Streptococcus E24 Inhibits The Adhesion And Colonization Of Staphylococcus Aureus In Respiratory Tract

ObjectiveThere are a large number of microorganisms on the surface of each organ of the human body.The microbiota can ensure the normal nutrition metabolism and maintain the microecological stability through the interaction with the host cells.Staphylococcus aureus is one of the important pathogenic bacteria causing hospital acquired pneumonia（hospital acquired pneumonia,HAP）,especially Methicillin-resistant Staphylococcus aureus（MRSA）has brought great trouble to clinical treatment,and colonization is the first step that it usually causes invasive disease.Previous antibiotic therapies tend to disrupt the microecological balance of the respiratory tract and increase drug-resistant strains.The physiological Streptococci of human pharynx exhibit antagonism and colonization resistance to related bacteria and play a role in balancing mucosal flora and alleviating inflammation.Our research group isolated the Streptococcal E24 in the oropharynx of healthy children,this research through the inhibition of S.aureus in vitro and in vivo experiment,adhesion human bronchial epithelial cells（16HBE）and in mice respiratory engraftment of related research,explore E24 potential profit by nature.Methods（1）The growth curves of S.aureus and E24 strains were measured,and the concentration of S.aureus（1×10~5CFU/ml）and E24（1×10~7CFU/ml,1×10~8CFU/ml,1×10~9CFU/ml）were prepared.After co-incubation with human bronchial epithelial cells16HBE for 30,60,90,120,150 and 180 min,the bacterial adhesion cells were observed by plate counting method,and the adhesion morphology was observed by gram staining and scanning electron microscopy.Bacteria and cells were co-incubated for 2,4,6 and 8h,and cell activity was detected using MTT kit.After incubation for 12h,cell supernatant was collected and the levels of inflammatory cytokines IL-6,IL-8 and TNF-αwere detected by ELISAkit.（2）The mice were given S.aureus（10~5CFU/ml）and E24（10~8CFU/ml,10~9CFU/ml）by aerosol inhalation.Mouse throat swabs were collected at 1-day intervals and plate-counted,α-hemolytic Streptococcus was also identified by 16Sr RNA,dynamic monitoring of both colonization and inhibitory colonization.At the end of the experiment,the pharyngeal swabs of mice were collected for high-throughput sequencing,and the serum of mice was collected.The levels of inflammatory cytokines IL-6,IL-8 and TNF-αwere detected by ELISA kit,and the histopathological observation of the trachea and lung tissues of mice was conducted.Results（1）The logarithmic phase of strain E24 was 4～14h,and the stationary phase was14～22h;The logarithmic phase of S.aureus growth is 4～18h,and the stationary phase is18～24h.The adhesive force of E24 reached the highest at 1×10~9CFU/ml for 120min.When the concentration of S.aureus was 1×10~5CFU/ml,the adhesion reached the highest value at180 min.When E24 and S.aureus were co-cultured with 16HBE cells for 120 min and 180min,each concentration of E24 inhibited the adhesion rate of S.aureus to varying degrees,and the inhibition was most obvious at the concentration of 1×10~9CFU/ml for 180min（P<0.0001）.At the corresponding concentration,the cell viability of S.aureus group decreased at 4h（P<0.05）,while the cell viability of high dose E24 induced an increase（P<0.01）.When E24 and 16HBE cells were co-cultured for 60 min,the thalli were attached around or on the surface of cells in granular form,and when 120 min,the thalli were attached around cells in chain form,which was more clear under scanning electron microscope.S.aureus resulted in increased levels of cytokines（P<0.0001）,and E24effectively reduced TNF-αlevels（P<0.0001）.（2）Mice were continuously atomized with S.aureus for 14 days,and then atomized with different concentrations of E24 for 8 days.Compared with mice treated with S.aureus alone,the colonization density of S.aureus decreased on days 19,21 and 23,and the inhibition was most obvious on days 21 and 23,（at 21 days,P<0.0001 in the medium dose group of S.aureus+E24,P<0.001 in the high dose group of S.aureus+E24,at 23 days,P<0.0001 in the medium dose group of S.aureus+E24 and P<0.0001 in the high dose group of S.aureus+E24）.The results of serum inflammatory factor detection suggest that high dose E24 can effectively reduce the serum TNF-αlevel in mice（P<0.05）.Pathological examination showed that Streptococcus E24 alleviated the inflammatory response caused by S.aureus and maintained the balance of respiratory tract microecology to a certain extent.ConclusionStreptococcus E24 can inhibit the adhesion of Staphylococcus aureus to bronchial epithelial cells 16HBE,and inhibit the colonization of Staphylococcus aureus in mouse respiratory tract in animal experiments.Moreover,Streptococcus E24 can play a certain immunomodulatory role and maintain the microecological balance of respiratory tract,which has a potential beneficial effect.