Search Results (18)

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

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

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

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

Marine finfish aquaculture is affected by diverse infectious diseases, and they commonly occur as co-infection. Some of the most frequent and prevalent Gram-negative bacterial pathogens of the finfish aquaculture include Piscirickettsia salmonis, Aeromonas salmonicida, Yersinia ruckeri, Vibrio anguillarum and Moritella viscosa. To prevent co-infections in aquaculture, polyvalent or universal vaccines would be ideal. Commercial polyvalent vaccines against some of these pathogens are based on whole inactivated microbes and their efficacy is controversial. Identification of common antigens can contribute to the development of effective universal or polyvalent vaccines. In this study, we identified common and unique antigens of P. salmonis, A. salmonicida, Y. ruckeri, V. anguillarum and M. viscosa based on a reverse vaccinology pipeline. We screened the proteome of several strains using complete available genomes and identified a total of 154 potential antigens, 74 of these identified antigens corresponded to secreted proteins, and 80 corresponded to exposed outer membrane proteins (OMPs). Further analysis revealed the outer membrane antigens TonB-dependent siderophore receptor, OMP assembly factor BamA, the LPS assembly protein LptD and secreted antigens flagellar hook assembly protein FlgD and flagellar basal body rod protein FlgG are present in all pathogens used in this study. Sequence and structural alignment of these antigens showed relatively low percentage sequence identity but good structural homology. Common domains harboring several B-cells and T-cell epitopes binding to major histocompatibility (MHC) class I and II were identified. Selected peptides were evaluated for docking with Atlantic salmon (Salmo salar) and Lumpfish MHC class II. Interaction of common peptide-MHC class II showed good in-silico binding affinities and dissociation constants between −10.3 to −6.5 kcal mol⁻¹ and 5.10 × 10⁻⁹ to 9.4 × 10⁻⁶ M. This study provided the first list of antigens that can be used for the development of polyvalent or universal vaccines against these Gram-negative bacterial pathogens affecting finfish aquaculture. Full article

Currently, aquaculture production of rainbow trout (Oncorhynchus mykiss) is a multibillion dollar industry; nevertheless, the development of this sector has not been exempt from pitfalls related to the recurrent presence of pathogens of bacterial origin. This is the case of Yersinia ruckeri, the etiologic agent of the infectious pathology known as Enteric Red Mouth Disease (ERM), causing serious economic losses that can be as high as 30–70% of production. Although several studies have been performed regarding pathogen features and virulence factors, more information is needed about the host defense mechanism activation after infection. Given this perspective, this study aimed to evaluate rainbow trout’s short-term innate immune response against infection with Y. ruckeri. A series of factors linked to the innate immune response were evaluated, including determination of hematological parameters, oxidative stress biomarkers, and analysis of the expression of immune-related genes. Results showed a significant decrease in several hematological parameters (white blood cell count, hematocrit, neutrophils, monocytes, lymphocytes, and thrombocytes) and oxidative stress indicators (SOD) between the control and infected groups. In addition, there were significant differences in the level of gene expression between infected individuals and the control group. Most of these genes (il-1β, il-8, il-10, tnf-α1, tnf-α2, socs3, mmp-9, cath, hsp-70, saa, fer, pcb) were upregulated within the first 24 h following infection. Results from this study showed more insights into the short-term immune response of rainbow trout to infection with Y. ruckeri, which may be useful for the establishment of biomarkers that may be used for the early detection of ERM. Full article

Background: The IPNV is one of the most common viral pathogens of rainbow trout (Oncorhynchus mykiss), while Y. ruckeri infections are widespread among bacterial agents. The current study aimed to determine the influence of IPNV and Y. ruckeri co-infection on a non-specific immune response. Methods: Two experiments were conducted. The first experiment determined the changes in non-specific immunity parameters upon the simultaneous occurrence of IPNV and Y. ruckeri infection. In the second experiment, infection with the IPNV was performed two weeks before Y. ruckeri infection. The level of total protein, gamma globulins, the activity of lysozyme and ceruloplasmin, as well as the metabolic activity and potential killing activity of phagocytes were measured: 0, 24 h, 72 h, 7 days, 14 days, and 21 days after co-infection. Results: A differentiated effect on the parameters of the non-specific immune response was shown between single infections with the IPNV and Y. ruckeri as well as co-infection with these pathogens. Conclusions: The immune response in the course of a co-infection depended on the time between infections. IPNV infection causes lysozyme activity suppression, which may lead to secondary bacterial infections. Full article

Yersinia ruckeri causes outbreaks of enteric redmouth disease in salmon aquaculture all over the world. The transient antibiotic tolerance exhibited by bacterial persisters is commonly thought to be responsible for outbreaks; however, the molecular factors underlying this behavior have not been explored in Y. ruckeri. In this study, we investigated the participation of the RNA chaperone Hfq from Y. ruckeri in antibiotic persistence. Cultures of the hfq-knockout mutant (Δhfq) exhibited faster replication, increased ATP levels and a more reductive environment than the wild type. The growth curves of bacteria exposed to sublethal concentrations of ampicillin, oxolinic acid, ciprofloxacin and polymyxin B revealed a greater susceptibility for the Δhfq strain. The time-kill curves of bacteria treated with the antibiotics mentioned above and florfenicol, using inoculums from exponential, stationary and biofilm cultures, demonstrated that the Δhfq strain has significant defects in persister cells production. To shed more light on the role of Hfq in antibiotic persistence, we analyzed its dependence on the (p)ppGpp synthetase RelA by determining the persister cells production in the absence of the relA gene. The ΔrelA and ΔrelAΔhfq strains displayed similar defects in persister cells formation, but higher than Δhfq strain. Similarly, stationary cultures of the ΔrelA and ΔrelAΔhfq strains exhibited comparable levels of ATP but higher than that of the Δhfq strain, indicating that relA is epistatic over hfq. Taken together, our findings provide valuable information on antibiotic persistence in Y. ruckeri, shedding light on the participation of Hfq in the persistence phenomenon. Full article

To uncover the relationship between skin bacterial flora and pathogen infection, we developed a percutaneous infection model using zebrafish and Yersinia ruckeri, a pathogen causing enteric redmouth disease in salmon and in trout. Pathogen challenge, either alone or together with pricking by a small needle, did not cause infection of the fish. However, cold stress given by water temperature shift from the optimum 28 °C for zebrafish to 20 °C caused fatal infection of injured fish following pathogen challenge. We investigated the effects of cold stress, injury, and pathogen challenge, alone and in combination, on fish skin bacterial flora using 16S rDNA metagenomics. We found that cold stress drastically altered the skin bacterial flora, which was dominated by Y. ruckeri on infected fish. In addition, fish whose intrinsic skin bacterial flora was disrupted by antibiotics had their skin occupied by Y. ruckeri following a challenge with this pathogen, although the fish survived without injury to create a route for invasion into the fish body. Our results suggest that the intrinsic skin bacterial flora of fish protects them from pathogen colonization, and that its disruption by stress allows pathogens to colonize and dominate their skin. Full article

Yersinia pseudotuberculosis, Y. enterocolitica and Y. pestis are pathogenic bacteria capable of causing disease in humans by growing extracellularly in lymph nodes and during systemic infections. While the capacity of these bacteria to invade, replicate, and survive within host cells has been known for long, it is only in recent years that their intracellular stages have been explored in more detail. Current evidence suggests that pathogenic Yersinia are capable of activating autophagy in both phagocytic and epithelial cells, subverting autophagosome formation to create a niche supporting bacterial intracellular replication. In this review, we discuss recent results opening novel perspectives to the understanding of intimate host-pathogens interactions taking place during enteric yersiniosis and plague. Full article

YerA41 is a Myoviridae bacteriophage that was originally isolated due its ability to infect Yersinia ruckeri bacteria, the causative agent of enteric redmouth disease of salmonid fish. Several attempts to determine its genomic DNA sequence using traditional and next generation sequencing technologies failed, indicating that the phage genome is modified in such a way that it is an unsuitable template for PCR amplification and for conventional sequencing. To determine the YerA41 genome sequence, we performed RNA-sequencing from phage-infected Y. ruckeri cells at different time points post-infection. The host-genome specific reads were subtracted and de novo assembly was performed on the remaining unaligned reads. This resulted in nine phage-specific scaffolds with a total length of 143 kb that shared only low level and scattered identity to known sequences deposited in DNA databases. Annotation of the sequences revealed 201 predicted genes, most of which found no homologs in the databases. Proteome studies identified altogether 63 phage particle-associated proteins. The RNA-sequencing data were used to characterize the transcriptional control of YerA41 and to investigate its impact on the bacterial gene expression. Overall, our results indicate that RNA-sequencing can be successfully used to obtain the genomic sequence of non-sequencable phages, providing simultaneous information about the phage–host interactions during the process of infection. Full article