROM AUTHOR]

Alteration of respiratory components as a function of pressure is a common strategy developed in deep-sea microorganisms, presumably to adapt to high hydrostatic pressure (HHP). While the electron transport chain and terminal reductases have been extensively studied in deep-sea bacteria, little is known about their adaptations for ATP generation. In this study, we showed that the deep-sea bacterium Photobacterium profundum SS9 exhibits a more pronounced piezophilic phenotype when grown in minimal medium supplemented with glucose (MG) than in the routinely used MB2216 complex medium. The intracellular ATP level varied with pressure, but with opposite trends in the two culture media. Between the two ATPase systems encoded in SS9, ATPase-I played a dominant role when cultivated in MB2216, whereas ATPase-II was more abundant in the MG medium, especially at elevated pressure when cells had the lowest ATP level among all conditions tested. Further analyses of the ΔatpI, ΔatpE1 and ΔatpE2 mutants showed that disrupting ATPase-I induced expression of ATPase-II and that the two systems are functionally redundant in MB2216. Collectively, we provide the first examination of the differences and relationships between two ATPase systems in a piezophilic bacterium, and expanded our understanding of the involvement of energy metabolism in pressure adaptation. [ABSTRACT FROM AUTHOR]

In enhancing oil recovery, more and more new water‐soluble polymers are developed to replace the high toxicity and low stability acrylamide (ACR) monomer. The common replacement monomer is acrylic acid (AA) and 2‐acrylamido‐2‐methylamido‐2‐methyl‐1‐propanesulfonic acid (AMPS), which are considered safe and efficient. In this study, AA, ACR and AMPS caused remarkable cytotoxicity in Photobacterium phosphoreum, the rat pheochromocytoma cells (PC12) and the Human neuroblastoma cells (SK‐N‐SH). ACR is much more lethal than AA and AMPS in PC12 and SK‐N‐SH cells, meanwhile, the toxicity of AA and AMPS decreases with the decrease of acid. Furthermore, similar to ACR, AA, and AMPS can induce severe DNA double‐strand breakage in PC12 and SK‐N‐SH cells. Both AA and ACR can cause cell cycle arrest in the G0/G1 phase in PC12 and SK‐N‐SH cells. In addition, like ACR, AA, and AMPS can generate reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and mitochondrial‐dependent apoptosis in both PC12 and SK‐N‐SH cells. The acute toxicity of AA and AMPS is lower than ACR, however, the decline in acute toxicity in monomers does not mean toxic‐free. We should focus on the toxicity of AA and ACR and reduce occupational contact to protect employee occupational health. [ABSTRACT FROM AUTHOR]

In this study, we identified and analyzed a novel genomic island (GI), named pdd GI-1, located on chromosome II of certain strains of the marine pathogen Photobacterium damselae subsp. damselae (Pdd). This GI shares structural similarities with other GIs found in Vibrio species, such as the Vibrio seventh pandemic island-II (VSP-II) of V. cholerae. The pdd GI-1 island is a mosaic of gene blocks that encode functions related to ROS defense, anaerobic energy metabolism, and restriction-modification (RM) systems. Notably, pdd GI-1 also includes a complete vibrioferrin siderophore system, enabling the bacteria to thrive in low-iron environments. Vibrioferrin was chemically identified from cell-free supernatants of Pdd RG91. Additionally, a pvsD mutant deficient in vibrioferrin biosynthesis was generated and analyzed. The results suggest that Pdd strains harbouring pdd GI-1 gain a distinct growth advantage under iron-limited conditions. These findings, along with previous research, highlight the significant heterogeneity in iron assimilation systems among Pdd strains. • Strains of Photobacterium damselae subsp damselae harbor the genomic island pdd GI-1. • Pdd GI-1 encodes a complete gene cluster for vibrioferrin synthesis and transport. • Vibrioferrin was chemically identified from cell-free supernatants of Pdd RG91. • A vibrioferrin deficient mutant was generated and analyzed. • The mutant shows reduced growth under iron-restricted conditions and reduced virulence. [ABSTRACT FROM AUTHOR]

ZIF-67, as a typical MOF material, is considered a new type of high-potential adsorbent due to its ample surface area and tunable surface chemistry, which has the potential to interact with other contaminants in unforeseen ways, resulting in combined toxicity. To further elucidate this possibility, we chose typical bisphenol compound (BP) which is widely used in commercial manufacturing, to explore the combined toxicity with MOF. MOF showed a high adsorption capacity for BPAF (> 80 %) and the weakest adsorption capacity for BPA (< 10 %), and DFT confirmed the different interaction strengths of MOF for BPs. The difference in adsorption capacity for BPs resulted in different amounts of free BPs, contributing to combined toxicity. Based on flow cytometry and TEM, the results showed that membrane damage was reduced and the ability of ZIF-67 to enter the cell was decreased in the low-concentration ZIF-67 mixing group, and the ability of ZIF-67 to enter the cell was increased in the high-concentration ZIF-67 mixing group, and the membranes were severely damaged. RT-PCR and biochemical indicators measurements helped to explain the underlying toxicity mechanism. This study is of practical significance for the development of environmental guidelines for mixed contaminant effects and accurate risk assessments. [Display omitted] • Combined effects of ZIF-67 and bisphenol compounds (BPA, BPS and BPAF) to Photobacterium phosphoreum. • MOF showed a high adsorption capacity for BPAF and the weakest adsorption capacity for BPA. • The difference in adsorption capacity for BPs resulted in different amounts of free BPs, contributing to the combined toxicity. • Oxidative damage and luminescence damage are the main reasons for combined toxicity. [ABSTRACT FROM AUTHOR]

Abstract Background Invertebrates are a very attractive subject for studying host-microbe interactions because of their simple gut microbial community and host diversity. Studying the composition of invertebrate gut microbiota and the determining factors is essential for understanding their symbiotic mechanism. Cephalopods are invertebrates that have similar biological properties to vertebrates such as closed circulation system, an advanced nervous system, and a well-differentiated digestive system. However, it is not currently known whether their microbiomes have more in common with vertebrates or invertebrates. This study reports on the microbial composition of six cephalopod species and compares them with other mollusk and marine fish microbiomes to investigate the factors that shape the gut microbiota. Results Each cephalopod gut consisted of a distinct consortium of microbes, with Photobacterium and Mycoplasma identified as core taxa. The gut microbial composition of cephalopod reflected their host phylogeny, the importance of which was supported by a detailed oligotype-level analysis of operational taxonomic units assigned to Photobacterium and Mycoplasma. Photobacterium typically inhabited multiple hosts, whereas Mycoplasma tended to show host-specific colonization. Furthermore, we showed that class Cephalopoda has a distinct gut microbial community from those of other mollusk groups or marine fish. We also showed that the gut microbiota of phylum Mollusca was determined by host phylogeny, habitat, and diet. Conclusion We have provided the first comparative analysis of cephalopod and mollusk gut microbial communities. The gut microbial community of cephalopods is composed of distinctive microbes and is strongly associated with their phylogeny. The Photobacterium and Mycoplasma genera are core taxa within the cephalopod gut microbiota. Collectively, our findings provide evidence that cephalopod and mollusk gut microbiomes reflect host phylogeny, habitat, and diet. It is hoped that these data can contribute to future studies on invertebrate–microbe interactions.

Abstract Background Sharks play essential roles in ocean food webs and human culture, but also face population declines worldwide due to human activity. The relationship between sharks and the microbes on and in the shark body is unclear, despite research on other animals showing the microbiome as intertwined with host physiology, immunity, and ecology. Research on shark-microbe interactions faces the significant challenge of sampling the largest and most elusive shark species. We leveraged a unique sampling infrastructure to compare the microbiomes of two apex predators, the white (Carcharodon carcharias) and tiger shark (Galeocerdo cuvier), to those of the filter-feeding whale shark (Rhincodon typus), allowing us to explore the effects of feeding mode on intestinal microbiome diversity and metabolic function, and environmental exposure on the diversity of microbes external to the body (on the skin, gill). Results The fecal microbiomes of white and whale sharks were highly similar in taxonomic and gene category composition despite differences in host feeding mode and diet. Fecal microbiomes from these species were also taxon-poor compared to those of many other vertebrates and were more similar to those of predatory teleost fishes and toothed whales than to those of filter-feeding baleen whales. In contrast, microbiomes of external body niches were taxon-rich and significantly influenced by diversity in the water column microbiome. Conclusions These results suggest complex roles for host identity, diet, and environmental exposure in structuring the shark microbiome and identify a small, but conserved, number of intestinal microbial taxa as potential contributors to shark physiology.

The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic-acid-containing molecules. Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST) was shown previously to have α2-6-specific, but weak, sialidase activity. Here, we develop a high-throughput blue-white colony screening method to identify Pd2,6ST mutants with improved α2-6-sialidase activity from mutant libraries generated by sequential saturation mutagenesis. A triple mutant (Pd2,6ST S232L/T356S/W361F) has been identified with 100-fold improved activity, high α2-6-sialyl linkage selectivity, and ability to cleave two common sialic acid forms, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). It is a valuable tool for sialoglycan structural analysis and functional characterization. The sequential saturation mutagenesis and screening strategy developed here can be explored to evolve other linkage-specific neoglycosidases from the corresponding glycosyltransferases.

Infectious diseases caused by marine bacterial pathogens inflict increasing economic losses to fisheries and aquaculture, while also posing a growing risk to public health and affected species conservation. In this study, four wild marine fish species were collected at five fishing sites in Israel, divided into two regions—north (Acre, Haifa, Shefayim) and center-south (Tel-Aviv and Ashdod), and screened for Photobacterium damselae. An initial screening was carried out using PCR analysis with specifically designed primers on DNA extracted from livers and kidneys. P. damselae-positive samples had their 16S rRNA amplicons sequenced. Later, an attempt to specify relevant sub-species was performed, using a three-layered gene screen: Car, ureC and toxR. Of 334 fish samples, 47 (14%) were found to be P. damselae-positive, of which 20 were identified as P. damselae subsp. piscicida (Phdp), two as P. damselae subsp. damselae (Phdd) and 25 could not be identified to subspecies. Our results strengthen the view that fish residing in a polluted environment are receptive of pathogenic microorganisms. To assess how the presence of pathogens may affect population management and conservation, this research should be followed by studies aimed at: (i) quantifying levels of pollutants that may affect pathogen emergence, and (ii) creating a standard pollution-level index as a basis for setting criteria, above which authorities should take measures of precaution. [ABSTRACT FROM AUTHOR]

This study investigated the effects of two commercial protective cultures, one containing Lactobacillus sakei (now Latilactobacillus sakei) and the other containing Staphylococcus carnosus and L. sakei, in vacuum-packaged minced beef (ground beef) to advance the understanding of this biopreservation approach for fresh meat shelf-life extension. The protective cultures' effects on spoilage-related bacterial profiles over time were evaluated using both culture-dependent and culture-independent methods in premium (containing 7.7% fat) and standard (containing 19.2% fat) beef mince stored at 4 °C for 12 days. The culture-dependent method showed that in premium mince, the mixed culture containing S. carnosus and L. sakei significantly suppressed the growth of Enterobacteriaceae and Pseudomonas spp. The culture containing only L. sakei exhibited a slight inhibitory effect against these spoilage bacteria. In contrast, neither protective culture inhibited the spoilage bacteria in standard mince. The 16S rRNA gene sequencing indicated bacterial community changes by protective cultures. Photobacterium spp., a potentially important group of meat spoilage bacteria that were usually undetected in culture-dependent studies, were found to be abundant in this study. The protective cultures' impact on other meat quality aspects was also assessed. The culture containing S. carnosus and L. sakei lowered the pH of both premium and standard mince more than the culture containing only L. sakei, and the mixed culture slightly decreased the redness of premium mince. These findings support the use of protective cultures for bacterial spoilage control and shelf-life extension of fresh red meat, but their application may be limited to lean or low-fat products. [ABSTRACT FROM AUTHOR]

"Pirot 'ironed' sausage" (Pis) is a traditional, fermented sausage, made from different types of meat (beef and chevon), without additives or starter cultures. The physical–chemical properties (pH, water activity, fats, moisture, and protein contents) were examined in the initial meat batter stuffing and during ripening. Total bacterial diversity was examined at different time points using both culturable (traditional) and non-culturable (NGS sequencing) approaches. During the ripening, a decrease in pH value, aw, and moisture content was observed, as well as an increase in protein and fat content. At least a two-fold significant decrease was noted for colorimetric values during the ripening period. The dominance of Proteobacteria and Firmicutes was observed in the non-culturable approach in all studied samples. During the ripening process, an increase in Firmicutes (from 33.5% to 63.5%) with a decrease in Proteobacteria (from 65.4% to 22.3%) was observed. The bacterial genera that were dominant throughout the ripening process were Lactobacillus, Photobacterium, Leuconostoc, Weissella, and Lactococcus, while Carnobacterium, Brochothrix, and Acinetobacter were found also, but in negligible abundance. Among the culturable bacteria, Latilactobacillus sakei (Lactobacillus sakei) and Leuconostoc mesenteoides were present in all stages of ripening. [ABSTRACT FROM AUTHOR]

Photobacterium damselae subspecies damselae, an abundant, generalist marine pathogen, has been reported in various cetaceans worldwide. We report a bottlenose dolphin in the eastern Mediterranean Sea that was found stranded and dead. The dolphin had a severe case of chronic suppurative pneumonia and splenic lymphoid depletion caused by this pathogen. [ABSTRACT FROM AUTHOR]

Silver pomfret has been widely cultured in China due to its high economic value. Photobacterium damselae subsp. damselae (PDD) is a Gram-negative bacterium that has been shown to infect many fish species. To increase knowledge of the molecular mechanisms of the host defense against PDD , we conducted transcriptome analysis of head kidney in silver pomfret at 24 h and 72 h post-infection (hpi) via Illumina sequencing. The de novo assembly resulted in the identification of 79,063 unigenes, with 59,386 (75.11%) successfully annotated in public databases (NR, NT, KO, Swiss-Prot, Pfam, GO, and KOG databases). Comparison of gene expression profiles between PBS-injected fish (sham control) and PDD- challenged fish revealed 329 and 570 differentially expressed genes (DEGs) were screened at 24 hpi and 72 hpi, respectively. The DEGs were enriched in multiple immune-related pathways such as Hepatitis C, Gastric acid secretion, CAMs and Leukocyte transendothelial migration pathways, Primary immunodeficieny, ECM-receptor interaction, PI3K-Akt signaling pathway. The data obtained in the present study offers valuable information for acute immune response of silver pomfret challenged with PDD , which will facilitate further investigations on strategies against Photobacterium spp. infection in teleosts. • Silver pomfret head kidney transcriptome was sequenced following acute infection with PDD. • 329 and 570 DEGs were differentially expressed between control and PDD -infected fish, respectively. • The DEG KEGG analysis revealed that 134 and 208 pathways were significantly assigned at 24 and 72 hpi, respectively. • Quantitative PCR analysis of the selected DEGs confirmed that the generated RNA-seq data are a reliable resource. [ABSTRACT FROM AUTHOR]

The immunomodulatory effect of different dietary arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) ratios was evaluated in gilthead sea bream juveniles after intraperitoneal injection (i.p.) with formalin‐killed Photobacterium damselae subsp. piscicida (Phdp). Fish were fed with four isoproteic (47% crude protein) and isolipidic (18% crude lipids) diets with the following long‐chain polyunsaturated fatty acids (LC‐PUFA) ratios: Diet A (2.0% ARA: 0.2% EPA: 0.1% DHA); Diet B (1.0% ARA: 0.4% EPA: 0.4% DHA); Diet C (0% ARA: 0.6% EPA: 0.6% DHA); Diet D (0% ARA: 0.3% EPA: 1.5% DHA). After being fed for 56 days with the experimental diets, fish from each dietary treatment were randomly selected and i.p. injected with 0.2 ml of killed Phdp (1 × 108 CFU ml−1) or phosphate‐buffered saline (control group). Four and twenty‐four hours (i.e., twice) after the i.p., the haematological profile, plasma humoral innate immune parameters, and the intestine expression of immune‐related genes were assessed. Results showed that fish fed diet D (high DHA content) had increased complement activity and higher intestinal expression of cyclooxygenase 2 (cox2) and interleukin 1β (il‐1β) at 4 and 24 hours after i.p., as well as increased intestinal tumour necrosis factor α (tnfα) expression at 4 hours. On the other hand, fish fed with diet A (high ARA content) showed higher plasma bactericidal activity against Phdp than fish fed with diet D. Overall, the results of this study suggest a positive modulatory effect of DHA in response to the pathogenic agent. [ABSTRACT FROM AUTHOR]

The occurrence of pathogenic bacteria has emerged as a plausible key component of summer mortalities in mussels. In the current research, four bacterial isolates retrieved from moribund Greenshell࣪ mussels, Perna canaliculus , from a previous summer mortality event, were tentatively identified as Vibrio and Photobacterium species using morpho-biochemical characterization and MALDI-TOF MS and confirmed as V. celticus, P. swingsii, P. rosenbergii , and P. proteolyticum using whole genome sequencing. These isolates were utilized in a laboratory challenge where mussels were injected with cell concentrations ranging from 105 to 109 CFU/mussel. Of the investigated isolates, P. swingsii induced the highest mortality. Additionally, results from quantitative polymerase chain reaction analysis, focusing on known virulence genes were detected in all isolates grown under laboratory conditions. Photobacterium rosenbergii and P. swingsii showed the highest expression levels of these virulence determinants. These results indicate that Photobacterium spp. could be a significant pathogen of P. canaliculus , with possible importance during summer mortality events. By implementing screening methods to detect and monitor Photobacterium concentrations in farmed mussel populations, a better understanding of the host–pathogen relationship can be obtained, aiding the development of a resilient industry in a changing environment. [ABSTRACT FROM AUTHOR]

The energetic costs of being in stressful conditions require the involvement of hormones associated with metabolic support, which may also influence immune function. The present work aimed to explore the links between tryptophan nutrition and metabolic responses in European seabass (Dicentrarchus labrax) held under space-confined conditions, and subsequently submitted to an immune challenge. To study that, two dietary treatments were evaluated, i.e. control diet (CTRL) and CTRL-based diet supplemented with tryptophan (0.3%; TRP) to fish under space-confinement conditions (10 kg/m 3 ) or not (5 kg/m 3 ). Dietary treatments were offered for 15 days after which fish were intraperitoneally injected (i.p.) with Photobacterium damselae piscicida. Liver was sampled before the immune challenge (at end of the nutritional trial, 0 h) and at 4, 24, 48 and 72 h post-injection. Fish-fed TRP exhibited distinct metabolic profiles compared to those fed CTRL diets, particularly in energy metabolism and stress response. CTRL-fed fish in space-confined conditions showed a gradual reduction of lipid oxidative enzyme activity post-injection, pointing to acute stress-induced lipid catabolism feedback. In contrast, stressed fish fed TRP under same rearing conditions presented reduced glucose levels and cortisol production but unchanged 3-hydroxiacil-CoA dehydrogenase (EC 1.1.1.35, HOAD) activity patterns, suggesting an inhibitory and modulatory role of tryptophan in stress response. Discriminant analysis revealed that fish fed TRP under space-confined conditions resembled fish CTRL-fed under non-stressful conditions. These findings suggest that tryptophan dietary supplementation for stressed fish modulates their metabolic responses and potentially mitigates the negative effects of rearing stressful conditions after subsequent acute stress induced by an immune challenge., Competing Interests: Declarations. Ethics approval: The experimental trial was directed by trained scientists (following FELASA category C recommendations) and conducted according to the guidelines on the protection of animals used for scientific purposes from the European directive 2010/63/UE. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Chemokines play a crucial role in immune responses by facilitating the migration of cells expressing corresponding chemokine receptors along concentration gradients. Photobacterium damselae subsp. Damselae (PDD) and Nocardia seriolae (NS) are known to induce substantial mortality in silver pomfret populations, yet there exists a dearth of research regarding the immune response of CCLs in PDD- or NS-infected silver pomfret. In our investigation, we identified 10 PaCCLs, which include one fish-specific CCL (PaCCL44). Phylogenetic analysis revealed considerable diversity in CCL types and copy numbers among various teleost fishes. Notably, silver pomfret lacks specific CCL genes, with most PaCCLs exhibiting heightened expression levels in immune-related organs such as the spleen and kidney, and some being expressed in mucosal immune-related organs like the skin and gills. Transcriptome analysis conducted on silver pomfret infected with NS and PDD elucidated that the expression changes of PaCCLs primarily manifested in the spleen during the initial stages of NS infection, shifting to the kidney in later stages. Conversely, the expression changes of PaCCLs following PDD infection predominantly occurred in the kidney. In vitro studies using silver pomfret spleen cell lines demonstrated an early peak in PaCCLs expression during infection, followed by gradual decline with NS treatment and rapid diminishment with PDD treatment. These findings suggest that PaCCLs primarily support the innate immunity of silver pomfret, potentially exhibiting chemotactic effects in the early infection stages, such as the synergistic action of PaCCL4 and PaCCL25, and later serving as direct antibacterial agents. NS invasion is characterised by a chronic infection affecting multiple organs, whereas PDD primarily inflicts severe damage to the kidney. PaCCL19a and PaCCL19b are specific to PDD, and their expression levels may decrease in the later stages of infection due to PDD immune escape. These data offer initial insights into understanding the mechanism underlying the innate immune response of the CCL gene family in silver pomfret and provide theoretical underpinnings for fish culture practices., (© 2024 John Wiley & Sons Ltd.)

Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain's metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of Photobacterium ganghwense C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain's complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of Photobacterium ganghwense C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process. [ABSTRACT FROM AUTHOR]

Simple Summary: The present study examined and confirmed the presence of Photobacterium bacteria in aquaculture units in Greece, where the sector plays a crucial role in the national economy. The majority of these bacteria are characterized by high pathogenicity and antibiotic resistance, indicating the need for appropriate management strategies and systematic surveillance.. Bacteria belonging to the species Photobacterium damselae are pathogens of cultured marine fish, causing diseases of high importance, such as Pasteurellosis. Thus, they are considered a major threat to the aquaculture sector. Despite the great importance of fish mariculture for the Greek economy, the distribution and abundance of these bacteria are not well documented in aquaculture units in Greece. Keeping this in mind, the scope of the present study was to investigate the presence, antibiotic profile, and virulence of Photobacterium bacteria originating from a representative sample of mariculture units throughout Greece. Samples were collected from diseased fish belonging to three different cultured fish species, namely Sparus aurata, Dicentrarchus labrax, and Pagrus pagrus, from both the Aegean and the Ionian Sea. Tissue samples were cultured in agar media, and bacteria were molecularly identified using both bacterial universal and species-specific primer pairs for Photobacterium spp. Additionally, the identified strains were characterized for the presence of virulence genes as well as antibiotic profiles. According to the results, the aforementioned bacteria are distributed in the Greek aquaculture units and are characterized by high pathogenicity based on the abundance of virulence genes. Furthermore, the majority of the detected strains exhibit some level of antibiotic resistance. In summary, our results indicate the need for systematic surveillance and study of their antibiotic profiles in Greek aquaculture since these bacteria constitute a major threat to the sector. [ABSTRACT FROM AUTHOR]

Propane is a major component of liquefied petroleum gas, a major energy source for off-grid communities and industry. The replacement of fossil fuel-derived propane with more sustainably derived propane is of industrial interest. One potential production route is through microbial fermentation. Here we report, for the first time, the isolation of a marine bacterium from sediment capable of natural propane biosynthesis. Propane production, both in mixed microbial cultures generated from marine sediment and in bacterial monocultures was detected and quantified by gas chromatography–flame ionization detection. Using DNA sequencing of multiple reference genes, the bacterium was shown to belong to the genus Photobacterium. We postulate that propane biosynthesis is achieved through inorganic carbonate assimilation systems. The discovery of this strain may facilitate synthetic biology routes for industrial scale production of propane via microbial fermentation. [ABSTRACT FROM AUTHOR]

The use of phytogenic extracts is considered a sustainable strategy for the prevention of fish diseases, including Alliaceae as a potential option due to their variety of bioactive compounds. In this study, we analyzed the antibacterial and antiparasitic potential of propyl-propane-thiosulfinate (PTS) and propyl-propane-thiosulfonate (PTSO) from onions. The in vitro activity against Pseudomonas anguilliseptica, Tenacibaculum maritimum, and Photobacterium damselae of both compounds was tested. In addition, the viability of Sparicotyle chrysophrii larvae was evaluated. Moreover, a diet that consisted of a blend of PTS/PTSO (ALLIUM) was used. A total of 90 gilthead sea bream juveniles were tested against P. damselae subsp. Piscicida after 12 weeks of dietary administration. Furthermore, 150 fish with a rate of 10–15 parasites/fish were fed for 21 days and the number of gill parasites was recorded. All strains were sensitive to both compounds. PTSO showed the highest inhibitory effect against all target strains, while PTS showed higher effectiveness against S. chrysophrii. Fish from ALLIUM group presented the highest probability of survival, increasing up to 91.1%, whereas in the control group, the probability of survival was 66.7%. The number of parasites in the gilthead sea bream decreased in the ALLIUM group over time. These results suggest the inclusion of PTS and PTSO in feed as a natural strategy to prevent antibacterial and antiparasitic fish diseases. [ABSTRACT FROM AUTHOR]