| Diseases of the mare reproductive system can lead to a decrease in horse reproductive efficiency,thereby affecting the transformation,upgrading,and sustainable development of the horse industry.At present,vaginal Lactobacilli(primarily Lactobacillus)play an important role in maintaining the balance of vaginal microbiota and preventing and treating vaginal bacterial infections.In this study,a Lactobacillus salivarius strain,named Y20,was isolated from the vaginal mucus of healthy Mongolian horses using traditional microbiological methods.It was identified as Lactobacillus salivarius by modern biotechnological methods.The biological characteristics and prebiotic properties of this strain were analyzed in depth.Finally,the whole genome sequencing and comparative genome analysis were carried out through the second-generation and third-generation high-throughput sequencing technology.This study focuses on the vaginal secretions of healthy Mongolian horses,and the main research content and results are as follows:(1)Screening and Identification of Lactic Acid Bacteria in the Vagina of Mongolian Horses.80 strains of lactic acid producing bacteria were isolated through selective screening medium(bromocresol purple screening medium,litmus milk chromogenic medium).Subsequently,8 candidate Lactic Acid Bacteria were further screened through acid resistance tests,growth performance tests,and acid production tests.Through morphological identification of these 8 strains,combined with acid resistance,growth performance,and acid production tests,a Lactobacillus Y20 with excellent acid resistance,growth performance,and acid production was comprehensively screened.The strain Y20 was finally identified as Lactobacillus salivarius and named as Lactobacillus salivarius Y20 by comprehensive evaluation of physiological and biochemical,Biolog Gene III microbial identification system and molecular biology.(2)Detection and analysis of the biological characteristics of Lactobacillus salivarius Y20.Lactobacillus salivarius Y20 has good heat resistance,as it could still survive at75 ℃.Y20 has good bacteriostatic performance,and has bacteriostatic effects on Escherichia coli,Staphylococcus aureus,Salmonella,Gardnerella vaginalis,beta hemolytic streptococcus,and Streptococcus equi subspecies zooepidemicus.Among them,the inhibitory effect on Staphylococcus aureus is the best,with an bacteriostatic diameter of 20.4233 ± 0.1297 mm.Y20 has good hydrogen peroxide production ability and excellent antioxidant ability,with a DPPH free radical clearance rate of 73.77%,a T-AOC value of67.49 U/m L,and a T-SOD value of 59.66 U/m L.Y20 showed sensitivity to carbenicillin,guaiacillin,cefradine and doxycycline.It was non-hemolytic,possessed good autoaggregation and biofilm formation abilities,and was preliminarily determined as a safe strain.(3)Genome-wide sequencing analysis of Lactobacillus salivarius Y20.The genome of Lactobacillus salivarius Y20 was assembled into a circular chromosome(size of 1.74 Mb,GC content of 33.01%)and two plasmids.Within the circular chromosome,1,602 genes,six transposons,two CRISPRs,three genomic islands,and one prophage were predicted.Functional annotation results showed that KEGG annotated the most genes(800)to metabolism-related pathways.GO annotation screened 17 genes related to antioxidant activity(such as thioredoxin).COG annotation assigned 105 genes to carbohydrate transport and metabolism,131 genes to amino acid transport and metabolism,and 19 genes to secondary metabolite biosynthesis,transport,and catabolism,etc.Pfam annotation identified 2,203 genes associated with protein families and domains.CAZy annotated to 5classes of enzymes related to carbohydrate enzymes,and 241 enzymes were annotated in total..The number of secreted proteins predicted to have signal peptide structures was 62,the number of proteins predicted to be associated with transmembrane structures was 35,and the number of secreted proteins was 27.Three bacteriocins were identified in the Y20 genome,one of which had low similarity to the identified bacteriocins,suggesting the possibility of a novel bacteriocin..(4)Comparative genomic analysis of Lactobacillus salivarius Y20.The comparative genomic analysis of Lactobacillus salivarius Y20 with Lactobacillus salivarius IBB3154(from hens),JCM1046(from pigs),LPM01(from humans),2D(from horses),and S01(from fish)was performed.The results of collinearity showed that the corresponding regions of Y20 and 2D had more reverse matching regions.Y20 exhibited the highest number of single nucleotide polymorphisms(SNPs)with IBB3154(29,845 SNPs),while the lowest number was observed with 2D.Y20 had the highest number of insertion sites with S01 and the lowest number with 2D.Similarly,Y20 had the highest number of deletion sites with S01 and the lowest number with 2D..In Structural variation(SV)statistics,the inverted site coverage area of horse derived Lactobacillus salivary Y20 and2 D is particularly large,ranging from 0.4 to 1.4 Mbp.Population evolutionary analysis revealed 65 genes specific to Y20,of which immune-related genes and unique genes between Y20 and other strains were analyzed,the Y20-specific immune gene was RAMP protein csm5,and single nucleotide variants of immune genes common to Y20 and Lactobacillus faecalis salivarius of equine origin were also enumerated;by species evolutionary tree analysis,Y20 and Lactobacillus salivarius 2D of equine origin were found to be in the same branch.In conclusion,Lactobacillus salivarius Y20 is a horse-derived vaginal lactic acid bacterium with excellent probiotic properties.It serves as a potential candidate strain for the development of future equine probiotic formulations. |
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