Study On Adhesion Characteristics Of Symbiotic Bacteria C-17 And D-19 In Upper Respiratory Tract And Their Inhibition On Pathogen Adhesion

ObjectiveThe respiratory tract is an open channel between the human body and the outside world,which is always stimulated by various microorganisms and external environmental factors.There are reports of mortality related to respiratory infections as the fifth leading cause of death overall.Antibiotics are the most commonly used treatment for respiratory tract infections.However,extensive use of antibiotics may lead to disorders of the respiratory commensal flora,resulting in reduced immunity and increasing the chance of pathogenic bacteria infection.Respiratory commensals maintain a symbiotic relationship with the body and play an important role in the balance of the immune system within the host.Respiratory commensals can inhibit the growth of other pathogens in the respiratory tract through antagonistic action.So bacterial therapy may be a new alternative to the prevention of viral respiratory infections,addressing the problem of resistant strains caused by the abuse of antibiotics.Respiratory commensals,already colonized in healthy respiratory ways,have unique advantages as candidate strains for probiotics.Respiratory probiotics should be selected from the respiratory symbiont flora for in-depth and comprehensive research.The adhesion ability of bacterial strains is an important indicator for the screening of probiotic strains.In this study,two upper respiratory tract commensal strains isolated from the oropharynx of healthy children were identified as novel bacteria of Streptococcus,named C-17 and D-19,which showed broad-spectrum antagonism against common pathogenic bacteria.The purpose of this study was to explore the adhesion properties of two upper respiratory commensals C-17 and D-19 with broad-spectrum antagonistic effects and their adhesion inhibition on pathogens,so as to provide basic research for their research and development as microecology of the respiratory tract.Methods（1）The auto aggregation ability,biofilm forming ability and hydrophobicity of C-17 and D-19 were measured by spectrophotometry,bath method and salting out coagulation method respectively.The adhesion ability of C-17 and D-19 to 16-HBE cells was observed under scanning electron microscope and ordinary microscope.These indexes were used to evaluate the adhesion ability of C-17 and D-19 to 16-HBE cells.（2）The adhesion properties of C-17 and D-19 were determined by plate counting,that is,different times,different concentrations,different inactivation modes,different growth stages,different p H values,different chemical reagents,different sugar solutions,and metabolites on the adhesion properties of C-17 and D-19.（3）The adhesion inhibition effect of C-17 and D-19 on S.aureus and S.aeruginosa was first assessed by fluorescence labeling and plate counting,that is,competition inhibition,exclusion inhibition and replacement inhibition.The effect of different treatment factors（different chemical reagents）on the adhesion inhibition of pathogenic bacteria was then determined by plate counting.Results（1）Strains C-17 and D-19 have good automatic aggregation ability,biofilm forming ability,hydrophobicity and adhesion.And their automatic aggregation ability,biofilm forming ability,hydrophobicity and adhesion performance are positively correlated.（2）The adhesion ability of strains C-17 and D-19 reached the maximum at 2 h,and the adhesion ability did not increase significantly more than 2 h（P>0.05）;When the concentration is 1×10~8 CFU/ml,the adhesion performance is the largest,and when the concentration is greater than 1×10~8 CFU/ml,the adhesion performance does not increase significantly（P>0.05）;After inactivation at different temperatures（65℃for 30 min and100℃for 10 min）,the adhesion of strains C-17 and D-19 decreased significantly compared with the control group（P<0.05）;When the growth stage was the stable stage,the adhesion performance was the largest,which was greater than that in the logarithmic stage and decline stage（P<0.05）;Compared to p H4.0,5.0,6.0,8.0,maximum adhesion performance at p H7.0（P<0.05）;After treatment with 0.25 mg/ml trypsin,5 mol/l Licl,and 60 mg/ml sodium hyperiodate,compared with the control group,their adhesion performance was significantly decreased（P<0.05）;After adding mannose,glucose,fructose and lactose solution respectively,compared with the control group,their adhesion performance decreased significantly（P<0.05）,especially glucose for strain C-17 and lactose for strain D-19.Compared with the supernatant of culture medium,there was no significant difference in the adhesion ability of strains C-17 and D-19 after suspension in fresh liquid medium or PBS buffer（P>0.05）.（3）Strains C-17 and D-19 obtained by fluorescence labeling and plate counting can reduce the adhesion of Staphylococcus aureus and Pseudomonas aeruginosa to 16-HBE cells by competition,exclusion and replacement compared with the control group,the adhesion ability of Staphylococcus aureus and Pseudomonas aeruginosa to 16-HBE cells increased significantly after 25 mg/ml trypsin,5 mol/l Licl and 60 mg/ml sodium periodate treatment（P<0.05）.Conclusions（1）The auto-aggregation,hydrophobicity,biofilm formation and adhesion properties of strains C-17 and D-19 had a positive correlation,and the size of their in vitro adhesion capacity,namely both strains were relatively high to 16-HBE cells.（2）The adhesion of strains C-17 and D-19 to 16-HBE cells will be affected by time,their own body concentration,p H,growth stage,temperature,and body surface composition,and their body metabolites have no effect on their adhesion to 16-HBE cells.（3）Strains C-17 and D-19 could inhibit the adhesion of S.aureus and P.aeruginosa to 16-HBE cells through competition,exclusion,and replacement.At the same time,their surface-related proteins and polysaccharide components play an important role in their adhesion process.