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

Abstract

Staphylococcus aureus is a common pathogen that can cause a range of infections in humans, including skin infections, pneumonia, and sepsis. It is also a leading cause of foodborne illness. In recent years, the use of antibiotics to treat S. aureus infections has become less effective due to the emergence of antibiotic-resistant strains. Therefore, there is a need to find alternative treatments for S. aureus infections. One potential alternative is the use of natural compounds, such as lingzhi mushroom polysaccharides (LMPs). In this study, we investigated the effects of LMPs on the gut microbiota of mice infected with S. aureus.

Introduction

Staphylococcus aureus is a Gram-positive bacterium that is commonly found on the skin and in the nasal cavity of humans. It can cause a range of infections, from mild skin infections to severe infections such as pneumonia, sepsis, and endocarditis. S. aureus is also a leading cause of foodborne illness, with outbreaks occurring in a variety of foods, including meat, dairy products, and baked goods (Bergdoll, 1989). The emergence of antibiotic-resistant strains of S. aureus, such as methicillin-resistant S. aureus (MRSA), has made treatment of these infections more difficult (Chambers and DeLeo, 2019). Therefore, there is a need to find alternative treatments for S. aureus infections.

One potential alternative treatment is the use of natural compounds, such as lingzhi mushroom polysaccharides (LMPs). Lingzhi mushroom, also known as Ganoderma lucidum, is a medicinal mushroom that has been used in traditional Chinese medicine for centuries (Wachtel-Galor et al., 2011). LMPs are the main active components of lingzhi mushroom, and have been shown to have a range of biological activities, including immune modulation, anti-tumor activity, and anti-inflammatory effects (Zhang et al., 2016). In addition, LMPs have been shown to have antibacterial activity against a range of Gram-positive and Gram-negative bacteria (Wang et al., 2016).

The gut microbiota is a complex ecosystem of microorganisms that plays an important role in human health. The gut microbiota is involved in a range of physiological processes, including digestion, metabolism, and immune regulation (Belkaid and Hand, 2014). Alterations in the gut microbiota have been associated with a range of diseases, including inflammatory bowel disease, obesity, and diabetes (Turnbaugh et al., 2006). Therefore, maintaining a healthy gut microbiota is important for overall health.

In this study, we investigated the effects of LMPs on the gut microbiota of mice infected with S. aureus. We hypothesized that LMPs would modulate the gut microbiota and improve the outcome of S. aureus infection.

Materials and Methods

Animal model

Male C57BL/6 mice (6-8 weeks old) were obtained from Shanghai Laboratory Animal Center (Shanghai, China). Mice were housed in a specific pathogen-free facility and provided with water and standard chow ad libitum. All animal procedures were approved by the Animal Care and Use Committee of Fudan University.

Experimental design

Mice were randomly divided into four groups (n=6 per group): control, LMPs, S. aureus, and LMPs+S. aureus. Mice in the S. aureus and LMPs+S. aureus groups were intragastrically inoculated with 1x10^8 CFU of S. aureus in 0.2 ml of sterile saline. Mice in the LMPs and LMPs+S. aureus groups were intragastrically administered 100 mg/kg LMPs in 0.2 ml of sterile saline once per day for 7 days. Mice in the control group were intragastrically administered 0.2 ml of sterile saline once per day for 7 days.

Sample collection

Mice were sacrificed on day 7 after infection or treatment. Fecal samples were collected and stored at -80°C until analysis. The small intestine and colon were aseptically removed and homogenized in sterile saline. Serial dilutions were plated on blood agar plates to determine the bacterial load.

16S rRNA gene sequencing

Total DNA was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The V3-V4 region of the 16S rRNA gene was amplified using the primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The amplicons were purified and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA).

Data analysis

The raw sequencing data were processed using the QIIME pipeline (version 1.9.1). The sequences were quality filtered, denoised, and clustered into operational taxonomic units (OTUs) at a sequence similarity threshold of 97%. The taxonomy of each OTU was assigned using the Greengenes database (version 13.8). Alpha diversity was calculated using the Shannon index, and beta diversity was calculated using the weighted UniFrac distance metric. Differential abundance analysis was performed using the DESeq2 package in R.

Results

LMPs reduce S. aureus colonization in the gut

Mice in the S. aureus and LMPs+S. aureus groups had higher bacterial loads in the small intestine and colon compared to the control and LMPs groups (Figure 1A and 1B). However, mice in the LMPs+S. aureus group had lower bacterial loads in the small intestine and colon compared to the S. aureus group (Figure 1A and 1B). These results suggest that LMPs can reduce S. aureus colonization in the gut.

LMPs modulate the gut microbiota composition

To investigate the effects of LMPs on the gut microbiota composition, we performed 16S rRNA gene sequencing on the fecal samples. Alpha diversity was not significantly different between the groups (Figure 2A). However, beta diversity analysis showed that the gut microbiota composition was significantly different between the groups (Figure 2B). Differential abundance analysis showed that LMPs treatment significantly increased the relative abundance of Lactobacillus and decreased the relative abundance of Bacteroides compared to the control group (Figure 3A and 3B). S. aureus infection significantly decreased the relative abundance of Lactobacillus and increased the relative abundance of Enterococcus compared to the control group (Figure 3A and 3C). LMPs treatment in combination with S. aureus infection increased the relative abundance of Lactobacillus compared to the S. aureus group (Figure 3A).

Discussion

In this study, we investigated the effects of LMPs on the gut microbiota of mice infected with S. aureus. We found that LMPs reduced S. aureus colonization in the gut and modulated the gut microbiota composition. LMPs treatment increased the relative abundance of Lactobacillus and decreased the relative abundance of Bacteroides, while S. aureus infection decreased the relative abundance of Lactobacillus and increased the relative abundance of Enterococcus. LMPs treatment in combination with S. aureus infection increased the relative abundance of Lactobacillus compared to the S. aureus group.

Lactobacillus is a beneficial bacterium that is commonly found in the human gut microbiota. Lactobacillus has been shown to have probiotic effects, such as immune modulation, inhibition of pathogen growth, and improvement of gut barrier function (Liu et al., 2018). Bacteroides is a gut commensal that is involved in carbohydrate metabolism and immune regulation (Coyne and Comstock, 2008). Enterococcus is a facultative anaerobe that is commonly found in the human gut and can cause infections in immunocompromised individuals (Arias et al., 2010). The decrease in Lactobacillus and increase in Enterococcus in the S. aureus group suggests that S. aureus infection disrupts the gut microbiota composition and reduces the abundance of beneficial bacteria. The increase in Lactobacillus in the LMPs+S. aureus group suggests that LMPs treatment can modulate the gut microbiota and promote the growth of beneficial bacteria.

In conclusion, our study provides evidence that LMPs can reduce S. aureus colonization in the gut and modulate the gut microbiota composition. These findings suggest that LMPs may have potential as a natural treatment for S. aureus infections. Further studies are needed to investigate the mechanisms underlying the effects of LMPs on the gut microbiota and to determine the optimal dosing and duration of LMPs treatment.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81773511) and the Shanghai Municipal Natural Science Foundation (No. 19ZR1438700).

Conflict of Interest

The authors declare no conflict of interest.

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Figure Legends

Figure 1. Bacterial loads in the small intestine (A) and colon (B) of mice. Data are presented as mean ± SEM.

Figure 2. Alpha diversity (A) and beta diversity (B) of the gut microbiota. Alpha diversity was measured using the Shannon index. Beta diversity was measured using the weighted UniFrac distance metric. Data are presented as mean ± SEM.

Figure 3. Relative abundance of bacterial taxa. Differential abundance analysis was performed using the DESeq2 package in R. (A) Lactobacillus, (B) Bacteroides, (C) Enterococcus. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01.