Metagenomic Studies On Intestinal Microbial Composition And Antibiotic Resistance Of Nine Species Of Primates And Construction Of Analysis System

BackgroundIntestinal microbiome is a complex system that is closely related to human activities.With the widespread use of antibiotics in clinical practice and animal husbandry,a large number of drug-resistant bacteria and even superbacteria are produced.Primates are the closest phylogenetic relatives to humans,and systematic analysis of the composition and drug resistance of their gut microbes will help us understand the influence of the host lifestyle and geography environment on the characteristics of gut microbes and drug resistance,but a large number of microorganisms cannot be cultured in a laboratory condition.Metagenomic sequencing does not require isolation and culture,and can directly extract the total DNA of samples to obtain a lot of useful information.However,the current metagenomic analysis software has a complex installation environment and time-consuming operation process,and no software that can comprehensively analyze the structure and drug resistance of intestinal microorganisms.Objectives1.To study the structure and functional characteristics of intestinal microflora of nine primates with different lifestyles and geographical conditions,and to explore the differences in intestinal microflora of Trachypithecus leucocephalus（T.leucocephalus）adapted to the karst environment.2.To study the characteristics of intestinal microbial resistance in primates with different lifestyles,and to explore the potential correlation between microbes and drug-resistance gene composition.3.To construct a microbial structure and the resistance system framework to provide a reference basis for microorganisms and resistance analysis.MethodsFirstly,we collected 150 fecal samples of nine primates from five sites in Guangxi,extracted the total DNA of the samples,and sequenced them using PE150 strategy on Illumina Hiseq x-ten sequencing platform to obtain the offline data.The Fast QC quality control was carried out,and the host sequence was filtered by BBMAP.Kraken2 was used for species annotation,and software R was used to calculate and visualize the Alpha diversity index of the nine groups of animals.PCA was used to obtain the similarity of intestinal microorganisms among sample groups,and LEf Se analysis was used to obtain the key flora of each group of animals.Species and functions were annotated through HUMAn N2 to mine for potential functional differences and to compare the results of species annotation with those of Kraken2.Secondly,potential antibiotic resistance genes（ARGs）were pre-screened from short reads and long reads by usearch,and then resistance information was obtained from the SARG（V2）database by Blast X comparison.PCA and NMDS were used to obtain the similarity of ARG types and ARG subtypes between samples respectively.In addition,four sets of intestinal microbiota metagenomic data from primates were downloaded from the NCBI SRA database for drug resistance analysis and compared with our data.Spearman correlation analysis was performed on the resistant genes relative abundance that is greater than 0 in at least one sample.Procrustes inferred the correlation between genus-level microorganisms and ARGs in the intestinal microorganism of nine groups of animals.The vegan package,ggplot2 package,corrplot package,Hmisc package were used to analyze the correlation between ARGs and ARGs as well as ARGs and microorganisms and visualized the network on the Gephi platform（https://gephi.org/）.Finally,based on Linux platform,the software used in this study was encapsulated through Python3 to construct the intestinal microbial composition and drug resistance analysis system.Results1.The research results of intestinal microbiota（1）65 phyla were annotated by Kraken2 and 14 phyla were annotated by HUMAn N2;The PCA results showed that the intestinal microbiota of primates living in the same location had great similarity;The diversity index and richness index of intestinal microorganism were the highest in T.leucocephalus and Trachypithecus cristatus（T.cristatus）.（2）The results of STAMP analysis showed that eleven kinds of bacteria related to high temperature and cold resistance with the high relative abundance in the T.leucocephalus group.Moreover,the key flora of T.leucocephalus about cellulose digestion and degradation were obtained by LEf Se analysis;（3）A total of 340 pathways were obtained in this study,among which 288pathways were annotated in limestome langurs and 330 pathways were annotated in other 7 apes and monkeys.Ten biological functional pathways specific to limestome langurs were identified by Venn diagram analysis,and the relative abundance of glucose metabolism-related pathways in the three langurs group was lower than other groups.2.Drug resistance study results（1）A total of 18 antibiotic types were annotated in 150 samples,with relative abundances of 3.82x10^-6-4.47x10^-1,which was relatively low compared with the data download in public databases(1.124x10^-5-1.863).（2）Tetracycline antibiotics show high relative abundance in captive primates.The relative abundance of ARG types in captive animals is generally higher than that in the wild.The relative abundance of ARG types in T.leucocephalus is the lowest,but we also annotate a variety of ARG types.The composition of ARGs was similar among species with the same lifestyle.（3）A total of 521 ARGs were identified in nine primates,with the relative abundance ratio ranging from 1.19x10^-6to 2.3x10^-1.Procrustes and network co-occurrence analysis showed that the bacterial community structure plays an important role in the composition of ARGs,and 34 genura of bacteria may be the potential hosts of 204 ARG subtypes.3.Results of system constructionBased on Linux platform,several mainstream microorganism and drug resistance analysis softwares were integrated,supplemented by Python automation scripts and relevant databases,and the framework of microorganism and drug resistance information analysis system was successfully constructed.Conclusion1.microorganism studiesThe specific flora related to the high temperature and high cold resistance in T.leucocephalus are high and the majority of key flora are related to cellulose digestion and degradation;The relative abundance of glucose metabolism-related pathways of the three langurs（T.leucocephalus,T.francoisi,T.cristatus）is relatively low.Primates of the same diet tend to have consistent richness and diversity.Also,primates of the same genus tend to have similar richness and diversity,while geographical differences have little effect on the richness of intestinal microbial diversity.2.Drug resistance studiesThe relative abundance of ARG types are generally higher in captive than in wild primates;Tetracycline antibiotics are relatively abundant in captive primates,so they should be used less.Co-occurrence network analysis showed that 34 genera of bacteria may be the potential hosts of 204 ARG subtypes.3.Study on system constructionThe system process is assisted by Python and R language to call the software related to each step for analysis,which realizes the microbiota composition,function,resistance analysis,and the visualization of the results of the Metagenomic data.The analysis system can meet the current requirements for the analysis of microbial composition,function and drug resistance.