| The gut flora of horses is abundant,varied,and intricate.It contains multiple genes that enhance the energy extraction from food and synthesizes several biologically active molecules with microbial relevance.The understanding of the dwarf horse gut microbiome is currently limited due to the absence of a genome set,which restricts the extraction of macrogenomic data.The equine industry has extensively used antibiotics for disease treatment,growth,development,and health improvement in horses.However,their irrational use has resulted in the development of microbial resistance,leading to dysbiosis of the microbiota that negatively affects the host’s health.Despite this,the genetic profile of antibiotic resistance in equine feces remains largely unknown.This research analyzed the fecal samples of 118 horses belonging to three breeds,namely Debao pony,Ningqiang pony,and Guanzhong horse,from two provinces of China.The study utilized 16 S rRNA and metagenomic sequencing techniques to examine the composition and potential functions of the fecal microbiota in Chinese southern ponies(Debao and Ningqiang ponies)and northern large horses(Guanzhong horses).Moreover,the research also investigated the presence of antibiotic resistance genes in the gut microbiota of horses.The objective of this study was to enhance our knowledge on the microbiota in ponies,including its composition and diversity.Additionally,the study aimed to create a detailed overview of the antibiotic resistance genes present in horse fecal microbiota.Furthermore,a new and highly transferable resistance gene with high minimum inhibitory concentration was identified.The results of this study can be utilized as a valuable resource for promoting healthy breeding practices in horses,constructing reference genomes for the horse gut microbiota,and optimizing the use of antibiotics for treating horse diseases.The following are the specific experimental findings:(1)Analysis of the diversity of fecal microbiota in dwarf horses based on 16 S rRNA sequencing technology.We conducted 16 S rRNA sequencing analysis to examine the fecal microbial diversity of the De Ba,Ni Qi,and Gu Zh groups.The alpha diversity analysis showed that the Ni Qi group had a more abundant and homogeneous faecal microbiota.Moreover,the beta diversity analysis indicated that the faecal microbiota of the De Ba and Ni Qi groups were similar.The analysis of microbial community structure revealed that the F to B ratio was significantly higher in the Gu Zh group(6.66 ± 1.28)than in the De Ba group(1.04 ± 0.55)and the Ni Qi group(1.36 ± 0.41)(P < 0.01).Analysis of genus differences revealed that the relative abundance of Streptococcus and Prevotella was significantly higher in the dwarf horse group(De Ba and Ni Qi)than in the Gu Zh group;the Coprococcus pair abundance was significantly higher in the tall horse group(Gu Zh)than in the dwarf horse group(De Ba and Ni Qi).Analysis of the correlation between genera with >1% abundance and horse height six genera had the highest correlation with body height and may have contributed to horse body height.Functional predictions revealed that the microbial biota of the dwarf horse(De Ba and Ni Qi)group may function more similarly to that of the taller horses.(2)Exploring the composition and function of the microbiome in ponies’ horse feces based on metagenomics.Metagenomics was used to analyze the composition of the microbiome in pony horse feces,including bacteria,archaea,fungi,and viruses.Bacteria were found to be the dominant microbial group,with the phyla Firmicutes and Bacteroidetes being the most abundant in the horse gut.Beta diversity analysis revealed significant differences in the microbiome composition among the three groups of horses,with the Ni Qi group being intermediate between the De Ba and Gu Zh groups.The high-horse group(Gu Zh)was significantly enriched in the phylum Fibrobacteres.At the genus level,De Ba was significantly enriched in the genera Streptococcus and Prevotella,while Gu Zh was significantly enriched in the genera Treponema and Fibrobacter,which can promote cellulose degradation and utilization.The phylum Euryarchaeota was significantly enriched in the De Ba group.Functional annotation of the equine gut microbiome metagenomics revealed that the pathways significantly enriched in the Gu Zh group of KEGG metabolic pathways were all associated with diseases,which may indicate that Guanzhong horses may be at potential risk of multiple diseases;the pathways significantly enriched in the De Ba and Ni Qi groups were dominated by fatty acid and sugar metabolism.COG bifunctional analysis revealed that equine gut microflora functions are mainly involved in host vital activities and basal energy metabolism.The analysis of carbohydrate-active enzymes revealed that the main functions of the significantly enriched glycoside hydrolase family were all involved in the degradation of cellulose,glucan and xylan;In the role of horse manure microorganisms in carbon cycling,it was found that high horses(Gu Zh)are stronger than dwarfs(De Ba)for carbon land use,and we hypothesize that De Ba dwarfs produce large amounts of methane in the process of fiber decomposition,as evidenced by the significant enrichment of Euryarchaeota in the De Ba group.(3)Mining of antibiotic resistance genes in horse feces microorganismsUsing metagenomics technology,the predicted genes were annotated to the NCBI AMRFinder database,and a total of 3,264 antibiotic resistance genes were annotated,of which 2,420 were new antibiotic resistance genes,which greatly expanded the existing ARG repository.In these new ARG,the mechanism of more than 90% of ARG is the inactivation of antibiotics and the protection of target proteins,while less than 10% of ARG is to prevent the penetration of antibiotics.Tracing the source of antibiotic resistance genes,it was found that these ARG were carried by microorganisms from 23 phyla and 146 genera.In addition,we also found 447 novel moble ARGs located in plasmids,mainly macrolide,streptomycin,aminoglycoside and β-lactam antibiotics.Due to the richness of macrolide antibiotics and the frequent use of macrolide antibiotics in China,we constructed expression plasmids to test the activity of resistance genes of three new macrolide antibiotics.We found that the MIC of the recombinant vector carrying Abc-f(9)gene to tilmicosin increased from 64 μg/ml to 256 μg/ml,which greatly enhanced its resistance to macrolides.Although the sensitivity of the recombinant vector carrying Abc-f(9)gene to tylosin was similar to that of the control,MIC>512 μg/ml,the recombinant vector carrying Abc-f(9)gene had higher resistance to tylosin.The MIC of tilmicosin and tylosin in the recombinant vector carrying Abcf(10)and Abc-f(34)genes was close to the control.This study utilized 16 SrRNA sequencing technology and metagenomics to examine the gut microbial composition and potential functions of gut microbes in Chinese dwarf horses.The profile of ARGs in equine gut microbes was analyzed and the activity of antibiotic resistance genes was verified using the minimum inhibitory concentration test.Our study establishes the groundwork and provides data to support future research on the composition of the gut microbiota in horses.This research can serve as a reference for maintaining the health of horses and treating diseases,as well as offering valuable recommendations for the regulation of antibiotics usage. |
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