Research Article Bacterial Atlas of Mouse Gut Microbiota

Background . Mouse model is one of the most widely used animal models for exploring the roles of human gut microbiota, a complex system involving in human immunity and metabolism. However, the structure of mouse gut bacterial community has not been explored at a large scale. To address this concern, the diversity and composition of the gut bacteria of 600 mice were characterized in this study. Results . The results showed that the bacteria belonging to 8 genera were found in the gut microbiota of all mouse individuals, indicating that the 8 bacteria were the core bacteria of mouse gut microbiota. The dominant genera of the mouse gut bacteria contained 15 bacterial genera. It was found that the bacteria in the gut microbiota were mainly involved in host ’ s metabolisms via the collaborations between the gut bacteria. The further analysis demonstrated that the composition of mouse gut microbiota was similar to that of human gut microbiota. Conclusion . Our study presented a bacterial atlas of mouse gut microbiota, providing a solid basis for investing the bacterial communities of mouse gut microbiota.


Background
The human gut microbiota plays key roles in human homeostasis, thus largely affecting human health [1]. The composition of gut microbiota develops dynamically in the first 2-3 years of life, which can affect risk factors related to adult health [2]. Increasing evidence has linked the human gut microbiota to diseases. The altered microbial communities are associated with obesity [3], gastrointestinal cancer [4], and type 2 diabetes [5]. To characterize human gut microbiota, murine models have been widely used, due to the extensive similarities in anatomy, physiology, and genetics [6]. In mice, the dysbiosis of gut microbiota leads to severe diseases. Cognitive dysfunction is associated with the abnormal composition of the gut microbiota of mice [7]. The gut microbiota of mice influences the pathogenesis of malaria [8] and is identified as an important mediator of acute pancreatitis [9]. There are numerous lines of evidence suggesting that the gut microbiota of mice takes great effects on major depressive disorder [10] and type 1 diabetes [11]. In the gut microbiota of mice, Bacteroides thetaiotaomicron can affect the immune system because it is able to recapitulate the effects of the entire conventional microbiota and notably induces Treg pathways [12]. Chryseomonas, Veillonella, and Streptococcus may be the initial source of the atherosclerotic [13], while Akkermansia muciniphila, a mucindegrading bacterium, elicits beneficial effects on metabolism and reduces atherosclerotic lesion formation [14]. As reported, type 2 diabetes is associated with a reduced abundance of butyrate producing bacteria and an increased abundance of Lactobacillus sp. [15]. Although the gut microbiota of mice plays very important roles in health, the structure of gut microbiota of mice has not been extensively explored.
The colonization of the gut is influenced by many complex environmental factors, such as host genetics, age, diet, lifestyle, diseases, and antibiotic use [16]. Based on microbial metagenome sequencing of some mice fed with low-fat or high-fat diets, the dominate genera in mice gut microbiota are Faecalibacterium, Coprobacillus, Odoribacter, Anaerotruncus, Desulfovibrio, Enterococcus, Marvinbryantia, Pseudoflavonifractor, Coprococcus, Parabacteroides, Blautia, Eubacterium, Ruminococcus, Roseburia, Lactobacillus, Alistipes, Prevotella, Butyrivibrio, Clostridium, and Bacteroides [17]. An increased ratio of the major phyla Firmicutes and Bacteroidetes (FIR/BAC ratio) and depletion of several bacterial species such as Akkermansia muciniphila can promote the development of obesity in mice [18]. Generally, the mouse gut microbiota can be divided into two enterotypes, including Ruminococcus enterotype and Bacteroides enterotype, a reproducible pattern of variation in the microbiota, according to their composition and community structure properties [19,20]. At present, however, the diversity and abundance of the gut bacteria in healthy mice have not been characterized at a large scale.
To address this issue, the composition of the gut microbiota of 600 healthy mice was explored in the present investigation. The results revealed that at the genus level, the core bacteria of mouse gut microbiota contained 8 bacteria, and the dominant bacteria consisted of 15 bacteria. There was a similarity between mouse and human gut bacterial communities.

Sample Collection.
A total of 300 ICR (Institute of Cancer Research) female and 300 ICR male mice (8 weeks old) purchased form Zhejiang Academy of Medical Sciences were raised in a sterilized condition for 3 days to stabilize the composition of gut microbiome. The mice were raised for another 7 days in the same sterilized condition. On day 3 and day 10, the feces of each mouse were collected for later use.

Sequencing and Sequence Analysis of Bacterial 16S rRNA.
The bacterial genomic DNA was extracted directly from the fecal samples with FastDNA® SPIN Kit (MP Biomedicals, USA) according to the manufacturer's manuals. Subsequently, the bacterial 16S rRNA gene was amplified by PCR using gene-specific primers (515F, 5 ′ -GTGCCAGCMGCCGCGG-3 ′ ; 907R, 5′-CCGTCAA TTCMTTTRAGTTT-3′) (M = A/C; R = A/G). The libraries of bacterial 16S rRNA gene were generated using NEB Next®Ultra™DNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendation. The libraries were sequenced on an Illumina MiSeq platform.
Sequence analysis was performed by the UPARSE software package using the UPARSE-OTU and UPARSE-OTUref algorithms. All primers, spacers, low-quality fragments, and the sequences shorter than 50 bp were removed. The remaining sequences were further processed with the pre.cluster command and chimera.uchime command in Mothur. All sequences were denoised and screened for chimeric sequences. Then, the sequences were assigned to the same operational taxonomic units (OTUs) by 97% sequence similarity. The uclust was used to annotate taxonomic information for each OTU. Mothur was used to analyze the community richness, community diversity, and rarefaction curve.

Principal Coordinate Analysis.
To compare two or more microbial communities, principal coordinate analysis (PCoA) was performed. The Bray-Curtis dissimilarity algorithm was applied for PCoA. The vegan of package R (version 3.4.4) (https://www.r-project.org/) was used.

KEGG (Kyoto Encyclopedia of Genes and Genomes)
Pathway Analysis. The bacterial 16S gene sequencing data were clustered with Greengenes database (http://greengenes .secondgenome.com) using closed reference OTU picking. The internal standardization was conducted to obtain the normalized species abundance. Subsequently, the abundance of three levels of KEGG Orthology and pathway was obtained according to the relationship between the Greengenes database and the copy number of KEGG Orthology.

Bacterial Communities in Mice.
To determine the composition of microbiota in the gut of mice, a total of 600 ICR (Institute of Cancer Research) mice (300 female and 300 male mice), one of the most commonly used laboratory mice, were subjected to the analysis of bacterial community (Figure 1(a)). On day 3 and day 10 after mouse raise, the bacterial 16S rRNA sequencing of mouse feces was performed (Figure 1(a)). The results of transmission electron microscopy showed that the bacteria of mouse gut were rod-shaped, short rod-shaped, and spherical-shaped (Figure 1(b)). The sequencing analysis of bacterial 16S rRNA gene of 1,200 mice yielded a total of 33,390,144 reads (Table S1). Based on these reads, 2478 operational taxonomic units (OTUs) were identified (GenBank accession no. PRJNA721276) (Table S1). All OTUs were defined by 97% similarity. The rarefaction curves of all samples approached plateaus (Figure 1(c)), indicating that the sequencing data represented the gut microbiome of mice.

The Dominant Bacteria and Core Bacteria in the Gut
Microbiota of Mice. To determine the dominant bacteria and the core bacteria in the gut microbiota of mice, the composition of the gut microbiota of 600 ICR mice was characterized. The results of the principal coordinate analysis of mouse gut microbiota showed that there was no significant difference of gut microbiota composition on day 3 and day 10 ( Figure 2(a)), indicating that the gut microbiota was stable and the data were reliable. However, the dots representing the gut microbiota of 600 mice were scattered in different locations (Figure 2(a)), showing the existence of individual differences of gut microbiota.
To determine the dominant bacteria in the gut microbiota of mice, the bacteria of 600 mice were analyzed. The results revealed that at the phylum level, dominant bacteria in the gut microbiota of mice mainly included Firmicutes (55.75%), Bacteroidetes (37.02%), Proteobacteria (4.05%), Actinobacteria (1.98%), and Tenericutes (1.09%), while the abundance of other bacteria was less than 1% (Figure 2(b)). The most dominant genus was Bacteroidales S24-7 group_norank (23.89%), followed by Lactobacillus (22.98%), Faecalibaculum (11.17%),        Each dot represented the stucture of gut microbiota of mice on day 3 (blue) or day 10 (red). There was no statistically significant difference of gut microbiota composition on day 3 and day 10 (p < 0:01). (b) Relative abundance of gut bacteria of mice. "Uncultured" represented the bacteria that could not be cultured. "Unclassified" indicated the sequences that could not be classified. "Norank" represented that there was no classification information or classification name. Bacteria with a relative abundance of less than 1% were classified as "Others." (c) The core bacteria of mouse gut microbiota at the genus level. (d) The core bacteria of mouse gut microbiota at the species level. "Uncultured" represented the bacteria that could not be cultured.        "Uncultured" indicated the bacteria that could not be cultured. "Unclassified" showed the sequences that could not be classified. "Norank" represented that there was no classification information or classification name. Only the bacteria with a relative abundance of more than 1% were listed.  (Figure 2(b)).
Collectively, these results revealed that the bacteria belonging to 8 genera were the core bacteria of the mouse gut microbiota. The dominant genera of the mouse gut bacteria contained 15 bacterial genera.

Bacterial Composition in the Gut Microbiota of Male and
Female Mice. To compare the bacterial composition of male and female mice, the gut microbiota of mice were analyzed. The results showed that the female mice had a total of 1,041 OTUs, which could be classified into 27 phyla, 70 classes, 138 orders, 254 families, 626 genera, and 841 species (Figure 3(a)). The male mice contained a total of 1038 OTUs, which were classified into 30 phyla, 75 classes, 141 orders, 256 families, 624 genera, and 833 species (Figure 3(a)).
Taken together, these findings revealed that the dominant bacteria in the gut microbiota of male and female mice contained of 16 and 15 genera of baceria, respectively. The core bacteria in the gut microbiota of male and female mice consisted of 11 and 9 genera, respectively.

Functional Profiles of the Bacteria in the Gut Microbiota of
Mice. To characterize the functions of the bacteria in the gut microbiota of mice, KEGG analysis was performed. The results exhibited that at level 1, the bacteria in gut microbiota of mice involved in metabolism (61.99%), none (11.51%), organismal systems (13.45%), genetic information processing (4.80%), environmental information processing (4.12%), human diseases (2.14%), and cellular processes (1.99%), indicating that the bacteria in the gut microbiota of mice mainly functioned in host's metabolism (Figure 4(a)).
At level 2, the gut bacteria of mice played important roles in global and overview maps (22.94%), energy metabolism (18.08%), endocrine system (12.85%), carbohydrate metabolism (8.08%), amino acid metabolism (3.89%), membrane transport ( Figure 5: Similarity between human and mouse gut microbiota. (a) The dominant bacteria of mouse and human gut microbiota at the genus level. "uncultured" represented the bacteria that could not be cultured. "unclassified" indicated the bacteria that could not be classified. "norank" represented that there was no classification information or classification name. Bacteria with a relative abundance of less than 1% were classified as "others." (b) The dominant bacteria of mouse and human gut microbiota at the species level. (c) Comparison of the core bacteria in human and mouse gut microbiota. 17 Cellular Microbiology glycan biosynthesis and metabolism (1.09%), cellular community-prokaryotes (1.01%), and signal transduction (1.00%) (Figure 4(a)). At level 3, the main functions of mouse gut bacteria included sulfur metabolism (15.81%), adipocytokine signaling pathway (12.52%), metabolic pathways (9.09%), biosynthesis of secondary metabolites (4.11%), biosynthesis of antibiotics (3.09%), microbial metabolism in diverse environments (2.22%), biosynthesis of amino acids (2.13%), carbon metabolism (1.47%), and ribosome (1.34%) (Figure 4(a)). These data showed that the main functions of the bacteria in the gut microbiota of mice were associated with metabolisms.
To explore the functions of the dominant bacteria and the core bacteria in mouse gut microbiota, the dominant and the core bacteria were subjected to the KEGG analysis. The results showed that the dominant bacteria were mainly involved in the metabolisms, including carbohydrate metabolism, amino acid metabolism, energy metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, lipid metabolism, glycan biosynthesis and metabolism, xenobiotic biodegradation and metabolism and metabolism of terpenoids and polyketides (Figure 4(b)). At the same time, the analysis indicated that the core bacteria in the mouse gut microbiota mainly took part in the metabolisms, including carbohydrate metabolism, amino acid metabolism, energy metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, glycan biosynthesis and metabolism, lipid metabolism, metabolism of terpenoids and polyketides, and xenobiotic biodegradation and metabolism (Figure 4(c)). These data revealed that the involvement of metabolisms was the major role of gut bacteria.
Taken together, these findings presented that the bacteria in the gut microbiota mainly took part in the metabolisms by the collaborations between the gut bacteria.

Similarity between Human and Mouse Gut Microbiota.
To explore the similarity and difference between mouse and human gut microbiota, the dominant and core bacteria in human and mouse gut microbiota were compared. The sequencing data of human gut microbiota were obtained from the NCBI database (Table S4), including 1,053 human fecal samples. At the genus level, the dominant bacteria in the human gut microbiota contained Bacteroides (19.73%), Blautia (8.81%), Bifidobacterium (7.56%), Faecalibacterium (5.85%), Fusicatenibacter (2.65%), Anaerostipes (2.00%), Lachnoclostridium (1.08%), and Alistipes (1.05%) (Figure 5(a)). Among these bacteria, Alistipes and Bacteroides were the dominant bacteria in the gut microbiota of mice, while the remaining bacteria were the dominant bacterium unique in human gut microbiota. At the genus level, the human gut microbiota was partially similar to that of mouse gut microbiota.
Based on the NCBI database (Table S4), the human gut microbiota analysis showed that only two genera of bacteria were present in all human fecal samples. The bacteria were Bacteroides and Blautia, accounting for 19.73% and 8.81%, respectively. Bacteroides belonged to the core bacteria of mouse gut microbiota, while Blautia was unique in the human gut microbiota (Figure 5(c)). At the species level, there was no core species of bacteria in human gut microbiota.
Taken together, the findings indicated that the structure of mouse gut microbiota was similar to that of human gut microbiota.

Discussion
It is well known that the gut microbiota plays important roles in human health by affecting metabolisms. The gut microbiota participates in energy metabolism via inducing the expression of genes related to lipid and carbohydrate metabolism, whose dysbiosis can lead to obesity [21]. The structure of gut microbiota is always changed in patients with inflammatory bowel disease [22]. The gut microbiota is able to promote dysbiosis, barrier failure, colorectal cancer, and inflammation [23]. Multiple neurological diseases, such as autism spectrum disorder, are related to gut microbiota, which regulates behaviors 18 Cellular Microbiology through production of neuroactive metabolites [24]. Many investigations demonstrate that the functions of gut microbiota depend on the structure of gut bacteria [25,26]. The dysbiosis of gut microbiota can promote or boost susceptibility to metabolic disorders [26]. Therefore, the gut microbiota has attracted more and more attentions. As mammalian models, rodents, especially mouse, are widely to explore the roles of gut microbiota [27]. For the better genetic and physiological similarities to humans, mouse model is most commonly employed. The mouse model has the ability to control environmental factors more easily in experiments to minimize changes in baseline gut microbiota between individuals. Although many investigations focus on mouse gut microbiota, the structure of gut microbiota of mouse has not been explored at a large scale [28]. Based on the analysis of mouse gut bacteria at a large scale, our findings revealed that the core bacteria of the mouse gut microbiota included 8 bacteria at the genus level, while the dominant genera of the mouse gut bacteria contained 15 bacteria. Compared with the previous study [29], 4 of 8 genera of core bacteria (Bacteroidales S24-7 group_norank, Lachnospiraceae NK4A136 group, Lachnospiraceae_ uncultured, and Ruminiclostridium 9) and 10 of 15 genera of dominant bacteria (Bacteroidales S24-7 group_norank, Faecalibaculum, Lachnospiraceae NK4A136 group, Lachnospiraceae_ uncultured, Escherichia-Shigella, Enterorhabdus, Ruminococcaceae UCG-014, Ruminiclostridium, Roseburia, and Mollicutes RF9_norank) revealed in this study were novel. The remaining core and dominant bacteria of this investigation were consistent with the previous data [29]. In this study, the results revealed that the exclusive core genera in female mice was Ruminococcaceae UCG-014, while Parabacteroides, Ruminococcaceae_ uncultured, and Lachnoclostridium were the exclusive core genera in male mice. The dominant bacteria in the gut microbiota of male and female mice contained of 16 and 15 genera of baceria, respectively. These data indicated that there were differences of the core and dominant microbiota between male and female mice. The structure of the gut microbiota of mice, including the core bacteria and the dominant bacteria, was similar to that of human being. Therefore, our study provided a solid basis for the investigations of gut microbiota. Our findings revealed that two bacteria at the genus level, Alistipes and Bacteroides, were shared by mouse and human microbiota, suggesting the importance of Alistipes and Bacteroides in mammalian gut microbiota. Alistipes, one of the genus members of the Bacteroidetes phylum, is highly relevant to dysbiosis and metabolic diseases [30]. Various species of gut bacteria belonging to the genus Alistipes have been isolated from patients with appendicitis and abdominal and rectal abscess [31]. It is found that Alistipes is pathogenic in patients with colorectal cancer or depression [32,33]. However, Alistipes also has protective effects against some diseases, such as liver fibrosis, colitis, cancer immunotherapy, and cardiovascular disease [30]. These data demonstrate that Alistipes in gut microbiota plays important regulatory roles in metabolic diseases. Bacteroides, a dominant bacterial genus in gut microbiota of mouse and human being, can product sphingolipid, which is essential for the maintenance of the symbiotic relationship between gut microbiota and mammalian hosts [34]. Evidences indicate that Bacteroides is related to host-and diet-derived glycans and has tremendous capability to utilize complex recalcitrant glycans to sustaining gut microbial symbiosis [35]. These findings show that Bacteroides can confer health benefit to the host, therefore helping prevent or delay diseases. Nevertheless, some species of Bacteroides are pathogenetic. B. fragilis and B. thetaiotaomicron are commonly isolated anaerobic pathogens, which are often found in the patients with gangrenous and perforated appendicitis [36,37]. In this context, Alistipes and Bacteroides might be biomarker bacteria for metabolic diseases as well as bacterial indicators for human health.

Data Availability
Sequencing data was stored at NCBI database (PRJNA721276).

Conflicts of Interest
The authors declare that they have no competing interests.