Metagenomic analysis of rat stool
Analysis of rat intestinal diversity
Diversity refers to a set of indices or metrics used to quantify the distribution of taxa abundance. Species diversity can be divided into Alpha (α), Beta (β) and Gama (γ) diversity (Chistoserdovai. 2010). Among them, α diversity is mainly concerned with the number of species in a uniform local habitat, β diversity is concerned with the number of species in the same locality and different groups; γ diversity is concerned with the number of species in different localities and even in different climates. In this study, α diversity and β diversity were used to analyze the diversity of rats feces.
It can be seen from Fig. 1 that all samples have similar α diversity on day 0 and samples of the blank group; on day 30, the α diversity of the blank group did not change significantly, and the α diversity of the control group decreased significantly (Two-sided Wilcoxon signed-rank test, p < 0.05), the alpha diversity of the probiotic intervention group was significantly increased (Two-sided Wilcoxonsigned-rank test, p < 0.05), which proved that the occurrence of mastitis would lead to the decrease of intestinal diversity in rats, and after the intervention of probiotics, it can increase the diversity and abundance of intestinal alpha.The analysis method of this experiment uses principal coordinate analysis (PCoA), which aims to clearly reflect the similarities and differences in the species composition of each sample, indicating that the 4 groups of rat intestinal flora β are diverse The difference in sex. The calculation method in this experiment is the bray-cuis distance calculation, the confidence interval is 95%, and the contribution rates of the first principal coordinate (PCoAl) and the second principal coordinate (PCoA2) are 28.61% and 25.63% respectively; the results found that 4 groups of rat samples The separation is not obvious, and each group of samples has a high degree of dispersion. It shows that there are obvious individual differences in the species composition abundance and evolutionary relationship of the rats samples.
Horizontal community structure of intestinal bacterial species in rats
The relative levels of specific bacterial species in rat stool samples in this study are shown in Fig. 2. On day 0, the composition of the feces of the rats in each group was similar, mainly Lactobacillus (60–70%), Bacteroides (10–15%) and Lachnospiraceae (5–10%); when the rat breast tissue was challenged, Lactobacillus, The abundances of Bacteroides and Lachnospiraceae all declined, and this result was consistent with the diversity results. Significant changes were found in the stool samples of rats in each group on the 30th day. In the stool samples of rats in the control group, Lactobacillus murinus (9.71%), Lactobacillus johnsonii (8.19%), Lactobacillus reuteri (11.65%), Lactobacillus animalis (4.21%) were significantly reduced, while Parasutterella excrementihominis (3.47%), Enterococcus faecalis (1.2%) Escherichia coli (2.4%), Bacteroides massiliensis (1.1%) significantly increased, while in the Probio-M9 intervention group, Lactobacillus murinus (9.71%), Lactobacillus johnsonii (10.69%), Lactobacillus reuteri (14.21%), Lactobacillus animalis (5.16%), Eubacterium (3.2%), Parabacteroides graminis (3.4%), Bacteroides fragilis (7.2%) significantly increased, while Alipis shahii (2.8%), Brevibacterium massiliense (2.6%), Enterococcus faecalis (2.1%), Desulfovibrio desulfuricans (1.9%) and Erysipelotrichaceae bacterium (1.2%) decreased significantly.
LEfse analysis of different strains
As shown in the Fig. 3, There was a high abundance of Desulfovibrio desulfuricans (P < 0.01), Alistipes indistinctus (P = 0.02), Brevibacterium massiliense (P = 0.02), Alistipes Shahii (p = 0.02), Bacteroides finegoldii (P = 0.02), Erysipelotrichaceae bacterium 21 3 (P = 0.04), Escherichia coli (P = 0.02), Parasutterella excrementihominis (P = 0.04), Enterococcus faecalis (P < 0.01) in MG groups, While Lactobacillus hominis (P < 0.01), Lactobacillus reuteri (P < 0.01), Lactobacillus murinus (P < 0.01), Lactobacillus Johnsonii (P = 0.02), Eubacterium (P = 0.03), Parabacteroides Goldsteinii (P = 0.04), Lactobacillus Johnsonii (P = 0.02), Eubacterium (P = 0.03), Bacteroides fragilis (P = 0.04) was enriched in M9PG groups.
Path analysis
In this study, MetaCyc database was used to conduct difference pathway analysis of metacomgenome. As shown in the Fig. 4, most of the differential pathways were significantly upregulated in the M9PG and M9TG groups on day 30. Compared with the MG group, the common upregulated pathway in other groups was glycolysis. Superpathway of purine nucleotide salvage, Superpathway of L-alanine biosynthesis.
The human intestine is one of the densest and most diverse microbial communities. The intestinal microbiome includes a large number of organisms such as bacteria, viruses, fungi, archaea and protozoa. Bacteroides are potential settlers of the colon and account for the main part of the intestinal flora. These gram-negative obligate anaerobes play multiple roles in the human intestinal flora and are the main players in maintaining the intestinal microbial food web. Bacteroides have been proven to be symbiotic bacteria, not only acting as a "provider" for the host and other microorganisms living nearby, but also helping the host by providing many health benefits. However, certain types of bacteroides may play a dual role of beneficial and pathogenic depending on their location in the host. Bacteroides are usually "friendly" when they live in the intestines, but when they live in other places, they tend to become opportunistic pathogens (Zhu et al. 2019). Entering the normal sterile tissue through the intestinal mucosa will eventually produce different discomforts. The research results show that the by-products of Bacteroides fragilis in the metabolic process have significantly improved the barrier function of epithelial cells and mucus, have significant antibacterial properties, can inhibit the adhesion of intestinal pathogens in the body, and improve the intestinal microenvironment of the body stability (Chattopadhyay et al. 2021). In the mastitis MG group in this study, the content of bacteroides decreased significantly. After the intervention of probiotics, the content of bacteroides fragilis increased significantly.
Enterococcus faecalis and Escherichia coli are the bacteria species that are significantly increased in the control groups in this experiment. Enterococcus is widely present in nature and is a normal flora that exists in the intestines of humans and animals. Current studies have shown that enterococcal infection can have a significant impact on the body. Enterococcus, as an important pathogen, can cause urinary tract infections, skin and soft tissue infections, and more serious abdominal infections, sepsis, meningitis and other diseases. Escherichia coli is widely present in the intestines of humans and animals. When the intestinal flora is imbalanced or the immune system is disturbed, Escherichia coli can become pathogenic bacteria and cause intestinal diseases or other tissue and organ diseases (Franz et al. 2011). Studies have found that, Escherichia coli mainly causes epidemic infant diarrhea, adult pleurisy, arthritis, sepsis, etc. After the intervention of probiotics, the content of E. faecium and E. coil decreased significantly.
Lactobacillus reuteri mainly exists in the intestines of vertebrates and mammals. It is harmless to humans and animals and has good biocompatibility. It competes with harmful bacteria that adhere to the intestines to reduce the risk of harmful bacteria reproduction, maintain the stability of the intestinal flora, balance the composition of the intestinal flora; Lactobacillus reuteri also has the effect of improving allergies and enhancing human immunity (Martinson et al. 2020). Studies have shown that Lactobacillus reuteri produces lactic acid, acetic acid and other substances after glycolysis and pentose phosphate metabolism. The metabolites can also inhibit harmful bacteria and play a probiotic effect.
Purine itself does not exist in nature, but its derivatives are widely present in nature. Adenine and guanine are important components of nucleic acid. Studies have found that disrupting the biosynthesis of purine in organisms will lead to the growth of a large number of pathogenic bacteria, such as Escherichia coli, Bacillus anthracis, staphylococcus aureus, etc. Mastitis can cause DNA damage of microorganisms in the body, such as superpathway of purine deoxyribonucleosides degradation, adenine and adenosine salvage III et al.) and down-regulate energy metabolism (e.g., glycolysis, lactose and galactose degradation pathways, etc.). After probiotic intervention, the DNA repair pathway of gut microbes can be up-regulated.
Energy supply produced by metabolism plays an important role in the dynamic regulation of organisms. Human cells can play their normal physiological functions mainly by mitochondrial aerobic oxidation pathway and glycolysis metabolic pathway derived from organelles. The Glycolytic pathway, also known as the EMP pathway, is a series of reactions in which glucose and glycogen are degraded into pyruvate accompanied by ATP production. It is a universal pathway for glucose degradation in all biological organisms. The glycolysis pathway can be performed under both anaerobic and aerobic conditions and is a common metabolic pathway for the aerobic or anaerobic decomposition of glucose. After probio-M9 intervention, the energy metabolism pathway, including glycolysis pathway, amino acid pathway and fatty acid pathway, were up-regulated.
Non-targeted Metabonomics analysis of rat blood
UPLC-Q TOF-MS chromatogram analysis
The serum sample were analyzed by UPLC-Q-TOF/MS, and data were collected in positive and negative ion modes. The total ion chromatogram of serum samples of NG, MG, M9PG and M9TG was shown in Fig. 5. The results showed that the ionic peak of each metabolite had a complete shape and could be well separated, indicating that the conditions of chromatography and mass spectrometry were suitable for the detection and analysis of the samples.
Multivariate statistical analysis
Use unsupervised statistical model Principal Component Analysis (PCA), Which is a multivariate statistical analysis method. Partial least square discriminant analysis (PLS-DA) is a supervised statistical method of discriminant analysis, which uses partial least squares regression to establish a model of the relationship between metabolite expression and sample category, so as to realize the prediction of sample category. The PL-SDA scores of the four groups of serum samples were shown in Figs. 6 and 7.
In the PCA and PLS-DA score plots of serum samples, each group was completely separated. MG and NG were completely separated, indicating that compared with NG group (Fig. 6), the metabolites in serum of MG group were changed, which proved that the establishment of mastitis model was successful. NG、M9PG and M9TG were completely separated, and compared with MG, the samples of other groups were gradually inclined to NG (Fig. 7). These results indicate that Probio-M9 has certain anti-inflammatory effect, and M9PG has better anti-inflammatory effect than M9TG. Relevant parameters of the PLS-DA model were in Table1.
Table 1 Evaluation parameters of PLS-DA model
Differential metabolite identification
This study combined multivariate and univariate statistical analysis. Finally, the differential metabolites obtained by the screening criteria of p value<0.05, fold chang≥2 and VIP value>1 were searched and identified in the database of Metlin, MassBank, Pubchem and HMDB(Table 2). It was determined that 27 metabolites in serum samples were significantly different metabolites for Mastitis rats induced by S. aureus.
Table 2 The changing trend of significantly different metabolites in the positive ion mode with four groups
Table 3 The changing trend of significantly different metabolites in the negative ion mode with four groups
Compared with NG, 12 significantly different metabolites were significantly decreased and 16 significantly different metabolites were significantly increased in MG (Tables 2 and 3),After oral administration of Probio-M9༌The contents of the 27 significantly different metabolites all changed significantly and all tended to NG. The results show that probiotics regulate the metabolic disorder caused by rat mastitis to a certain extent, so as to achieve the purpose of treating mastitis.
Analysis of serum differential metabolite pathway
The metabolic pathways of 27 significantly different metabolites were analyzed by Metabo Analyst website (https://www.metaboanalyst.ca/MetaboAnalyst/home.xhtml). The results showed that the major metabolic pathways were sphingolipid metabolism, linoleic acid metabolism, steroid hormone biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, retinol metabolism, arachidonic acid metabolism (Fig. 8). They were involved in six significantly different metabolites, including phosphatidylcholine, linoleate, Betaine, retinoate, arachidonate, and glutamate, According to the analysis. It is suggested that the therapeutic mechanism of Probio-M9 on S.aureus-induced mastitis may be through regulating the metabolic disorder caused by mastitis, and by regulating the significantly different metabolites to restore it to normal, thereby playing the role of treating mastitis.
Correlation analysis of blood metabolites and intestinal flora
The 33 strains of intestinal bacteria and 54 blood differential metabolites enriched in feces were selected for Spearman correlation analysis. As shown in Fig. 9, Parabacteroides goldsteinii is positively correlated with succinic acid; Bacteroides dosei is significantly negatively correlated with Palmitic acid, 2-Nonen-1-ol and oleic acid, and is significantly negatively correlated with 1-hexadecanoyl-2-(9Z, 12Z). Rumenococcus active is negatively correlated with palmitic acid and 2-stearoyl-sn-glycerol-3-phosphocholine, Lactobacillus reuteri is negatively correlated with the blood metabolites Lys-gly-lys and 1-stearoyl-sn-glycerol- 3-Phosphoethanolamine is positively correlated. It shows that palmitic acid metabolism is related to Rumenococcus active, Bacteroides dosei. After the intervention of probiotics, through the regulation of intestinal flora, the body's metabolites are then adjusted.
When the breast is infected by S.aureus, the metabolic pathways involved are shown in Fig. 10, the metabolites involved in the glycerophospholipid metabolism pathway, such as phosphocholine, glycerophosphocholine, and phosphatidylcholine, were all significantly affected. Phosphatidylcholine can inhibit lipid peroxidation to prevent cell membrane damage. It also has the effect of emulsifying and decomposing fats, reducing the content of neutral fats and removing peroxides. In addition, glycerophosphate is an important part of glycerolipid metabolism, and is related to other energy metabolism pathways (Lin et al. 2008). The results showed that when S.aureus stimulates breast tissue, the energy demand provided by fatty acid oxidation may increase, and the content of glycerophosphate, glycerophosphatecholine, phosphatidylcholine and phosphocholine increased significantly. After feeding Probio-M9, Glycerophosphoric acid and other metabolites are significantly reduced, thereby regulating the metabolic pathway of glycerophospholipids.
Choline is an important nutrient used in the synthesis of cell membrane phospholipids and is an important source of circulating lipoprotein components and betaine in mammals (Thomas et al., 2016). Betaine is an important osmotic regulator in many biological tissues, and can be used by most mammalian tissues to adjust cell size (Ueland et al. 2011); Betaine almost comes from the oxidation of cereals, vegetables or dietary choline in the beet family. First, betaine is formed by oxidation of choline, then demethylated to N, N-Dimethylglycine, and oxidized to sarcosine, and finally to glycine. This pathway is particularly important for the body's osmotic regulation and the source of methyl groups. In this study, compared with the NG group, the betaine concentration in the MG group decreased, indicating that the pathogenic bacteria stimulated the body's osmotic regulation mechanism. The decrease in blood betaine concentration is considered to be related to increased risk of stress, metabolic syndrome, lipid disorders, and vascular disease (Xi. 2017; Moyes et al. 2015; Sundekilde et al. 2013; Chandrasekharan et al. 2016). Compared with the MG group, the betaine in blood metabolites under the intervention of Probio-M9 was significantly related elevated, has a certain regulatory effect.
Fatty acids play an important role in the body's biofilm synthesis, cell energy supply and signal transduction pathways effect. According to whether it needs to be taken from food, it can be divided into essential and non-essential fatty acids. Among them, linoleic acid is a typical essential fatty acid. It is an important beneficial metabolite from bacteria. A series of positions and geometric isomers of linoleic acid constitute conjugated linoleic acid. Conjugated linoleic acid plays an important role in anti-cancer, anti-inflammatory, anti-atherosclerosis, regulating the immune system, antagonizing intestinal pathogenic bacteria and maintaining the homeostasis of intestinal flora. Arachidonic acid is an important precursor substance of eicosanoid, which can be produced by linoleic acid; and eicosanoid is an inflammatory mediator, such as prostaglandins and leukotrienes, which can affect the body's inflammation and immune response (Peng, 2018; Puppel et al. 2016; Sakemi et al. 2011; Yang et al. 2015). The content of linoleic acid in the blood metabolites of the model group in this study was significantly reduced, while arachidonic acid and leukotrienes were significantly increased; pathogenic bacteria severely disrupted the body’s fatty acid metabolism. After oral administration of probiotics, the content of linoleic acid increased significantly, while the content of arachidonic acid and leukotrienes decreased significantly.
The metabolic pathway of glutathione also involves glutamate, which can also be derived from D-glutamine. D-glutamine is derived from the pathway of glutamate metabolism. Glutamate can also be derived from alanine. The metabolic pathways of aspartate and glutamate produce glutamine and succinate (Gall et al. 2015). Among them, because mucosal cells catabolize glutamate to produce ATP, glutamate is the main available energy for intestinal epithelial cells; at the same time, glutamate is also involved in the synthesis of proteins and peptides that maintain normal intestinal structure and function. The results showed that when Staphylococcus aureus induced mastitis, compared with the control group, glutathione was significantly up-regulated, indicating that when Staphylococcus aureus stimulates breast tissue, its related metabolic pathways will be activated and the cells will Produce a lot of antioxidant metabolites, such as inositol and glutathione, and the glutathione content in the probiotic group will be reduced, thereby eliminating excessive oxygen free radicals and maintaining immunity. The normal operation of the system can promote the restoration of cell homeostasis. After the intervention of probiotics, the content of glutamate increased significantly to maintain intestinal function.
Sphingolipids are an important component of biological membranes, which have the function of protecting intracellular substances and also play an important role in signal transduction of cell life activities. Sphingosine-1-phosphate (S1P) is a kind of bioactive lipid with important physiological functions. It is widely present in blood, lymph, red blood cells, neutrophils, platelets and other body fluids and cells. S1P is produced by sphingomyelin through a multi-step enzymatic reaction and is regulated by a variety of enzymes. Sphingomyelin generates ceramide under the catalysis of sphingomyelinase (SMase), sphingosine under the catalysis of ceramidase (CDase), and S1P under the catalysis of sphingosine kinase (SPHK) (Gao et al. 2019). While forming S1P, cells can be dephosphorylated by S1P phosphorylase to produce sphingosine, thereby ensuring the physiological environment of the human body. S1P can be used as a second messenger to directly act on intracellular targets, or it can be transported outside the cell (Liu et al. 2012), a series of downstream signaling pathways are activated to produce important physiological functions and participate in various physiological functions (Baranowski et al. 2011; Zhang et al. 2017; Dai et al. 2017). In this study, Probio-M9 can significantly regulate S1P and sphingosine in the blood, thereby achieving the purpose of treating mastitis.