Flavobacterium psychraquaticum sp. nov., isolated from water system of Atlantic salmon (Salmo salar) smolts cultured in Chile.

Strain LB-N7T, a novel Gram-negative, orange, translucent, gliding, rod-shaped bacterium, was isolated from water samples collected from an open system of Atlantic salmon (Salmo salar) smolts in a fish farm in Chile during a flavobacterial infection outbreak in 2015. Phylogenetic analysis based on 16S rRNA sequences (1337 bp) revealed that strain LB-N7T belongs to the genus Flavobacterium and is closely related to the type strains Flavobacterium ardleyense A2-1T (98.8 %) and Flavobacterium cucumis R2A45-3T (96.75 %). The genome size of strain LB-N7T was 2.93 Mb with a DNA G+C content 32.6 mol%. Genome comparisons grouped strain LB-N7T with Flavobacterium cheniae NJ-26T, Flavobacterium odoriferum HXWNR29T, Flavobacterium lacisediminis TH16-21T and Flavobacterium celericrescens TWA-26T. The calculated digital DNA-DNA hybridization values between strain LB-N7T and the closest related Flavobacterium strains were 23.3 % and the average nucleotide identity values ranged from 71.52 to 79.39 %. Menaquinone MK-6 was the predominant respiratory quinone, followed by MK-7. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The primary polar lipids detected included nine unidentified lipids, two amounts of aminopospholipid and phospholipids, and a smaller amount of aminolipid. Phenotypic, genomic, and chemotaxonomic data suggest that strain LB-N7T (=CECT 30406T=RGM 3221T) represents as a novel bacterial species, for which the name Flavobacterium psychraquaticum sp. nov. is proposed.

Comparative analysis of chlorine-resistant bacteria after chlorination and chloramination in drinking water treatment plants.

Chlorine-resistant bacteria (CRB) in drinking water treatment plants (DWTPs) jeopardize water quality and pose a potential risk to human health. However, the specific response of CRB to chlorination and chloramination remains uncharacterized. Therefore, we analyzed 16 S rRNA sequencing data from water samples before and after chlorination and chloramination taken between January and December 2020. Proteobacteria and Firmicutes dominated all finished water samples. After chloramination, Acinetobacter, Pseudomonas, Methylobacterium, Ralstonia, and Sphingomonas were the dominant CRB, whereas Ralstonia, Bacillus, Acinetobacter, Pseudomonas, and Enterococcus were prevalent after chlorination. Over 75% of the CRB e.g. Acinetobacter, Pseudomonas, Bacillus, and Enterococcus were shared between the chlorination and chloramination, involving potentially pathogens, such as Acinetobacter baumannii and Pseudomonas aeruginosa. Notably, certain genera such as Faecalibacterium, Geobacter, and Megasphaera were enriched as strong CRB after chloramination, whereas Vogesella, Flavobacterium, Thalassolituus, Pseudoalteromonas, and others were enriched after chlorination according to LEfSe analysis. The shared CRB correlated with temperature, pH, and turbidity, displaying a seasonal pattern with varying sensitivity to chlorination and chloramination in cold and warm seasons. These findings enhance our knowledge of the drinking water microbiome and microbial health risks, thus enabling better infectious disease control through enhanced disinfection strategies in DWTPs.

Forty-nine metagenomic-assembled genomes from an aquatic virome expand Caudoviricetes by 45 potential new families and the newly uncovered Gossevirus of Bamfordvirae.

Twenty complete genomes (29-63 kb) and 29 genomes with an estimated completeness of over 90 % (30-90 kb) were identified for novel dsDNA viruses in the Yangshan Harbor metavirome. These newly discovered viruses contribute to the expansion of viral taxonomy by introducing 46 potential new families. Except for one virus, all others belong to the class Caudoviricetes. The exception is a novel member of the recently characterized viral group known as Gossevirus. Fifteen viruses were predicted to be temperate. The predicted hosts for the viruses appear to be involved in various aspects of the nitrogen cycle, including nitrogen fixation, oxidation and denitrification. Two viruses were identified to have a host of Flavobacterium and Tepidimonas fonticaldi, respectively, by matching CRISPR spacers with viral protospacers. Our findings provide an overview for characterizing and identifying specific viruses from Yangshan Harbor. The Gossevirus-like virus uncovered emphasizes the need for further comprehensive isolation and investigation of polinton-like viruses.

Gliding motility proteins GldJ and SprB contribute to Flavobacterium columnare virulence.

Flavobacterium columnare causes columnaris disease in fish. Columnaris disease is incompletely understood, and adequate control measures are lacking. The type IX secretion system (T9SS) is required for F. columnare gliding motility and virulence. The T9SS and gliding motility machineries share some, but not all, components. GldN (required for gliding and for secretion) and PorV (involved in secretion but not required for gliding) are both needed for virulence, implicating T9SS-mediated secretion in virulence. The role of motility in virulence is uncertain. We constructed and analyzed sprB, sprF, and gldJ mutants that were defective for motility but that maintained T9SS function to understand the role of motility in virulence. Wild-type cells moved rapidly and formed spreading colonies. In contrast, sprB and sprF deletion mutants were partially defective in gliding and formed nonspreading colonies. Both mutants exhibited reduced virulence in rainbow trout fry. A gldJ deletion mutant was nonmotile, secretion deficient, and avirulent in rainbow trout fry. To separate the roles of GldJ in secretion and in motility, we generated gldJ truncation mutants that produce nearly full-length GldJ. Mutant gldJ563, which produces GldJ truncated at amino acid 563, was defective for gliding but was competent for secretion as measured by extracellular proteolytic activity. This mutant displayed reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Fish that survived exposure to the sprB deletion mutant or the gldJ563 mutant exhibited partial resistance to later challenge with wild-type cells. The results aid our understanding of columnaris disease and may suggest control strategies.IMPORTANCEFlavobacterium columnare causes columnaris disease in many species of freshwater fish in the wild and in aquaculture systems. Fish mortalities resulting from columnaris disease are a major problem for aquaculture. F. columnare virulence is incompletely understood, and control measures are inadequate. Gliding motility and protein secretion have been suggested to contribute to columnaris disease, but evidence directly linking motility to disease was lacking. We isolated and analyzed mutants that were competent for secretion but defective for motility. Some of these mutants exhibited decreased virulence. Fish that had been exposed to these mutants were partially protected from later exposure to the wild type. The results contribute to our understanding of columnaris disease and may aid development of control strategies.

Structure-function analysis of PorXFj, the PorX homolog from Flavobacterium johnsioniae, suggests a role of the CheY-like domain in type IX secretion motor activity.

The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

Characterization of a GH20 ß-N-Acetylhexosaminidase from Flavobacterium algicola Suitable to Synthesize Lacto-N-triose II.

ß-N-Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto-N-triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family ß-N-acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward pNP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM pNP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from pNP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM pNP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 ß-N-acetylhexosaminidases and promoting the high-value utilization of whey powder.

Three novel species of the genus Flavobacterium, Flavobacterium odoriferum sp. nov., Flavobacterium fragile sp. nov. and Flavobacterium luminosum sp. nov., isolated from activated sludge.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria were isolated from activated sludge samples. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that the three strains, designated HXWNR29T, HXWNR69T and HXWNR70T, had the highest sequence similarity to the type strains Flavobacterium cheniae NJ-26T, Flavobacterium channae KSM-R2A30T and Flavobacterium amniphilum KYPY10T with similarities of 97.66 %, 98.66 and 98.14 %, respectively. The draft genomes of these three strains were 2.93 Mbp (HXWNR29T), 2.69 Mbp (HXWNR69T) and 2.65 Mbp (HXWNR70T) long with DNA G+C contents of 31.84 %, 32.83 % and 34.66 %, respectively. These genomes contained many genes responsible for carbohydrate degradation and antibiotic resistance. The major fatty acids (>5 %) included iso-C15 : 0, iso-C15 : 0 3-OH and iso-C17 : 0 3-OH. The major menaquinone was MK-6 for all the three strains. The average nucleotide identity (ANI; 72.7-88.5 %) and digital DNA-DNA hybridization (dDDH; 19.6-35.3 %) results further indicated that these three strains represented three novel species within the genus Flavobacterium, for which the names Flavobacterium odoriferum sp. nov. (type strain HXWNR29T = KCTC 92446T = CGMCC 1.61821T), Flavobacterium fragile sp. nov. (type strain HXWNR69T = KCTC 92468T = CGMCC 1.61442T) and Flavobacterium luminosum sp. nov. (type strain HXWNR70T = KCTC 92447T = CGMCC 1.61443T) are proposed.

Nutrient enrichment increases virulence in an opportunistic environmental pathogen, with greater effect at low bacterial doses.

Eutrophication of aquatic ecosystems is associated with an increased risk of pathogen infection via increased pathogen growth and host exposure via increased pathogen doses. Here, we studied the effect of nutrients on the virulence of an opportunistic bacterial pathogen of fish, Flavobacterium columnare, in challenge experiments with rainbow trout fingerlings. We hypothesized that removing all nutrients by washing the bacteria would reduce virulence as compared to unwashed bacteria, but adding nutrients to the tank water would increase the virulence of the bacterium. Nutrient addition and increase in bacterial dose increased virulence for both unwashed and washed bacteria. For unwashed bacteria, the addition of nutrients reduced the survival probability of fish challenged with low bacterial doses more than for fish challenged with higher bacterial doses, suggesting activation of bacterial virulence factors. Washing and centrifugation reduced viable bacterial counts, and the addition of washed bacteria alone did not lead to fish mortality. However, a small addition of nutrient medium, 0.05% of the total water volume, added separately to the fish container, restored the virulence of the washed bacteria. Our results show that human-induced eutrophication could trigger epidemics of aquatic pathogens at the limits of their survival and affect their ecology and evolution by altering the dynamics between strains that differ in their growth characteristics.

Unveiling the microbiome of hydroponically cultivated lettuce: impact of Phytophthora cryptogea infection on plant-associated microorganisms.

Understanding the complex interactions between plants and their associated microorganisms is crucial for optimizing plant health and productivity. While microbiomes of soil-bound cultivated crops are extensively studied, microbiomes of hydroponically cultivated crops have received limited attention. To address this knowledge gap, we investigated the rhizosphere and root endosphere of hydroponically cultivated lettuce. Additionally, we sought to explore the potential impact of the oomycete pathogen Phytophthora cryptogea on these microbiomes. Root samples were collected from symptomatic and nonsymptomatic plants in three different greenhouses. Amplicon sequencing of the bacterial 16S rRNA gene revealed significant alterations in the bacterial community upon P. cryptogea infection, particularly in the rhizosphere. Permutational multivariate analysis of variance (perMANOVA) revealed significant differences in microbial communities between plants from the three greenhouses, and between symptomatic and nonsymptomatic plants. Further analysis uncovered differentially abundant zero-radius operational taxonomic units (zOTUs) between symptomatic and nonsymptomatic plants. Interestingly, members of Pseudomonas and Flavobacterium were positively associated with symptomatic plants. Overall, this study provides valuable insights into the microbiome of hydroponically cultivated plants and highlights the influence of pathogen invasion on plant-associated microbial communities. Further research is required to elucidate the potential role of Pseudomonas and Flavobacterium spp. in controlling P. cryptogea infections within hydroponically cultivated lettuce greenhouses.

Br - nanoconjugate enhances the antibacterial efficacy of nimboloide against Flavobacterium columnare infection in Labeo rohita: A nanoinformatics approach.

BACKGROUND: The bacterial pathogen, Flavobacterium columnare causes columnaris disease in Labeo rohita globally. Major effects of this bacterial infection include skin rashes and gill necrosis. Nimbolide, the key ingredient of the leaf extract of Azadirachta indica possesses anti-bacterial properties effective against many microorganisms. Nano-informatics plays a promising role in drug development and its delivery against infections caused by multi-drug-resistant bacteria. Currently, studies in the disciplines of dentistry, food safety, bacteriology, mycology, virology, and parasitology are being conducted to learn more about the wide anti-virulence activity of nimbolide. METHODS: The toxicity of nimbolide was predicted to determine its dosage for treating bacterial infection in Labeo rohita. Further, comparative 3-D structure prediction and docking studies are done for nimbolide conjugated nanoparticles with several key target receptors to determine better natural ligands against columnaris disease. The nanoparticle conjugates are being designed using in-silico approaches to study molecular docking interactions with the target receptor. RESULTS: Bromine conjugated nimbolide shows the best molecular interaction with the target receptors of selected species ie L rohita. Nimbolide comes under the class III level of toxic compound so, attempts are made to reduce the dosage of the compound without compromising its efficiency. Further, bromine is also used as a common surfactant and can eliminate heavy metals from wastewater. CONCLUSION: The dosage of bromine-conjugated nimbolide can be reduced to a non-toxic level and thus the efficiency of the Nimbolide can be increased. Moreover, it can be used to synthesize nanoparticle composites which have potent antibacterial activity towards both gram-positive and gram-negative bacteria. This material also forms a good coating on the surface and kills both airborne and waterborne bacteria.

Surface colonization by Flavobacterium johnsoniae promotes its survival in a model microbial community.

Flavobacterium johnsoniae is a ubiquitous soil and rhizosphere bacterium, but despite its abundance, the factors contributing to its success in communities are poorly understood. Using a model microbial community, The Hitchhikers of the Rhizosphere (THOR), we determined the effects of colonization on the fitness of F. johnsoniae in the community. Insertion sequencing, a massively parallel transposon mutant screen, on sterile sand identified 25 genes likely to be important for surface colonization. We constructed in-frame deletions of candidate genes predicted to be involved in cell membrane biogenesis, motility, signal transduction, and transport of amino acids and lipids. All mutants poorly colonized sand, glass, and polystyrene and produced less biofilm than the wild type, indicating the importance of the targeted genes in surface colonization. Eight of the nine colonization-defective mutants were also unable to form motile biofilms or zorbs, thereby suggesting that the affected genes play a role in group movement and linking stationary and motile biofilm formation genetically. Furthermore, we showed that the deletion of colonization genes in F. johnsoniae affected its behavior and survival in THOR on surfaces, suggesting that the same traits are required for success in a multispecies microbial community. Our results provide insight into the mechanisms of surface colonization by F. johnsoniae and form the basis for further understanding its ecology in the rhizosphere. IMPORTANCE: Microbial communities direct key environmental processes through multispecies interactions. Understanding these interactions is vital for manipulating microbiomes to promote health in human, environmental, and agricultural systems. However, microbiome complexity can hinder our understanding of the underlying mechanisms in microbial community interactions. As a first step toward unraveling these interactions, we explored the role of surface colonization in microbial community interactions using The Hitchhikers Of the Rhizosphere (THOR), a genetically tractable model community of three bacterial species, Flavobacterium johnsoniae, Pseudomonas koreensis, and Bacillus cereus. We identified F. johnsoniae genes important for surface colonization in solitary conditions and in the THOR community. Understanding the mechanisms that promote the success of bacteria in microbial communities brings us closer to targeted manipulations to achieve outcomes that benefit agriculture, the environment, and human health.

Complete genome sequence and potential pathogenic assessment of Flavobacterium plurextorum RSG-18 isolated from the gut of Schlegel's black rockfish, Sebastes schlegelii.

Flavobacterium plurextorum is a potential fish pathogen of interest, previously isolated from diseased rainbow trout (Oncorhynchus mykiss) and oomycete-infected chum salmon (Oncorhynchus keta) eggs. We report here the first complete genome sequence of F. plurextorum RSG-18 isolated from the gut of Schlegel's black rockfish (Sebastes schlegelii). The genome of RSG-18 consists of a circular chromosome of 5,610,911 bp with a 33.57% GC content, containing 4858 protein-coding genes, 18 rRNAs, 63 tRNAs and 1 tmRNA. A comparative analysis was conducted on 11 Flavobacterium species previously reported as pathogens or isolated from diseased fish to confirm the potential pathogenicity of RSG-18. In the SEED classification, RSG-18 was found to have 36 genes categorized in 'Virulence, Disease and Defense'. Across all Flavobacterium species, a total of 16 antibiotic resistance genes and 61 putative virulence factors were identified. All species had at least one phage region and type I, III and IX secretion systems. In pan-genomic analysis, core genes consist of genes linked to phages, integrases and matrix-tolerated elements associated with pathology. The complete genome sequence of F. plurextorum RSG-18 will serve as a foundation for future research, enhancing our understanding of Flavobacterium pathogenicity in fish and contributing to the development of effective prevention strategies.

Advanced treatment of antibiotic-polluted wastewater by a consortium composed of bacteria and mixed cyanobacteria.

This study constructed a cyanobacteria-bacteria consortium using a mixture of non-toxic cyanobacteria (Synechococcus sp. and Chroococcus sp.) immobilized in calcium alginate and native bacteria in wastewater. The consortium was used for the advanced treatment of sulfamethoxazole-polluted wastewater and the production of cyanobacterial lipid. Mixed cyanobacteria increased the abundances of denitrifying bacteria and phosphorus-accumulating bacteria as well as stimulated various functional enzymes in the wastewater bacterial community, which efficiently removed 70.01-71.86% of TN, 91.45-97.04% of TP and 70.72-76.85% of COD from the wastewater. The removal efficiency of 55.29-69.90% for sulfamethoxazole was mainly attributed to the upregulation of genes encoding oxidases, reductases, oxidoreductases and transferases in two cyanobacterial species as well as the increased abundances of Stenotrophomonas, Sediminibacterium, Arenimonas, Novosphingobium, Flavobacterium and Hydrogenophaga in wastewater bacterial community. Transcriptomic responses proved that mixed cyanobacteria presented an elevated lipid productivity of 33.90 mg/L/day as an adaptive stress response to sulfamethoxazole. Sediminibacterium, Flavobacterium and Exiguobacterium in the wastewater bacterial community may also promote cyanobacterial lipid synthesis through symbiosis. Results of this study proved that the mixed cyanobacteria-bacteria consortium was a promising approach for advanced wastewater treatment coupled to cyanobacterial lipid production.

Persistence of heterologous Flavobacterium psychrophilum genetic variants in microcosms simulating fish farm and hatchery environments.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, causes substantial economic losses in salmonid farms and hatcheries. Some multilocus sequence types (ST) of F. psychrophilum are more likely to be associated with fish farms and hatcheries, but it is unclear if these patterns of association represent genetic lineages that are more adapted to aquaculture environments. Towards elucidating the disease ecology of F. psychrophilum, the culturability of 10 distinct F. psychrophilum STs was evaluated for 13 weeks in three microcosms including sterilized well water, sterilized well water with commercial trout feed, or sterilized well water with raceway detritus. All STs remained culturable in each of the microcosms for at least 8 weeks, with bacterial concentrations often highest in the presence of raceway detritus. In addition, most (e.g., 90%) STs remained culturable for at least 13-weeks. Significant differences in log10 cfus were observed among STs, both within and between microcosms, suggesting potential variability in environmental persistence capacity among specific variants. Collectively, results highlight the ability of F. psychrophilum to not only persist for weeks under nutrient-limited conditions but also thrive in the presence of organic substrates common in fish farms and hatchery-rearing units.

Efficacy of whole-cell-based monovalent and bivalent vaccines against Streptococcus iniae and Flavobacterium covae in fingerling Asian seabass (Lates calcarifer).

Streptococcosis and columnaris caused by Streptococcus spp. and Flavobacterium spp. have been recognized as critical problems in Asian seabass aquaculture development because they cause severe mortality. In this study, we identified various isolates of S. iniae and F. covae from diseased Asian seabass farmed in Thailand for use as candidates for vaccine development. The efficacy of the vaccines was evaluated by challenge tests and immune parameter analyses in fish that received whole-cell-based monovalent and bivalent vaccines containing S. iniae (Sin) and F. covae (Fco) delivered by top-dressed feed (TD) and intraperitoneal injection (IP). The results showed that all vaccinated groups exhibited increased antibody titers compared with control fish that peaked on day 28 after booster administration with high detection levels in the Sin-IP and Fco-IP groups. Moreover, the immune responses to the injected monovalent vaccines (Sin-IP and Fco-IP) were better than the responses in the other vaccinated groups. The hematological and innate immunological parameters were significantly increased by Sin-IP and Fco-IP, particularly lysozyme activity, nitroblue tetrazolium (NBT) activity, bactericidal activity, and white blood cell numbers, and immune-related genes, including IgM, MHC-IIα, TCRß and CD4, were significantly upregulated in the head kidney, whole blood and spleen (P < 0.05). After experimental challenge, survival in the Sin-IP and Fco-IP groups was significantly higher than that in the Sin-TD, Fco-TD, Sin + Fco-TD, and Sin + Fco-IP groups, with 80.0 % and 60.0 % survival after S. iniae and F. covae infection, respectively. In contrast, survival after bacterial challenge in the control groups was 10 % in each group. Histopathological analysis revealed that Sin-IP- and Fco-IP-vaccinated fish exhibited significantly more goblet cells in the intestines and melanomacrophage centers (MMCs) in the head kidney and spleen than those in the other groups (P < 0.05). Overall, the results of our study indicated that the monovalent vaccines Sin-IP and Fco-IP provoked better vaccine efficacy and immune responses than their orally administered counterparts, and these results are consistent with those from the immunological assays that showed significantly increased responses after immunization.

Early divergent responses to virulent and attenuated vaccine isolates of Flavobacterium covae sp. nov. In channel catfish, Ictalurus punctatus.

Columnaris disease continues to inflict substantial losses among freshwater cultured species since its first description one hundred years ago. The experimental and anecdotal evidence suggests an expanded range and rising virulence of columnaris worldwide due to the warming global climate. The channel catfish (Ictalurus punctatus) are particularly vulnerable to columnaris. A recently developed live attenuated vaccine (17-23) for Flavobacterium columnare (now Flavobacterium covae sp. nov.) demonstrated superior protection for vaccinated catfish against genetically diverse columnaris isolates. In this study, we aimed to elucidate the molecular mechanisms and patterns of immune evasion and host manipulation linked to virulence by comparing gene expression changes in the host after the challenge with a virulent (BGSF-27) or live attenuated F. covae sp. nov. vaccine (17-23). Thirty-day-old fry were accordingly challenged with either virulent or vaccine isolates. Gill tissues were collected at 0 h (control), 1 h, and 2 h post-infection, which are two critical time points in early host-pathogen interactions. Transcriptome profiling of the gill tissues revealed a larger number (518) of differentially expressed genes (DEGs) in vaccine-exposed fish than those exposed to the virulent pathogen (321). Pathway analyses suggested potent suppression of early host immune responses by the virulent isolate through a higher expression of nuclear receptor corepressors (NCoR) responsible for antagonizing macrophage and T-cell signaling. Conversely, in vaccinated fry, we observed induction of Ca2+/calmodulin-dependent protein kinase II (CAMKII), responsible for clearing NCoR, and commensurate up-regulation of transcription factor AP-1 subunits, c-Fos, and c-Jun. As in mammalian systems, AP-1 expression was connected with a broad immune activation in vaccinated fry, including induction of CC chemokines, proteinases, iNOS, and IL-12b. Relatedly, divergent expression patterns of Src tyrosine kinase Lck, CD44, and CD28 indicated a delay or suppression of T-cell adhesion and activation in fry exposed to the virulent isolate. Broader implications of these findings will be discussed. The transcriptomic differences between virulent and attenuated bacteria may offer insights into how the host responds to the vaccination or infection and provide valuable knowledge to understand the early immune mechanisms of columnaris disease in aquaculture.

Phenotypic and genomic characterization of a non-pathogenic Epilithonimonas ginsengisoli isolated from diseased farmed rainbow trout (Oncorhynchus mykiss) in Chile.

Flavobacterial infection associated with diseased fish is caused by multiple bacterial species within the family Flavobacteriaceae. In the present study, the Chilean isolate FP99, from the gills of a diseased, farmed rainbow trout (Oncorhynchus mykiss), was characterized using phenotypic and genomic analyses. Additionally assessed was pathogenic activity. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that isolate FP99 belonged to the genus Epilithonimonas, an average nucleotide identity value of 100% was detected with the Chilean isolate identified as Epilithonimonas sp. FP211-J200. In silico genome analysis, mechanisms for toxins production, and superantigens, adhesion, or other genes associated with virulence were not detected. However, genes encoding proteins for antibiotic resistance were found, including the chrA gene and the nucleotide sequence that encodes for multiple antibiotic resistance MarC proteins. Furthermore, the blaESP-1 gene (87.85% aminoacidic sequence identity), encoding an extended-spectrum subclass B3 metallo-ß-lactamase and conferring carbapenem-hydrolysing activity, and the tet(X) gene, which encodes a monooxygenase that catalyses the degradation of tetracycline-class antimicrobials were carried by this isolate. Phenotyping analyses also supported assignment as E. ginsengisoli. Challenge trials against healthy rainbow trout resulted in no observed pathogenic effect. Our findings identify for the first time the species E. ginsengisoli as associated with fish farming, suggesting that this isolate may be a component of the microbiota of the freshwater system. Notwithstanding, poor environmental conditions and any stressors associated with aquaculture situations or lesions caused by other pathogenic bacteria, such as F. psychrophilum, could favour the entry of E. ginsengisoli into rainbow trout.

Analysis of programmed frameshifting during translation of prfB in Flavobacterium johnsoniae.

Ribosomes of Bacteroidia fail to recognize Shine-Dalgarno (SD) sequences due to sequestration of the 3' tail of the 16S rRNA on the 30S platform. Yet in these organisms, the prfB gene typically contains the programmed +1 frameshift site with its characteristic SD sequence. Here, we investigate prfB autoregulation in Flavobacterium johnsoniae, a member of the Bacteroidia. We find that the efficiency of prfB frameshifting in F. johnsoniae is low (∼7%) relative to that in Escherichia coli (∼50%). Mutation or truncation of bS21 in F. johnsoniae increases frameshifting substantially, suggesting that anti-SD (ASD) sequestration is responsible for the reduced efficiency. The frameshift site of certain Flavobacteriales, such as Winogradskyella psychrotolerans, has no SD. In F. johnsoniae, this W. psychrotolerans sequence supports frameshifting as well as the native sequence, and mutation of bS21 causes no enhancement. These data suggest that prfB frameshifting normally occurs without SD-ASD pairing, at least under optimal laboratory growth conditions. Chromosomal mutations that remove the frameshift or ablate the SD confer subtle growth defects in the presence of paraquat or streptomycin, respectively, indicating that both the autoregulatory mechanism and the SD element contribute to F. johnsoniae cell fitness. Analysis of prfB frameshift sites across 2686 representative bacteria shows loss of the SD sequence in many clades, with no obvious relationship to genome-wide SD usage. These data reveal unexpected variation in the mechanism of frameshifting and identify another group of organisms, the Verrucomicrobiales, that globally lack SD sequences.

Varying Flavobacterium psychrophilum shedding dynamics in three bacterial coldwater disease-susceptible salmonid (Family Salmonidae) species.

Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and is responsible for substantial losses in farm and hatchery-reared salmonids (Family Salmonidae). Although F. psychrophilum infects multiple economically important salmonids and is transmitted horizontally, the extent of knowledge regarding F. psychrophilum shedding rates and duration is limited to rainbow trout (Oncorhynchus mykiss). Concurrently, hundreds of F. psychrophilum sequence types (STs) have been described using multilocus sequence typing (MLST), and evidence suggests that some variants have distinct phenotypes, including differences in host associations. Whether shedding dynamics differ among F. psychrophilum variants and/or salmonids remains unknown. Thus, three F. psychrophilum isolates (e.g., US19, US62, and US87) in three MLST STs (e.g., ST13, ST277, and ST275) with apparent host associations for coho salmon (O. kisutch), Atlantic salmon (Salmo salar), or rainbow trout were intramuscularly injected into each respective fish species. Shedding rates of live and dead fish were determined by quantifying F. psychrophilum loads in water via quantitative PCR. Both live and dead Atlantic and coho salmon shed F. psychrophilum, as did live and dead rainbow trout. Regardless of salmonid species, dead fish shed F. psychrophilum at higher rates (e.g., up to ~108-1010 cells/fish/hour) compared to live fish (up to ~107-109 cells/fish/hour) and for a longer duration (5-35 days vs 98 days); however, shedding dynamics varied by F. psychrophilum variant and/or host species, a matter that may complicate BCWD management. Findings herein expand knowledge on F. psychrophilum shedding dynamics across multiple salmonid species and can be used to inform future BCWD management strategies.IMPORTANCEFlavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, both of which cause substantial losses in farmed and hatchery-reared salmon and trout populations worldwide. This study provides insight into F. psychrophilum shedding dynamics in rainbow trout (Oncorhynchus mykiss) and, for the first time, coho salmon (O. kisutch) and Atlantic salmon (Salmo salar). Findings revealed that live and dead fish of all fish species shed the bacterium. However, dead fish shed F. psychrophilum at higher rates than living fish, emphasizing the importance of removing dead fish in farms and hatcheries. Furthermore, shedding dynamics may differ according to F. psychrophilum genetic variant and/or fish species, a matter that may complicate BCWD management. Overall, study results provide deeper insight into F. psychrophilum shedding dynamics and will guide future BCWD management strategies.

Systemic immune response of rainbow trout exposed to Flavobacterium psychrophilum infection.

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is one of the most serious bacterial diseases leading to significant economic loss for rainbow trout (Oncorhynchus mykiss) aquaculture. However, little is known about the systemic immune response of rainbow trout against F. psychrophilum infection. This study investigated the immune response of rainbow trout to F. psychrophilum infection using multiple experiments, including bacterial load detection, phagocyte activity assessment, enzyme activity evaluation, and gene expression profiling. Results showed that the spleen index and intestinal pathogen load reached a peak at 3 days post-infection, with strong pro-inflammatory gene expression observed in rainbow trout. Leukocytes RBA and PKA were significantly elevated in the spleen, blood and intestine at 7 days post-infection. Heat map analysis demonstrated that the spleen had a more substantial pro-inflammatory response compared to the intestine post-infection and exhibited higher expression levels of immune-related genes, including IgM, il1ß, il6, cd4, cd8a, cd8b, c1q, chathelicidin, inos, and lysozyme. Both Th1 and Th2 polarized responses in the spleen were activated, with Th2 (il4/13a, gata3) (FC > 4) being more intense than Th1 (tnfα, t-bet) (FC > 2). Tight junction proteins exhibited down-regulation followed by up-regulation post-infection. Collectively, the results of this study expand our current understanding of the immune response of rainbow trout post F. psychrophilum infection but also provide new avenues for investigation in salmonid aquaculture.