Modern animal production has been changing in recent years due to the problems of bacterial resistance derived from the overuse of antimicrobials for prophylactic and growth promotion purposes [40, 41]. In this regard, many investigations have focused on probiotics, prebiotics, enzymes, acidifiers, extract plants, and some metals (copper and zinc) as feed additives, given their antimicrobial properties and effects in promoting growth, mainly . Therefore, in the present study, copper acetate (CA), an organic source of copper, curcumin (CR), and a mixture of curcumin with copper acetate (CA-CR), were evaluated. Selection of CA was based on its advantages over inorganic sources since it has been described that inorganic sources tend to dissociate in the upper part of the gastrointestinal tract, causing a decrease in the availability of copper due to its interaction with other metals (chelation) and therefore a reduction in its activity [18, 43]. In contrast, the solubility of organic sources of copper is higher in weak acid enviroments, making their dissolution slower and increasing their availability and activity . Furthermore, lower fecal copper excretion rates have been reported in broilers exposed to an organic source of copper compared to inorganic sources . Although the copper ion is known to be more effective against Gram-positive bacteria , dietary supplementation with CA significantly reduced more than 18% of the concentration of S. Typhimurium in both trials compared to PC group (Table 2). Copper ion has been reported to cause damage at the bacterial membrane level due to its adhesion to membranes and the generation of reactive oxygen species . Additionally, it can be associated with the functional groups of proteins and enzymes, leading to the inactivation or inhibition of some cellular processes, as well as having a direct negative effect on the genetic material of bacteria [45, 46].
In the case of the group treated with the solid dispersion of curcumin (CR), which was previously described by our research group and is characterized by being more soluble and permeable , the concentration of S. Typhimurium significantly decreased by more than 35% (more than 2 log) with respect to the PC group after ten days of treatment (Table 2). These results are due to the antimicrobial action of curcumin, which in general, is associated with damage to the bacterial membrane and inhibition of bacterial cell proliferation [47, 48]. Furthermore, it has been published that curcumin can induce some physical and mechanical changes of the S. Typhimurium flagellar filament, causing a decrease in motility, adherence, and invasion of the host cells, which results in a reduction or elimination of its virulence . Likewise, curcumin has been reported to decrease bacterial cell division processes since it interacts with the FtsZ protein, a cytoskeleton protein essential for this process . Meanwhile, the treatment containing the mixture of CA and CR (CA-CR) was slightly more effective in reducing the S. Typhimurium concentration compared to the group treated with CR and the PC group (2% and 37%, respectively). These results suggest that physical mixtures between CR and CA do not improve or enhance the pharmacological effects of curcumin compared to the complexes that it can form with heavy metals, including copper. These complexes that are chemically synthesized have been shown to have a better effect than curcumin itself and even decrease the toxicity of metals [16, 51, 52].
After oral infection with Salmonella, this pathogen must overcome the conditions of the gastrointestinal tract to interact with the intestinal epithelium . Invasion of epithelial layers by S. Typhimurium is known to increase intestinal permeability in both in vivo and in vitro models since the expression of some markers such as claudin-1, occludin, and mucin-2, mRNA levels of zonula occludens-1 and E-cadherin was reduced [53, 54]. Although the markers mentioned above were not determined in the present study, FITC-d, a large molecule (3–5 kDa) that, under normal intestinal health conditions, does not leak through the epithelium, was used. However, when there is damage to the epithelium, the permeability of FITC-d increases so that it can be quantified in serum . In the present study, all treated groups showed lower serum FITC-d concentrations compared to the PC group (Table 2). However, only the group treated with CR had significantly lower concentrations when compared to PC and turned out to have serum FITC-d concentrations comparable to the NC group. Perhaps, this result is due to the ability of CR to restore the intestinal barrier function and the expression of proteins associated with the tight junctions, the proliferation-regeneration of the intestinal epithelium, and its antimicrobial action, resulting in decreased paracellular permeability as has been previously reported [56, 57]. Regarding the treatments with CA and CA-CR, although the S. Typhimurium counts decreased significantly compared to the PC group, the serum FITC-d concentration only decreased numerically since it has been described that the production of reactive oxygen species by copper affects not only bacteria but also epithelial cells .
The chicken gut microbiota is composed of several microorganisms that are involved in digestion and metabolism, regulation of enterocytes, vitamin synthesis, and development and regulation of the host immune system . However, the cecum is by far the most densely colonized microbial habitat in chickens . Despite the absence of any clinical signs of Salmonella infection, the composition of the microbiota is affected but, the changes in the cecal microbiota are quite weak [61, 62], which supports our results since no significant differences in alpha (measured by the observed OTUs) and beta diversity was observed in the cecal samples at ten days post-S. Typhimurium challenge, which means that there were no changes in the relationship of the number of different species per sample (richness) and in the diversity of the microbial community between different samples, respectively . Notwithstanding the above, the taxonomic composition showed some significant differences at the family and genus levels when the groups were compared.
At the family level, Enterococcaceae was significantly higher in the PC group when compared to the other treatment groups. Enterococcaceae, one of the six families of the order Lactobacillales , is comprised of the genera Enterococcus, Bavariicoccus, Catellicoccus, Melissococcus, Pilibacter, Tetragenococcus, and Vagococcus . So this decrease in Enterococcaceae in other dietary treatment groups as compared to the PC group could be due to copper since it has been described that it alters the intestinal microbiota and decreases the counts of lactic acid bacteria . In contrast, Salmonella infection is known to increase the relative abundance of Enterococcaceae, Lactobacillaceae, Clostridiaceae, Lachnospiraceae, Erysipelotrichaceae, Peptostreptococcaceae, and Ruminococcaceae, but decrease that of Enterobacteriaceae . Furthermore, the Clostridiaceae was significantly higher in chickens treated with CR when compared to other groups. Clostridiaceae is one of the responsible families for converting polysaccharides into short-chain fatty acids (SCFAs) . It has been described that SCFAs such as acetate, propionate, and butyrate, are important maintaining the intestinal homeostasis due to their immunomodulatory capacity, maintenance of metabolism, proliferation, differentiation and promotion at low pH, favoring beneficial bacteria, and reducing the growth and viability of pathogenic bacteria , thus supporting our findings and relating to the antimicrobial activity of the treatments.
In terms of genu level, Salmonella, Coprobacillus, Eubacterium, and Clostridium were significantly enriched in the PC group, which is closely related to the severity of the Salmonella infection process. Coprobacillus, Clostridium, and Eubacterium have an important role in the production of SCFAs essential amino acids and the digestion of non-starch polysaccharides, which stimulate the production of SCFAs for metabolic balance [68, 70]. Likewise, it has been reported that the reduction of Clostridium and the maintenance of Eubacterium and Coprobacillus levels could be related to the effectiveness of the treatments since they represent a positive effect in the maintenance of intestinal homeostasis [70–72]. Finally, high levels of Salmonella are related to colonization in ceca tonsils  and are perfectly related to the severity of the infection. Furthermore, the genus Faecalibacterium and Enterococcus were significantly enriched in the group treated with CR. After infection with Salmonella, this pathogenic bacteria alter the intestinal microbiota, causing a decrease in bacteria of the genus Blautia, Enorma, Faecalibacterium, Shuttleworthia, Sellimonas, Intestinimonas, and Subdoligranulum, as well as an increase in the abundance of Butyricicoccus, Erysipelatoclostridium, Oscillibacter and Flavonifractor . However, in the case of the group treated with CR, the increase in Faecalibacterium, a genus of bacteria responsible for the production of butyrate and related to health benefits in poultry, could be mainly due to the prebiotic effect of curcumin, like other substances with the same activity . It has been described that CR could act as a factor of promotion, proliferation, growth, and survival for the beneficial bacteria of the intestinal microbiota from its biotransformation . Finally, the bacterial genus that belong to Erysipelotrichaceae and Lachnospiraceae were significantly enriched in the CA-CR and CA groups, respectively. It has been published that in chickens infected with Salmonella this genus of bacteria decreases markedly, which could negatively affect the diversity and development of intestinal bacteria . In the specific case of CA and CA-CR, copper is known to increase the relative abundance of these bacterial genera, which are the most active microbial components in the healthy gut and are responsible for preventing the production of inflammatory cytokines and induce intestinal production of SCFAs by fermenting carbohydrates [76, 77].