Identification Of Broad-spectrum Antagonistic Bacteria 4-2,4-19 And Its Safety Evaluation

Objective Probiotics are active microorganisms that produce beneficial effects in the human body.The probiotic preparations currently studied are proteases such as Bifidobacterium and Streptococcus thermophilus that act on indirect micro-ecosystems and function in the body.However,the research and replacement of probiotics that play a beneficial role in the respiratory tract microecological system and play a beneficial role in the respiratory tract.Streptococcus A is a normal flora in the human oropharynx.Related studies have used probiotic preparations containing Streptococcus a pores in conjunction with other drugs to treat respiratory infectious diseases.Compared with antibiotics alone,it produces better results.4-2,4-19.In this study,the species names and taxonomy of the two strains of Streptococcus were identified,and the safety during use was evaluated.Methods(1)The bacterial genomic DNA extraction kit was used to extract the genomes of strains 4-2 and 4-19.The 16 S r RNA and conserved gene fragments were amplified by PCR,and the 16 S r RNA and conserved gene sequences of the two strains were obtained by sequencing.The Gen Bank database was used to align the 16 S r RNA sequences of the two strains.The complete genomes of the two strains were subjected to whole gene sequencing.At the same time,the colony morphology,morphology under microscope and physiological and biochemical characteristics of the strains were detected.(2)A 7-day acute toxicity test(multi-dose toxicity study)and a 28-day sub-acute toxicity test(multi-dose toxicity study)were performed on experimental animals orally.During the test,the body weight and feed intake of the experimental animals were monitored.After the test,blood of the experimental animals was used to determine the blood routine,and throat swabs of the experimental animals were collected for high throughput sequencing.Histopathological examinations were performed on all major organs.The sensitivity of the two strains to various antibiotics commonly used in the genus Streptococcus was determined.Results(1)By comparing the sequencing results of 16 S r RNA genes,the results of the two strains are similar,and both belong to the retardation group in Streptococcus.Both strains have the highest similarity to Streptococcus oralis subsp.Dentisani strain 7747,with a Per.Ident value of 99.03%,and are similar to Streptococcus pseudopneumoniae strain 108,Streptococcus mitis strain ATCC 49456,Streptococcus mitis strain NS51,Streptococcus mitis strain ATCC 49456.,Streptococcus pneumoniae strain NBRC 102642,Streptococcus pneumoniae strain ATCC 33400,Per.Ident values were all greater than 98%.The analysis of the 16 S r RNA gene and the conserved gene evolutionary tree also confirmed this result.At the same time,Streptococcus pneumoniae ATCC 33400,Streptococcus pseudopneumoniae ATCC BAA-960,Streptococcus oralis ATCC 35037,Streptococcus mitis ATCC 49456,and Streptococcus dentisani DSM 27089 were 4-2,4-19 neighboring strains.After culturing,the two strains were observed as round bulges,white,small colonies with alpha hemolysis at the tip size.Light and electron microscopy showed typical streptococcal morphology.Biochemical and fatty acid results showed that strains 4-19 differed from their neighboring strains to varying degrees.The DDH hybridization results of the whole genome sequencing results showed that the values of strains 4-2 and 4-19 reached 99.6%,which was much higher than the critical value of 70%.However,the values of 4-19 and other streptococci in the streptococcus group were less than 70%.(2)In the acute and subacute toxicity tests,no significant reductions in feed intake,body weight and relative organ weight were found between the dose groups and the control group.There were no significant changes in the blood routine items between the dose group and the control group in the subacute toxicity experiment.In pathological examination,no necrosis,fibrosis,infiltration of inflammatory cells,loss of normal structure,and atrophy were found in all organs of male animals in the high-dose group.The results of high-throughput sequencing revealed that the male group given the high dose group of 4-19 significantly increased the number of actinomycetes and the number of proteobacteria significantly decreased compared with the control group.In the drug sensitivity test,strains 4-19 were sensitive to 10 antibiotics commonly used in streptococci.Conclusion(1)The research results show that the strains 4-2,4-19 are different from the known streptococci.It is a new species in the genus Streptococcus that has not been discovered.We named it streptococcus shenyangsis.(2)In the acute and subacute toxicity tests,it is safe to give the experimental animals two oral doses.