Oral administration of a new copper (I) complex with coumarin as ligand: modulation of the immune response and the composition of the intestinal microbiota in Onchorhynchus mykiss.

[Cu(NN1)2]ClO4 is a copper (I) complex, where NN1 is an imine ligand 6-((quinolin-2-ylmethylene) amino)-2H-chromen-2-one obtained by derivatization of natural compound coumarin, developed for the treatment of infectious diseases that affect salmonids. In previous research, we showed that the Cu(I) coordination complex possesses antibacterial activity against Flavobacterium psychrophilum, providing protection against this pathogen in rainbow trout during challenge assays (with an RPS of 50%). In the present study, the effects of administering [Cu(NN1)2]ClO4 to Oncorhynchus mykiss over a 60-days period were evaluated with regard to systemic immune response and its potential to alter intestinal microbiota composition. In O. mykiss, an immunostimulatory effect was evident at days 30 and 45 after administration, resulting in an increment of transcript levels of IFN-γ, IL-12, TNF-α, lysozyme and perforin. To determine whether these immunomodulatory effects correlated with changes in the intestinal microbiota, we analyzed the metagenome diversity by V4 16S rRNA sequencing. In O. mykiss, both [Cu(NN1)2]ClO4 and commercial antibiotic florfenicol had comparable effects at the phylum level, resulting in a predominance of proteobacteria and firmicutes. Nonetheless, at the genus level, florfenicol and [Cu(NN1)2]ClO4 complex exhibited distinct effects on the intestinal microbiota of O. mykiss. In conclusion, our findings demonstrate that [Cu(NN1)2]ClO4 is capable of stimulating the immune system at a systemic level, while inducing alterations in the composition of the intestinal microbiota in O. mykiss.

[Cu(NN1)2]ClO4, a Copper (I) Complex as an Antimicrobial Agent for the Treatment of Piscirickettsiosis in Atlantic Salmon.

Piscirickettsia salmonis is the pathogen that most affects the salmon industry in Chile. Large quantities of antibiotics have been used to control it. In search of alternatives, we have developed [Cu(NN1)2]ClO4 where NN1 = 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one. The antibacterial capacity of [Cu(NN1)2]ClO4 was determined. Subsequently, the effect of the administration of [Cu(NN1)2]ClO4 on the growth of S. salar, modulation of the immune system and the intestinal microbiota was studied. Finally, the ability to protect against a challenge with P. salmonis was evaluated. The results obtained showed that the compound has an MIC between 15 and 33.9 µg/mL in four isolates. On the other hand, the compound did not affect the growth of the fish; however, an increase in the transcript levels of IFN-γ, IL-12, IL-1ß, CD4, lysozyme and perforin was observed in fish treated with 40 µg/g of fish. Furthermore, modulation of the intestinal microbiota was observed, increasing the genera of beneficial bacteria such as Lactobacillus and Bacillus as well as potential pathogens such as Vibrio and Piscirickettsia. Finally, the treatment increased survival in fish challenged with P. salmonis by more than 60%. These results demonstrate that the compound is capable of protecting fish against P. salmonis, probably by modulating the immune system and the composition of the intestinal microbiota.

Analysis of microbiota-host communication mediated by butyrate in Atlantic salmon.

Butyrate is a microbiota-produced metabolite, sensed by host short-chain fatty acid receptors FFAR2 (Gpr43), FFAR3 (Gpr41), HCAR2 (Gpr109A), and Histone deacetylase (HDAC) that promotes microbiota-host crosstalk. Butyrate influences energy uptake, developmental and immune response in mammals. This microbial metabolite is produced by around 79 anaerobic genera present in the mammalian gut, yet little is known about the role of butyrate in the host-microbiota interaction in salmonid fish. To further our knowledge of this interaction, we analyzed the intestinal microbiota and genome of Atlantic salmon (Salmo salar), searching for butyrate-producing genera and host butyrate receptors. We identified Firmicutes, Proteobacteria, and Actinobacteria as the main butyrate-producing bacteria in the salmon gut microbiota. In the Atlantic salmon genome, we identified an expansion of genes orthologous to FFAR2 and HCAR2 receptors, and class I and IIa HDACs that are sensitive to butyrate. In addition, we determined the expression levels of orthologous of HCAR2 in the gut, spleen, and head-kidney, and FFAR2 in RTgutGC cells. The effect of butyrate on the Atlantic salmon immune response was evaluated by analyzing the pro and anti-inflammatory cytokines response in vitro in SHK-1 cells by RT-qPCR. Butyrate decreased the expression of the pro-inflammatory cytokine IL-1ß and increased anti-inflammatory IL-10 and TGF-ß cytokines. Butyrate also reduced the expression of interferon-alpha, Mx, and PKR, and decreased the viral load at a higher concentration (4 mM) in cells treated with this molecule before the infection with Infectious Pancreatic Necrosis Virus (IPNV) by mechanisms independent of FFAR2, FFAR3 and HCAR2 expression that probably inhibit HDAC. Moreover, butyrate modified phosphorylation of cytoplasmic proteins in RTgutGC cells. Our data allow us to infer that Atlantic salmon have the ability to sense butyrate produced by their gut microbiota via different specific targets, through which butyrate modulates the immune response of pro and anti-inflammatory cytokines and the antiviral response.

Protective Effect of [Cu(NN1)2](ClO4) Complex in Rainbow Trout Challenged against Flavobacterium psychrophilum.

Previously, we reported an in vitro evaluation regarding antibacterial effects against F. psychrophilum by a new Cu (I) complex, [Cu(NN1)2](ClO4). This study presents the results of an in vivo evaluation of [Cu(NN1)2](ClO4) added as a dietary supplement against F. psychrophilum in rainbow trout. The results showed that the administration of [Cu(NN1)2](ClO4) at 29 and 58 µg/g of fish for 15 days does not affect the growth of rainbow trout. On the other hand, the amount of copper present in the liver, intestine, and muscle of rainbow trout was determined. The results showed that the amount of copper in the liver, when compared between treated fish and control fish, does not change. While, in the intestine, an increase in the fish fed at 58 µg/g of fish was observed. In muscle, a slight decrease at 29 µg/g was obtained. Additionally, copper concentrations in the pond water after 15 days of feeding with the [Cu(NN1)2](ClO4) complex showed the highest levels of copper. Finally, the effect of the administration of [Cu(NN1)2](ClO4) for 15 days at 58 µg/g of fish was evaluated against F. psychrophilum, where a 75% survival was obtained during 20 days of challenge.

In Vitro Effect of Copper (I) Complex [Cu(NN1)2](ClO4) on Vibrio harveyi BB170 Biofilm Formation.

Biofilm formation in pathogenic bacteria is an important factor of resistance to antimicrobial treatments, allowing them to survive for a long time in their hosts. In the search for new antibiofilm agents, in this work we report the activity of a copper (I) complex, [Cu(NN1)2]ClO4, synthesized with Cu (I) and NN1, an imine ligand 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one, a derivate of natural compound coumarin. The antibacterial and antibiofilm capacity was evaluated in Vibrio harveyi BB170 used as model bacteria. Antibacterial activity was measured in vitro by minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and half-maximal inhibitory concentration (IC50) determination. Antibiofilm capacity of copper (I) complex was analyzed by different concentrations of IC50 values. The results showed that the sub-IC50 concentration, 12.6 µg/mL of the copper (I) complex, was able to reduce biofilm formation by more than 75%, and bacterial viability was reduced by 50%. Inverted and confocal laser scanning microscopy showed that the [Cu(NN1)2]ClO4 complex affected the biofilm structure. Therefore, the copper (I) complex is effective as an antibiofilm compound in V. harveyi BB170.

Key cytokines of adaptive immunity are differentially induced in rainbow trout kidney by a group of structurally related geranyl aromatic derivatives.

Filifolinone is a semi-synthetic terpenoid derivative obtained from Heliotropium filifolium that increases the expression level of pro-inflammatory and anti-inflammatory cytokines in kidney cells of salmon. Because cytokines are produced in response to a foreign organism and by distinct other signals modulating immune responses, we further studied the potential immunomodulatory effects of a group of structural related terpenoid derivatives from H. filifolium on salmonids to determine the relationship between the chemical structure of the derivatives and their ability to modify cytokine expression and the lymphoid content. The resin and four 3H-spiro 1-benzofuran-2,1'-cyclohexane derivatives were tested in vivo in rainbow trout (Oncorhynchus mykiss) by quantifying the transcript levels of antiviral and T helper-type cytokines and T and B cells in the kidney. Three of the four terpenoids differ only in the C-7'substituent of the cyclohexane and the presence of the ketone group at this position in Filifolinone appeared responsible of an important up-regulation of IFN-α1, IFN-γ, IL-4/13A and IL-17D in the kidney of the treated trout. In addition, the absence of a methoxy group in carbon 7 of the benzene ring, found in all compounds but not in Folifolinoic acid, produced a significant reduction of IFN-γ, IL-12 and IL-4/13A transcripts. B cells were not affected by the compound treatment but Filifolinoic acid and the resin induced a significant reduction of T cells. Altogether, results showed that immunomodulating responses observed in the trout by effect of 3H-spiro 1-benzofuran-2,1'-cyclohexane derivatives is related to the presence of the ketone group in the carbon 7' and the methoxy group in carbon 7 of the benzene ring, being Filifolinone the most active immunostimulatory compound identified.