Isolation,Screening And Safety Evaluation Of Broad Spectrum Antagonists 1-2 And 3-21 From Respiratory Tract

ObjectiveIn order to find the antagonistic strains which have antibacterial properties and can antagonize common pathogens in respiratory tract,the safety of antagonistic strains was studied by acute and subacute general toxicity assay in vivo of mice.Methods（1）Methods:The oropharyngeal swabs were collected from 19 healthy children aged 4-6,and then coated on 5%sheep blood agar plate.After 24 hours of incubation at37℃,108 dominant strains with grass green hemolytic ring were selected.According to the color,size,shape,surface and edge,35 strains without grass green hemolytic ring were selected.To detect their antagonistic effects against staphylococcus aureus,E.coli and Pseudomonas aeruginosa using oxford cup method for initial screening after three generations of isolation and purification.Afterwards,the same methods were used to detect their antagonistic effects on Klebsiella pneumoniae,Acinetobacter baumannii,Proteus vulgaris,Haemophilus influenzae,Candida albicans and Streptococcus pneumoniae for secondary screening.Finally,the strains with good inhibitory effect on many pathogenic bacteria were screened,and their genomes and 16S r RNA fragments were sequenced and compared,and then the taxonomic status of antagonistic strains was determined by genome-wide sequence analysis.Its biological characteristics were studied by morphological features,culture characteristics and biochemical reaction.（2）Kunming mice were randomly divided into control group（normal saline）,low dose group（1x10⁸ CFU/ml）,medium dose group（1x10⁹CFU/ml）and high dose group(1x10¹⁰CFU/ml).The general acute toxicity test lasted for 7 days and the general subacute toxicity test lasted for 28 days.During the experiment,the weight and feed intake of mice were recorded every day.After the experiment,the blood routine test of mice in subacute toxicity high-dose group was collected to detect the blood routine,and the main organs were taken for histopathological examination.Results（1）In the preliminary screening experiment,27 of 108 strains with grass green hemolytic ring had obvious inhibitory effect on Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa.The 35 strains without grass green hemolytic ring had no inhibitory effect on the three pathogenic bacteria.Through further screening,7 of 27strains had obvious inhibitory effect on Klebsiella pneumoniae,Acinetobacter baumannii,Proteus vulgaris,Haemophilus influenzae,Candida albicans and Streptococcus pneumoniae.The strains were labeled as 1-2、1-3、3-21、3-4、1-3、11-4、19-4 respectively.16S r RNA sequence alignment showed that 1-2 and 1-3 were homologous to Streptococcus,3-21 was a subspecies of Streptococcus,3-4 and 11-4 were two subspecies of oral Streptococcus,and 7-3 and 19-4 were tardive Streptococcus.The results of susceptibility test to common antibiotics showed that 1-2 and 3-21 had lower drug resistance,which was more suitable for potential probiotics.The whole-genome online hybridization results showed that the similarity between strains 1-2 and 3-21 and their adjacent species was less than 70%.Under ordinary light microscope and electron microscope,it could be observed that strains 1-2 and 3-21 were gram-positive,flagella-free,spore free,and long-chain arranged coccus.After plate culture,it was observed that the two strains were both round and raised,white.Needle-size microscopic colonies with an alpha hemolytic ring.（2）In general acute and subacute toxicity tests,there were no significant differences in feed intake,weight and relative organ weight between the two groups.In the general subacute toxicity test,there was no significant difference in routine blood index between each dose group and the control group.The result of HE staining showed that there was no histopathological change in the high dose group.The result of high throughput showed that the control group,1-2 high-dose group and 3-21 high-dose group were dominated by Firmicutes and Proteus at phylum level as well as Streptococcus,pasteurella and Mycobacterium at genus level.Conclusions（1）The results showed that strains 1-2 and 3-21 showed different degree of inhibition to various pathogenic bacteria.（2）In the acute and subacute toxicity test,bacterial fluid containing two strains was safe to be administered to experimental animals by pharyngeal drip irrigation.