Abstract:A large number of commensal microorganisms inhabit the human and animal guts. These commensals participate in host life activities throughout the whole life course. Revealing the dynamic changes of the commensals, and the mutual dependency and competitive relationships between microorganisms and host will give us a comprehensive view on the life's essence of higher organisms. This special issue present domestic researchers' new progress and opinions in the field of gut microbes form the following five aspects:gut microbes and disease, gut microbial community structure, interaction between gut microbes and host, gut microbial resource and methods to study gut microbes.

Abstract:[Objective] To investigate the effect of streptomycin sulfate on the symptoms and gut microbiota of chronic Parkinson's disease mice. [Methods] Forty C57BL/6 mice were randomly divided into the control group, streptomycin sulfate control group, Parkinson's disease (PD) model group and streptomycin sulfate treated PD model group. PD mice were induced by injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg) combined with probenecid (250 mg/kg) at the first 5 weeks of the experiment; the streptomycin sulfate treated PD mice began to drink the streptomycin aqueous solution (500 μg/mL) on the first day of the experiment until the end of the experiment. A variety of experimental methods, such as rotarod test, pole test, immunohistochemistry, real-time PCR, and high-throughput sequencing were used to detect the related symptoms and indicators of each group. [Results] Compared with the control, chronic PD mice showed motor impairment (P<0.01), dopaminergic neurons in the nigrostria and their fibers in the striatum reduced significantly (P<0.01), intestinal dysfunction and inflammation, along with the structure of gut microbiota changed significantly:the ratio of Firmicutes/Bacteroides and the abundance of Family Ruminococcaceae increased (P<0.01), whereas the abundance of Family Prevotellaceae and Genus Prevotellaceae_UCG-001 decreased (P<0.01). The intervention of streptomycin sulfate significantly improved the exercise capacity of chronic PD mice (P<0.05), relieved the decrease of dopaminergic neurons and fibers in the nigrostriatal system (P<0.05), and improved the intestinal dysfunction and inflammation, meanwhile reduced the ratio of the Firmicutes/Bacteroides and the abundance of Family Ruminococcaceae, Rikenellaceae and Lactobacillaceae and increased the abundance of Family Prevotellaceae and Genus Prevotellaceae_UCG-001.[Conclusion] Streptomycin sulfate can relieve the symptoms associated with PD mice and affect the gut microbiota of PD mice.

Abstract:[Objective] To explore the immune regulatory effect of gut microbiota in experimental autoimmune encephalomyelitis (EAE), we studied the change of gut microbiota and its correlation with the production of IL-17 and IFN-γ at different times. [Methods] Female C57BL/6 mice were divided randomly into 2 groups:the normal group and EAE. EAE was induced with MOG_35-55 mixed with complete Freund's adjuvant. The weight and neurological scores of 2 groups were observed. The 16S rDNA V3/V4 region of DNA of gut microbiota was indentified by the Illumina Mi Seq high-throughput sequencing.IL-17 and IFN-γ in the blood were detected by ELSIA. [Results] The production of IL-17 and IFN-γ reached the peak on day 21. The abundance of Alistipes, Blautia and Lachnospiraceae_NK4A136_group in EAE were different from the normal group on day 14. However, the abundance of Allobaculum, Eubacterium, Helicobacter were significantly changed on day 30. According to LefSe analysis, the microbial strains that were mainly affected on 7 day, 14 day and 21 day gradually decreased, and dropped the least on 21 day. Odoribacter played an important role on 21 day. Compared with the normal group, the abundance and diversity of gut microbiota in EAE changed. The abundance of Prevotellaceae_NK3B31_group in EAE was lower than the normal group, and related negatively with the production of IFN-γ (r=-0.537, P<0.01). Prevotellaceae_NK3B31_group maybe the key bacteria that caused the demyelination of MS. [Conclusion] The abundance and diversity of gut microbiota played the important role at the different times in the EAE group. The production of IL-17 and IFN-γ leaded to the inflammatory of EAE that induced by MOG_35-55.

Abstract:[Objective] To explore the possible role of intestinal bacteria in the occurrence and development of type 2 diabetes mellitus (T2DM) and cardiovascular complications, we analyzed the difference of intestinal flora between T2DM patients, T2DM patients with cardiovascular complications and healthy volunteers, and the association of intestinal flora with blood glucose and blood lipid. [Methods] A total of 251 healthy controls, 160 diabetic patients with cardiovascular complications and 295 diabetic patients were collected. From each group 30, 30 and 40 subjects were randomly selected for blood biochemical index analysis and metagenome detection of intestinal flora. [Results] The levels of serum alanine aminotransferase, alkaline phosphatase, triglyceride, fasting blood glucose, insulin and glycosylated hemoglobin in diabetic cardiovascular complications group and T2DM group were significantly higher than the healthy control group (P<0.05). The α-diversity of intestinal microflora of diabetic patients and diabetic patients with cardiovascular complications was significantly decreased, and the intestinal microbial communities were unbalanced compared with that of the healthy controls. The majority of dominant bacteria in the healthy control group were from Bacteroidetes and Firmicutes, whereas the majority of dominant bacteria in the diabetic cardiovascular complications group and the diabetic patients group were from Bacteroidetes, Proteobacteria and Actinomycetes. Compared with the healthy controls, the proportion of Firmicutes was significantly decreased, and the proportions of Actinomycetes and Proteobacteria were significantly increased in the diabetic patients with cardiovascular complications and diabetic patients. At the species level, fasting blood glucose had a positive correlation with Lactobacillus mucosae, Escherichia coli and Bacteroides fragilis, and a negative correlation with Eubacterium ramulus, Roseburia inulinivorans, Roseburia hominis, Eubacterium eligens and Ruminococcus callidus, low density lipoprotein cholesterol had a positive correlation with Streptococcus cristatus and a negative correlation with Lactobacillus amylovorus, and total cholesterol had a positive correlation with Streptococcus sanguinis. [Conclusion] T2DM patients and T2DM patients with cardiovascular complications had obvious abnormal glucose and lipid metabolism and intestinal flora imbalance. Intestinal flora disorder may play an important role in the pathogenesis and progression of diabetes.

Abstract:[Objective] To understand the diversity characteristics and possible causes of gut bacterial composition of healthy Tibetan and Han population in the same age group living in the same environment. [Methods] Healthy male students including 20 Tibetan and 20 Han aged 20-24 in the same major of the same campus volunteered for this study. Total DNA was extracted from fresh feces collected at the same day from male healthy Tibetan and Han students in the same grade. The V3-V4 region of 16S rRNA were amplified by PCR then sequenced on Illumina MiSeq platform. The sequencing results were analyzed with bioinformatic softwares combined with Dietary status questionnaire to study the relationship between gut bacterial diversity and dietary habits of healthy Tibetan and Han students. [Results] A total of 2880 OTUs were obtained from 40 samples. Alpha and beta diversity analysis showed that male students from different ethnic groups had significantly different gut bacterial compositions under the same environment. Prevotella 9, Bacteroides, Lachnoclostridium and other 15 genera are shared by both Tibetan and Han students. Meanwhile, the composition and abundance of 10 genera, such as Prevotella 9, Faecalibacterium, Succinivibrio etc., are the biomarkers that have significant influence on the difference of intestinal bacteria composition between the two ethnic students. Further cluster analysis showed that all the student samples were divided into three enterotypes:ET F, ET P, mixed ET F and ET B. The follow-up survey results showed that different enterotypes corresponded to different dietary habits based on fat and starch. [Conclusion] Living in the same environment, the composition and differences of gut bacteria of healthy Tibetan and Han students may be closely related to genetic factors and dietary habits.

Abstract:[Objective] The purpose of this study was to study the intestinal microbial diversity of the caracal cats (Caracal caracal). [Methods] Fecal samples from 7 wild adult caracal cats (2 males and 5 females) were collected, 2 of them were raised in Ji'nan Wildlife Park and 5 of them were raised in Weihai Wildlife Park. High-throughput sequencing of the 16S rRNA gene V3-V4 region of caracal cats was carried out. [Results] A total of 1458741 valid sequences of the V3-V4 region of the 16S rRNA gene was obtained, with an average of 208392 valid sequences and an average sequence length of 433 bp. By classifying with 97% sequence similarity, an average of 233 operational taxonomic units (OTUs) were obtained. These OTUs were all classified into the bacterial domain, including 13 phyla, 26 classes, 43 orders, 75 families, and 119 genera. Among them, the most abundant bacteria are Firmicutes (accounting for 61.7% of all OTUs), Actinobacteria (12.42%), Bacteroidetes (7.79%), Fusobacteroidetes (7.79%) and Proteobacteria (7.53%). The most abundant families were Peptostreptococcaceae (accounting for about 16.15% of all OTUs), Clostridiaceae_I (14.78%), Lachnospiraceae (13.13%) and Coriobacteriaceae (12.31%), etc. The most abundant genera were Collinsella (11.44%), Peptoclostridium (10.91%), Clostridium sensu stricto 1 (10.3%), Bacteroides (7.41%) and Peptostreptococcus (5.21%), etc. An average of 15.35% of the OTUs in the gut microbiota of the 7 caracal cats was unclassified at the genus level. Cluster analysis showed that the samples from the same park were clustered into one branch. [Conclusion] Characteristics of the intestinal microbiota of the caracal cats were described and the results would provide scientific information for the rescue feeding and digestive physiology research of the caracal cats.

Abstract:[Objective] To explore the effect of reducing dietary protein level on plasma hormones associated with growth and microbiota in digesta of ileum and cecum in weaned piglets. [Methods] Fifty-four selected weaned piglets with similar body weight were assigned to three groups with three diets, each diet contained 20% CP, 17% CP or 14% CP supplemented with Lys, Met, Thr and Trp, according to NRC (2012) recommendation. At day 10, 25 and 45, blood samples were collected from jugular vein of six piglets in each group for analysis of blood parameters and hormones associated with growth. At day 45, we collected the digesta from ileum and cecum for microbiota and its metabolites analysis. [Results] At day 25 and 45, diets with 17% CP and 14% CP decreased the concentration of blood urea nitrogen compared to control group (P<0.05). Low protein diets with 14% CP increased the cholesterol concentration at day 45 and increased the concentration of triglyceride at day 25 (P<0.05). But dietary protein level had no effect on glucagon, growth hormone, insulin, T₃ and T₄ in different time point (P>0.05). In phylum level, Firmicutes were dominant bacteria in ileum and cecum, however dietary protein level had no significant effect on the relative abundance of Firmicutes (P>0.05). Reducing dietary protein level tended to increase the relative abundance of Lactobacillus and decreased the relative abundance of Clostridium_sensu_stricto_1, but data was not significant (P>0.05). Reducing dietary protein by 3% or 6% significantly decreased the concentration of ammonia in ileum and cecum (P<0.05). [Conclusion] Low-protein diet with 17% CP or 14% CP had no effect on the concentration of hormones associated with growth, whereas, it decreased the blood urea nitrogen and ammonia in digesta and tended to increase the relative abundance of Lactobacillus. The findings suggest that reducing dietary protein level increase the utilization of nitrogen and contribute to intestinal health.

Abstract:[Objective] To analyze the composition of endosymbiosis microbes of Martianus dermestoides at different stages, we took adult and larva as the research objects. [Methods] By the extraction of endosymbiosis microbes of Martianus dermestoides and high-throughput sequencing analysis, we obtained the composition and changes of microflora in different samples. [Results] At different insect stages, the endosymbiosis microbes abundance and diversity of Martianus dermestoides changed greatly with the development of the insect, the number of bacteria in the adult was relatively small, and the diversity was significantly lower than that in the larva. Different insect states had also significant differences in the composition of several specific microorganisms. Among the adult, Vagococcus and Lactobacillus were dominant bacterial genus; Lactobacillus in larvae was the dominant bacterial genus. The change of fungi was not obvious, and Saccharomyces spp. was dominant. [Conclusion] Our findings provide related information of biotic resources of Martianus dermestoides. Meanwhile, the result can also furnish the theoretical basis for the development and utilization of Martianus dermestoides endosymbiosis microorganism resources.

Abstract:[Objective] To verify if a corn-rich diet could change mice gut microbiota structure and enhance the host carbohydrate metabolism related gut flora genes by using high-throughput sequencing. [Methods] We fed mice for 10 weeks with either normal feedstuff or corn-rich feedstuff (containing 1/4 corn and 3/4 normal feedstuff), and compared the gut microbiome changes by high-throughput sequencing of fecal DNA. [Results] Mice in the two diet groups showed similar body weight after 10 weeks feeding. Fecal DNA of all mice generated high efficiency results, and different diet caused diversity difference of gut flora. The abundances of Bifidobacteriales-B. pseudolongum branch and Coriobacteriia-Collinsella/Enterorhabdus branch under Actinobacteria phylum were significantly higher in corn-rich diet group. Correspondingly, the abundances of phosphoglycerate mutase gene, and functional pathways and modules, including primary and secondary bile acid biosynthesis, as well as glucose transfer to pyruvate module and core module involving three-carbon compounds, were also increased in corn-rich diet group. [Conclusion] Corn-rich diet could promote the abundances of probiotics such as Bifidobacterium in gut microbiota, and raise glucose/lipid metabolic related gene and pathway abundances in microbiome, such influence could potentially improve the host metabolism status.

Abstract:[Objective] This study aimed to investigate the diversity and sex-specific differences in the intestinal microbiota of cheetah (Acinonyx jubatus). [Methods] Fecal samples were collected from 9 healthy adult cheetahs (4 male and 5 female), and intestinal microbiota were assessed through high-throughput sequencing of the V3-V4 region of 16S rRNA gene. [Results] In total, 599349 effective tags were obtained, with an average length of 405 bp. At 97% sequence similarity, 268 operational classification units (OTUs) were obtained and classified into the bacterial domain, including 10 phyla, 21 classes, 35 orders, 72 families, and 144 genera. Based on relative abundance, the 5 most abundant bacterial phyla were Firmicutes (accounting for approximately 42.29% of the total OTUs), Actinobacteria (31.54%), Fusobacteria (16.66%), Proteobacteria (5.30%), and Bacteroidetes (4.19%). The low abundance of phylum Bacteroidetes was the primary feature of the intestinal microbial community of the cheetah. The 5 most abundant families were Coriobacteriaceae (31.28%), Peptostreptococcaceae (17.66%), Fusobacteriaceae (15.46%), Lachnospiraceae (12.40%), and Clostridiaceae_1 (6.93%). The 5 most abundant genera were Collinsella (30.16%), Fusobacterium (15.46%), Peptoclostridium (11.46%), Blautia (8.28%), and Clostridium_sensu_stricto_1 (6.39%). Approximately 2.32% of the total OTUs were unclassified strains at the genus level. Alpha diversity analysis indicated that the Shannon indices of the intestinal microbiota ranged 2.93-4.41; Simpson indices, 0.72-0.91. The observed species and Shannon indices of the intestinal microbiota of male cheetahs were slightly greater than those of female cheetah. Beta diversity analysis revealed that the difference between sexes was greater than that within groups. However, cluster analysis revealed that intestinal microbes of cheetahs of the same sex were not clustered. [Conclusion] The present results show the intestinal microbial diversity of cheetah, and indicate that intestinal microbiota of cheetah do not differ significantly on the basis of sex, thus providing a scientific basis for studies on the conservation of cheetahs and their digestive physiology and for rescuing and feeding cheetahs.

Abstract:The gut microbiota regulates intestinal homeostasis, and also participates in the modulation of brain function and behaviors of hosts. Disorders of gut microbiota are associated with the dysfunction of host nervous system, causing the development of neurodegenerative diseases. Thus, better understanding the role of gut microbiota in the gut-brain axis can provide new strategies for the diagnosis and treatment of nervous system diseases by the targeted modulation of gut microbiota composition and function. In recent years, studies on the interaction between gut microbiota and brain function have been reported, but the underlying mechanisms are still not fully understood. Therefore, combining with the latest research findings from our laboratory, we review here the modulatory effects of gut microbiota on nervous system and the potential role of interaction between gut microbiota and host in the modulation of brain function and behaviors of hosts, aiming to deepen our understanding about the role of gut microbiota in the modulation of brain function and behaviors in hosts.

Abstract:[Objective] Ascosphaera apis is a lethal fungal pathogen for Apis cerana cerana larvae. microRNA can participate in host-pathogen interaction processes by inhibition or degradation of mRNA via targeting at post-transcriptional level. The aim of this study was to analyze the differentially expressed miRNAs (DEmiRNAs) and their target genes in the 6-day-old larval gut of A. c. cerana under A. apis stress and reveal DEmiRNAs' roles in the stress response process. [Methods] Normal and A. apis-challenged 6-day-old larval guts of A. c. cerana (AcCK and AcT) were sequenced using Illumina MiSeq platform, followed by prediction and analysis of DEmiRNAs and their target genes using related softwares. Target genes of DEmiRNAs were annotated to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases with Blast. Regulation networks between DEmiRNAs and target mRNAs were constructed using Cytoscape. Stem-loop RT-PCR and qPCR were conducted to verify the reliability of sequencing data. [Results] Deep sequencing of larval gut samples generated 537 miRNAs, the length of which was distributed between 16 nt and 35 nt. The first base bias of miRNAs with various length had apparent difference. The expression of 10 novel miRNAs were validated using Stem-loop RT-PCR. There were 54 DEmiRNAs in AcCK vs. AcT comparison group, including 31 up-regulated and 23 down-regulated miRNAs, which can respectively link 6170 and 8199 target genes. GO classification suggested that target genes of up-and down-regulated miRNAs were respectively involved in 47 and 47 terms, and the largest ones were binding, cellular process, and catalytic activity. KEGG pathway enrichment analysis demonstrated that target genes of up-and down-regulated miRNAs were respectively engaged in 134 and 126 pathways, and the mostly enriched ones were endocytosis and protein processing in endoplasmic reticulum. Analysis of regulation networks revealed that very complex regulation relationships existed between DEmiRNAs and corresponding target mRNAs; 31 miRNAs could bind 51 mRNAs associated with ubiquitin mediated proteolysis, and 18 miRNAs can bind 14 Jak-STAT signaling pathway-associated mRNAs; a total of 16 miRNAs, such as miR-1277-x, miR-26-x, miR-27-y, miR-30-x and miR-6052-x, can participate in regulating both of the above-mentioned immune pathways. Finally, three DEmiRNAs were randomly selected for qPCR, the result verified the reliability of our transcriptome sequencing data. [Conclusion] We first provided the expression profile and differential expression information of A. c. cerana miRNAs during the late stage of A. apis stress, revealed the complex interactions between A. apis and host at transcriptome level. As the core of regulation networks, miR-6052-x and miR-1277-x were likely to participate in host immune defense by affecting apoptosis, while miR-26-x and miR-30-x may join host responses to A. apis stress via regulation of Jak-STAT signaling pathway. Key DEmiRNAs screened in our study are expected to be used as potential molecular targets for chalkbrood control.

Abstract:[Objective] Investigating the relationship between Lactobacillus animalis LGM and transcription factors (T-bet, GATA3, ROR-γt and Foxp3) gene expressions in vivo or in vitro and observing the effects of L. animalis LGM gavage on DSS-induced colitis.[Methods] L. animalis LGM was isolated from pig colon contents using Hungate rolling tube technique. Bacterial supernatants were extracted and incubated Caco-2 cells with LPS (2 μg/mL) simultaneously. Bacterial suspension was adjusted to 1×10⁹ CFU/0.2 mL and gavaged to DSS-induced mice. Gene expressions of transcription factors (T-bet, GATA-3, ROR-γt and Foxp3) and intracellular cytokines (IL-4, IL-17, INF-γ and IL-10) were analyzed by real-time PCR. [Results] In Caco-2 cells culture experiment, compared to control and LPS groups, L. animalis LGM supernatants treatment significantly downregulated the GATA-3, IL-4, IL-17 and TGF-β gene expressions and upregulated Foxp3 and ROR-γt gene expressions (P<0.05). In DSS-induced colitis mice, compared to the DSS group, L. animalis LGM administration restored the colonic length and increased ROR-γt and Foxp3 gene expressions (P<0.05). [Conclusion] L. animalis LGM isolated from pig colon contents in the present study have distinctive effects on transcription factors. ROR-γt and Foxp3 expressions were significantly upregulated both in Caco-2 cells and DSS-induced colitis mice. L. animalis LGM showed protection on DSS-induced colitis and gut inflammation.

Abstract:Intestinal Segmented Filamentous Bacteria (SFB) is a Gram-positive Clostridium spp. with selective colonization of species, as shown by experiments. SFB mainly colonize on the surface of intestinal epithelial cells at the end of the ileum, to regulate the maturation of host immune system, stimulate the specific differentiation of CD4+ Th17 cells and promote the secretion of intestinal surface immunoglobulin A (sIgA). SFB play very important roles in preventing pathogenic microbial infection and induction of occurrence and development of autoimmune diseases. Although SFB can be detected in many vertebrates, few studies have been reported on human SFB. Studies have shown that SFB can be detected in human intestinal samples, and the investigation of clinical samples shows that there is a certain correlation of SFB in human intestinal tract with immune regulation and disease symptoms. However, due to the extremely low abundance of SFB in human intestinal tract and the possible existence of different SFB strains in the same individual, isolation and pure culture of SFB single bacteria are still the only way to further study the immune regulation function of human SFB.

Abstract:Thousands of bacteria are colonized in the intestinal tract of animals. Lactobacillus reuteri is a probiotic in animal gut and model strain for studying the evolutionary relationship between gut microbiota and host. [Objective] To study genetic diversity and host specificity of different isolates of L. reuteri, we analyzed 132 strains of L. reuteri including 116 strains isolated from human, poultry, rodents and pigs downloaded from NCBI database and 16 strains isolated from cow, sheep and horse in Inner Mongolia. [Methods] Seven housekeeping genes including ddl, pkt, leuS, gyrB, dltA, rpoA, and recA were used as targets in MLST (multilocus sequence typing) technology to study the genetic diversity of 132 L. reuteri and evolutionary relationship between strains and host habitats. [Results] All 132 strains of L. reuteri were assigned into 63 STs and 6 clonal complexes. Recombination analysis revealed that individual recombination events occurred during the evolution of L. reuteri. The eBURST and MSTree analysis showed that L. reuteri from different sources experienced distinct evolutionary processes. The 132 L. reuteri isolates were classified into 5 clusters by phylogenetic analysis, according to their correlation with the sources. [Conclusion] L. reuteri from different sources had high host specificity, indicating that L. reuteri may experience different evolutionary processes to adapt the distinct living environments.

Abstract:[Objective] To provide some theoretical references for further research and development of alkaline cellulase via obtaining a novel alkaline resistance cellulase in bacteria from intestines of earwig, and heterologously express, characterize the functions of the enzyme. [Methods] First, we isolated the bacterial strains from earwig gut samples in Nanyang Baotianman National Reserve Area, Henan province, China by primarily screening according to Congo red plate methods. Then, we obtained and identified the bacterial strains with high alkaline cellulase activities by phenotypic and genotypic characteristics. We designed degenerate primes according to the known endoglucanase gene sequences in GenBank to carry out PCR, analyzed the cloned sequence, and expressed the enzyme in Escherichia coli BL21. [Results] We obtained one bacterial strain with high alkaline cellulase activities named strain Q5. The bacterium was classified to be Bacillus methylotrophicus. The full length of a cellulase gene cDNA (1500 bp) (GenBank KR067575) coding region was successfully cloned. The homogeneous analysis demonstrated that the deduced amino acid of the gene showed 98% similarities with the alkaline β-1,4 endoglycosidase from Bacillus sp. 2190 (ALE32753.1). The activity of the recombined endoglucanase was 3.46 U/mL, which was 1.69 times higher than that of the wild Bacillus methylotrophicus Q5 (2.05 U/mL). The highest cellulase activity reached 4.99 U/mL after orthogonal experiment. The properties of the recombined enzyme were determined. The optimum temperature and pH value were 50℃ and pH 8.5, respectively. The enzyme maintained over 80% of the original enzyme activity at pH 8.0 and pH 9.0 after incubated at 50℃ for 48 h. The enzyme was stable below 50℃ and the activity decreased sharply above 60℃. The activity of this enzyme was activated by 10 mmol/L Ca²⁺和Mg²⁺. The values of K_m and V_max were 2.217 mol/mL and 9.606 μmol/(min·L), respectively. The enzyme showed obviously inhibiting the growth of the cotton pathogenic fungi Verticillum dahliae. [Conclusion] It was the first time we got an alkaline resistance endoglucanasegene from Bacillus methylotrophicus isolated from guts of earwig. Our findings will lay a theoretical foundation for the application in alkaline environments.

Abstract:In recent years, microbiome study based on high-throughput gene sequencing has greatly deepened the understanding of the relationship between gut microbiota and health. However, gene sequencing method cannot directly determine the functional activity of microorganisms. It is difficult to identify the key functional molecules in microorganisms. Application of this single technology cannot answer some key scientific questions which members of gut microbiota and how they affect the host. The disadvantages of singles-omics research are obvious, and it is imperative to perform more multi-omics studies. Metabolomics of the gut microbiota takes all small molecular metabolites of microbial community as the research object. It can discover the key metabolites of intestinal microorganisms with the pathophysiological changes of host. It provides clues for the study of microbial-host interaction mechanism and becomes an important complement to gut functional metagenomics. Integration study of gut functional metagenomics and metabolomics have made a lot of progress in host physiology, disease pathology and other aspects, showing a promising future. However, there are some crucial problems in multi-omics study of gut functional metagenomics and metabolomics, such as misapplication of various methods, resulting in inconsistency between relevant conclusions and biological knowledge. In order to help the proper application of multi-omics study on gut functional metagenomics and metabolomics, this paper reviewed the principles, advantages, disadvantages and application scope of various multi-omics data integration analysis methods, and gave some application suggestions.

Abstract:[Objective] The intestinal flora represents the microbial flora located in the human intestine, and its composition has been related to the occurrence of various diseases in humans. For example, the intestinal flora plays an important role in the pathogenesis of gestational diabetes mellitus (GDM). Therefore, in this study, we evaluated and compared the intestinal flora in pregnant women according to ethnicity (Mongolian or Han) and GDM status using PacBio SMRT sequencing. [Methods] Ninety-seven fecal samples from pregnant women with or without GDM and of Han or Mongolian ethnicity were subjected to full-length 16S rRNA sequencing using the PacBio SMRT system. [Results] Overall, the microbial flora compositions of the pregnant women were similar during the same pregnant stage, and various microbes exhibited different degrees of correlation with core flora levels. We identified 44 species at the species level. The relative abundance of Akkermansia muciniphila was significantly higher in the non-GDM pregnant women than in pregnant women with GDM in the Han population, while no differences were found in the Mongolian women with and without GDM. The relative abundance of Bacteroides uniformis in the intestinal flora of healthy Han pregnant women was significantly higher than that in healthy Mongolian pregnant women, while no differences were found in the GDM groups between ethnicities. Moreover, the functional prediction results showed that the functional composition of the four groups of flora was highly similar, and most of the functional genes were related to energy metabolism. There was no significant difference between the GDM patients and controls in the Han population, but the relative abundance of flora related to inorganic ion transport in Mongolian GDM patients was significantly higher than that of healthy Mongolian pregnant women. [Conclusion] The intestinal flora is generally relatively stable during the same pregnant stage, and ethnic differences do not have a significant impact on the flora during pregnancy. However, changes in some low-abundance organisms such as Akkermansia muciniphila may contribute to alterations in some metabolic activities of the intestinal flora, including those related to intestinal nutrient absorption, which could influence the development of GDM. The results of this study can help elucidate the role of intestinal flora in GDM pathogenesis.