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. 2024 Mar 6;14(4):102. doi: 10.1007/s13205-024-03938-3

Intestinal microbial diversity of swines fed with different sources of lithium

Leandro de Souza Lopes 1, Juliana Soares da Silva 1, José Maria Rodrigues da luz 1, Marliane de Cássia Soares da Silva 1,✉, Helena Santiago Lima 1, Gabriel Cipriano Rocha 2, Hilário Cuquetto Mantovani 1,3, Maria Catarina Megumi Kasuya 1,✉
PMCID: PMC10917731  PMID: 38464613

Abstract

A drug that is widely used in the treatment of psychiatric disorder is lithium (Li) salts. The people who make therapeutic use of this drug develop a series of side effects. Through metataxonomic data, this study assessed the impacts of lithium, as Li carbonate or Li-enriched mushrooms, on the microbial composition of the ileum, colon, and feces of piglets. Employing Bray–Curtis metric, no differences were observed among the treatments evaluated. Nevertheless, the alpha diversity indices showed differences in the Simpson, Shannon, and Chao-1 indices in the colon and Chao-1 in the feces in the diets with Li compared with the diets without Li. The taxa with the highest relative abundance varied among the ileum, colon, and feces, with a predominance of the phyla Firmicutes, Bacteroidota, and Proteobacteria in diets with Li. Many groups of microorganisms that are important for the health of the host (e.g., Lactobacillus, Ruminococcaceae, Enterorhabdus, Muribaculaceae, and Coprococcus) had their relative abundance increased in animals that received diets with the recommended dose of lithium. Furthermore, there was an increase in the abundance of Prevotellaceae and Bacteroidales (in the diet with Li-enriched mushroom) and Clostridia, Ruminococcus, Burkholderia, and Bacteroidales (diets with Li carbonate) at the recommended dosages. This is the first study to show the effects of Li carbonate and Li-enriched mushrooms on the intestinal microbiota of piglets. Thus, the effects of lithium on the body may be related to its ability to change the composition of the intestinal microbiota.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-03938-3.

Keywords: Disorder, Piglets, Metataxonomy, Dysbiosis, Organic acids

Introduction

The human gastrointestinal tract harbors a microbial community estimated at 100 trillion microorganisms composed of the most diverse groups of bacteria, archaea, fungi, and viruses (Thursby and Juge 2017; Rinninella et al. 2019) with more than 3 million genes (about 130 times more than the human genome) and producing thousands of metabolites (Valdes et al. 2018). Microbial phyla of the digestive tract were found with a predominance of the taxa Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia, while the first two represent 90% of the microbiota present in the intestine (Vasilescu et al. 2022). The same authors also point out that Firmicutes are represented by genera such as Clostridium, Lactobacillus, Bacillus, Enterococcus and Ruminococcus, of which the former represents 95% of this phylum in the human gastrointestinal tract.

The gut microbiota produces thousands of metabolites that can influence the host (Valdes et al. 2018). In addition, it can modulate immune responses, digest ingested, produce intestinal hormones, perform neurological signaling, and modify the action of drugs and toxins in the host (Fan and Pedersen 2021). Through bidirectional communication, the nervous system communicates with the enteric system and its microorganisms (brain–intestinal microbiota axis), which can produce neuroactive compounds such as neurotransmitters, hormones, amino acids, and short-chain fatty acids, all of which can influence the host’s metabolism (Morais et al. 2021). The same authors also point out that the intestinal microbiota can additionally influence the integrity of the intestinal barrier that controls the passage of signaling molecules from the intestinal lumen to blood circulation. The integrity of the intestinal barrier can be interrupted in some neuropsychiatric diseases that are related to the abundance of some groups of microorganisms present in the intestine. Many studies have shown that the composition of the gut microbiota in individuals with various neurological diseases is different compared to healthy individuals (Jiang et al. 2015; Morais et al. 2021).

Factors such as age, sex, childbirth, exposure to stress, and diet can cause changes in the composition of the gut microbiota. In the latter case, the gut microbiota can vary greatly between individuals who have a high-protein diet (meat) and individuals who eat a diet rich in carbohydrates and fiber (vegetables, legumes, mushrooms) (Rinninella et al. 2019). Some studies have shown that the gut microbiota can be affected by some neuropsychiatric disorders (anxiety and depression) and consequently affect gastrointestinal functions (Huang et al. 2022; Koloski et al. 2012; Gracie et al. 2018). Likewise, some drugs used to treat these disorders can positively modify the gut microbiota, protecting the intestinal mucosa and regulating immune cells (Maier et al. 2018; Song et al. 2020; Huang et al. 2022).

One drug that is widely used in the treatment of disorders such as uni- and bipolar disorder and in the prevention of suicide is lithium salts, mainly in its lithium carbonate (Li2CO3) form (Cammarota et al. 2020). The same authors emphasize that the administration of these salts, mainly in the form of carbonate, could cause side effects such as nausea, diarrhea, excessive urination and thirst, hand tremor, weight gain, cognitive impairment, sexual dysfunction, dermatological problems, and kidney dysfunctions. Regarding Li toxicity, this depends on the species affected and the time of exposure (Shahzad et al. 2017).

Edible mushrooms, in addition to providing several health benefits, are also considered probiotics, as they induce the growth or action of microorganisms that contribute to the well-being of their host (Jayachandran et al. 2017). Previous studies have shown that mushrooms, when grown in a substrate with the presence of selenium, and/or other minerals, convert selenium into a more bioavailable and less toxic form for the organism (da Silva et al. 2010; Kora 2020). |Moreover, lithium is more bioavailable and bioaccessible when the source is Li-enriched mushroom (de Assunção et al. 2012; de Souza Lopes et al. 2022). This demonstrates the biotechnological potential of Li-enriched mushrooms as an alternative source of this mineral and can even minimize the toxic effects for people who make therapeutic use of this element.

The United States Environmental Protection Agency (EPA) and some authors recommend a daily Li dose of approximately 1 µg for a 70-kg adult (Szklarska and Rzymski 2019). Among these health benefits, lithium (Li) is known for its ability to stimulate the production of neural stem cells (Zhang et al. 2019), protect against oxidative stress, stimulate the immune system, produce a calming effect, have a neuroprotective effect (Szklarska and Rzymski 2019), in addition to its potential to prevent and treat some types of cancer (Ge and Jakobsson 2019). The therapeutic serum concentration of Li in humans is in the range of 0.5 to 1 mmol L−1. Signs of mild toxicity are seen in the range of 1.8 to 2.5 mmol L−1, and values greater than 2.5 mmol L−1 can lead to severe toxicity (Won and Kim 2017).

Lithium influences and/or can change the diversity and composition of the gut microbiota (Lieb 2004; Cussotto et al. 2019; Huang 2022). However, these authors did not analyze the microbiota subjected to different sources and dosages of lithium. So, the objective of this study was to evaluate the diversity and composition of the intestinal microbiota in the ileum, colon, and feces of piglets submitted to different dosages (recommended, therapeutic and overdose) and sources of Li (no Li, Li-enriched mushroom and Li2CO3).

Material and methods

Inoculum and enriched mushrooms

The Pleurotus djamor strain (PLO 13) used in the study belongs to the collection of fungi of the Laboratório de Associações Micorrízicas, Departamento de Microbiologia, Instituto de Biotecnologia Aplicada à Agropecuária—BIOAGRO/Universidade Federal de Viçosa—UFV. The isolate PLO 13 was grown in Petri dishes containing 20 mL of potato-dextrose-agar culture medium and kept at 25 ± 1 °C. After 7 days, ¼ of the plate colonized by the fungus was transferred to each 280 mL pot containing 130 g of cooked and autoclaved sorghum grain.

The substrate used for the production of the mushrooms was a mixture of coffee husk and sugarcane bagasse (9:1, v/v). The coffee husks were boiled in water for 2 h and centrifuged at 1500 g for 1 min. The sugar cane bagasse was immersed in a 2% calcium hydroxide solution (w:v) for 12 h and centrifuged at 1500 g for 1 min. Next, 1 kg of this mixture was placed in a polypropylene bag and autoclaved for 1 h, at 121 °C. Then PLO 13 was inoculated into the substrate with 50 mL lithium chloride, at a concentration of 40 g L−1. After about 25 days, the mushrooms were harvested, dehydrated to constant weight, and ground. The quantification of Li content in the mushrooms was done by flame atomic emission spectrometry (Tedesco et al. 1995).

Animal assay

The animal test was carried out in partnership with the Department of Animal Science at UFV in the swine sector. All methods involving the handling of piglets followed the ethical principles of animal research (CONCEA 2019) and were previously approved by the Commission of Ethics in the Use of Animal Production of the Universidade Federal de Viçosa (Protocol No. 02/2021). Piglets were chosen as the animal model for this work because of their metabolic and physiological similarities with humans (Yang et al. 2016), and since lithium is used in the treatment of psychiatric diseases, the results of this study can be extrapolated to humans.

Twenty-four 28-day-old female piglets were used (Sus Domesticus, AGPIC 415 × Camborough) (Agroceres PIC, Patos de Minas, MG, Brazil). The animals were distributed in 7 pens, with 4 animals per pen, in a completely randomized design, in a room in which the temperature was maintained within the thermoneutral zone during the experimental period. The piglets had free access to feed and water throughout the five experimental periods (28–33 days of age).

To verify the effect of lithium in its different forms (carbonate or enriched mushroom) and dosages (therapeutic or recommended), the following treatments were performed (Table 1). All treatments were administered three times a day, that is, the values mentioned (Table 1) were divided into three doses. Treatments M + LiT and M also received empty capsules three times a day, so that all animals could be under the same stress conditions (Lopes et al. 2023). This process was repeated during five experimental days. Then the animals were electrically stunned and exsanguination was performed to collect samples of the ileum, colon, and feces. Intestinal (colon and ileum) and fecal samples were collected shortly after slaughter, which was performed by electronarcosis stunning and brachiocephalic trunk bleeding. Two copper electrodes were placed in the temporal fossa and another electrode was fixed in the region close to the animals’ hearts at the beginning of the stunning. The alternating voltage and current were 94 and 456 V and 0.30 and 4.90 amperes with a frequency of 60 Hz for 1 s (Carrascal et al. 2021).

Table 1.

Diets and amount of lithium provided for each pig

Diets Source of lithium Lithium content (mg/day)
Control No lithium 0
LiT 300 mg of Li2CO3 56*
M + LiT 122 g of Li-enriched mushroom flour 56
M 122 g of non-enriched mushroom flour 0
LiR 5.7 mg of Li2CO3 1**
M + LiR 2.2 g of Li-enriched mushroom flour 1
LiS 600 mg of Li2CO3 (Li overdose) 113
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*Recommended and **therapeutic dosage (Szklarskaa and Rzymski 2019)

Extraction, sequencing and analysis of sequences

Total DNA extraction from the samples was performed according to the methodology proposed by Stevenson and Weimer (2007). The quality and quantity of extracted DNA were measured using nano DropTM Plate, and stored at − 20 °C until use.

DNA was sequenced using the Illumina method (Callahan et al. 2016). The sequences were demultiplexed and trimmed to remove primers, barcodes, and adapters. All reads with a maximum expected error of one or more were removed to keep only high-quality sequences. Then all chimeras and singletons were removed. The remaining sequences were clustered into Amplicon Sequence Variants (ASVs). Each ASV was annotated using a pre-trained algorithm (classify-sklearn) using the SILVA V.138 database. All sequences annotated as organelles (mitochondria, chloroplasts) were removed from the upstream analyses. All analyses were performed using Qiime2, version 2020.8 (Bolyen et al. 2019).

Concentration of organic acids

The quantification of organic acids was performed with fecal samples from piglets. For analysis, stool samples (~ 200 mg) were homogenized in 800 µL of Milli-Q water with the aid of vortex and centrifuged at 12,000 g for 10 min. The supernatant was removed and the other steps were performed as described by Siegfried et al. (1984). The samples were analyzed by high-performance liquid chromatography (HPLC), using a Dionex Ultimate 3000 Dual chromatograph coupled to a Shodex RI-101 refractive index (IR) detector maintained at 40 °C, and a Phenomenex Rezex ROA ion exclusion column, 300 × 7.8 mm maintained at 40 °C. The mobile phase used was 5 mM sulfuric acid (H2SO4) with a flow of 0.7 mL min−1. Acetic, propionic, butyric, and lactic acids were used as standards in the calibration curve. The measurements were performed in duplicate.

Statistical analysis

Differences among groups were evaluated by the ANOVA or Kruskal–Wallis tests, followed by Bonferroni’s post hoc test (P ≤ 0.05). The figures were generated in the GraphPad Prism program (GraphPad Software, San Diego California USA) (Prism 1994). Normality was tested by the Shapiro–Wilk test for all variables analyzed. Alpha diversity indices were calculated using PAST software (Hammer et al. 2001), and group differences were analyzed in Minitab, v. 5, using the Kruskal–Wallis test. Beta diversity analyses to compare microbial composition were assessed at the level of ASVs. The non-metric multidimensional scale (nMDS) was used in beta diversity analyses based on the Bray–Curtis paired distance and nonparametric similarity analysis (ANOSIM), with a permutation number of 10,000 and the aid of the PAST software (Hammer et al. 2001). Differences in the relative abundance of ASVs were evaluated by the Kruskal–Wallis test, using the software STAMP, v. 2.1.3 (Statistical Analysis of Taxonomic and Functional Profiles) (Parks et al. 2014). For analyses of relative abundance at the phylum, family, and genus levels, the Wilcoxon test was used to detect intra-group differences after the intervention, differences among groups were analyzed by the Kruskal–Wallis test. The P values were adjusted using Benjamini–Hochberg’s False Discovery Rate (FDR). Values of P < 0.05 and P_FDR < 0.05 were considered significant in all analyses. The analyses were performed in the Minitab program, version 5.

The beta diversity determined by the Bray–Curtis metric (dissimilarity matrix, which considers the presence and relative abundance of the species that make up the community) was used to evaluate the variation of the microbial community in the different treatments. Species richness or biological variability within each community was assessed by alpha diversity calculated by the Chao-1 diversity index, Shannon diversity index (takes into account richness and equity), and Simpson dominance index (takes into account the evenness of species).

Intergroup genus-level differences in each treatment were analyzed by the linear discriminant analysis (LDA) effect size method (LEfSe) (Thomas et al. 2011) with default settings at https://huttenhower.sph.harvard.edu/galaxy/root.

Results and discussion

Sequencing of DNA

A total of 10,639,088 crude sequences were generated with an average length of 467 bp in all samples. After cutting, quality filtering, and chimera removal, 9,878,563 high-quality bacterial sequences were obtained. The Good’s coverage in the samples was > 97% indicating that the sequencing efforts sufficiently covered the diversity of bacterial communities in the ileum, colon, and feces of piglets. The summary of sequence counts and ASVs that passed through the filtering, cleaning, and normalizing steps is shown in Supplemental Table S1. These steps for processing raw reads are important to ensure the quality of the sequencing data that will be used in the follow-up analyses (Gołębiewski and Tretyn 2020).

Amplicon sequencing of the 16S rRNA gene has been used since 1980 in studies of microbial ecology for phylogenetic classification, taxonomic identifications, and analyses of changes in the microbial community in space and time (Moore and Woese 2014). Furthermore, the technique allows evaluating the presence of cultivable and non-cultivable microorganisms in samples of biological materials (Gołębiewski and Tretyn 2020). Thus, the sequencing of the 16S rRNA gene of the microbiota associated with the gastrointestinal tract of swine may provide new knowledge on the diversity and dynamics of the microbial community after a diet of mushrooms and Li-enriched mushrooms and check or validate the potential of the medicinal use of this element as a food supplement.

Influence of lithium on the composition of the microbial community

This is the first study to analyze the composition of the microbial community of piglets treated with different doses and sources of lithium in the form of lithium carbonate and mushroom enriched with the same element. By the taxonomic analysis of bacterial communities from the ileum, colon, and feces compartments of piglets, 19,339 ASVs were observed, belonging to 36 phyla, 88 classes, 175 orders, 273 families, 562 genera (Supplementary Table S2 to S4).

Through beta diversity analysis and inference using the Bray–Curtis dissimilarity index, it was possible to observe that the bacterial communities were not grouped by treatments (P > 0.05) (Fig. 1). In the present study, the experimental period of 5 days may have been insufficient to modify the microbial communities, resulting in that the differences may not have been detected by the nMDS analysis. In a study in which the intestinal microbiota of rats treated with Li2CO3 was compared to that of a control group (who did not receive Li), differences were observed among groups after 28 days of treatment (Cussoto et al. 2019).

Fig. 1.

Fig. 1

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Non-metric multidimensional scale (nMDS) plots of Bray–Curtis dissimilarity index for bacterial communities of the ileum (a), colon (b), and feces (c). Control (feed only), mushroom flour enriched at the therapeutic dose (300 mg of Li2CO3) mixed with the feed (M + LiT), unenriched mushroom flour mixed with the feed (M), lithium carbonate at the therapeutic dosage (300 mg of Li2CO3) supplied in capsules (LiT), enriched mushroom flour at the recommended dosage (1 mg of Li2CO3) supplied in capsules (M + LiR), lithium carbonate at the recommended dosage (1 mg of Li2CO3) supplied in capsules (LiR), and overdose of lithium carbonate (600 mg of Li2CO3) supplied in capsules (LiS)

Alpha diversity varied only in the colon and feces samples (Dunnet’s test, P < 0.05). The Simpson’s diversity index showed maximum values observed in the communities of the treatments M + LiT, LiT, and LiS and minimum values in the communities of the control and the treatments M, M + LiR, and LiR (Table 2). Therefore, therapeutic dosages (M + LiT and LiT), regardless of the form (enriched mushroom or Li2CO3) and overdose (LiS) can cause an increase in colon microbial diversity in piglets. Both the mushroom (M) and the recommended dosages (M + LiR and LiR) did not influence the microbial diversity. The Shannon diversity index in the colon showed the highest diversity values for all treatments that received Li (M + LiT, LiT, M + LiR, LiR, LiS), regardless of the form and dose (Table 2).

Table 2.

Alpha diversity indices in the ileum, colon, and feces of piglets

Index Diets
Control M + LiT M LiT M + LiR LiR LiS
Ileum
Simpson 1-D 0.75 0.80 0.82 0.85 0.73 0.74 0.66
Shannon 2.65 2.72 2.80 2.90 2.80 2.62 2.35
Chao-1 472.50 536.25 384.33 389.40 617.91 702.90 548.25
Colon
Simpson 1-D 0.90b 0.98a 0.90b 0.98a 0.96b 0.96b 0.98a
Shannon 3.90b 5.36a 4.40b 5.15a 5.00a 5.94a 5.00a
Chao-1 707.50b 1133.75a 850.60b 1059.33a 1153.64a 921.30b 972.70b
Feces
Simpson 1-D 0.95 0.99 0.95 0.97 0.97 0.97 0.97
Shannon 4.75 5.50 4.82 5.00 4.80 5.00 4.90
Chao-1 775.85b 1061.20a 904b 886b 871b 990.50b 880b
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For each index, means followed by different letters on the same line differed at the 5% significance level determined by Dunnet’s test

Maximum values of Chao-1 richness indices in the colon and feces were observed in the microbial communities of the M + LiT, M + LiR, and LiT treatments, the two latter only in the colon. Thus, Li influenced the species richness of the colon at the therapeutic dosage (M + LiT and LiT) and recommended dosage (M + LiR) and feces in the treatments (M + LiT) (Table 2). In studies, performed with rats, it was found that animals treated with Li (about 150 mg kg−1) increased the diversity of the gut microbiota (Shannon and Chao indices) compared to the control group that did not receive Li (Cussotto et al. 2019; Huang et al. 2022).

Firmicutes was the predominant bacterial phylum in the ileum, colon, and feces in all treatments, not differing among treatments (P > 0.05) (Fig. 2a, Supplemental Table S2). Proteobacteria and Actinobacteriota were the other predominant phyla in the ileum microbial community (Fig. 2a), except for the LiR treatment in which the phylum Bacteroidota had the second highest relative frequency (2.09%) followed by Actinobacteriota (1.25%). In the colon, the predominant bacterial phyla were Firmicutes (81.61%) and Bacteroidota (14.54%) (Fig. 2b).

Fig. 2.

Fig. 2

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Phylum-level bacterial composition in the ileum (a), colon (b), and feces (c) of piglets. Each bar represents the average composition of the bacterial community in the control (feed only), mushroom flour enriched at the therapeutic dose (300 mg of Li2CO3) mixed with the feed (M + LiT), unenriched mushroom flour mixed with the feed (M), lithium carbonate at the therapeutic dosage (300 mg of Li2CO3) supplied in capsules (LiT), enriched mushroom flour at the recommended dosage (1 mg of Li2CO3) supplied in capsules (M + LiR), lithium carbonate at the recommended dosage (1 mg of Li2CO3) supplied in capsules (LiR), and overdose of lithium carbonate (600 mg of Li2CO3) supplied in capsules (LiS). The ten microbial phyla with the highest relative frequencies were evaluated in this figure. The other phyla are grouped under “others”

In the feces, the most abundant phyla were also Firmicutes and Bacteroidota (Supplemental Table S2). This greater abundance of Spirocaeota in treatments M + LiT and M may be related to a greater proportion of fibers that were present mixed in the feed of these animals, since only these two treatments received 122 g of mushroom flour (Table 1) and this phylum is responsible for fiber degradation (Arora et al. 2022). There was no difference in bacterial composition among treatments at the phylum level (P > 0.05).

The findings of this study at the level of the most abundant phyla agree with works in the literature in which the most abundant phyla in piglets are Firmicutes and Proteobacteria in the ileum (Yang et al. 2016) and Firmicutes, Bacteroidota, and Proteobacteria in fecal samples (Costa et al. 2014; Mach et al. 2015; Gardiner et al. 2020). In the literature, the phyla with the highest abundance in the colon were Firmicutes, Proteobacteria, and Bacteroidota (Quan et al. 2018).

Some studies have shown a decrease in Actinobacteriota abundance in individuals with some types of stress-related disorders (Reber et al. 2016; Malan-Muller et al. 2018). In this study, it was observed that Actinobacteriota were among the ten phyla with the highest relative abundance in the ileum, colon, and feces and that in the M + LiT and LiS treatments, the colon is where the greatest abundance of this phylum occurs, differing from both the other treatments (P < 0.05, Fig. 2). This is very interesting because both M + LiT and LiS treatments can enrich this group of microorganisms in the gut of individuals with some type of stress-related depression. In a study with rats treated with Li2CO3, an increase was verified in the phylum Actinobacteriota when compared to animals that did not receive Li2CO3 (Cussotto et al. 2019). This is an interesting finding since M + LiT corresponds to a therapeutic dosage of Li, and this dosage in the form of an enriched mushroom seems to favor the development of this phylum in the colon of swine, differing even from the LiT treatment that have the same dosage of Li, but without the mushrooms.

At the family level, Lactobacillaceae was the most representative in the ileum (Fig. 3a) and did not differ among treatments. The second most representative families were Enterobacteriaceae (control, M and LiT), Streptococcaceae (M + liT and M + LiR), Lachnospiraceae (LiR), and Pasteurellaceae (LiS) (Fig. 3a, Supplemental Table S3). In the ileum, Peptostreptococcaceae had a relative frequency almost seven times higher in the M treatment compared to the other treatments with the highest frequencies (P < 0.05) not only differing from the control, but also from M + LiT, LiT, and M. Peptostreptococcaceae is generally considered as a normal commensal bacterium, and its proportion is higher in the gut microbiota of healthy animals than in those with some gut microbiota dysbiosis, indicating that Peptostreptococcaceae may help maintain intestinal homeostasis (Leng et al. 2016; Fan et al. 2017). An increase in this family was related to the introduction of Ganoderma lucidum in the diet of rats (Diling et al. 2020). This increase in the population of this family in the M treatment in piglets may be associated with the various benefits associated with the consumption of mushrooms, such as stimulating the biosynthesis of tryptophan which is one of the essential amino acids, having antioxidant effects, as well as being a precursor of the neurotransmitter serotonin, a sedative drug that regulates the circadian rhythm and improves sleep (Diling et al. 2020).

Fig. 3.

Fig. 3

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Family-level bacterial composition in the ileum (a), colon (b), and feces (c) of piglets. Each bar represents the average composition of the bacterial community in the control (feed only), mushroom flour enriched at the therapeutic dose (300 mg of Li2CO3) mixed with the feed (M + LiT), unenriched mushroom flour mixed with the feed (M), lithium carbonate at the therapeutic dosage (300 mg of Li2CO3) supplied in capsules (LiT), enriched mushroom flour at the recommended dosage (1 mg of Li2CO3) supplied in capsules (M + LiR), lithium carbonate at the recommended dosage (1 mg of Li2CO3) supplied in capsules (LiR), and overdose of lithium carbonate (600 mg of Li2CO3) supplied in capsules (LiS). The ten microbial families with the highest relative frequencies are evaluated in this figure. The other families are grouped under “others”

Generally in the colon, the families with the highest relative frequency were Lactobacillaceae, Lachnospiraceae, and Prevotellaceae, which are responsible for the degradation of carbohydrates and proteins, in which the penultimate had greater abundance in treatment M that had mushroom flour added to the feed. The addition of mushrooms to the diet of rats led to the enrichment of intestinal Lachnospiraceae (Li et al. 2021). Although members of Lachnospiraceae are among the main producers of short-chain fatty acids, different taxa of Lachnospiraceae are also associated with different intestinal dysbiosis in humans (Vacca et al. 2020). The increased abundance of Lachnospiraceae may also be associated with a decrease in swine pathogens such as Clostridium difficile (Umu et al. 2015). This impact of Lachnospiraceae on host physiology is often inconsistent across different studies. In the colon of treatment M, the family Oscillospiraceae had a higher abundance when compared to the other treatments, but did not differ from them significantly (P > 0.05) (relative frequency of 4.5%) (Fig. 3b). Some members of the Oscillospiraceae family are beneficial to intestinal health due to the production of butyrate, which can be used as an energy source by the host and is also a strong candidate as a probiotic (Yang et al. 2021).

In feces, the most representative families were Muribaculaceae, Lactobacillaceae, Prevotellaceae, Lachnospiraceae, Ruminococcaceae (Fig. 3C). The microbial community of Lactobacillaceae varied among the treatments in which the animals that received the lowest doses of Li (1 mg) both in the form of Li2CO3 (LiR) and in the enriched mushroom form (M + LiR) were the only ones that did not differ from the microbial community of Lactobacillaceae in the control (P < 0.05). The family Lactobacillaceae is known to positively influence host health and has proven effects on intestinal permeability and the immune system and inhibits the growth of harmful bacteria (Oscarsson et al. 2021). This may indicate that both the mushroom and therapeutic or overdose dosages of Li can influence the composition of fecal Lactobacillaceae in piglets. The Rikenellaceae family had its frequency about three times higher in the feces of treatment M when compared to the other treatments (P > 0.05). This family is considered to be protective against cardiovascular and metabolic diseases as well as markers of healthy aging in humans (Tavella et al. 2021).

The Muribaculaceae family was detected only in the feces and colon of the animals (Fig. 3C). This family is known to degrade a variety of complex carbohydrates (Lagkouvardos et al. 2019). Muribaculaceae and Prevotellaceae together represented the families with the highest abundance in the colon of healthy piglets (Tang et al. 2020).

The ten bacterial genera with the highest relative abundance in the ileum, colon, and feces are represented in Fig. 4. The predominant group in the ileum were Lactobacillus, Escherichia–Shigella, and Streptococcus (Fig. 4a, Supplemental Table S4). In the colon, Lactobacillus was also one of the most abundant, followed by the genera Prevotella and Subdoligranulum (Fig. 4b). In the feces, there was a predominance of Muribaculaceae, Prevotella, and Lactobacillus (Fig. 4c). Some studies that have sought to identify the profile of the microbial community in pig feces found that Lactobacillus (20.95%), Prevotella (6.41%), Treponema (2.48%), Oscillospira (1.96%), Clostridium (1.69%), Ruminococcus (1.36%), Holdemania (1.28%), Streptococcus (1.15%), Bacteroides (1.06%), and Coprococcus (0.87%) were the top ten genera of greatest relative abundance, representing the gut bacteria of different breeds of piglets (Xiao et al. 2016; Yang et al. 2018; Wang et al. 2022). These results suggest that some bacteria such as Prevotella, Ruminococcus, Lactobacillus, and Clostridium are relatively constant in the intestinal microbiota of piglets and that these variations in diversity are linked to factors such as age, diet, sex, and antibiotic use (Wang et al. 2022).

Fig. 4.

Fig. 4

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Genus-level bacterial composition in the ileum (a), colon (b), and feces (c) of piglets. Each bar represents the average composition of the bacterial community in the control (feed only), mushroom flour enriched at the therapeutic dose (300 mg of Li2CO3) mixed with the feed (M + LiT), unenriched mushroom flour mixed with the feed (M), lithium carbonate at the therapeutic dosage (300 mg of Li2CO3) supplied in capsules (LiT), enriched mushroom flour at the recommended dosage (1 mg of Li2CO3) supplied in capsules (M + LiR), lithium carbonate at the recommended dosage (1 mg of Li2CO3) supplied in capsules (LiR), and overdose of lithium carbonate (600 mg of Li2CO3) supplied in capsules (LiS). The ten microbial families or genus with the highest relative frequencies are evaluated in this figure. The other genera are grouped under “others”

Within the genus Lactobacillus, only in the feces was there a difference among treatments (P < 0.05) (Fig. 4c). The treatments M + LiT, M, LiT, and LiS differed from the others. Apparently, therapeutic dosages of Li, regardless of the form (enriched mushroom or Li2CO3), mushroom meal (M) and Li overdose (LiS) can cause a decrease in the Lactobacillus population in the intestine of piglets according to fecal samples. This same trend was also observed in the colon, but there was no significant difference among treatments (Fig. 4b).

The genera Lactobacillus and Streptococcus are important members of the intestinal microbiota as they metabolize carbohydrates and produce lactic acid that can be used as an energy source by the host, in addition to being involved in immune, metabolic, and intestinal homeostasis functions (Dempsey and Corr 2022). There are studies with rats demonstrating that the abundance of Lactobacillus in the intestine is related to beneficial effects on anxiety and depression-like behaviors in addition to improving the intestinal barrier against pathogens and toxins (Malan-Muller et al. 2018; Peirce and Alviña 2019).

Although the overgrowth of Escherichia–Shigella is usually associated with dysbiosis leading animals to diarrhea (Menezes-Garcia et al. 2020; Luo et al. 2022), in this study, this symptom was not verified in pigs, except in the LiS treatment. Despite being associated with many diseases, Escherichia–Shigella is not exclusively pathogenic, being part of the natural intestinal microbial community of animals such as piglets and humans where they establish mutualistic relationships that maintain intestinal homeostasis (Martinson and Walk 2020).

Prevotella is among the ten most abundant genera in the colon and feces of piglets, not differing statistically among treatments. This genus has the ability to digest complex carbohydrates and the genetic and enzymatic potential to break down plant fibers, being present in greater abundance in populations with this diet when compared to diets rich in protein and fat (Precup and Vodnar 2019). There are studies showing that a decreased abundance of the genus Prevotella was associated with depressed individuals (Wingfield et al. 2021) and/or with some other illness related to mood disorders when this genus was compared to the control (Tomizawa et al. 2021). Contrary to the benefits of these groups in minimizing the symptoms of mood-related diseases, there are studies relating the abundance of the genus Faecalibacterium to patients with depression (Jiang et al. 2015; Chang et al. 2022). This genus was also among the ten genera with the highest relative abundance only in the colon and feces and there was no difference in abundance among treatments. Regardless of the route it was administered to animals, lithium does not seem to influence the composition of the Faecalibacterium community.

Muribaculaceae was the group with the highest relative abundance in feces. This family is a recently discovered group of bacteria commonly found in the gastrointestinal tract of mammals that acts in the fermentation of complex polysaccharides, being one of the most important groups that produce propionate (Smith et al. 2021). The LiS overdose treatment produced (which received 600 mg of Li2CO3) symptoms of intoxication such as tremors, diarrhea, and loss of appetite in animals. Diarrhea is caused by bacterial pathogens such as Escherichia, Shigella, Salmonella, Campylobacter, Clostridium difficile, and Aeromonas (Li et al. 2021). In this study, no members of the genera Salmonella or Aeromonas were found, but Escherichia–Shigella, Campylobacter, and Clostridium were present in the tissues of pigs. The last two (despite not being among the ten with the highest abundance) had higher relative abundance in the ileum in the LiS treatment, but did not differ significantly from the other treatments. Nevertheless, it is important to highlight that there might not have been enough time for the microbiota to undergo a noticeable change as the animals were treated with Li2CO3 for only 5 days.

To filter the differences among bacterial communities among treatments and among the ileum, colon, and feces, we used Venn diagrams to analyze ASVs that were shared (Fig. 5). We found 19,339 ASVs distributed in all samples, being 4,319 exclusive to the ileum, 5,162 to the colon, and 6,343 to the feces. We found that 1,262 ASVs are shared among the ileum, colon, and feces (Fig. 5a). The largest number of recorded ASVs was in the feces.

Fig. 5.

Fig. 5

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Venn diagrams showing the number of bacterial ASVs: a total ASVs shared among the ileum, colon, and feces; b ASVs from the control group shared among the ileum, colon, and feces; c M + LiT treatment ASVs shared among the ileum, colon, and feces; d M-treatment ASVs shared among the ileum, colon, and feces; e ASVs from LiT treatment shared among the ileum, colon, and feces; f M + LiR treatment ASVs shared among the ileum, colon, and feces; d ASVs from LiR treatment shared among the ileum, colon, and feces; g ASVs from LiS treatment shared among the ileum, colon, and feces; h M-treatment ASVs shared among the ileum, colon, and feces; i comparison of the number of ASVs from M + LiT and LiT treatments in the ileum; j comparison of the number of ASVs from M + LiT and LiT treatments in the colon; k comparison of the number of ASVs from M + LiT and LiT treatments in feces

The Venn diagram was performed only for the treatments M + LiT (56 mg of Li in the form of enriched mushroom) and LiT (Li in the form of Li2CO3) to verify if there was a change in the number of ASVs depending on the source of Li that was supplied to the animals (Fig. 5i–k). In all areas analyzed and feces, the number of ASVs was higher in the M + LiT treatment (Fig. 5i-k) when compared to LiT, especially in the ileum where the difference is almost double (Fig. 5i). This analysis was performed because Li in the mushroom form was more bioavailable and generated less free radicals than Li2CO3 (Lopes et al. 2023). Thus, Li-enriched mushroom is a source of Li that favors a greater diversity of microorganisms in the gastrointestinal tract of piglets than Li2CO3 (Fig. 5i–k). According to Porter and Bernot (2010), lithium reduces microbial respiration rates by inhibiting aerobic and anaerobic metabolism. Thus, the effect of Li on microbial growth and diversity is related to energy metabolism. Nevertheless, until now there have been no studies confirming this effect. According to Plotnikov et al. (2023), studies of the effects of Li on microorganisms show contradictory results with both inhibition and stimulation of bacterial growth. These contradictions may be due to the concentration and physical–chemical conditions of the environment. A study carried out in saline reserves of the Salar de Atacama showed different diversity of bacteria and archaea with different survival strategies and lithium resistance (Cubillos et al. 2018). Lithium can replace Na+, K+, Mg+2, and Ca+2 ions in the microbial cell (Shahzad et al. 2017). Sodium and potassium are important for the transport of nutrients across the cell membrane. Magnesium is a cofactor of energy metabolism enzymes. Calcium acts as an intracellular signaling agent and enzymatic cofactor. An overexpression of molecular chaperones was observed in the lithium-tolerant bacterium Rhodococcus sp. (Urbano et al. 2013). These enzymes influence the degradation of damaged proteins related to transcription and translation processes (Belfiore et al. 2017). Cellular accumulation of solutes compatible with osmotic stress, glutamine, glycerol, and glycine is also a survival strategy for Rhodococcus spp. to lithium (Belfiore et al. 2017). Tolerance to Li was also observed during fungal growth in different chemical forms and doses of Li (De Assunção et al. 2012; Nunes et al. 2014). Therefore, the positive stimulation of lithium on the microbial diversity of the intestine of pigs receiving feed with this mineral may influence these different survival strategies and resistance to Li.

LEfSe and LDA analysis based on ASVs

To verify which treatments enriched the intestinal microbiota of the piglets, LEfSe and LDA analyses were performed (Fig. 6). In these analyses, it can be observed that some genera showed differential abundance among treatments (P < 0.05).

Fig. 6.

Fig. 6

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The LEfSe analysis taxa with different abundances as biomarkers per treatment group. Taxa abundant as biomarkers in the ileum (a), colon (b), and feces (c). Differences between taxa were evaluated by the Kruskal–Wallis test with alpha set at 0.05 for significant differences

The LDA scores indicated that the relative abundances of taxa such as Enterorhabdus, Muribaculaceae (ileum), Ruminococcaceae (colon), Lactobacillus, and Coprococcus (feces) were more enriched in the M + LiR treatment (mushroom enriched with the recommended dosage). Studies have shown a lower abundance of these taxa in rats and humans with some mood disorders, anxiety, and/or depression compared to the control group (Burokas et al. 2017; Heym et al. 2019; Wang et al. 2020; Zheng et al. 2021; Zou et al. 2021).

In the LiS treatment, genera such as Weissella and Olsenella were enriched in the colon. As already mentioned in this treatment, the animals presented diarrhea. These genera are not associated with diarrhea, on the contrary, there is work showing a decrease in Weissella in rats with diarrhea (Zhuang et al. 2018). In the M + LiT treatment, while there was enrichment of Prevotellaceae in the colon and feces. Decline in Prevotellaceae is associated with anxiety disorders in humans (Chen et al. 2019). There was enrichment of Bacteroidales, Ruminococcus, and Clostridia in the therapeutic Li treatment (LiT) in colon and feces, respectively. Bacteroidales and Clostridia groups are associated with depressed individuals and healthy individuals, respectively (Liu et al. 2020). A decrease in the population of Ruminococcus was associated with human patients with depression and bipolar disorder in addition to being a genus with low richness in individuals with inflammatory bowel diseases leading to diarrhea (Liu et al. 2020; Daliri et al. 2020; Zou et al. 2021).

In the feces, there was enrichment of Burkholderia in which its low and high abundance were found in depressed rats and humans with depression, respectively (Chen et al. 2019). Bacteroidales and Micrococcaceae had increased relative abundance in humans with depression when compared to healthy humans (Liu et al. 2020; Fontana et al. 2020).

An increase in the relative abundance of some groups of microorganisms that are considered biomarkers for some psychiatric diseases was show in this study. Studies have shown that altering the structure and composition of the intestinal microbiota affects the development of diseases since this microbiota produces metabolites that can act directly on the central and peripheral nervous systems (Zou et al. 2021). Furthermore, some important groups such as Lactobacillus and Ruminococcus were in greater relative abundance (P < 0.05) in treatments that received Li. These results show that this element can have therapeutic effects through the modulation of the intestinal microbiota, and that mushrooms enriched with this element can be an alternative source of Li, enriching certain beneficial groups of microorganisms such as Lactobacillus which had their relative abundance increased only in the M + LiR treatment. This modulation may be due to different survival strategies and Li resistance of microorganisms (Porter and Bernot 2010; Cubillos et al. 2018; Belfiore et al. 2017).

Production of short-chain organic acids (SCOA)

A difference in the production of short-chain organic acids among some treatments was observed in this study (Table 3). This variation is related to the relative abundance of some groups of microorganisms in the gastrointestinal tract of piglets. Furthermore, a relationship between the abundance of the microbial community and the production of organic acids has been observed in this study. The production of lactic acid, for example, is directly related to the abundance of Lactobacillus (major producers of lactic acid) in the feces, in which the treatments M + LiT, M, LiT, and LiS were the ones that showed the lowest values of abundance of this genus and the production of lactic acid (Table 3). Li in therapeutic dosages, regardless of the source, seems to influence the production of these acids in piglets. The treatment that received only mushroom flour also had reduced lactic acid production. This may be related to the type of substrate that is degraded by Lactobacillus, in which this genus is known to degrade carbohydrates (Dempsey and Corr 2022) and this treatment (M) had mushroom flour added which is rich in fiber.

Table 3.

Profile of organic acids (SCOA) in pig feces according to the experimental groups

SCOA (g L−1) Diets
Control M + LiT M LiT M + LiR LiR LiS
Acetic 0.55a 0.32 0.10 0.34 0.37a 0.38a 0.33
Propionic 0.25a 0.13 0.05 0.14 0.17a 0.18a 0.13
Butyric 0.16a 0.10 0.03 0.10 0.13a 0.13a 0.10
Lactic 0.11a 0.04 0.01 0.04 0.04 0.06a 0.02
Total SCOA 1.07a 0.57 0.19 0.62 0.72a 0.75a 0.58
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Means not labeled with the letter a lowercase are significantly different from the treatment mean at 5% significance by Dunnet’s test

The therapeutic level of Li dosages, regardless of the form (unenriched mushroom or enriched mushroom or Li2CO3) and overdose (LiS), decrease the production of these organic acids when compared to the control. In a study with rats, Li increased the production of propionate, butyrate, and acetate acids and the biosynthesis pathways of organic acids were upregulated after treatment with this element (Cussotto et al. 2019; Huang et al. 2022). Another hypothesis is that Li at these dosages can increase the absorption of these acids by the host, and thus less acid is quantified in the analyses. The other acids such as acetic, propionic, and butyric are quite widespread within the phyla Firmicutes and Bacteroidetes (Venkataraman et al. 2016). The family Lachnospiraceae and Ruminococcaceae have received more attention because they are very abundant in the human colon, comprising 10 to 20% of the total bacteria and producing butyric acid (Vital et al. 2014). These acids can improve gut health through several local effects, ranging from maintaining intestinal barrier integrity, producing mucus, and protecting against inflammation to reducing the risk of colorectal cancer (Silva et al. 2020). In piglets the energy contribution of short-chain organic acids to the basal metabolic rate is estimated to be 10–30% (Bergman 1990; Rhouma et al. 2021).

Conclusion

This is the first work that shows the effects not only of Li, but also of Li-enriched mushrooms on the composition of microbial communities in swine ileum, colon, and feces. Li can influence the abundance and richness of some taxa of bacteria in the intestines of these animals. Li at the recommended dosage (enriched mushroom) and therapeutic dosage (enriched mushroom and Li2CO3) can exert its effects via population modulation of some specific groups of microorganisms, such as Lactobacillus spp. and Ruminococcus spp., in which the richness of these genera is associated with healthy individuals. It is noteworthy that the overdose dosage resulted in only poor enrichment of a few genera and these are little reported in the literature as being important for the intestinal homeostasis of the host. Future studies over a longer period should be performed to verify these changes in microbial composition. In addition, the results of this study will help in the development of drugs or therapies using mushrooms enriched with lithium to control psychiatric disorders, anxiety, and stress. Furthermore, this study will contribute to the understanding of lithium metabolism in different organs and tissues of animals.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 21 KB) (21.4KB, docx)
Supplementary file2 (DOCX 18 KB) (18KB, docx)
Supplementary file3 (DOCX 18 KB) (18.5KB, docx)
Supplementary file4 (DOCX 19 KB) (18.6KB, docx)

Acknowledgements

The authors are grateful to the Cordenação de Aperfeiçoamento de Pessoal de Nível superior (Capes-0001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for their financial support.

Author contributions

Conceptualization, validation, investigation, writing, formal analysis: L. S. Lopes, J. S.Silva, M. C. S. da Silva, H. S. Lima, G. C. Rocha, H. C. Mantovani, M. C. M. Kasuya. Data processing and analysis: L. S. Lopes, J. S.Silva, J. M. R. Luz, M. C. S. da Silva, H. S. Lima, G. C. Rocha, H. C. Mantovani, M. C. M. Kasuya. The investigation, writing—review and editing: L. S. Lopes, J. S.Silva, J. M. R. Luz, M. C. S. da Silva, H. S. Lima, G. C. Rocha, H. C. Mantovani, M. C. M. Kasuya.

Data availability

The authors declare the data availability for interested persons.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the study reported in this paper.

Ethical approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of National Council for the Control of Animal Experimentation (CONCEA, Law number 11,794, of October 8, 2008) and Commission of Ethics in the Use of Animal Production of the Universidade Federal de Viçosa (Protocol No. 02/2021). All participants of the QDA and Napping panels were provided with consent forms before taking part in this study.

Contributor Information

Marliane de Cássia Soares da Silva, Email: marliane.silva@ufv.br.

Maria Catarina Megumi Kasuya, Email: mkasuya@ufv.br.

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Supplementary Materials

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Data Availability Statement

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