| BackgroundThe bacterial biofilm refers to the bacterial community composed by the bacteria and their secreting extracellular polymer after the bacteria attach to the materials,whether the materials being living or non-living.The bacterial biofilm is the floating state where the bacteria exist,which enables them to be more resistant to antibiotics.According to the NIH,about 80 percent of the chronically infectious diseases can be attributed to the so-called "bacterial biofilm".The continuous existence of the pathogens leads to the intractability and the recurrence of the infections,which remains to be a difficulty and poses great challenges to clinical treatment.Qingkailing Injection is composed of Cholic acid,Mother of Pearl(powder),Hyodeoxycholic Acid,Gardenia jasminoides,Buffalo horn(powder),Isatis root,Baicalin,Honeysuckle.It can clear away the heat and the toxic material,eliminate phlegm and free channels and induce resuscitation.Therefore,Qingkailing Injection is effective in the treatment of fever,coma,hemiplegia in the wind,unconsciousness,acute hepatitis and upper respiratory infectious diseases.It is an incomprehensive and non-objective method to evaluate Qingkailing Injection's anti-bacteria effect by its capacity to kill the suspending bacteria as most of the bacterium reside in the form of biofilm.Therefore,this study aims to explore Qingkailing Injection's effect on the bacterial biofilm and the possible mechanisms.ObjectiveThis study explores the effect of Qingkailing Injection on Staphylococcus aureus,methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa,Escherichia coli biofilm,as well as the mechanisms against Staphylococcus aureus biofilm.This study provides experimental basis for Qingkailing's validity in treating bacterial infections and provide new conceptions for the mechanisms and the evaluation for the effects of antibacterial medicine.MethodThe first partQingkailing Injection's effect on Staphylococcus aureus,methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa,Escherichia coll biofilmThe microplate assay were adopted to establish the models of Staphylococcus a ureus,methicillin-resistant Staphylococcus aureus,Pseudomonas aeruginosa,Escherichia coli bacterial biofilm in the forming and the mature stage.The control group,Qingkailing injection group,penicillin group(methicillin group)were set up.Crystal violet staining was employed to detect its inhibitory effect on the total amount of bacterial biofilm.WST or XTT staining were used to measure its effect against the living bacterium in the biofilm.The second partThe mechanism of Qingkailing Injection on Staphylococcus aureus biofilm1.The mature biofilm was cultured in the filter membranes.The control group,the Qingkailing inject ion group,the penicillin group were set up.The Qingkailing group and the penicillin group were treated for 24 hours after the administration,then they are transferred to the LB plate containing penicillin.The biological membrane was located upward,the filter membrane of the same size as the biological membrane was put above the biological membrane,thus a sandwich was formed.The filter paper coated with the media was also put above the sandwich for 4 hours at 37?.The filter paper was transferred to the LB plane for 18—24 hours at 37?,and the inhibitory effect was observed.The antibiotics that penetrated the biofilm were used as the indicators to evaluate the integrity of the biofilms.Meanwhile,the effects of the treatments were also evaluated.2.The BioFlux biofilm systerm was used for cultivating the mature biofilm,the control group,the Qingkailing Injection group and the penicillin groupwere set up.The effects of the drugs against the bacterial biofilms were evaluated at 0min,10min,20min,30min,1h,3h,4h,7h after the treatment.3.Mature biofilms were cultured in glass placed in 6 hole plate.The control group,the Qingkailing Injection group,the penicillin group were set up.SEM samples were prepared after the treatment to observe the effects of drugs on bacteria via scanning electron microscope.4.Mature biofilms were cultured in 15mL centrifuge tubes,the control group,the Qingkailing Injection group,the penicillin group were set up.The TEM samples were prepared after the treatment to observe the effects of drugs on bacterial ultrastructure via the transmission electron microscope.ResultsThe first partQingkailing Injection's effect on Staphylococcus aureus,metlhicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa,Escherichia coli biofilms1.Compared with the control group,the Qingkailing Injection at the concentration of 22500-352mg/L can reduce the amount of total biofilm and living bacteria in biofilm,while penicillin at the concentration of 20-0.039mg/L can reduce the amount of total biofilm and living bacteria in biofilm at the period of the formation of Staphylococcus aureus biofilm.Compared with the control group,Qingkailing Injection at the concentration of 22500-44mg/L had no effect on the amount of total biofilm,and at the concentration of 22500-1406mg/L can reduce the living bacteria in the biofilm;penicillin at the concentration of 20-0.039mg/L had no effect on the of total amount of the biofim and live bacteria in biofilm at the mature period of of Staphylococcus aureus biofilm.2.For the formation of methicillin resistant Staphylococcus aureus biofilm,Compared with the control group,Qingkailing Injection at the concentration of 22500mg/L can reduce the total amount of the biofilm,and at 22500-11250mg/L can reduce theliving bacteria in the biofilm.Methicillin at the concentration of 20-10mg/L can reduce the total amount of biofilm,and at 20mg/L can reduce the living bacteria in the biofilm.Compared with the control group,Qingkailing Injection had no effect on the total concentration of the biofilm,and can reduce the living bacteria in the biofilm from the range of 22500 to 2813mg/L at the mature period of the methicillin-resistant Staphylococcus aureus biofilm Methicillin at the concentration of 20-0.039mg/L had no effect on the biofilm and the living bacteria.3.The formation and mature stage of Pseudomonas aeruginosa,Escherichia coli biofilm,Compared with the control group,Qingkailing Injection at the concentration of 22500-44mg/L had no effect on the total amount of the biofilm and the living bacteria in biofilm at the forming and mature stage of Pseudomonas aeruginosa,Escherichia coli biofilm,.Penicillin at the concentration of 20-0.039mg/L had no effect on the amount of total biofilm and the living bacteria in the biofilm.The second partThe mechanism of Qingkailing Injection on Staphylococcus aureus biofilm1.There are no inhibition zone at control group,indicating that penicillin in LB plate was not penetrate to the bacterial biofilm,Qingkailing group had inhibition zone,which indicated that penicillin in LB plate penetrated into the bacterial biofilm.It also served as an indirect evidence of Qingkailing Injection can damage the bacterial biofilm.The observation that the penicillin group had no inhibition zone suggested that penicillin in LB plate is not penetrate to the bacterial biofilm,which was indirect evidence of penicillin's failure to destroy bacterial biofilm.2.By the BioFlux biof ilm systerm analysis,The Qingkailing Injection group's light density of bacterial biofilm decreased after the treatment for 20 minutes Treatment for 1-3 h,biofilm appeared loose as they gradually spread out and the biofilm structure was destroyed,with wall growth biofilm gradually became into the suspending bacteria.Treatment for 4h-7h,bacterial biof i lm dispersed with wall growth,and thay were full of suspending bacteria within the tube.The penicillin group did not change significantly,and they were with wall growth,structural integrity,and were not destroyed.There were less suspending bacteria in tube.The observations suggested that Qingkailing Injection can damage the mature biofilm structure,make the bacteria in suspension,which was not seen in penicillin.3.Seen from the scanning electron microscopy,the control group showed that the biofilm bacteria was closely arranged,with uniform size,bacterial structure intact,no obvious death cell.And the bacterial surf ace was surrounded by the extracellular matrix and the extracellular matrix were distributed in bacteria,which was identified as a typical biofilm structure.In the Qingkail ing Injection group,the bacterial biofilm structure was loose,and were of all sizes.There were large-sized bacteria,and the wizened bacteria due to the spilling of the contents from the swelling bacteria.The bacteria in the penicillin group were almost normal,with the structure of biofilm being sparse and the arrangement of bacterial biofilm surfacebeing disordered.4.Transmission electron microscopy showed that in the control group,the bacterial cell wall was smooth,and the cytoplasmic evenly-distributed.The newly-born cell walls at the mitosis stage were visible.In the Qingkailing Injection group,the cell wall of bacteria blurred.The cytoplasm was unevenly-distributed,aggregated,and even showed plasmolysis and the vague cell walls.In the penicillin group,the integrity of the bacterial cell walls were not destroyed,and the newly-born cell walls at the mitosis stage were almost invisible,and there were dark staining in the cytoplasm.ConclusionIn the formation of Staphylococcus aureus and wethicillin-resistant Staphylococcus aureus biofilm,Qingkailing Injection can reduce the amount of the living bacteria biomass,inhibited the adhesion of bacterial biofilms.In the mature stage,there seems to be no effect of Qingkailing Injection on the total amount of the biofilm but it can reduce the amount of the living bacteria in biofilm.Thus,we assume that Qingkailing Inject ion may exert its anti-bacterial effect by penetaring the mature bacterial biofilm.The results showed that the difference in the anti-bacterial mechanisms between the Qingkailing Injection and the penicillin.Qingkailing Injection can increase the drug permeability of bacterial biofilm,destroy bacteria biofi lm structure thus damaging thebacterial cytoplasm and cell wall in the biofilm,therefore leading to the death of the bacteria.Qingkailing Injection has the effect of destroying the bacterial biofilm,causing the death of the bacteria in the biofilm.However,the site that the Qingkailing Injection targeted at the bacteria needs more investigations. |
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