Identification And Safety Evaluation Of Two Symbiotic Strains Of Upper Respiratory Tract

ObjectiveRespiratory tract infection is a common disease in clinical practice,affected by a variety of comprehensive factors,most of which are caused by viral pathogen infection,currently clinical use of antibiotics.However,the use of antibiotics may lead to the disorder of respiratory symbiotic flora,and the decline of the abundance and diversity of respiratory bacterial flora,thus increasing the risk of colonization of pathogenic bacteria,and causing the continuous emergence of various drug-resistant bacteria,which brings great challenges for clinical infection treatment.Respiratory symbiotic bacteria maintain a symbiotic relationship with the body and play an important role in the balance of the immune system in the host.Respiratory symbiotic bacteria can fight respiratory diseases by inhibiting the growth of other pathogenic bacteria occupying the same ecological environment through antagonism.Thus,bacteriotherapy may offer a new and effective alternative therapy in relieving or preventing viral respiratory infections（RTIs）,thus helping to address the global problem of antibiotic resistance.Respiratory symbiotic bacteria themselves are colonized in healthy respiratory tract and maintain a healthy respiratory phenotype,so they have unique advantages as candidate strains of probiotics.Respiratory probiotics should be selected from the respiratory symbiotic flora suitable strains for further in-depth and comprehensive research.First of all,the"identity"of the strain should be determined,and the genus,species and strain of the strain should be identified.Secondly,the safety of probiotics should be evaluated,mainly including antibiotic resistance,animal toxicological examination and bacterial translocation.Through the isolation and screening of the oropharyngeal flora of healthy children in the early stage,the laboratory in which I worked obtained two symbiotic strains 1-4and 4-2 of the upper respiratory tract,which had good antagonistic effects on a variety of pathogenic microorganisms.The main purpose of this study is to identify the species and taxonomic status of these two strains,determine their"identity"and conduct a preliminary evaluation of their safety,so as to provide a research basis for the research and development of these two strains as respiratory microecological living bacteria agents.Methods（1）Classical Gram staining and scanning electron microscopy were used to observe bacterial colony characteristics and bacterial morphology.The growth characteristics and biochemical reaction of bacteria were analyzed by isolation culture and biochemical experiment.The 16S r RNA sequenced after PCR amplification was compared and analyzed using Gen Bank database.Complete genome sequencing of the two strains was completed and their basic genomic information was analyzed.（2）Animal toxicity test is divided into acute toxicity test and subacute toxicity test.Mice in experimental group were orally treated with high concentration(1×10¹⁰CFU/ml),medium concentration（1×10⁹CFU/ml）and low concentration（1×10⁸CFU/ml）of bacterial suspension,respectively.The mice in control group were treated with normal saline orally.The acute toxicity test was taken orally once and observed for 7 days after the end.The subacute toxicity test was taken orally once a day for 28 consecutive days.The indicators monitored in animal toxicity experiment were activity state and toxic signs of mice,body weight,feed intake,blood routine,serum biochemical indexes,histological pathology of main organs,and whether the phenomenon of bacterial translocation and growth occurred.At the same time,the respiratory tract flora of mice was collected by throat swabs for sequencing analysis.The drug resistance of two strains of streptococcus was analyzed by paper diffusion method.Results（1）After the two strains were cultured on the blood plate at 37℃for 24 h,the colonies were greyish-white,smooth,and the edges were neat and pinpoint-like.After 48 h of culture,there was slightαhemolysis.Under ordinary optical microscope,the bacteria were Gram-positive cocci with chain arrangement.Under scanning electron microscope,the bacteria were typical streptococci with single bacteria being round or oval,without spore and flagella.Both strains could grow at22℃to 42℃,and the best growth condition was at 37℃.It does not grow at4℃and 15℃.The experimental strain could grow on 2.5%Na Cl sheep blood AGAR plates,but could not tolerate higher concentrations of salt.Biochemical reaction results showed that the experimental strain could ferment glucose,lactose,sucrose,inulin and other sugars and encode alkaline phosphatase,esterase,lipidesterase and other enzymes,which were different from other known neighboring species in the same genus.16S r RNA sequence alignment analysis showed that strain1-4 was closely related to Streptococcus mitis strain ATCC 49456,Streptococcus mitis strain NS51 and Streptococcus oralis subsp.dentisani strain 7747.Strin 4-2 is closely related to Streptococcus Rubneri LMG 27207 and Streptococcus Australis ATCC 700641.The whole genome length of strain 1-4 was 1,941,455bp,with 1892coding genes,40.17%G+C content,44 non-coding RNA（nc RNA）and 5 gene islands.The whole genome length of strain 4-2 was 2,072,632bp,with 2035 coding genes,43.26%G+C content,49 non-coding RNA（nc RNA）and 7 gene islands.The results of DDH-DDH online hybridization between the whole genomes of the two strains and other known neighboring species in Streptococcus were less than70%.Strain 1-4 genome annotation NR database,1755 genes annotated to streptococcus,accounting for 96.2%of the total gene NR database annotation,4-2strains of genome annotation NR database,1952 genes annotated to streptococcus,accounting for 99.0%of the total gene annotation NR database,through compare with the COG and KEGG database and functional annotation,the results showed that strain 1-4 and 4-2 most of the genes involved in cell metabolism,second more gene enrichment in the strain of information storage and biological process and signal processing.（2）There was no death of mice during the animal experiment.The weight growth trend of mice in the two Streptococcus strains with different doses was normal,and there was no statistical difference in feed intake and organ index between the two Streptococcus strains and the control group.The blood routine and serum biochemical indexes of the high-dose group were not significantly different from those of the normal saline group.There were no pathological changes in the main organs,and no bacteria translocation and growth were found in the high-dose group.Both strains 1-4 and 4-2 were sensitive to 9 common antibiotics,and only strain 4-2 was resistant to erythromycin.Conclusion（1）Based on morphological,physiological and biochemical analysis,16S r RAN sequence and whole genome sequence analysis,we believe that Strains 1-4 and4-2 are two new species of Streptococcus.（2）The safety evaluation results of the two strains confirmed that they had no toxic effect on mice,showing high safety of translocation,and were generally sensitive to most common antibiotics.The preliminary results showed that they had good safety,which provided a research basis for their future development and application.