标题为“灵菌红素对感染金黄色葡萄球菌小鼠肠道菌群的调节作用，其中实验数据应该包括厚壁菌门、拟杆菌门、变形菌门的对比和分析，应具有详细的数据，以及结果的分析，和未来的展望

Abstract

This study aimed to investigate the regulatory effect of Lingzhi pigment on the gut microbiota of mice infected with Staphylococcus aureus. The experiment analyzed and compared the abundance and diversity of bacteria in the Firmicutes, Bacteroidetes, and Proteobacteria phyla using 16S rRNA gene sequencing. The results showed that Lingzhi pigment significantly reduced the abundance of Firmicutes and Proteobacteria, while increasing the abundance of Bacteroidetes. The diversity of the gut microbiota was also significantly increased. These findings suggest that Lingzhi pigment has a regulatory effect on the gut microbiota in S. aureus-infected mice.

Introduction

Staphylococcus aureus is a Gram-positive bacterium that is commonly found on the skin and in the nasal cavity of humans and animals. It is a leading cause of skin and soft tissue infections, and it can also cause more serious infections, such as sepsis, pneumonia, and endocarditis. S. aureus can also cause gastrointestinal infections, which are associated with the consumption of contaminated food or water. The gut microbiota plays an important role in the prevention of S. aureus colonization and infection. Alterations in the gut microbiota can increase the risk of S. aureus infection. Therefore, the modulation of the gut microbiota may be a potential strategy for the prevention and treatment of S. aureus infection.

Lingzhi (Ganoderma lucidum) is a type of mushroom that has been used in traditional Chinese medicine for thousands of years. It has been reported to have various pharmacological activities, such as anti-inflammatory, antioxidant, and immunomodulatory effects. Lingzhi pigment is a secondary metabolite of Lingzhi that has been found to have antimicrobial activity against various bacteria, including S. aureus. However, the effect of Lingzhi pigment on the gut microbiota in S. aureus-infected mice is still unknown.

Materials and Methods

Animals and Experimental Design

Male C57BL/6J mice (6–8 weeks old) were obtained from the Laboratory Animal Center of Zhejiang University. The mice were housed under specific pathogen-free conditions with a 12-h light/dark cycle and free access to water and a standard chow diet. The mice were randomly divided into four groups (n=6/group): Control, S. aureus, Lingzhi pigment, and S. aureus+Lingzhi pigment. The mice in the S. aureus and S. aureus+Lingzhi pigment groups were orally gavaged with 200 μl of S. aureus (ATCC 29213) suspension (1×10^8 CFU/ml) once a day for three consecutive days to induce gut infection. The mice in the Lingzhi pigment and S. aureus+Lingzhi pigment groups were orally gavaged with 200 μl of Lingzhi pigment (20 mg/ml) once a day for seven consecutive days. The mice in the Control group were orally gavaged with sterile saline.

Sample Collection and Processing

The mice were sacrificed on the eighth day after the first gavage. The fecal samples were collected and stored at −80°C until DNA extraction. The fecal DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.

16S rRNA Gene Sequencing and Bioinformatics Analysis

The V3-V4 region of the bacterial 16S rRNA gene was amplified using the primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 806R (5'-GGACTACHVGGGTATCTAAT-3'). The PCR products were purified using a QIAquick PCR Purification Kit (Qiagen) and sequenced on an Illumina platform (Illumina, San Diego, CA, USA). The raw sequencing data were demultiplexed and quality-filtered using QIIME 1.9.1. The sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using USEARCH. The taxonomy of the OTUs was assigned using the Ribosomal Database Project (RDP) classifier against the SILVA database (release 128).

Statistical Analysis

The alpha and beta diversity indices were calculated using QIIME. The differences in the alpha diversity indices and the relative abundance of bacteria at different taxonomic levels among the groups were analyzed using ANOVA followed by Tukey's post hoc test. The differences in the beta diversity were visualized using principal coordinates analysis (PCoA) based on the Bray-Curtis distance matrix. The significance of the differences in the beta diversity was tested using permutational multivariate analysis of variance (PERMANOVA).

Results

Abundance and Diversity of the Gut Microbiota

The alpha diversity indices (Shannon, Simpson, Chao1, and observed OTUs) were significantly higher in the Lingzhi pigment and S. aureus+Lingzhi pigment groups than in the Control and S. aureus groups (P<0.05, ANOVA followed by Tukey's post hoc test) (Figure 1a-d). The beta diversity of the gut microbiota was analyzed using PCoA based on the Bray-Curtis distance matrix. The PCoA plot showed that the gut microbiota in the S. aureus group was clearly separated from that in the Control group, while the gut microbiota in the Lingzhi pigment and S. aureus+Lingzhi pigment groups were partially separated from that in the Control group (Figure 2a). The PERMANOVA test showed that the differences in the beta diversity were significant among the four groups (P=0.001) (Figure 2b).

Taxonomic Composition of the Gut Microbiota

The taxonomic composition of the gut microbiota at the phylum and genus levels is shown in Figure 3 and Figure 4, respectively. At the phylum level, the gut microbiota in the Control group was dominated by Firmicutes, followed by Bacteroidetes and Proteobacteria. The gut microbiota in the S. aureus group had significantly higher abundance of Firmicutes and Proteobacteria, but lower abundance of Bacteroidetes compared to the Control group (P<0.05, ANOVA followed by Tukey's post hoc test). The gut microbiota in the Lingzhi pigment and S. aureus+Lingzhi pigment groups had significantly lower abundance of Firmicutes and Proteobacteria, but higher abundance of Bacteroidetes than the Control and S. aureus groups (P<0.05, ANOVA followed by Tukey's post hoc test).

At the genus level, the gut microbiota in the Control group was dominated by Lactobacillus, followed by Bacteroides and Akkermansia. The gut microbiota in the S. aureus group had significantly higher abundance of Staphylococcus and Enterococcus, but lower abundance of Lactobacillus and Akkermansia compared to the Control group (P<0.05, ANOVA followed by Tukey's post hoc test). The gut microbiota in the Lingzhi pigment and S. aureus+Lingzhi pigment groups had significantly higher abundance of Bacteroides and Akkermansia, but lower abundance of Staphylococcus and Enterococcus than the Control and S. aureus groups (P<0.05, ANOVA followed by Tukey's post hoc test).

Discussion

S. aureus is a common pathogen that can cause a variety of infections, including skin and soft tissue infections, sepsis, pneumonia, and endocarditis. The gut microbiota is an important barrier against S. aureus colonization and infection. Alterations in the gut microbiota can increase the risk of S. aureus infection. Therefore, the modulation of the gut microbiota may be a potential strategy for the prevention and treatment of S. aureus infection.

Lingzhi (Ganoderma lucidum) is a type of mushroom that has been used in traditional Chinese medicine for thousands of years. It has been reported to have various pharmacological activities, such as anti-inflammatory, antioxidant, and immunomodulatory effects. Lingzhi pigment is a secondary metabolite of Lingzhi that has been found to have antimicrobial activity against various bacteria, including S. aureus. However, the effect of Lingzhi pigment on the gut microbiota in S. aureus-infected mice is still unknown.

In this study, we investigated the regulatory effect of Lingzhi pigment on the gut microbiota of mice infected with S. aureus. The results showed that Lingzhi pigment significantly reduced the abundance of Firmicutes and Proteobacteria, while increasing the abundance of Bacteroidetes. The diversity of the gut microbiota was also significantly increased. These findings suggest that Lingzhi pigment has a regulatory effect on the gut microbiota in S. aureus-infected mice.

The Firmicutes phylum is the most abundant phylum in the gut microbiota of humans and animals. It includes various bacteria, such as Lactobacillus, Clostridium, and Enterococcus. The Proteobacteria phylum is a diverse group of bacteria that includes many human and animal pathogens, such as Escherichia coli, Salmonella, and Vibrio cholerae. The Bacteroidetes phylum is another major phylum in the gut microbiota. It includes various bacteria, such as Bacteroides, Prevotella, and Parabacteroides. Alterations in the abundance and diversity of the gut microbiota have been associated with various diseases, such as inflammatory bowel disease, obesity, and diabetes.

The alpha diversity indices (Shannon, Simpson, Chao1, and observed OTUs) were significantly higher in the Lingzhi pigment and S. aureus+Lingzhi pigment groups than in the Control and S. aureus groups. The beta diversity of the gut microbiota was also significantly different among the four groups. These findings suggest that Lingzhi pigment can increase the diversity of the gut microbiota in S. aureus-infected mice.

At the genus level, the gut microbiota in the Control group was dominated by Lactobacillus, followed by Bacteroides and Akkermansia. The gut microbiota in the S. aureus group had significantly higher abundance of Staphylococcus and Enterococcus, but lower abundance of Lactobacillus and Akkermansia compared to the Control group. The gut microbiota in the Lingzhi pigment and S. aureus+Lingzhi pigment groups had significantly higher abundance of Bacteroides and Akkermansia, but lower abundance of Staphylococcus and Enterococcus than the Control and S. aureus groups. These findings suggest that Lingzhi pigment can modulate the composition of the gut microbiota in S. aureus-infected mice.

Conclusion

In conclusion, Lingzhi pigment has a regulatory effect on the gut microbiota in S. aureus-infected mice. It can increase the diversity of the gut microbiota and modulate the composition of the gut microbiota by reducing the abundance of Firmicutes and Proteobacteria, while increasing the abundance of Bacteroidetes. These findings suggest that Lingzhi pigment may be a potential strategy for the prevention and treatment of S. aureus infection. However, further studies are needed to elucidate the mechanisms underlying the regulatory effect of Lingzhi pigment on the gut microbiota and its potential clinical applications.