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Frontiers in Microbiology 2019Histamine poisoning is the most common cause of human foodborne illness due to the consumption of fish products. An enzyme-based amperometric biosensor was developed to...
Histamine poisoning is the most common cause of human foodborne illness due to the consumption of fish products. An enzyme-based amperometric biosensor was developed to be used as a screening tool to detect histamine and histamine-producing bacteria (HPB) in tuna. It was developed by immobilizing histidine decarboxylase and horseradish peroxidase on the surface of screen-printed electrodes through a cross-linking procedure employing glutaraldehyde and bovine serum albumin. The signal generated in presence of histamine at the surface of the electrode was measured by chronoamperometry at in presence of a soluble redox mediator. The sensitivity of the electrode was 1.31-1.59 μA/mM, with a linear range from 2 to 20 μg/ml and detection limit of 0.11 μg/ml. In this study fresh tuna filets purchased in supermarkets in different days ( = 8) were analyzed to detect HPB. Samples with different concentration of histamine were analyzed with culture-based counting methods, biosensor and HPLC and also a challenge test was made. Recovery of histamine from cultures and tuna samples was also assessed. The presence of , , and was detected using culture- and PCR-based methods. At the time of purchase these tuna samples had histamine concentrations from below the limit of detection (LOD) to 60 μg/g. HPLC and biosensor methods provided similar results in the range from zero to 432 μg/g (correlation coefficient, = 0.990) and the recovery of histamine from cultures and tuna samples was very high (mean bias -12.69 to 1.63%, with root-mean-square error <12%). These results clearly show that fresh tuna is commonly contaminated with strong HPB. The histamine biosensor can be used by the Food Business Operators as a screening tool to detect their presence and to determine whether their process controls are adequate or not.
PubMed: 31507542
DOI: 10.3389/fmicb.2019.01844 -
Frontiers in Immunology 2021The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central...
The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from subsp. or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes' immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher and expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including and . This study showed the ability of methionine surplus to improve leucocytes' response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.
Topics: Animals; Apoptosis; Bass; Cells, Cultured; Culture Media; Head Kidney; Immunity, Innate; Immunomodulation; Leukocytes; Lipopolysaccharides; Methionine; Photobacterium; Tryptophan
PubMed: 33790917
DOI: 10.3389/fimmu.2021.660448 -
Environmental Research Oct 2023Polycyclic aromatic hydrocarbons found in crude oil can impair fish health following sublethal exposure. However, the dysbiosis of microbial communities within the fish...
Polycyclic aromatic hydrocarbons found in crude oil can impair fish health following sublethal exposure. However, the dysbiosis of microbial communities within the fish host and influence it has on the toxic response of fish following exposure has been less characterized, particularly in marine species. To better understand the effect of dispersed crude oil (DCO) on juvenile Atlantic cod (Gadus morhua) microbiota composition and potential targets of exposure within the gut, fish were exposed to 0.05 ppm DCO for 1, 3, 7, or 28 days and 16 S metagenomic and metatranscriptomic sequencing on the gut and RNA sequencing on intestinal content were conducted. In addition to assessing species composition, richness, and diversity from microbial gut community analysis and transcriptomic profiling, the functional capacity of the microbiome was determined. Mycoplasma and Aliivibrio were the two most abundant genera after DCO exposure and Photobacterium the most abundant genus in controls, after 28 days. Metagenomic profiles were only significantly different between treatments after a 28-day exposure. The top identified pathways were involved in energy and the biosynthesis of carbohydrates, fatty acids, amino acids, and cellular structure. Biological processes following fish transcriptomic profiling shared common pathways with microbial functional annotations such as energy, translation, amide biosynthetic process, and proteolysis. There were 58 differently expressed genes determined from metatranscriptomic profiling after 7 days of exposure. Predicted pathways that were altered included those involved in translation, signal transduction, and Wnt signaling. EIF2 signaling was consistently dysregulated following exposure to DCO, regardless of exposure duration, with impairments in IL-22 signaling and spermine and spermidine biosynthesis in fish after 28 days. Data were consistent with predictions of a potentially reduced immune response related to gastrointestinal disease. Herein, transcriptomic-level responses helped explain the relevance of differences in gut microbial communities in fish following DCO exposure.
Topics: Animals; Gastrointestinal Microbiome; Gadus morhua; Petroleum; Fishes; Microbiota; Water Pollutants, Chemical
PubMed: 37399986
DOI: 10.1016/j.envres.2023.116516 -
Bioengineering (Basel, Switzerland) Feb 2022Our current study aimed to adapt a bioluminescent bacteria-based bioassay to monitor the bioeffects of gold nanoparticles (AuNPs). Luminous marine bacteria and AuNPs...
Our current study aimed to adapt a bioluminescent bacteria-based bioassay to monitor the bioeffects of gold nanoparticles (AuNPs). Luminous marine bacteria and AuNPs modified with polyvinylpyrrolidone were employed; low-concentration (≤10 g/L) bioeffects of AuNPs were studied. Bioluminescence intensity was used as an indicator of physiological activity in bacteria. Two additional methods were used: reactive oxygen species (ROS) content was estimated with a chemiluminescent luminol method, and bacterial size was monitored using electron microscopy. The bacterial bioluminescent response to AuNPs corresponded to the "hormesis" model and involved time-dependent bioluminescence activation, as well as a pronounced increase in the number of enlarged bacteria. We found negative correlations between the time courses of bioluminescence and the ROS content in bacterial suspensions, demonstrating the relationship between bioluminescence activation and bacterial ROS consumption. The combined effects of AuNPs and a beta-emitting radionuclide, tritium, revealed suppression of bacterial bioluminescent activity (as compared to their individual effects) and a reduced percentage of enlarged bacteria. Therefore, we demonstrated that our bacteria-based bioluminescence assay is an appropriate tool to study the bioeffects of AuNPs; the bioeffects can be further classified within a unified framework for rapid bioassessment.
PubMed: 35200414
DOI: 10.3390/bioengineering9020061 -
Vavilovskii Zhurnal Genetiki I Selektsii Dec 2023The light emitted by a luminescent bacterium serves as a unique native channel of information regarding the intracellular processes within the individual cell. In the...
The light emitted by a luminescent bacterium serves as a unique native channel of information regarding the intracellular processes within the individual cell. In the presence of highly sensitive equipment, it is possible to obtain the distribution of bacterial culture cells by the intensity of light emission, which correlates with the amount of luciferase in the cells. When growing on rich media, the luminescence intensity of individual cells of brightly luminous strains of the luminescent bacteria Photobacterium leiognathi and Ph. phosporeum reaches 104-105 quanta/s. The signal of such intensity can be registered using sensitive photometric equipment. All experiments were carried out with bacterial clones (genetically homogeneous populations). A typical dynamics of luminous bacterial cells distributions with respect to intensity of light emission at various stages of batch culture growth in a liquid medium was obtained. To describe experimental distributions, a phenomenological model that links the light of a bacterial cell with the history of events at the molecular level was constructed. The proposed phenomenological model with a minimum number of fitting parameters (1.5) provides a satisfactory description of the complex process of formation of cell distributions by luminescence intensity at different stages of bacterial culture growth. This may be an indication that the structure of the model describes some essential processes of the real system. Since in the process of division all cells go through the stage of release of all regulatory molecules from the DNA molecule, the resulting distributions can be attributed not only to luciferase, but also to other proteins of constitutive (and not only) synthesis.
PubMed: 38213711
DOI: 10.18699/VJGB-23-102 -
Fish and Shellfish Immunology Reports Dec 2023The effects of ssp (Phdp) on immune responses and intestinal ultrastructure of following infection and their amelioration by the probiotic bacteria and were...
The effects of ssp (Phdp) on immune responses and intestinal ultrastructure of following infection and their amelioration by the probiotic bacteria and were evaluated. Pathogen growth inhibition in coculture with each probiotic and its virulence against were confirmed with an LC of 10 CFU mL. Phdp administration to at sublethal levels resulted in depletion of superoxide dismutase, glutathione reductase, glutathione transferase and phenoloxidase activities, extensive lipid peroxidation and reduced survival. Following a combined administration of each probiotic and the pathogen, enzyme activities and survival were significantly higher, while lipid peroxidation was reduced, compared to the infected group with no probiotic treatment ( < 0.05). The transmission electron microscopy study revealed that pathogen infection resulted in disarranged and fragmented microvilli, formation of empty or pathogen containing cytoplasmic vacuoles and damaged mitochondria. In the probiotic-treated and Phdp-infected series, intestinal cells showed normal appearance, except for the presence of pathogen-containing vacuoles and highly ordered but laterally stacked microvilli. The results of the present study indicate that Phdp induces cell death through an oxidative stress response and probiotics enhance immune responses to protect it against the Phdp induced damage.
PubMed: 37671319
DOI: 10.1016/j.fsirep.2023.100113 -
Frontiers in Microbiology 2023The mud crab, , holds great commercial significance as a marine crustacean widely cultivated in the Indo-Pacific region. Understanding the core gut microbiota of aquatic...
INTRODUCTION
The mud crab, , holds great commercial significance as a marine crustacean widely cultivated in the Indo-Pacific region. Understanding the core gut microbiota of aquatic animals is crucial for their overall health and growth, yet the core gut microbiota of mud crab remains poorly characterized.
METHODS
In this study, we gathered gut samples from mud crabs across five locations within Sanmen Bay, China. Through the utilization of high-throughput sequencing, we delved into the composition of the gut microbial community and identified the core gut microbiome of mud crab.
RESULTS
Our results demonstrate that the gut microbial diversity of mud crab did not exhibit significant variation among the five sampling sites, although there were some differences in community richness. At the phylum level, we identified 35 representative phyla, with Firmicutes, Proteobacteria, Bacteroidota, and Campilobacterota as the dominant phyla. Among the 815 representative genera, we discovered 19 core genera, which accounted for 65.45% of the total sequences. These core genera were distributed across 6 phyla, and among them, exhibited the highest average relative abundance.
DISCUSSION
has probiotic activity and may play a crucial role in enhancing the immune response of the host and maintaining the diversity of the gut microbiota. Moreover, we observed a positive correlation between the relative abundance of core genera and the stability of the gut microbial community. Furthermore, our findings revealed distinct differences in gut microbial composition and specific taxa between the sexes of mud crab. These differences subsequently influenced the functionality of the gut microbial community. Overall, our investigation sheds light on the core gut microbiota of mud crab, emphasizing the importance of core gut microbial communities in maintaining the health and growth of these commercially significant marine crustaceans.
PubMed: 37727291
DOI: 10.3389/fmicb.2023.1243334 -
PLoS Pathogens Nov 2022Small antibacterial effectors, including lysozymes, lectins, and antimicrobial peptides, are key regulators of intestinal immunity. However, whether there is...
Small antibacterial effectors, including lysozymes, lectins, and antimicrobial peptides, are key regulators of intestinal immunity. However, whether there is coordination among them during regulation is an interesting, but largely unknown, issue. In the present study, we revealed that small effectors synergistically regulate peptidoglycan-derived intestinal immunity in the kuruma shrimp, Marsupenaeus japonicus. A C-type lysozyme (LysC) was screened as a responsive factor for the intestine-bacteria interaction. LysC functions to restrict intestinal bacteria, mainly by cleaving Photobacterium damselae peptidoglycan to generate muropeptides which are powerful stimulators that induce anti-lipopolysaccharides factor B1 (AlfB1), an effective bactericidal peptide. The muropeptides also induce a C-type lectin (Ctl24), which recognizes peptidoglycan and coats bacteria. By counteracting LysC-mediated muropeptide release and AlfB1's bactericidal activity, Ctl24 prevents the continuous elimination of intestinal bacteria. Therefore, this study demonstrates a mechanism by which small immune effectors coordinate to achieve intestinal homeostasis, and provides new insights into peptidoglycan-derived intestinal immunity in invertebrates.
Topics: Animals; Peptidoglycan; Penaeidae; Cell Wall; Intestines; Lectins, C-Type
PubMed: 36417479
DOI: 10.1371/journal.ppat.1010967 -
ACS Chemical Biology May 2018The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic-acid-containing molecules. Photobacterium damselae...
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.
Topics: Bacterial Proteins; High-Throughput Screening Assays; Hydrogen-Ion Concentration; Kinetics; Mutagenesis; Mutation; Neuraminidase; Photobacterium; Sialyltransferases; Substrate Specificity
PubMed: 29543427
DOI: 10.1021/acschembio.8b00002 -
International Journal of Molecular... Apr 2022The marine bacterium subsp. () causes photobacteriosis in fish and important financial losses in aquaculture, but knowledge of its virulence factors is still scarce....
The marine bacterium subsp. () causes photobacteriosis in fish and important financial losses in aquaculture, but knowledge of its virulence factors is still scarce. We here demonstrate that an unstable plasmid (pPHDPT3) that encodes a type III secretion system (T3SS) is highly prevalent in strains from different geographical origins and fish host species. We found that pPHDPT3 undergoes curing upon in vitro cultivation, and this instability constitutes a generalized feature of pPHDPT3-like plasmids in strains. pPHDPT3 markers were detected in tissues of naturally-infected moribund fish and in the colonies grown directly from the fish tissues but were undetectable in a fraction of the colonies produced upon the first passage of the primeval colonies on agar plates. Notably, cured strains exhibited a marked reduction in virulence for fish, demonstrating that pPHDPT3 is a major virulence factor of . The attempts to stabilize pPHDPT3 by insertion of antibiotic resistance markers by allelic exchange caused an even greater reduction in virulence. We hypothesize that the existence of a high pressure to shed pPHDPT3 plasmid in vitro caused the selection of clones with off-target mutations and gene rearrangements during the process of genetic modification. Collectively, these results show that pPHDPT3 constitutes a novel, hitherto unreported virulence factor of that shows a high instability in vitro and warn that the picture of virulence genes has been historically underestimated, since the loss of the T3SS and other plasmid-borne genes may have occurred systematically in laboratories for decades.
Topics: Animals; Fish Diseases; Fishes; Gram-Negative Bacterial Infections; Photobacterium; Plasmids; Type III Secretion Systems; Virulence; Virulence Factors
PubMed: 35563122
DOI: 10.3390/ijms23094729