Search Results (34)

Knowledge about the composition (volatile and non-volatile) and functionality of natural extracts from Mediterranean plants serves as a basis for their further application. In this study, five selected plants were used for the extraction of plant metabolites. Leaves and flowers of Critmum maritimum, Rosmarinus officinalis, Olea europea, Phylliera latifolia and Mellisa officinalis were collected, and a total of 12 extracts were prepared. Extractions were performed under microwave-assisted conditions, with two solvent types: water (W) and a hydroalcoholic (ethanolic) solution (HA). Detailed extract analysis was conducted. Phenolics were analyzed by detecting individual bioactive compounds using high-performance liquid chromatography and by calculating total phenolic and total flavonoid content through spectrophotometric analysis. Higher concentrations of total phenolics and total flavonoids were obtained in the hydroalcoholic extracts, with the significantly highest total phenolic and flavonoid values in the rosemary hydroalcoholic extract (3321.21 mg_GAE/L) and sea fennel flower extract (1794.63 mg_QE/L), respectively; and the lowest phenolics in the water extract of olive leaves (204.55 mg_GAE/L) and flavonoids in the water extracts of sea fennel leaves, rosemary, olive and mock privet (around 100 mg_QE/L). Volatile organic compounds (VOC) were detected using HS-SPME/GC–MS (Headspace Solid-Phase Microextraction coupled with Gas Chromatography-Mass Spectrometry), and antioxidant capacity was estimated using DPPH (2,2-diphenyl-1-picrylhydrazyl assay) and FRAP (Ferric Reducing Antioxidant Power) methods. HS-SPME/GC–MS analysis of samples revealed that sea fennel had more versatile profile, with the presence of 66 and 36 VOCs in W and HA sea fennel leaf extracts, 52 and 25 in W and HA sea fennel flower extracts, 57 in rosemary W and 40 in HA, 20 in olive leaf W and 9 in HA, 27 in W mock privet and 11 in HA, and 35 in lemon balm W and 10 in HA extract. The lowest values of chlorophyll a were observed in sea fennel leaves (2.52 mg/L) and rosemary (2.21 mg/L), and chlorophyll b was lowest in sea fennel leaf and flower (2.47 and 2.25 mg/L, respectively), while the highest was determined in olive (6.62 mg/L). Highest values for antioxidant activity, determined via the FRAP method, were obtained in the HA plant extracts (up to 11,216 mg_AAE/L for lemon balm), excluding the sea fennel leaf (2758 mg_AAE/L) and rosemary (2616 mg_AAE/L). Considering the application of these plants for fresh fish preservation, antimicrobial activity of water extracts was assessed against Vibrio fischeri JCM 18803, Vibrio alginolyticus 3050, Aeromonas hydrophila JCM 1027, Moraxella lacunata JCM 20914 and Yersinia ruckeri JCM 15110. No activity was observed against Y. ruckeri and P. aeruginosa, while the sea fennel leaf showed inhibition against V. fisheri (inhibition zone of 24 mm); sea fennel flower was active against M. lacunata (inhibition zone of 14.5 mm) and A. hydrophila (inhibition zone of 20 mm); and rosemary and lemon balm showed inhibition only against V. fisheri (inhibition zone from 18 to 30 mm). This study supports the preparation of natural extracts from Mediterranean plants using green technology, resulting in extracts rich in polyphenolics with strong antioxidant potential, but with no clear significant antimicrobial efficiency at the tested concentrations. Full article

The fish pathogen Yersinia ruckeri forms biofilms on abiotic surfaces, contributing to recurrent infections in aquaculture. Increasing evidence suggests that the RNA chaperone Hfq and small non-coding RNAs (sRNAs) are key regulators of bacterial biofilm formation. However, the regulatory mechanisms mediated by these factors remain largely unexplored in Y. ruckeri. In this study, we investigated the roles of Hfq and the Hfq-dependent sRNAs RprA, ArcZ, and RybB in the biofilm formation of Y. ruckeri. We first characterized the sRNAome of biofilm-forming cells, identifying the conserved RprA, ArcZ, and RybB, among the upregulated sRNAs. We then evaluated motility, biofilm formation, and architecture in strains lacking either hfq (Δhfq) or these sRNAs (ΔsRNA). Our results reveal that both Δhfq and ΔsRNA strains exhibit significant alterations in biofilm and motility phenotypes, including changes in bacterial morphology and extracellular matrix. Furthermore, expression analyses indicate that these sRNAs modulate the transcription of key regulatory factors, flagellar and phosphodiesterase genes, ultimately influencing intracellular cyclic di-GMP levels, a key second messenger in biofilm formation. Together, our findings demonstrate that Hfq and its associated sRNAs play critical regulatory roles in Y. ruckeri biofilm formation by controlling the expression of genes involved in motility, bacterial envelope proteins, and c-di-GMP metabolism. Full article

The bacterium Yersinia ruckeri causes enteric redmouth disease in salmonids and hence has substantial economic implications for the farmed fish industry. The Norwegian Y. ruckeri outbreak isolate NVH_3758 carries a relatively uncharacterized plasmid, pYR4, which encodes both type 4 pili and a type 4 secretion system. In this study, we demonstrate that pYR4 does not impose a growth burden on the Y. ruckeri host bacterium, nor does the plasmid contribute to twitching motility (an indicator of type 4 pilus function) or virulence in a Galleria mellonella larval model of infection. However, we show that pYR4 is conjugative. We also reveal, through mutagenesis, that pYR4 encodes a functional post-segregational killing system, HigBA, that is responsible for plasmid maintenance within Y. ruckeri. This is the first toxin–antitoxin system to be characterized for this organism. Whilst further work is needed to elucidate the virulence role of pYR4 and whether it contributes to bacterial disease under non-laboratory conditions, our results suggest that the plasmid possesses substantial stability and transfer mechanisms that imply importance within the organism. These results add to our understanding of the mobile genetic elements and evolutionary trajectory of Y. ruckeri as an important commercial pathogen, with consequences for human food production. Full article

Aquaculture is expected to play a vital role in solving the challenge of sustainably providing the growing world population with healthy and nutritious food. Pathogen outbreaks are a major risk for the sector, so early detection and a timely response are crucial. This can be enabled by monitoring the pathogen levels in aquaculture facilities. This paper describes a photonic biosensing platform based on silicon nitride waveguide technology with integrated active components, which could be used for such applications. Compared to the state of the art, the current system presents improvements in terms of miniaturization of the Photonic Integrated Circuit (PIC) and the development of wafer-level processes for hybrid integration of active components and for material-selective chemical and biological surface modification. Furthermore, scalable processes for integrating the PIC in a microfluidic cartridge were developed, as well as a prototype desktop readout instrument. Three bacterial aquaculture pathogens (Aeromonas salmonicida, Vagococcus salmoninarum, and Yersinia ruckeri) were selected for assay development. DNA biomarkers were identified, corresponding primer-probe sets designed, and qPCR assays developed. The biomarker for Aeromonas was also detected using the hybrid PIC platform. This is the first successful demonstration of biosensing on the hybrid PIC platform. Full article

Postbiotics are innovative tools in animal husbandry, providing eco-friendly solutions for disease management within the industry. In this study, a new postbiotic product was evaluated for its impact on the health of rainbow trout (Oncorhynchus mykiss). In vivo studies were conducted to assess the safety of the Weissella cibaria strains used in postbiotic production. Additionally, this study evaluated the impact of diet supplementation with 0.50% postbiotics on growth performance during a 30-day feeding trial; the gut microbial communities, immunomodulation, and protection against Yersinia ruckeri infection were evaluated. The strains did not harm the animals during the 20-day observation period. Furthermore, the effect of postbiotics on growth performance was not significant (p < 0.05). The treated group showed a significant increase in acid-lactic bacteria on the 30th day of the feeding trial, with counts of 3.42 ± 0.21 log CFU/mL. Additionally, there was an up-regulation of the pro-inflammatory cytokine IL-1β in head kidney samples after 48 h of feed supplementation, whereas cytokines IL-10, IL-8, INF-γ, and TNF-α were down-regulated. The findings indicate that rainbow trout fed with postbiotics saw an improvement in their survival rate against Y. ruckeri, with a 20.66% survival improvement in the treated group. This study proves that incorporating postbiotics from two strains of W. cibaria previously isolated from rainbow trout into the diet of fish has immunomodulatory effects, enhances intestinal microbial composition, and improves fish resistance against Y. ruckeri. Full article

Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance. Full article

This research investigates the potential of lactic acid bacteria (LAB) from freshwater salmonids as prospective probiotics for application in aquaculture. LAB and pathogenic bacteria were obtained from mucus and tissues of Oncorhynchus mykiss and Salmo trutta from fish farms in northeast Spain that had not used antibiotics for the six months preceding the study. Isolates were identified using Gram staining and sequencing of 16S rRNA and ITS-1. To assess the safety of the LAB, antibiotic susceptibility tests (ASTs) against 23 antimicrobials were performed. In vitro antagonism assays were conducted to evaluate the inhibitory effects of living LAB using the agar diffusion test method and their metabolites using the agar well diffusion method. The assays targeted six specific pathogens: Aeromonas salmonicida subsp. salmonicida, Carnobacterium maltaromaticum, Vagococcus salmoninarum, Yersinia ruckeri, Lactococcus garvieae, and the marine pathogen Vibrio jasicida. Additionally, a toxicity assay was conducted on embryonic eggs of S. trutta. The ASTs on probiotic LAB candidates revealed varied responses to antimicrobials, but no resistance to oxytetracycline or florfenicol, which are two antibiotics commonly used in aquaculture, was detected. The in vitro assays indicate that LAB exhibit antagonistic effects against pathogens, primarily when directly stimulated by their presence. In applications involving embryonic eggs or larvae, certain live strains of LAB were found to have adverse effects, with some isolates resulting in higher mortality rates compared to the control group or other isolates. Furthermore, the potential pathogenicity of certain LAB strains, typically considered safe in salmonids, warrants deeper investigation. Full article

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed and conserved across species. We have previously shown that in teleost fish, PACAP not only possesses direct antimicrobial properties but also immunomodulatory effects against the bacterial pathogens Flavobacterium psychrophilum and Pseudomonas aeruginosa using in vitro and in vivo experiments. These previous results suggest PACAP can be used as an alternative to antibiotics to prevent and/or treat bacterial infections in the aquaculture industry. To accomplish this goal, more studies are needed to better understand the effect of PACAP on pathogens affecting fish in live infections. In the present study, the transcripts PACAP, PRP/PACAP, and VPAC2 receptor were examined in rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri, which exhibited an increase in their expression in the spleen when compared to healthy fish. Synthetic Clarias gariepinus PACAP-38 has direct antimicrobial activity on Y. ruckeri and inhibits up to 60% of the bacterial growth when the peptide is at concentrations between 50 and 100 µM in TSB. The growth inhibition increased up to 90% in the presence of 12.5 µM of PACAP-38 when salt-free LB broth was used instead of TSB. It was also found to inhibit Y. ruckeri growth in a dose-dependent manner when the rainbow trout monocyte/macrophage-like cell line (RTS11) was pre-treated with lower concentrations of the peptide (0.02 and 0.1 µM) before going through infection. Differential gene expression was analyzed in this in vitro model. Overall, the results revealed new evidence to support the role of PACAP as an antimicrobial and immunomodulatory peptide treatment in teleosts. Full article

IL-10 and IL-6 play important roles in protecting against inflammation and clearing pathogens from the body. In this study, homologous compounds of IL-10 and IL-6 were identified in channel catfish, and their immune responses were analyzed. The CDS sequences of IL-10 and IL-6 were 549 bp and 642 bp, respectively, and showed the highest homology with Ameiurus melas. In addition, the expression of the IL-10 and IL-6 genes was ubiquitous in 10 tissues examined. IL-10 is highly expressed in the liver and slightly expressed in the gill. The high expression of the IL-6 gene was observed in the spleen, heart, and gonad, with the lowest levels in the liver. LPS, Poly(I:C), PHA, and PMA showed a highly significant increase in IL-10 and IL-6 expression 48 h after CCK stimulation (p < 0.01). Otherwise, Yersinia ruckeri, Streptococcus iniae, channel catfish virus, and deltamethrin induced IL-10 and IL-6 expression, varying in intensity between different organs. Our results suggest that IL-10 and IL-6 are involved in the immune response of the host against the pathogen. Full article

Weissella cibaria is a lactic acid bacteria that has been receiving great interest in the last decade; however, its research into aquaculture remains insufficient. In this context, the resistance to pH 3–5, bile salts (10–20%), hydrophobicity, and carbohydrate metabolisms of two W. cibaria strains isolated from rainbow trout were evaluated. At the same time, the postbiotic products of both W. cibaria were produced under different conditions, and their stability to heat (95–130 °C) and proteolytic enzymes were determined. The low pH sensitivity of two pathogens (Aeromonas salmonicida subsp. salmonicida and Yersinia ruckeri) was also recorded. Both strains of W. cibaria survived pH and bile salt treatments. Neither expressed enzymatic activity or hydrophobic characteristics with the executed methods. Treatment with heat, enzymes, and transfer to vegetal broth significantly reduced (p < 0.05) the postbiotic product’s antibacterial activity. On the other hand, no significant difference (p < 0.05) was reported in antibacterial characteristics when postbiotics were produced by coculturing both strains. Low pH does not significantly influence pathogen growth (p < 0.05). Our findings suggest that the studied strains do not adjust as probiotics, but the postbiotic product obtained from them could become a promising tool as a supplement food for fish aquaculture applications. Full article

YTH domain-containing genes are important readers of N⁶-methyladenosine (m⁶A) modifications with ability to directly affect the fates of distinct RNAs in organisms. Despite their importance, little is known about YTH domain-containing genes in teleosts until now. In the present study, a total of 10 YTH domain-containing genes have been systematically identified and functionally characterized in rainbow trout (Oncorhynchus mykiss). According to the phylogenetic tree, gene structure and syntenic analysis, these YTH domain-containing genes could be classified into three evolutionary subclades, including YTHDF, YTHDC1 and YTHDC2. Of them, the copy number of OmDF1, OmDF2, OmDF3, and OmDC1 were duplicated or even triplicated in rainbow trout due to the salmonid-specific whole-genome duplication event. The three-dimensional protein structure analysis revealed that there were similar structures and the same amino acid residues that were associated with cage formation between humans and rainbow trout, implying their similar manners in binding to m⁶A modification. Additionally, the results of qPCR experiment indicated that the expression patterns of a few YTH domain-containing genes, especially OmDF1b, OmDF3a and OmDF3b, were significantly different in liver tissue of rainbow trout under four different temperatures (7 °C, 11 °C, 15 °C, and 19 °C). The expression levels of OmDF1a, OmDF1b and OmDC1a were obviously repressed in spleen tissue of rainbow trout at 24 h after Yersinia ruckeri infection, while increased expression was detected in OmDF3b. This study provides a systemic overview of YTH domain-containing genes in rainbow trout and reveals their biological roles in responses to temperature stress and bacterial infection. Full article

The use of antibiotics in aquaculture leads to the proliferation of multidrug-resistant bacteria, and an urgent need for developing new alternatives to prevent and control disease has, thus, arisen. In this scenario, postbiotics represent a promising tool to achieve this purpose; thus, in this study, isolation and selection of bacteria to further produce and evaluate their postbiotics antibacterial activity against fish pathogens was executed. In this respect, bacterial isolates from rainbow trout and Nile tilapia were obtained and tested in vitro against Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida. From 369 obtained isolates, 69 were selected after initial evaluation. Afterwards, additional screening was carried out by spot-on-lawn assay to finally select twelve isolates; four were identified as Pediococcus acidilactici, seven as Weissella cibaria, and one as Weissella paramesenteroides by matrix assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS). Selected bacteria were used to obtain postbiotic products to test their antagonistic activity through coculture challenge and broth microdilution assays. The influence of incubation time prior to postbiotic production on antagonistic behavior was also recorded. Two isolates identified as W. cibaria were able to significantly reduce (p < 0.05) A. salmonicida subsp. salmonicida’s growth in the coculture challenge up to 4.49 ± 0.05 Log CFU/mL, and even though the reduction in Y. ruckeri was not as effective, some inhibition on the pathogen’s growth was reported; at the same time, most of the postbiotic products obtained showed more antibacterial activity when obtained from broth cultures incubated for 72 h. Based on the results obtained, the preliminary identification of the isolates that expressed the highest inhibitory activity was confirmed by partial sequencing as W. cibaria. Through our study, it can be concluded that postbiotics produced by these strains are useful to inhibit the growth of the pathogens and could, thereby, be applicable in further research to develop suitable tools as feed additives for disease control and prevention in aquaculture. Full article

Yersinia ruckeri is an important fish pathogen causing enteric redmouth disease. Antibiotics have traditionally been used to control this pathogen, but concerns of antibiotic resistance have created a need for alternative interventions. Presently, chlorate and certain nitrocompounds were tested against Y. ruckeri as well as a related species within the genus, Y. aleksiciae, to assess the effects of these inhibitors. The results reveal that 9 mM chlorate had no inhibitory effect against Y. ruckeri, but inhibited growth rates and maximum optical densities of Y. aleksciciae by 20–25% from those of untreated controls (0.46 h⁻¹ and 0.29 maximum optical density, respectively). The results further reveal that 2-nitropropanol and 2-nitroethanol (9 mM) eliminated the growth of both Y. ruckeri and Y. aleksiciae during anaerobic or aerobic culture. Nitroethane, ethyl nitroacetate and ethyl-2-nitropropionate (9 mM) were less inhibitory when tested similarly. Results from a mixed culture of Y. ruckeri with fish tank microbes and of Y. aleksiciae with porcine fecal microbes reveal that the anti-Yersinia activity of the tested nitrocompounds was bactericidal, with 2-nitropropanol and 2-nitroethanol being more potent than the other tested nitrocompounds. The anti-Yersinia activity observed with these tested compounds warrants further study to elucidate the mechanisms of action and strategies for their practical application. Full article

One of the major challenges in detecting waterborne pathogens is the low concentration of the target bacteria in water. In this study, we applied the coagulation and foam concentration method to obtain DNA from water samples collected from upstream, near an estuary. The DNA samples were analyzed using 16S rRNA gene sequencing to clarify the microbial community shifts and to identify potentially pathogenic bacteria. Bacterial communities changed as the river flowed downstream, most likely influenced by land use and human activities such as the discharge of wastewater-treatment plant effluent. Based on the 16S rRNA gene amplicon sequencing, potentially pathogenic bacteria were detected with greater than 0.1% of their relative abundances. Among these, Yersinia ruckeri and Pseudomonas alcaligenes were widely detected in the river water. In addition, digital PCR (dPCR) was used to quantify major waterborne pathogens. Shiga toxin-producing Escherichia coli (STEC), Shigella spp., and Campylobacter jejuni were all below the limit of detection. In contrast, general E. coli, which has the beta-D-glucuronidase gene (uidA) were detected by dPCR (copies/100 mL) at similar levels to those measured using the culture-based method (as colony forming units/100 mL). These results suggest that the coagulation and foam concentration method is useful for concentrating microbes and obtaining DNA from river water samples for environmental monitoring. Full article

Marine bio-sourced chitosan nanoparticles (CSNP) are antimicrobial and immunomodulatory agents beneficial for fish medicine. Herein, dietary CSNP was investigated for the amelioration of the systemic inflammatory responses of an induced fish model. One hundred and forty-four rainbow trout were assigned to one pathogen-free and non-supplemented group (negative control), and three challenged groups: non-supplemented (positive control), CSNP-preventive, and CSNP-therapeutic. After a feeding experiment extended for 21 days, the organosomatic indices (OSI) and molecular aspects were assessed. After a challenge experiment extended for further 28 days, CSNP-therapeutic intervention was assessed on fish survival and systemic inflammatory responses on pathology, histo-morphology, and molecular aspects. With CSNP administration, OSI nonsignificantly decreased and the relative expression of targeted inflammatory-mediator genes was significantly increased. The CSNP-therapeutic fish showed an RPS of 80% as compared to the positive control group, and CSNP-therapeutic administration retained the highest gene expression augmentation up to 28 days after the challenge. Notably, the splenic reticulin fibers framework of the CSNP-therapeutic group retained the highest integrity among the groups during the infection. After recovery, reticulin fibers density in the CSNP-therapeutic samples was significantly higher than in the negative control group, which indicates high innate immunity. Thus, CSNP showed promising biotherapeutic features enhancing fish resistance against infections. Full article